CN113789357A

CN113789357A - Industrial preparation method for producing cordycepin and pentostatin by combined fermentation

Info

Publication number: CN113789357A
Application number: CN202111208114.1A
Authority: CN
Inventors: 陈开云; 冯成勇
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2021-12-14

Abstract

The invention provides an industrial preparation method for producing cordycepin and pentostatin by combined fermentation, which uses cordycepin and antibioticsStreptomyces is used as an original strain, and is subjected to test tube slant culture, suspension culture, mutagenesis, combined seed culture, fermentation culture, high-speed centrifugation of fermentation liquor, macroporous resin purification, recrystallization and other technologies and supercritical CO₂The fluid drying technology and the full-automatic control technology are combined, and the cordycepin and the streptomyces antibioticus are subjected to combined fermentation under the synergistic effect of the two technologies to produce cordycepin and pentostatin, so that the non-chemical synthesis and the biosynthesis of non-cordyceps militaris extraction are simultaneously carried out on the cordycepin and the pentostatin; the cordycepin and pentostatin compound produced by the combined fermentation of the technology of the invention has the advantages of mild reaction conditions, relatively simple operation, low product cost, high yield, high purity, low residual solvent, low energy consumption, high productivity and little environmental pollution, is suitable for industrial mass production, and has good economic benefit and application prospect.

Description

Industrial preparation method for producing cordycepin and pentostatin by combined fermentation

Technical Field

The invention belongs to the technical field of biological metabolism synthesis and purification, and particularly relates to an industrial preparation method for producing cordycepin and pentostatin through combined fermentation.

Background

Cordycepin is also called as Cordyceps sinensis extract, cordycepin, or 3' -deoxyadenosine, and has chemical formula C₁₀H₁₃N₅O₃Molecular weight is 251.25; is the first nucleoside antibiotic isolated from fungi.

In the 50 s of the 20 th century, wild cordyceps militaris is found in Changbai mountain of Jilin, and researches prove that the component efficacy of the wild cordyceps militaris is far superior to that of the wild cordyceps militaris, so that the cordyceps militaris is named; in 1951, German scientists Cunningham and the like discover that a core ingredient of cordyceps militaris, namely cordycepin, has a plurality of pharmacological activities of antibiosis, anti-inflammation, antivirus, antitumor, immunoregulation and the like for the first time; for decades, the academics have intensively developed a great deal of research on cordycepin; in 1997, cordycepin has been used in three-phase clinical trials in the united states for the treatment of patients with acute pre-B and pre-T lymphoblastic leukemia; in 2017, the professor wang cheng tree of Chinese academy of sciences published the latest research results on cordycepin on line in Cell electronics, Cell chemical biology: the research completely analyzes the biosynthesis mechanism of cordycepin in cordyceps militaris (Cordycepsmilitaris), and simultaneously discovers that the cordyceps militaris can synthesize an anticancer drug, namely pentostatin for the first time, and the compound is used for protecting the structural stability of the synthesized cordycepin.

Research results show that the cordycepin has the biological activities of lung and kidney protection, high blood pressure, high blood sugar, high blood fat and high blood fat. Therefore, cordycepin is concerned by students in the fields of anti-aging, health care, new drug development and the like.

Pentostatin, which is a white crystal with a molecular formula: c₁₁H₁₆N₄O₄Molecular weight: 268.27, respectively; it was first isolated from Streptomyces antibioticus broth by Woo et al in 1974.

Pentostatin is a purine nucleotide analog antibiotic, and is separated from cordyceps militaris fermentation liquor and bacterial streptomycete antibiotic. Pentostatin, also known as 2' -deoxycoformycin, binds to and inhibits Adenine Deaminase (ADA), an enzyme essential for purine metabolism; ADA activity is greatest in cells of the lymphatic system, where T cells have higher activity than B cells, and T cell malignancies have higher ADA activity than B cell malignancies. Inhibition of ADA by pentastatin appears to result in increased intracellular dATP levels, which may block DNA synthesis by inhibiting ribonucleotide reductase. The agent may also inhibit RNA synthesis and may selectively deplete CD26+ lymphocytes.

Pentostatin lyophilized powder for injection developed by Nipent corporation of America is effective for acute and chronic lymphocytic leukemia, non-Hodgkin lymphoma, cutaneous T cell lymphoma and hairy cell leukemia; the product is still very effective for patients with anti-interferon. The complete remission rate (CR) of patients with cell leukemia, whether splenectomies exist or not or whether the patients receive a-interferon treatment or not, reaches 33 to 92 percent after the patients use the product. After almost all patients are treated by the composition for 2 courses of treatment, the peripheral blood cell count is improved, and the most obvious improvement is about 4 months of treatment on average. All patients with CR remained stable for an average of 6 years and no further treatment was required. The complete remission rate of the B cell chronic lymphocytic leukemia is only 4 percent, and the partial remission rate is 15 to 27 percent.

Patent No. 202110749628.1 application of a Cordyceps militaris pharmaceutical composition in preparation of preparations for preventing and/or treating Xinguan and resisting respiratory viruses discloses application experiments of cordycepin and pentostatin composition in preventing and treating Xinguan and resisting respiratory viruses. 4 patients with continuous positive coronavirus nucleic acid (the positive of throat swab nucleic acid detection is more than 14 days every day) are randomly selected in an experiment, and the concentrated cordyceps militaris extracting solution is diluted by 200mL of physiological saline, so that the cordyceps militaris extracting solution contains 5ug/mL of cordycepin and 2ug/mL of pentostatin. The nasal cavity was washed 3 times a day using a type a nasal irrigator manufactured by yangzhou strong medical devices ltd. In the control group, 2 patients who were continuously positive for the new coronavirus nucleic acid were randomly selected and then the nose was washed with physiological saline 3 times a day. The clinical effect of transferring virus nucleic acid detection to negative and discharging is obtained 48-72 hours after the cordyceps militaris extracting solution is washed nose, and the time for transferring virus nucleic acid detection to negative is more than 14 days after the physiological saline is washed nose. Is the clinical test result of the effect of quickly eliminating the new coronavirus in the cordyceps militaris extracting solution (containing cordycepin and pentostatin) nasal irrigation treatment in the sixth embodiment of the invention. Clinical tests prove that the cordycepin and pentostatin-containing cordyceps militaris extracting solution can effectively clear new coronavirus in nasopharynx when being used for nasal irrigation. The virus titers of the cells treated by the combination of the cordyceps militaris extracting solution and the cordycepin and the pentostatin are respectively (2.12 +/-0.55) multiplied by 105PFU/mL and (0.5 +/-0.167) multiplied by 105PFU/mL, namely, the virus clearance rate of the cordyceps militaris extracting solution is 98.6 percent, and the virus clearance rate of the combination of the cordycepin and the pentostatin is 99.9 percent.

Disclosure of Invention

The invention aims to provide an industrial preparation method for producing cordycepin and pentostatin by combined fermentation aiming at the defects of the prior art, and the industrial preparation method for producing cordycepin and pentostatin by combined fermentation can well solve the problems.

In order to meet the requirements, the technical scheme adopted by the invention is as follows: the industrial preparation method for producing the cordycepin and the pentostatin by combined fermentation comprises the following steps:

s1: culturing cordyceps militaris: selecting high-yield, high-quality and early-maturing cordyceps militaris sporocarp, carrying out surface disinfection by using 75% alcohol, cleaning surface liquid medicine by using sterile water, suspending the liquid medicine above a container filled with a comprehensive culture medium, carrying out static culture at the temperature of 28-30 ℃ for 5-7 days, picking single or multiple colonies in an inoculation box when starburst-shaped cordyceps militaris colonies appear on the surface of the culture medium, placing the colonies in a test tube inclined plane culture medium for culture, and purifying after cordyceps militaris mycelia are fully distributed on the inclined plane;

s2: culturing antibiotic streptomycete: taking a strain with a preservation number as follows: the CGMCC No.1349 streptomyces antibioticus is subjected to static culture at 28-30 ℃ for 5-7 days, when streptomyces antibioticus colonies appear on the surface of a culture medium, a single colony or a plurality of colonies are selected from an inoculation box and placed in a test tube slant culture medium for culture, and the cordyceps militaris mycelia are purified after being fully distributed on the slant;

s3: respectively placing a cordyceps militaris bacterium culture medium and a streptomyces antibioticus culture medium in a constant-temperature incubator at 28-30 ℃ for culture, observing at regular time, sequentially transferring newly grown hyphae to a new culture medium by using an inoculating needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane for preservation of two strains;

