CN114431070B - Cordyceps militaris large-scale production method and application - Google Patents

Abstract

The invention relates to the technical field of production and application of cordyceps militaris, in particular to a large-scale production method and application of cordyceps militaris, wherein a Chinese caterpillar fungus tannin strain of silkworm pupa is taken as a strain, a culture mother strain is obtained after tissue rejuvenation, a culture bacterial solution is further prepared by solid culture and liquid amplification, and cordyceps militaris with high cordycepin, N6- (2-hydroxyethyl) adenosine and ergosterol contents in sporocarp is obtained by culture after inoculation; the scale production of the cordyceps militaris sporocarp based on the solid culture medium is designed by controlling the technical key points and key parameters of each link in the production process, the scale production is realized, the production process is simple and feasible, the parameters are easy to control, the effective components with good activity can be obtained, and the effective components play a role in resisting lung cancer by specifically regulating the estradiol metabolism and play a role in resisting liver cancer by specifically regulating the expression of glyceraldehyde-3-phosphate dehydrogenase.

Description

Cordyceps militaris large-scale production method and application

Technical Field

The invention belongs to the technical field of extraction of cordyceps militaris production agents, and particularly relates to a large-scale production method and application of cordyceps militaris.

Background

Cordyceps militaris, having a scientific name of (l.exfr.) link.in 1833, is Cordyceps militaris (l.ex Fr.) link, is a fungus of the genus Cordyceps of the family ergomycetaceae, is also called Cordyceps militaris, is a type of Cordyceps militaris, and is described in the national chinese herbal medicine compilation: the fruiting body and body of Cordyceps militaris can also be used as Cordyceps sinensis medicine. Record of Chinese medicine sea: cordyceps militaris (also called as cordyceps militaris (Jilin), has sweet and mild nature and taste, enters the lung and kidney meridians, has the characteristics of tonifying kidney yang and benefiting lung yin, and has the effects of tonifying kidney yang, benefiting essence, stopping bleeding and reducing phlegm. The wild cordyceps militaris is discovered in Jilin province in 1958 in China for the first time, and the patent of the invention of culturing the cordyceps militaris by using a solid culture medium is applied by the national institute of silkworm industry science in Jilin province in 1987.

With the continuous improvement of the traditional technology and the application of new technology, the cordyceps militaris is developed in a rapid and drastic way in aspects of strain breeding, process control, harvesting and storage and the like, so that the cultivation level and the industrialization level of the cordyceps militaris are improved continuously, for example, the genome map of the cordyceps militaris is more precise and real due to the upgrading of gene sequencing technology, scientists obtain more information by reading the genome of the cordyceps militaris, and continuously and deeply excavate key biological information of the cordyceps militaris, so as to guide the research and production of the cordyceps militaris. Researches find that the growth and metabolism processes of cordyceps militaris are influenced by factors such as strain sources, cultivation modes and environments, so that the activity and the content of metabolites of cordyceps militaris are different. In addition, from the perspective of drug development, it is still necessary to search for specific targets related to disease treatment by using cordyceps militaris active ingredients as lead compounds based on multi-level comprehensive analysis and through experiments and clinical demonstration.

Modern researches prove that the cordyceps militaris plays roles of resisting tumors, insomnia and oxidation, reducing blood sugar, improving immunity, resisting inflammation, protecting liver and the like, and is mainly realized by regulating the function of a human body through multi-target interaction of adenosine compounds, cordyceps polysaccharide, sterol compounds and the like. Therefore, the key active ingredients of the cordyceps militaris fruiting body for inhibiting the growth of the lung cancer and the liver cancer cells are excavated, the action mechanism of the cordyceps militaris fruiting body for inhibiting the growth of the cancer cells is clarified, effective and safe applicable natural medicines are provided for treating the lung cancer and the liver cancer, and the cordyceps militaris active ingredient is taken as a lead compound, so that the key specific target of the disease is further searched, and the cordyceps militaris active ingredient has great application value for diagnosing and preventing the disease.

At present, a plurality of research and development, production and marketing research units and enterprises related to the cordyceps militaris exist, the agricultural cultivation technology of the cordyceps militaris is basically mature, the edible and medicinal bases and application research of the cordyceps militaris are wide, but the quality of cordyceps militaris raw materials is different, and cordyceps militaris brand products are still lacking. The problems of high pollution rate, poor quality and low yield in the production process are still the problems to be solved at the present stage. Therefore, the method has great significance in selecting the cordyceps militaris strain with excellent quality, developing the production process research of cost reduction, efficiency improvement and environmental protection. In the past research, the cordyceps militaris strain c.militaris (HN), which is separated from Guizhou Guian accurate medicine, inc., and identified by microbiological research institute of Chinese academy of sciences, was found to be capable of culturing differentiable cordyceps militaris on the components of dead silkworm pupae after silk reeling, and the cordyceps militaris fruiting body was obtained by artificial culture. The invention creates a mother seed culture for large-scale production by selecting high-quality cordyceps militaris Haining plant sporocarp obtained by artificial culture, adopting a tissue rejuvenation method, selecting a single colony from a solid culture medium to be transformed into a liquid culture medium for strain amplification and the like, and provides a large-scale production and quality control method for solving the problems of pollution control, poor quality of sporocarp products, low yield of sporocarp and the like in the cordyceps militaris culture process.

Disclosure of Invention

The invention provides a large-scale production method and application of cordyceps militaris aiming at the defects of the prior art.

The method is realized by the following technical scheme:

a first object of the present invention is to provide: a large-scale production method of Cordyceps militaris comprises the following steps:

step one, breeding a silkworm chrysalis cordyceps sinensis Hainin strain C.militaris (HN):

1) Seed selection: selecting the silkworm chrysalis cordyceps sinensis tannin plant fruiting bodies which are robust in growth, have no condensed water or condensed water, are clear and transparent in water drop and have no white aerial hyphae at the root and grow to be 2-3 cm high;

2) Culturing strains: in an aseptic environment, adopting an aseptic inoculation hook to hook off the root of the sporocarp, taking out the sporocarp, placing the sporocarp on a sterilized gauze, clamping the root of the sporocarp by using an aseptic forceps, sequentially wiping the surface of the sporocarp by 75% alcohol, cleaning residual alcohol on the surface of the sporocarp by using aseptic water, removing the epidermis of the sporocarp by using an aseptic scalpel, selecting a part with the length of 1-1.5 cm at the top of the sporocarp, dividing the part into 3-5 granules per 1cm, placing the small granules of the sporocarp on a PDA solid culture medium by using an aseptic inoculation hook, keeping the interval of the granules of the sporocarp about 3.5cm, and carrying out shading inversion culture in a biochemical incubator at the temperature of 22 ℃ until hyphae appear;

3) And (3) strain purification: in an aseptic environment, using an inoculating loop to select a loop of hyphae obtained in a strain culture link, adopting continuous plate streaking to separate and purify strains, and performing inverted culture in a biochemical incubator at the temperature of 21-23 ℃ until the hyphae grow to the height of about 0.5cm;

step two strain preparation

According to the proportion that the inoculation amount of each 120mL of liquid culture medium is 1cm multiplied by 1cm, a flat single bacterial colony obtained by purifying the bacterial strain in the first step is picked by a sterile scalpel and cut up, the bacterial colony is quickly transferred into the liquid culture medium in a conical flask, the bacterial colony is uniformly distributed by slight shaking, the mouth of the flask is tightened, the bacterial colony is cultured in a shaking table at 22 ℃ and 170rpm/min in a dark place, and qualified culturable strains are obtained when hyphae are snow white, uniform, dense, strong in penetrating power and free from peculiar smell and grow to be larger than 1/2 area of the plane of the culture medium;

cultivation in three steps

1) Initial culture of spawn running: adopting a full-automatic cap opening, inoculating and covering integrated machine, quickly injecting 2mL of bacterial liquid into a culture bottle, covering, culturing in the dark for 3-5 days at the temperature of 15-18 ℃ and the relative air humidity of 63-68%, keeping hyphae in a snow white color, uniformly and densely growing, having strong penetrating power and no peculiar smell, continuously culturing until the hyphae fully grow over the whole culture medium, and entering a light culture strain to culture in the early stage of differentiation when a plurality of dome-shaped bulges with different quantities and sizes are generated on the material surface, wherein the culture medium with less pollution in the stage can be recycled and used as a culture medium for secondary culture after being further sterilized and crushed; the temperature is 15-18 ℃, the growth of other mixed bacteria can be well inhibited, and the pollution rate is reduced; the culture bottle adopts transparent high-temperature and high-pressure resistant glass or a pv material as a production container, the container is circular and consists of three parts, namely a base, a culture medium fall-preventing plate and a bottle body, the diameter of the bottle is 6.5cm, the total height of the bottle is 12cm, the height of the base is 3cm, the fall-preventing plate is a concentric circle with the outer diameter of 6.5cm and the inner diameter of 5cm, and the height of the bottle body is 9cm; the base, the fall-stopping plate and the bottle body are detachable, and the top of the bottle is provided with a small vent hole; the culture bottle is filled with 40g of strain culture medium, the height is about 2cm, and the culture bottle has the advantages of shortened bottom penetrating time for growth and faster hypha differentiation in the later period.

