CN104285820A - Selenium-rich cordyceps militaris strain and application thereof - Google Patents
Selenium-rich cordyceps militaris strain and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of microbial fungi, and relates to a selenium-rich cordyceps militaris strain CM001; the wild cordyceps militaris strain which is newly separated in the nature is selected as an original strain; the original strain is subjected to ultraviolet mutation, and the strain can be obtained by separating and screening; the selenium-rich cordyceps militaris strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 18, 2014 with the preservation registration number of CGMCC No.9460, is named as CM001 and is named as cordyceps militaris in a classified way. Under the artificial culture conditions, the cordyceps militaris strain has the tolerance for high-concentration selenium, and can carry out biotransformation in a body; the hyphae yeast, the growth matrix or the sporocarp of the selenium-rich cordyceps militaris strain can be used as raw materials of medicines, foods and drinks, or can be prepared into powder, tablets, freeze-drying powder, capsules or electuary.
Description
Technical field
The invention belongs to microbial fungi field, relate to strain selenium-enriched cordceps militaris bacterial classification and an application thereof.
Background technology
Selenium is the necessary trace element of human body, can free radical in purged body, effectively defends the infringement of radical pair cell, strengthens immunity of organisms.Chinese Soclety of Nutrition selenium is classified as 15 kinds every day one of diet nutrient, the selenium of 50 ~ 250 micrograms should be taken in everyone every day, and, study and find that the selenium taking in 200 ~ 400 micrograms every day also has the effect of Tumor suppression, therefore selenium is called as " king of anti-cancer ", be internationally recognizedly press down cancer agent, there is the effects such as cancer-resisting, treatment hepatopathy and anti-oxidant, detoxication and toxicant eliminating function.2003 FDA (Food and Drug Adminstration) (FDA) express: " selenium can reduce suffer from cancer risk " and " selenium can produce anticancer change effect in human body ", and approve that but selenium is cancer agent, and allow the cancer resistant effect of businessman to selenium to make mark.Research shows, the distribution of selenium in natural environment is uneven, and according to incompletely statistics, the whole world has 42 countries to lack selenium.China's majority is for lacking selenium area, and wherein lack selenium, Pin Xi county up to 72% in 22 provinces in the whole nation, nearly 700,000,000 populations in area of about 2/3 are in scarce selenium state, and therefore mending selenium has become the important measures improving our people's fitness.
At present, the mode of mending selenium has nature mend selenium and manually mend selenium, and naturally mending selenium is the food etc. that the high self-sow of wild, natural Se content got by food, and what supplement is Organic Selenium; Artificial benefit selenium is the artificial all kinds of benefit selenium products added of picked-up, mainly be divided into inorganic selenium and Organic Selenium two class, inorganic selenium is mainly sodium selenite, there is very large toxin, not easily be absorbed by the body, availability is low, and Organic Selenium is compared with the inorganic seleniums such as sodium selenite, there is edible safety, has no side effect, absorption rate is high, be of high nutritive value advantages such as (as high-caliber vitamins, high-quality albumen etc.).It is desirable first-selection of mending selenium.
Cordyceps militaris (Cordyceps militaris) is Ascomycotina, ergot Zoopagales, the type sepecies of Clavicipitaceae, Cordyceps, a kind of integration of drinking and medicinal herbs, the precious tonic of the tradition that can throughout the year take, " Xinhua's book on Chinese herbal medicine outline " is recorded: effect that the sweet property of Cordyceps militaris taste is put down, useful lung kidney mends marrow hemostasis and phlegm, " national Chinese herbal medicine compilation " is recorded: Cordyceps militaris can substitute Cordyceps sinensis and be used as medicine, and has the multiple efficacies such as immunity moderation systemic-function, antitumor, antifatigue, anti-ageing, radioresistance.Selenium in fungi is after transforming in body, major part exists with Organic Selenium form, as the form such as selenium polysaccharide, selenoprotein exists, it is anti-oxidant, biologically active is high, very easily utilize in absorption of human body, can as the best food materials mending selenium, but the Se content in common fruiting bodies of cordyceps militaris is on the low side, be approximately 0.1-20mg/kg, and its to the dosis tolerata of selenium and biological transformation ratio lower, in the medium of selenium concentration >20mg/kg, the growth of mycelia and fruit body is all subject to serious suppression, even dead.