s4: preparation of bacterial suspension: respectively taking several dominant bacterial strain slopes cultured for one week, respectively adding physiological saline containing synergistic isonicotin, scraping thallus on the slopes by using an inoculating loop, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to resist oscillation, filtering the mixture on a funnel with a layer of sterile mirror wiping paper after oscillation to obtain monospore suspension of the bacterial strain, counting spores by using a hemocytometer, and adjusting the concentration of the spores to be about 10⁶Per ml;

s5: ultraviolet mutagenesis: respectively taking 5ml of prepared cordyceps militaris and streptomyces antibioticus monospore suspension into a sterile plate with the diameter of 9mm, opening a 15W ultraviolet lamp for preheating, putting the plate containing the bacterial suspension on a magnetic stirrer at a certain distance from a lamp tube, opening a plate cover, stirring and irradiating to ensure that cells uniformly absorb ultraviolet light waves, operating under a red light background to avoid light restoration, immediately transferring the thalli subjected to ultraviolet mutagenesis into the sterile test tube, immersing the thalli in ice water for 30min to inhibit enzyme activity and inhibit restoration, and adding the bacterial suspension subjected to ice bath after irradiation into a PDA culture medium for post-culture according to the principle of delay phenomenon to improve the mutation rate;

s6: carrying out normal-pressure room-temperature plasma mutagenesis: respectively uniformly coating 10 mu L of spore suspension with proper concentration of ultraviolet mutagenized cordyceps militaris and streptomyces antibioticus on a sterilized mutagenesis cup, then mutagenizing under the optimal condition, putting the mutagenesis cup into a 2mL centrifuge tube after mutagenesis, adding 1mL of sterile ultrapure water, coating the mutagenesis cup on an ISP2 culture medium after shaking for 1min, culturing for 6-8 d at 30 ℃, observing colonies with color, texture and character which generate brightness difference change compared with a control when the colonies grow to be about 0.5cm in diameter, screening, and selecting the strains with the highest yield for preservation for subsequent culture;

s7: the liquid shake flask strain culture medium is PDA, PH7, initial PH value is 5.0, 250mL are prepared totally, split charging 250mL triangular flask, each 30mL, sterilizing at 121 deg.C for 30 minutes, cooling to room temperature for standby;

s8: preparing liquid shake flask strains: respectively subpackaging cordyceps militaris or streptomyces antibioticus liquid culture medium into 500ml triangular flasks, adding 200ml of culture solution into each flask, sealing the flasks by adding 12 layers of gauze and a layer of kraft paper, sterilizing for 13-30 minutes, inoculating the mother seeds into the triangular flasks by using an inoculating hook under the aseptic condition, connecting each inclined plane to more than 10 bottles, performing shake culture at 28 ℃ for about 5-7 days after inoculation, and performing inoculation after uniform pellets are formed;

s9: preparing a seed culture medium according to a formula standard, respectively pumping the seed culture medium into No. 1-8 500L first-stage seed culture tanks, and sterilizing by using a tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

s10: first-order seed culture: after the first-stage seed culture tank is sterilized, when the temperature of a culture medium in the tank is reduced to 28-30 ℃, respectively inoculating the triangular flask fermentation liquor into a No. 1-8 500L first-stage seed fermentation tank through an aseptic inoculation system, performing seed culture by using the seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 28-30 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7.2, the rotation speed of the seed tank is 100r/min and the ventilation amount is 0.5-1 vvm/min after 0-2 days, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.5-1 vvm/min after 2 days, and collecting seed liquid after the fermentation culture.

S11: preparing a seed culture medium according to a formula standard, respectively filling the seed culture medium into No. 1-8 1000L secondary seed fermentation tanks, and sterilizing by using a retort steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

s12: secondary seed culture: after the second-stage seed culture tank is sterilized, when the temperature is reduced to 25-28 ℃, respectively inoculating first-stage seed fermentation liquor fermented for 5-7 days into a 1000L second-stage seed fermentation tank with the number of 1-8 through an aseptic inoculation system, performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 25-28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 150r/min and the ventilation amount is 0.8-1.4 vvm/min after 2 days, and collecting seed liquid after the fermentation culture;

s13: accurately weighing the culture medium materials according to the formula in batches, putting the culture medium materials into a 40000L multiplied by 3 culture medium preparation tank, pumping purified water according to the formula, and stirring for 30 min;

s14: pumping fermentation medium into No. 1-10 100000L fermentation single tank or No. 1-3 medium liquid supplementing tank in batches, filling the tank with coefficient of 0.7, sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

s15: after single-tank sterilization is finished, cooling culture medium in the tank to 28 ℃ by using chilled water, respectively inoculating secondary seed fermentation liquor fermented for 5-7 days from No. 1-8 to a No. 1-10 100000L fermentation tank through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 28 ℃, the tank pressure is 0.03MPa, the fermentation culture is carried out for 120 hours under the conditions of pH7, the stirring rotation speed is 130r/min, the ventilation amount is 0.6-1.4 vvm/min, and after 180 hours, the fermentation tank rotation speed is 100r/min, and the ventilation amount is 1.4 vvm/min;

s16: supplementing liquid in real time according to the culture medium quantity standard in the fermentation period; when the culture medium supplement reaches the standard and the fermentation time reaches the standard, finishing the fermentation to prepare a fermentation mixed solution of the fruiting body, the cordycepin and the pentostatin;

s17: pressing the prepared mycelium and fruiting body, cordycepin and pentostatin fermentation mixed liquor into a No. 1-2 40000L fermentation liquor temporary storage tank;

s18: pressing the obtained fermentation liquid into coarse filtration equipment, and filtering to obtain mycelium, fruiting body filter cake and filtrate;

s19: pumping the filtrate into a membrane ultrafiltration unit for ultrafiltration, and filtering out impurities in the fermentation liquor to obtain a mixed crude solution of cordycepin and pentostatin;

s20: centrifuging the filtered concentrated solution with automatic scraper centrifuge for desolventizing to obtain waste such as fruiting body;

s21: pumping the prepared mixed crude solution of cordycepin and pentostatin into a membrane nanofiltration concentration unit for concentration to prepare a mixed concentrated solution of cordycepin and pentostatin and wastewater;

s22: pumping the prepared cordycepin and pentostatin mixed concentrated solution into an automatic scraper centrifuge for centrifugation and desolventization to prepare a cordycepin and pentostatin mixed crude product filter cake A;

s23: sending the prepared mixed crude product filter cake A of the cordycepin and the pentostatin into a No.1 extraction kettle, pumping a lipophilic solvent which is 3 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, pumping deionized water which is 6 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, extracting the mixture for 2 to 3 times, and collecting and combining the extract liquor;

s24: performing liquid-liquid centrifugal extraction on the extract liquor, and separating an organic phase and a water phase to obtain an organic phase containing pentostatin and a water phase containing cordycepin;

s25: filtering the organic phase by a bag filter, concentrating the filtrate by a No.1 concentrator, centrifugally desolventizing, and recovering the solvent to prepare a crude pentostatin dry filter cake A;

s26: pumping the water phase into a flocculation precipitation tank, adding a flocculating agent, stirring for 10-30 min, precipitating for 3-6 h, filtering by a bag filter, concentrating by a No. 2 concentrator, centrifuging, desolventizing, and recovering the solvent to obtain a cordycepin crude product dry filter cake A;

s27, respectively feeding the cordycepin crude product dry filter cake A and the pentostatin crude product dry filter cake A into an extraction kettle of No. 2 or No. 3 3000L, respectively adding acetone solvent with the weight 3 times of that of the dry filter cake, adding decolorizing agent with the weight 0.03 time of that of the dry filter cake, extracting and decolorizing for 2-3 times at the temperature of 40-60 ℃, filtering, combining the extract solutions, and respectively preparing cordycepin and pentostatin extract solutions;

s28, pumping the cordycepin extract and the pentostatin extract into a concentrator for concentration and recovering the solvent to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution;

s29, pumping the cordycepin crude product concentrated solution and the pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and solvent recovery to respectively prepare a cordycepin crude product filter cake B and a pentostatin crude product filter cake B;

s30: mixing the cordycepin crude product filter cake B with the crystallized cordycepin mother liquor filter cake, putting into a No.1 column feeding solution preparation tank, and dissolving with 30-60% ethanol by volume fraction to prepare column feeding solution; the solute concentration of the upper column liquid is 5-10 g/L;

s31: the prepared upper column liquid pump is used for automatically controlling the adsorption of a macroporous resin column system, and the adsorption flow rate is 2-3 BV; eluting with 30-60% ethanol; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

s32: pumping the adsorption effluent and the eluate into a concentrator respectively for concentration, and recovering the solvent to obtain a cordycepin crude product concentrated solution;

s33: pumping the obtained cordycepin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing, and recovering ethanol solvent to obtain cordycepin crude product filter cake;