2) Strain differentiation early stage culture: culturing for 3-10 days under the conditions that the temperature is 18-23 ℃, the relative humidity of air is more than or equal to 85 percent and the illumination intensity is 200-250 Lx, and continuing to culture for 1-3 days after hypha on the surface of the culture medium gradually changes from white to orange, thus entering the original basal period; the light is irradiated for more than 12 hours every day in the light culture process, but the light cannot be continuously irradiated, so that the dark culture for 6 hours every day is ensured, and the direct irradiation of sunlight is prevented;

3) Strain differentiation primordial stage culture: culturing for 10 days by adopting temperature difference stimulation under the condition that the relative air humidity is 68-72 percent to form primordium, keeping the temperature at 20-23 ℃ after a large amount of primordium is formed, gradually increasing the relative air humidity to 85-90 percent, and prolonging the illumination time to 15 hours; continuing culturing for 1-3 days until the top of the primordium begins to appear a cone-shaped sporocarp and the sporocarp enters the early growth stage; the temperature difference stimulation is divided into a daytime part and a nighttime part, the daytime temperature is 18-23 ℃, the illumination intensity is 50-100 Lx, and the time is 12-14h; the temperature at night is 5-8 ℃, and the time is 6-10h;

4) The fruiting body is grown in the early stage: the fruiting body in the early growth stage is orange yellow and 1-2 cm high, and is irradiated by scattered light or a fluorescent lamp 500Lx for 18h every day in an environment with the temperature of 22-25 ℃ and the air relative humidity of 85-90%, the illumination is discontinued, the fruiting body is cultured for 20-30 days, the fruiting body is 2-4 cm high and is in a long cone shape, and then the fruiting body can be transferred to the later growth stage;

5) And (3) fruiting body growth later stage: culturing for 10-20 days under the conditions that the temperature is 20-23 ℃, the relative air humidity is 85% -90%, the illumination intensity is 1000Lx, and the illumination time is 18h every day until the sporocarp is not thickened and increased, the head part begins to expand, the sporocarp is in a strip shape, grows robustly, is bright orange yellow and is good in growth;

step four harvesting and storing

1) Picking fruit bodies: the fruiting body of Cordyceps militaris is orange, the head is enlarged and has an ascocarp shell, and yellow powder appears on the surface, and the fruiting body is mature and picked in time; the collected culture medium can be recycled for secondary cultivation;

2) And (3) drying and storing the sporocarps: the picked cordyceps militaris sporocarp is freeze-dried and then stored in a dry and cool place.

The preparation method of the PDA solid culture medium comprises the following steps: collecting 37g of Potato glucose Agar (i.e. Potato Dextrose Agar) and 5g of silkworm chrysalis powder, adding 800mL of purified water, heating to dissolve, stirring, adding water to 1L, subpackaging as required, and sterilizing at 121 deg.C for 30min for use.

The preparation method of the culture medium adopted by the strain purification comprises the following steps: collecting 37g of potato glucose agar (PDA), adding 1L of water, heating to dissolve, stirring thoroughly, sterilizing at 121 deg.C for 30min for use.

The preparation method of the liquid culture medium comprises the following steps: collecting glucose 20g, peptone 5g, yeast extract 5g, and MgSO ₄ 1g, 1.5g potassium dihydrogen phosphate, adding purified water to 1L, stirring to dissolve completely, packaging, and sterilizing at 121 deg.C for 30min.

The preparation method of the strain culture medium comprises the following steps: taking 8kg of silkworm chrysalis, 12kg of rice, 20kg of purified water, 30g of monopotassium phosphate and 10g of sodium dihydrogen phosphate for later use, grinding the silkworm chrysalis and the rice into powder, uniformly mixing, adding the purified water containing the monopotassium phosphate and the sodium dihydrogen phosphate, fully stirring the homogenate until the pH value is 6-6.5, and sterilizing at 121 ℃ for 30min.

In the strain preparation, in the liquid culture process, if a bacterial colony is turbid and does not have flocculent appearance, the strain is polluted, and the bacterial colony needs to be immediately placed into an autoclave for sterilization so as to avoid expanded pollution.

The temperature of the dark culture is 15-18 ℃, and the relative humidity of air is 65%; in the culture process, a polluted culture medium is cleaned in time, for example, hyphae obviously contain mixed bacterial spots such as penicillium, saccharomycetes and the like or grow badly, and the culture medium is rotten to be in a water stain shape and has peculiar smell and the like.

The strain is cultured in a differentiation primordial stage, and ventilation is carried out for 15-20 min in the morning, in the middle and at night or for 30min in the morning and at night every day during the culture period.

In the early growth stage of the sporocarp, considering that the cordyceps militaris sporocarp has phototaxis, the irradiation direction of a light source is adjusted to ensure that the sporocarp keeps growing upwards and vertically.

The quality identification method of the culturable strains with qualified quality comprises the following steps: inoculating the amplified bacterial liquid on a newly prepared sterile culture medium in an ultra-clean workbench, wherein the amount of the inoculated bacterial liquid is 2 mL/bottle, inoculating 10-20 bottles each time, performing dark culture in an incubator with the temperature of 22 ℃ and the humidity of 65%, sealing the bacterial liquid after each inoculation, and storing the bacterial liquid in a refrigerator with the temperature of-20 ℃; observing the growth condition of the strain after 3 days, and determining the batch of bacterial liquid as qualified bacterial liquid after determining that the strain grows well and no other mixed bacteria grow, so that the strain can be used for production inoculation; the qualified bacteria liquid is characterized in that hyphae are snow white, uniform, dense, strong in penetrating power and free of peculiar smell, and the growth of bacterial colonies is larger than 1/2 area of the plane of a culture medium.

The second object of the present invention is to provide: an ergosterol compound in the fruiting body of Cordyceps militaris and Hainin can specifically regulate estradiol metabolism of cancer cells and GAPDH expression of cancer cells, thereby realizing the application of cancer diagnosis or treatment in the preparation of medicines.

The third object of the present invention is to provide: an application of ergosterol compound in fruiting body of Cordyceps militaris (L.) Link in inhibiting cancer cell growth by specifically regulating estradiol (E2) in cancer cell and regulating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is disclosed.

A fourth object of the present invention is to provide: an application of ergosterol compounds in fruiting body of Cordyceps militaris and Hainin strain in regulating E2 metabolism in lung cancer cell is provided.

A fifth object of the present invention is to provide: an application of ergosterol compounds in fruiting body of Cordyceps militaris (L.) Link in up-regulating GAPDH expression in hepatocarcinoma cell is provided.

The ergosterol compound has a molecular formula of C ₂₈ H ₄₄ O, specifically named (3R, 9R,10S,13S,14S, 17S) -17- ((2S, 5R, E) -5,6-dimethylhept-3-en-2-yl) -10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta [ a]Phenanthrene-3-ol, abbreviated as "DI", has the following chemical structural formula:

a method for extracting DI from Cordyceps militaris fruiting body comprises the following steps:

s1, crushing: coarse crushing the dry silkworm cordyceps militaris and hening plant fruiting body into 40 meshes, and then carrying out ultrafine crushing into 500 meshes to obtain powder;

s2, extraction: uniformly stirring the powder and water according to the mass ratio of 1 to 12, soaking for 24h, extracting for 3h at 90 ℃, cooling to room temperature, performing centrifugal separation, adding a precipitate into a 12-time 95% ethanol aqueous solution, leaching for 24h, performing centrifugal separation to extract a supernatant, concentrating to remove ethanol, and performing freeze drying to constant weight to obtain a dried substance;

s3, purification: dissolving the dried product with 100% ethanol to 100mg/mL, separating with 0.25 μm microporous membrane by chromatography, collecting 25.4-30min components, concentrating to remove the eluting solvent, and drying at high temperature;

s4, crystallization: adding a good solvent into the purified product, fully dissolving, naturally volatilizing at 26 ℃, keeping the temperature and standing, carrying out suction filtration on the obtained crystal, and cleaning with n-hexane at 4 ℃ to obtain a single crystal; the good solvent is a mixed solvent of n-hexane and dichloromethane.

The chromatographic column for chromatographic separation is DAC (150 × 250mm C18 μm), the ultraviolet detection wavelength is 282nm, and the flow rate is 600 ml/min ^-1 (ii) a The elution mode is that 98% methanol water solution is eluted for 40min isocratically.

Has the beneficial effects that:

the invention takes the silkworm chrysalis cordyceps sinensis tannin strain as a strain, obtains a culture mother strain after tissue rejuvenation, further prepares a culture bacterial solution through solid culture to liquid amplification, and obtains the cordyceps militaris with higher cordycepin, N6- (2-hydroxyethyl) adenosine and ergosterol contents in sporocarp after inoculation and culture; the culture method has the advantages of short production period, high production efficiency, realization of large-scale production, simple and feasible production process, easy control of parameters and capability of obtaining effective components with good activity.

Because the strains in the centrifugal tube are directly picked for strain expansion preparation by the traditional method, the strains in the centrifugal tube cannot be judged whether to be polluted by naked eyes, which increases the risk of contamination to subsequent culture; the invention adopts the condition that the growth vigor of the hyphae on the flat plate, whether other mixed bacteria are polluted or not and the like is directly observed by naked eyes, thereby preliminarily judging the vitality of the strains and having the advantage of controlling the pollution from the source.

