A method for liquid fermentation of Cordyceps militaris for increasing cordycepin production

Abstract

The invention discloses a Cordyceps militaris liquid fermentation method for increasing the yield of cordycepin, and belongs to the technical field of biological fermentation. The invention can significantly increase the yield of cordycepin in the Cordyceps militaris fermentation liquid by adding an appropriate amount of rotenone after the Cordyceps militaris is fermented for a period of time. Compared with the yield of 189.66 mg / L of traditional fermentation without adding rotenone, the yield after adding rotenone is as high as 804.69 mg / L, that is, the total yield of cordycepin is increased by 324.28%. The method has a simple process and does not need to improve the enzyme activity by means of genetic modification or the like; the production cost is low, and there is no need to continuously feed adenine and other substrates required for the synthesis of cordycepin, and the total fermentation time of cordycepin is short, and the production efficiency is significantly improved, which is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to a Cordyceps militaris liquid fermentation method for improving the yield of cordycepin, and belongs to the technical field of biological fermentation. Background Art

[0002] Cordyceps militaris is a model species of the Ascomycota, Hypomorphales, Clavicipitaceae, and Cordyceps genus. It is also called northern cordyceps and northern cordyceps. It is an important medicinal fungus of insects. Traditional Chinese medicine believes that Cordyceps militaris can both replenish lung yin and kidney yang, and is a Chinese medicine that can balance and regulate yin and yang at the same time. Compared with Cordyceps sinensis, Cordyceps militaris has the following two advantages: on the one hand, the fruiting technology of Cordyceps militaris under artificial conditions is more mature than Cordyceps sinensis; on the other hand, the analysis of genes related to the biosynthesis of cordycepin in Cordyceps militaris is relatively clear. CmCNS1 and CmCNS2, two genes directly involved in the synthesis and metabolism of cordycepin, were discovered in Cordyceps militaris in 2017. Cordycepin, also known as cordycepin, has a chemical name of 3'-deoxyadenosine, and is a nucleoside antibiotic isolated from Cordyceps militaris. In the past two decades, people have made groundbreaking achievements in the application research of cordycepin in various anti-tumor activity tests, improvement of aging caused by oxidative stress, enhancement and regulation of immune function, inhibition of fat cell differentiation to prevent obesity, inflammation inhibition, production of IL-10 by human peripheral blood mononuclear cells, anti-leukemia activity and mechanism of action. The diverse pharmacological effects of cordycepin have been recognized, and the current price of high-purity cordycepin in the domestic market has exceeded 6,000 yuan / gram, so it has great development value and broad market prospects.

[0003] There are three common ways to obtain cordycepin: one is to extract it from natural Cordyceps militaris fruiting bodies; the second is to extract it after artificially breeding Cordyceps militaris fruiting bodies; the third is to use modern biotechnology to ferment Cordyceps militaris mycelium and then extract it from the fermented mycelium or culture medium. Regardless of whether it is natural Cordyceps militaris fruiting bodies or artificially bred Cordyceps militaris fruiting bodies, the cordycepin content of their fruiting bodies is relatively low, and the process is complicated and the production cost is high, which cannot meet the market needs. The cordycepin produced by liquid deep fermentation technology, which has the advantages of short production cycle and low cost, has basically the same biological activity as the cordycepin obtained from wild Cordyceps militaris, and has become the most economical method to obtain cordycepin. Although the technology of producing cordycepin by liquid deep fermentation of Cordyceps militaris has made certain progress in recent years, the cordycepin produced by this technology still cannot meet the increasing cordycepin production capacity demand. Therefore, how to reduce production costs or further increase cordycepin production and ultimately improve economic benefits by optimizing the production process has become a technical problem that needs to be solved in the cordyceps products industry.

[0004] In addition to optimizing the cordycepin extraction process and selecting high-yield Cordyceps militaris strains, the industry generally focuses on optimizing fermentation conditions by controlling temperature, light, pH, salt ions, nutrients, precursors and hormones, thereby affecting cordycepin metabolism and ultimately increasing cordycepin production. The fermentation methods based on these optimized fermentation conditions are still traditional liquid fermentation methods, such as:

[0005] Literature: Xia Yongliang. Research on the mechanism of cordycepin biosynthesis. Doctoral dissertation, University of Chinese Academy of Sciences, 2014. The synthetic gene cluster of cordycepin was reported, and a method for achieving efficient cordycepin biosynthesis was revealed. The method is to heterologously express the synthetic gene cluster of cordycepin in Cordyceps militaris into fungi that contain or can self-generate 3'-AMP, so that it can produce cordycepin. However, the disadvantage is that the specific yield or increase of cordycepin is not given.

