CN101707966B - Living Chinese caterpillar fungi artificially cultivated by using larvae as hosts and method for cultivating same - Google Patents
Living Chinese caterpillar fungi artificially cultivated by using larvae as hosts and method for cultivating same Download PDFInfo
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- CN101707966B CN101707966B CN2009100492984A CN200910049298A CN101707966B CN 101707966 B CN101707966 B CN 101707966B CN 2009100492984 A CN2009100492984 A CN 2009100492984A CN 200910049298 A CN200910049298 A CN 200910049298A CN 101707966 B CN101707966 B CN 101707966B
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Abstract
The invention provides a living Chinese caterpillar fungi artificially cultivated by using larvae as hosts, which comprise insect larvae and one or two about two centimeter living Chinese caterpillar fungus fruiting bodies grown on the head of each the larva. The cultivating process comprises the steps of: invasion of the Chinese caterpillar fungi into the living insect larvae, in vivo growth of the hyphae in the living insect larvae, inactivation of the larvae, growth of the Chinese caterpillar fungus fruiting bodies on the heads and the like; and in the whole process, not only the insect larvae and the hyphae are grown in the polypide, but also the Chinese caterpillar fungus fruiting bodies are grown on the heads, the obtained Chinese caterpillar fungi have the same appearance and almost the same nutrient content as the wild Chinese caterpillar fungi, so that the Chinese caterpillar fungi obtained by the method are the real living Chinese caterpillar fungi obtained by artificial cultivation.
Description
Technical field
The present invention relates to a kind of living Chinese caterpillar of artificial culture, relating in particular to a kind of is the living Chinese caterpillar and the breeding method thereof of host's artificial culture with the larva.
Background technology
Cordyceps sinensis (Cordyceps Sinensis (Berk.) Sacc.) claim CORDYCEPS, Chinese caterpillar fungus again, and it is that section ergot fungus cordyceps sinensis colonizes in the ossify complex of body of the aweto fruit body that forms on the larva of host's Hepialidae insect and larva.Whenever midsummer, on the forest grassy marshland or lawn of high height above sea level, the bat moth is stayed worm's ovum on the floral leaf, behind the moth ovum adult, creeps in the moist loose soil, absorbs nourishing and growing of plant roots and stems.Spherical ascospore can infect Chinese caterpillar fungus bat width of cloth moth larvae, sprouts mycelia in the larva body.In addition, when eating the leaf of aweto fungus, the larva of Chinese caterpillar fungus bat moth also might become Chinese caterpillar fungus.
The stroma Dan Sheng of wild Chinese caterpillar fungus, rare 2-4, elongated cylindrical, long 4-7 centimetre, about 3 millimeters of diameter; Surface dark-brown or sepia have thin vertical wrinkle, and expand slightly on top, and matter is pliable and tough, the section off-white color.The little raw meat of gas, mildly bitter flavor, contain crude protein, several amino acids, cordycepic acid (D-mannitol), cordycepin (3 '-deoxyadenosin, cordycepin), ergosterol, galactomannans, uracil, adenine etc.
Cordyceps sinensis not only has regulating action to the function of the various internal organs of human body, also has the adjusting functions of immune system; Regulate the respiratory system function; Improve the body tolerance to cold and lessen fatigue; Improve the heart hypoxia-bearing capability, reduce consumption and the anti-arrhythmia of heart oxygen; Can alleviate noxious material to the damage of liver and the generation of liver fibrosis; Can alleviate the renal lesions of chronic disease, improve renal function, alleviate the infringement of toxicant kidney; Strengthen marrow and generate blood platelet, red blood cell and leukocytic ability; Reduce cholesterol and triglycerides in the blood, improve high-density lipoprotein (HDL), thereby alleviate atherosclerotic.Containing cordycepin in the Cordyceps sinensis, is the main component of its performance antitumor action.Cordycepic acid also has the raising plasma osmolarity, causes in the in-house moisture intravasation, thereby alleviates tissue edema, replenishes blood plasma.
Cordyceps militaris (cordyceps.Militaris (L.:Fr.) Link) has another name called northern Chinese caterpillar Fungus, is Ascomycotina (Ascomycotina) ergot Zoopagales (Clavicipitales), Clavicipitaceae (Cordycepps) fungi.The stroma list is given birth to or had several to send from the insect head, sends from joint sometimes, and is orange-yellow, branch scarcely, high 2-5 centimetre, head clavate, long 1-2 centimetre, slightly 0.3-0.5 centimetre.Domesticly found first in Jilin Province in 1958, be tested and appraised, think that it and Cordyceps sinensis are same genus, name and be pupa grass (C.militaris (L.) Link), be born on the pupal cell of lepidopterous insects in softwood forest, broad leaved forest or the mixed forest surface soil layer, also have and find to infect on pupa, cocoon, adult or the larva body of insects such as coleoptera and diptera.Detect through the Norman Bethune Medical University and to show, some nutrition of Cordyceps militaris and drug ingedient as: cordycepin, Cordyceps sinensis polysaccharide, protein etc. also are higher than Cordyceps sinensis, aspect pharmacological function and clinical effectiveness with the Cordyceps sinensis basically identical.Present medical research proves that Cordyceps militaris not only contains rich in protein and amino acid, and contains the trace element of more than 30 kind of needed by human body.
