阅读说明：本技术 一种利用固体培养基获得高产虫草素的方法 (Method for obtaining high-yield cordycepin by using solid culture medium ) 是由 刘晓红 于 2020-08-10 设计创作，主要内容包括：本发明涉及虫草素提取技术领域,具体公开了一种利用固体培养基获得高产虫草素的方法,包括制备固体培养基和蚕蛹液,蚕蛹液涂布,条件差异培养提高培养基中虫草素含量,去除子实体,检测培养基中虫草素含量。本发明的方法在培养蛹虫草的过程中,大大提高了固体培养基中虫草素的积累量。(The invention relates to the technical field of cordycepin extraction, and particularly discloses a method for obtaining high-yield cordycepin by using a solid culture medium. The method greatly improves the accumulation of cordycepin in the solid culture medium in the process of culturing the cordyceps militaris.)

1. A method for obtaining high-yield cordycepin by using a solid culture medium is characterized by comprising the following steps of:

s1, preparing a solid culture medium and a silkworm pupa liquid, and sterilizing;

the formula of the solid culture medium is as follows: KH (Perkin Elmer)₂PO₄1-5g，MgSO₄1-3g, vitamin B12-7 g, vitamin B21-5 g, glucose 2-3g, peptone 2-4g, silkworm chrysalis meal 4-6g, and rice 4-12gg, 2-4g of corn grit, 2-6g of buckwheat flour, 2-5g of coconut shell particles and 300ml of distilled water, and the pH value is adjusted to 6.2-8.3;

the silkworm pupa liquid is prepared by mixing silkworm pupa powder, glycine, adenine and distilled water;

subpackaging the above solid culture medium and pupa Bombycis solution, sterilizing at 121 deg.C for 20-30 min;

s2, adding silkworm pupa liquid into the solid culture medium before inoculation, and uniformly coating;

s3, increasing the cordycepin content in the culture medium by condition differential culture:

inoculating the prepared cordycepin-producing strain in a solid culture medium, culturing at 14-20 deg.C in the dark for 6-10 days, regulating the culture temperature to 18-30 deg.C, continuously culturing in the dark for 14-20 days, and performing day and night alternate culture;

the day and night alternate culture comprises the following specific steps: culturing at 18-30 deg.C under illumination of 200 and 500lx for 12-14h in the daytime and at 10-12 deg.C for 10-12h in the dark at night;

the day and night alternate culture time is 8-12 days;

then adjusting the illumination to 1000-;

s4, removing the fruiting body in the culture medium in the S2, and detecting the cordycepin content in the culture medium.

2. The method for obtaining cordycepin with high yield according to claim 1, wherein in S1, the coconut shell particles in the solid medium are particles with a particle size of 5-7mm prepared by crushing coconut shells.

3. The method for obtaining cordycepin with high yield according to claim 1, wherein in S1, the mixing ratio of the silkworm chrysalis meal, the glycine, the adenine and the distilled water is 1 g: 1 g: 1 g: 100-.

4. The method for obtaining cordycepin with high yield according to claim 3, wherein the solid medium is dispensed in culture pots in an amount of 18 to 22 g/pot in S1.

5. The method for obtaining cordycepin with high yield according to claim 4, wherein in S2, the addition amount of the silkworm pupa liquid is 40-60 μ l/tank.

6. The method for obtaining cordycepin with high yield according to claim 1, wherein in S3, the strain is Cordyceps militaris.

Technical Field

The invention relates to the technical field of cordycepin extraction, and particularly relates to a method for obtaining high-yield cordycepin by using a solid culture medium.

Background

Cordycepin is 3' -deoxyadenosine, and is a natural bioactive substance extracted from Cordyceps militaris. The cordycepin has a molecular formula of CsHyON and a relative molecular mass of 251.25, can be dissolved in water and ethanol, is a nucleic acid adaptor containing nitrogen glycoside, and belongs to purine alkaloids. Cordycepin is a new nucleoside drug, and has the functions of inhibiting tumor, resisting plasmodium and inhibiting mRNA translation in the 70 th 20 th century, and the research in the 90 th 20 th century shows that the addition of Adenosine Deaminase (ADA) inhibitor plays an important role in the expression of the antitumor activity of the adenosine deaminase. Cordycepin can interfere the synthesis of RNA and DNA of gene cells, inhibit the division of abnormal cells such as cancer cells and the like, and can be used as a tool for distinguishing different RNA polymerases in the cells; meanwhile, cordycepin also shows extremely strong antifungal activity, HIV-I virus resistance activity and clostridium selective inhibition activity, which draws high attention of the medical field, and has been in the end of the three years of the United states as a new anticancer and antiviral drug.