The extraction method is simple, the operation is easy, the parameters are easy to control, and effective components with good activity can be obtained, the effective components can play a role in resisting lung cancer by participating in regulating the metabolism of estradiol, and participate in regulating and controlling GAPDH expression to play a role in resisting liver cancer, so that better activities of resisting liver cancer and resisting lung cancer are displayed; according to the invention, the main active ingredients of the cornsterol compounds in the cultured cordyceps militaris and henning plant sporocarp exert the anti-tumor effect are found by specifically regulating the relationship between estradiol and glyceraldehyde-3-phosphate dehydrogenase in lung cancer cells and liver cancer cells, so that the anti-tumor active ingredients of the cordyceps militaris and henning plant sporocarp realize an anti-lung cancer application mechanism of the anti-tumor active ingredients of the cordyceps militaris and henning plant by regulating the estradiol metabolism of the lung cancer cells and an anti-liver cancer application mechanism of regulating the expression of glyceraldehyde-3-phosphate dehydrogenase in the liver cancer cells, the application basis of the cordyceps militaris and henning plant sporocarp and the preparation for preparing lung cancer, liver cancer diagnosis preparations and treatment preparations is laid, and a path is provided for exploring the generation mechanism of the lung cancer and the liver cancer.

Drawings

FIG. 1 is an HPLC chromatogram for detecting the content of cordycepin and N6- (2-hydroxyethyl) adenosine in the dry product of the Chinese caterpillar fungus Haining plant fruiting body in the experimental example 1;

FIG. 2 is an HPLC chromatogram for detecting the ergosterol content in the dry product of the fruiting body of the Hainin plant of Cordyceps militaris in Experimental example 1;

FIG. 3 is an ESI-MS spectrum of cordycepin in the dry product of the Cordyceps militaris Haining plant fruiting body in Experimental example 1;

FIG. 4 is an ESI-MS spectrum of N6- (2-hydroxyethyl) gland in a dry product of a cordyceps militaris Haining plant fruiting body in Experimental example 1;

FIG. 5 is an ESI-MS spectrum of ergosterol in the dried product of the fruiting body of Chinese caterpillar fungus Haining plant in Experimental example 1;

FIG. 6 is the structural formula of the extract DI of the plant fruiting body of Cordyceps militaris (L.) Link of example 2;

FIG. 7 is an ESI-MS spectrum of the second extract of the plant of Chinese caterpillar fungus Hainin of example 2;

FIG. 8 shows example 2 of extract DI from the fruit body of Chinese caterpillar fungus Hainin plant ¹ H NMR spectrum;

FIG. 9 shows example 2 of extract DI from the fruit body of Chinese caterpillar fungus Hainin plant ¹³ C NMR spectrum;

FIG. 10 is DEPT NMR spectrum of the extract DI of the plant fruiting body of Cordyceps militaris (L.) Link of example 2;

FIG. 11 is the HSQC NMR spectra of the extract DI from the plant entity of the Cordyceps militaris Hainin plant of example 2;

FIG. 12 is the HMBC NMR spectrum of the extract DI of the fruiting body of the Cordyceps militaris Hainin plant of example 2.

Detailed Description

The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.

Example 1

A large-scale production method of Cordyceps militaris comprises the following steps:

first step Medium preparation

The preparation method of the PDA solid culture medium comprises the following steps: adding 800mL of purified water into 37g of PDA and 5g of silkworm chrysalis powder, heating and dissolving, uniformly stirring, supplementing water to 1L, subpackaging according to requirements, and sterilizing at 121 ℃ for 30min for later use;

the preparation method of the culture medium adopted by the strain purification comprises the following steps: heating and dissolving 37g PDA in 1L water, stirring thoroughly, packaging as required, sterilizing at 121 deg.C for 30min;

the preparation method of the liquid culture medium comprises the following steps: collecting glucose 20g, peptone 5g, yeast extract 5g, and MgSO ₄ 1g, 1.5g of monopotassium phosphate, adding purified water to 1L, stirring, fully dissolving, subpackaging, sterilizing at 121 ℃ for 30min for later use;

the preparation method of the strain culture medium comprises the following steps: taking 8kg of silkworm chrysalis, 12kg of rice, 20kg of purified water, 30g of monopotassium phosphate and 10g of sodium dihydrogen phosphate for later use, grinding the silkworm chrysalis and the rice into powder, uniformly mixing, adding the purified water containing the monopotassium phosphate and the sodium dihydrogen phosphate, and fully stirring the homogenate until the pH value is 6-6.5;

the second step of breeding the family silkworm chrysalis cordyceps sinensis Hainin strain C.militaris (HN)

1) Seed selection: selecting the silkworm chrysalis cordyceps sinensis tannin plant fruiting bodies which are robust in growth, have no condensed water or condensed water, are clear and transparent in water drop and have no white aerial hyphae at the root and grow to be 2-3 cm high;

2) Culturing strains: in a sterile environment, using a sterile inoculating hook to hook off the root of a sporocarp, taking the sporocarp out, placing the sporocarp on sterilized gauze, using sterile forceps to clamp the root of the sporocarp, sequentially wiping the surface of the sporocarp by 75 percent alcohol, cleaning residual alcohol on the surface of the sporocarp by sterile water, removing the epidermis of the sporocarp by a sterile scalpel, selecting a part with the length of 1-1.5 cm at the top of the sporocarp, dividing the part into 3-5 granules per 1cm, using the sterile inoculating hook to place the small granules of the sporocarp on a PDA solid culture medium, wherein the interval of the granules of the sporocarp is about 3.5cm, and carrying out shading inversion culture in a biochemical incubator at the temperature of 22 ℃ until hyphae appear;

3) And (3) strain purification: placing the slant of the mother seed test tube in a workbench in a sterile environment, disinfecting both hands, entering the workbench, igniting an alcohol lamp, wiping an inoculating loop with an alcohol cotton ball before using, burning the inoculating loop with the part of the inoculating handle together, opening the slant test tube opening after sterilizing, picking a ring of strains from the slant (mother seeds) by using the inoculating loop, quickly marking the strains on the inoculating loop on a flat plate, and inversely culturing in a biochemical incubator at 21-23 ℃ until the height of the strains is about 0.5cm;

third step of strain preparation

Strain amplification; according to the proportion that the inoculation amount of each 120mL of liquid culture medium is 1cm multiplied by 1cm, a single bacterial colony which is obtained by purification and has the quality reaching the standard is picked by an aseptic scalpel and cut up, the single bacterial colony is quickly transferred into the liquid culture medium in a conical flask, the bacterial colony is evenly distributed by slight shaking, the mouth of the flask is tightened, the bacterial colony is cultured in a shaking table at 22 ℃ and 170rpm/min in a dark place, the obtained bacterial liquid is inoculated into a solid culture medium to test the quality of the bacterial colony, the hypha is snow white, even, dense, strong in penetrating power and free from peculiar smell, the bacterial colony grows more than 1/2 area of the plane of the culture medium, and the culturable bacterial colony with qualified quality is obtained; in the liquid culture process, if the bacterial colony is turbid and does not appear flocculent, the strain is polluted and needs to be immediately placed into an autoclave for sterilization so as to avoid expanding pollution;

the fourth step of cultivation

1) Preparing a culture flask: the culture bottle adopts transparent high-temperature and high-pressure resistant glass or a pv material as a production container, the container is circular and consists of three parts, namely a base, a culture medium fall-preventing plate and a bottle body, the diameter of the bottle is 6.5cm, the total height of the bottle is 12cm, the base is 3cm, the fall-preventing plate is a concentric circle with the outer diameter of 6.5cm and the inner diameter of 5cm, and the height of the bottle body is 9cm; the base, the fall-preventing plate and the bottle body are detachable, small vent holes are formed in the top of the bottle, air circulation is facilitated, and germ pollution can be isolated, a full-automatic bottle washing and filling integrated machine is adopted, each bottle of strain cultivation culture medium is about 2cm high and can not exceed 3cm, and high-pressure sterilization is carried out at 121 ℃ for 30min in an automatic damp-heat sterilization cabinet;

2) Initial culture of spawn running: adopting a full-automatic integrated machine of uncovering, inoculating and covering, quickly injecting 2mL of bacterial liquid into a culture bottle, then automatically covering, culturing in the dark for 3-5 days under the conditions that the temperature is 15-18 ℃ and the relative air humidity is 63-68%, keeping hyphae in a snowy white color, uniformly, densely, highly penetrating and odorless, continuously culturing until the hyphae all overgrow the whole culture medium, and entering into the light culture strain to culture in the differentiation early stage when a plurality of hillock-shaped bulges with different quantities and sizes are generated on the material surface; in the culture process, a polluted culture medium is cleaned in time, for example, hyphae obviously contain mixed bacterial spots or poor growth of penicillium, saccharomycetes and the like, and the culture medium is rotten to be in a water stain shape and has peculiar smell and the like.

