CN1109103C - Artificial culture of jiuzhou caterpillar fungus and the application of its sporophore - Google Patents
Artificial culture of jiuzhou caterpillar fungus and the application of its sporophore Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The present invention relates to a method for artificially culturing cordyceps kyushuensis, and the application of sporocarps thereof. In the method, wild cordyceps kyushuensis carries out monospore or single colony purification, the purified strains are transplanted back to a culture medium which is raised under an aseptic condition and takes larvae of living greenish-brown hawk moths as the major constituents, natural infection is carried out under optimal culture conditions, and sporocarps which are identical to natural cordyceps kyushuensis are cultured. The method overcomes the problem of serious strain abortion existing in the prior art, and has the advantages of stable strain, short periodicity, capability of continuous and bulk production, and high quantity of effective nutrient component. Pure sporocarps are added into medicine to make capsules and tablets, or used as the raw material of medicined diet, health food, oral liquid, beverage, tonic wine, etc. The dosage is low, but the drug effects are obvious.
Description
The present invention relates to a kind of artificial culture method of microbial fungi, the particularly new Chinese record species of finding first in China---the artificial culture method of Cordyceps kyushuensis (Cordyceps KyushuensisY.Kobayasi) and the application of sporophore thereof.
Cordyceps is commonly used and common fungi, and wild Chinese caterpillar fungus has multiple pharmacologic action, and tonic effect has become traditional precious Chinese medicine, extremely human consumer's favor.But because the very shortage of its wild resource, and growing environment is special, and wild amount is limited, adds excessively and excavates, and far can not satisfy the demands.For this reason, many scientific workers are being devoted to the artificial culture of Chinese caterpillar fungus, attempt to solve the problem of existing wild Chinese caterpillar fungus shortage of resources.Artificial culture method according to some Cordyceps sinensis fungus of relevant bibliographical information mainly contains: " industrial process of Cordyceps mycelium " (patent No. 85107991) and " cordyceps species 814 seed selections and zymotechnique thereof " (patent No. 85102231), all be to carry out deep layer with its isolating pure strain to cultivate, obtain mycelium with zymotechnique, up to the present, it has only been finished asexual generation, still can not artificial culture." artificial culturing method of silkworm chrysalis Cordyceps sinensis " (patent No. 87106987) is to utilize the wheel skill bacterium of Cordyceps militaris (L.) Link. to be inoculated on the solid medium, treats that mycelia is long to a certain degree putting into silkworm chrysalis again, allows its natural infection and grows sporophore.Its incubation time is long, and sporophore and pupal cell one is reinstated when being used as medicine, because of pupal cell is big, sporophore only account for its about 1/5th, the intravital effective constituent major part of pupa has been transformed on the sporophore, historical facts or anecdotes border effect is affected." method of artificial culture Brazil Chinese caterpillar fungus " (90109380.7) are to utilize Brazilian Cordyceps to be inoculated on the silkworm, and obtain worm, bacterium combination.Its application still is not pure sporophore." Chinese caterpillar fungus obviously increases the spleen weight of mouse, and discovery causes composition that spleen heavily increases at stroma, and polypide is invalid according to " Tianjin pharmacy " 98 years the 10th the 1st phase of volume." visible Chinese caterpillar fungus be used as medicine drug action mainly in sporophore, be not polypide.China in " China is by the hair spore and finish the technology of perfect stage Cordyceps sinensis " (88103453) is the anamorph of Cordyceps sinensis by the hair spore, its bacterial classification will be through the generation spore that cultivates one's ability of some months, contaminate host's Hepialus oblifurcus Chu et Wang larva with asexual spore again, after 10 degree left and right sides subzero treatment, 18 ℃ of left and right sides heat insulating culture, grew stroma through two months, whole culture cycle is longer.Because the abortion phenomenon in its spore exists, thereby germination rate is low, inoculation back parasitic rate is also low, and sporophore is difficult to form, and therefore influences it and applies.