s34: mixing a crude pentostatin filter cake and a crystallized pentostatin mother solution dry filter cake A, B, putting into a No. 2 column feed preparation tank, and dissolving with ethanol with the volume fraction of 70-80% to prepare column feed; the solute concentration of the upper column liquid is 5-15 g/L;

s35: pumping the prepared upper column liquid into a No. 2 macroporous resin column system for adsorption, wherein the flow rate of the upper column liquid is 2-3 BV; eluting with deionized water and 70-80% ethanol by volume; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

s36: adsorbing the effluent and the eluent by a No. 2 macroporous resin column system, pumping the effluent and the eluent into a concentrator for concentration, and recovering the solvent to prepare a crude pentostatin concentrated solution;

s37: pumping the obtained pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering an ethanol solvent to obtain a pentostatin crude product filter cake;

s38: feeding the obtained cordycepin filter cake into a No.1 crystallization kettle, dissolving with a solvent, recrystallizing at low temperature, and filtering to obtain cordycepin wet crystal and mother liquor;

s39: pumping the prepared cordycepin mother liquor into a concentrator for concentration, centrifuging for desolventizing, and recovering the solvent to prepare a cordycepin mother liquor dry filter cake;

s40: feeding the obtained crude pentostatin filter cake into a freezing crystallization kettle, dissolving the crude pentostatin filter cake by using a solvent, crystallizing at a low temperature, and filtering crystals to obtain pentostatin crude crystals and pentostatin mother liquor;

s41: pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a dry filter cake A of the pentostatin mother liquor;

s42: feeding the obtained crude pentostatin crystal into a No. 2 crystallization kettle, dissolving the crude pentostatin crystal by using a solvent, recrystallizing at low temperature, and filtering the crystal to obtain a wet pentostatin crystal and a mother solution;

s43: pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a pentostatin mother liquor dry filter cake B;

s44: combining the pentostatin mother solution dry filter cake B with the pentostatin crude product filter cake to prepare upper column solution;

s45: the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake are sent into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare cordycepin crystal powder with the content of more than or equal to 98 percent and cordycepin powder with low content;

s46, feeding the prepared pentostatin wet crystal into a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare pentostatin crystal powder with the content of more than or equal to 98 percent;

s47: and respectively feeding the prepared cordycepin crystal powder with the content of more than or equal to 98 percent and the prepared cordycepin powder with the low content into a batch V-shaped mixer for batch mixing to prepare a cordycepin crystal powder product with the content of more than or equal to 98 percent and a cordycepin powder product with the low content.

S48: and (3) feeding the prepared pentostatin crystal powder with the concentration of more than or equal to 98 percent into a batch V-shaped mixer for batch mixing to prepare a pentostatin crystal powder product with the concentration of more than or equal to 98 percent.

The industrial preparation method for producing cordycepin and pentostatin by combined fermentation has the advantages that:

1) the cordycepin and pentostatin produced by the invention adopt a non-cordyceps militaris extraction process and a non-chemical synthesis process, and a brand new process route is opened up for the industrial production of cordycepin and pentostatin.

2) The process for producing cordycepin and pentostatin of the invention saves a large amount of resources and solves the problem of insufficient extraction raw materials of cordycepin and pentostatin because cordycepin and pentostatin are not extracted from cordyceps militaris.

3) The invention utilizes the purine metabolic mechanism of a protector-protector (protector-prot) between cordycepin and pentostatin to simultaneously produce cordycepin and pentostatin, and the production cost is not less than half of the unit cost of extracting and purifying cordycepin and pentostatin from cordyceps militaris, so the product cost is low.

4) The invention designs 8 sets of full-automatic control primary seed fermentation tanks, 8 sets of full-automatic control secondary seed fermentation tanks, 3 sets of full-automatic control culture medium preparation tanks, 3 sets of full-automatic control culture medium supplement tanks and 10 sets of full-automatic control process fermentation tanks so as to meet the requirement of circular production, wherein a single industrial fermentation tank corresponds to a corresponding single secondary seed fermentation tank, a single secondary seed fermentation tank corresponds to a corresponding single primary seed fermentation tank, and 10 sets of fermentation systems perform continuous circular fermentation. Therefore, the method for producing the cordycepin and the pentostatin compound has the advantages of high productivity and continuous production.

5) In the method for preparing cordycepin and pentostatin, a modern full-automatic control technology is adopted to carry out preparation of the culture medium, control of the filling amount of the culture medium, control of the temperature, control of the ventilation quantity, control of the fermentation time, automatic cleaning of each tank, automatic control of sterilization and the like, thereby ensuring the product quality, improving the product yield, optimizing and saving the energy consumption and saving a large amount of labor force.

6) In the process for preparing the cordycepin and pentostatin fermentation liquor, cordyceps militaris and streptomyces antibioticus are subjected to ultraviolet irradiation strain mutagenesis and ARTP (normal pressure room temperature plasma) mutagenesis, an elicitor is added into a suspension culture medium, precursor substances such as L-phenylalanine, arachidonic acid and the like are added into the culture medium, and high-yield cordycepin and pentostatin compounds are obtained, wherein the yield of the cordycepin in the fermentation liquor reaches more than 3.5g/L, and the yield of the pentostatin reaches more than 100 mg/L.

7) The invention adopts the membrane separation and membrane concentration device, and the sewage discharged by the device can be used for plant irrigation besides generating part of solid waste, thereby avoiding the discharge of a large amount of polluted water and reducing the environmental pollution.

8) The invention adopts an MVR evaporator or a full-electric evaporator for vacuum concentration or membrane concentration, desolventizing by an automatic scraper centrifuge and recovering the solvent, improves the recovery rate of the solvent, achieves the recovery rate of the solvent above 90 percent, and greatly reduces the energy consumption of the product and the pollution of air.

9) The invention adopts centralized linkage control power, steam, cooling water and sterile air supply, and utilizes an automatic control technology to automatically control and distribute the use time and supply quantity of the power, the steam, the cooling water and the sterile air according to the time difference of the circulating fermentation of 10 sets of fermentation production systems, thereby improving the utilization rate of equipment, reducing the purchase cost of the equipment and reducing the energy consumption of the equipment.

10) According to the invention, a crystallization mother liquid extract generated in the cordycepin or pentostatin crystallization step is sent into a column feeding liquid preparation tank to be combined with a crude product filter cake to prepare column feeding liquid, and higher cordycepin or pentostatin in the crystallization mother liquid extract is adsorbed, eluted and recrystallized by macroporous resin again, so that the yield of cordycepin or pentostatin is increased by more than 98%.

11) The invention prepares cordycepin or pentostatin compound by adopting supercritical CO₂The fluid device is used for simultaneously drying, removing residual solvent and sterilizing, the product has low solvent residue and uniform color, and the sanitation index meets the relevant standard.

12) The method uses cordyceps militaris and streptomyces antibioticus as starting strains, and adopts the technologies of respectively mutagenesis, test tube slant culture, combined seed culture, fermentation culture, high-speed centrifugation of fermentation liquor, membrane filtration, membrane concentration, centrifugal extraction separation, extraction decoloration, macroporous resin purification, recrystallization and the like, and supercritical CO₂The fluid drying technology and the full-automatic control technology are combined, and the cordycepin and the streptomyces antibioticus are subjected to combined fermentation under the synergistic effect of the two technologies to produce cordycepin and pentostatin, so that the non-chemical synthesis and the biosynthesis of non-cordyceps militaris extraction are simultaneously carried out on the cordycepin and the pentostatin; the cordycepin and pentostatin compound produced by the combined fermentation of the technology of the invention has the advantages of mild reaction conditions, relatively simple operation, low product cost, high yield, high purity, low residual solvent, low energy consumption, high productivity and little environmental pollution, is suitable for industrial mass production, and has good economic benefit and application prospect.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the accompanying drawings and specific embodiments.

In the following description, references to "one embodiment," "an embodiment," "one example," "an example," etc., indicate that the embodiment or example so described may include a particular feature, structure, characteristic, property, element, or limitation, but every embodiment or example does not necessarily include the particular feature, structure, characteristic, property, element, or limitation. Moreover, repeated use of the phrase "in accordance with an embodiment of the present application" although it may possibly refer to the same embodiment, does not necessarily refer to the same embodiment.

Certain features that are well known to those skilled in the art have been omitted from the following description for the sake of simplicity.

