CN104774888A - Cordycepin fermentation solid medium and preparation method and application thereof - Google Patents

Abstract

The invention discloses a cordycepin fermentation solid medium and its preparation method and application. The medium is prepared by the following steps: 1) adding glucose, yeast extract, peptone, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and magnesium sulfate , Tween 80, the solvent is water, after fully stirring and dissolving, the pH value of the solution is adjusted to 5.5-6.0 to obtain a fermentation culture liquid; 2) the fermentation culture liquid in step 1) is mixed with expanded perlite evenly, and the Put it into a fermenter and steam sterilize at 121°C for 20 minutes to obtain a solid medium for cordycepin fermentation. The fruit bodies provided by this method are large in size, good in synchronization and strong in vitality, and the growth of Cordyceps militaris and the accumulation of cordycepin are promoted by spraying and supplementing cordycepin precursor adenine and triolein, which can improve the production of cordycepin. output and quality.

Description

A kind of cordycepin fermentation solid medium and its preparation method and application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a solid medium for cordycepin fermentation and its preparation method and application.

Background technique

Cordycepin is one of the main active ingredients in Cordyceps. Cordycepin has a variety of biological activities, such as anti-tumor, anti-proliferation, anti-metastasis, antibacterial, antiviral, immune regulation, and anti-inflammation. The preparation of cordycepin mainly includes chemical synthesis and biosynthesis. Due to the high production cost of chemically synthesized cordycepin, complex synthesis process, low yield and difficult product purification, cordycepin is mainly prepared by biosynthesis. There are two ways to prepare cordycepin by biosynthesis: one is to obtain Cordyceps fruiting body by solid fermentation, and then extract it; the other is to directly extract from the fermentation broth through liquid fermentation of Cordyceps. Due to the advantages of liquid fermentation over solid fermentation in terms of fermentation scale, cell growth rate, growth density and controllability, the extraction of cordycepin by liquid fermentation of Cordyceps sinensis has become the main preparation method of cordycepin.

Contents of the invention

The technical problem to be solved by the invention is to provide a solid medium for cordycepin fermentation that can produce a large amount of fruiting bodies.

The technical problem to be solved by the present invention is to provide the preparation method of the above-mentioned solid culture medium.

The final technical problem to be solved by the present invention is to provide the application of the above-mentioned solid medium.

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

A preparation method for cordycepin fermentation solid medium, which comprises the steps of:

1) Glucose 40-50g/L, yeast extract 3-10g/L, peptone 5-15g/L, potassium dihydrogen phosphate 0.2-1.0g/L, dipotassium hydrogen phosphate 0.2-1.0g/L, magnesium sulfate 0.2 ～1.0g/L, Tween 80 0.5～5.0g/L, the solvent is water, fully stir and dissolve, adjust the pH value of the solution to 5.5～6.0, and obtain the fermentation medium;

2) Mix the fermentation culture solution in step 1) with the expanded perlite evenly, put it into a fermenter after spray drying, and steam sterilize at 121° C. for 20 minutes to obtain a solid medium for cordycepin fermentation.

In step 1), glucose 42g/L, yeast extract 6g/L, peptone 10g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, Tween 802g /L, the solvent is water, and the pH value of the solution is adjusted to 5.8 after fully stirring and dissolving to obtain a fermentation broth.

In step 2), the particle size of the expanded perlite is 4-8 mm, preferably 6 mm.

In step 2), the volume-to-mass ratio of the fermentation broth to the expanded perlite is 30-50ml/g, preferably 40ml/g.

In step 2), the water content of the spray-dried material is controlled at 2.5-5ml/g, preferably 4ml/g.

The cordycepin fermentation solid medium prepared by the above preparation method is also within the protection scope of the present invention.

The application of the above-mentioned cordycepin fermentation solid medium in the fermentative production of cordycepin is also within the protection scope of the present invention.

