CN104145719A - Cordyceps sinensis mycelium fermentation production method - Google Patents
Cordyceps sinensis mycelium fermentation production method Download PDFInfo
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- CN104145719A CN104145719A CN201410449700.9A CN201410449700A CN104145719A CN 104145719 A CN104145719 A CN 104145719A CN 201410449700 A CN201410449700 A CN 201410449700A CN 104145719 A CN104145719 A CN 104145719A
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Abstract
The invention provides a cordyceps sinensis mycelium fermentation production method. The method includes the steps of preparation of a special culture medium for cordyceps sinensis mycelium liquid state fermentation, optimization of a mycelium provenance, fermentation of a strain in the culture medium and the like, wherein optimization of the mycelium provenance includes the procedures that cordyceps sinensis strain mycelia obtained after more than ten times of artificial passage are inoculated to the artificial solid culture medium, superior fruit bodies are selected, and a preferred China hirsutella rhossiliensis strain is separated from the fruit bodies. The cordyceps sinensis mycelium fermentation production method has the advantages that through strain selection, the cordyceps sinensis strain with strain character advantages is obtained, the screening effect is good, the problem of strain character recession can be effectively solved, and meanwhile the method also can serve as a China hirsutella rhossiliensis strain passage preservation method.
Description
Technical field
The present invention relates to a kind of bacterial classification cultural method of aweto, particularly a kind of aweto mycelium fermentation production method, the invention belongs to medicine and field of health care food.
Background technology
Cordyceps sinensis (Ophiocordyceps sinensis (Berk.) G.H.Sung et al.) belongs to Ascomycota (Ascomycota), Hypocreales (Hypocreales), nematode grass section (Ophiocordycipitaceae), line Cordyceps (Ophiocordyceps) in biosystematics.Cordyceps sinensis is the rare rare traditional Chinese medicine of China's special product, is that aweto parasitizes a kind of entomogenous fungi complex forming in Hepialus insect larva body.The historical supplementary Amplifications of the Compendium of Materia Medica that begins to be loaded in of medication of Cordyceps sinensis, claim it to have " invigorate the lung and the kidney, hemostasis and phlegm effect ", modern age, pharmacology and clinical research showed, Cordyceps sinensis, to regulating the various diseases such as body's immunity, treatment chronic nephritis, chronic hepatitis, chronic bronchitis, hyperlipidemia and sex dysfunction to have significant curative effect, has wide market prospects.
Cordyceps sinensis natural resources are on the brink of extinction at present, are listed in Key Protected species (II level).The rareness of resource and the growth of demand have aggravated 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 traditional Chinese medicine rise in price amplitudes, is called " soft gold " by people.Since last century the eighties, scientific workers attempt to solve by following approach a difficult problem for Cordyceps Resources scarcity, the needs that Cordyceps sinensis increased to meet people.The one, the mycelial exploitation of the pseudo-phorozoon of Cordyceps sinensis.Up to now, from Cordyceps sinensis, isolate and comprised Chinese Mortierella, China's Paecilomyces varioti, Paecilomyces hepiali chen, Chinese caterpillar fungus head is full mould, bat moth is satisfied mould, the miscellaneous bacterias such as Scytalidium hepiali, effect of the physicochemical property of the tunning of these bacterium and some pharmacological functions and Cordyceps sinensis is approximate, some enterprises carry out large scale fermentation production using the substitute as Cordyceps sinensis by above-mentioned bacterial classification, as the recovering capsule of being bestowed by heaven, paecilomyces hepiall chen, zhiling capsules, Ningxinbao Capsules etc., but the fungi that these products use differs greatly at biology and Cordyceps sinensis, in clinical efficacy, differ larger, thereby be difficult to substitute natural cordyceps, more can not serve as real Cordyceps sinensis, the 2nd, develop other Chinese caterpillar fungus and replace Cordyceps sinensis, as Cordyceps militaris, although this product as the new resources listing that gets the Green Light, its indication and Cordyceps sinensis are inconsistent, also not by consumer is accepted, the 3rd, artificially cultivating cordyceps, cultivate at the form Fermented Cordyceps consistent with natural cordyceps with active ingredient and accept in market the most, this approach is to solve the Cordyceps Resources basic method that supply falls short of demand and means, but because artificial culture natural cordyceps relates to, subject is many, research cycle long, Some Key Technologies is left to be desired and relate to " medicine registration management way " office makes the difficult problem of declaring of No. 28, and the Cordyceps sinensis that real artificial industrialization is cultivated appears on the market still not within the foreseeable future, the 4th, by modern biotechnology, utilize aweto strain to carry out mycelium fermentation, realize the industrialization of aweto, this is to solve at present the most real ways and means of Cordyceps Resources scarcity.
Cordyceps sinensis is that aweto parasitizes the bacterium worm complex forming in Hepialus insect body, and the main medical active composition of Cordyceps sinensis comes from the metabolite of aweto.Modern Pharmaceutical Chemistry and pharmaceutical research show that aweto mycelium and natural cordyceps have close chemical composition, have similar pharmacological action.Therefore, carry out the liquid submerged fermentation of aweto and produce aweto mycelium to replace natural Cordyceps sinensis, not only can alleviate the pressure of wild cordyceps resource, also can meet the demand of people to Cordyceps sinensis health care function.
Aweto is that one is had a liking for low form fungi, and its growth temperature is lower than 20 DEG C.In the time that cultivation temperature is 20 DEG C-22 DEG C, aweto mycelium is grown extremely slowly and bacterium colony forms little; When cultivation temperature is during higher than 23 DEG C, mycelium is grown hardly.Meanwhile, aweto is also higher to the requirement of medium nutrition, though under the suitableeest cultivation temperature condition on common fungi culture medium mycelial growth also very slow, mycelia yield is low.By batch production fermentation pattern produce aweto mycelium because fermentation temperature is low, poor growth, cultivation cycle be long, because staining of miscellaneous bacteria ends in failure, fermentation costs is high conventionally.At present, domestic existing Duo Jia unit has carried out the liquid culture studies of aweto, but its fermentation mycelium product is not all real aweto after molecular biology identification.