The inventor once disclosed the tame method of Cordyceps militaris (L.) Link. (having another name called Cordyceps militaris) in " method of artificially cultivating north Chinese caterpiller fungus daughter vaccine " (93105056.1), " seed selection of cordyceps militaris excellent species and high yield cultivating method " (94110061.8), " Cordyceps militaris (L.) Link. high yield cultivating method " patent documentations such as (97105853.0).This method utilization is the solid medium of major ingredient with the rice, successfully cultivates Cordyceps militaris (L.) Link., but its matrix is single, the nutritional status that has broken away from primary object can not satisfy the needs of sporophore growth, spawn degeneration fully, the problem that pharmacologically active goes down can occur, effectively nutrient composition content is low.Therefore, above-mentioned artificial culture method can not guarantee that sometimes bacterial classification is stable, has increased difficulty for the work of carrying out cultured continuously Cordyceps sporophore.296 pages of " fungus system " 2000 the 19th the 2nd phases of volume " new Chinese record species-kyushu aweto " disclosed in addition, it is a kind of Cordyceps on greenish brown hawk moth (Clanis Bilineata Walker) the larva body that parasitizes in Shenyang Eastern Mountain Area collection, is accredited as kyushu aweto (Cordyceps Kyushuensis Y.Kobayasi).Its outward appearance and morphological specificity are summarized as follows: sporophore is born on the shoestring (rhizomorph) that forms from killing bean hawkmoth larva, shoestring white.Stroma is orange red, high 1.7-8.6cm, and diameter 0.2-0.6cm gives birth in the perithecium half, and is avette, 367-495 * 260-316 μ m.Ascus is cylindrical, diameter 3.5-4.8 μ m.Secondary thecaspore short cylindrical, 3.9-5.4 * 1.0-1.2 μ m.But the report that does not still have kyushu aweto artificial culture method on the at present domestic and international open source literature.
Purpose of the present invention just is to provide a kind of artificial culture method of new Chinese record species-Cordyceps kyushuensis and the application of sporophore thereof, this method has effectively overcome existing can not carrying out of existing in the Chinese caterpillar fungus technology problems such as total man worker cultivates, incubation time is grown, the spawn degeneration phenomenon is serious of cultivating, it is stable to have bacterial classification, with short production cycle, can be continuously, produce in batches, the effective high advantage of nutrient composition content, adopt pure sporophore to be used as medicine, its consumption is few, and the preparation medicinal effect is remarkable.
The object of the present invention is achieved like this: this method is earlier the wild bacterium of Cordyceps kyushuensis (Cordyceps Kyushuensis Y.Kobaygasi) to be carried out monospore or single bacterium colony purifying, bacterial strain behind the purifying is made on the substratum that the liquid spawn tieback is main component to the live body killing bean hawkmoth larva of raising down with the aseptic condition of surface sterilization or is on the substratum that is mixed of main component with live body killing bean hawkmoth larva lyophilized powder, paddy rice, nutritive medium, natural infection under suitable culture condition continues to cultivate until growing the kyushu aweto sporophore;
Employed substratum live body killing bean hawkmoth larva is that 3 orders-5 of raising under the aseptic condition make larva, its surface sterilization is inserted liquid spawn, natural infection 30-40 days, treat that mycelia is full of in its body, cover 2-3 centimetre of habitat soil, continue to cultivate until growing the kyushu aweto sporophore through sterilization.
The employed substratum substratum that is mixed, the weight ratio of each component is respectively in per hundred parts of substratum that are mixed: live body killing bean hawkmoth larva lyophilized powder 3-5 part that aseptic condition is raised down, paddy rice 97-95 part, nutritive medium is 1.4 times of above-mentioned raw materials gross weight, be mixed and insert liquid spawn behind the medium sterilization, natural infection under the thermophilic culture condition treats that mycelia is covered with media surface substantially and extends downwards, give the scattered light irradiation, ventilate, until growing the kyushu aweto sporophore.