3) Strain differentiation early stage culture: slowly raising the temperature to 18-23 ℃ and the relative air humidity to be more than or equal to 85%, and performing light culture for 3-10 days under the condition that the illumination intensity is set to be 200-250 Lx, and continuing to culture for 1-3 days after hypha on the material surface of the culture medium gradually changes from white to orange, so as to enter the original culture period; the light is irradiated for more than 12 hours every day in the light culture process, but the light cannot be continuously irradiated, so that the dark culture for 6 hours every day is ensured, and the direct irradiation of sunlight is prevented;

4) Strain differentiation primordial stage culture: culturing for 10 days by adopting temperature difference stimulation under the condition that the relative air humidity is 68-72 percent to form primordium, keeping the temperature at 20-23 ℃ after a large amount of primordium is formed, gradually increasing the relative air humidity to 85-90 percent, and prolonging the illumination time to 15 hours; continuing culturing for 1-3 days until the top of the primordium begins to appear a cone-shaped sporocarp and the sporocarp enters the early growth stage; the temperature difference stimulation is divided into a daytime part and a nighttime part, the daytime temperature is 18-23 ℃, the illumination intensity is 50-100 Lx, and the time is 12-14h; the temperature at night is 5-8 ℃, and the time is 6-10h; in the culture process, ventilation is respectively carried out for 15-20 min in the morning, in the middle of the day and at night or for 30min in the morning and at night, parameters need to be strictly controlled, and if the illumination is too strong or the ventilation is insufficient, the phenomena that primordium is densely differentiated and fungus quilts are formed but stroma is not grown easily occur; if the humidity is too high, the phenomenon that the formation of the stroma is influenced by the excessive growth of aerial hyphae appears;

5) The fruiting body is grown in the early stage: the fruiting body is orange yellow at the early growth stage and is 1-2 cm high, the fruiting body is placed in an environment with the temperature of 22-25 ℃ and the relative air humidity of 85-90%, scattered light or fluorescent lamp 500Lx is adopted to irradiate for 18 hours every day, discontinuous illumination is carried out, the fruiting body is cultured for 20-30 days, the fruiting body is 2-4 cm high and is in a long cone shape, and then the fruiting body can be transferred to the later growth stage; in the culture process, the irradiation direction of the light source is adjusted to ensure that the fruiting body grows upwards in consideration of phototaxis of the fruiting body of Cordyceps militaris;

6) And (3) fruiting body growth later stage: culturing for 10-20 days under the conditions that the temperature is 20-23 ℃, the relative air humidity is 85% -90%, the illumination intensity is 1000Lx, and the illumination time is 18h every day until the sporocarp is not thickened and increased, the head part begins to expand, the sporocarp is in a strip shape, grows robustly, is bright orange yellow and is good in growth;

the fifth step of harvesting and storing

1) Picking fruit bodies: the cordyceps militaris fruiting body is orange, the head is expanded and has a seed capsule shell, the surface is mature when yellow powder appears, and the cordyceps militaris fruiting body is picked in time by adopting a full-automatic cover opening, inverted picking and culture medium cleaning integrated machine; the collected culture medium can be repeatedly used for secondary cultivation;

2) And (3) drying and storing the sporocarps: freeze drying the picked Cordyceps militaris fruiting body, and storing in dry and cool place;

in the embodiment, 1064kg of silkworm chrysalis and 1596kg of rice are put into fruit bodies of silkworm chrysalis (C.militaris HN), and after optimized cultivation, the fruit bodies are put into 75050 bottles on a shelf, and the production cycle is less than 60 days, so that 158kg of dried fruit bodies of silkworm chrysalis can be obtained totally. The production is based on controlling pollution from a source, meanwhile, effective control is carried out on the whole cultivation process for cooperation, the culture medium sporocarp grows and differentiates faster through good control on the cultivation environment, small buds of the sporocarp can grow out in culture bottles on shelves, the obtained cordyceps militaris sporocarp is uniform in color and orange, the cultivation period can be shortened by about 20-30 days, the pollution rate in the cultivation process is less than 1%, about 12g of fresh cordyceps militaris sporocarp can be obtained in each bottle, the cultivation efficiency is improved by more than 20%, the target components of cordycepin, N6- (2-hydroxyethyl) adenosine and ergosterol in the sporocarp are high, and the ergosterol compound which is good in anti-tumor activity and has a lung cancer and liver cancer differential application mechanism is obtained. The method can obtain the cordyceps militaris with high quality and high yield.

Experimental example 1

In the experimental example, the quality of the fruiting bodies of the silkworm cordyceps militaris and the henning plant is preliminarily judged by detecting the contents of cordycepin, N6- (2-hydroxyethyl) adenosine and ergosterol in the fruiting bodies of the cordyceps militaris from several sources; the number of the cultivated silkworm cordyceps militaris Haining plant fruiting body produced in the embodiment is 'A', the number of the Tibetan cordyceps militaris fruiting body is 'B', the number of the Liaoning cordyceps militaris fruiting body is 'C';

1) Ergosterol assay

Preparation of a test solution: precisely weighing 1.010g of A, 1.014g of B and 1.017g of C, respectively adding 50ml of 95% ethanol, refluxing in a constant temperature water bath at 85 ℃ for 1h, filtering, and temporarily storing the filtrate; extracting the residue with the above method for 2 times, mixing filtrates, concentrating under reduced pressure to less than 10mL (no more than 60 deg.C), diluting with ethanol to 10mL, filtering with membrane, and measuring the filtrate;

preparation of control solutions: accurately weighing 5.2mg ergosterol standard, dissolving with ethanol and diluting to 25mL to obtain 0.20592mg/mL standard control solution;

chromatographic conditions are as follows: a chromatographic column: c18 column (250 mm. Times.4.6 mm 5 μm, GRACE), mobile phase: 98% methanol-water, flow rate: 1mL/min, column temperature: 25 ℃, sample introduction: 20 μ L, ultraviolet detection wavelength: 282nm;

2) Detection of cordycepin and N6- (2-hydroxyethyl) adenosine components

Preparing a test sample: collecting oven-dried Cordyceps militaris sporophore1.013g of material A, 1.015g of material B and 1.012g of material C are crushed at ultralow temperature; adding appropriate amount of ultrapure water into the superfine powder, heating and extracting for 3 times, each for 3 hr, mixing extractive solutions, and concentrating. Dispersing with appropriate amount of water, adding 95% ethanol to reach a concentration of 80%, mixing, precipitating in a refrigerator at 4 deg.C overnight, collecting supernatant, concentrating, drying, and weighing; adding 15% methanol-water to dissolve to a concentration of 200 mg/mL ^-1 Filtering with microporous membrane, and separating with high performance liquid chromatography;

chromatographic conditions are as follows: a chromatographic column: c18 (250 mm. Times.4.6 mm 5 μm, GRACE), mobile phase: water-methanol (volume ratio 85: 260nm; sample introduction amount: 20 μ L column temperature: 25 ℃;

3) Content comparison calculation

The area normalization method and the external standard method have the following formulas:

the cordycepin and N6- (-2 hydroxyethyl) adenosine contents (%) = target peak area/total peak area x 100%;

ergosterol content (%) = target peak area/control peak area × control concentration/sample concentration × dilution factor × 100%;

4) Results

As can be seen from the figure 1 and the figure 2, the HPLC method is adopted to carry out comparative analysis with cordycepin, N6- (2-hydroxyethyl) adenosine and ergosterol in cordyceps militaris from other two sources, and the mass spectrum identification results of cordycepin, N6- (2-hydroxyethyl) adenosine and ergosterol are used as auxiliary materials to confirm, based on the HPLC peak area preliminary measurement, the quality of the home silkworm chrysalis cordyceps militaris produced in the example 1 is relatively best, the cordycepin content is respectively 4.00 times of the fruit body of Tibetan cordyceps militaris and 2.30 times of the fruit body of Liaoning cordyceps militaris, the N6- (2-hydroxyethyl) adenosine content is respectively 1.05 times of the fruit body of Tibetan cordyceps militaris and 2.40 times of the fruit body of Liaoning cordyceps militaris, and the ergosterol content is respectively 5.60 times of the fruit body of Tibetan cordyceps militaris and 1.80 times of the fruit body of Liaoning cordyceps militaris.

Example 2

A method for extracting (3R, 9R,10S,13S,14S, 17S) -17- ((2S, 5R, E) -5,6-dimethylhept-3-en-2-yl) -10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta [ a ] phenanthren-3-ol (short for DI) from cordyceps militaris comprises the following steps:

s1, crushing: coarsely pulverizing dry plant fruiting body of silkworm Cordyceps militaris and Hainin to 40 mesh, and micronizing to 500 mesh to obtain powder;

s2, extraction: uniformly stirring the powder and water according to the mass ratio of 1 to 12, soaking for 24h, extracting for 3h at 90 ℃, cooling to room temperature, performing centrifugal separation, adding a precipitate into a 12-time 95% ethanol aqueous solution, leaching for 24h, performing centrifugal separation to extract a supernatant, concentrating to remove ethanol, and performing freeze drying to constant weight to obtain a dried substance;

s3, purification: dissolving the dried product with 100% ethanol to 100mg/mL, separating with 0.25 μm microporous membrane by chromatography, collecting 25.4-30min components, concentrating to remove the eluting solvent, and drying at high temperature;

s4, crystallization: adding the purified product into a mixed solvent of n-hexane and dichloromethane in a certain proportion, fully dissolving, placing at 26 ℃ for natural volatilization, keeping the temperature and standing, carrying out suction filtration on the obtained crystal, and cleaning with 4 ℃ n-hexane to obtain a single crystal;

the chromatographic column for chromatographic separation is DAC (150 × 250mm C18 μm), the ultraviolet detection wavelength is 282nm, and the flow rate is 600 ml/min ^-1 . The elution mode is that 98% methanol water solution is eluted for 40min isocratically;

measuring the product obtained in the step S4 by using an X single crystal diffractometer;

as a result: ergosterol compounds in the molecular entities of Hainin plants of Cordyceps militaris of Bombyx mori are (3R, 9R,10S,13S,14S, 17S) -17- ((2S, 5R, E) -5,6-dimethylhept-3-en-2-yl) -10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta [ a]Phenanthrene-3-ol, its structural formula is shown in figure 3, its ESI-MS spectrum is shown in figure 7, ¹ the H NMR spectrum is shown in figure 8, ¹³ the C NMR spectrum is shown in FIG. 9, the DEPT NMR spectrum is shown in FIG. 10, the HSQC NMR spectrum is shown in FIG. 11, and the HMBC NMR spectrum is shown in FIG. 12.