With respect to animal, plant, fungi is variation and degeneration easily, in the liquid incubation of aweto, the general multistage fermented and cultured pattern of inclined-plane kind to liquid strain that adopt, slant strains goes down to posterity through tube repeatedly, spawn degeneration serious (planting approximately 15 generations of tube from the mother who starts most to separate just falls into a decline), on slant medium, growing, it is slow to show as mycelial growth, easy aging self-dissolving, after the production of hybrid seeds, in liquid medium within, fermentation mycelium is sparse, the large surperficial dead smooth of bacterium ball particle, 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: the one, bacterial strain causes the decline of bacterial classification because the forfeiture of physiological function gene causes the genetics characteristic of self to change; The 2nd, the bacterial classification decline that the human factors such as passage number, condition of culture, envirment factor cause.In incubation, it a little less than bacterial classification decline, mold fungi filament growth potential, is a large bottleneck of restriction aweto fermentation industrialization.Therefore, in the fermentation production process of aweto, take the measure of selecting of optimizing to recover the good characteristic of bacterial classification, cause biomass and low this bottleneck problem of active constituent content with the bacterial classification decline solving in aweto fermentation industrialization process, realize sustainable, the steady production of aweto.
The principle that aweto strain is selected is to obtain as much as possible strong stress resistance, bacterial classification that hereditary capacity is good.In habitat, non-plateau, can carry out on a large scale screening test, and separate from fruit body the aweto strain obtaining by vegetative mode, by liquid state fermentation special culture media and the cultural method mentioned in the present invention, winter worm summer herb thallus biomass yield and adenosine content are all planted the suitable of test tube strains with mother.
At present; in triage techniques (patent of invention CN103404368A) literary composition of aweto strain, introduce the screening and culturing method of artificial breeding cordyceps species; the object of this invention is the sustainable production problem that solves bacterial classification in Chinese caterpillar fungus artificial cultivation, produces bacterial classification guarantee is provided for the scale sustainability of Cordyceps sinensis medicinal material.It is characterized in that utilizing cordyceps species to infect Chinese caterpillar fungus host, after infecting, continue to cultivate 1-2 age, render to screening base, after transitional period, hardening period, gather the cordyceps militaris sporocarp of sexual spore maturation in the wild, utilize ripe sexual spore to carry out the kind screening of Cordyceps Militaris.Have the difference of matter with the present invention, concrete difference is: 1. goal of the invention and principle difference.Foregoing invention content is to utilize host insect as screening vector, relate to the infection processs of Cordyceps Militaris to the bright bat insect of host complexity, infect and successfully continue afterwards to cultivate, grow and can produce ripe anisosporous stroma from polypide, utilize ripe sexual spore to carry out separation, the screening of Cordyceps Militaris by the mode of sexual propagation.Object is to produce bacterial classification guarantee is provided for the artificial scale sustainability of Cordyceps sinensis medicinal material.The present invention utilizes the liquid-spawn inoculation of Anamorph of Cordyceps Sinensis on screening and culturing base, in conventional environment, utilize climatic cabinate to cultivate and grow fruit body, utilize fruit body to carry out bacterial screening by vegetative mode, object is to solve the bacterial classification kind decline problem that aweto strain occurs in liquid state fermentation incubation.2. screening cycle difference.Cycle in foregoing invention content is calculated, from host insect infects cordyceps species, start to need 3-5 to anisosporous maturation, cultivation 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. condition of culture difference.In 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 large, height above sea level higher than 2000m, rarefaction of air, oxygen content is low, air pressure is low.And method in the present invention to geographical environment without particular/special requirement, all can turn out fruit body by the inventive method in the laboratory of tool climatic cabinate.
Patent of invention CN101695255B relates to a kind of method of being cultivated Cordyceps sinensis stroma by hair spore with China.It is characterized in that full egg, the beef peptone of getting maize pulp, analysis for soybean powder, wheat berry, grain, sorghum grain, dried silkworm chrysalis meal or pulverizing are soaked in water; Former raw material are soaked and growth hormone hybrid filtering obtains filtrate and on the medium of artificial preparation, 1800 meters of height above sea level are above, connect asexual bacterial classification by hair spore, the ascocarpous stroma of output length in batches in day and night temperature >=15 DEG C, environment without industrial pollution.The Cordyceps sinensis stroma biological transformation ratio of cultivating is in 40% left and right, containing adenosine C
10h
13n
5o
4be greater than 0.02%, containing cordycepin C
10h
13n
5o
3be greater than 0.10%.Be from the difference of matter of the present invention: 1. principle and object are different.Foregoing invention content can find out from its summary of the invention, and object is on the medium of artificial preparation, to cultivate long ascocarpous Cordyceps sinensis stroma, belongs to the sexual category of breeding.And the present invention be directed to the kind decline technical problem running in Cordyceps sinensis strains liquid fermentation production process, by the seed selection operation of bacterial classification, make aweto strain keep female good characteristic of planting to belong to pierre category.2. formula and condition of culture difference.Formula in the formula of mentioning in foregoing invention content and ratio and the present invention and ratio all have the difference of matter, research is found, aweto growing state on different culture media is totally different, and different carbon sources, nitrogenous source, vitamin content, microelement match and C/N ratio all can affect the formation of fruit body primordium and whether grow fruit body.The present invention, without Special Geographic condition, all can carry out in common lab, and foregoing invention content characteristic is more than height above sea level 1800m, and day and night temperature is more than or equal to 15 DEG C, in the Qinghai Tibet plateau cordyceps main producing region environment without industrial pollution, carries out.
In sum, patent of invention CN103404368A, CN101695255B are compared with the present invention, all different from selection, composition proportion, technology category and the condition of culture etc. of inventive principle, object, mode of reproduction, purposes, culture matrix.Compared with above two patents of invention, the present invention to aweto strain select have easy and simple to handle, be not subject to regional limits, pollution rate is low, success rate is high, the advantages such as the cycle is short, the bacterial classification of selecting rear separation is used further to liquid state fermentation, thalline biomass and adenosine content and female plant close, solve the bacterial classification kind decline problem in Cordyceps sinensis strains liquid fermentation process, thalline biomass and active constituent content are ensured, being applicable to Cordyceps sinensis commerial scale sustainable development ferment produces, and the present invention is to reducing cordyceps sinensis mycelium powder cost, promotion mycelium powder really moves towards consumers in general and has important value and significance.