[0006] Literature: Tang Jiapeng, Wang Jianxiong. Plant growth regulators promote the production of cordycepin in liquid surface culture of Cordyceps militaris. The technology of adding different concentrations of plant growth regulators to increase cordycepin production was reported, but the production cycle is relatively long.

[0007] Chinese Patent: Chen Ping et al., application number 202110338257.8 provides a method for overexpressing cordycepin in recombinant Cordyceps militaris, and isolating and extracting cordycepin from the fermentation broth of the strain. The method is to overexpress and recombinant adenylate succinate synthase gene, adenylate succinate lyase gene or 5'-nucleotidase gene in Cordyceps militaris. The fermentation broth of the transformant strain is then separated and purified to obtain cordycepin. However, the disadvantage is that the genetic modification technology of overexpression and recombination is complex and difficult to operate, and the yield of cordycepin in the fermentation broth is low. Summary of the invention

[0008] The purpose of the present invention is to provide a cordycepin production method which can significantly increase the cordycepin yield in Cordyceps militaris fermentation broth, does not require genetic modification, does not require the addition of cordycepin precursors, has a simple production process and a relatively short fermentation time in view of the various deficiencies in the existing cordycepin fermentation technology.

[0009] The invention provides application of rotenone in promoting the yield of cordycepin in Cordyceps militaris.

[0010] In one embodiment, the application is to use rotenone to promote the production of cordycepin during the fermentation process of Cordyceps militaris.

[0011] In one embodiment, the rotenone is added to the fermentation medium of Cordyceps militaris at a final concentration of 1-10 mg / L.

[0012] In one embodiment, the rotenone is added to the fermentation medium using dimethyl sulfoxide as solvent.

[0013] In one embodiment, the Cordyceps militaris is purchased from China General Microorganism Culture Collection Center with a collection number of CCGMC 3.14242.

[0014] The invention also provides a Cordyceps militaris fermentation method for improving the yield of cordycepin, which comprises fermenting Cordyceps militaris strains, the strain preservation number of which is CCGMC 3.14242, in a fermentation medium containing rotenone.

[0015] In one embodiment, the fermentation medium contains 20 g / L glucose, 10 g / L of an equal mixture of animal tissue pepsin hydrolysate and trypticase, and pH=5.6±0.1.

[0016] In one embodiment, the final concentration of rotenone is 1-10 mg / L.

[0017] In one embodiment, the fermentation is carried out at 20-28° C. and 150-250 r / min for at least 5 days.

[0018] In one embodiment, the fermentation is first carried out in a fermentation medium for 1 to 5 days, and then rotenone with a final concentration of 1 to 10 mg / L is added to the medium to continue the fermentation.

[0019] In one embodiment, the method is performed by the following steps:

[0020] 1) inoculating Cordyceps militaris strains into a seed culture medium containing glucose and potato extract powder, and fermenting in a shaking flask at 25-28° C. and a rotation speed of 180-220 r / min for 3-5 days to obtain Cordyceps militaris seed liquid;

[0021] 2) After the Cordyceps militaris seed liquid prepared in step 1) is mixed with a cantilever electric stirrer, the mycelium with a particle size of less than 0.1 mm is prepared into a mycelium suspension with a concentration of 1.0-2.0 g / L (based on the wet weight of the mycelium) as the seed liquid; the seed liquid is transferred and inoculated into a liquid fermentation medium at a volume ratio of 0.5-1.0%, and fermented at 25-28° C. and a rotation speed of 180-220 r / min for 1-5 days;

[0022] 3) After adding rotenone with a final concentration of 1-10 mg / L, the fermentation was continued for 10-15 days at 25-28° C. and a rotation speed of 180-220 r / min.

[0023] In one embodiment, the seed culture medium contains: 20 g / L glucose, 6 g / L potato extract powder, and pH = 5.6±0.2.

[0024] The invention also provides application of the method in producing products containing cordycepin.

