CN104145719B - A kind of Cordyceps fungus mycelium fermentation production method - Google Patents
A kind of Cordyceps fungus mycelium fermentation production method Download PDFInfo
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Abstract
The present invention provides a kind of Cordyceps fungus mycelium fermentation production method, comprises the preparation of Cordyceps sinensis strains liquid fermentation special culture media, the preferred of mycelium provenance and be linked in substratum by bacterial classification the steps such as fermentation; Wherein, the preferred of mycelium provenance selects advantage sporophore for be inoculated on artificial solid medium by the Cordyceps strain mycelium after more than 10 times manually go down to posterity, and isolates preferred China by hair spore bacterial classification from sporophore. A kind of Cordyceps fungus mycelium fermentation production method that the present patent application provides, be it is advantageous that and selected by bacterial classification, obtain having kind of a Cordyceps strain for property advantage. Aforesaid method screening effect is good, it is possible to effectively solve kind of a property decline problem, and meanwhile, the method can also go down to posterity method for preserving as a kind of China pilose spore bacterial classification.
Description
Technical field
The present invention relates to a kind of spawn culture method of Cordyceps fungus, in particular to a kind of Cordyceps fungus mycelium fermentation production method, the invention belongs to medicine and field of health care food.
Background technology
Cordyceps sinensis (Ophiocordycepssinensis (Berk.) G.H.Sungetal.) belongs to Ascomycota (Ascomycota), Hypocreales (Hypocreales), nematode grass section (Ophiocordycipitaceae), line Cordyceps (Ophiocordyceps) in taxology. The rare rare traditional Chinese medicine of China of Cordyceps sinensis system special product is the parasitic a kind of entomogenous fungi complex body formed in Hepialus insect larva body of Cordyceps fungus. The medication history of Cordyceps sinensis begins to be loaded in " A Supplement to the Compendium of Materia Medica ", it is claimed to have " effect of tonifying lung benefit kidney, hemostasis and phlegm ", modern age pharmacology and clinical study show, the multiple diseases such as adjustment body's immunity, treatment chronic nephritis, chronic hepatitis, chronic bronchitis, hyperlipidaemia and sexual dysfunction are had significant curative effect by Cordyceps sinensis, have wide market outlook.
Current Cordyceps sinensis natural resource are on the brink of extinction, are listed in special-protection-by-the-State wildlife species (II grade). the rareness of resource and the growth of demand exacerbate the contradiction that supply falls short of demand, cause price suddenly long, and recent two decades carrys out the hat that Cordyceps sinensis price amount of increase is various Chinese medicinal materials price increase amplitude, is called " soft gold " by people. since the 80's of last century, scientific workers attempt to be solved a difficult problem for Cordyceps Resources scarcity by following approach, to meet the needs that Cordyceps sinensis is increased by people. one is the exploitation of the pseudo-anamorph mycelium of Cordyceps sinensis. up to now, isolate from Cordyceps sinensis and comprised China's mortierella, Paecilomyces sinensis, peacilomyce hepiahi, Chinese caterpillar fungus head is satisfied mould, bat moth is satisfied mould, the miscellaneous bacterias such as Scytalidium hepiali, effect of the physico-chemical property of the tunning of these bacterium and some pharmacological functions and Cordyceps sinensis is similar to, above-mentioned bacterial classification is carried out large scale fermentation and produces using the surrogate as Cordyceps sinensis by some enterprises, such as recovering capsule of being bestowed by heaven, JINSHUIBAO capsule, to curing capsule, Ningxinbao Capsules etc., but the fungi that these products use differs greatly at biology and Cordyceps sinensis, clinical efficacy differ bigger, thus it is difficult to substitute natural cordyceps, more can not as real Cordyceps sinensis, two is that other Chinese caterpillar fungus of exploitation replaces Cordyceps sinensis, and such as Cordyceps militaris (L.) Link., although this product gets the Green Light listing as new resources, but its indication and Cordyceps sinensis are inconsistent, also not by human consumer is accepted, three is artificially cultivating cordyceps, cultivate the artificial Cordyceps sinensis form and effective constituent are consistent with natural cordyceps to accept in market the most, this approach solves the Cordyceps Resources basic method that supply falls short of demand and means, but due to artificial culture natural cordyceps relate to that subject is many, research cycle length, part gordian technique need to be improved and relate to " drug registration management method " office make No. 28 declare a difficult problem, the Cordyceps sinensis that real artificial industrialization is cultivated appears on the market still not within the foreseeable future,Four is by modern biotechnology, utilizes Cordyceps strain to carry out mycelium fermentation, it is achieved the industrialization of Cordyceps fungus, and this solves Cordyceps Resources the most real deficient method and means at present.
Cordyceps sinensis is the parasitic bacterium worm complex body formed in Hepialus insect body of Cordyceps fungus, and the main medical active composition of Cordyceps sinensis comes from the meta-bolites of Cordyceps fungus. Modern Pharmaceutical Chemistry and pharmaceutical research show that Cordyceps mycelium and natural cordyceps have close chemical composition, have similar pharmacological action. Therefore, the liquid submerged fermentation carrying out Cordyceps fungus produces Cordyceps mycelium to replace natural Cordyceps sinensis, is possible not only to the pressure alleviating wild cordyceps resource, also can meet people to the demand of Cordyceps sinensis health care function.
Cordyceps fungus is a kind of addicted to low temperature modification fungi, and its growth temperature is lower than 20 DEG C. When culture temperature is 20 DEG C-22 DEG C, Cordyceps mycelium grows extremely slowly and bacterium colony is formed little; When culture temperature is higher than 23 DEG C, mycelium does not almost grow. Meanwhile, Cordyceps fungus is also higher to the requirement of substratum nutrition, even if on common fungi culture medium, mycelial growth is also very slow when the suitableeest culture temperature, mycelia yield is low. Produce Cordyceps mycelium owing to leavening temperature is low, poor growth, culture cycle are long by batch production fermentation pattern, usually end in failure due to staining of miscellaneous bacteria, fermentation costs height. At present, domestic existing many units have carried out the liquid culture studies of Cordyceps fungus, but its fermentation mycelium product is not all real Cordyceps fungus after molecular biology identification.