Summary of the invention
Aweto strain repeatedly go down to posterity tube cultivate and liquid state fermentation incubation in, bacterial classification occur decline, have a strong impact on the expression of winter worm summer herb thallus biomass and adenosine content.Therefore, the invention provides a kind of fermentation method for producing of aweto.
Aweto strain screening technique of the present invention comprises the steps:
Silkworm chrysalis or the tussah chrysalis of not sprouting wings are processed to 5-10min with tissue refiner, and the pupa skin of not smashing if having discards pupa skin, and gained tissue juice is silkworm chrysalis or tussah chrysalis tissue juice.
The preparation (raw material umber is parts by weight) of solid matrix: take rice 10-40 part, corn flour 10-30 part, wheat 15-35 part, Chinese sorghum 10-30 part, oat meal 5-15 part, wheat bran 5-15 part.
The preparation of nutrient solution: glucose 10-30g/L, sucrose 10-20g/L, peptone 10-20g/L, yeast extract 5-20g/L, composite aminophenol powder 5-20g/L, potassium dihydrogen phosphate 0.1-0.5g/L, dipotassium hydrogen phosphate 0.1-0.5g/L, magnesium sulfate 0.1-0.5g/L, Cobastab
15mg-50mg/L, Cobastab
25mg-50mg/L, Cobastab
610mg-50mg/L, silkworm chrysalis or tussah chrysalis tissue juice 50-150g/L.
Aweto is selected the preparation of medium: by solid matrix and nutrient solution 1:1.4-1.8 preparation by weight proportion.
With the above screening and culturing base of can vial splendid attire, with sealed membrane sealing, 121 DEG C of sterilizing 30min, be cooled to room temperature, connect aweto liquid spawn, the amount of every 100 grams of screening and culturing bases access Cordyceps sinensis liquid spawn is 10-30ml, and bacterial classification is evenly distributed on media surface.Described liquid spawn is will after the aweto access shaking flask of pure culture, to pass through 10-18 days gained of shaking flask liquid culture by sterile working; Described can glass bottle height × diameter=12cm × 10cm; Described sealed membrane is 15cm × 15cm, and central authorities are circle, the aperture 0.2 μ m vapor-permeable type filter screen of diameter 2cm, 80 grams of every bottled solid matrixs.
Postvaccinal vial is placed in to climatic cabinate, and preference temperature is 15-20 DEG C, and relative air humidity is 50%-75%, and lucifuge is cultivated 25-30 days.
After mycelia is covered with whole medium plane, give light stimulation, daylight time every day is not less than 10 hours, and intensity of illumination is 150-500LX, temperature 12-18 DEG C, daytime, the cultivation temperature temperature difference was not less than 3 DEG C, relative air humidity 50%-75%.Mycelia produces crocus pigment after light is processed, and forms button in media surface, continues to cultivate button and grows into fruit body.
From fruit body root tweezer cordyceps sporophore, fresh fruit body is organized to separation with tweezers, can obtain screening aweto strain.Described cordyceps sporophore is closely cylindrical, taupe, the slightly thick 1.5-5mm of base portion, is about 1.5-6cm.
Another object of the present invention is to utilize bacterial classification that the present invention selects that a kind of liquid cultural method of aweto is provided sustainably, wherein also relates to a kind of Cordyceps sinensis strains liquid fermentation special culture media.
The liquid cultural method of aweto provided by the invention comprises the steps:
Cordyceps sinensis strains liquid fermentation special culture media comprises: monose or disaccharide 1-30 gram, compound nitrogen source 1-200 gram, compound amino acid 0-10 gram, peptone 0-30 gram, fish meal 0-20 gram, dusty yeast 0-20 gram, vitamin B1 10-80mg, vitamin B2 10-80mg, dipotassium hydrogen phosphate 0-1 gram, potassium dihydrogen phosphate 0-1 gram, magnesium sulfate 0-1 gram, animal oil 0-5 gram, water is settled to 1000ml.
Above-mentioned monose or disaccharide are glucose, sucrose; Above-mentioned compound nitrogen source is corn, bean powder, wheat bran, dried silkworm chrysalis meal, tussah chrysalis, plain chocolate or oat meal; Above-mentioned compound amino acid is glutamic acid, glycine; Above-mentioned animal oil is yellow mealworm worm oil or silkworm chrysalis oil.Described Cordyceps sinensis strains liquid fermentation special culture media optimization formula is: glucose 5-30 gram, compound nitrogen source 50-200 gram, glutamic acid 0-5 gram, glycine 0-5 gram, peptone 5-30 gram, fish meal 5-20 gram, dusty yeast 5-20 gram, vitamin B1 10-50mg, vitamin B2 10-50mg, dipotassium hydrogen phosphate 0-0.5 gram, potassium dihydrogen phosphate 0-0.5 gram, magnesium sulfate 0-0.5 gram, yellow mealworm worm oil 0-4 gram, water is settled to 1000ml.
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, glutamic acid 1-5 gram, glycine 1-5 gram, peptone 15-30 gram, fish meal 10-20 gram, dusty yeast 10-20 gram, vitamin B1 20-50mg, vitamin B2 20-50mg, dipotassium hydrogen phosphate 0.2-0.5 gram, potassium dihydrogen phosphate 0.2-0.5 gram, magnesium sulfate 0.1-0.3 gram, yellow mealworm worm oil 2-4 gram, water is settled to 1000ml.
By the boiling twice that adds water of above-mentioned compound nitrogen source by weight ratio, each 20 minutes, after filtered through gauze, get supernatant to container, add again other composition, after heating for dissolving, divide and be 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 gone out liquid nutrient medium of bacterium of step (5) is accessed to the teat glass inclined-plane aweto strain of length × wide=200mm × 20mm in superclean bench, be placed on shaking table and cultivate.Described liquid amount is that 10 DEG C-20 DEG C of 1/10-3/5, initial pH value 5-7.5, cultivation temperature, rotating speed 120-200rpm, the lucifuge of triangular pyramidal bottle capacity cultivated the liquid seeds that can obtain in fast growing period for 12-16 days.