According to an embodiment of the application, an industrial preparation method for producing cordycepin and pentostatin by combined fermentation is provided, which comprises the following steps:

The separation, mutagenesis and preservation method of the cordyceps militaris strain and the streptomyces antibioticus strain comprises the following steps:

culturing cordyceps militaris: selecting high-yield, high-quality and early-maturing cordyceps militaris sporocarp, carrying out surface disinfection by using 75% alcohol, cleaning surface liquid medicine by using sterile water, suspending the liquid medicine above a container filled with a comprehensive culture medium, carrying out static culture at the temperature of 28-30 ℃ for 5-7 days, and picking single or multiple colonies in an inoculation box to place the single or multiple colonies in a test tube slant culture medium for culture when starburst-shaped cordyceps militaris colonies appear on the surface of the culture medium. Purifying after the cordyceps militaris hyphae are covered with the inclined plane; culturing antibiotic streptomycete: taking a strain with a preservation number as follows: the CGMCC No.1349 streptomyces antibioticus is subjected to static culture at 28-30 ℃ for 5-7 days, and when streptomyces antibioticus colonies appear on the surface of a culture medium, a single colony or a plurality of colonies are picked in an inoculation box and placed in a test tube slant culture medium for culture; purifying after the cordyceps militaris hyphae are covered with the inclined plane; respectively adding a cordyceps militaris bacterium culture medium and a streptomyces antibioticus culture medium,culturing in a constant temperature incubator at 28-30 ℃; observing at regular time, sequentially transferring the newly grown hyphae to a new culture medium by using an inoculating needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane to preserve two strains; preparation of bacterial suspension: respectively taking several inclined planes of the dominant strains cultured for one week, respectively adding physiological saline containing synergistic isonicotin, scraping thalli on the inclined planes by using an inoculating loop, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to resist oscillation, and filtering the mixture by using a funnel with a layer of sterile mirror wiping paper after the oscillation is finished to obtain monospore suspension of the strains; counting spores with a hemocytometer (hemocytometer) to adjust the spore concentration to about 10⁶Per ml; ultraviolet mutagenesis: respectively taking 5ml of the prepared cordyceps militaris and streptomyces antibioticus monospore suspension into an aseptic plate with the diameter of 9 mm; a 15W ultraviolet lamp is turned on to preheat so as to stabilize light waves; putting the plate containing the bacterial suspension on a magnetic stirrer, keeping a certain distance from a lamp tube, opening a dish cover, stirring and irradiating to ensure that cells uniformly absorb ultraviolet light waves; the top was operated in a red background, avoiding photorepair. The thallus after ultraviolet mutagenesis is immediately transferred into a sterile test tube and immersed in ice water for 30min to inhibit enzyme activity and repair. According to the principle of delay phenomenon, adding the irradiated bacterial suspension subjected to ice bath into a PDA culture medium for post-culture so as to improve mutation rate; carrying out normal-pressure room-temperature plasma mutagenesis: respectively taking 10 mu L of spore suspension with proper concentration of ultraviolet mutagenized cordyceps militaris and streptomyces antibioticus, uniformly coating the spore suspension on a sterilized mutagenesis cup, then mutagenizing under the optimal condition, putting the mutagenesis cup into a 2mL centrifuge tube after mutagenesis, adding 1mL of sterile ultrapure water, coating the mutagenesis cup on an ISP2 culture medium after shaking for 1min, culturing for 6-8 days at 30 ℃, observing colonies with color, texture and character changed in brightness difference compared with a control when the colonies grow to be about 0.5cm in diameter, and screening. The strain with the highest yield is selected for preservation and used for subsequent culture.

The preparation and industrial fermentation method of the strain comprises the following steps:

the liquid shake flask strain culture medium is PDA, PH7, initial PH value is 5.0, 250mL are prepared totally, split charging 250mL triangular flask, each 30mL, sterilizing at 121 deg.C for 30 minutes, cooling to room temperature for standby; preparing liquid shake flask strains: respectively subpackaging cordyceps militaris or streptomyces antibioticus liquid culture medium into 500ml triangular flasks, adding 200ml of culture solution into each flask, sealing the flasks by adding a layer of kraft paper on 12 layers of gauze, sterilizing for 13-30 minutes, inoculating mother seeds into the triangular flasks by using an inoculating hook under an aseptic condition, connecting each inclined plane to more than 10 bottles, performing shake cultivation at 28 ℃ for about 5-7 days after inoculation, forming uniform pellets, and then using for inoculation, preparing a seed culture medium according to a formula standard, respectively pumping the seed culture medium into No. 1-8 500L first-stage seed culture tanks, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min; first-order seed culture: after the first-stage seed culture tank is sterilized, when the temperature of a culture medium in the tank is reduced to 28-30 ℃, respectively inoculating the triangular flask fermentation liquor into a first-stage seed fermentation tank of No. 1-8 500L through an aseptic inoculation system, performing seed culture by using the seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 28-30 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7.2, the rotation speed of the seed tank is 100r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.5-1 vvm/min after 2 days, collecting seed liquid after the fermentation culture, preparing the seed culture medium according to a formula standard, respectively filling the seed culture medium into a second-stage seed fermentation tank of No. 1-8L, sterilizing by using a steam sterilization method in the seed tank, wherein the sterilization temperature is not lower than 121 ℃, and the sterilization time is not lower than 30 min; secondary seed culture: after the second-stage seed culture tank is sterilized, when the temperature is reduced to 25-28 ℃, respectively inoculating first-stage seed fermentation liquor fermented for 5-7 days into a 1000L second-stage seed fermentation tank with the number of 1-8 through an aseptic inoculation system, performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 25-28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5 days under the condition that the pH is 7, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 150r/min and the ventilation amount is 0.8-1.4 vvm/min after 2 days, collecting seed liquid after the fermentation culture, accurately weighing the culture medium materials according to a formula, filling the culture medium materials into a 40000L multiplied by 3 culture medium preparation tank in batches, and stirring the purified water according to the formula for 30 min; pumping fermentation medium into No. 1-10 100000L fermentation single tank or No. 1-3 medium liquid supplementing tank in batches, filling the tank with coefficient of 0.7, sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min; after single-tank sterilization is finished, cooling culture medium in the tank to 28 ℃ by using chilled water, respectively inoculating the secondary seed fermentation liquor fermented for 18 hours from No.1 to No. 8 to a No.1 100000L fermentation tank through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 28 ℃, the tank pressure is 0.03MPa, the fermentation culture is carried out for 120 hours under the condition that the pH is 7, the stirring rotation speed is 130r/min, the ventilation amount is 0.8-1.4 vvm/min, and after 180 hours, the fermentation tank rotation speed is 100r/min and the ventilation amount is 1 vvm/min; supplementing liquid in real time according to the culture medium quantity standard in the fermentation period; and when the culture medium feeding reaches the standard and the fermentation time reaches the standard, ending the fermentation to prepare a fermentation mixed solution of the fruiting body, the cordycepin and the pentostatin.

The steps for purifying cordycepin and pentostatin are as follows:

pressing the prepared fruiting body, cordycepin and pentostatin fermentation mixed solution into a No. 1-2 40000L fermentation liquid temporary storage tank; pressing the obtained fermentation liquid into coarse filtration equipment, and filtering to obtain mycelium, fruiting body filter cake and filtrate; pumping the filtrate into a membrane ultrafiltration unit for ultrafiltration, and filtering out impurities in the fermentation liquor to obtain a mixed crude solution of cordycepin and pentostatin; centrifuging the filtered concentrated solution with automatic scraper centrifuge for desolventizing to obtain waste such as fruiting body; pumping the prepared mixed crude solution of cordycepin and pentostatin into a membrane nanofiltration concentration unit for concentration to prepare a mixed concentrated solution of cordycepin and pentostatin and wastewater; pumping the prepared cordycepin and pentostatin mixed concentrated solution into an automatic scraper centrifuge for centrifugation and desolventization to prepare a cordycepin and pentostatin mixed crude product filter cake A; sending the prepared mixed crude product filter cake A of the cordycepin and the pentostatin into a No.1 extraction kettle, pumping a lipophilic solvent which is 3 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, pumping deionized water which is 6 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, extracting the mixture for 2 to 3 times, and collecting and combining the extract liquor; performing liquid-liquid centrifugal extraction on the extract liquor, and separating an organic phase and a water phase to obtain an organic phase containing pentostatin and a water phase containing cordycepin; filtering the organic phase by a bag filter, concentrating the filtrate by a No.1 concentrator, centrifugally desolventizing, and recovering the solvent to prepare a crude pentostatin dry filter cake A; pumping the water phase into a flocculation precipitation tank, adding a flocculating agent, stirring for 10-30 min, precipitating for 3-6 h, filtering by a bag filter, concentrating by a No. 2 concentrator, centrifuging, desolventizing, and recovering the solvent to obtain a cordycepin crude product dry filter cake A; respectively feeding the cordycepin crude product dry filter cake A and the pentostatin crude product dry filter cake A into an extraction kettle of No. 2 or No. 3 3000L, respectively using an acetone solvent with the weight 3 times of that of the dry filter cake, adding a decolorizing agent with the weight 0.03 time of that of the dry filter cake, carrying out extraction and decolorization for 2-3 times at the temperature of 40-60 ℃, filtering, and combining extract liquor to respectively prepare cordycepin and pentostatin extract liquor; pumping the cordycepin extract and the pentostatin extract into a concentrator for concentration, and recovering the solvent to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution; pumping the cordycepin crude product concentrated solution and the pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and solvent recovery to respectively prepare a cordycepin crude product filter cake B and a pentostatin crude product filter cake B; mixing the cordycepin crude product filter cake B with the crystallized cordycepin mother liquor filter cake, putting into a No.1 column feeding solution preparation tank, and dissolving with 30-60% ethanol by volume fraction to prepare column feeding solution; the solute concentration of the upper column liquid is 5-10 mg/L; the prepared upper column liquid pump is used for automatically controlling the adsorption of a macroporous resin column system, and the adsorption flow rate is 2-3 BV; eluting with 30-60% ethanol; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent; pumping the adsorption effluent and the eluate into a concentrator respectively for concentration, and recovering the solvent to obtain a cordycepin crude product concentrated solution; pumping the obtained cordycepin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing, and recovering ethanol solvent to obtain cordycepin crude product filter cake; mixing a crude pentostatin filter cake and a crystallized pentostatin mother solution dry filter cake A, B, putting into a No. 2 column feed preparation tank, and dissolving with ethanol with the volume fraction of 70-80% to prepare column feed; the solute concentration of the upper column liquid is 5-15 mg/L; pumping the prepared upper column liquid into a No. 2 macroporous resin column system for adsorption, wherein the flow rate of the upper column liquid is 2-3 BV; eluting with deionized water and 70-80% ethanol by volume; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent; adsorbing the effluent and the eluent by a No. 2 macroporous resin column system, pumping the effluent and the eluent into a concentrator for concentration, and recovering the solvent to prepare a crude pentostatin concentrated solution; pumping the obtained pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering an ethanol solvent to obtain a pentostatin crude product filter cake; feeding the obtained cordycepin filter cake into a No.1 crystallization kettle, dissolving with a solvent, recrystallizing at low temperature, and filtering to obtain cordycepin wet crystal and mother liquor; pumping the prepared cordycepin mother liquor into a concentrator for concentration, centrifuging for desolventizing, and recovering the solvent to prepare a cordycepin mother liquor dry filter cake; feeding the obtained crude pentostatin filter cake into a freezing crystallization kettle, dissolving the crude pentostatin filter cake by using a solvent, crystallizing at a low temperature, and filtering crystals to obtain pentostatin crude crystals and pentostatin mother liquor; pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a dry filter cake A of the pentostatin mother liquor; feeding the obtained crude pentostatin crystal into a No. 2 crystallization kettle, dissolving the crude pentostatin crystal by using a solvent, recrystallizing at low temperature, and filtering the crystal to obtain a wet pentostatin crystal and a mother solution; pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a pentostatin mother liquor dry filter cake B; combining the pentostatin mother solution dry filter cake B with the pentostatin crude product filter cake to prepare upper column solution; the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake are sent into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare cordycepin crystal powder with the content of more than or equal to 98 percent and cordycepin powder with low content; feeding the prepared pentostatin wet crystal into a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare pentostatin crystal powder with the content of more than or equal to 98 percent; respectively feeding the prepared cordycepin crystal powder with the concentration of more than or equal to 98 percent and the prepared cordycepin powder with the low concentration into a batch V-shaped mixer for batch mixing to prepare a cordycepin crystal powder product with the concentration of more than or equal to 98 percent and a cordycepin powder product with the low concentration, and feeding the prepared pentostatin crystal powder with the concentration of more than or equal to 98 percent into the batch V-shaped mixer for batch mixing to prepare a pentostatin crystal powder product with the concentration of more than or equal to 98 percent.

The separation, mutagenesis and preservation method of the cordyceps militaris strain and the streptomyces antibioticus comprises the following steps:

a. culturing cordyceps militaris: selecting high-yield, high-quality and early-maturing cordyceps militaris sporocarp, carrying out surface disinfection by using 75% alcohol, cleaning surface liquid medicine by using sterile water, suspending the liquid medicine above a container filled with a comprehensive culture medium, carrying out static culture at the temperature of 28-30 ℃ for 5-7 days, picking single or multiple colonies in an inoculation box when starburst-shaped cordyceps militaris colonies appear on the surface of the culture medium, placing the colonies in a test tube inclined plane culture medium for culture, and purifying after cordyceps militaris mycelia are fully distributed on the inclined plane;

b. culturing antibiotic streptomycete: taking a strain with a preservation number as follows: the CGMCC No.1349 streptomyces antibioticus is subjected to static culture at 28-30 ℃ for 5-7 days, when streptomyces antibioticus colonies appear on the surface of a culture medium, a single colony or a plurality of colonies are selected from an inoculation box and placed in a test tube slant culture medium for culture, and the cordyceps militaris mycelia are purified after being fully distributed on the slant;

c. respectively placing a cordyceps militaris bacterium culture medium and a streptomyces antibioticus culture medium in a constant-temperature incubator at 28-30 ℃ for culture, observing at regular time, sequentially transferring newly grown hyphae to a new culture medium by using an inoculating needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane for preservation of two strains;

d. preparation of bacterial suspension: respectively taking several dominant bacterial strain slopes cultured for one week, respectively adding physiological saline containing synergistic isonicotin, scraping thallus on the slopes by using an inoculating loop, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to resist oscillation, filtering the mixture on a funnel with a layer of sterile mirror wiping paper after oscillation to obtain monospore suspension of the bacterial strain, counting spores by using a hemocytometer, and adjusting the concentration of the spores to be about 10⁶Per ml;

e. ultraviolet mutagenesis: respectively taking 5ml of prepared cordyceps militaris and streptomyces antibioticus monospore suspension into a sterile plate with the diameter of 9mm, opening a 15W ultraviolet lamp for preheating, putting the plate containing the bacterial suspension on a magnetic stirrer at a certain distance from a lamp tube, opening a plate cover, stirring and irradiating to ensure that cells uniformly absorb ultraviolet light waves, operating under a red light background to avoid light restoration, immediately transferring the thalli subjected to ultraviolet mutagenesis into the sterile test tube, immersing the thalli in ice water for 30min to inhibit enzyme activity and inhibit restoration, and adding the bacterial suspension subjected to ice bath after irradiation into a PDA culture medium for post-culture according to the principle of delay phenomenon to improve the mutation rate;

f. carrying out normal-pressure room-temperature plasma mutagenesis: respectively taking 10 mu L of spore suspension with proper concentration of ultraviolet mutagenized cordyceps militaris and streptomyces antibioticus, uniformly coating the spore suspension on a sterilized mutagenesis cup, then mutagenizing under the optimal condition, putting the mutagenesis cup into a 2mL centrifuge tube after mutagenesis, adding 1mL of sterile ultrapure water, coating the mutagenesis cup on an ISP2 culture medium after shaking for 1min, culturing for 6-8 days at 30 ℃, observing colonies with color, texture and character changed in brightness difference compared with a control when the colonies grow to be about 0.5cm in diameter, screening, and selecting the strains with the highest yield for low-temperature preservation for subsequent culture.

In the step a, the ethanol accounts for 75% of the volume fraction;

in step c, the cordyceps militaris potato culture medium comprises: 20-30 g of glucose, 5-8 g of peptone, 200mL of 20% potato leachate, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 4-6 g of yeast powder, 1000mL of distilled water and 6.5-7 of PH, wherein the streptomyces antibioticus culture medium comprises: 4-6 g of glucose, 4-6 g of yeast powder, 8-10 g of malt extract powder, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 20g of agar, 1000mL of distilled water and 6.5-7 of PH;

in the step e, the ultraviolet irradiation time is respectively 0min, 0.5min, 1min, 1.5min, 2min, 2.5min and 5 min;

in the step f, culturing the ISP2 culture medium for 6-8 days at 30 ℃;

in the steps a to f, the streptomyces antibioticus strain is preserved in China general microbiological culture collection center (CGMCC), and the preservation number is as follows: CGMCC No. 1349.