Because the substratum that adopted of artificial culture method of the present invention is that the live body killing bean hawkmoth larva with artificial breeding under the aseptic condition is a main component, this larva body is big, nutritious, and there is not the intravital assorted bacterium of wild live larva in the body, so when wild bacterium is carried out the purifying breeding, not only can guarantee to select the pure strain identical, and continue to grow the artificial culture kyushu aweto sporophore identical in the culturing process with natural kyushu aweto sporophore in inoculation with wild kyushu aweto.This method is compared with other Chinese caterpillar fungus artificial culture method, with short production cycle, bacterial classification is stablized, and significantly improves the infection rate of Cordyceps kyushuensis, and it is cultivated surviving rate and grow the sporophore rate and all reaches more than 100%, transformation efficiency is up to 168.3%, also lays good basis for continuous batch production, high yield.Can prevent effectively that also pharmacologically active from going down.Through Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences and the test of new Chinese medicine development center, Liaoning Province, its result shows: the stroma that artificial culture grows is through section and microscopic examination, its formalness and the internal feature all feature with natural kyushu aweto are close, see table 1 for details:
Table 1
The natural kyushu aweto artificial culture of feature kyushu aweto
Stroma length 1.7-8.0cm 6-12cm
The color and luster of stroma is orange red orange-yellow to shallow orange red
The stroma head shape is cylindrical cylindrical to club shape to club shape
Perithecium is measured 367-500 * 250-316 μ m 350-550 * 200-300 μ m
Ascus width 3.5-5 μ m 3-4.5 μ m
Secondary thecaspore is measured 3.9-5.4 * 1-1.2 μ m 3.5-4.8 * 1-1.2 μ m
The nutritive ingredient difference of the kyushu aweto sporophore that Cordyccps-militaris-(L.)-link. Sporophore that artificial culture grows and artificial culture grow is bigger: see following table for details:
Table 2
Nutrition Cordyceps militaris (L.) Link. Cordyceps sinensis kyushu aweto
Amino acid mg/100g 19,362 1779.87 24083
Cordyceps polysaccharide mg/g 13.2 11.2 19.75
Cordycepin % 0.23 0.07 0.35
Vitamin A mg/kg 1.25 0.315 3.00
Se mg/kg 0.20 0.14 0.39
SOD U/mg 35.7 8.68 42.4
This shows effective nutrient composition content height of artificial culture kyushu aweto sporophore.Adopt pure sporophore to be used as medicine and be used for healthcare products, not only strengthen the medicinal and health-care effect of tonifying lung, kidney-nourishing, hemostasis and phlegm of natural kyushu aweto, the effective disease that causes of treatment lung, kidney two void is set upright (promoting to regulate immunologic function), eliminating evil (suppress tumor growth and improve increase in life span), attenuation pharmacological actions such as (alleviate the secondary damage of chemotherapy and promote hematopoietic function) but also have to tumor animal.Obviously anticancer growth sets up, and further improves immune function of human body.Because of effective nutrient composition content height of the kyushu aweto sporophore of artificial culture, so consumption is few, can guarantee under the situation of curative effect, reduce the pharmacy cost.Just because of this, this artificial culture kyushu aweto has been put into the emphasis key scientific and technological projects of the Department of Science and Technology and has carried out the research of first class national new drug, its sporophore can be made into kyushu aweto capsule and kyushu aweto tablet, also can be used as raw materials such as herbal cuisine and healthcare products, oral liquid, beverage and tonic wine.
Below describe the present invention in detail with implementation method in conjunction with the accompanying drawings.
Fig. 1 is wild kyushu aweto morphological specificity figure.
Fig. 2 is the kyushu aweto morphological specificity figure with killing bean hawkmoth larva substratum artificial culture.
Fig. 3 is the kyushu aweto morphological specificity figure with the substratum artificial culture that is mixed.
Shown in Fig. 1-2, the morphological specificity of artificial culture kyushu aweto and wild kyushu aweto is close, sees Fig. 1-2 for details.Cultural method of the present invention is different with other Cordyceps artificial culture method, its principal feature is that the wild Cordyceps kyushuensis that will collect is earlier organized or spore separation, carry out single bacterium colony or monospore purifying again, examine under a microscope after cultivating mycelia, the purifying bacterial strain that will meet the Cordyceps kyushuensis feature is made liquid spawn, tieback is mixed on the substratum that is mixed that the paddy metric system becomes to the substratum of the live body killing bean hawkmoth larva of raising down with the aseptic condition of surface sterilization or with above-mentioned killing bean hawkmoth larva lyophilized powder again, natural infection treats that mycelia is full of substratum, can grow the sporophore identical with wild kyushu aweto under suitable culture condition.
In the aforesaid method, the killing bean hawkmoth larva of raising under aseptic condition should select for use 3 orders-5 to make (about seven days vegetative period be one make) live larva tieback liquid spawn.In 100 weight parts were mixed substratum, the weight ratio of each component was respectively: live body killing bean hawkmoth larva lyophilized powder 3-5 part that aseptic condition is raised down, contain paddy rice 97-95 parts such as the seed of Job's tears, broken corn, rice, and nutritive medium is 1.4 times of above-mentioned raw materials gross weight.Used nutritive medium is prepared by the following method: contain the pure powder 1-1.5 of kyushu aweto sporophore part in the nutritive medium of 1,000 weight parts, KH
2PO
42-2.5 part, K
2HPO
41-1.5 part, MgSO
41-1.5 part, surplus is a water.Used liquid spawn is that the inoculation behind the purifying is obtained in liquid nutrient medium, and per hundred weight parts contain killing bean hawkmoth larva lyophilized powder 3-5 part in its liquid nutrient medium, potato 10-15 part, and sugared 1-2 part, vitamins B 0.001-0.002 part, surplus is a water.