Liquid seeds is prepared optimal conditions: the teat glass inclined-plane aweto strain usage amount of described length × wide=200mm × 20mm is prop up/400ml of 4-7 liquid nutrient medium; Liquid amount is the 1/5-3/5 of triangular pyramidal bottle capacity; Initial pH value is 5.5-6.5; Cultivation temperature is preferably 15-19 DEG C; Rotating speed is preferably 180rpm.
Cordyceps sinensis strains liquid fermentation is cultivated: by under initial pH5-7, inoculum concentration 5%-25% is aweto liquid spawn access aweto special culture media.Preference temperature 15-18 DEG C, rotating speed 150-200rpm, liquid amount 1/5-3/5, lucifuge shaken cultivation reach fermentation termination for 8-12 days and can obtain aweto mycelium.
Cordyceps sinensis strains liquid fermentation is cultivated optimum condition: initial pH value 5.5-7, and inoculum concentration 10-20%, cultivation temperature 16-18 DEG C, rotating speed 170-190rpm, shaking flask liquid amount are 1/5-2/5; Described fermentation termination be judged as content of reducing sugar lower than 1.5%, ammoniacal nitrogen is lower than 200 μ g/ml;
Cordyceps sinensis strains liquid fermentation is cultivated most preferably condition: initial pH value 5.5-6.5, and inoculum concentration 10-20%, cultivation temperature 16-18 DEG C, rotating speed 180rpm, shaking flask liquid amount are 2/5; Shaken cultivation 8-12 days, fermentation termination be judged as content of reducing sugar lower than 1.5%, ammoniacal nitrogen is lower than 200 μ g/ml;
Above-mentioned liquid cultural method can amplify step by step according to 10 times of liquid culture base unit weight (volume), till reaching the needed mycelia scale of construction.
Technique effect of the present invention is: a kind of aweto mycelium fermentation production method that the present patent application provides, and it is advantageous that by bacterial classification and select, obtain having the aweto strain of kind of sexual clorminance.Said method screening effect is good, can effectively solve kind of a property decline problem, and meanwhile, the method can also be as a kind of Hirsutella sinensis bacterial classification method for preserving that goes down to posterity.
Embodiment:
Embodiment 1: aweto mycelium liquid state fermentation is produced
Material therefor and explanation: the new isolated strains of Cordyceps sinensis (female kind) is aweto through molecular biology identification, is numbered DC-0; Obtain respectively 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 as starting strain goes down to posterity to cultivate;
Silkworm chrysalis or tussah chrysalis: commercially available fresh living silkworm chrysalises or tussah chrysalis, for the preparation of tissue juice.
The preparation of aweto screening and culturing base: solid matrix (rice 10-40 part, corn flour 10-30 part, wheat 15-35 part, Chinese sorghum 10-30 part, oat meal 5-15 part, wheat bran 5-15 part) and nutrient solution (tussah chrysalis tissue juice 50-150g/L, glucose 10-30g/L, sucrose 10-20g/L, peptone 10-20g/L, yeast extract 5-20g/L, composite aminophenol powder 5-20g/L, potassium dihydrogen phosphate 0.1-0.5g/L, dipotassium hydrogen phosphate 0.1-0.5g/L, magnesium sulfate 0.1-0.5g/L, Cobastab
15mg-50mg/L, Cobastab
25mg-50mg/L, Cobastab
610mg-50mg/L) according to the ratio preparation of 1:1.6.
Cordyceps sinensis strains liquid fermentation special culture media: glucose 15-30 gram, compound nitrogen source 80-200 gram, glutamic acid 1-5 gram, glycine 1-5 gram, peptone 15-30 gram, fish meal 10-20 gram, dusty yeast 10-20 gram, vitamin B1 20-50mg, vitamin B2 20-50mg, dipotassium hydrogen phosphate 0.2-0.5 gram, potassium dihydrogen phosphate 0.2-0.5 gram, magnesium sulfate 0.1-0.3 gram, yellow mealworm worm oil 2-4 gram, water is settled to 1000ml.
Glass preserving jar: glass bottle height × diameter=12cm × 10cm; Described sealed membrane is 15cm × 15cm, and central authorities are the vapor-permeable type filter core of 0.2 μ m, the circular filter core that diameter is 2cm, 80 grams of every bottled solid matrixs.
Sealed membrane: length × wide=15cm × 15cm, central authorities for diameter be that 2cm, circle, aperture are 0.2 μ m vapor-permeable type filter screen.
Cordyceps sinensis strains liquid fermentation special culture media: 20 grams of glucose, 30 grams of dried silkworm chrysalis meals, 30 grams of bean powderes, 30 grams of corns, 2 grams, glutamic acid, 2 grams of glycine, 15 grams of peptones, 10 grams of fish meal, 15 grams of dusty yeasts, vitamin B1 20mg, vitamin B2 20mg, 0.4 gram of dipotassium hydrogen phosphate, 0.4 gram of potassium dihydrogen phosphate, 0.2 gram, magnesium sulfate, 3 grams of yellow mealworm worm oil, water is settled to 1000ml.
The preparation of Cordyceps sinensis strains liquid fermentation special culture media: dried silkworm chrysalis meal, bean powder, corn are put into digester twice of the boiling that add water, each 20 minutes, after filtered through gauze, get supernatant to container, then add other nutrient component, after heating for dissolving, moisturizing is to 1000ml.
1, different passage number bacterial strain comparation and assessment
Initial pH5.5-6.5, is connected to aweto strain in medium by inoculum concentration 10%, and shaking flask liquid amount is volume 2/5.Then 16 DEG C of cultivation temperature, shaking speed 180rpm, lucifuge is cultivated 8-12 days (to content of reducing sugar in culture fluid lower than 1.5%, ammoniacal nitrogen during lower than 200 μ g/ml), obtains aweto mycelium.The Cordyceps strain of different passage numbers has been carried out to fermentation test, and three every group parallel, taking mycelial biomass and adenosine content as investigating index.