At present, the cordycepin is obtained by a sporocarp mostly, and then the method is quite expensive, and the cordycepin can be obtained in a fermentation culture medium of cordyceps militaris, the content of the cordycepin is not lower than that of the sporocarp, the cordycepin is obtained by culture fermentation, but most of the cordycepin is obtained by liquid fermentation through culture medium fermentation, the liquid fermentation is easy to be polluted, and although the pollution rate can be reduced by solid culture, the content of the cordycepin obtained by the solid culture is low and is usually lower than 1.8 mg/g. Therefore, the invention provides a method for obtaining high-yield cordycepin by using a solid culture medium.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for obtaining high-yield cordycepin by using a solid culture medium, which greatly improves the cordycepin content in the solid culture medium by coating silkworm pupa liquid on the surface of the solid culture medium and performing condition differential culture.

The invention provides a method for obtaining high-yield cordycepin by using a solid culture medium, which comprises the following steps of:

s1, preparing a solid culture medium and a silkworm pupa liquid, and sterilizing;

the formula of the solid culture medium is as follows: KH (Perkin Elmer)₂PO₄1-5g，MgSO₄1-3g of vitamin B12-7 g, vitamin B21-5 g, 2-3g of glucose, 2-4g of peptone, 4-6g of silkworm chrysalis powder, 4-12g of rice, 2-4g of corn grit, 2-6g of buckwheat powder, 2-5g of coconut shell particles, 100ml of distilled water and 300ml of water, and the pH value is adjusted to 6.2-8.3;

the silkworm pupa liquid is prepared by mixing silkworm pupa powder, glycine, adenine and distilled water;

subpackaging the above solid culture medium and pupa Bombycis solution, sterilizing at 121 deg.C for 20-30 min;

s2, adding silkworm pupa liquid into the solid culture medium before inoculation, and uniformly coating;

s3, increasing the cordycepin content in the culture medium by condition differential culture:

inoculating the prepared cordycepin-producing strain in a solid culture medium, culturing at 14-20 deg.C in the dark for 6-10 days, regulating the culture temperature to 18-30 deg.C, continuously culturing in the dark for 14-20 days, and performing day and night alternate culture;

the day and night alternate culture comprises the following specific steps: culturing at 18-30 deg.C under illumination of 200 and 500lx for 12-14h in the daytime and at 10-12 deg.C for 10-12h in the dark at night;

the day and night alternate culture time is 8-12 days;

then adjusting the illumination to 1000-;

s4, removing the fruiting body in the culture medium in the S2, and detecting the cordycepin content in the culture medium.

Preferably, the coconut shell particles in the solid medium are particles obtained by crushing coconut shells into particles with the particle size of 5-7 mm.

Preferably, the mixing dosage ratio of the silkworm chrysalis meal, the glycine, the adenine and the distilled water is 1 g: 1 g: 1 g: 100-.

Preferably, in S1, the solid culture medium is dispensed into culture tanks, and the dispensing amount is 18-22 g/tank.

Preferably, in S2, the addition amount of the silkworm pupa liquid is 40-60 μ l per tank.

Preferably, in S3, the strain is cordyceps militaris.

Compared with the prior art, the invention has the beneficial effects that:

1. the method provided by the invention effectively improves the cordycepin content in the solid culture medium, and the average content of cordycepin in the solid culture medium is up to 7.06 mg/g.

2. Various substances in the formula of the solid culture medium used in the invention cooperate with each other to promote the accumulation of cordycepin, wherein, the addition of the coconut shells can increase the porosity inside the solid culture medium, thereby not only being beneficial to the growth of cordyceps militaris in space, but also providing a carbon source.

3. According to the invention, the silkworm pupa liquid prepared by mixing the silkworm pupa powder, the glycine, the adenine and the distilled water is added to the surface of the solid culture medium before inoculation, so that the early generation of cordycepin is promoted, and the cordycepin content in the solid culture medium is finally improved.

4. The invention also improves the cordycepin content in the solid culture medium by condition differential culture.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a chromatogram of a cordycepin standard of the present invention;

FIG. 2 is a chromatogram of cordycepin obtained from the solid medium of example 1 according to the present invention;

FIG. 3 is a graph comparing the unit contents of cordycepin obtained in examples 1 to 3 of the present invention and comparative examples 1 to 3.

Detailed Description

The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.