[0025] Beneficial effects:

[0026] 1) The present invention can significantly increase the yield of cordycepin in the fermentation liquid of Cordyceps militaris by adding an appropriate amount of rotenone after the Cordyceps militaris is fermented for a period of time. Compared with the yield of 189.66 mg / L in the traditional fermentation without adding rotenone, the yield after adding rotenone is as high as 804.69 mg / L, and the total yield of cordycepin is increased by 324.28%.

[0027] 2) The present invention improves the activity of intracellular 3-phosphoglyceraldehyde dehydrogenase and increases [NADH / NAD + ] level, increase the transcription level of cordycepin biosynthesis genes CmCNS1-3 and increase the proportion of adenine (adenylic acid) in nucleotide metabolism, thus comprehensively improving cordycepin anabolism from these three dimensions.

[0028] 3) The fermentation process of the present invention is simple, and there is no need to improve the enzyme activity by means of genetic modification or the like; the production cost is low, and there is no need to continuously feed adenine and other substrates required for the synthesis of cordycepin; and the method of the present invention has a short fermentation time, a simple process, and significantly improved production efficiency compared to the existing method, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a result diagram of the effect of different concentrations of rotenone on cordycepin yield and biomass in the manner of Examples 1 to 3; wherein 00 represents the DMSO group without rotenone added in Example 2; 01, 02, 05, 10 represent the experimental groups with rotenone added in DMSO as solvent at final concentrations of 1 mg / L, 2 mg / L, 5 mg / L and 10 mg / L, respectively.

[0030] Figure 2 This is the result graph of the effect of rotenone on the cordycepin fermentation curve and sugar consumption curve.

[0031] Figure 3 In the embodiment of Example 4, rotenone has an effect on the intracellular [NADH / NAD + ] levels and the results of the effects on 3-phosphoglyceraldehyde dehydrogenase activity.

[0032] Figure 4 This is the result of fluorescence quantitative PCR verification of the effect of rotenone on the transcription level of the intracellular cordycepin synthesis gene CmCNS1-3 of Cordyceps militaris.

[0033] Figure 5 This is a graph showing the effects of rotenone on genes related to nucleotide metabolism in Cordyceps militaris based on transcriptome analysis.

[0034] Figure 6 This is a result diagram based on transcriptome analysis showing the effects of rotenone on genes related to glycolysis, gluconeogenesis and anaerobic respiration in Cordyceps militaris.

[0035] Figure 7 This is a result diagram of the effect of rotenone on genes related to the tricarboxylic acid cycle in Cordyceps militaris based on transcriptome analysis.

[0036] Figure 8 This is a graph showing the results of transcriptome quality control using the method of Example 5. DETAILED DESCRIPTION

[0037] The preparation and addition method of rotenone mother liquor is as follows: weigh different weights of rotenone powder respectively in a dry environment, then use dimethyl sulfoxide to fully dissolve the rotenone powder and fix the volume, so that the rotenone concentration is accurate to 2g / L, 4g / L, 10g / L and 20g / L (error ≤1%); filter with a 0.22μm nylon microporous filter to obtain the rotenone mother liquor; when adding rotenone to the fermentation medium, the rotenone mother liquor needs to be slowly added while gently shaking or stirring the medium.

[0038] Detection method:

[0039] Use three layers of non-woven fabric to filter all fermented products, the filtrate is the crude fermentation broth sample, and the filter residue is the crude mycelium sample; after high-speed centrifugation of the crude fermentation broth sample, the supernatant is taken and filtered through a 0.22 μm polyethersulfone microporous filter to obtain the fermentation broth sample to be tested.

[0040] Biomass detection: The crude mycelium sample was washed three times with single distilled water and then dried in a 60°C forced air drying oven. The weight when dried to a constant weight was the mycelium biomass.

[0041] Cordycepin content detection: The cordycepin content in the fermentation broth to be tested was quantified by high performance liquid chromatography (NY / T 2116-2012), and the supernatant was filtered through a 0.22 μm polyethersulfone microporous filter to obtain the test sample; the mobile phase was adjusted to pH = 4.0 ± 0.1 with phosphoric acid before degassing or use, and the chromatographic column was the Shim-pack GIST C18 chromatographic column (model: 227-30017-07) of Shimadzu Corporation of Japan; the reducing sugar content test kit (article number: HYT-1-Y) of Suzhou Keming Biotechnology Co., Ltd. was used to quantify the reducing sugar content in the fermentation broth to be tested, and the detection instrument was the SpectraMax ABS Plus of Meigu Molecular Instrument (Shanghai) Co., Ltd.