As follows by systematic study result that artificial kyushu aweto sporophore is carried out:
Drug efficacy study shows:
Artificial kyushu aweto sporophore can make S with the dosage gastric infusion of 300mg/kg, 600mg/kg 10 days
180The spleen index of solid tumor tumor-bearing mice, carbon element are cleaned up immune indexes such as the SRBC hemolysin, M phagocytic index of index and phagocytic index, serum hemolysin and splenocyte mediation and are obviously increased, and compare P＜0.05 or 0.01 with the lotus knurl.
Artificial kyushu aweto sporophore can make S with the dosage gastric infusion of 300mg/kg, 600mg/kg 10 days
180The TNF-β and the IL-2 of solid tumor tumor-bearing mice have obvious facilitation, and obviously to be better than with the lentinan be the powerful health (P＜0.01) of Chinese medicine contrast medicine of compound.
Artificial kyushu aweto sporophore can make S with the dosage gastric infusion of 300mg/kg, 600mg/kg 10 days
180Solid tumor tumor-bearing mice inhibition rate of tumor growth is respectively 20.83% and 30.72%; Increase in life span to S180 ascitic type BALB/C mice is respectively 193% and 185%; To endoxan effect similar 180%.
Artificial kyushu aweto sporophore is with the dosage gastric infusion of 300mg/kg, 600mg/kg 10 days, and the reduction of blood leukocytes around the caused by cyclophosphamide mouse is had the certain protection effect, P＜0.05 of comparing with control group.
In addition, artificial kyushu aweto sporophore can make the thymus gland of normal mouse immune organ and spleen weight obviously increase; And can make hydrogen examine due to thymic weight, T lymphocyte esterase dyeing rate, the chicken red blood cell phagocytic index of immunologic hypofunction mouse obviously increase; Normal mouse hypoxia endurance time and low temperature water-bath swimming time are obviously prolonged.
Preliminary clinical study result
With the oral dosage successive administration 28 days of 1.2G every day, and course of treatment of the second week beginning chemotherapy.The result shows: 30 routine middle and advanced stage cancerous swelling patients' digestive tube side reaction number, leukopenia number, the NK cytoactive routine number average that raises obviously is better than the high poly-golden Portugal of contrast element.
Table 3 30 routine patient tumors distribution situations (seeing Table 3)
Table 3
Other totals of group lung cancer gastrointestinal cancer malignant lymphoma mammary cancer
Artificial kyushu aweto sporophore group 19 6311 30
The plain group 75133 19 in high poly-golden Portugal
Pharmacological research shows: artificial kyushu aweto sporophore not only can suppress the tumor-bearing mice tumor growth, prolong the tumor animal survival time, also tumor animal had clear and definite immunoregulation effect, particularly obviously be better than clinical widely used vegetable polysaccharides para-immunity conditioning agent at present on the regulating effect to the TNF of tumor animal and IL-2, prove that this medical instrument has the double pharmacological action that suppresses tumor growth and promote the immunomodulatory function.
Chemical research shows: artificial kyushu aweto sporophore contains more nucleoside compound, its content apparently higher than similar Cordyceps sinensis fungus and artifact, its main active ingredient cordycepin has the obvious suppression tumor growth and with endoxan the obvious synergistic effect is arranged, and its content is more than 10 times of Cordyceps militaris (L.) Link. fungus powder.It is strong that artificial kyushu aweto sporophore and clinical strengthening vital QI to eliminate pathogenic factors class compound preparation commonly used and other artificial aweto metal products are compared the controllability with dosage little (1.35g/ day), medicinal material and preparation, easily characteristics such as accepted by the world market.
Preliminary toxicological study shows: artificial kyushu aweto sporophore toxicity is less, drug safety.
Acute toxicity test shows: artificial kyushu aweto sporophore is 10g/kg to the maximum tolerated dose of mouse stomach administration, 444 times of the daily dosage portion that is equivalent to be grown up.