Summary of the invention
The object of the invention is the defect such as low for the dosis tolerata of current common cordyceps militaris link bacterial strain to selenium, biological transformation is low and a strain selenium-enriched cordceps militaris (Cordyceps militaris) bacterial classification CM001 is provided, this bacterial strain is under culture conditions, can the selenium of enduring high-concentration, and carry out biological transformation in vivo, the fruit body Se content of this strain growth can up to 139mg/kg.
Bacterial strain provided by the present invention chooses Cordyceps militaris (Cordyceps militaris) wild-type strain that natural world is newly separated as starting strain, after ultraviolet mutagenesis process, the bacterial classification that separation screening obtains, this bacterial strain has been preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; preservation registration number is CGMCC No.9460; called after CM001, Classification And Nomenclature is Cordyceps militaris (Cordyceps militaris) on July 18th, 2014.
The strain characteristic of Cordyceps militaris provided by the present invention (Cordyceps militaris) bacterial classification CM001: the mycelia grown on solid culture medium is flourishing, and in white, through illumination after 2 ~ 5 days, mycelia transfers yellow to gradually.Fruit body feature: the high 3 ~ 14cm of stroma, diameter 1 ~ 7mm, yellow, there is golden yellow conidial powder time ripe.This bacterial classification is identified through Protocols in Molecular Biology, comprises sequence in ITS-PCR, sequencing and Genbank more right, and the autoploidy of this bacterial classification and Cordyceps militaris (northern Chinese caterpillar Fungus) is 100%.
In the fruit body of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001, Se content can be 0 ~ 139ug/g.
In the fruit body of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001, cordycepin content is greater than 0.6%, and adenosine content is greater than 0.1%, and Cordyceps sinensis polysaccharide content is greater than 6%.
The seed selection of above-mentioned Cordyceps militaris (Cordyceps militaris) bacterial classification CM001:
(1) field acquisition cordyceps militaris link bacterial strain, adopts tissue isolation to carry out Pure strain separation screening; The single bacterium colony filtered out, after strain idenfication, carries out grass experiment; Reservation can grow the bacterial strain of fruit body, as the starting strain of strain improvement.
(2) starting strain is prepared into bacteria suspension, is placed in culture dish, liquid layer thickness is 2 ~ 4mm, and use 15w power UV fluorescent tube to irradiate, irradiation distance is 28 ~ 32cm, irradiation time 10s ~ 20min.After being disposed, the vessel black cloth containing bacteria suspension being wrapped cultivation, then carries out separation screening.
(3) by the bacterial strain of UV mutation separation screening acquisition, inoculate in solid state cultivation medium, first carry out light culture 3 ~ 15 days, cover with after medium until mycelia, proceed to illumination cultivation, intensity of illumination is 100 ~ 500Lux, relative air humidity 75 ~ 90%, temperature 18 ~ 24 DEG C, cultivates 50 ~ 60 days, mature sporophore gather after kept dry.Detect dried fruiting body yield and composition as screening index: fruiting body yield is more than or equal to 1.5g/ bottle, Se content is more than or equal to 35ug/g, Cordyceps sinensis polysaccharide is more than or equal to 2.5%, cordycepin is more than or equal to 0.3%, Chinese caterpillar fungus adenosine is more than or equal to 0.055%.Filter out Cordyceps militaris (Cordyceps militaris) the bacterial classification CM001 being suitable for industrial production.The formula of described solid state cultivation medium: cereal crops 20 ~ 60g, then add 30 ~ 70 milliliters of nutrient solutions and mix thoroughly, described cereal crops are one in rice, sorghum rice, wheat or corn or mixing; The formula of described nutrient solution: glucose 1 ~ 10g, sucrose 1 ~ 10g, dusty yeast 1 ~ 10g, peptone 1 ~ 10g, potassium dihydrogen phosphate 1 ~ 1.5g, dipotassium hydrogen phosphate 0.1 ~ 0.3g, citric acid eucalyptus 0 ~ 1g, magnesium sulfate 0.3 ~ 0.6g, VB11 ~ 3 slice, sodium selenite 0 ~ 40mg, 1000 milliliters, water.