Relative to animal, plant, fungi easily makes a variation and degenerates, in the liquid culturing process of Cordyceps fungus, general employing inclined-plane kind is to the multistage fermentation culture pattern of liquid strain, slant strains goes down to posterity through repeatedly turning pipe, spawn degeneration relatively serious (from female kind of separation turn pipe about 15 generation just start decline), on slant medium, growth perfonnance is that mycelial growth is slow, easy aging self-dissolving, after the production of hybrid seeds, in liquid medium within, fermentation mycelium is sparse, bacterium ball particle big surface aurora are sliding, it is poor that bacterium ball mass transfer passes oxygen, biomass yield is low, active constituent content is low. cause a lot of because have of this kind of phenomenon, substantially can be divided into two classes: one is bacterial strain causes the decline of bacterial classification owing to the forfeiture of physiological function gene causes the genetics characteristic of self to change, two is the bacterial classification decline that the human factors such as passage number, culture condition, environmental factor cause. in culturing process, bacterial classification fails, thread fungus mycelial growth gesture is weak is the big bottleneck restricting Cordyceps fungus fermentation industrialization. therefore, the fermentation production process of Cordyceps fungus is taked the measure of selecting optimized recover the good characteristic of bacterial classification, biomass and active constituent content this bottleneck problem low is caused, it is achieved sustainable, the stably manufactured of Cordyceps fungus with the bacterial classification decline solved in Cordyceps fungus fermentation industrialization process.
The principle that Cordyceps strain is selected be obtain strong stress resistance as much as possible, bacterial classification that hereditary property is excellent. The primary border of not high can be carried out shaker test on a large scale, and sporophore, the Cordyceps strain obtained it is separated by the mode of vegetative propagation, by the liquid state fermentation special culture media mentioned in the present invention and cultural method, winter worm summer herb thallus biomass yield and adenosine content all plant the suitable of test tube strains with female.
At present; triage techniques (patent of invention CN103404368A) one literary composition of Cordyceps strain describes the screening and culturing method of artificial breeding cordyceps species; the object of this invention is the Sustainable Production problem solving bacterial classification in Chinese caterpillar fungus artificial culture, for the mass-producing sustainability of Cordyceps sinensis medicinal material produces offer bacterial classification guarantee.It is characterized in that utilizing cordyceps species Chinese caterpillar fungus host to be infected, continue after infection to cultivate 1-2 age, render to screening base, after transitional period, exercise phase, gather the Cordyceps sporophore of sexual spore maturation in the wild, utilize ripe sexual spore to carry out the kind screening of Cordyceps. Having the difference of matter with the present invention, concrete difference is: 1. goal of the invention and principle are different. Foregoing invention content utilizes host insect as screening vector, relate to Cordyceps to the infection processs of the bright bat insect complexity of host, infect and successfully continue to cultivate afterwards, from polypide, grow the stroma that can produce ripe sexual spore, utilize ripe sexual spore to be carried out separation, the screening of Cordyceps by the mode of sexual propagation. Object produces for the artificial mass-producing sustainability of Cordyceps sinensis medicinal material to provide bacterial classification guarantee. The present invention utilizes the liquid-spawn inoculation of Cordyceps sinensis anamorph in screening culture medium, conventional environment utilizes growth cabinet cultivate and grow sporophore, sporophore is utilized to carry out bacterial screening by the mode of vegetative propagation, it is intended that to solve the bacterial classification kind decline problem that Cordyceps strain occurs in liquid state fermentation culturing process. 2. screen the cycle different. Being calculated in cycle in foregoing invention content, from host insect infects after cordyceps species, maturation to sexual spore needs 3-5, and culture cycle is extremely long. The screening cycle of the present invention is 55-65 days, and easy and simple to handle, controlled, the cycle is short. 3. culture condition is different. Foregoing invention content must carry out in Qinghai Tibet plateau cordyceps main producing region, utilize local special geographical environment, as temperature all the year round on the low side, day and night temperature big, height above sea level higher than 2000m, air rarefaction, oxygen level is low, air pressure is low. And the method in the present invention to geographical environment without particular requirement, all can turn out sporophore in the laboratory of tool growth cabinet by the inventive method.
Patent of invention CN101695255B relates to a kind of method that China is cultivated Cordyceps sinensis stroma by hair spore. The full egg, the beef peptone that it is characterized in that getting Semen Maydis grit, analysis for soybean powder, wheat groat, grain, sorghum grain, dried silkworm chrysalis meal or pulverizing are soaked in water; Primary material is soaked and growth hormone hybrid filtering obtain filtrate and on the substratum of artificial preparation, height above sea level more than 1800 meters, day and night temperature >=15 DEG C, without the environment of industrial pollution connecting asexual bacterial classification by hair spore, the stroma of output eldest son's cystocarp in batches. The Cordyceps sinensis stroma biological transformation ratio cultivated about 40%, containing adenosine C10H13N5O4It is greater than 0.02%, containing cordycepin C10H13N5O3It is greater than 0.10%. It is from the difference of matter of the present invention: 1. principle and object are different. Foregoing invention content it may be seen that object is the Cordyceps sinensis stroma cultivating eldest son's cystocarp on the substratum of artificial preparation, belongs to Sexual breed category from its summary of the invention. And the present invention be directed in Cordyceps sinensis strains liquid fermentation production process the kind decline technical problem run into, operated by the seed selection of bacterial classification, make Cordyceps strain keep female good characteristic planted to belong to pierre category. 2. formula and culture condition are different. Formula in the formula mentioned in foregoing invention content and ratio and the present invention and ratio all have the difference of matter, research finds, Cordyceps fungus growing state on different culture media is totally different, and different carbon source, nitrogenous source, vitamin contents, microelement match and carbon-nitrogen ratio all can affect the formation of fruit body primordium and whether grow sporophore.The present invention, without the need to special geographical conditions, all can carry out in common lab, and foregoing invention content characteristic is that, at more than height above sea level 1800m, day and night temperature is more than or equal to 15 DEG C, carries out without in the Qinghai Tibet plateau cordyceps main producing region environment of industrial pollution.
In sum, patent of invention CN103404368A, CN101695255B are compared with the present invention, all different from the selection of inventive principle, object, mode of reproduction, purposes, culture medium, composition proportion, technology category and culture condition etc. compared with above two patents of invention, the present invention Cordyceps strain is selected have easy and simple to handle, do not limit by region, pollution rate is low, success ratio height, the advantages such as the cycle is short, after selecting, the bacterial classification of separation is used further to liquid state fermentation, Fungal biodiversity and adenosine content and female plant close, solve the decline problem of the bacterial classification kind in Cordyceps sinensis strains liquid fermentation process, ensure that Fungal biodiversity and active constituent content, it is applicable to Cordyceps sinensis commerial scale sustainable development ferment produce, and the present invention is to reduction cordyceps sinensis mycelium powder cost, promote mycelium powder really to move towards consumers in general and there is important value and significance.