The different algebraically bacterial strain of table 1 rating test
As can be drawn from Table 1, same bacterial classification is used further to fermenting and producing after reaching the 15th generation and 15 generations, and now thalline biomass and active constituent content are all on the low side, has significance (P<0.05).Passage number is more, and bacterial strain good characteristic is lost more, causes strain fermentation ability decline, is embodied in thalline biomass and adenosine synthesis capability all goes down.
2, bacterial strain comparation and assessment before and after screening
In the large production of reality, for solving the problem of the bacterial strain decline occurring in fermentation process, ensure the normal industrialization production of bacterial strain, aweto is selected.In his-and-hers watches 1, bacterial strain DC-15, DC-20, DC-30 sieve and select respectively, and method is as follows:
1) silkworm chrysalis or the tussah chrysalis of not sprouting wings are processed to 5-10min with refiner, the pupa skin of not smashing if having discards pupa skin, and gained tissue juice is silkworm chrysalis or tussah chrysalis tissue juice.
2) preparation of aweto screening and culturing base: solid matrix (15 parts, rice, 15 parts of corn flour, 15 parts of wheats, 15 parts of Chinese sorghums, 10 parts of oat meals, 10 parts, wheat bran) and nutrient solution (tussah chrysalis tissue juice 100g/L, glucose 15g/L, sucrose 10g/L, peptone 10g/L, yeast extract 10g/L, composite aminophenol powder 10g/L, potassium dihydrogen phosphate 0.4g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.2g/L, Cobastab
120mg/L, Cobastab
220mg/L, Cobastab
630mg/L) according to the ratio preparation of 1:1.6.
3) with the above screening and culturing base of glass preserving jar splendid attire, with sealed membrane sealing, 121 DEG C of sterilizing 30min, be cooled to room temperature, connect aweto liquid spawn, the amount of every 100 grams of screening and culturing bases access Cordyceps sinensis liquid spawn is 10-30ml, and bacterial classification is evenly distributed on media surface.
4) above-mentioned postvaccinal vial is placed in to climatic cabinate, temperature is 16 DEG C, and relative air humidity is 60%, and lucifuge is cultivated 30 days.
5) mycelia is covered with after whole medium plane, give light stimulation, 12 hours daylight time every day, intensity of illumination is 200LX, 16 DEG C of day temperatures, 12 DEG C of diurnal temperatures (daytime, the cultivation temperature temperature difference was not less than 3 DEG C), relative air humidity 60%, mycelia produces crocus pigment after light is processed, and continues to cultivate and treats that former base is differentiated to form button, till button grows into fruit body.
6) with tweezers from the fruit body root cordyceps sporophore of gathering, fresh fruit body is organized to separation, can obtain aweto strain.
Strain number after screening is: DC-15-FZ (the screening isolated strains of DC-15 bacterial strain, the first generation), DC-20-FZ (the screening isolated strains of DC-20 bacterial strain, the first generation), DC-30-FZ (the screening isolated strains of DC-30 bacterial strain, the first generation).At initial pH5.5-6.5, inoculum concentration 10% is connected to aweto strain in medium, and shaking flask liquid amount is volume 2/5.Then 16 DEG C of cultivation temperature, shaking speed 180rpm, lucifuge is cultivated 8-12 days (to content of reducing sugar in culture fluid lower than 1.5%, ammoniacal nitrogen during lower than 200 μ g/ml), obtains aweto mycelium.To before screening with screening after Cordyceps strain carried out fermentation test, three every group are parallel, taking mycelial biomass and adenosine content as investigating index, the results are shown in Table 2.
Different strains rating test before and after table 2 screening
As can be seen from Table 2, the bacterial strain of different algebraically adopt the bacterial strain that separates after the screening technique screening 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 is compared with DC-30-FZ, 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 screening technique of the present invention can be fine solution bacterial strain decline problem, thereby solve bacterial classification sustainable use and the production problem on actual large production.
Embodiment 2: screening is front to be tested with the rear bacterial strain comparation and assessment of screening parallel verified
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 technique is as follows:
Wherein, the preparation of aweto screening and culturing base: solid matrix (10 parts, rice, 30 parts of corn flour, 15 parts of wheats, 30 parts of Chinese sorghums, 15 parts of oat meals, 5 parts, wheat bran) and nutrient solution (tussah chrysalis tissue juice 50g/L, glucose 10g/L, sucrose 10g/L, peptone 20g/L, yeast extract 5g/L, composite aminophenol powder 5g/L, potassium dihydrogen phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, magnesium sulfate 0.1g/L, Cobastab
15mg/L, Cobastab
25mg/L, Cobastab
610mg/L) according to the ratio preparation of 1:1.4.
Other methods of operating are identical with embodiment 1; In his-and-hers watches 1, bacterial strain DC-15, DC-20, DC-30 screen respectively.
Strain number after screening is: DC-15-FZ (the screening isolated strains of DC-15 bacterial strain, the first generation), DC-20-FZ (the screening isolated strains of DC-20 bacterial strain, the first generation), DC-30-FZ (the screening isolated strains of DC-30 bacterial strain, the first generation).At initial pH 5.5-6.5, inoculum concentration 10% is connected to aweto strain in medium, and shaking flask liquid amount is volume 2/5.Then 16 DEG C of cultivation temperature, shaking speed 180rpm, lucifuge is cultivated 8-12 days (to content of reducing sugar in culture fluid lower than 1.5%, ammoniacal nitrogen during lower than 200 μ g/ml), obtains aweto mycelium.To before screening with screening after Cordyceps strain carried out fermentation test, three every group are parallel, taking mycelial biomass and adenosine content as investigating index, the results are shown in Table shown in 3.
Table 3 screens the front and rear different strains rating test of screening
As can be seen from Table 3, the bacterial strain of different algebraically adopt the bacterial strain that separates after the screening technique screening 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 is compared with DC-30-FZ, 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 screening technique of the present invention can be fine solution bacterial strain decline problem, thereby solve bacterial classification sustainable use and the production problem on actual large production.