Example 3

A specific application mechanism of ergosterol compounds in the fruiting bodies of silkworm pupa cordyceps sinensis and quinine plants in resisting lung cancer and liver cancer; specifically, the method comprises the following steps: an application of ergosterol compounds in fruiting body of Cordyceps militaris (L.) Link in inhibiting cancer cell growth by specifically regulating estradiol (E2) in cancer cell and regulating glyceraldehyde-3-phosphate dehydrogenase (GAPDH);

the ergosterol compound has a molecular formula of C ₂₈ H ₄₄ O, specifically named (3R, 9R,10S,13S,14S, 17S) -17- ((2S, 5R, E) -5,6-dimethylhept-3-en-2-yl) -10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta [ a]Phenanthrene-3-ol, referred to as DI, and its chemical structural formula is shown in FIG. 6;

the specific regulation is as follows: after the lung cancer cells are treated by DI, the content of E2 in the lung cancer cells is increased, and the content of GAPDH is not obviously changed; after the liver cancer cells are treated by DI, the E2 content in the liver cancer cells has no obvious change, but the GAPDH content is increased; by utilizing the specificity, the application of DI in the treatment of lung cancer and liver cancer can be realized, and the DI can be further used for preparing a preparation for regulating estradiol or estrogen metabolism in lung cancer cells and a preparation for regulating GAPDH or energy metabolism in liver cancer cells, wherein the preparations comprise but are not limited to tablets, capsules, powders, granules, pills, solutions, suspensions, syrups, oral lozenges, sublingual lozenges, injections, ointments, suppositories, inhalants and other various dosage forms.

Example 4

Example 2 application of the extracted effective component DI in regulating E2 metabolism in cancer cells and GAPDH expression in cancer cells;

relationship of DI to E2 and GAPDH verifies:

1. investigation of DI on lung cancer and liver cancer cell inhibitory concentration

1) The human lung cancer A549 cells and liver cancer HepG2 cells were recovered by a conventional method, and the harvested cells were subjected to A5% CO assay at 37 ℃ in a DMEM high-glucose complete medium containing 10% FBS ₂ Standing and culturing in a constant-temperature incubator; when the cells grow to 80% -90% and are fused, carrying out pancreatin digestion and passage by 0.25% -EDTA;

2) MTT method for detecting cell proliferation

(1) Taking A549 and HepG2 cells in logarithmic growth phase at 1X 10 ⁴ Inoculating each well separatelyAdding 100 mu L of the mixture into each hole of a 96-hole plate respectively; after the cells adhere to the wall, changing to the action medicine with corresponding concentration (plating in the afternoon of the previous day, adding medicine in the morning of the next day), continuing culturing for 24h, 48h and 72h, and observing under an inverted microscope;

(2) setting a zero-adjusting group, a blank control group and a DI group (10, 25, 50, 75 and 100 mu g/mL), wherein each group is provided with 3 multiple wells; wherein, the zero-adjusting group is a hollow hole, and the cell group is not added with any substance in the hole; the blank control group is prepared by not adding DI in the fruiting body of the silkworm pupa cordyceps sinensis, and the rest steps are the same as those of the DI group;

(3) after the culture is finished, removing the culture medium, adding 100 mu L of MTT solution (the final concentration is 0.5 mg/mL) into each hole, incubating for 4h at 37 ℃, discarding the MTT solution, carefully flushing for 2-3 times by PBS, adding 200 mu L of DMSO into each hole, shaking for 10min at room temperature, uniformly mixing to fully dissolve crystals, and measuring the absorbance by a 490nm full-automatic enzyme standard instrument. The proliferation inhibition rate was calculated as follows;

proliferation inhibition (%) = (OD blank-ODDI group)/(OD blank-OD zeroing group) × 100%;

2. cell drug treatment

1) Cell culture and plating

2) Adding medicine: after culturing the cells in a 12-well plate for 24h, setting a blank control group, adding medicines according to the sample concentration of 50 mu g/mL and 75 mu g/mL, repeating two multiple wells for each concentration, and culturing for 72h;

3. cell extract-repeated freezing and thawing method

1) Sucking out the culture medium in the culture plate, digesting the cells by trypsin, and then adding a proper amount of culture medium to wash the cells on the culture plate;

2) Collecting the cell suspension in a centrifuge tube, centrifuging at 2-8 deg.C at 1000 Xg for 5min, removing the culture medium by suction, and washing the cells with precooled PBS for 3 times;

3) Adding a proper amount of precooled PBS for resuspending the cells; culture plates in 12 wells (about 1X 10) ⁶ Individual cells), 70-150 μ L PBS was added to each well to resuspend the cells;

4) Repeatedly freezing and thawing the sample at-20 deg.C or-80 deg.C and room temperature, and repeating the freezing and thawing process for several times (at least 3 times) until the cells are completely lysed; the suspension may also be sonicated using a sonicator to lyse the cells;

5) Centrifuging at 1500 × g at 2-8 deg.C for 10min, removing cell debris, collecting supernatant, and storing at-20 deg.C or-80 deg.C to avoid repeated freeze thawing.

4. Detection by ELISA method

Estradiol (E2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ELISA kits were purchased from Wuhan Gene Mei Biotech, inc., and the E2 and GAPDH contents were detected according to the procedures of the kits.

1) Dilution and sample loading of standard: setting 10 holes of a standard substance hole on an enzyme-labeled coating plate, respectively adding 100 mu L of a standard substance into the first hole and the second hole, then adding 50 mu L of a standard substance diluent into the first hole and the second hole, and uniformly mixing; then respectively adding 100 mu L of the diluent from the first hole and the second hole into the third hole and the fourth hole, respectively adding 50 mu L of the standard substance diluent into the third hole and the fourth hole, and uniformly mixing; then respectively taking 50 mu L of the diluent in the third hole and the fourth hole and discarding, respectively taking 50 mu L of the diluent in the fifth hole and the sixth hole, respectively adding 50 mu L of the diluent in the fifth hole and the sixth hole, and uniformly mixing; respectively taking 50 mu L of the diluent from the fifth hole and the sixth hole after uniformly mixing, respectively adding the diluent into the seventh hole and the eighth hole, respectively adding 50 mu L of the standard substance diluent into the seventh hole and the eighth hole, respectively taking 50 mu L of the diluent from the seventh hole and the eighth hole after uniformly mixing, respectively adding the diluent into the ninth hole and the tenth hole, respectively adding 50 mu L of the standard substance diluent into the ninth hole and the tenth hole, respectively taking 50 mu L of the diluent from the ninth hole and the tenth hole after uniformly mixing, and discarding; (lung cancer concentration gradient E2:0pg/mL, 31.25pg/mL, 62.5pg/mL, 125pg/mL, 250pg/mL, 500pg/mL; GAPDH:0pg/mL, 5pg/mL, 10pg/mL, 20pg/mL, 40pg/mL, 80pg/mL; liver cancer concentration gradient: 0pg/mL, 15pg/mL, 30pg/mL, 60pg/mL, 120pg/mL, 180 pg/mL);

2) Sample adding of a sample to be tested: respectively arranging a blank reference hole and a sample hole to be detected; adding 40 mu L of sample diluent into a sample hole to be detected on the enzyme-labeled coated plate, and then adding 10 mu L of sample to be detected (the final dilution of the sample is 5 times); adding a sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;

sample preparation: acting different concentrations of DI (50 μ g/mL, 75 μ g/mL) on lung cancer and liver cancer cells, and collecting cell extractive solutions after 72h;

setting a group: blank control group, zero adjustment group, 50 μ g/mL, 75 μ g/mL, each concentration repeat two more wells;

3) Adding an enzyme: adding 50 mu L of enzyme-labeled reagent into each hole except for blank holes;

4) And (3) incubation: sealing the plate with sealing plate film, and incubating at 37 deg.C for 30min;

5) Preparing liquid: diluting 20 times of the concentrated washing liquid with 20 times of distilled water for later use;

6) Washing: carefully uncovering the sealing plate film, discarding liquid, patting dry on absorbent paper, filling each hole with washing liquid (350 mu L), standing for 30s, discarding the washing liquid, patting dry on the absorbent paper, and washing the plate for 5 times in this way;

7) Color development: adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, shaking gently, mixing, incubating at 37 deg.C in dark for 10min;

8) And (4) terminating: adding 50 mu L of stop solution into each well to stop the reaction (at the moment, the blue color immediately turns to yellow);

9) And (3) determination: using blank hole zero setting group, measuring absorbance (OD value) of each hole in sequence at the wavelength of 450 nm; the determination should be carried out within 15min after the addition of the stop solution;

5. calculation of the results of the experiment

Drawing a standard curve: in an Excel worksheet, taking the concentration of a standard substance as an abscissa and taking a corresponding OD value as an ordinate, drawing a linear regression curve of the standard substance, substituting the OD value of a sample into an equation, calculating the concentration of the sample, and multiplying the concentration by a dilution multiple to obtain the actual concentration of the sample;

6. results and analysis

1 finding the optimum concentration of DI acting corresponding to the action by the calculated inhibition ratio

1) Effect of different concentrations of DI on proliferation of Lung cancer and liver cancer cells

2 standard curve

1) Spectrophotometric value (OD 450) (Lung cancer cell treatment)

2) Spectrophotometric value (OD 450) (liver cancer cell treatment)

	0pg/mL	15pg/mL	30pg/mL	60pg/mL	120pg/mL	180pg/mL
							E2	0.082	0.124	0.16	0.294	0.45	0.635
GAPDH	0.253	0.291	0.379	0.533	0.781	0.993

3 content detection

Selecting two concentrations of 50 mug/mL and 75 mug/mL which have good cell state and DI inhibition cancer cell growth activity from low to high, and carrying out content detection on the two concentrations and a blank control group. The inhibition rates of 50 mu g/mL and 75 mu g/mL lung cancer cells A549 are 63.35 percent and 67.73 percent respectively, and the inhibition rates of 50 mu g/mL and 75 mu g/mL liver cancer cells HepG2 are 57.88 percent and 82.28 percent respectively.