Though natural cs or Cordyceps militaris are medicinal and be of high nutritive value, its limited amount and gather difficulty, people have carried out extensive exploration aspect the artificial culture Cordyceps militaris for this reason.Chinese invention patent application 97105853.9 discloses a kind of high-yield cultivating method of cordyceps, adopt Cordyceps militaris Paecilomyces varioti bacterial classification (Paecilomyces mlitaris (Kob.) Br.etSm) to be incubated on the synthetic medium of fine quality rice Ensure Liquid liquid, 23 ± 2 ℃ of pH8-8.5, temperature, lucifuge was cultivated 7-10 days, after mycelia is covered with medium, carry out surperficial mycelium stimulation and thermal stimulation, the illumination that shines such as penetrates at mode, through obtaining mature sporophore in 20-25 days.Chinese invention patent application 03133807.0 discloses a kind of Cordyceps militaris food and Cordyceps militaris matrix and preparation method thereof, it is inoculated in cordyceps liquid in the middle of the medium made from rice, milk and potato etc., through making after lucifuge fermented and cultured and astigmatic fermented and cultured and the light filling fermented and cultured.Chinese invention patent application 200610117998.9 discloses a kind of industrial method for culturing north winter of worm summer herb with high-content of cordycepin, this method adopts bacterial strain Cordyceps militaris C0B201, the CGMCC No.1823 of screening to be inoculated in the improvement potato dextrose agar, 20 ± 1 ℃ of temperature, humidity 65-95%, intensity 1500-3500lx illumination was cultivated 45-60 days down in 9 hours, obtained the northern Chinese caterpillar Fungus of worm summer herb with high-content of cordycepin.These methods are Cordyceps militaris and the edible and medical fungi planting type basically identical that main medium carries out artificial culture with rice or potato.
Chinese invention patent application 94112056.2 discloses the high-yield quick artificial culturing method of a kind of Cordyceps militaris, its conidium with cordyceps is made the steamed stuffed bun suspension, dip in insect needle or analog and to get bacterium liquid, thrust silkworm chrysalis, treat nutrient hypha fully growth in vivo, after silkworm chrysalis is stiff, covers one deck thereon or stiff pupa is inserted on the porose material of easily preserving moisture, under suitable temperature, humidity, light, cultivate and to obtain a large amount of Cordyceps militaris with porous material.
Chinese invention patent 97113190.2 discloses the artificial culture method of Chinese caterpillar funguses such as a kind of Cordyceps militaris, the constituent that this method adopts Isaria japonica Yasuda, worm flower Isaria, Cordyceps militaris and mountain, Kaohsiung Chinese caterpillar fungus to be inoculated in silkworm chrysalis is on the liquid nutrient medium of main component, or on the silkworm chrysalis that obtains with the artificial feed aseptic breeding of silkworm, under suitable environment, cultivate the fruit body identical with natural cs.
It is host's fruiting bodies of cordyceps militaris and breeding method thereof with the yellow meal worm that Chinese invention patent application 200510101348.0 discloses a kind of, adopt cordyceps after 0.05-0.2ml is injected in the yellow meal worm shirtfront, the yellow meal worm that is infected is placed dark cultivation of preserving moisture in the middle of the aseptic medium, its condition is: relative moisture 50-100%, temperature 18-25 ℃, to the yellow meal worm body surface white hypha appears; Carry out light induction then, intensity of illumination requires at 1000-2000lx, to body surface mycelia yellowing, produces cordycep militaris fruiting body entity primodium at the pupal cell table, continues to cultivate the fruiting bodies of cordyceps militaris of gathering 50-60 days when pupal cell begins atrophy.Chinese invention patent application 200810013831.7 discloses a kind of breeding method of silkworm chrysalis Cordyceps sinensis, forms silkworm chrysalis Cordyceps sinensis with the wild natural cordyceps bacterial strain spore inoculating that originates in Qinghai-Tibet Platean artificial culture to organic living silkworm chrysalises.
The cultivation method of artificial Chinese caterpillar fungus mainly is divided into the rice to be medium and to be host's fruiting bodies of cordyceps militaris two big classes with the silkworm chrysalis.With rice or potato is the Cordyceps militaris that main medium carries out artificial culture, and it has only fruit body and does not have insect bodies, can only can be regarded as " Cordyceps Militaris mushroom ".And be the Cordyceps militaris that the host cultivates with the silkworm chrysalis, be Cordyceps militaris with the pupa parasitism, the difference of essence is arranged with wild Chinese caterpillar fungus in appearance, infect larva with Cordyceps Militaris under the wild environment and the process of growing also has very big-difference in its body.In addition, these methods cultivate the cordyceps militaris sporocarp that obtains mostly many grow thickly mussily on medium or pupal cell.Still not occurring at present is the host fully with the insect larvae, at head growth 1-2 root, and more than the long 2cm, the living Chinese caterpillar similar to wild Chinese caterpillar fungus.