The specific application method is to wash the Cordyceps militaris strain grown on the PDA slant medium with 5ml sterile physiological saline to make a bacterial suspension, and inoculate it into a sterilized shake flask containing 50ml of fermentation broth. in 27°C, 180rpm constant temperature shaker culture for 4 days, add 450ml of sterile water, mix well, then inoculate 1wt% into the sterilized cordycepin fermented solid medium, fully stir to make the bacteria evenly distributed in the In the solid medium for cordycepin fermentation, add water at a rate of 1 wt% every 24 hours to maintain relative humidity, and stir and turn over the material. At the same time, sterile air with a relative humidity of 80% is introduced from the bottom of the fermenter with an air ratio of 0.5min ^-1 . After culturing at 27°C for 4 days, the aeration ratio was reduced to 0.5h ^-1 , and the culture medium containing 0.5g/L adenine and 2-6g/L (preferably 4g/L) triolein was sprayed with 5wt% every 5 days. The solution was used to produce a large number of fruiting bodies, and the other conditions remained unchanged. After 21 days of further cultivation, the fermentation ended; 1.5 times the volume of solid material was added, stirred and turned over at 80°C for 2 hours, and the filtrate was collected by filtration; the boiling process was carried out three times, and the filtrate was combined. Concentrate to solid volume to obtain cordycepin extract.

The above-mentioned Cordyceps militaris strains are any Cordyceps militaris strains that have the ability to produce cordycepin, for example, they can be purchased from China Industrial Microorganism Culture Collection Center with the number CICC 14014.

Through the adsorption of the culture solution on the 20-mesh expanded perlite, the technology takes advantage of solid fermentation and overcomes its defects, and uses the rich specific surface area of the 20-mesh expanded perlite to achieve the purpose of mass production of fruiting bodies. It is well known that cordycepin is usually enriched in the top of the fruiting bodies of Cordyceps militaris.

Through literature research and research, we found that the immediate precursor of cordycepin is adenine, and another structural unit of cordycepin is 3'-deoxyribose. In the fermentation of Cordyceps militaris, we found that adding adenine to the fermentation medium can greatly increase the production of cordycepin. Moreover, we also found that the content of cordycepin in the Cordyceps militaris fermentation broth further increased after adding vegetable oil.

The traditional culture method of Cordyceps militaris is to use the fermentation medium with glucose, peptone and yeast extract as the main components. These components can only meet the basic growth and development needs of Cordyceps militaris, and the yield of cordycepin is low. After adding adenine, a certain amount of cordycepin precursor is provided, so that the yield of cordycepin is increased. However, the 3'-deoxyribose unit is synthesized through the fatty acid metabolic pathway, through acetyl CoA, and then through the secondary metabolic pathway, which is different from the previous understanding that 3'-deoxyribose is synthesized through the pentose phosphate pathway. Therefore, it is difficult to increase the content of cordycepin in the absence of acetyl CoA supply. Glycerides such as vegetable oil can be decomposed into a large amount of acetyl CoA through fatty acid metabolism in Cordyceps militaris, which can further increase the production of cordycepin.

Beneficial effects of the present invention:

1. The 20-mesh expanded perlite used in the present invention is used as a support medium, and its specific surface area is 10m ² /g (in a fermenter with an inner diameter of 10 meters, the gas-liquid interface is only 78.5m ² under non-stirring conditions, which is only equivalent to 8g20 mesh expanded perlite), the porosity reaches 50-90%, which is conducive to mass transfer, fruiting body growth and spore production, with good synchronization.

2. According to the different oxygen supply conditions required for the growth of Cordyceps militaris and the accumulation of cordycepin, the aeration ratio is used to change it, which has fewer influencing factors and is simpler and more convenient.

3. The present invention adds adenine and triolein to provide two precursors of cordycepin, which is more conducive to the accumulation of cordycepin.

Description of drawings

Fig. 1 is the figure of influence of different mediums on the content of cordycepin, wherein abscissa 1 is embodiment 4, 2 is embodiment 5, 3 is embodiment 6, 4 is comparative example 1, 5 is comparative example 2, and ordinate represents Cordycepin content in the measured fermentation broth or cordycepin extract.

Fig. 2 is the figure of influence of different culture media on fruiting body concentration, and wherein abscissa 1 is embodiment 4, and 2 is embodiment 5, and 3 is embodiment 6, and 4 is comparative example 1, and 5 is comparative example 2, and ordinate represents Measured fruiting body concentration.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

Example 1: Preparation of solid medium for Cordyceps militaris fermentation.