i. the liquid shake flask strain culture medium is PDA, PH7, initial PH value is 5.0, 250mL are prepared totally, split charging 250mL triangular flask, each 30mL, sterilizing at 121 deg.C for 30 minutes, cooling to room temperature for standby;

ii. Preparing liquid shake flask strains: separately packaging Cordyceps militaris or Streptomyces antibioticus liquid culture medium into 500ml triangular flasks, adding 200ml culture solution into each flask, sealing with 12 layers of gauze and a layer of kraft paper, sterilizing for 13-30 min, inoculating the mother strain into the triangular flasks with an inoculating hook under aseptic condition, allowing each slant to connect more than 10 bottles, performing shake culture at 28 deg.C for about 5-7 days, and allowing uniform pellets to form for inoculation,

iii, preparing a seed culture medium according to a formula standard, respectively pumping the seed culture medium into No. 1-8 500L first-stage seed culture tanks, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

iv, primary seed culture: after the first-stage seed culture tank is sterilized, when the temperature of a culture medium in the tank is reduced to 28-30 ℃, respectively inoculating the triangular flask fermentation liquor into a No. 1-8 500L first-stage seed fermentation tank through an aseptic inoculation system, performing seed culture by using the seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 28-30 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7.2, the rotation speed of the seed tank is 100r/min and the ventilation amount is 0.8-1.4 vvm/min for 0-2 days, after 2 days, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min, and collecting seed liquid after the fermentation culture,

v, preparing a seed culture medium according to a formula standard, respectively filling the seed culture medium into No. 1-8 1000L secondary seed fermentation tanks, and sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

vi, secondary seed culture: after the second-stage seed culture tank is sterilized, when the temperature is reduced to 25-28 ℃, respectively inoculating the first-stage seed fermentation liquor fermented for 5-7 days into a 1000L second-stage seed fermentation tank with the number of 1-8 through an aseptic inoculation system, performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 25-28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 150r/min and the ventilation amount is 0.8-1.4 vvm/min after 2 days, and collecting seed liquid after the fermentation culture,

vii, accurately weighing the culture medium materials in batches according to the formula, then putting the culture medium materials into a 40000L multiplied by 3 culture medium preparation tank, pumping purified water according to the formula, and stirring for 30 min;

viii, pumping the fermentation medium into a No. 1-10 100000L fermentation single tank or a No. 1-3 medium liquid supplementing tank in batches, filling the fermentation medium into the tank by using a filling coefficient of 0.75, and sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

ix, after single-tank sterilization is finished, cooling a culture medium in the tank to 28 ℃ by using chilled water, respectively inoculating the secondary seed fermentation liquor fermented for 18 hours from 1 to 8 to a 100000L fermentation tank from 1 to 10 through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1 to 5 percent, the fermentation temperature is 28 ℃, the tank pressure is 0.03MPa, the fermentation culture is carried out for 120 hours under the conditions of pH7, the stirring rotation speed is 130r/min, the ventilation amount is 0.8 to 1.4vvm/min, and after 180 hours, the fermentation tank rotation speed is 100r/min, and the ventilation amount is 0.8 to 1.4 vvm/min;

x, supplementing liquid in real time according to the standard of the culture medium amount in the fermentation period; and when the culture medium feeding reaches the standard and the fermentation time reaches the standard, ending the fermentation to prepare a fermentation mixed solution of the fruiting body, the cordycepin and the pentostatin.

In the step i, the cordyceps militaris potato culture medium (modified by PDA) is as follows: 20-30 g of glucose, 5-8 g of peptone, 200mL of 20% potato extract, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 4-6 g of yeast powder, 1000mL of distilled water and 6.5-7 of pH; the streptomyces antibioticus culture medium (modified by PDA) is as follows: 4-6 g of glucose, 4-6 g of yeast powder, 8-10 g of malt extract powder, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 20g of agar, 1000mL of distilled water and 6.5-7 of PH;

in step ii, after inoculation, carrying out shake cultivation at 28 ℃ for about 5-7 days at a rotation speed of 120 revolutions per minute;

in the step iv, the fermentation temperature is 28-30 ℃, the tank pressure is 0.10Mpa, the pH is 7.2, the fermentation culture is carried out for 5-7 days, the rotation speed of the seeding tank is 100r/min and the ventilation volume is 0.8-1.4 vvm/min in 0-2 days, and after 2 days, the rotation speed of the seeding tank is 120r/min and the ventilation volume is 0.8-1.4 vvm/min;

in the step vi, the inoculation amount is 1-5%, the fermentation temperature is 25-28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is carried out for 5-7 days under the condition that the pH is 7, the rotating speed of a seeding tank is 120r/min for 0-2 days, the ventilation volume is 0.8-1.4 vvm/min, and after 2 days, the rotating speed of the seeding tank is 150r/min, and the ventilation volume is 0.8-1.4 vvm/min;

in the step vi, the inoculation amount is 1-5%, the fermentation temperature is 25-28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is carried out for 5-7 days under the condition that the pH is 7, the rotating speed of a seeding tank is 120r/min and the ventilation volume is 0.8-1.4 vvm/min after 0-2 days, the rotating speed of the seeding tank is 150r/min and the ventilation volume is 0.8-1.4 vvm/min after 2 days, and seed liquid is collected after the fermentation culture;

in step vii, the optimized medium formula is as follows: 90g of cane sugar, 25-30 g of bean curd jelly, 10g of peptone, 18g of yeast extract powder, 0.3mg of L-phenylalanine, 0.05mg of sodium acetate, 3-5 g of sodium chloride, 0.5-1 g of magnesium sulfate, 0.02mg of sodium benzoate, 0.1mg of arachidonic acid, 0.05mg of cinnamic acid, 40g of zinc acetate, 1000mL of distilled water and 7 of PH;

in the steps i, iii, v and viii, the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

in the step ix, the inoculation amount is 1-5%, the fermentation temperature is 28 ℃, the tank pressure is 0.03Mpa, the fermentation culture is carried out for 120h under the conditions that the pH is 7, the stirring rotating speed is 130r/min, the ventilation volume is 0.8-1.4 vvm/min, and after 180h, the rotating speed of a fermentation tank is 100r/min, and the ventilation volume is 1 vvm/min; the fermentation time is 240 h;

in the step x, when the pH value of the fermentation liquor is lower than 6.2, starting to introduce ammonia, wherein the pH value is 6.3-6.5 before 100h, the pH value is 6.2-6.3 after 100h, and stopping introducing ammonia 8h before tank placing; the liquid supplementing standard is that total sugar is supplemented according to the residual sugar value of the fermentation liquid, the residual sugar value is controlled to be 8.0-9.0% before 100 hours of fermentation, the residual sugar value is controlled to be 7.0-8.0% after 100 hours-150 hours of fermentation, and the residual sugar value is controlled to be 5.0% after 150 hours and 6 hours before tank discharge.

The method for purifying cordycepin and pentostatin comprises the following steps:

n1, pressing the prepared mycelium and fruiting body, cordycepin and pentostatin fermentation mixed solution into a No. 1-2 40000L fermentation liquid temporary storage tank;

n2, pressing the prepared fermentation liquor into a coarse filtration device for filtration to prepare mycelium, a fruiting body filter cake and filtrate;

n3, pumping the filtrate into a membrane ultrafiltration unit for ultrafiltration, and filtering out impurities in the fermentation liquor to obtain a mixed crude solution of cordycepin and pentostatin;

n4, centrifuging and desolventizing the filtered concentrated solution by using an automatic scraper centrifuge to prepare wastes such as sporocarp and the like;

n5, pumping the prepared mixed crude solution of the cordycepin and the pentostatin into a membrane nanofiltration concentration unit for concentration to prepare a mixed concentrated solution of the cordycepin and the pentostatin and wastewater;

n6, pumping the prepared cordycepin and pentostatin mixed concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing to prepare a cordycepin and pentostatin mixed crude product filter cake A;

n7, feeding the prepared mixed crude product filter cake A of the cordycepin and the pentostatin into a No.1 extraction kettle, pumping a lipophilic solvent which is 3 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, pumping deionized water which is 6 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, extracting the mixture for 2 to 3 times, and collecting and combining extract liquor;

n8, carrying out liquid-liquid centrifugal extraction on the extract liquor, and separating an organic phase and a water phase to obtain an organic phase containing pentostatin and a water phase containing cordycepin;

n9, filtering the organic phase by a bag filter, concentrating the filtrate by a No.1 concentrator, centrifuging, desolventizing, and recovering the solvent to prepare a crude pentostatin dry filter cake A;

n10, pumping the water phase into a 6000L flocculation precipitation tank, adding a flocculating agent, stirring for 10-30 min, precipitating for 6h, filtering by a bag filter, concentrating the filtrate by a No. 2 concentrator, desolventizing by a concentrated solution centrifuge, and recovering the solvent to obtain a cordycepin crude product dry filter cake A;