Summary of the invention
It is the living Chinese caterpillar of host's artificial culture with the larva that one object of the present invention is to provide a kind of.The live body insect larvae head that infects through Cordyceps Militaris grows cordyceps militaris sporocarp, and it is similar to the wild Chinese caterpillar fungus process of growth, possesses the form and the composition of wild Chinese caterpillar fungus simultaneously.
It is the living Chinese caterpillar breeding method of host's artificial culture with the larva that another object of the present invention is to provide a kind of.Through cultivating earlier Cordyceps Militaris liquid, again Cordyceps Militaris bacterium liquid being infected insect larvae, the mycelia of cultivation grow in the larva body, larva is ossify and step such as cultivation living Chinese caterpillar after, the insect larvae head grows the 1-2 root, length is greater than the living Chinese caterpillar fruit body of 2cm.
Insect larvae of the present invention is selected from Arthropoda, barley worm, silkworm and the psychid of Insecta.
Barley worm (Barley pest) belongs to Arthropoda, Insecta, coleoptera, plan goes on foot the Staphylinidae insect, is main feed with wheat bran, gains the name thus.Its larva removes and to contain thick protein 51%, outside the fat 29%, also contains trace elements such as various saccharides, amino acid, vitamin, hormone, enzyme and phosphorus, iron, potassium, sodium and calcium, is of high nutritive value, also as the bait of pet fish.
Silkworm belongs to Arthropoda, Insecta, Lepidoptera, various in style, as: silkworm, tussah, eri silkworm, cassava silkworm, Coriaria sinica silkworm, Hui Lican, giant silkworm, amber silkworm, wild silkworm, chestnut silkworm, Philosamia cynthia, Chinese tallow tree silkworm and willow silkworm etc., preferentially select silkworm (Bombyx mori Linaeus).To should be understood to only be explanation that " silkworm " carried out and unrestricted to cited concrete silkworm kind among the present invention, and the silkworm kind specifically do not listed of other that can meet also can be used as the host that Cordyceps Militaris infects, grows and breaks up for those skilled in the art.
Psychid belongs to Arthropoda, Insecta, Lepidoptera, and is various in style, as: big nest psychid, little nest psychid, the color psychid of wax, black pigment used by women in ancient times to paint their eyebrows psychid, by psychid, line loose psychid, tea tree tea bagworm, the white capsule psychid of tea tree and eurycyttarus nigriplaga etc.To should be understood to only be explanation that " psychid " carried out and unrestricted to cited concrete psychid kind among the present invention, and the psychid kind specifically do not listed of other that can meet also can be used as the host that Cordyceps Militaris infects, grows and breaks up for those skilled in the art.
After Cordyceps Militaris infected above-mentioned insect larvae, Chinese caterpillar fungus hypha was grown in the larva body, and larva is ossify after mycelia is covered with polypide, and cordyceps militaris sporocarp grows from the larva head.
A kind of is the living Chinese caterpillar of host's artificial culture with the larva, comprises insect larvae and the 1-2 root that grows in the larva head, and length is the above living Chinese caterpillar fruit body of 2cm.
Another kind of is the living Chinese caterpillar of host's artificial culture with the larva, comprises insect larvae and the 1-2 root that grows in the larva head, and length is the living Chinese caterpillar fruit body of 2-5cm.
Cordycepin 〉=0.5 of containing in the middle of polypide and the living Chinese caterpillar fruit body ‰.
Another kind of is the living Chinese caterpillar of host's artificial culture with the larva, comprises insect larvae and the 1-2 root that grows in the larva head, and length is the living Chinese caterpillar fruit body of 2-5cm, cordycepin 〉=0.5 ‰ that its polypide and living Chinese caterpillar fruit body contain.
Another kind of is the living Chinese caterpillar of host's artificial culture with the larva, comprises insect larvae and the 1-2 root that grows in the larva head, and length is the living Chinese caterpillar fruit body of 2-5cm, cordycepin 〉=1.0 ‰ that its polypide and living Chinese caterpillar fruit body contain.
Cordycepic acid 〉=4.5% that contains in the middle of polypide and the living Chinese caterpillar fruit body.
Another kind of is the living Chinese caterpillar of host's artificial culture with the larva, comprises insect larvae and the 1-2 root that grows in the larva head, and length is the living Chinese caterpillar fruit body of 2-5cm, cordycepic acid 〉=4.5% that its polypide and living Chinese caterpillar fruit body contain.
Another kind of is the living Chinese caterpillar of host's artificial culture with the larva, comprise insect larvae and the 1-2 root that grows in the larva head, length is the living Chinese caterpillar fruit body of 2-5cm, the cordycepin that its polypide and living Chinese caterpillar contain 〉=0.5 ‰, cordycepic acid 〉=4.5% that polypide and living Chinese caterpillar fruit body contain.