Summary of the invention
Cordyceps strain turns pipe and cultivates and in liquid state fermentation culturing process, decline occurs in bacterial classification repeatedly going down to posterity, and seriously affects the expression of winter worm summer herb thallus biomass and adenosine content. Therefore, the present invention provides the fermentation method for producing of a kind of Cordyceps fungus.
The Cordyceps strain screening method of the present invention comprises the steps:
The silkworm chrysalis do not sprouted wings or cocoon chrysalis tissue refiner being processed 5-10min, if there being the pupa skin do not smashed, abandoning pupa skin, gained tissue juice is silkworm chrysalis or cocoon chrysalis tissue juice.
The preparation (raw material part number is weight part number) of solid substrate: take rice 10-40 part, Semen Maydis powder 10-30 part, wheat 15-35 part, Chinese sorghum 10-30 part, oatmeal 5-15 part, wheat bran 5-15 part.
The preparation of nutritive medium: glucose 10-30g/L, sucrose 10-20g/L, peptone 10-20g/L, yeast extract paste 5-20g/L, compound amino acid powder 5-20g/L, potassium primary phosphate 0.1-0.5g/L, dipotassium hydrogen phosphate 0.1-0.5g/L, magnesium sulfate 0.1-0.5g/L, vitamins B15mg-50mg/L, vitamins B25mg-50mg/L, vitamins B610mg-50mg/L, silkworm chrysalis or cocoon chrysalis tissue juice 50-150g/L.
Cordyceps fungus selects the preparation of substratum: by solid substrate and nutritive medium by weight proportion 1:1.4-1.8 prepare.
By the can above screening culture medium of vial splendid attire, with sealing film sealing, 121 DEG C of sterilizing 30min, it is cooled to room temperature, connects Cordyceps fungus liquid spawn, the amount of every 100 grams of screening culture medium access Cordyceps sinensis liquid spawn is 10-30ml, and bacterial classification is evenly distributed on media surface. Described liquid spawn be by aseptic technique by after the Cordyceps fungus of pure culture access shaking flask by shaking flask liquid culture 10-18 days gained; Described can glass bottle height �� diameter=12cm �� 10cm; Described sealing film is 15cm �� 15cm, and central authorities are circle, 0.2 ��m, the aperture air-permeable filter screen of diameter 2cm, every bottled solid substrate 80 grams.
Vial after inoculation is placed in growth cabinet, and optimal temperature is 15-20 DEG C, and relative air humidity is 50%-75%, and lucifuge is cultivated 25-30 days.
Being covered with after whole substratum plane until mycelia, give light stimulation, daytime every day, light application time was not less than 10 hours, and intensity of illumination is 150-500LX, temperature 12-18 DEG C, and daytime, the culture temperature temperature difference was not less than 3 DEG C, relative air humidity 50%-75%.Mycelia produces safran pigment after optical processing, forms button in media surface, continues to cultivate button and grows into sporophore.
With tweezers from sporophore root tweezer Cordyceps sporophore, fresh sporophore is carried out separate tissue, screening Cordyceps strain can be obtained. Described Cordyceps sporophore is closely cylindrical, beige, the slightly thick 1.5-5mm in base portion, long about 1.5-6cm.
Another object of the present invention is the liquid cultural method that the bacterial classification utilizing the present invention to select provides a kind of Cordyceps fungus sustainably, wherein also relates to a kind of Cordyceps sinensis strains liquid fermentation special culture media.
The liquid cultural method of Cordyceps fungus provided by the invention comprises the steps:
Cordyceps sinensis strains liquid fermentation special culture media comprises: monose or 1-30 gram, disaccharide, compound nitrogen source 1-200 gram, aminoacids complex 0-10 gram, peptone 0-30 gram, fish meal 0-20 gram, yeast powder 0-20 gram, VITMAIN B1 10-80mg, Lin Suanna Vitamin B2 Sodium Phosphate 10-80mg, dipotassium hydrogen phosphate 0-1 gram, potassium primary phosphate 0-1 gram, 0-1 gram, magnesium sulfate, 0-5 gram, animal oil, is settled to 1000ml with water.
Above-mentioned monose or disaccharide are glucose, sucrose; Above-mentioned compound nitrogen source is corn, bean powder, wheat bran, dried silkworm chrysalis meal, cocoon chrysalis, pure milk or oatmeal; Above-mentioned aminoacids complex is L-glutamic acid, glycine; Above-mentioned animal oil is tenebrio molitor worm oil or silkworm chrysalis oil. Described Cordyceps sinensis strains liquid fermentation special culture media screening formulation is: glucose 5-30 gram, compound nitrogen source 50-200 gram, 0-5 gram, L-glutamic acid, glycine 0-5 gram, peptone 5-30 gram, fish meal 5-20 gram, yeast powder 5-20 gram, VITMAIN B1 10-50mg, Lin Suanna Vitamin B2 Sodium Phosphate 10-50mg, dipotassium hydrogen phosphate 0-0.5 gram, potassium primary phosphate 0-0.5 gram, 0-0.5 gram, magnesium sulfate, tenebrio molitor worm oil 0-4 gram, is settled to 1000ml with water.
Described Cordyceps sinensis strains liquid fermentation special culture media is particularly preferably filled a prescription and is: glucose 15-30 gram, compound nitrogen source 80-200 gram, 1-5 gram, L-glutamic acid, glycine 1-5 gram, peptone 15-30 gram, fish meal 10-20 gram, yeast powder 10-20 gram, vitamin B12 0-50mg, Lin Suanna Vitamin B2 Sodium Phosphate 20-50mg, dipotassium hydrogen phosphate 0.2-0.5 gram, potassium primary phosphate 0.2-0.5 gram, 0.1-0.3 gram, magnesium sulfate, tenebrio molitor worm oil 2-4 gram, is settled to 1000ml with water.
Add water boiling twice by above-mentioned compound nitrogen source by weight ratio, each 20 minutes, gets supernatant liquor in container after filtered through gauze, add other composition again, after heating for dissolving, divide and it is filled in triangular pyramidal bottle, 121 DEG C, high pressure steam sterilization 30min after cotton plug beyond the Great Wall.