1) Glucose 42g/L, yeast extract 6g/L, peptone 10g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, Tween 80 2g/L, The solvent is water, and the pH value of the solution is adjusted to 5.8 after fully stirring and dissolving to obtain a fermentation culture solution.

2) Mix and stir the fermentation medium in step 1) and the expanded perlite with a particle size of 6mm according to the volume-to-mass ratio of 40ml/g. After spray drying, the water content of the material is controlled at 4ml/g and put into a fermenter at 121°C. Steam sterilize for 20 minutes to obtain a solid culture medium for cordycepin fermentation.

Example 2: Preparation of Cordyceps militaris fermentation medium.

1) Glucose 42g/L, yeast extract 6g/L, peptone 10g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, Tween 80 2g/L, The solvent is water, and the pH value of the solution is adjusted to 5.8 after fully stirring and dissolving to obtain a fermentation culture solution.

2) Mix and stir the fermentation medium in step 1) and the expanded perlite with a particle size of 4mm according to the volume-to-mass ratio of 30ml/g. After spray drying, the water content of the material is controlled at 5ml/g and put into a fermenter at 121°C. Steam sterilize for 20 minutes to obtain a solid culture medium for cordycepin fermentation.

Example 3: Preparation of Cordyceps militaris fermentation medium.

1) Glucose 42g/L, yeast extract 6g/L, peptone 10g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, Tween 80 2g/L, The solvent is water, and the pH value of the solution is adjusted to 5.8 after fully stirring and dissolving to obtain a fermentation culture solution.

2) The fermentation culture liquid in step 1) is mixed with the expanded perlite with a particle size of 8mm according to the volume to mass ratio of 50ml/g. After spray drying, the water content of the material is controlled at 2.5ml/g and loaded into a fermenter. 121 ℃ steam sterilization for 20 minutes to obtain a solid medium for cordycepin fermentation.

Example 4: Application of Cordyceps militaris solid fermentation medium in increasing the yield of Cordycepin militaris.

1. The Cordyceps militaris (No.: CICC14014) purchased from the China Industrial Microbial Strain Preservation Management Center is preserved in an ampoule tube and is in a freeze-dried state, and the activity of the strain needs to be restored before the experiment. In the ultra-clean workbench, wipe the ampoule tube with absorbent cotton soaked in 70% alcohol, heat the top of the ampoule tube on the flame, and drop a few drops of sterile water to the heated place to crack the glass. Knock off the top of the cracked ampoule tube with tweezers, add 0.5ml of 0.9% physiological saline, shake to dissolve the freeze-dried bacteria and form a suspension. Take 0.2ml of the bacterial cell suspension and add it to the slant medium, and incubate at a constant temperature of 25°C for 7 days.

2. Wash the strains grown on the PDA slant medium with 5ml of sterile normal saline to make a bacterial suspension, inoculate into a sterilized shaker flask containing 50ml of fermentation medium, at 27°C, Cultivate in a constant temperature shaker at 180rpm for 4 days, add 450ml of sterile water, mix evenly, and then inoculate 1 wt% into the sterilized solid medium of Example 1, stir well to make the bacteria evenly distributed in the medium, every At 24 hours, add water at a ratio of 1wt% to maintain an appropriate relative humidity, and stir and turn over the material. At the same time, feed sterile air with a relative humidity of 80% from the bottom of the fermenter with an aeration ratio of 0.5min ^-1 . After culturing at 27°C for 4 days, aerate Gradient drop is 0.5h ^-1 , every 5 days is sprayed with 5wt% culture solution containing 0.5g/L adenine and 4g/L triolein to make it produce a large number of fruiting bodies, and the rest of the conditions remain unchanged, and then Fermentation ended after 21 days of cultivation. Add water 1.5 times the volume of the solid material, stir and turn over at 80°C for 2 hours, and collect the filtrate by filtration. The water boiling process is carried out three times, and the filtrates are combined and concentrated to the solid volume to obtain the cordycepin extract.