Another kind of is the living Chinese caterpillar of host's artificial culture with the larva, comprise insect larvae and the 1-2 root that grows in the larva head, length is the living Chinese caterpillar fruit body of 2-5cm, the cordycepin that its polypide and living Chinese caterpillar contain 〉=1.0 ‰, cordycepic acid 〉=4.5% that polypide and living Chinese caterpillar fruit body contain.
Above-mentioned various be the living Chinese caterpillar of host's artificial culture with the larva, breeding method is followed successively by: the Cordyceps Militaris bacterium liquid that will cultivate in advance infects insect larvae, cultivates the living Chinese caterpillar fruit body again, obtains living Chinese caterpillar at last.Mycelia grows in live body insect larvae body, larva is all ossify because of Chinese caterpillar fungus hypha is covered with and head length goes out processes such as live body cordyceps militaris sporocarp passing through successively in the middle of the Chinese caterpillar fungus incubation.
Cordyceps Militaris bacterium liquid culture medium should comprise carbon source, nitrogenous source, trace element and vitamin etc.
In the artificial culture process, the general carbon source that is combined as with glucose, sucrose, maltose, starch, pectin or its arbitrary proportion, and preferentially select small molecular sugar classes such as glucose, sucrose.
Required nitrogenous source can be selected organic nitrogen compounds such as amino acid, peptone, soybean cake powder and dried silkworm chrysalis meal in the Cordyceps Militaris process of growth, also can the selective chlorination ammonium, inorganic nitrogen compound such as sodium nitrate, diammonium hydrogen phosphate.Wherein, utilize effect best with organic nitrogen especially.
Satisfy the needs of Chinese caterpillar fungus to trace element by add inorganic salts in the middle of medium, elements such as phosphorus, potassium, calcium and magnesium are the most important.
Owing to can not synthesize essential vitamins voluntarily in the Chinese caterpillar fungus hypha process of growth, should in the middle of medium, add a certain amount of vitamin.
In addition, the height of temperature also has bigger influence to the growth of Chinese caterpillar fungus.Generally speaking, mycelial growth temperature is 6 ℃-30 ℃, temperature the suitableeest growth in the time of 18 ℃-22 ℃.The sporophore growth temperature is controlled at 10 ℃-25 ℃, and the suitableeest production temperature then is generally 15 ℃-23 ℃.The original hase differentiation phase is owing to need stimulate by the bigger temperature difference, and the temperature difference generally should remain on 5 ℃-10 ℃.
Moisture is the somatic important component part of Cordyceps Militaris.Vegetative stage, moisture content in medium remains on 60%-65%, and relative air humidity remains on 60%-70%; In the sporophore growth stage, moisture content in medium will reach 65%-70%, and relative air humidity remains on 80%-90%.In the cultivating process of Chinese caterpillar fungus, be also noted that ventilation.
Spore germination and vegetative stage do not need illumination, should keep dark surrounds.But be transformed into the scattered light that generative growth phase need become clear, intensity of illumination is 1000-2400lx.
Cultivate Cordyceps Militaris:
A kind of Cordyceps Militaris culture fluid, each composition is composed as follows in every 1000ml liquid: glucose 1-2% (w/v), potassium dihydrogen phosphate 0.1-0.2% (w/v), magnesium sulfate 0.1-0.15% (w/v), milk powder 0.5% (w/v), beef extract 1% (w/v), potato filtered fluid 200ml and 2 tablets of vitamin B compounds.
Cordyceps Militaris is inoculated in above-mentioned in the middle of the cordyceps culture fluid of high-temperature sterilization, cultivates after 5-7 days for 20-30 ℃ and obtain Cordyceps Militaris bacterium liquid.
Another kind of Cordyceps Militaris culture fluid, each composition is composed as follows in every 1000ml liquid: glucose 1-2% (w/v), potassium dihydrogen phosphate 0.1-0.2% (w/v), magnesium sulfate 0.1-0.15% (w/v), milk powder 0.5% (w/v), beef extract 1% (w/v), potato filtered fluid 200ml and 2 tablets of vitamin B compounds.
Cordyceps Militaris is inoculated in above-mentioned in the middle of the Cordyceps Militaris culture fluid of high-temperature sterilization, 10-18 ℃ of shake-flask culture obtains Cordyceps Militaris bacterium liquid after 45 days.
In the middle of the above-mentioned Cordyceps Militaris culture fluid, every tablet of vitamin B compound contains 3mg vitamin B1,1.5mg vitamin B2,0.2mg vitamin B6,10mg vitamin PP and 1mg calcium pantothenate.Also contain auxiliary materials such as calcium bicarbonate, starch, dextrin, tartaric acid and sodium carboxymethyl starch.
Cordyceps Militaris infects the live body insect larvae:
A kind of Cordyceps Militaris infects the method for live body insect larvae, at temperature 22-30 ℃, under the environment of relative moisture 60-70%, insect larvae is fed more than 15 days in the middle of Cordyceps Militaris liquid is mixed in feed by elder generation, with above-mentioned Cordyceps Militaris bacterium liquid spray live body insect larvae, make Cordyceps Militaris infect larva afterwards.
According to different insect larvaes, prepare different feeds.With the barley worm is example, can sneak into a certain amount of Cordyceps Militaris liquid in feed in advance.