Long term toxicity test shows: continuous 16 weeks give artificial kyushu aweto sporophore 1.2g/kg.d and 0.6g/kg.d (be equivalent to clinical consumption 53.3 times and 26.7 times) to rat oral gavage, to rat general signs, action behavior, grow, biochemical analysis and all no abnormal changes of pathologic finding such as hemogram checking, liver, renal function, prompting this product long-term prescription safety.
In sum, artificial kyushu aweto sporophore and preparation thereof have aboundresources, and pharmacological action is clear and definite, main active substances is clear substantially, characteristics such as toxicity meets the existing new drug approval requirement of China, and medicinal material and preparation controllability are strong are very promising strengthening vital QI to eliminate pathogenic factors tumor aid treatment medicines.Not only can make China in the Cordyceps sinensis fungus development field, continue position oneself at the forefront if succeed in developing, also can create suitable social benefit and economic benefit.
Specific implementation method of the present invention and use as follows:
Implementation method 1:
The wild kyushu aweto sporophore of gathering is carried out surface sterilization, peel off skin, expose marrow, get on the substratum plate that the 0.2-0.3cm tissue block is placed on sterilization with the sterilization blade, cultivate through 7 days thermophilics, healthy and strong mycelia is put on the test tube slant picking, thermophilic was cultivated 7-10 days again, roughly select according to the mycelial growth situation, be connected on the plate, inserted sheet around inoculation point allows its growth again, about 5 days, take out cover glass, examine under a microscope the mycelia form, the staying of mycelia feature that meets this bacterial strain produced liquid spawn, tieback is to the killing bean hawkmoth larva body again, can grow the be selected pure strain identical with wild kyushu aweto sporophore. this liquid spawn is that the bacterial strain behind the purifying is inserted in the 100 milliliters of liquid substratum for 10 milligrams, contains killing bean hawkmoth larva lyophilized powder 3-5 part in per hundred parts of liquid nutrient mediums, potato 10-15 part, sugar 1-2 part, vitamins B
10.001-0.002 part, surplus is a water.
Implementation method 2:
Selected pure strain is inoculated into through 3 of surface sterilization for 1 milliliter makes on the killing bean hawkmoth larva body, allow its natural infection about 30 days, treat that mycelia is full of in its body, 2 centimetres in the habitat soil of covering through sterilizing, about 20 days, just grow orange red to orange-yellow kyushu aweto sporophore from the larva body.
Implementation method 3:
Selected pure strain is inoculated into through 4 of surface sterilization for 1.5 milliliters makes on the killing bean hawkmoth larva body, allow its natural infection about 35 days, treat that mycelia is full of in its body, 2.5 centimetres in the habitat soil of covering through sterilizing, about 20 days, just grow orange red to orange-yellow kyushu aweto sporophore from the larva body.
Implementation method 4:
Selected bacterial classification is inoculated into through 5 of surface sterilization for 2 milliliters makes on the killing bean hawkmoth larva body, allow its natural infection about 40 days, treat that mycelia is full of in its body, cover, about 20 days, be convenient to grow on the larva body its sporophore through 3 centimetres in the habitat soil of sterilization.
Implementation method 5:
With 3 kilograms of killing bean hawkmoth larva lyophilized powders, 10 kilograms of the seeds of Job's tears, 12 kilograms of broken corns, 75 kilograms in rice, mix the Cans of packing into (or Plastic Bottle, in the bag) every bottled 30 grams, add 40 milliliters of nutritive mediums, make the substratum that is mixed, 2 milliliters of liquid spawns are inserted in the sterilization back, and thermophilic was cultivated about 20 days, treat that mycelia is covered with media surface substantially and also extends downwards, give light scattering (about 2500LUX), ventilate (the dazzling ventilation of bottleneck film) door, window staggers every day 3-4 time, each 20-30 minute, indoor humidity is 85-90%, the 25-30 days sporophores of can gathering.
Implementation method 6:
With 4 kilograms of killing bean hawkmoth larva lyophilized powders, 9 kilograms of the seeds of Job's tears, 11 kilograms of broken corns, 76 kilograms in rice mixes in the bottle of packing into (or bag), every bottled amount 40 grams, 56 milliliters of Ensure Liquid liquid, 3 milliliters of bacterial classifications are inserted in the sterilization back, and the thermophilic lucifuge was cultivated about 23 days, treat that mycelia is covered with media surface and extension downwards gives light scattering, as follows implementation method 5.