n11, respectively feeding the cordycepin crude product dry filter cake A and the pentostatin crude product dry filter cake A into an extraction kettle of No. 2 or No. 3 3000L, respectively using an acetone solvent with the weight 3 times of that of the dry filter cake, adding a decolorizing agent with the weight 0.03 time of that of the dry filter cake, carrying out extraction and decolorization for 2-3 times at the temperature of 40-60 ℃, filtering, combining extract liquor, and respectively preparing cordycepin extract liquor and pentostatin extract liquor;

n12, pumping the cordycepin extract and the pentostatin extract into a concentrator for concentration, and recovering the solvent to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution;

n13, pumping the cordycepin crude product concentrated solution and the pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and solvent recovery to respectively prepare a cordycepin crude product filter cake B and a pentostatin crude product filter cake B;

n14, combining the cordycepin crude product filter cake B with the crystallized cordycepin mother liquor filter cake, putting into a No.1 column feeding solution preparation tank, and dissolving with 30-60% ethanol by volume fraction to prepare column feeding solution; the solute concentration of the upper column liquid is 5-10 g/L;

n15, fully automatically controlling the adsorption of the prepared upper column liquid pump by a macroporous resin column system, wherein the adsorption flow rate is 2-3 BV; eluting with 30-60% ethanol; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

n16, pumping the adsorption effluent and the eluent into a concentrator respectively for concentration, and recovering the solvent to prepare a cordycepin crude product concentrated solution;

n17, pumping the prepared cordycepin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing, and recovering an ethanol solvent to prepare a cordycepin crude product filter cake;

n18, mixing a crude pentostatin filter cake with a crystallized dry pentostatin mother solution filter cake A, B, putting into a No. 2 column solution preparation tank, and dissolving with ethanol with the volume fraction of 70-80% to prepare column solution; the solute concentration of the upper column liquid is 5-15 g/L;

n19, pumping the prepared column feeding liquid into a No. 2 macroporous resin column system for adsorption, wherein the flow rate of the column feeding liquid is 2-3 BV; eluting with deionized water and 70-80% ethanol by volume; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

n20, adsorbing the effluent and the eluent by a No. 2 macroporous resin column system, pumping the effluent and the eluent into a concentrator for concentration, and recovering the solvent to prepare a crude pentostatin concentrated solution;

n21, pumping the prepared crude pentostatin concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing, and recovering an ethanol solvent to prepare a crude pentostatin filter cake;

n22, feeding the prepared cordycepin filter cake into a No.1 crystallization kettle, dissolving the cordycepin filter cake with a solvent, recrystallizing at a low temperature, and filtering crystals to obtain cordycepin wet crystals and a mother solution;

n23, pumping the prepared cordycepin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a cordycepin mother liquor dry filter cake;

n24, feeding the prepared crude pentostatin filter cake into a freezing crystallization kettle, dissolving the crude pentostatin filter cake by using a solvent, crystallizing at a low temperature, and filtering crystals to obtain pentostatin crude crystals and pentostatin mother liquor;

n25, pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering a solvent to prepare a pentostatin mother liquor dry filter cake A;

n26, feeding the prepared crude pentostatin crystals into a No. 2 crystallization kettle, dissolving the crude pentostatin crystals by using a solvent, recrystallizing at a low temperature, and filtering the crystals to obtain wet pentostatin crystals and mother liquor;

n27, pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering a solvent to prepare a pentostatin mother liquor dry filter cake B;

n28, combining the dry filter cake B of the pentostatin mother solution with the filter cake of the pentostatin crude product to prepare upper column solution;

n29, feeding the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain cordycepin crystal powder with the content of more than or equal to 98 percent and cordycepin powder with low content;

n30, feeding the prepared pentostatin wet crystal into a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare pentostatin crystal powder with the content of more than or equal to 98 percent;

n31, respectively sending the prepared cordycepin crystal powder with the content of more than or equal to 98 percent and the prepared cordycepin powder with the low content into a batch V-shaped mixer for batch mixing to prepare a cordycepin crystal powder product with the content of more than or equal to 98 percent and a cordycepin powder product with the low content,

and N32, feeding the prepared pentostatin crystal powder with the concentration of more than or equal to 98 percent into a batch V-shaped mixer for batch mixing to prepare a pentostatin crystal powder product with the concentration of more than or equal to 98 percent.

In the step N2, the coarse filtration equipment is an automatic scraper centrifuge, a bag filter or a plate and frame filter press; the productivity is 4000L/h;

in the step N3, the membrane ultrafiltration unit is a full-automatic control ultrafiltration unit, and the capacity is 4000L/h;

in step N4, the model of the automatic scraper centrifuge is PGZ-1000;

in the step N5, the membrane nanofiltration concentration unit is a full-automatic control nanofiltration concentration unit, and the capacity is 4000L/h;

in step N6, the model of the automatic scraper centrifuge is PGZ-1000;

in step N7, the solvent is dichloromethane or ethyl acetate solvent; the solvent extraction temperature is 40-60 ℃;

in the step N9, the liquid-liquid centrifugal extraction equipment is CTL350-N, and the capacity is 1000-3000L/h;

in step N10, the flocculant is a ZTC1+1 natural clarifier; the flocculation precipitation temperature is 60-80 ℃; the precipitation time is 4-6 h;

in step N11, the decolorant is activated clay or activated carbon;

in the steps N9, N10, N12, N16, N20, N24, N29 and N31, the concentrator is an MVR evaporator or a full-electric evaporator or a membrane concentrator set; the concentration temperature of the MVR evaporator or the full-electric evaporator is 55-60 ℃; the vacuum degree is 0.07-0.085 Mpa; the capacity of the equipment is 500-4000L/h;

in step N13, the model of the automatic scraper centrifuge is PGZ-1000;

in the step N14, the solvent is ethanol or methanol with the volume fraction of 30-60% and the PH of 4-6; the solute concentration of the upper column liquid is 5-12 g/L;

in the step N15, the macroporous resin system is a full-automatic control macroporous resin column system; the macroporous resin is D001, HD-8, 711 or ML-7 macroporous resin; the macroporous resin has an adsorption flow rate of 2-3 BV; the elution solvent is ethanol with the volume fraction of 30-60%; the flow rate of the eluent is 3-5 BV;

in the step N18, the solute concentration of the upper column liquid is 5-15 g/L;

in step N19, the macroporous resin is HZ202 or D201 or D241 or D261; the adsorption flow rate is 3-4 BV/h; respectively using deionized water, PH 4-6 and 85-95% ethanol or methanol in volume fraction as eluent; the elution flow rate of the deionized water is 2-3 BV/h; the gradient elution flow rate of the ethanol or the methanol is 3-5 BV/h;

in the step N22, the crystallization solvent is ethanol or methanol with the volume fraction of 30-60%; stirring for 10-30 min each time; the crystallization temperature is 40-50 ℃→ 5 ℃; the crystallization time is 12-24 h each time; the crystallization kettle is a full-automatic control 8000L crystallization kettle;

in the step N24, the crystallization solvent is ethanol or methanol with volume fraction of 75-85%; stirring for 10-30 min each time; the crystallization temperature is 40-50 ℃→ -15 ℃; the crystallization time is 12-24 h each time; the crystallization kettle is a full-automatic control 8000L crystallization kettle;

in the step N26, the crystallization solvent is ethanol or methanol or acetone with volume fraction of 75-85%; stirring for 10-30 min each time; the crystallization temperature is 40-50 ℃→ 5 ℃; the crystallization time is 12-24 h each time; the crystallization kettle is a full-automatic control 8000L crystallization kettle;

in the step N28, the mother liquor extract is a crystallized mother liquor extract with the cordycepin content more than or equal to 50%;

in the steps N29 and N30, the supercritical drying device is a 200L supercritical drying device with double-extraction double-separation electric heating full-automatic control; supercritical drying and residual solvent removal pressure of 20-30 Mpa, drying temperature of 40-60 ℃, CO₂The flow rate is 1500-4500L/h, and the drying time is 360-480 min;

in the step N31, the batch V-shaped mixer is V-1000; the batch mixing time is 30-60 min.