A kind of barley worm larvae feeds comprises 5-20% (w/w) corn flour, 1-9% (w/w) Cordyceps Militaris bacterium liquid and vitamin B compound, and its surplus is supplied by wheat bran, wherein drops into 10 tablets of vitamin B compounds in the middle of the per kilogram Cordyceps Militaris bacterium liquid.
Another kind of barley worm larvae feeds comprises 10-20% (w/w) corn flour, 1-7% (w/w) Cordyceps Militaris bacterium liquid and vitamin B compound, and its surplus is supplied by wheat bran, wherein drops into 10 tablets of vitamin B compounds in the middle of the per kilogram Cordyceps Militaris bacterium liquid.
Another kind of barley worm larvae feeds comprises 80% (w/w) wheat bran, 15% (w/w) corn flour, 5% (w/w) Cordyceps Militaris bacterium liquid and vitamin B compound, wherein drops into 10 tablets of vitamin B compounds in the middle of the per kilogram Cordyceps Militaris bacterium liquid.
In the middle of the above-mentioned feed, every tablet of vitamin B compound contains 3mg vitamin B1,1.5mg vitamin B2,0.2mg vitamin B6,10mg vitamin PP and 1mg calcium pantothenate.Also contain auxiliary materials such as calcium bicarbonate, starch, dextrin, tartaric acid and sodium carboxymethyl starch.
Silkworm feed is generally mulberry leaf, Cordyceps Militaris liquid is sprayed on feeds silkworm larvae behind the mulberry leaf more than 15 days, with above-mentioned Cordyceps Militaris bacterium liquid spray live body silkworm larvae, makes Cordyceps Militaris infect larva afterwards.
The psychid feed is generally Folium Pterocaryae, Cordyceps Militaris liquid is sprayed on feeds the psychid larva behind the Folium Pterocaryae more than 15 days, with above-mentioned Cordyceps Militaris bacterium liquid spray live body psychid larva, makes Cordyceps Militaris infect larva afterwards.
Cultivate living Chinese caterpillar:
It is 10-30 ℃ that barley worm, silkworm or the psychid larva of infecting through Cordyceps Militaris places temperature, cultivates more than 60 days under the condition of relative moisture 85-95%, obtains the insect larvae head and grows the 1-2 root, the living Chinese caterpillar fruit body of long 2-5cm.Along with the continuation of incubation time prolongs, fruit body still can continued growth and is made its length surpass 5cm.
The beneficial effect that technical solution of the present invention realizes
The Chinese caterpillar fungus that the present invention cultivates for the Cordyceps Militaris host with the insect larvae is made up of insect larvae and the fruit body that grows in the larva head.Incubation comprises that Cordyceps Militaris infects that the larva of live body barley worm, silkworm or psychid, mycelia grow in the larva body, larva is ossify and head grows processes such as cordyceps militaris sporocarp, existing insect larvae of whole process and mycelia grow in polypide, cordyceps militaris sporocarp is arranged again, gained Chinese caterpillar fungus outward appearance is identical with wild cordyceps, the nutrient component basically identical.
The present invention adopts earlier the larva with Cordyceps Militaris forage feed barley worm, silkworm or psychid, the mode of bacterium liquid spray larva has been realized Cordyceps Militaris to the infecting of the larva of active barley worm, silkworm or psychid again, and infection rate reaches more than 85%.The larva of barley worm, silkworm or psychid after infecting only grows the 1-2 root at the larva head through cultivating, and length is the real living Chinese caterpillar that obtains by artificial culture greater than the cordyceps militaris sporocarp of 2cm.
Term involved in the present invention is identical with its universal in this area.
Described " Chinese caterpillar fungus " comprises Cordyceps sinensis and Cordyceps militaris.
Described " Cordyceps Militaris " is aweto (Cordyceps Sinensis (Berk.) Sacc.) or cordyceps (Cordyceps.Militaris (L.:Fr.) Link).
Described " living Chinese caterpillar " is for after Cordyceps Militaris infects the live body insect larvae, again through cultivating a kind of Chinese caterpillar fungus that obtains.
Described " cordyceps militaris sporocarp " or " fruit body " are for growing in the Cordyceps sinensis (Cordyceps Sinensis (Berk.) Sacc.) or Cordyceps militaris (Cordyceps.Militaris (L.:Fr.) Link) fruit body of insect larvae head." living Chinese caterpillar fruit body " is for after Cordyceps Militaris infects the live body insect larvae, again through cultivating the cordyceps militaris sporocarp that grows in the larva head that obtains.
Described " barley worm larvae feeds ", wherein various components are that radix calculates with the gross weight of feed all, and insufficient section is supplied with wheat bran, and the gross weight that makes feed is 100% (w/w); The weight of vitamin B compound is not counted in the gross weight of feed.
Described " vitamin B compound ", its listed auxiliary material only are abundant disclosed necessity of vitamin B complex tablet and to enumerating of its component and non-limiting.It is a kind of carbon source that various auxiliary materials should be understood to, as: starch, dextrin, tartaric acid or sodium carboxymethyl starch, or trace element, as: calcium bicarbonate and sodium carboxymethyl starch.These components content in the middle of culture solution is extremely low, and it does not have main effect to thalli growth.