The preparation of liquid seeds: the liquid nutrient medium of bacterium of step (5) having been gone out accesses the glass test tube inclined-plane Cordyceps strain of length �� wide=200mm �� 20mm in Bechtop, is placed on shaking table and cultivates. Described liquid amount is that the 1/10-3/5 of triangular pyramidal bottle capacity, initial pH value 5-7.5, culture temperature 10 DEG C-20 DEG C, rotating speed 120-200rpm, lucifuge are cultivated and can be obtained the liquid seeds being in fast growing period in 12-16 days.
Liquid seeds prepares optimal conditions: the glass test tube inclined-plane Cordyceps strain usage quantity of described length �� wide=200mm �� 20mm is that 4-7 props up/400ml liquid nutrient medium; Liquid amount is the 1/5-3/5 of triangular pyramidal bottle capacity; Initial pH value is 5.5-6.5; Culture temperature is preferably 15-19 DEG C; Rotating speed is preferably 180rpm.
Cordyceps sinensis strains liquid fermentation cultivate: by under initial pH5-7, inoculum size 5%-25% by Cordyceps fungus liquid spawn access Cordyceps fungus special culture media.Optimal temperature 15-18 DEG C, rotating speed 150-200rpm, liquid amount 1/5-3/5, lucifuge reach fermentation termination for shaking culture 8-12 days and can obtain Cordyceps mycelium.
Cordyceps sinensis strains liquid fermentation cultivates optimum condition: initial pH value 5.5-7, inoculum size 10-20%, culture temperature 16-18 DEG C, rotating speed 170-190rpm, shaking flask liquid amount be 1/5-2/5; The judgement of described fermentation termination is reducing sugar content lower than 1.5%, ammonia-state nitrogen is lower than 200 �� g/ml;
Cordyceps sinensis strains liquid fermentation cultivates most preferably condition: initial pH value 5.5-6.5, inoculum size 10-20%, culture temperature 16-18 DEG C, rotating speed 180rpm, shaking flask liquid amount be 2/5; Shaking culture 8-12 days, the judgement of fermentation termination was reducing sugar content lower than 1.5%, ammonia-state nitrogen is lower than 200 �� g/ml;
Above-mentioned liquid cultural method can amplify step by step according to 10 times of liquid nutrient medium amount (volume), till reaching the required mycelia scale of construction.
The technique effect of the present invention is: a kind of Cordyceps fungus mycelium fermentation production method that the present patent application provides, and it is advantageous that and is selected by bacterial classification, obtains having kind of a Cordyceps strain for property advantage. Aforesaid method screening effect is good, it is possible to effectively solve kind of a property decline problem, and meanwhile, the method can also go down to posterity method for preserving as a kind of China pilose spore bacterial classification.
Embodiment:
Embodiment 1: Cordyceps mycelium liquid state fermentation is produced
Material therefor and explanation: the new isolated strains of Cordyceps sinensis (female kind) is Cordyceps fungus through molecular biology identification, is numbered DC-0; The 10th generation bacterial strain (being numbered DC-10), the 15th generation bacterial strain (being numbered DC-15), the 20th generation bacterial strain (being numbered DC-20), the 30th generation bacterial strain (being numbered DC-30) is obtained respectively by starting strain Secondary Culture of this female kind;
Silkworm chrysalis or cocoon chrysalis: commercially available fresh living silkworm chrysalises or cocoon chrysalis, for the preparation of tissue juice.
The preparation of Cordyceps fungus screening culture medium: solid substrate (rice 10-40 part, Semen Maydis powder 10-30 part, wheat 15-35 part, Chinese sorghum 10-30 part, oatmeal 5-15 part, wheat bran 5-15 part) and nutritive medium (cocoon chrysalis tissue juice 50-150g/L, glucose 10-30g/L, sucrose 10-20g/L, peptone 10-20g/L, yeast extract paste 5-20g/L, compound amino acid powder 5-20g/L, potassium primary phosphate 0.1-0.5g/L, dipotassium hydrogen phosphate 0.1-0.5g/L, magnesium sulfate 0.1-0.5g/L, vitamins B15mg-50mg/L, vitamins B25mg-50mg/L, vitamins B610mg-50mg/L) prepare according to the ratio of 1:1.6.
Cordyceps sinensis strains liquid fermentation special culture media: glucose 15-30 gram, compound nitrogen source 80-200 gram, 1-5 gram, L-glutamic acid, glycine 1-5 gram, peptone 15-30 gram, fish meal 10-20 gram, yeast powder 10-20 gram, vitamin B12 0-50mg, Lin Suanna Vitamin B2 Sodium Phosphate 20-50mg, dipotassium hydrogen phosphate 0.2-0.5 gram, potassium primary phosphate 0.2-0.5 gram, 0.1-0.3 gram, magnesium sulfate, tenebrio molitor worm oil 2-4 gram, is settled to 1000ml with water.
Glass preserving jar: glass bottle height �� diameter=12cm �� 10cm; Described sealing film is 15cm �� 15cm, and central authorities are the air-permeable filter core of 0.2 ��m, and diameter is the circular filter core of 2cm, every bottled solid substrate 80 grams.
Sealing film: length �� wide=15cm �� 15cm, central is 0.2 ��m of air-permeable filter screen for diameter is 2cm, circle, aperture.
Cordyceps sinensis strains liquid fermentation special culture media: glucose 20 grams, dried silkworm chrysalis meal 30 grams, bean powder 30 grams, corn 30 grams, 2 grams, L-glutamic acid, glycine 2 grams, peptone 15 grams, fish meal 10 grams, yeast powder 15 grams, vitamin B12 0mg, Lin Suanna Vitamin B2 Sodium Phosphate 20mg, dipotassium hydrogen phosphate 0.4 gram, potassium primary phosphate 0.4 gram, 0.2 gram, magnesium sulfate, tenebrio molitor worm oil 3 grams, is settled to 1000ml with water.
The preparation of Cordyceps sinensis strains liquid fermentation special culture media: dried silkworm chrysalis meal, bean powder, corn are put into steam cooker and added water boiling twice, each 20 minutes, get supernatant liquor after filtered through gauze in container, then add other nutritive ingredient, after heating for dissolving, moisturizing is to 1000ml.
1, different passage number bacterial strain comparation and assessment
Initial pH5.5-6.5, is connected in substratum by inoculum size 10% by Cordyceps strain, and shaking flask liquid amount is the 2/5 of volume. Then culture temperature 16 DEG C, shaking speed 180rpm, lucifuge cultivate 8-12 days (to nutrient solution reducing sugar content lower than 1.5%, ammonia-state nitrogen lower than 200 �� g/ml time), obtain Cordyceps mycelium. The Cordyceps strain of different passage number is carried out fermentation test, has often organized three parallel, taking mycelial biomass and adenosine content as inspection target.