Implementation method 7: with 5 kilograms in killing bean hawkmoth larva powder, 8 kilograms of the seeds of Job's tears, 10 kilograms of broken corns, 77 kilograms in rice, mix, in the bottle of packing into (or bag), every bottled amount 50 grams, 70 milliliters of Ensure Liquid liquid, 4 milliliters of bacterial classifications are inserted in the sterilization back, and thermophilic was cultivated about 25 days, treat that mycelia is covered with charge level and also extends downwards, give light scattering, as follows implementation method 5.
Implementation method 8:
The kyushu aweto sporophore 60 ℃ of following drying and crushing, is crossed No. six sieve series and become pure powder, add the alcohol granulation about 80%, 60 ℃ of dryings, whole grain, encapsulated, promptly get the kyushu aweto capsule with packing behind cobalt 60 irradiation-sterilizes, its main component sees Table 4.
Implementation method 9:
With pure powder 300 grams of kyushu aweto sporophore.N.F,USP MANNITOL powder 256.85 grams, Magnesium Stearate 2 grams mix in mixing machine; and menthol 0.15 gram is dissolved in makes tackiness agent and mixed powder system or suitable softwood in the ethanol; and in nodulizer, make suitable particle, dry under 50 ℃, after 6 hours; whole grain; be pressed into the pincushion sheet that labelled amount is 560mg with tabletting machine, after the assay was approved, carry out the bubble eye with aluminium type wrapping machine and pack; carry out cobalt 60 irradiation-sterilizes again, packing promptly gets the kyushu aweto sheet.
Its main component sees Table 5:
The kyushu aweto capsule uses through 30 routine middle and terminal cancer patients, if treatment group, with kyushu aweto capsule gram every days 1.2, divide and take for three times, the intramuscular injection next day that control group giving 4 milligrams of plain injection liquids of high poly-golden grape, time is that a month treatment leukopenia number is 40%, is starkly lower than control group 84%, P＜0.005; The NK cytoactive raises, and the treatment group is 83%, apparently higher than control group 53%, and P＜0.025.To the digestive tube side reaction, the treatment group is starkly lower than control group and is respectively 3 3.3% and 78.9%, P＜0.005.
The kyushu aweto sheet is checked through Chinese Academy Of Preventive Medicine Research Institute Of Nutrition And Food Hygiene, think and have the immunity of adjusting, the effect of antifatigue and regulating blood fat, the clinical immunologic hypofunction that is used for, blood fat increases, and is engaged in the crowd that need regain one's strength after mental manual work and the motion.Contain clothes or swallow, every day the 4-6 sheet.
The main effective constituent of table 4 kyushu aweto sheet
Cordycepin Cordyceps polysaccharide cordycepic acid SOD total amino acid content
(mg/g) (％) (mg/g) (U/mg) (mg/100g)
1.45 26.98 209 1.54 9,115
VA VE VD3(mg/kg) Fe Zn Ca Se(mg/kg)
0.288 1.84×10
3 1.05 58.0 38.87?302.3?0.018
The capsular main effective constituent of table 5 nine divisions of China in remote antiquity worm
Cordycepin total points sugar cordycepic acid SOD total amino acid content
(mg/g) (％) (mg/g) (U/mg) (mg/100g)
2.26 17.70 87.9 42.4 21,562
VA VE VD3(mg/kg) Fe Zn Ca Se(mg/kg)
0.288 1.84×10
3 1.05 58.0 38.87 302.3?0.018
Claims (5)
1, a kind of artificial culture method of Cordyceps kyushuensis, it is characterized in that earlier the wild bacterium of Cordyceps kyushuensis (Cordyceps Kyushuensis Y.Kobaygasi) being carried out monospore or single bacterium colony purifying, bacterial strain behind the purifying is made on the substratum that the liquid spawn tieback is main component to the live body killing bean hawkmoth larva of raising down with the aseptic condition of surface sterilization or is on the substratum that is mixed of main component with live body killing bean hawkmoth larva lyophilized powder, paddy rice, nutritive medium, natural infection under suitable culture condition continues to cultivate until growing the kyushu aweto sporophore;
Employed substratum live body killing bean hawkmoth larva is that 3 orders-5 of raising under the aseptic condition make larva, its surface sterilization is inserted liquid spawn, natural infection 30-40 days, treat that mycelia is full of in its body, cover 2-3 centimetre of habitat soil, continue to cultivate until growing the kyushu aweto sporophore through sterilization.