Described " Cordyceps Militaris bacterium liquid and cultural method thereof " is that conventional microorganism is cultivated, list the abundant disclosed needs of realizing breeding method involved in the present invention just to satisfying at this, the concrete composition of bacterium liquid culture medium and the concrete mode of cultivation can not limit the present invention.Those skilled in the art can be by regulating medium composition or use mode such as solid culture medium instead and realize cultivation Cordyceps Militaris.As: potassium dihydrogen phosphate can directly be selected from various dihydric phosphate (H
2PO
4 -), or pass through to phosphate, hydrophosphate (HPO
4 2-) the solution mode that adds alkaline solution prepares; Cobastab is a vitamin B group, and it can directly drop into culture solution by single kind or multiple this family vitamin that obtains after fermentation or the chemosynthesis, or mixes the back according to certain proportioning and drop into culture solution; Use the constituent of silkworm chrysalis, as: individuality, tissue juice and dried silkworm chrysalis meal etc., one or more of replacement milk powder, potato and beef extract provide necessary protein, trace element, glycogen and lipid etc. for thalli growth.
Description of drawings
Fig. 1 cultivates the living Chinese caterpillar that obtains behind the barley worm larva for cordyceps infects, wherein, and " 1 " expression living Chinese caterpillar fruit body, " 2 " expression polypide;
Fig. 2 cultivates the living Chinese caterpillar that obtains behind the barley worm larva for cordyceps infects, wherein, and " 3 " expression living Chinese caterpillar fruit body, " 4 " expression polypide;
Fig. 3 cultivates the living Chinese caterpillar that obtains behind the silkworm larvae for cordyceps infects, wherein, and " 5 " expression living Chinese caterpillar fruit body, " 6 " expression polypide;
Fig. 4 cultivates the living Chinese caterpillar that obtains behind the psychid larva for cordyceps infects, wherein, and " 7 " expression living Chinese caterpillar fruit body, " 8 " expression polypide.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with accompanying drawing.The embodiment of the invention is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.
The used reagent of the present invention is not if clearly indicate, then all available from Sigma-aldrich (Sigma-Aldrich).
Prepare every 1000ml culture fluid according to component ratio.Wherein, glucose 2% (w/v), potassium dihydrogen phosphate 0.2% (w/v), magnesium sulfate 0.15% (w/v), milk powder 0.5% (w/v) (the bright dairy industry in Shanghai Co., Ltd), beef extract 1% (w/v) (Invitrogen company), potato filtered fluid 200ml and 2 tablets of vitamin B compounds (Shanghai nine good fortune pharmaceutcal corporation, Ltds).
Add the 200g potato in every 1000ml water, boil to filter after half an hour and make the potato filtered fluid.
Cordyceps (Academy of Agricultural Sciences, Shanghai City) is inoculated in above-mentioned in the middle of the cordyceps culture fluid of high-temperature sterilization, 20-30 ℃ obtained cordyceps bacterium liquid after shake-flask culture 5-7 days.
Be made into live body barley worm larvae feeds with dropping into 10 tablets of vitamin B compounds in the middle of 88% (w/w) wheat bran, 10% (w/w) corn flour, 2% (w/w) cordyceps bacterium liquid (embodiment 1 cultivates) and the per kilogram cordyceps bacterium liquid.
At temperature 22-30 ℃, under the relative moisture 60-70% environment, with this forage feed barley worm larva (rare insect breeds the research center in south China) 16 days.Afterwards, the cordyceps bacterium liquid that embodiment 1 is cultivated sprays active barley worm larva, makes cordyceps infect larva.
Be made into live body barley worm larvae feeds with dropping into 10 tablets of vitamin B compounds in the middle of 77% (w/w) wheat bran, 15% (w/w) corn flour, 8% (w/w) cordyceps bacterium liquid (embodiment 1 cultivates) and the per kilogram cordyceps bacterium liquid.
At temperature 22-30 ℃, under the relative moisture 60-70% environment, with this forage feed barley worm larva 25 days.Afterwards, the cordyceps bacterium liquid that embodiment 1 is cultivated sprays active barley worm larva, makes cordyceps infect larva.
Be made into live body barley worm larvae feeds with dropping into 10 tablets of vitamin B compounds in the middle of 80% (w/w) wheat bran, 15% (w/w) corn flour, 5% (w/w) cordyceps bacterium liquid (embodiment 1 cultivates) and the per kilogram cordyceps bacterium liquid.
At temperature 22-30 ℃, under the relative moisture 60-70% environment, with this forage feed barley worm larva 19 days.Afterwards, the cordyceps bacterium liquid that embodiment 1 is cultivated sprays active barley worm larva, makes cordyceps infect larva.