The different algebraically bacterial strain rating test of table 1
As can be drawn from Table 1, same bacterial classification is used further to fermentative production after reaching the 15th generation and 15 generations, and now Fungal biodiversity and active constituent content are all on the low side, there is significance (P < 0.05). Passage number is more many, and bacterial strain good characteristic is lost more many, causes strain fermentation ability to fail, and is embodied in Fungal biodiversity and adenosine synthesis capability all goes down.
2, bacterial strain comparation and assessment before and after screening
In reality is produced greatly, for solving in fermenting process the problem of the bacterial strain decline occurred, ensure that the normal industrialization of bacterial strain is produced, Cordyceps fungus has been selected. In his-and-hers watches 1, bacterial strain DC-15, DC-20, DC-30 carry out sieve and select respectively, and method is as follows:
1) silkworm chrysalis do not sprouted wings or cocoon chrysalis refiner being processed 5-10min, if there being the pupa skin do not smashed, abandoning pupa skin, gained tissue juice is silkworm chrysalis or cocoon chrysalis tissue juice.
2) preparation of Cordyceps fungus screening culture medium: solid substrate (15 parts, rice, Semen Maydis powder 15 parts, wheat 15 parts, Chinese sorghum 15 parts, oatmeal 10 parts, 10 parts, wheat bran) and nutritive medium (cocoon chrysalis tissue juice 100g/L, glucose 15g/L, sucrose 10g/L, peptone 10g/L, yeast extract paste 10g/L, compound amino acid powder 10g/L, potassium primary phosphate 0.4g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.2g/L, vitamins B120mg/L, vitamins B220mg/L, vitamins B630mg/L) prepare according to the ratio of 1:1.6.
3) by the above screening culture medium of glass preserving jar splendid attire, with sealing film sealing, 121 DEG C of sterilizing 30min, it is cooled to room temperature, connects Cordyceps fungus liquid spawn, the amount of every 100 grams of screening culture medium access Cordyceps sinensis liquid spawn is 10-30ml, and bacterial classification is evenly distributed on media surface.
4) vial after above-mentioned inoculation being placed in growth cabinet, temperature is 16 DEG C, and relative air humidity is 60%, and lucifuge cultivates 30 days.
5) after mycelia covers with whole substratum plane, give light stimulation, daytime every day light application time 12 hours, intensity of illumination is 200LX, day temperature 16 DEG C, diurnal temperature 12 DEG C (daytime, the culture temperature temperature difference was not less than 3 DEG C), relative air humidity 60%, mycelia produces safran pigment after optical processing, continues cultivation and treats that former base is differentiated to form button, till button grows into sporophore.
6) gather Cordyceps sporophore from sporophore root with tweezers, fresh sporophore is carried out separate tissue, Cordyceps strain can be obtained.
Bacterial strain after screening is numbered: DC-15-FZ (the screening and separating bacterial strain of DC-15 bacterial strain, the first-generation), DC-20-FZ (the screening and separating bacterial strain of DC-20 bacterial strain, the first-generation), DC-30-FZ (the screening and separating bacterial strain of DC-30 bacterial strain, the first-generation).At initial pH5.5-6.5, Cordyceps strain is connected in substratum by inoculum size 10%, and shaking flask liquid amount is the 2/5 of volume. Then culture temperature 16 DEG C, shaking speed 180rpm, lucifuge cultivate 8-12 days (to nutrient solution reducing sugar content lower than 1.5%, ammonia-state nitrogen lower than 200 �� g/ml time), obtain Cordyceps mycelium. To before screening with screening after Cordyceps strain carried out fermentation test, often organize three parallel, taking mycelial biomass and adenosine content as inspection target, the results are shown in Table 2.
Table 2 screens front and back different strains rating test
As can be seen from Table 2, the bacterial strain that the bacterial strain of different algebraically is separated after adopting the screening of the screening method in the present invention for fermentation mycelial biomass with adenosine content compared with DC-0 (female kind), DC-10, there is not significant difference (P>0.05). DC-15 and DC-15-FZ, DC-20 and DC-20-FZ, DC-30 and DC-30-FZ compare, and on biomass and adenosine content, the bacterial strain after screening is all significantly better than the bacterial strain (P<0.05) before screening. Therefore, adopt the screening method of the present invention can solve bacterial strain decline problem very well, thus solve the bacterial classification sustainable use in actual big production and production problem.
Embodiment 2: the rear bacterial strain comparation and assessment parallel verified test with screening before screening
First group of parallel test:
Adopt the DC-0 bacterial classification identical with embodiment 1; In his-and-hers watches 1, bacterial strain DC-15, DC-20, DC-30 screen respectively, and screening method is as follows:
Wherein, the preparation of Cordyceps fungus screening culture medium: solid substrate (10 parts, rice, Semen Maydis powder 30 parts, wheat 15 parts, Chinese sorghum 30 parts, oatmeal 15 parts, 5 parts, wheat bran) and nutritive medium (cocoon chrysalis tissue juice 50g/L, glucose 10g/L, sucrose 10g/L, peptone 20g/L, yeast extract paste 5g/L, compound amino acid powder 5g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, magnesium sulfate 0.1g/L, vitamins B15mg/L, vitamins B25mg/L, vitamins B610mg/L) prepare according to the ratio of 1:1.4.
Other working method are identical with embodiment 1; In his-and-hers watches 1, bacterial strain DC-15, DC-20, DC-30 screen respectively.
Bacterial strain after screening is numbered: DC-15-FZ (the screening and separating bacterial strain of DC-15 bacterial strain, the first-generation), DC-20-FZ (the screening and separating bacterial strain of DC-20 bacterial strain, the first-generation), DC-30-FZ (the screening and separating bacterial strain of DC-30 bacterial strain, the first-generation). At initial pH5.5-6.5, Cordyceps strain is connected in substratum by inoculum size 10%, and shaking flask liquid amount is the 2/5 of volume. Then culture temperature 16 DEG C, shaking speed 180rpm, lucifuge cultivate 8-12 days (to nutrient solution reducing sugar content lower than 1.5%, ammonia-state nitrogen lower than 200 �� g/ml time), obtain Cordyceps mycelium. To before screening with screening after Cordyceps strain carried out fermentation test, often organize three parallel, taking mycelial biomass and adenosine content as inspection target, the results are shown in Table shown in 3.