[0042] Example 1

[0043] The Cordyceps militaris strain preserved by the China General Microbiological Culture Collection Center is used for fermentation, and the strain preservation number is CCGMC 3.14242.

[0044] The seed culture medium used potato glucose water (article number: HB0233-4) produced by Qingdao Haibo Biological Company, the main ingredients of which are: glucose 20g / L, potato extract powder 6g / L;

[0045] The liquid fermentation medium used was Sabouraud glucose liquid medium (article number: HB0253-71) produced by Qingdao Haibo Biological Company. The main components of the medium were: 20 g / L glucose, 10 g / L equal mixture of animal tissue pepsin hydrolysate and trypsin casein, pH = 5.6 ± 0.2.

[0046] The Cordyceps militaris mycelium block was inoculated in a 250mL shaking flask containing 100mL seed culture medium, and fermented in a rotary shaking flask cabinet at a fermentation temperature of 25°C and a rotation speed of 200r / min for 4 days to obtain the seed mycelium culture of the Cordyceps militaris strain. After the culture was mixed with a blender, the mycelium with a particle size less than 0.1mm was adjusted to a mycelium suspension with a concentration of 1.0g / L (based on the wet weight of the mycelium) with sterile water, and then 1mL of the mycelium suspension was inoculated in a 250mL shaking flask containing 100mL fermentation medium. The fermentation temperature was 25°C and the rotation speed was 200r / min. Fermentation was 15 days, during which 0.5-1.0mL of fermentation liquid was collected every day for detection. After the fermentation was completed, the remaining fermentation was harvested for detection.

[0047] The results are as follows Figure 1 , Figure 2 As shown in the CK group, the biomass after fermentation was 919.90±181.767 mg, and the cordycepin production was 189.66±53.48 mg / L.

[0048] Example 2

[0049] The strain, seed culture medium, liquid fermentation medium and inoculation method are the same as those in Example 1, except that the fermentation temperature is controlled at 25°C, the rotation speed is 200 r / min, the fermentation is performed for 1 day, and then 50 μL of dimethyl sulfoxide is added to the fermentation liquid at one time, and the fermentation is continued for 14 days under the same conditions, during which 0.5-1.0 mL of the fermentation liquid is collected every day for detection. After the fermentation is completed, the remaining fermentation product is harvested for detection.

[0050] The results are as follows Figure 1 , Figure 2As shown in the 0 mg / L group, the biomass was 910.67±153.47 mg and the cordycepin production was 188.21±20.38 mg / L. It can be seen that the addition of 0.5‰ dimethyl sulfoxide will not have a significant effect on the fermentation of Cordyceps militaris.

[0051] Example 3

[0052] The composition of the strain, seed culture medium and liquid fermentation culture medium and the inoculation method are the same as those in Example 1, except that the fermentation temperature is controlled to be 25° C., the rotation speed is 200 r / min, and the fermentation is carried out for 1 day. Then, 50 μL of rotenone mother solution (solvent is dimethyl sulfoxide) containing different concentrations (2 g / L, 4 g / L, 10 g / L and 20 g / L) is added to the fermentation liquid at one time, so that the final concentration of rotenone is 1 mg / L, 2 mg / L, 5 mg / L and 10 mg / L, respectively. Fermentation is continued for 14 days under the same conditions. After the fermentation is completed, the remaining fermented products are harvested respectively.

[0053] The results are as follows Figure 1 , Figure 2 As shown in the 1 mg / L, 2 mg / L, 5 mg / L and 10 mg / L groups, the biomass at each concentration was 977.10±52.16 mg, 998.43±146.40 mg, 1024.27±35.95 mg and 909.53±23.31 mg, and the cordycepin production was 501.87±52.44 mg / L, 654.64±81.58 mg / L, 804.69±29.45 mg / L and 738.36±17.72 mg / L, respectively.