1) Elisa test of the spectrophotometric value (OD 450) of the cell sample after drug treatment

2) Calculated sample concentration

Thus, it can be seen that: the inhibition of DI on the growth of cancer cells is higher than that of 10 mug/mL, the inhibition rate of the cancer cells is also higher as the concentration of DI is increased, and the difference is obvious.

After cancer cells are treated by DI in the sporocarp of the cordyceps militaris haining plant of different concentrations for 72 hours, the DI group is compared with a zero adjustment group and a blank control group, the content of E2 in the lung cancer cells is increased, the content of the liver cancer cells is not changed obviously, and the difference is obvious.

After cancer cells are treated by DI in the fruiting bodies of the silkworm cordyceps militaris Hainin plants with different concentrations for 72 hours, compared with a zero adjustment group and a blank control group, the DI group has the advantages that the content of GAPDH in lung cancer cells is not greatly changed, the content of GAPDH in liver cancer cells is obviously increased, and the difference is obvious.

Description of the drawings: DI in the silkworm cordyceps militaris and Hainin plant fruiting body can play a role in resisting lung cancer by participating in regulating the metabolism of estradiol, and the liver cancer resisting function can play a role in resisting liver cancer by regulating glyceraldehyde-3-phosphate dehydrogenase.

Experimental example 2

In order to test whether DI in the molecular entity of the silkworm chrysalis cordyceps sinensis tannin plant truly plays a role in resisting lung cancer by participating in the regulation of estradiol metabolism and regulates the function of glyceraldehyde-3-phosphate dehydrogenase in resisting liver cancer, a third-generation gene sequencing technology is adopted to sequence and assemble a human lung cancer cell A549 and liver cancer cell HepG2 complete transcription group, whether the estradiol 17 beta-dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase participating in estradiol metabolism have specific mutation in the original A549 and HepG2 is further analyzed, and the specificity mechanism of DI is verified on the gene and protein level;

1. method of producing a composite material

1 cancer cell whole transcriptome sequencing

Single molecule real-time sequencing of human Lung cancer cell A549 and liver cancer cell HepG2 transcriptome Using Pacific Bioscience (Pacific Bioscience) PacBio RS II sequencing System (

Sequencing); extracting total RNA according to a UNIQ-10 column type Trizol total RNA extraction kit; total RNA was sequenced according to Procedure and Checklist-Isoform Sequencing (Iso-Seq) ^TM )Using the

Constructing an SMRT library by using a PCR cDNA Synthesis Kit operation process; setting the relevant parameters of the upper computer by a calculating software Binding Call-master Version 2.3.1.1; push buttonPreparing on-machine Sequencing according to the Kit procedures of Pacifiaac Biosciences DNA/Polymerase Binding Kit P6v2, magBead Kit, DNA Sequencing Reagent 4.0 and the like; obtaining a high-quality full-length transcript through PacBio RS II single-molecule real-time sequencing;

2 analysis of enzyme mutation

Comparing the obtained transcriptome data of the human lung cancer cell A549 and the liver cancer cell HepG2 with 4729 enzymes related to human diseases discovered by research, extracting the enzymes with the homology rate of 50-97 percent and no less than 70 amino acids, further comparing the amino acid sequence of the enzymes with NCBI, selecting the mutated protein according to the comparison result of the NCBI, and further searching whether the mutated protein has the mutation of estradiol 17 beta-dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase;

2. results

1 mutation analysis of the enzyme estradiol 17 beta-dehydrogenase based on the sequencing of the human lung carcinoma cell A549 complete transcriptome

1.1 transcript statistics

Number of transcripts	45146
		Total length of transcript	59559kb
Maximum transcript Length	7497bp
		Minimum transcript Length	200bp
Average transcript Length	1328bp
		Average GC content	48.36％
ContigN50	1490bp

Comparing the transcriptome assembly results with 4729 enzymes, 2660 enzymes (of which, mitochondria 4, non-mitochondria 2656) were detected, 192 enzymes were found to have mutations;

1.2 mutation of estradiol 17 beta-dehydrogenase

1.3 Data comparing amino acid sequence of estradiol 17 beta-dehydrogenase of A549 cells with NCBI

>Query＝c22055/f2p2/2293

MARTVVLITGCSSGIGLHLAVRLASDPSQSFKGIDRQGQGGREGRSPWRLEGKSDLPPLPKPPVYATLRDLKTQGRLWEAARALACPPGSLETLQLDVRDSKSVAAARERVTEGRVDVLGEPPGSIWAPRSLLRPALKPTCSQAQGARGDRPC*g*c*gglvgplVSAVCNaglgllgplealgEDAVASVLDVNVVGTVRMLQAFLPDMKRRGSGRVLVTGSVGGLMG

PREDICTED:estradiol 17-beta-dehydrogenase 1 isoform X8[Homo sapiens]

Sequence ID:XP_011523034.1Length:180Numberof Matches:1

Related Information

Gene-associated gene details

Range 1:1 to 179GenPeptGraphicsNext MatchPrevious Match

Query 1

MARTVVLITGCSSGIGLHLAVRLASDPSQSFKGIDRQGQGGREGRSPWRLEGKSDLPPLP 60

MARTVVLITGCSSGIGLHLAVRLASDPSQSFKGIDRQGQGGREGRSPWREGKSDLPPLP

Sbjct 1

MARTVVLITGCSSGIGLHLAVRLASDPSQSFKGIDRQGQGGREGRSPWRPEGKSDLPPLP 60

Query 61

KPPVYATLRDLKTQGRLWEAARALACPPGSLETLQLDVRDSKSVAAARERVTEGRVDVLG 120

KPPVYATLRDLKTQGRLWEAARALACPPGSLETLQLDVRDSKSVAAARERVTEGRVDVL

Sbjct 61

KPPVYATLRDLKTQGRLWEAARALACPPGSLETLQLDVRDSKSVAAARERVTEGRVDVL-119

Query 121

EPPGSIWAPRSLLRPALKPTCSQAQGARGDRPC*G*C*GGLVGPLVSAVCNAGLGLLGPL 180

VCNAGLGLLGPL

Sbjct 120-----------------------------------------VCNAGLGLLGPL 131

Query 181

EALGEDAVASVLDVNVVGTVRMLQAFLPDMKRRGSGRVLVTGSVGGLM 228

EALGEDAVASVLDVNVVGTVRMLQAFLPDMKRRGSGRVLVTGSVGGLM

Sbjct 132

EALGEDAVASVLDVNVVGTVRMLQAFLPDMKRRGSGRVLVTGSVGGLM 179

2 mutation analysis of glyceraldehyde-3-phosphate dehydrogenase based on HepG2 Whole transcriptome sequencing of human Lung cancer cells

HepG2 sequencing results were as follows:

2.1 transcript Assembly statistics

Number of transcripts	46512
		Total length of transcript	69890247kb
Maximum transcript Length	8656bp
		Minimum transcript Length	203bp
Average transcript Length	1502bp
		Average GC content	48.37％
ContigN50	1651bp

Comparing the transcriptome assembly results with 4729 enzymes, 2660 enzymes (of them, 4 mitochondria and 2656 non-mitochondria) were detected, and 340 enzymes were found to have mutations.

2.2 mutation of glyceraldehyde-3-phosphate dehydrogenase

2.3 amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase of HepG2 alignment data with NCBI

Query＝c174868/f1p5/1054

HGKFHGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPLLTPHVRHGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

glyceraldehyde-3-phosphate dehydrogenase isoform 2[Homo sapiens]

Sequence ID:ref|NP_001243728.1|Length:293Number of Matches:1

See 1 more title(s)

Related Information

Gene-associated gene details

Map Viewer-aligned genomic context

Identical Proteins-Identical proteins to NP_001243728.1

Range 1:11 to 293GenPeptGraphicsNext MatchPrevious Match

Query 1

HGKFHGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHL 60

HGKFHGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHL

Sbjct 11

HGKFHGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHL 70

Query 61

QGGAKRVIISAPLL-TPHVRHGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVE 119

QGGAKRVIISAP P

GVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVE

Sbjct 71

QGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVE 130

Query 120

GLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMA 179

GLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMA

Sbjct 131

GLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMA 190

Query 180

FRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHS 239

FRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHS

Sbjct 191

FRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHS 250

Query 240

STFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE 282

STFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

Sbjct 251

STFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE 293

Through third-generation gene sequencing detection, specific mutation of estradiol 17 beta-dehydrogenase which is a key enzyme for E2 metabolism exists in A549 cells, and specific mutation of glyceraldehyde-3-phosphate dehydrogenase exists in HepG2 cells.