Table 3 is different strains rating test rear with screening before screening
As can be seen from Table 3, the bacterial strain that the bacterial strain of different algebraically is separated after adopting the screening of the screening method in the present invention mycelial biomass with adenosine content compared with DC-0 (female kind), DC-10, there is not significant difference (P>0.05). DC-15 and DC-15-FZ, DC-20 and DC-20-FZ, DC-30 and DC-30-FZ compare, and on biomass and adenosine content, the bacterial strain after screening is all significantly better than the bacterial strain (P<0.05) before screening.Therefore, adopt the screening method of the present invention can solve bacterial strain decline problem very well, thus solve the bacterial classification sustainable use in actual big production and production problem.
2nd group of parallel test:
Adopt the DC-0 bacterial classification identical with embodiment 1; In his-and-hers watches 1, bacterial strain DC-15, DC-20, DC-30 screen respectively, and screening method is as follows:
Wherein, the preparation of Cordyceps fungus screening culture medium: solid substrate (40 parts, rice, Semen Maydis powder 10 parts, wheat 35 parts, Chinese sorghum 10 parts, oatmeal 5 parts, 15 parts, wheat bran) and nutritive medium (cocoon chrysalis tissue juice 150g/L, glucose 30g/L, sucrose 20g/L, peptone 10g/L, yeast extract paste 20g/L, compound amino acid powder 20g/L, potassium primary phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, vitamins B150mg/L, vitamins B250mg/L, vitamins B650mg/L) prepare according to the ratio of 1:1.8.
Other operations of test are identical with embodiment 1.
Bacterial strain after screening is numbered: DC-15-FZ (the screening and separating bacterial strain of DC-15 bacterial strain, the first-generation), DC-20-FZ (the screening and separating bacterial strain of DC-20 bacterial strain, the first-generation), DC-30-FZ (the screening and separating bacterial strain of DC-30 bacterial strain, the first-generation). At initial pH5.5-6.5, Cordyceps strain is connected in substratum by inoculum size 10%, and shaking flask liquid amount is the 2/5 of volume. Then culture temperature 16 DEG C, shaking speed 180rpm, lucifuge cultivate 8-12 days (to nutrient solution reducing sugar content lower than 1.5%, ammonia-state nitrogen lower than 200 �� g/ml time), obtain Cordyceps mycelium. To before screening with screening after Cordyceps strain carried out fermentation test, often organize three parallel, taking mycelial biomass and adenosine content as inspection target, the results are shown in Table 4.
Table 4 is different strains rating test rear with screening before screening
As can be seen from Table 4, the bacterial strain that the bacterial strain of different algebraically is separated after adopting the screening of the screening method in the present invention mycelial biomass with adenosine content compared with DC-0 (female kind), DC-10, there is not significant difference (P>0.05). DC-15 and DC-15-FZ, DC-20 and DC-20-FZ, DC-30 and DC-30-FZ compare, and on biomass and adenosine content, the bacterial strain after screening is all significantly better than the bacterial strain (P<0.05) before screening. Therefore, adopt the screening method of the present invention can solve bacterial strain decline problem very well, thus solve the bacterial classification sustainable use in actual big production and production problem.
The application of embodiment 3 screening method in bacterial classification goes down to posterity
Prepare Cordyceps sinensis strains liquid fermentation special culture media: glucose 20 grams, dried silkworm chrysalis meal 30 grams, corn 30 grams, pure milk 50 grams, 2 grams, L-glutamic acid, glycine 2 grams, peptone 15 grams, fish meal 10 grams, yeast powder 15 grams, vitamin B12 0mg, Lin Suanna Vitamin B2 Sodium Phosphate 20mg, dipotassium hydrogen phosphate 0.4 gram, potassium primary phosphate 0.4 gram, 0.2 gram, magnesium sulfate, tenebrio molitor worm oil 3 grams.
Dried silkworm chrysalis meal, corn being put into steam cooker add water boiling twice, each 20 minutes, get supernatant liquor in container, then add other composition after filtered through gauze, after heating for dissolving, moisturizing is to 1000ml.
Adopting the mother of the DC-0 in embodiment 1 to plant, initial pH5.5-6.5, Cordyceps strain is connected in substratum by inoculum size 10%, and shaking flask liquid amount is the 2/5 of volume. Then culture temperature 16 DEG C, shaking speed 180rpm, lucifuge cultivate 8-12 days (to nutrient solution reducing sugar content lower than 1.5%, ammonia-state nitrogen lower than 200 �� g/ml time), obtain Cordyceps mycelium.The Cordyceps strain of different passage number is carried out fermentation test, has often organized three parallel, taking mycelial biomass and adenosine content as inspection target, the results are shown in Table 5.
The different algebraically bacterial strain rating test of table 5
As can be drawn from Table 5, same bacterial classification passage number is more than after 15 generations, it is used further to fermentative production, now thalli growth gesture and the equal significance of active constituent content (P < 0.05) on the low side, passage number is more many, bacterial strain part excellent genes is lost, and causes strain fermentation ability to fail, and Fungal biodiversity and adenosine synthesis capability go down.
Adopt the application of the screening method described in embodiment 1,2 in bacterial classification goes down to posterity.
In table 5, bacterial strain DC-15 is as initial strain, carries out research of going down to posterity, and method is as follows:
1) silkworm chrysalis do not sprouted wings or cocoon chrysalis refiner being processed 5-10min, if there being the pupa skin do not smashed, abandoning pupa skin, gained tissue juice is silkworm chrysalis or cocoon chrysalis tissue juice.
2) preparation of Cordyceps fungus screening culture medium: solid substrate (15 parts, rice, Semen Maydis powder 15 parts, wheat 15 parts, Chinese sorghum 15 parts, oatmeal 10 parts, 10 parts, wheat bran) and nutritive medium (cocoon chrysalis tissue juice 100g/L, glucose 15g/L, sucrose 10g/L, peptone 10g/L, yeast extract paste 10g/L, compound amino acid powder 10g/L, potassium primary phosphate 0.4g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.2g/L, vitamins B120mg/L, vitamins B220mg/L, vitamins B630mg/L) prepare according to the ratio of 1:1.6.
3) by the above screening culture medium of glass preserving jar splendid attire, with sealing film sealing, 121 DEG C of sterilizing 30min, it is cooled to room temperature, connects Cordyceps fungus liquid spawn, the amount of every 100 grams of screening culture medium access Cordyceps sinensis liquid spawn is 10-30ml, and bacterial classification is evenly distributed on media surface.