It is 20-30 ℃ that the barley worm larva of infecting through embodiment 2-4 cordyceps places temperature, cultivates under the condition of relative moisture 90-95%, can grow the 1-2 root at barley worm larva head, and the living Chinese caterpillar fruit body of long 2-5cm obtains barley worm larva living Chinese caterpillar.(seeing Fig. 1 and Fig. 2)
The content of fruit body cordycepic acid can be selected 2005 editions, and " Chinese pharmacopoeia is about the detection method of mannitol.Have several different methods detect cordycepin in the fruit body (special product research [J], 2008,4,75-78), mostly by the HPLC method detect (Northwest University's journal (natural science edition) [J], 2003,33,569-571).
Present embodiment is cultivated the Chinese caterpillar fungus that obtains and is measured through edible fungus quality supervision and test test center of the Ministry of Agriculture (Shanghai), and the Cordyceps militaris that cultivation obtains and the cordycepic acid content of wild cordyceps are approaching, and its cordycepin content is far above wild Chinese caterpillar fungus.(seeing Table 1)
Chinese caterpillar fungus cultivated by table 1 and wild Chinese caterpillar fungus compares
Prepare every 1000ml culture fluid according to component ratio.Glucose 2% (w/v), potassium dihydrogen phosphate 0.2% (w/v), magnesium sulfate 0.15% (w/v), milk powder 0.5% (w/v) (the bright dairy industry in Shanghai Co., Ltd), beef extract 1% (w/v) (Invitrogen company), potato filtered fluid 200ml and 2 tablets of vitamin B compounds (Shanghai nine good fortune pharmaceutcal corporation, Ltds).
Add the 200g potato in every 1000ml water, boil to filter after half an hour and make the potato filtered fluid.
Aweto (Qinghai Province's agriculture and animal husbandry academy of sciences) is inoculated in above-mentioned in the middle of the Cordyceps Militaris culture fluid of high-temperature sterilization, 10-18 ℃ of shake-flask culture obtains Cordyceps Militaris bacterium liquid after 45 days.
Be made into live body barley worm larvae feeds with dropping into 10 tablets of vitamin B compounds in the middle of 88% (w/w) wheat bran, 10% (w/w) corn flour, 2% (w/w) Cordyceps Militaris bacterium liquid (embodiment 6 cultivates) and the per kilogram Cordyceps Militaris bacterium liquid.
At temperature 22-30 ℃, under the relative moisture 60-70% environment, with this forage feed barley worm larva 16 days.Afterwards, the aweto bacterium liquid that embodiment 6 is cultivated sprays active barley worm larva, makes cordyceps infect larva.At 10-30 ℃, to cultivate more than 60 days under the condition of relative moisture 85-95%, barley worm larva head can grow the 1-2 root, and the living Chinese caterpillar fruit body of long 2-5cm obtains barley worm larva living Chinese caterpillar.
At temperature 22-30 ℃, under the relative moisture 60-70% environment, there are the mulberry leaf of Cordyceps Militaris liquid to feed silkworm larvae (Zhejiang silkworm industry research institute) 18 days this sprinkling.Afterwards, the cordyceps bacterium liquid that embodiment 1 is cultivated sprays active silkworm larvae, makes cordyceps infect larva.
It is 20-30 ℃ that the barley worm larva of infecting through cordyceps places temperature, cultivates under the condition of relative moisture 90-95%, can grow the 1-2 root at barley worm larva head, the living Chinese caterpillar fruit body of long 2-5cm.At 10-30 ℃, to cultivate more than 60 days under the condition of relative moisture 85-95%, barley worm larva head can grow the 1-2 root, and the living Chinese caterpillar fruit body of long 2-5cm obtains the silkworm larvae living Chinese caterpillar.(see figure 3)
Embodiment 9 live body psychid larvae cultivation living Chinese caterpillars
At temperature 22-30 ℃, under the relative moisture 60-70% environment, there are the mulberry leaf of Cordyceps Militaris liquid to feed psychid larva (Academy of Agricultural Sciences, Shanghai City) 16 days this sprinkling.Afterwards, the cordyceps bacterium liquid that embodiment 1 is cultivated sprays active psychid larva, makes cordyceps infect larva.