[0054] Example 4

[0055] The composition of the strain, seed culture medium and liquid fermentation culture medium and the inoculation method were the same as those in Example 1, except that the fermentation temperature was 25° C., the rotation speed was 200 r / min, and the fermentation was performed for 5 days. Then, ultrapure water and 50 μL of rotenone mother solution with a concentration of 10 g / L were added to the fermentation liquid at one time (the two groups were named CK group and Rotenone group, respectively), and the fermentation was continued for 6 hours under the same conditions. After the fermentation was completed, the remaining fermentation product was harvested.

[0056] Use three layers of non-woven fabric to filter all the fermented products, and the filter residue is the crude mycelium sample; wash the crude mycelium sample three times with single distilled water and then use filter paper to absorb the moisture as much as possible, and then weigh and package the mycelium according to the subsequent required usage.

[0057] The NADH / NAD+, NADPH / NADP+ and GAPDH enzyme activities of mycelium were detected using the NAD+ / NADH detection kit (WST-8 method, catalog number: S0175) and NADP+ / NADPH detection kit (WST-8 method, catalog number: S0179) of Shanghai Biotech Co., Ltd. and the 3-phosphoglyceraldehyde dehydrogenase kit (catalog number: GAPDH-1-Y) of Suzhou Keming Biotechnology Co., Ltd. The results are as follows Figure 3 As shown, the NADH / NAD+ ratios of the Rotenone group and the CK group were 1.452±0.068 and 0.598±0.082, the NADPH / NADP+ ratios were 1.342±0.313 and 0.561±0.150, and the GAPDH enzyme activities of the Rotenone group and the CK group were 425.864±8.296nmol / min / g and 675.083±16.366nmol / min / g, respectively - the NADH / NAD+ ratio, NADPH / NADP+ ratio and GAPDH enzyme activity of the Rotenone group were increased by 142.809%, 139.216% and 58.521% respectively compared with the CK group.

[0058] Example 5

[0059] The composition of the strain, seed culture medium and liquid fermentation culture medium and the inoculation method are the same as those in Example 1, except that the fermentation temperature is 25° C., the rotation speed is 200 r / min, and the fermentation is carried out for 1 day. Then, ultrapure water and 50 μL of rotenone mother solution with a concentration of 10 g / L are added to the fermentation liquid at one time (so that the final concentration of rotenone in the fermentation liquid is 5 mg / L). The two groups are named CK group and Rotenone group, respectively, and the fermentation is continued for 3.5 days under the same conditions. After the fermentation is completed, the remaining fermentation product is harvested.

[0060] Use three layers of non-woven fabric to filter all the fermented products, and the residue is the crude mycelium sample; wash the crude mycelium sample three times with single distilled water, and then use filter paper to absorb as much water as possible, and then freeze the mycelium in liquid nitrogen. TM Kit (Cat. No.: 15596026) and SuperScript TM II Reverse Transcription Kit (Cat. No.: 18064014) was used for RNA extraction and reverse transcription; Illumina's TruSeq Stranded mRNA Library Prep Kit (Cat. No.: RS-122-2101) and HiSeq X Ten Sequencing System were used for cDNA library construction and transcriptome sequencing.

[0061] Use Hunan Aikerui Biotechnology Co., Ltd. Green Premix Pro Taq HS qPCR Kit (Cat. No. AG11701) was used for quantitative PCR; the quantitative upstream primer sequence of CmCNS1 gene was 5'-GCGAACCCACGATCTGCACAAGCCCTACA-3', and the downstream primer sequence was 5'-CCATCATGGACTGCGTGAGGTGCGGATACA-3'; the quantitative upstream primer sequence of CmCNS2 gene was 5'-TCGCAGCCCTGCTCCATGACATTGACATTTCTCACA-3', and the downstream primer sequence was 5'-GCATCTCCA CCAGCGGAAAGAAACAGCTCTTCTTCA-3'; the quantitative upstream primer sequence of the CmCNS3 gene is 5'-CACGCTGCGGCTTGCTGACGACGAC-3', and the downstream primer sequence is 5'-GCCGTGATACGCCGCTAGTTCCTGATGACC-3'; the quantitative upstream primer sequence of the internal reference gene (CmActin) is 5'-CAACAACTTCCTGACGGGC-3', and the downstream primer sequence is 5'-TCCTTGGGCTTCTGCTGAC-3'.