4) vial after above-mentioned inoculation being placed in growth cabinet, temperature is 16 DEG C, and relative air humidity is 60%, and lucifuge cultivates 30 days.
5) after mycelia covers with whole substratum plane, give light stimulation, daytime every day light application time 12 hours, intensity of illumination is 200LX, day temperature 16 DEG C, diurnal temperature 12 DEG C (daytime, the culture temperature temperature difference was not less than 3 DEG C), relative air humidity 60%, mycelia produces safran pigment after optical processing, continues cultivation and treats that former base is differentiated to form button, till button grows sporophore.
6) gather Cordyceps sporophore from sporophore root with tweezers, fresh sporophore is carried out separate tissue, the Cordyceps strain (China is by hair spore bacterial strain) of screening can be obtained.
7) by above-mentioned steps 6) described bacterial classification, repeat above-mentioned steps 1)-step 6) and method go down to posterity. Collect respectively and go down to posterity the 5th time (to by name: DC-5-CF), the 10th time (to by name: the DC-10-CF) and 15th time (to being called: the bacterial classification of sporophore DC-15-CF) gone down to posterity; At initial pH5.5-6.5, Cordyceps strain is connected in substratum by inoculum size 10%, and shaking flask liquid amount is the 2/5 of volume. Then culture temperature 16 DEG C, shaking speed 180rpm, lucifuge cultivate 8-12 days (to nutrient solution reducing sugar content lower than 1.5%, ammonia-state nitrogen lower than 200 �� g/ml time), obtain Cordyceps mycelium. To before screening with screening after Cordyceps strain carried out fermentation test, often organize three parallel, taking mycelial biomass and adenosine content as inspection target, the results are shown in Table 6.
Table 6 goes down to posterity effect expedition
As can be seen from Table 6, adopt the method for sporophore screening to carry out going down to posterity bacterial strain that obtained bacterial classification is separated mycelial biomass with adenosine content compared with DC-0 (female kind), DC-10, there is not significant difference (P > 0.05).Therefore, the screening method of employing the present invention carries out going down to posterity and can solve bacterial strain kind decline problem very well, allows the good characteristic of bacterial strain be continued, thus solves the bacterial classification sustainable use problem in actual production.
Embodiment 4 Cordyceps fungus mycelium fermentation is produced
Material therefor and explanation: the new separating and preserving bacterial classification of Cordyceps sinensis (female kind) is Cordyceps fungus through molecular biology identification, is numbered DC-0; Obtain the 10th generation bacterial strain (being numbered DC-10), the 15th generation bacterial strain (being numbered DC-15), the 20th generation bacterial strain (being numbered DC-20), the 30th generation bacterial strain (being numbered DC-30) taking this female kind respectively as starting strain Secondary Culture, analogized for the n-th generation bacterial strain (being numbered DC-n) with this;
Silkworm chrysalis or cocoon chrysalis: commercially available fresh living silkworm chrysalises or cocoon chrysalis, for the preparation of tissue juice.
The preparation of Cordyceps fungus screening culture medium: solid substrate (rice 10-40 part, Semen Maydis powder 10-30 part, wheat 15-35 part, Chinese sorghum 10-30 part, oatmeal 5-15 part, wheat bran 5-15 part) and nutritive medium (cocoon chrysalis tissue juice 50-150g/L, glucose 10-30g/L, sucrose 10-20g/L, peptone 10-20g/L, yeast extract paste 5-20g/L, compound amino acid powder 5-20g/L, potassium primary phosphate 0.1-0.5g/L, dipotassium hydrogen phosphate 0.1-0.5g/L, magnesium sulfate 0.1-0.5g/L, vitamins B15mg-50mg/L, vitamins B25mg-50mg/L, vitamins B610mg-50mg/L) prepare according to the ratio of 1:1.6.
Cordyceps sinensis strains liquid fermentation special culture media: glucose 15-30 gram, compound nitrogen source 80-200 gram, 1-5 gram, L-glutamic acid, glycine 1-5 gram, peptone 15-30 gram, fish meal 10-20 gram, yeast powder 10-20 gram, vitamin B12 0-50mg, Lin Suanna Vitamin B2 Sodium Phosphate 20-50mg, dipotassium hydrogen phosphate 0.2-0.5 gram, potassium primary phosphate 0.2-0.5 gram, 0.1-0.3 gram, magnesium sulfate, tenebrio molitor worm oil 2-4 gram, is settled to 1000ml with water.
Glass preserving jar: glass bottle height �� diameter=12cm �� 10cm; Described sealing film is 15cm �� 15cm, and central authorities are the air-permeable filter core of 0.2 ��m, and diameter is the circular filter core of 2cm, every bottled solid substrate 80 grams.
Sealing film: length �� wide=15cm �� 15cm, central is 0.2 ��m of air-permeable filter screen for diameter is 2cm, circle, aperture.
Cordyceps sinensis strains liquid fermentation special culture media: glucose 20 grams, dried silkworm chrysalis meal 30 grams, bean powder 30 grams, corn 30 grams, 2 grams, L-glutamic acid, glycine 2 grams, peptone 15 grams, fish meal 10 grams, yeast powder 15 grams, vitamin B12 0mg, Lin Suanna Vitamin B2 Sodium Phosphate 20mg, dipotassium hydrogen phosphate 0.4 gram, potassium primary phosphate 0.4 gram, 0.2 gram, magnesium sulfate, tenebrio molitor worm oil 3 grams, is settled to 1000ml with water.
The preparation of Cordyceps sinensis strains liquid fermentation special culture media: dried silkworm chrysalis meal, bean powder, corn are put into steam cooker and added water boiling twice, each 20 minutes, get supernatant liquor after filtered through gauze in container, then add other nutritive ingredient, after heating for dissolving, moisturizing is to 1000ml.
Fermentation process: the bacterial strain of separation was passaged to respectively the 5th generation, 10th generation, 15th generation, 20th generation, 25th generation, obtain DC-0, DC-5, DC-10, DC-15, DC-20, DC-25 is for bacterial classification, then these bacterial classifications are utilized to prepare liquid spawn by liquid seeds preparation method in summary of the invention respectively, at initial pH5.5-6.5, Cordyceps strain is connected in liquid specific substratum by inoculum size 10%, shaking flask liquid amount is the 2/5 of volume, then culture temperature 16 DEG C, shaking speed 180rpm, within 8-12 days, (to nutrient solution, reducing sugar content is lower than 1.5% in lucifuge cultivation, when ammonia-state nitrogen is lower than 200ug/ml), often parallel for bacterial classification three, obtain Cordyceps mycelium.Taking mycelial biomass and adenosine content as inspection target, the results are shown in Table 7.