Second 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 technique is as follows:
Wherein, the preparation of aweto screening and culturing base: solid matrix (40 parts, rice, 10 parts of corn flour, 35 parts of wheats, 10 parts of Chinese sorghums, 5 parts of oat meals, 15 parts, wheat bran) and nutrient solution (tussah chrysalis tissue juice 150g/L, glucose 30g/L, sucrose 20g/L, peptone 10g/L, yeast extract 20g/L, composite aminophenol powder 20g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, Cobastab
150mg/L, Cobastab
250mg/L, Cobastab
650mg/L) according to the ratio preparation of 1:1.8.
Other operations of test are identical with embodiment 1.
Strain number after screening is: DC-15-FZ (the screening isolated strains of DC-15 bacterial strain, the first generation), DC-20-FZ (the screening isolated strains of DC-20 bacterial strain, the first generation), DC-30-FZ (the screening isolated strains of DC-30 bacterial strain, the first generation).At initial pH5.5-6.5, inoculum concentration 10% is connected to aweto strain in medium, and shaking flask liquid amount is volume 2/5.Then 16 DEG C of cultivation temperature, shaking speed 180rpm, lucifuge is cultivated 8-12 days (to content of reducing sugar in culture fluid lower than 1.5%, ammoniacal nitrogen during lower than 200 μ g/ml), obtains aweto mycelium.To before screening with screening after Cordyceps strain carried out fermentation test, three every group are parallel, taking mycelial biomass and adenosine content as investigating index, the results are shown in Table 4.
Table 4 screens the front and rear different strains rating test of screening
As can be seen from Table 4, the bacterial strain of different algebraically adopt the bacterial strain that separates after the screening technique screening 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 is compared with DC-30-FZ, 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 screening technique of the present invention can be fine solution bacterial strain decline problem, thereby solve bacterial classification sustainable use and the production problem on actual large production.
The application of embodiment 3 screening techniques in bacterial classification goes down to posterity
Prepare Cordyceps sinensis strains liquid fermentation special culture media: 20 grams of glucose, 30 grams of dried silkworm chrysalis meals, 30 grams of corns, 50 grams of plain chocolates, 2 grams, glutamic acid, 2 grams of glycine, 15 grams of peptones, 10 grams of fish meal, 15 grams of dusty yeasts, vitamin B1 20mg, vitamin B2 20mg, 0.4 gram of dipotassium hydrogen phosphate, 0.4 gram of potassium dihydrogen phosphate, 0.2 gram, magnesium sulfate, 3 grams of yellow mealworm worm oil.
Dried silkworm chrysalis meal, corn are put into the digester boiling twice that adds water, each 20 minutes, after filtered through gauze, to get supernatant to container, then add other composition, after heating for dissolving, moisturizing is to 1000ml.
Adopt female kind of DC-0 in embodiment 1, initial pH5.5-6.5, inoculum concentration 10% is connected to aweto strain in medium, and shaking flask liquid amount is volume 2/5.Then 16 DEG C of cultivation temperature, shaking speed 180rpm, lucifuge is cultivated 8-12 days (to content of reducing sugar in culture fluid lower than 1.5%, ammoniacal nitrogen during lower than 200 μ g/ml), obtains aweto mycelium.The Cordyceps strain of different passage numbers has been carried out to fermentation test, and three every group parallel, taking mycelial biomass and adenosine content as investigating index, the results are shown in Table 5.
The different algebraically bacterial strain of table 5 rating test
As can be drawn from Table 5, same bacterial classification passage number exceeded after 15 generations, be used further to fermenting and producing, the now equal significance of thalli growth gesture and active constituent content (P<0.05) on the low side, passage number is more, bacterial strain part excellent genes is lost, and causes the decline of strain fermentation ability, and thalline biomass and adenosine synthesis capability go down.
The application of screening technique described in employing embodiment 1,2 in bacterial classification goes down to posterity.
Taking bacterial strain DC-15 in table 5 as initial strain, the research of going down to posterity, method is as follows:
1) silkworm chrysalis or the tussah chrysalis of not sprouting wings are processed to 5-10min with refiner, the pupa skin of not smashing if having discards pupa skin, and gained tissue juice is silkworm chrysalis or tussah chrysalis tissue juice.
2) preparation of aweto screening and culturing base: solid matrix (15 parts, rice, 15 parts of corn flour, 15 parts of wheats, 15 parts of Chinese sorghums, 10 parts of oat meals, 10 parts, wheat bran) and nutrient solution (tussah chrysalis tissue juice 100g/L, glucose 15g/L, sucrose 10g/L, peptone 10g/L, yeast extract 10g/L, composite aminophenol powder 10g/L, potassium dihydrogen phosphate 0.4g/L, dipotassium hydrogen phosphate 0.4g/L, magnesium sulfate 0.2g/L, Cobastab
120mg/L, Cobastab
220mg/L, Cobastab
630mg/L) according to the ratio preparation of 1:1.6.
3) with the above screening and culturing base of glass preserving jar splendid attire, with sealed membrane sealing, 121 DEG C of sterilizing 30min, be cooled to room temperature, connect aweto liquid spawn, the amount of every 100 grams of screening and culturing bases access Cordyceps sinensis liquid spawn is 10-30ml, and bacterial classification is evenly distributed on media surface.
4) above-mentioned postvaccinal vial is placed in to climatic cabinate, temperature is 16 DEG C, and relative air humidity is 60%, and lucifuge is cultivated 30 days.
5) mycelia is covered with after whole medium plane, give light stimulation, 12 hours daylight time every day, intensity of illumination is 200LX, 16 DEG C of day temperatures, 12 DEG C of diurnal temperatures (daytime, the cultivation temperature temperature difference was not less than 3 DEG C), relative air humidity 60%, mycelia produces crocus pigment after light is processed, and continues to cultivate and treats that former base is differentiated to form button, till button grows fruit body.
6) with tweezers from the fruit body root cordyceps sporophore of gathering, fresh fruit body is organized to separation, can obtain the aweto strain (China is by a hair spore bacterial strain) of screening.