[0062] The results of fluorescence quantitative PCR and transcriptome sequencing are as follows Figures 4 to 8 As shown, the transcription levels of cordycepin synthesis genes CmCNS1-3 in the Rotenone group increased by 501.35%, 1379.53% and 1677.89% respectively compared with the CK group; at the same time, the Rotenone group had a higher proportion of adenine (adenylic acid) synthesis in nucleotide metabolism compared with the CK group. The data showed that the addition of rotenone can upregulate genes related to adenylic acid synthesis and downregulate genes related to the synthesis of guanylic acid, uridylic acid and cytidylic acid, thereby making the nucleotide metabolic flow in Cordyceps militaris more biased towards adenine (adenylic acid), so that there is no need to add adenine and other specific substrates required for cordycepin metabolism during the fermentation process. In addition, compared with the CK group, the related genes in the tricarboxylic acid cycle pathway of the Rotenone group showed a significant downward trend, while the related genes in the glycolysis and anaerobic respiration pathways showed a significant upward trend in contrast.

[0063] Comparative Example 1:

[0064] Referring to the method in the paper "Glutathione Promotes the Synthesis of Cordycepin by Cordyceps militaris in Liquid Fermentation System", glutathione (GSH) was added to the fermentation medium, and the final concentration of GSH in the fermentation medium was controlled to be 0.5, 1.0, 2.0 and 3.0 g / L, respectively. The results showed that the biomass of fermentation for 15 days and the cordycepin content of fermentation for 20 days were 14.45 ± 0.63 g / L and 439.69 ± 12.43 mg / L, respectively. The transcription level of CmCNS1-2 in the group with 3.0 g / L GSH addition for 15 days was increased by 540.67 times and 25.81 times, respectively, compared with the CK group. It can be seen that this method is much lower than the present method in terms of the final yield of cordycepin and the transcription level of the cordycepin synthesis gene CmCNS2, and the dosage and cost of the addition are large, and the production cycle is as long as 20 days, which is 1.33 times that of the present invention.

[0065] Comparative Example 2:

[0066] Referring to the method in the paper "The Effect of Adding Precursors on the Yield of Cordycepin and Kinetic Characteristics", 1.5g / L adenosine, 2.5g / L phenylalanine, 1.5g / L adenine, 2.5g / L glycine and 2.5g / L L-glutamine were added to the fermentation medium respectively. The results showed that the cordycepin yields after 20 days of fermentation were 772.5mg / L, 661.7mg / L, 103.5mg / L, 149.6mg / L and 174.0mg / L respectively. The final yield of cordycepin was lower than that of this method, and the dosage and cost of the addition were large, and the production cycle was as long as 20 days, which was 1.33 times that of the present invention.

[0067] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of rotenone in promoting the fermentation of Cordyceps militaris to produce cordycepin.

2. The use according to claim 1, characterized in that: The application is to add rotenone at a final concentration of 1-10 mg / L into the fermentation medium of Cordyceps militaris.

3. The use according to claim 1, characterized in that: The rotenone is added into the fermentation medium with dimethyl sulfoxide as solvent.

4. The use according to claim 2, characterized in that: The rotenone is added to the fermentation medium with dimethyl sulfoxide as solvent at a final concentration of 1-10 mg / L.

5. The use according to any one of claims 1 to 4, characterized in that: The Cordyceps militaris preservation number is CGMCC3.14242.

6. A method for producing cordycepin by fermentation, characterized in that: Ferment the Cordyceps militaris in a fermentation medium containing rotenone at 20-28°C and 150-250r / min.

7. The method according to claim 6, characterized in that The fermentation medium contains glucose, animal tissue pepsin hydrolysate and trypticase.

8. The method according to claim 6, characterized in that The final concentration of rotenone is 1-10 mg / L.

9. The method according to any one of claims 6 to 8, characterized in that: The fermentation is firstly carried out in a fermentation medium for 1 to 5 days, and then rotenone with a final concentration of 1 to 10 mg / L is added to the medium to continue the fermentation.

10. The method according to any one of claims 6 to 8, characterized in that: First, Cordyceps militaris is cultured in a seed culture medium to obtain a seed solution, and then the seed solution is transferred to a fermentation culture medium for fermentation.

11. Use of the method according to any one of claims 6 to 10 in producing a product containing cordycepin.

Images