Table 7 fermentation test
Can obtain by table 7, Cordyceps fungus from the 15th generation biomass and the equal significance of adenosine content lower than the bacterial classification before the 10th generation, hypha form is based on bacterium ball, and in fermenting process, the formation of bacterium ball is unfavorable for mass transfer and passes oxygen, therefore biomass significance is lower than loose shape mycelia. therefore bacterial classification from the 15th generation is undertaken carrying out fermentation test more respectively after kind of property is recovered by the kind restoration methods in present disclosure, plant property and recover working method by enforcement described in claim book and summary of the invention, the bacterial strain numbering recovered through kind of property is respectively DC-15-HF, DC-20-HF, DC-25-HF, then these bacterial classifications are utilized to prepare liquid spawn by liquid seeds preparation method in summary of the invention respectively, at initial pH5.5-6.5, Cordyceps strain is connected in liquid specific substratum by inoculum size 10%, shaking flask liquid amount is the 2/5 of volume, then culture temperature 16 DEG C, shaking speed 180rpm, within 8-12 days, (to nutrient solution, reducing sugar content is lower than 1.5% in lucifuge cultivation, when ammonia-state nitrogen is lower than 200ug/ml), often organize three parallel, obtain Cordyceps mycelium. taking mycelial biomass and adenosine content as inspection target, the results are shown in Table 8.
Table 8 kind property recovers secondary fermentation test
Can obtain by table 8, female passage number of planting is undertaken fermenting after kind of property is recovered by the method in content of the present invention more than after 15 generations again, its Fungal biodiversity, adenosine content and hypha form all obtain recovery, every index plants DC-0 with female, DC-5, DC-10 there was no significant difference (P > 0.05). Therefore, the process of industrial fermentation production Cordyceps mycelium adopts the method for the present invention fermentation bacterial classification carries out kind of property and recover to solve very well bacterial strain decline problem in fermentation production process, thus solve the bacterial classification Sustainable Production problem in existing fermentative production, to reduction cordyceps sinensis mycelium powder cost, promote mycelium powder really to move towards ordinary populace and there is important value and significance.
Claims (7)
1. a Cordyceps fungus mycelium fermentation production method, comprises the preparation of Cordyceps sinensis strains liquid fermentation special culture media, the preferred of mycelium provenance and is linked in substratum by bacterial classification fermentation step; It is characterized in that: Cordyceps fungus mycelium provenance be preferably: by the Cordyceps fungus liquid-spawn inoculation of the Cordyceps fungus Mycelium culture after more than 10 times manually go down to posterity in screening culture medium, select advantage sporophore, the China isolating advantage from advantage sporophore by hair spore bacterial classification as Cordyceps fungus mycelium provenance; Described Cordyceps fungus mycelium provenance preferably include following step:
1) silkworm chrysalis tissue refiner being processed 5-10min, screen out the pupa skin do not smashed, gained tissue juice is silkworm chrysalis tissue juice;
2) preparation of solid substrate: take rice 10-40 part, Semen Maydis powder 10-30 part, wheat 15-35 part, Chinese sorghum 10-30 part, oatmeal 5-15 part, wheat bran 5-15 part; The preparation of nutritive medium: glucose 10-30g/L, sucrose 10-20g/L, peptone 10-20g/L, yeast extract paste 5-20g/L, compound amino acid powder 5-20g/L, potassium primary phosphate 0.1-0.5g/L, dipotassium hydrogen phosphate 0.1-0.5g/L, magnesium sulfate 0.1-0.5g/L, vitamins B15mg-50mg/L, vitamins B25mg-50mg/L, vitamins B610mg-50mg/L, silkworm chrysalis tissue juice 50-150g/L; The preparation of screening culture medium: by solid substrate and nutritive medium by weight proportion 1:1.4-1.8 prepare;
3) by the can above screening culture medium of vial splendid attire, with sealing film sealing, 121 DEG C of sterilizing 30min, it is cooled to room temperature, connecing Cordyceps fungus liquid spawn, the amount of every 100 grams of screening culture medium access Cordyceps fungus liquid spawn is 10-30ml, and bacterial classification is evenly distributed on screening culture medium surface;
4) vial after inoculation being placed in growth cabinet, optimal temperature is 15-20 DEG C, and relative air humidity is 50%-75%, and lucifuge is cultivated 25-30 days;
5) being covered with after whole screening and culturing base plane until mycelia, light stimulation, daytime every day, light application time was not less than 10 hours, temperature 12-18 DEG C, and daytime, the culture temperature temperature difference was not less than 3 DEG C, relative air humidity 50%-75%;
Mycelia produces safran pigment after optical processing, forms button on screening culture medium surface, continues to cultivate button and grows into sporophore;
6) sporophore that growth selection is vigorous, with tweezers from sporophore root tweezer Cordyceps sporophore, carries out separate tissue to fresh sporophore, obtains advantage China by hair spore bacterial classification as Cordyceps fungus mycelium provenance.
2. a kind of Cordyceps fungus mycelium fermentation production method according to claim 1, it is characterised in that described silkworm chrysalis be do not sprout wings, the pupa of the golden yellow not black change of body colour.
3. a kind of Cordyceps fungus mycelium fermentation production method according to claim 1, it is characterised in that described rice, Semen Maydis powder, wheat, Chinese sorghum, oatmeal, wheat bran be pollution-free, without the rotten raw material gone mouldy.
4. a kind of Cordyceps fungus mycelium fermentation production method according to claim 1, it is characterised in that described can glass bottle height �� diameter=12cm �� 10cm, fills solid substrate 80 grams in every bottle.
5. a kind of Cordyceps fungus mycelium fermentation production method according to claim 1, it is characterised in that described Cordyceps fungus liquid spawn be by aseptic technique by after the Cordyceps fungus mycelium access shaking flask of pure culture by shaking flask liquid culture 10-18 days gained.
6. a kind of Cordyceps fungus mycelium fermentation production method according to claim 1, it is characterised in that: described sealing film specification is 15cm �� 15cm, and the central circle for diameter 2cm, aperture are 0.2 ��m of air-permeable filter screen.
7. according to the arbitrary described Cordyceps fungus mycelium fermentation production method of claim 1-6, it is characterised in that growth cabinet intensity of illumination is 150LX-500LX.
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