The present invention also provides hypha fermentation thing or the growth substrate of above-mentioned Cordyceps militaris (Cordyceps militaris) bacterial classification CM001, crushed after being dried, can be used as the raw material of medicine, food and beverage, maybe can be prepared into powder, tablet, freeze dried powder, capsule or electuary, also can be used as feedstuff.
The present invention also provides the fruit body of above-mentioned Cordyceps militaris (Cordyceps militaris) bacterial classification CM001, dry or vacuum cooling drying after, make dry product or directly eat after pulverizing, also can be used as the raw material of medicine, health products, food, drinks, flavouring and beverage, maybe can be prepared into powder, tablet, freeze dried powder, capsule or electuary.There is raising immunity, effect of Tumor suppression.
The performance measurement of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001:
1, the toxicity test of bacterial classification
Acute toxicity experience is carried out to this bacterial classification fruit body, proves to belong to actual nontoxic level; The result of sperm malformation test and micronucleus test shows do not have mutagenicity; Subchronic toxicity test result shows it to organs such as animal growth, biochemical indicator, liver kidneys without impact.
Test is carried out according to relevant method in national standard " toxicological evaluation of food safety procedure and method ".
(1) acute toxicity testing: choose healthy NIH mouse 60, by 10g/kg gavage once, observes 14 days, and without dead mouse, LD50>10g/kg, by acute toxicity grading criteria evaluation, belongs to actual nontoxic level.
(2) micronucleus test: choose healthy NIH mouse 50, be divided into 5 groups at random, often organizes 10.If high, medium and low three dosage, and establish feminine gender and positive controls.Animal is contaminated 2 times continuously, interval 24h, after last contamination, 6h puts to death animal, observe the polychromatic erythrocyte number containing micronucleus, the results are shown in Table 1, result of the test shows, high, medium and low three dosage microkernel incidences (MN%) are respectively 1.8%, 1.6%, 1.9%, negative and positive controls is respectively 1.8% and 35%.Each dosage group microkernel incidence and negative control indifference, sample is formed micronucleus and PCE/RBC ratio has no significant effect.
Table 1 Micronuclei In The Mouse Bone Marrow result of the test
(3) sperm malformation test: choose healthy NIH male mice, be divided into 5 groups at random, often organizes 5.If high, medium and low three dosage, and establish feminine gender and positive controls.The continuous gavage of laboratory animal 5 days, every day 1 time, within after contamination the 35th day, put to death animal, get bilateral epididymal film-making dyeing, every mouse counts 1000 complete sperms, record distortion sperm number, the results are shown in Table 2, result of the test shows, high, medium and low three dosage rate of teratosperms are respectively 1.5%, 1.3%, 1.6%, negative and positive controls is respectively 25% and 1.8%.Sample acts on without obvious teratogenesis shape sperm.
Table 2 mouse inbred strain result
(4) subchronic toxicity test: healthy SD rat 40, is divided into 4 groups at random, often organizes 10.If high, medium and low three dosage, and establish negative control group (water), every day, gavage 1 time, tested 90 days, and duration of test observes the situations such as animal behavior, body weight, diet, morbidity and death.Carry out Pathomorphologic inspection after off-test, find no the pathological change of meaning, animal growth is without exception.