It is 20-30 ℃ that the barley worm larva of infecting through cordyceps places temperature, cultivates under the condition of relative moisture 90-95%, can grow the 1-2 root at barley worm larva head, the living Chinese caterpillar fruit body of long 2-5cm.At 10-30 ℃, to cultivate more than 60 days under the condition of relative moisture 85-95%, barley worm larva head can grow the 1-2 root, and the living Chinese caterpillar fruit body of long 2-5cm obtains psychid larva living Chinese caterpillar.(see figure 4)
Claims (7)
1. one kind is the method for host's artificial culture living Chinese caterpillar with the larva, be included in temperature 22-30 ℃, under the environment of relative moisture 60-70%, earlier with forage feed barley worm larva more than 15 days, afterwards with Cordyceps Militaris bacterium liquid spray live body barley worm larva, make Cordyceps Militaris infect described barley worm larva,, cultivate more than 60 days under the condition of relative moisture 85-95% afterwards in temperature 10-30 ℃;
Described Cordyceps Militaris is inoculated in the middle of the Cordyceps Militaris culture fluid of high-temperature sterilization, cultivates after 5-7 days for 20-30 ℃ and obtains described Cordyceps Militaris bacterium liquid;
The described culture fluid of every 1000ml consists of: glucose 1-2w/v%, potassium dihydrogen phosphate 0.1-0.2w/v%, magnesium sulfate 0.1-0.15w/v%, milk powder 0.5w/v%, beef extract 1w/v%, potato filtered fluid 200ml and 2 tablets of vitamin B compounds;
Described feed comprises 5-20w/w% corn flour and the described Cordyceps Militaris bacterium of 1-9w/w% liquid, and surplus is supplied by wheat bran; Wherein drop into 10 tablets of vitamin B compounds in the middle of the described Cordyceps Militaris bacterium of the per kilogram liquid, the weight of described vitamin B compound is not counted in the gross weight of described feed;
Every tablet of described vitamin B compound contains 3mg vitamin B1,1.5mg vitamin B2,0.2mg vitamin B6,10mg vitamin PP, 1mg calcium pantothenate and auxiliary material, and described auxiliary material is calcium bicarbonate, starch, dextrin, tartaric acid and sodium carboxymethyl starch.
2. according to claim 1 is the method for host's artificial culture living Chinese caterpillar with the larva, and described feed comprises 10-20w/w% corn flour and the described Cordyceps Militaris bacterium of 1-7w/w% liquid, and its surplus is supplied by wheat bran; Wherein drop into 10 tablets of vitamin B compounds in the middle of the described Cordyceps Militaris bacterium of the per kilogram liquid, the weight of described vitamin B compound is not counted in the gross weight of described feed.
3. according to claim 1 is the method for host's artificial culture living Chinese caterpillar with the larva, and described feed comprises 80w/w% wheat bran, 15w/w% corn flour and the described Cordyceps Militaris bacterium of 5w/w% liquid; Wherein drop into 10 tablets of vitamin B compounds in the middle of the described Cordyceps Militaris bacterium of the per kilogram liquid, the weight of described vitamin B compound is not counted in the gross weight of described feed.
One kind according to claim 1 be the living Chinese caterpillar that the method for host's artificial culture living Chinese caterpillar is cultivated with the larva, comprise barley worm larva and grow in the 1-2 root of described barley worm larva head that length is greater than the living Chinese caterpillar fruit body of 2cm.
5. living Chinese caterpillar according to claim 4 is characterized in that cordycepin 〉=0.5 ‰ that barley worm larva polypide and cordyceps militaris sporocarp contain.
6. living Chinese caterpillar according to claim 4 is characterized in that cordycepin 〉=1.0 ‰ that barley worm larva polypide and cordyceps militaris sporocarp contain.
7. living Chinese caterpillar according to claim 4 is characterized in that cordycepic acid 〉=4.5% that barley worm larva polypide and cordyceps militaris sporocarp contain.
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| CN102523912A (en) * | 2011-11-27 | 2012-07-04 | 桐乡市中泰虫草专业合作社 | Cordyceps militaris fruiting body with silkworm larvae as hosts and cultivation method of cordyceps militaris fruiting body |
| CN103477863B (en) * | 2012-06-07 | 2015-05-20 | 金勇� | Chinese caterpillar fungus-growing device |
| CN102792855B (en) * | 2012-09-13 | 2014-03-05 | 重庆市中药研究院 | Cordyceps sinensis host infection strain material and method |
| CN103340091B (en) * | 2013-06-20 | 2014-09-17 | 洞口县老年科技工作者协会 | Method for artificially cultivating xuefengensis |
| CN103636408A (en) * | 2013-12-18 | 2014-03-19 | 钱国琛 | Factory-like production method of silkworm cordyceps |
| CN103766139A (en) * | 2014-01-11 | 2014-05-07 | 金勇� | Method for producing worm grass |
| CN106550764A (en) * | 2015-09-30 | 2017-04-05 | 黄友鹰 | A kind of SOYBEAN IN HIGH-YIELD BREEDING method of Cordyceps sinensis |
| CN105602857B (en) * | 2016-01-05 | 2018-07-13 | 江苏农林职业技术学院 | A kind of optimization method that wild cicada fungus liquid spawn is manually cultivated |
| CN105900691A (en) * | 2016-05-24 | 2016-08-31 | 兰陵县政和菌业有限公司 | Technological method for cultivating cordyceps militaris by means of corncobs |
| CN106116754A (en) * | 2016-06-24 | 2016-11-16 | 北京黄安坨科技开发有限公司 | Cultivate the culture medium of Cordyceps militaris, the preparation of culture medium and the breeding method of Cordyceps militaris |
| CN106962025A (en) * | 2017-05-20 | 2017-07-21 | 天津市东方中滨农业科技有限公司 | A kind of method that utilization LED lamplight cultivates Cordyceps militaris |
| CN110637677A (en) * | 2019-08-12 | 2020-01-03 | 江门市山海堂虫草有限公司 | Culture medium for improving cordycepin of cordyceps militaris and preparation method thereof |
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