Embodiment 5: the application of Cordyceps militaris fermentation medium in improving Cordycepin content in Cordyceps militaris fermentation broth

1. The Cordyceps militaris (No.: CICC14014) purchased from the China Industrial Microbial Strain Preservation Management Center is preserved in an ampoule tube and is in a freeze-dried state, and the activity of the strain needs to be restored before the experiment. In the ultra-clean workbench, wipe the ampoule tube with absorbent cotton soaked in 70% alcohol, heat the top of the ampoule tube on the flame, and drop a few drops of sterile water to the heated place to crack the glass. Knock off the top of the cracked ampoule tube with tweezers, add 0.5ml of 0.9% physiological saline, shake to dissolve the freeze-dried bacteria and form a suspension. Take 0.2ml of the bacterial cell suspension and add it to the slant medium, and incubate at a constant temperature of 25°C for 7 days.

2. Wash the strains grown on the PDA slant medium with 5ml of sterile normal saline to make a bacterial suspension, inoculate into a sterilized shaker flask containing 50ml of fermentation medium, at 27°C, Cultivate in a constant temperature shaker at 180rpm for 4 days, add 450ml of sterile water, mix evenly, and then inoculate 1 wt% into the sterilized solid medium of Example 2, stir well to make the bacteria evenly distributed in the medium, At 24 hours, add water at a ratio of 1wt% to maintain an appropriate relative humidity, and stir and turn over the material. At the same time, feed sterile air with a relative humidity of 80% from the bottom of the fermenter with an aeration ratio of 0.5min ^-1 . After culturing at 27°C for 4 days, aerate The gradient is 0.5h ^-1 , every 5 days is sprayed with 5wt% culture solution containing 0.5g/L adenine and 2g/L triolein to make it produce a large number of fruiting bodies, and the rest of the conditions remain unchanged. Fermentation ended after 21 days of cultivation. Add water 1.5 times the volume of the solid material, stir and turn over at 80°C for 2 hours, and collect the filtrate by filtration. The water boiling process is carried out three times, and the filtrates are combined and concentrated to the solid volume to obtain the cordycepin extract.

Embodiment 6: the application of Cordyceps militaris fermentation medium in improving Cordycepin content in Cordyceps militaris fermentation liquid

1. The Cordyceps militaris (No.: CICC14014) purchased from the China Industrial Microbial Strain Preservation Management Center is preserved in an ampoule tube and is in a freeze-dried state, and the activity of the strain needs to be restored before the experiment. In the ultra-clean workbench, wipe the ampoule tube with absorbent cotton soaked in 70% alcohol, heat the top of the ampoule tube on the flame, and drop a few drops of sterile water to the heated place to crack the glass. Knock off the top of the cracked ampoule tube with tweezers, add 0.5ml of 0.9% physiological saline, shake to dissolve the freeze-dried bacteria and form a suspension. Take 0.2ml of the bacterial cell suspension and add it to the slant medium, and incubate at a constant temperature of 25°C for 7 days.

2. Wash the strains grown on the PDA slant medium with 5ml of sterile normal saline to make a bacterial suspension, inoculate into a sterilized shaker flask containing 50ml of fermentation medium, at 27°C, Cultivate in a constant temperature shaker at 180rpm for 4 days, add 450ml of sterile water, mix evenly, and then inoculate 1wt% into the sterilized solid medium of Example 3, stir well to make the bacteria evenly distributed in the medium, every At 24 hours, add water at a ratio of 1wt% to maintain an appropriate relative humidity, and stir and turn over the material. At the same time, feed sterile air with a relative humidity of 80% from the bottom of the fermenter with an aeration ratio of 0.5min ^-1 . After culturing at 27°C for 4 days, aerate Gradient drop is 0.5h ^-1 , every 5 days with 5wt% spray adding culture solution containing 0.5g/L adenine and 6g/L triolein to make it produce a large number of fruiting bodies, the rest of the conditions remain unchanged, and then Fermentation ended after 21 days of cultivation. Add water 1.5 times the volume of the solid material, stir and turn over at 80°C for 2 hours, and collect the filtrate by filtration. The water boiling process is carried out three times, and the filtrates are combined and concentrated to the solid volume to obtain the cordycepin extract.

Comparative example 1:

1. Glucose 42g/L, yeast extract 6g/L, peptone 10g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, Tween 80 0.5-5.0g /L, the solvent is water, and the pH value of the solution is adjusted to 5.8 after fully stirring and dissolving to obtain a fermentation broth.