2, the fruit body Determination of Selenium of bacterial classification
Measure according to the method for GB 5009.93-2010 national food safety standard " mensuration of selenium in food ".Testing result shows, the Se content scope that selenium-enriched cordceps militaris bacterial classification CM001 is containing the fruit body grown under the culture medium for cultivating of different selenium concentration can be 0-139ug/g.
3, the fruit body effective constituent determination of bacterial classification
Respectively the active constituent contents such as the Cordyceps sinensis polysaccharide in fruit body, cordycepin, Chinese caterpillar fungus adenosine are measured, the results are shown in Table 3.
The fruit body active ingredient of table 3 bacterial classification
| Analysis project | Testing result | Detection method |
| Cordyceps sinensis polysaccharide | 6％ | Phend-sulphuric acid |
| Cordycepin | 0.6％ | HPLC method |
| Chinese caterpillar fungus adenosine | 0.1％ | HPLC method |
4, the selenium-enriched cordceps militaris of the present invention's screening compares with common fruiting bodies of cordyceps militaris antitumaous effect
Mouse armpit subcutaneous vaccination tumour cell, after becoming tumor model, is divided into blank group, common Cordyceps militaris group and selenium-enriched cordceps militaris group at random, often organizes 10 mouse.Blank group physiological saline gavage, common Cordyceps militaris group and selenium-enriched cordceps militaris group are respectively by common and selenium-enriched cordceps militaris gavage, administration in continuous 20 days, within 11st day, de-cervical vertebra puts to death mouse, strip tumour to weigh, calculate tumour inhibiting rate, the results are shown in Table 4, illustrate that selenium-enriched cordceps militaris has Tumor suppression effect, effect is better than common Cordyceps militaris.
Table 4
| Group | Animal number | Knurl heavy (X ± S) | Tumour inhibiting rate % | P |
| Blank group | 10 | 1.83±0.36 | ? | ? |
| Common Cordyceps militaris | 10 | 1.09±0.23 | 40.4 | <0.05 |
| Selenium-enriched cordceps militaris | 10 | 0.76±0.29 | 58.5 | <0.05 |
The present invention also provides one to utilize Cordyceps militaris (Cordyceps militaris) bacterial classification CM001 to carry out the method for large-scale industrialized application production, and its step is as follows:
1, prepared by female kind: be inoculated in PDA slant medium by Cordyceps militaris (Cordyceps militaris) bacterial classification CM001, temperature 18 ~ 24 DEG C, relative air humidity 60 ~ 75%, light culture 4 ~ 7 days, chooses fast, the neat bacterial classification of mycelial growth as mother's kind.
2, level liquid bacterial classification preparation: the mother chosen planted and be inoculated in strain liquid medium, temperature 18 ~ 24 DEG C, relative air humidity 60 ~ 75%, concussion light culture 3 ~ 7 days, choose mycelium pellet neat, size is that the bacterial classification of 2 ~ 3mm is as level liquid bacterial classification.
3, secondary produces bacterial classification preparation: inoculate in strain liquid medium by level liquid bacterial classification in 3 ~ 10% ratios, temperature 18 ~ 24 DEG C, relative air humidity 60 ~ 75%, concussion light culture 3 ~ 7 days, choose mycelium pellet neat, size is that the bacterial classification of 2 ~ 3mm produces bacterial classification as secondary, produces for Cordyceps militaris factory culture.
4, inoculation and artificial cultivation: with sterile water by volume: sterile water: secondary produce bacterial classification=2 ~ 5:1 dilution proportion secondary produce bacterial classification, then inoculate in solid state cultivation medium, every bottle graft enters 10 ~ 15ml.First carry out light culture 3 ~ 15 days, cover with after medium, proceed to illumination cultivation until mycelia, intensity of illumination is 100 ~ 500Lux, relative air humidity 75 ~ 90%, temperature 18 ~ 24 DEG C, cultivates 50 ~ 60 days, gathers after fruit body maturation.