Experimental example 3

The method verifies whether the estradiol 17 beta-dehydrogenase is a key enzyme for the occurrence and development of the lung cancer and whether the glyceraldehyde-3-phosphate dehydrogenase is a key enzyme for the occurrence and development of the liver cancer, and lays a foundation for the anti-lung cancer application of the estradiol in the lung cancer cells through the specific regulation and control and the anti-liver cancer application of the glyceraldehyde-3-phosphate dehydrogenase in the liver cancer cells through the DI in the silkworm pupa cordyceps sinensis tannin plant entity.

Based on the third generation gene sequencing human complete transcriptome, randomly collecting 2 human lung cancers from Tianjin tumor hospital and 2 human liver cancer disease exceptions Zhou Xieyang for complete transcriptome sequencing, comparing the transcriptome data of lung cancer patients or liver cancer patients with 4729 enzymes related to human diseases found by research, extracting the enzyme with homology rate of 50-97% and not less than 70 amino acids, further comparing the amino acid sequence of the enzyme with NCBI, analyzing whether the mutation of estradiol 17 beta-dehydrogenase exists and the mutation condition according to the comparison result with NCBI, analyzing whether the mutation of glyceraldehyde-3-phosphate dehydrogenase exists and the mutation condition according to the comparison result with NCBI, and the result shows that 2 lung cancer cases all specifically have estradiol 17 beta-dehydrogenase mutation and 2 liver cancer cases all specifically have glyceraldehyde-3-phosphate dehydrogenase mutation. Detailed mutations are as follows:

lung cancer case 1:

the sequence mutation situation is:

Query＝c8524/f1p0/1959

MARTVVLITGCSSGIGLHLAVRLASDPSQSFKGIDRQGQGGREGRSPWRPEGKSDLPPLPKPPVYATLRDLKTQGRLWEAARALACPPGSLETLQLDVRDSKSVAAARERVTEGRVDVLGEPPGSIWAPRSLLRPALKPTCSQAQGARGDRPCgcgglvgplVSAVCNaglgllgplealgEDAVASVLDVNVVGTVRMLQAFLPDMKRRGSGRVLVTGSVGGLMG

PREDICTED:estradiol 17-beta-dehydrogenase 1 isoform X8[Homo sapiens]

Sequence ID:ref|XP_011523034.1|Length:180Numberof Matches:1

Related Information

Gene-associated gene details

Range 1:1 to 179GenPeptGraphicsNext MatchPrevious Match

Query 1

MARTVVLITGCSSGIGLHLAVRLASDPSQSFKGIDRQGQGGREGRSPWRPEGKSDLPPLP 60

MARTVVLITGCSSGIGLHLAVRLASDPSQSFKGIDRQGQGGREGRSPWRPEGKSDLPPLP

Sbjct 1

MARTVVLITGCSSGIGLHLAVRLASDPSQSFKGIDRQGQGGREGRSPWRPEGKSDLPPLP 60

Query 61

KPPVYATLRDLKTQGRLWEAARALACPPGSLETLQLDVRDSKSVAAARERVTEGRVDVLG 120

KPPVYATLRDLKTQGRLWEAARALACPPGSLETLQLDVRDSKSVAAARERVTEGRVDVL Sbjct 61

KPPVYATLRDLKTQGRLWEAARALACPPGSLETLQLDVRDSKSVAAARERVTEGRVDVL-119

Query 121

EPPGSIWAPRSLLRPALKPTCSQAQGARGDRPCGCGGLVGPLVSAVCNAGLGLLGPLEAL 180

VCNAGLGLLGPLEAL

Sbjct 120--------------------------------------VCNAGLGLLGPLEAL 134

Query 181

GEDAVASVLDVNVVGTVRMLQAFLPDMKRRGSGRVLVTGSVGGLM 225

GEDAVASVLDVNVVGTVRMLQAFLPDMKRRGSGRVLVTGSVGGLM

Sbjct 135

GEDAVASVLDVNVVGTVRMLQAFLPDMKRRGSGRVLVTGSVGGLM 179

lung cancer case 2:

the sequence mutation situation is as follows:

>Query＝c133537/f1p1/1595

MKFlldillllplliVCSLESFVKLFIPKRRKSVTGEIVLITGAGHGIGRLTAYEFAKLKSKLVLWDINKVKAEIGDVSILVNNAGVVYTSDLFATQDPQIEKTFEVNVLAHFWRRKELGTIRVIVCDFIKTTKAFLPAMTKNNHGHIVTVASAAGHVSVPFLLAYCSSKFAAVGFHKTLTDELAALQITGVKTTCLCPNFVNTGFIKNPSTSLGPTLEPEEVVNRLMHGILTEQKMIFIPSSIAFLTTLERILPERFLAVLKRKISVKFDAVIGYKMKAQ

estradiol 17-beta-dehydrogenase 11 precursor[Homo sapiens]

Sequence ID:NP_057329.3Length:300Number of Matches:1

See 10 more title(s)

Related Information

Gene-associated gene details

GEO Profiles-microarray expression data

Map Viewer-aligned genomic context

Identical Proteins-Identical proteins to NP_057329.3

Range 1:1 to 300GenPeptGraphicsNext MatchPrevious Match

Query 1

MKFLLDILLLLPLLIVCSLESFVKLFIPKRRKSVTGEIVLITGAGHGIGRLTAYEFAKLK 60

MKFLLDILLLLPLLIVCSLESFVKLFIPKRRKSVTGEIVLITGAGHGIGRLTAYEFAKLK

Sbjct 1

MKFLLDILLLLPLLIVCSLESFVKLFIPKRRKSVTGEIVLITGAGHGIGRLTAYEFAKLK 60

Query 61 SKLVLWDINK-------------------------------VKAEIGDVSILVNN84

SKLVLWDINK

VKAEIGDVSILVNN

Sbjct 61

SKLVLWDINKHGLEETAAKCKGLGAKVHTFVVDCSNREDIYSSAKKVKAEIGDVSILVNN 120

Query 85

AGVVYTSDLFATQDPQIEKTFEVNVLAHFWRRKELGTIRVIVCDFIKTTKAFLPAMTKNN 144

AGVVYTSDLFATQDPQIEKTFEVNVLAHFW

TTKAFLPAMTKNN

Sbjct 121

AGVVYTSDLFATQDPQIEKTFEVNVLAHFW-----------------TTKAFLPAMTKNN 163

Query 145

HGHIVTVASAAGHVSVPFLLAYCSSKFAAVGFHKTLTDELAALQITGVKTTCLCPNFVNT 204

HGHIVTVASAAGHVSVPFLLAYCSSKFAAVGFHKTLTDELAALQITGVKTTCLCPNFVNT

Sbjct 164

HGHIVTVASAAGHVSVPFLLAYCSSKFAAVGFHKTLTDELAALQITGVKTTCLCPNFVNT 223

Query 205

GFIKNPSTSLGPTLEPEEVVNRLMHGILTEQKMIFIPSSIAFLTTLERILPERFLAVLKR 264

GFIKNPSTSLGPTLEPEEVVNRLMHGILTEQKMIFIPSSIAFLTTLERILPERFLA

VLKR

Sbjct 224

GFIKNPSTSLGPTLEPEEVVNRLMHGILTEQKMIFIPSSIAFLTTLERILPERFLAVLKR 283

Query 265 KISVKFDAVIGYKMKAQ 281

KISVKFDAVIGYKMKAQ

Sbjct 284 KISVKFDAVIGYKMKAQ 300

liver cancer case 1:

the sequence mutation situation is as follows:

Query＝c307214/f1p0/1615

GRELNWDDYKKQEQLETARKFLYYEMGYKSRSEQLTDRSEISLLPSDIDRYKKRFHKFDADQKGFITIVDVQRVLESIMSKWMKYTPNSKSFENGQVELNEF

glycerol-3-phosphate dehydrogenase 2(mitochondrial),isoform CRA_a[Homo sapiens]

Sequence ID:gb|EAX11452.1|Length:709Number of Matches:1

Related Information

Gene-associated gene details

Range 1:578 to 684GenPeptGraphicsNext MatchPrevious Match

Query 1

GRELNWDDYKKQEQLETARKFLYYEMGYKSRSEQLTDRSEISLLPSDIDRYKKRFHKFDA 60

GRELNWDDYKKQEQLETARKFLYYEMGYKSRSEQLTDRSEISLLPSDIDRYK

KRFHKFDA

Sbjct 578

GRELNWDDYKKQEQLETARKFLYYEMGYKSRSEQLTDRSEISLLPSDIDRYKKRFHKFDA 637

Query 61

DQKGFITIVDVQRVLESIMSKWMKYTPNSKSFE-----NGQVELNEF 102

DQKGFITIVDVQRVLESI++T+E NGQVELNEF

Sbjct 638

DQKGFITIVDVQRVLESINVQMDENTLHEILNEVDLNKNGQVELNEF 684

liver cancer case 2:

the sequence mutation situation is:

>Query＝c104825/f1p2/1106

MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

PREDICTED:glyceraldehyde-3-phosphate dehydrogenase isoform X6[Colobus angolensis palliatus]