2. The Cordyceps militaris (No.: CICC14014) purchased from the China Industrial Microbial Strain Preservation Management Center is preserved in an ampoule tube and is in a freeze-dried state, and the activity of the strain needs to be restored before the experiment. In the ultra-clean workbench, wipe the ampoule tube with absorbent cotton soaked in 70% alcohol, heat the top of the ampoule tube on the flame, and drop a few drops of sterile water to the heated place to crack the glass. Knock off the top of the cracked ampoule tube with tweezers, add 0.5ml of 0.9% physiological saline, shake to dissolve the freeze-dried bacteria and form a suspension. Take 0.2ml of the bacterial cell suspension and add it to the slant medium, and incubate at a constant temperature of 25°C for 7 days.

3. Wash the strains grown on the PDA slant medium with 5ml of sterile normal saline to make a bacterial suspension, inoculate into a sterilized shaker flask containing 50ml of fermentation medium, at 27°C, Cultivate in a constant temperature shaker at 180rpm for 4 days, add 450ml of sterile water, mix well, then inoculate 1% w/w into the sterilized fermentation culture medium, stir well to make the bacteria evenly distributed in the medium, every 24h Add water at a ratio of 1% w/w to maintain a proper relative humidity, and stir and turn over the material. At the same time, the aseptic air with a relative humidity of 80% is introduced from the bottom of the fermenter with an air ratio of 0.5min ^-1 . After 4 days of cultivation at 27°C , the aeration ratio was reduced to 0.5h ^-1 , and the culture medium containing 0.5g/L adenine and 4g/L triolein was sprayed with 5% w/w every 5 days, and the rest of the conditions remained unchanged. After 21 days of culture The fermentation is over. The filtrate was collected by filtration to obtain a cordycepin extract.

Comparative example 2:

1. Glucose 42g/L, yeast extract 6g/L, peptone 10g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, Tween 80 2g/L, The solvent is water, and the pH value of the solution is adjusted to 5.8 after fully stirring and dissolving to obtain a fermentation culture solution.

2. Mix and stir the fermentation medium in step 1 and the expanded perlite with a particle size of 6mm according to the volume-to-mass ratio of 40ml/g. Sterilize for 20 minutes to obtain a solid medium for cordycepin fermentation.

3. The Cordyceps militaris (No.: CICC14014) purchased from the China Industrial Microbial Strain Preservation Management Center is preserved in an ampoule tube and is in a freeze-dried state, and the activity of the strain needs to be restored before the experiment. In the ultra-clean workbench, wipe the ampoule tube with absorbent cotton soaked in 70% alcohol, heat the top of the ampoule tube on the flame, and drop a few drops of sterile water to the heated place to crack the glass. Knock off the top of the cracked ampoule tube with tweezers, add 0.5ml of 0.9% physiological saline, shake to dissolve the freeze-dried bacteria and form a suspension. Take 0.2ml of the bacterial cell suspension and add it to the slant medium, and incubate at a constant temperature of 25°C for 7 days.

4. Wash the strains grown on the PDA slant medium with 5ml of sterile normal saline to make a bacterial suspension, inoculate into a sterilized shaker flask containing 50ml of fermentation medium, at 27°C, Cultivate in a constant temperature shaker at 180rpm for 4 days, add 450ml of sterile water, mix well, and then inoculate 1wt% into the sterilized solid medium for cordycepin fermentation, stir well to make the bacteria evenly distributed in the medium, every 24h Supplement water with 1wt% ratio to maintain appropriate relative humidity, and stir and turn over materials. At the same time, sterilized air with a relative humidity of 80% is introduced from the bottom of the fermenter with an aeration ratio of 0.5min ^-1 . After culturing at 27°C for 4 days, the aeration ratio Reduced to 0.5h ^-1 , the rest of the conditions remained unchanged, and the fermentation ended after another 21 days of cultivation. Add water 1.5 times the volume of the solid material, stir and turn over at 80°C for 2 hours, and collect the filtrate by filtration. The water boiling process is carried out three times, and the filtrates are combined and concentrated to the solid volume to obtain the cordycepin extract.