Described strain liquid culture medium prescription: glucose 1 ~ 10g, sucrose 1 ~ 10g, corn flour 1 ~ 5g, analysis for soybean powder 1 ~ 5g, dusty yeast 1 ~ 5g, peptone 1 ~ 6g, potassium dihydrogen phosphate 1 ~ 1.5g, dipotassium hydrogen phosphate 0.1 ~ 0.3g, citric acid eucalyptus 0 ~ 1g, magnesium sulfate 0.3 ~ 0.6g, VB11 ~ 3 slice, 1000 milliliters, water.
The formula of described solid state cultivation medium: cereal crops 20 ~ 60g, then add 30 ~ 50 milliliters of nutrient solutions and mix thoroughly, described cereal crops are one in rice, sorghum rice, wheat or corn or mixing; The formula of described nutrient solution: glucose 1 ~ 10g, sucrose 1 ~ 10g, dusty yeast 1 ~ 10g, peptone 1 ~ 10g, potassium dihydrogen phosphate 1 ~ 1.5g, dipotassium hydrogen phosphate 0.1 ~ 0.3g, citric acid eucalyptus 0 ~ 1g, magnesium sulfate 0.3 ~ 0.6g, VB11 ~ 3 slice, sodium selenite 0 ~ 40mg, 1000 milliliters, water.
Cordyceps militaris provided by the invention (Cordyceps militaris) bacterial classification CM001 is under culture conditions, can the selenium of enduring high-concentration, and carry out biological transformation in vivo, the Se content of its fruit body is higher, have and improve the effect such as immunity, Tumor suppression, make dry product or directly eat after pulverizing, also can be used as the raw material of medicine, health products, food, drinks, flavouring and beverage, or be prepared into powder, tablet, freeze dried powder, capsule or electuary.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Experimental technique in described embodiment, if no special instructions, is conventional method; Percentage composition in described embodiment, if no special instructions, is weight percentage.
Embodiment one
The fruit body artificial cultivation method of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001, comprises following several stages:
(1) prepared by female kind: be inoculated in PDA slant medium by Cordyceps militaris (Cordyceps militaris) bacterial classification CM001, temperature 22 DEG C, relative air humidity 65%, light culture 7 days, chooses fast, the neat bacterial classification of mycelial growth as mother's kind.
(2) level liquid bacterial classification preparation: the excellent mother chosen planted and be inoculated in strain liquid medium, temperature 22 DEG C, relative air humidity 65%, concussion light culture 7 days, choose mycelium pellet neat, size is about the bacterial classification of 2-3mm as level liquid bacterial classification.
(3) secondary produces bacterial classification preparation: inoculate in strain liquid medium by level liquid bacterial classification in 6% ratio, temperature 22 DEG C, relative air humidity 65%, concussion light culture 7 days, choose mycelium pellet neat, the bacterial classification that size is about 2-3mm produces bacterial classification as secondary, produces for Cordyceps militaris factory culture.
(4) inoculation and artificial cultivation: with sterile water by volume: sterile water: secondary produce bacterial classification=2 ~ 5:1 ratio sterile water dilution secondary produce bacterial classification, inoculate in solid state cultivation medium, every bottle graft enters 15ml.First carry out light culture 15 days, cover with after medium, proceed to illumination cultivation until mycelia, intensity of illumination is 400Lux, relative air humidity 85%, temperature 23 DEG C, cultivates 50 days, gathers after fruit body maturation.
Strain liquid culture medium prescription: glucose 10g, corn flour 5g, analysis for soybean powder 5g, dusty yeast 1g, peptone 2g, potassium dihydrogen phosphate 1.5g, dipotassium hydrogen phosphate 0.3g, magnesium sulfate 0.5g, orotic acid sheet, 1000 milliliters, water.