7) by above-mentioned steps 6) described bacterial classification, repeat above-mentioned steps 1)-step 6) and method go down to posterity.DC-5-CF), the 10th time (to by name: the DC-10-CF) and 15th time (to being called: the bacterial classification of fruit body DC-15-CF) going down to posterity collect respectively the 5th that goes down to posterity (to by name:; At initial pH5.5-6.5, inoculum concentration 10% is connected to aweto strain in medium, and shaking flask liquid amount is volume 2/5.Then 16 DEG C of cultivation temperature, shaking speed 180rpm, lucifuge is cultivated 8-12 days (to content of reducing sugar in culture fluid lower than 1.5%, ammoniacal nitrogen during lower than 200 μ g/ml), obtains aweto mycelium.To before screening with screening after Cordyceps strain carried out fermentation test, three every group are parallel, taking mycelial biomass and adenosine content as investigating index, the results are shown in Table 6.
Table 6 effect expedition that goes down to posterity
As can be seen from Table 6, adopt the method for fruit body screening to go down to posterity bacterial strain that obtained bacterial classification separates mycelial biomass with adenosine content compared with DC-0 (female kind), DC-10, there is not significant difference (P>0.05).Therefore, adopt screening technique of the present invention go down to posterity can be fine solution bacterial strain kind decline problem, allow the good characteristic of bacterial strain be continued, thereby solve the bacterial classification Sustainable Use Problems in actual production.
Embodiment 4 aweto mycelium fermentations are produced
Material therefor and explanation: the new separating and preserving bacterial classification of Cordyceps sinensis (female kind) is aweto through molecular biology identification, is numbered DC-0; Obtain respectively 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 as starting strain goes down to posterity to cultivate, n is for bacterial strain (being numbered DC-n) by that analogy;
Silkworm chrysalis or tussah chrysalis: commercially available fresh living silkworm chrysalises or tussah chrysalis, for the preparation of tissue juice.
The preparation of aweto screening and culturing base: solid matrix (rice 10-40 part, corn flour 10-30 part, wheat 15-35 part, Chinese sorghum 10-30 part, oat meal 5-15 part, wheat bran 5-15 part) and nutrient solution (tussah chrysalis tissue juice 50-150g/L, glucose 10-30g/L, sucrose 10-20g/L, peptone 10-20g/L, yeast extract 5-20g/L, composite aminophenol powder 5-20g/L, potassium dihydrogen phosphate 0.1-0.5g/L, dipotassium hydrogen phosphate 0.1-0.5g/L, magnesium sulfate 0.1-0.5g/L, Cobastab
15mg-50mg/L, Cobastab
25mg-50mg/L, Cobastab
610mg-50mg/L) according to the ratio preparation of 1:1.6.
Cordyceps sinensis strains liquid fermentation special culture media: glucose 15-30 gram, compound nitrogen source 80-200 gram, glutamic acid 1-5 gram, glycine 1-5 gram, peptone 15-30 gram, fish meal 10-20 gram, dusty yeast 10-20 gram, vitamin B1 20-50mg, vitamin B2 20-50mg, dipotassium hydrogen phosphate 0.2-0.5 gram, potassium dihydrogen phosphate 0.2-0.5 gram, magnesium sulfate 0.1-0.3 gram, yellow mealworm worm oil 2-4 gram, water is settled to 1000ml.
Glass preserving jar: glass bottle height × diameter=12cm × 10cm; Described sealed membrane is 15cm × 15cm, and central authorities are the vapor-permeable type filter core of 0.2 μ m, the circular filter core that diameter is 2cm, 80 grams of every bottled solid matrixs.
Sealed membrane: length × wide=15cm × 15cm, central authorities for diameter be that 2cm, circle, aperture are 0.2 μ m vapor-permeable type filter screen.
Cordyceps sinensis strains liquid fermentation special culture media: 20 grams of glucose, 30 grams of dried silkworm chrysalis meals, 30 grams of bean powderes, 30 grams of corns, 2 grams, glutamic acid, 2 grams of glycine, 15 grams of peptones, 10 grams of fish meal, 15 grams of dusty yeasts, vitamin B1 20mg, vitamin B2 20mg, 0.4 gram of dipotassium hydrogen phosphate, 0.4 gram of potassium dihydrogen phosphate, 0.2 gram, magnesium sulfate, 3 grams of yellow mealworm worm oil, water is settled to 1000ml.
The preparation of Cordyceps sinensis strains liquid fermentation special culture media: dried silkworm chrysalis meal, bean powder, corn are put into digester twice of the boiling that add water, each 20 minutes, after filtered through gauze, get supernatant to container, then add other nutrient component, after heating for dissolving, moisturizing is to 1000ml.
Fermentation process: the bacterial strain of separation is passaged to respectively to the 5th generation, the 10th generation, the 15th generation, the 20th generation, the 25th generation, obtain DC-0, DC-5, DC-10, DC-15, DC-20, DC-25 is for bacterial classification, then utilize respectively these bacterial classifications to prepare liquid spawn by liquid seeds preparation method in summary of the invention, at initial pH5.5-6.5, inoculum concentration 10% is connected to aweto strain in liquid specific medium, shaking flask liquid amount is 2/5 of volume, then 16 DEG C of cultivation temperature, shaking speed 180rpm, lucifuge is cultivated 8-12 days (to content of reducing sugar in culture fluid lower than 1.5%, ammoniacal nitrogen is during lower than 200ug/ml), in per generation,, three of bacterial classifications were parallel, obtain aweto mycelium.Taking mycelial biomass and adenosine content as investigating index, the results are shown in Table 7.
Table 7 fermentation test
Can be obtained by table 7, aweto from the 15th generation biomass and the equal significance of adenosine content lower than the bacterial classification before the 10th generation, mycelia form is taking bacterium ball as main, and the formation of bacterium ball is unfavorable for mass transfer and passes oxygen during the fermentation, therefore biomass significance is lower than loose shape mycelia.Therefore the bacterial classification from the 15th generation is undertaken after kind of property is recovered carrying out respectively fermentation test by the kind recovering method present disclosure again, plant property recovery operation method implements described in claims and summary of the invention, the strain number recovering through kind of property is respectively DC-15-HF, DC-20-HF, DC-25-HF, then utilize respectively these bacterial classifications to prepare liquid spawn by liquid seeds preparation method in summary of the invention, at initial pH5.5-6.5, inoculum concentration 10% is connected to aweto strain in liquid specific medium, shaking flask liquid amount is 2/5 of volume, then 16 DEG C of cultivation temperature, shaking speed 180rpm, lucifuge is cultivated 8-12 days (to content of reducing sugar in culture fluid lower than 1.5%, ammoniacal nitrogen is during lower than 200ug/ml), every group three parallel, obtain aweto mycelium.Taking mycelial biomass and adenosine content as investigating index, the results are shown in Table 8.