Determination of Cordycepin Content in Fermentation Broth

The content of cordycepin in the fermentation broth of Examples 4-7 was determined by high performance liquid chromatography. The fermentation broth was centrifuged and the supernatant was diluted 6 times with pure water, oscillated and mixed, and the cordycepin was detected. Chromatographic conditions: Chromatographic column: Ultimate AQ-C18 (4.6mm×250mm, 5μm), mobile phase: methanol: phosphate solution (10mmol/L KH ₂ PO ₄ solution) = 15:85, column temperature 30°C, flow rate 1ml/ min, the injection volume was 20 μL, and the detection wavelength was 260 nm.

Results: The content of cordycepin in the culture medium prepared in Example 4 was increased by about 200% and 243% respectively compared with those in Comparative Examples 1-2.

Fruiting body concentration detection:

Weigh the mass of perlite before preparing the solid medium, record it as M ₀ , record the liquid medium as 0; after the fermentation is finished, filter and dry, weigh the total mass of the fermented solid, record it as M _total ; the total volume of fermentation is V , the fruiting body concentration is recorded as ( _Mtotal -M ₀ )/V.

Results: The concentration of the fruiting bodies of the culture medium prepared in Example 4 was increased by about 118% and 140% compared with those in Comparative Examples 1-2, respectively.

Claims (8)

1. a preparation method for cordycepin fermentation solid medium, is characterized in that, it comprises the steps: 1) Glucose 40-50g/L, yeast extract 3-10g/L, peptone 5-15g/L, potassium dihydrogen phosphate 0.2-1.0g/L, dipotassium hydrogen phosphate 0.2-1.0g/L, magnesium sulfate 0.2 ～1.0g/L, Tween 800.5～5.0g/L, the solvent is water, fully stir and dissolve, adjust the pH value of the solution to 5.5～6.0, and obtain the fermentation medium; 2) Mix the fermentation culture solution in step 1) with the expanded perlite evenly, put it into a fermenter after spray drying, and steam sterilize at 121° C. for 20 minutes to obtain a solid medium for cordycepin fermentation. 2. the preparation method of cordycepin fermentation solid medium according to claim 1, is characterized in that, in step 1), with glucose 42g/L, yeast extract 6g/L, peptone 10g/L, potassium dihydrogen phosphate 0.5 g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, Tween 802g/L, the solvent is water, after fully stirring and dissolving, adjust the pH value of the solution to 5.8 to prepare the fermentation broth. 3. The method for preparing a solid medium for cordycepin fermentation according to claim 1, characterized in that, in step 2), the particle size of the expanded perlite is 4-8mm. 4. The preparation method of the cordycepin fermented solid medium according to claim 1, characterized in that, in step 2), the volume-to-mass ratio of the fermentation broth to expanded perlite is 30 to 50 ml/g. 5. The preparation method of the cordycepin fermentation solid medium according to claim 1, characterized in that, in step 2), the water content of the spray-dried material is controlled at 2.5-5ml/g. 6. The cordycepin fermentation solid medium prepared by the preparation method described in any one of claims 1-5. 7. The application of the cordycepin fermenting solid medium according to claim 6 in the fermentative production of cordycepin. 8. application according to claim 7, is characterized in that, the Cordyceps militaris bacterial classification that grows on the PDA slant medium is washed down with 5ml aseptic saline, makes bacterial suspension, is inoculated to the sterilized good In a shaker flask with 50ml of fermentation culture medium, cultivate it on a constant temperature shaker at 27°C and 180rpm for 4 days, add 450ml of sterile water, mix well, and then inoculate 1wt% into the sterilized cordycepin fermentation solid medium , fully stirred to make the bacteria evenly distributed in the solid medium for cordycepin fermentation, replenish water at a ratio of 1wt% every 24 hours to maintain relative humidity, and stir and turn over the material, and at the same time, pass sterile air with a relative humidity of 80% from the bottom of the fermenter , the aeration ratio is 0.5min ^-1 , after 4 days of culture at 27°C, the aeration ratio is reduced to 0.5h ^-1 , every 5 days with 5wt% spraying with 0.5g/L adenine and 2-6g/L triolein The culture solution of the ester makes it produce a large amount of fruiting bodies, and the other conditions remain unchanged. After 21 days of further cultivation, the fermentation ends; add 1.5 times the volume of solid material water, stir and turn the material at 80°C for 2 hours, filter and collect the filtrate; carry out the boiling process three times, The combined filtrates were concentrated to the solid volume to obtain the cordycepin extract.