Solid state cultivation culture medium prescription: rice 20g, sorghum rice 10g, add 40 milliliters of nutrient solutions again and mix (nutrient solution prescription: glucose 15g thoroughly, dusty yeast 1g, peptone 2g, potassium dihydrogen phosphate 1.5g, dipotassium hydrogen phosphate 0.3g, citric acid eucalyptus 1g, magnesium sulfate 0.5g, orotic acid sheet, sodium selenite 10mg, 1000 milliliters, water)
The mycelia that Cordyceps militaris provided by the present invention (Cordyceps militaris) bacterial classification CM001 grows and fruit body can be used for preparing antitumor, antiviral drug.
Embodiment two
The hypha fermentation thing that Cordyceps militaris (Cordyceps militaris) bacterial classification CM001 grows, after pulverizing, adds the lixiviate of 2 times of volume distilled water, add alcohol settling, after drying, obtain Chinese caterpillar fungus selenium polysaccharide crude product, can be used as primary raw material that is antitumor, antiviral drugs.
Embodiment three
After the fruit body of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001 adopts 45 DEG C of dryings, pack or box, sealing, direct-edible, or make tea and drink.
Embodiment four
After the fruit body of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001 adopts 45 DEG C of dryings, through nanometer pulverization, pack or box, sealing, direct-edible.
Embodiment five
After the fruit body of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001 adopts 45 DEG C of dryings, to steep in 50 degree of white wine more than 15 days, make selenium-enriched cordceps militaris health liquor and drink.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. a strain selenium-enriched cordceps militaris (Cordyceps militaris) belongs to bacterial classification, be preserved on 07 18th, 2014 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; preservation registration number is CGMCC No.9460; called after CM001, Classification And Nomenclature is Cordyceps militaris (Cordyceps militaris).
2. the seed selection of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001 as claimed in claim 1:
(1) field acquisition cordyceps militaris link bacterial strain, adopts tissue isolation to carry out Pure strain separation screening; The single bacterium colony filtered out, after strain idenfication, carries out grass experiment; Reservation can grow the bacterial strain of fruit body, as the starting strain of strain improvement;
(2) starting strain is prepared into bacteria suspension, is placed in culture dish, liquid layer thickness is 2 ~ 4mm, and use 15w power UV fluorescent tube to irradiate, irradiation distance is 28 ~ 32cm, irradiation time 10s ~ 20min; After being disposed, the vessel black cloth containing bacteria suspension being wrapped cultivation, then carries out separation screening;
(3) by the bacterial strain of UV mutation separation screening acquisition, inoculate in solid state cultivation medium, first carry out light culture 3 ~ 15 days, cover with after medium until mycelia, proceed to illumination cultivation, intensity of illumination is 100 ~ 500Lux, relative air humidity 75 ~ 90%, temperature 18 ~ 24 DEG C, cultivates 50 ~ 60 days, mature sporophore gather after kept dry; Detect dried fruiting body yield and composition as screening index: fruiting body yield is more than or equal to 1.5g/ bottle, Se content is more than or equal to 35ug/g, Cordyceps sinensis polysaccharide is more than or equal to 2.5%, cordycepin is more than or equal to 0.3%, Chinese caterpillar fungus adenosine is more than or equal to 0.055%; Filter out Cordyceps militaris (Cordyceps militaris) the bacterial classification CM001 being suitable for industrial production; The formula of described solid state cultivation medium: cereal crops 20 ~ 60g, then add 30 ~ 70 milliliters of nutrient solutions and mix thoroughly, described cereal crops are one in rice, sorghum rice, wheat or corn or mixing; The formula of described nutrient solution: glucose 1 ~ 10g, sucrose 1 ~ 10g, dusty yeast 1 ~ 10g, peptone 1 ~ 10g, potassium dihydrogen phosphate 1 ~ 1.5g, dipotassium hydrogen phosphate 0.1 ~ 0.3g, citric acid eucalyptus 0 ~ 1g, magnesium sulfate 0.3 ~ 0.6g, VB11 ~ 3 slice, sodium selenite 0 ~ 40mg, 1000 milliliters, water.