Table 8 kind of property is recovered after fermentation test
Can be obtained by table 8, female kind after passage number exceeded for 15 generations undertaken after kind of property is recovered fermenting by the method in content of the present invention again, its thalline biomass, adenosine content and mycelia form have all obtained recovery, indices and female DC-0 that plants, DC-5, DC-10 there was no significant difference (P>0.05).Therefore, industrial fermentation produce in the process of aweto mycelium, adopt method of the present invention fermentation is carried out with bacterial classification kind of property recover can fine solution fermentation production process in bacterial strain decline problem, thereby solve the sustainable production problem of bacterial classification in existing fermenting and producing, to reducing cordyceps sinensis mycelium powder cost, promotion mycelium powder really moves towards ordinary populace and has important value and significance.
Claims (8)
1. an aweto mycelium fermentation production method, comprise Cordyceps sinensis strains liquid fermentation special culture media preparation, mycelium provenance preferably and by bacterial classification be linked into the step such as ferment in medium; It is characterized in that: the preferred of mycelium provenance selected advantage fruit body for the aweto strain mycelium after 10 times manually go down to posterity is above inoculated on artificial solid culture medium, isolates preferred China by a mao spore bacterial classification from fruit body.
2. a kind of aweto mycelium fermentation production method according to claim 1, is characterized in that: described mycelium provenance preferably include following steps:
1) silkworm chrysalis or tussah chrysalis are processed to 5-10min with tissue refiner, screen out the pupa skin of not smashing, gained tissue juice is silkworm chrysalis or tussah chrysalis tissue juice;
2) preparation of solid matrix: take rice 10-40 part, corn flour 10-30 part, wheat 15-35 part, Chinese sorghum 10-30 part, oat meal 5-15 part, wheat bran 5-15 part; The preparation of nutrient solution: glucose 10-30g/L, sucrose 10-20 g/L, peptone 10-20 g/L, yeast extract 5-20 g/L, composite aminophenol powder 5-20 g/L, potassium dihydrogen phosphate 0.1-0.5 g/L, dipotassium hydrogen phosphate 0.1-0.5 g/L, magnesium sulfate 0.1-0.5 g/L, Cobastab
15mg-50mg/L, Cobastab
25mg-50mg/L, Cobastab
610mg-50mg/L, silkworm chrysalis or tussah chrysalis tissue juice 50-150g/L; The preparation of aweto screening and culturing base: by solid matrix and nutrient solution 1:1.4-1.8 preparation by weight proportion;
3) with the above screening and culturing base of can vial splendid attire, with sealed membrane sealing, 121 DEG C of sterilizing 30min, be cooled to room temperature, connect aweto liquid spawn, the amount of every 100 grams of screening and culturing bases access Cordyceps sinensis liquid spawn is 10-30ml, and bacterial classification is evenly distributed on media surface;
4) postvaccinal vial is placed in to climatic cabinate, preference temperature is 15-20 DEG C, and relative air humidity is 50%-75%, and lucifuge is cultivated 25-30 days;
5) after mycelia is covered with whole medium plane, light stimulation, daylight time every day is not less than 10 hours, temperature 12-18 DEG C, daytime, the cultivation temperature temperature difference was not less than 3 DEG C, relative air humidity 50%-75%;
Mycelia produces crocus pigment after light is processed, and forms button in media surface, continues to cultivate button and grows into fruit body;
6) select to be grown to vigorous fruit body, from fruit body root tweezer cordyceps sporophore, fresh fruit body is organized to separation with tweezers, obtain advantage aweto strain.
3. a kind of aweto mycelium fermentation production method according to claim 2, it is characterized in that described silkworm chrysalis or tussah chrysalis be do not sprout wings, the pupa of the golden yellow not black change of body colour.
4. a kind of aweto mycelium fermentation production method according to claim 2, is characterized in that described rice, corn flour, wheat, Chinese sorghum, oat meal, wheat bran are pollution-free, the rotten raw material going mouldy of nothing.
5. a kind of aweto mycelium fermentation production method according to claim 2, is characterized in that described can glass bottle height × diameter=12cm × 10cm, fills 80 grams of solid matrixs in every bottle.
6. a kind of aweto mycelium fermentation production method according to claim 2, is characterized in that described liquid spawn is will after the aweto access shaking flask of pure culture, to pass through 10-18 days gained of shaking flask liquid culture by sterile working.
7. a kind of aweto mycelium fermentation production method according to claim 2, is characterized in that described sealed membrane is 15cm × 15cm, and central authorities are circle, the aperture 0.2 μ m vapor-permeable type filter screen of diameter 2cm.
8. according to the arbitrary described a kind of aweto mycelium fermentation production method of claim 1-7, it is characterized in that climatic cabinate intensity of illumination is 150LX-500LX.
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| CN108865904A (en) * | 2018-08-30 | 2018-11-23 | 云南大学 | A kind of Hirsutella sinensis expands the Hirsutella sinensis and cultural method of culture medium and its culture |
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| CN110894468A (en) * | 2019-10-29 | 2020-03-20 | 浙江工业大学 | Fermentation culture method based on hirsutella sinensis metabolic pathway |
| CN110923151A (en) * | 2019-12-19 | 2020-03-27 | 广东省微生物研究所（广东省微生物分析检测中心） | Cordyceps sinensis anamorph solid culture medium and preparation method thereof |
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| CN113330984A (en) * | 2021-06-03 | 2021-09-03 | 徐州工程学院 | Artificial cultivation method for improving cordyceps sobolifera polysaccharide content in cordyceps sobolifera sporocarp |
| CN113348962A (en) * | 2021-06-03 | 2021-09-07 | 徐州工程学院 | Artificial cultivation method for improving cordycepic acid content in cordyceps sobolifera sporocarp |
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