Sequence ID:XP_011790527.1Length:303Number of Matches:1

Related Information

Gene-associated gene details

Range 1:1 to 303GenPeptGraphicsNext MatchPrevious Match

Query 1

MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTV 60

MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTV

Sbjct 1

MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTV 60

Query 61

KAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTT----------------104

KAENGKLVING+PITIFQERDPSKIKWGDAGAEYVVESTGVFTT

Sbjct 61

KAENGKLVINGSPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKYDNSLKIVSNASC 120

Query 105

---------KVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTG 155

KVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTG

Sbjct 121

TTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTG 180

Query 156

AAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKG 215

AAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKG

Sbjct 181

AAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKG 240

Query 216

ILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMA 275

ILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMA

Sbjct 241

ILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMA 300

Claims (10)

1. a large-scale production method of cordyceps militaris is characterized by comprising the following steps:

step one, cordyceps militaris Haining strainC. militaris Strain breeding

1) Seed selection: selecting silkworm cordyceps militaris Hainin plant sporocarp which grows robustly, has no condensed water or condensed water, has clear and transparent water drops and no white aerial hyphae at the root of the sporocarp and grows to be 2-3cm high;

2) Culturing strains: in an aseptic environment, adopting an aseptic inoculation hook to hook off the root of the sporocarp, taking out the sporocarp, placing the sporocarp on a sterilized gauze, clamping the root of the sporocarp by using an aseptic forceps, sequentially wiping the surface of the sporocarp by 75% alcohol, cleaning residual alcohol on the surface of the sporocarp by using sterile water, removing the epidermis of the sporocarp by using an aseptic scalpel, selecting a part with the length of 1 to 1.5cm at the top end of the sporocarp, dividing the part into 3~5 granules every 1cm, placing the sporocarp granules on a PDA solid culture medium by using the aseptic inoculation hook, wherein the interval of the sporocarp granules is about 3.5cm, and carrying out shading inversion culture in a biochemical culture box at the temperature of 22 ℃ until hypha appears;

3) And (3) strain purification: in an aseptic environment, using an inoculating loop to select a loop of hyphae obtained in a strain culture link, adopting continuous plate marking to carry out strain separation and purification, and carrying out inverted culture in a biochemical incubator at the temperature of 21-23 ℃ until the hyphae grow to the height of about 0.5cm;

step two strain preparation

According to the proportion that the inoculation amount of each 120mL of liquid culture medium is 1cm multiplied by 1cm, picking and chopping a flat single bacterial colony obtained by purifying the bacterial colony obtained in the first step by using an aseptic scalpel, quickly transferring the bacterial colony into the liquid culture medium in a conical flask, slightly shaking to uniformly distribute the bacterial colony, tightening a bottle opening, culturing the bacterial colony in a shaking table at 22 ℃ and 170rpm in a dark place, and obtaining the bacterial colony which is qualified and can be cultivated when hyphae are snow white, uniform, dense, strong in penetrating power and free from peculiar smell and the bacterial colony grows to be larger than 1/2 area of the plane of the culture medium;

cultivation in three steps

1) Initial spawn running culture: adopting a full-automatic cap opening, inoculating and covering integrated machine, quickly injecting 2mL of bacterial liquid into a culture bottle, covering, culturing in dark for 3~5 days at the temperature of 15-18 ℃ and the relative air humidity of 63-68%, keeping the hypha in a snowy white color, continuously culturing in a uniform and dense way with strong penetrating power and no peculiar smell until the hypha fully overgrows the whole culture medium, and culturing in the early stage of differentiation of the light culture strain when a plurality of dome-shaped bulges with different numbers and sizes are generated on the material surface; 40g of strain culture medium is filled in the culture bottle, and the height is about 2cm;

2) Strain differentiation early stage culture: placing the culture bottle in a condition that the temperature is 18-23 ℃, the relative air humidity is not less than 85%, and the illumination intensity is 200-250Lx, performing light culture for 3-10 days until hypha on the surface of the culture medium gradually changes from white to orange, and continuing to culture 1~3 days, so as to enter the original period; the light is irradiated for more than 12 hours every day in the light culture process, but the light cannot be continuously irradiated, so that the dark culture for 6 hours every day is ensured, and the direct irradiation of sunlight is prevented;

3) Strain differentiation primordial stage culture: culturing for 10 days by adopting temperature difference stimulation under the condition that the relative air humidity is 68-72 percent to form an primordium, keeping the temperature at 20-23 ℃ after a large amount of primordium is formed, gradually increasing the relative air humidity to 85-90 percent, and prolonging the illumination time to 15 hours; continuously culturing for 1~3 days until the top of primordium begins to appear a cone-shaped sporophore, and entering the early growth stage of the sporophore; the temperature difference stimulation is divided into a daytime temperature and a nighttime temperature, the daytime temperature is 18 to 23 ℃, the illumination intensity is 50 to 100Lx, and the time is 12 to 14 hours; the temperature at night is 5-8 ℃, and the time is 6-10h;

4) And (3) fruiting body growth earlier stage: the fruiting body in the early growth stage is orange yellow and is 1 to 2cm high, the fruiting body is placed in an environment with the temperature of 22 to 25 ℃ and the air relative humidity of 85 to 90 percent, discontinuous illumination for 500Lx is carried out for 18 hours by adopting scattered light or a fluorescent lamp every day, the fruiting body is cultured for 20 to 30 days, the fruiting body is 2 to 4cm high, and the fruiting body can be transferred to the later growth stage when the fruiting body is in a long cone shape;

5) And (3) fruiting body growth later stage: culturing for 10 to 20 days under the conditions that the temperature is 20 to 23 ℃, the relative air humidity is 85 to 90 percent, the illumination intensity is 1000Lx, and the illumination time is 18 hours every day until the sporocarp is not thickened and increased, the head begins to expand, and the sporocarp which is in a strip shape and strong in growth is orange yellow is good in growth;

step four harvesting and storing

1) Picking fruit bodies: the cordyceps militaris fruiting body is orange, the head is expanded and has a seed capsule shell, the surface is mature when yellow powder appears, and the cordyceps militaris fruiting body is picked in time by adopting a full-automatic cover opening, inverted picking and culture medium cleaning integrated machine; the harvested culture medium can be recycled for secondary cultivation;

2) Drying and storing the sporocarps: the picked cordyceps militaris sporocarp is subjected to freeze drying and then is stored in a dry and cool place.

2. The large-scale production method of cordyceps militaris, as claimed in claim 1, wherein the preparation method of said PDA solid medium is: taking 37g of PDA and 5g of silkworm chrysalis powder, adding 800mL of purified water, heating for dissolving, uniformly stirring, complementing water to 1L, subpackaging according to requirements, and sterilizing at 121 ℃ for 30min for later use.

3. The method for large-scale production of cordyceps militaris as claimed in claim 1, wherein the preparation method of the culture medium adopted for strain purification comprises: adding 1L of PDA into 37g of water, heating for dissolving, stirring thoroughly, sterilizing at 121 deg.C for 30min for use.

4. The large-scale production method of cordyceps militaris as claimed in claim 1, wherein the preparation method of the liquid culture medium comprises the following steps: collecting glucose 20g, peptone 5g, yeast extract 5g, and MgSO ₄ 1g, 1.5g potassium dihydrogen phosphate, adding purified water to 1L, stirring to dissolve completely, packaging, and sterilizing at 121 deg.C for 30min.

5. The method for large-scale production of cordyceps militaris as claimed in claim 1, wherein the preparation method of the strain culture medium comprises the following steps: taking 8kg of silkworm chrysalis, 12kg of rice, 20kg of purified water, 30g of monopotassium phosphate and 10g of sodium dihydrogen phosphate for later use, grinding the silkworm chrysalis and the rice into powder, uniformly mixing, adding the purified water containing the monopotassium phosphate and the sodium dihydrogen phosphate, fully stirring the slurry until the pH value is 6-6.5, sterilizing at 121 ℃ for 30min, and using.

6. The application of ergosterol compounds in the fruiting body of the Chinese silkworm pupa cordyceps sinensis line produced by the large-scale production method of cordyceps militaris as claimed in any one of claims 1 to 5 in the preparation of medicines for diagnosing or treating cancer by specifically regulating estradiol metabolism of cancer cells and regulating and controlling GAPDH expression of cancer cells.

7. The application of ergosterol compounds in the fruiting body of the Chinese silkworm pupa cordyceps sinensis, which is produced by the large-scale production method of cordyceps militaris as claimed in any one of claims 1 to 5, in inhibiting the growth of cancer cells by specifically regulating E2 and GAPDH in the cancer cells.

8. The use of ergosterol compounds in the fruiting body of Chinese silkworm pupa, cordyceps sinensis and quinine plants produced by the large-scale production method of cordyceps militaris as claimed in any one of claims 1 to 5 in regulating estradiol metabolism in lung cancer cells.

9. The use of ergosterol compounds in the fruiting body of the Chinese pupa Bombycis Cordyceps sinensis Hainin strain produced by the large-scale production method of Cordyceps militaris as claimed in any one of claims 1-5 in up-regulating GAPDH expression in hepatocarcinoma cells.

10. The use according to any one of claims 6 to 9, wherein the ergosterol compound is (3)R，9R，10S，13S，14S，17S）-17 - （（2S，5R，E) -5,6-dimethylhept-3-en-2-yl) -10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta [ a]Phenanthrene-3-ol, abbreviated as "DI", has the following structural formula:

。

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