3. the hypha fermentation thing of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001 as claimed in claim 1 or growth substrate, crushed after being dried, as the raw material of medicine, food and beverage, or be prepared into powder, tablet, freeze dried powder, capsule or electuary, or as feedstuff.
4. the fruit body of Cordyceps militaris (Cordyceps militaris) bacterial classification CM001 as claimed in claim 1, dry or vacuum cooling drying after, make dry product or directly eat after pulverizing, or as the raw material of medicine, health products, food, drinks, flavouring and beverage, or be prepared into powder, tablet, freeze dried powder, capsule or electuary.
5. utilize the Cordyceps militaris described in claim 1 (Cordyceps militaris) bacterial classification CM001 to carry out a method for large-scale industrialized application production, it is characterized in that, its step is as follows:
1), prepared by female kind: be inoculated in PDA slant medium by Cordyceps militaris (Cordyceps militaris) bacterial classification CM001, temperature 18 ~ 24 DEG C, relative air humidity 60 ~ 75%, light culture 4 ~ 7 days, chooses fast, the neat bacterial classification of mycelial growth as mother's kind;
2), level liquid bacterial classification preparation: the mother chosen is planted and is inoculated in strain liquid medium, temperature 18 ~ 24 DEG C, relative air humidity 60 ~ 75%, concussion light culture 3 ~ 7 days, choose mycelium pellet neat, size is that the bacterial classification of 2 ~ 3mm is as level liquid bacterial classification;
3), secondary produces bacterial classification preparation: inoculate in strain liquid medium by level liquid bacterial classification in 3 ~ 10% ratios, temperature 18 ~ 24 DEG C, relative air humidity 60 ~ 75%, concussion light culture 3 ~ 7 days, choose mycelium pellet neat, size is that the bacterial classification of 2 ~ 3mm produces bacterial classification as secondary, produces for Cordyceps militaris factory culture;
4), inoculation and artificial cultivation: with sterile water by volume: sterile water: secondary produce bacterial classification=2 ~ 5:1 dilution proportion secondary produce bacterial classification, then inoculate in solid state cultivation medium, every bottle graft enters 10 ~ 15ml; First carry out light culture 3 ~ 15 days, cover with after medium, proceed to illumination cultivation until mycelia, intensity of illumination is 100 ~ 500Lux, relative air humidity 75 ~ 90%, temperature 18 ~ 24 DEG C, cultivates 50 ~ 60 days, gathers after fruit body maturation;
Described strain liquid culture medium prescription: glucose 1 ~ 10g, sucrose 1 ~ 10g, corn flour 1 ~ 5g, analysis for soybean powder 1 ~ 5g, dusty yeast 1 ~ 5g, peptone 1 ~ 6g, potassium dihydrogen phosphate 1 ~ 1.5g, dipotassium hydrogen phosphate 0.1 ~ 0.3g, citric acid eucalyptus 0 ~ 1g, magnesium sulfate 0.3 ~ 0.6g, VB11 ~ 3 slice, 1000 milliliters, water;
The formula of described solid state cultivation medium: cereal crops 20 ~ 60g, then add 30 ~ 50 milliliters of nutrient solutions and mix thoroughly, described cereal crops are one in rice, sorghum rice, wheat or corn or mixing; The formula of described nutrient solution: glucose 1 ~ 10g, sucrose 1 ~ 10g, dusty yeast 1 ~ 10g, peptone 1 ~ 10g, potassium dihydrogen phosphate 1 ~ 1.5g, dipotassium hydrogen phosphate 0.1 ~ 0.3g, citric acid eucalyptus 0 ~ 1g, magnesium sulfate 0.3 ~ 0.6g, VB11 ~ 3 slice, sodium selenite 0 ~ 40mg, 1000 milliliters, water.
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