The employed substratum substratum that is mixed, the weight ratio of each component is respectively in per hundred parts of substratum that are mixed: live body killing bean hawkmoth larva lyophilized powder 3-5 part that aseptic condition is raised down, paddy rice 97-95 part, nutritive medium is 1.4 times of above-mentioned raw materials gross weight, be mixed and insert liquid spawn behind the medium sterilization, natural infection under the thermophilic culture condition treats that mycelia is covered with media surface substantially and extends downwards, give the scattered light irradiation, ventilate, until growing the kyushu aweto sporophore.
2, cultural method according to claim 1, it is characterized in that described liquid spawn is that the inoculation behind the purifying is obtained in liquid nutrient medium, contain killing bean hawkmoth larva lyophilized powder 3-5 part, potato 10-15 part in its per hundred weight part liquid nutrient mediums, sugar 1-2 part, vitamins B
10.001-0.002 part, surplus is a water.
3, cultural method according to claim 1 is characterized in that containing in per 1,000 weight parts of described nutritive medium the pure powder 1-1.5 of kyushu aweto sporophore part, KH
2PO
42-2.5 part, K
2HPO
41-1.5 part MgSO
41-1.5 part, surplus is a water.
4, the application of the kyushu aweto that makes according to the described cultural method of claim 1, it is characterized in that kyushu aweto sporophore drying, be ground into 100 order fine powders, use the ethanol granulation, dry below 60 ℃, whole grain, incapsulate, after cobalt 60 sterilizations, make the kyushu aweto capsule.
5, the application of the kyushu aweto that makes of cultural method according to claim 1, it is characterized in that with the kyushu aweto sporophore be major ingredient, be aided with N.F,USP MANNITOL, Magnesium Stearate, soluble saccharin, menthol again, produce the kyushu aweto tablet, raw materials used each components by weight is: 300 parts in the pure powder of kyushu aweto sporophore, 256.85 parts in N.F,USP MANNITOL, 1 part of soluble saccharin, 0.15 part of menthol, 2 parts of Magnesium Stearates utilize ethanol granulation, drying, compressing tablet to be finished product.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
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| CN00110494A CN1109103C (en) | 2000-06-06 | 2000-06-06 | Artificial culture of jiuzhou caterpillar fungus and the application of its sporophore |
| KR10-2001-0031114A KR100442766B1 (en) | 2000-06-06 | 2001-06-04 | Artificial method to culture Cordyceps Kyushuensis Y.Kobayas and the application for its sporocarp |
| JP2001171804A JP3665845B2 (en) | 2000-06-06 | 2001-06-06 | Artificial culture method of Kyushu insecticide |
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| CN00110494A CN1109103C (en) | 2000-06-06 | 2000-06-06 | Artificial culture of jiuzhou caterpillar fungus and the application of its sporophore |
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| CN1109103C true CN1109103C (en) | 2003-05-21 |
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| CN100434508C (en) * | 2006-11-08 | 2008-11-19 | 中山大学 | Method for increasing yield of Qinghai Tibet plateau cordyceps |
| CN101659578B (en) * | 2009-09-18 | 2012-10-17 | 鲁东大学 | Artificial culture method of Kyushu cordyceps sinensis and culture medium thereof |
| CN104871830B (en) * | 2015-06-15 | 2017-03-15 | 鲁东大学 | With bean worm as the method for carrier-planting Cordyceps militaris (L.) Link. |
| CN110786204A (en) * | 2019-11-28 | 2020-02-14 | 张翰熙 | Industrial production method of cordyceps sinensis |
| CN112889758A (en) * | 2021-01-27 | 2021-06-04 | 连云港市农业科学院 | Environmental control condition for artificial indoor culture of bean hawkmoth |
| CN114568208B (en) * | 2022-03-14 | 2023-03-14 | 刘晓红 | Artificial cultivation method for cordyceps sinensis sporocarp |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1167825A (en) * | 1996-05-27 | 1997-12-17 | 日原町 | Method for artificial culture of cordyceps, e.g. Cordyceps mititaris |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1167825A (en) * | 1996-05-27 | 1997-12-17 | 日原町 | Method for artificial culture of cordyceps, e.g. Cordyceps mititaris |
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