According to one embodiment of the application, the industrial preparation method for producing cordycepin and pentostatin by combined fermentation comprises the following steps:

culturing cordyceps militaris: selecting high-yield, high-quality and early-maturing cordyceps militaris sporocarp, carrying out surface disinfection by using 75% alcohol, cleaning surface liquid medicine by using sterile water, suspending the liquid medicine above a container filled with a comprehensive culture medium, carrying out static culture at the temperature of 28-30 ℃ for 5-7 days, and picking single or multiple colonies in an inoculation box to place the single or multiple colonies in a test tube slant culture medium for culture when starburst-shaped cordyceps militaris colonies appear on the surface of the culture medium. Purifying after the cordyceps militaris hyphae are covered with the inclined plane; culturing antibiotic streptomycete: taking a strain with a preservation number as follows: the antibiotic streptomycete CGMCC No.1349 is statically cultured for 5 to 7 days at the temperature of between 28 and 30 ℃, and when the antibiotic streptomycete colony appears on the surface of a culture medium, the antibiotic streptomycete is inoculated in an inoculation boxPicking single or multiple colonies to put the colonies in a test tube slant culture medium for culture. Purifying after the cordyceps militaris hyphae are covered with the inclined plane; the culture medium of the cordyceps militaris and the culture medium of the streptomyces antibioticus are respectively put into a constant-temperature incubator at the temperature of 28-30 ℃ for culture. Observing at regular time, sequentially transferring the newly grown hyphae to a new culture medium by using an inoculating needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane to preserve two strains; preparation of bacterial suspension: taking several inclined planes of the dominant strains which are cultured for one week respectively, adding physiological saline containing synergistic isonicotin respectively, scraping off thalli on the inclined planes by using an inoculating ring, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to resist and shake, and filtering the mixture by using a funnel with a layer of sterile mirror wiping paper after shaking to obtain a monospore suspension of the strains. Counting spores with a hemocytometer (hemocytometer) to adjust the spore concentration to about 10⁶Per ml; ultraviolet mutagenesis: respectively taking 5ml of the prepared cordyceps militaris and streptomyces antibioticus monospore suspension into an aseptic plate with the diameter of 9 mm. The 15W UV lamp was turned on to preheat and stabilize the light. The dish containing the bacterial suspension is placed on a magnetic stirrer, is away from a lamp tube by a certain distance, the dish cover is opened, and the cell is stirred and irradiated at the same time, so that the cell can uniformly absorb ultraviolet light waves. The top was operated in a red background, avoiding photorepair. The thallus after ultraviolet mutagenesis is immediately transferred into a sterile test tube and immersed in ice water for 30min to inhibit enzyme activity and repair. According to the principle of delay phenomenon, adding the irradiated bacterial suspension subjected to ice bath into a PDA culture medium for post-culture so as to improve mutation rate; ARTP (atmospheric room temperature plasma) mutagenesis: respectively taking 10 mu L of spore suspension with proper concentration of ultraviolet mutagenized cordyceps militaris and streptomyces antibioticus, uniformly coating the spore suspension on a sterilized mutagenesis cup, then mutagenizing under the optimal condition, putting the mutagenesis cup into a 2mL centrifuge tube after mutagenesis, adding 1mL of sterile ultrapure water, shaking for 1min, coating the mixture on an ISP2 culture medium, and culturing for 6-8 days at 30 ℃. And observing the colony with color, grain and character changed differently from the contrast when the colony grows to the diameter of about 0.5cm, and screening. The strain with the highest yield is selected for preservation and used for subsequent culture.

the liquid shake flask strain culture medium is PDA, PH7. The initial pH was 5.0. Preparing 250mL of the mixture, subpackaging the mixture into 250mL of triangular bottles, sterilizing the mixture for 30 minutes at 121 ℃ in each bottle, and cooling the mixture to room temperature for later use; preparing liquid shake flask strains: separately packaging Cordyceps militaris or Streptomyces antibioticus liquid culture medium into 500ml triangular flasks, adding 200ml culture solution into each flask, and sealing with 12 layers of gauze and one layer of kraft paper. After 13-30 minutes of sterilization, the mother seeds are inoculated into a triangular flask by an inoculation hook under the aseptic condition, and each inclined plane can be connected with more than 10 bottles. After inoculation, carrying out shake cultivation at 28 ℃ for about 5-7 days, and after uniform small balls are formed, the inoculation can be carried out. Preparing a seed culture medium according to a formula standard, respectively pumping the seed culture medium into No. 1-8 500L first-stage seed culture tanks, and sterilizing by using a tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min; first-order seed culture: after the first-stage seed culture tank is sterilized, when the temperature of a culture medium in the tank is reduced to 28-30 ℃, respectively inoculating the triangular flask fermentation liquor into a No. 1-8 500L first-stage seed fermentation tank through an aseptic inoculation system, performing seed culture by using the seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 28-30 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7.2, the rotation speed of the seed tank is 100r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 2 days, and collecting seed liquid after the fermentation culture. Preparing a seed culture medium according to a formula standard, respectively filling the seed culture medium into No. 1-8 1000L secondary seed fermentation tanks, and sterilizing by using a retort steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min; secondary seed culture: after the second-stage seed culture tank is sterilized, when the temperature is reduced to 25-28 ℃, respectively inoculating the first-stage seed fermentation liquor fermented for 5-7 days into a 1000L second-stage seed fermentation tank with the number of 1-8 through an aseptic inoculation system, performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 25-28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 150r/min and the ventilation amount is 0.8-1.4 vvm/min after 2 days, and collecting seed liquor after the fermentation culture. Accurately weighing the culture medium materials according to the formula in batches, putting the culture medium materials into a 40000L multiplied by 3 culture medium preparation tank, pumping purified water according to the formula, and stirring for 30 min; pumping fermentation medium into No. 1-10 100000L fermentation single tank or No. 1-3 medium liquid supplementing tank in batches, filling the tank with coefficient of 0.7, sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min; after single-tank sterilization is finished, cooling culture medium in the tank to 28 ℃ by using chilled water, respectively inoculating the secondary seed fermentation liquor fermented for 18 hours from No.1 to No. 8 to a No.1 100000L fermentation tank through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 28 ℃, the tank pressure is 0.03MPa, the pH value is 7, the fermentation culture is performed for 120 hours, the stirring rotation speed is 130r/min, the ventilation amount is 0.8-1.4 vvm/min, and after 180 hours, the fermentation tank rotation speed is 100r/min, and the ventilation amount is 0.8-1.4 vvm/min; supplementing liquid in real time according to the culture medium quantity standard in the fermentation period; and when the culture medium feeding reaches the standard and the fermentation time reaches the standard, ending the fermentation to prepare a fermentation mixed solution of the fruiting body, the cordycepin and the pentostatin.

The steps for purifying cordycepin and pentostatin are as follows:

pressing the prepared fruiting body, cordycepin and pentostatin fermentation mixed solution into a No. 1-2 40000L fermentation liquid temporary storage tank; pressing the obtained fermentation liquid into coarse filtration equipment, and filtering to obtain mycelium, fruiting body filter cake and filtrate; pumping the filtrate into a membrane ultrafiltration unit for ultrafiltration, and filtering out impurities in the fermentation liquor to obtain a mixed crude solution of cordycepin and pentostatin; centrifuging the filtered concentrated solution with automatic scraper centrifuge for desolventizing to obtain waste such as fruiting body; pumping the prepared mixed crude solution of cordycepin and pentostatin into a membrane nanofiltration concentration unit for concentration to prepare a mixed concentrated solution of cordycepin and pentostatin and wastewater; pumping the prepared cordycepin and pentostatin mixed concentrated solution into an automatic scraper centrifuge for centrifugation and desolventization to prepare a cordycepin and pentostatin mixed crude product filter cake A; sending the prepared mixed crude product filter cake A of the cordycepin and the pentostatin into a No.1 extraction kettle, pumping a lipophilic solvent which is 3 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, pumping deionized water which is 6 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, extracting the mixture for 2 to 3 times, and collecting and combining the extract liquor; performing liquid-liquid centrifugal extraction on the extract liquor, and separating an organic phase and a water phase to obtain an organic phase containing pentostatin and a water phase containing cordycepin; filtering the organic phase by a bag filter, concentrating the filtrate by a No.1 concentrator, centrifugally desolventizing, and recovering the solvent to prepare a crude pentostatin dry filter cake A; pumping the water phase into a flocculation precipitation tank, adding a flocculating agent, stirring for 10-30 min, precipitating for 3-6 h, filtering by a bag filter, concentrating by a No. 2 concentrator, centrifuging, desolventizing, and recovering the solvent to obtain a cordycepin crude product dry filter cake A; respectively feeding the cordycepin crude product dry filter cake A and the pentostatin crude product dry filter cake A into an extraction kettle of No. 2 or No. 3 3000L, respectively using an acetone solvent with the weight 3 times of that of the dry filter cake, adding a decolorizing agent with the weight 0.03 time of that of the dry filter cake, carrying out extraction and decolorization for 2-3 times at the temperature of 40-60 ℃, filtering, and combining extract liquor to respectively prepare cordycepin and pentostatin extract liquor; pumping the cordycepin extract and the pentostatin extract into a concentrator for concentration, and recovering the solvent to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution; pumping the cordycepin crude product concentrated solution and the pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and solvent recovery to respectively prepare a cordycepin crude product filter cake B and a pentostatin crude product filter cake B; mixing the cordycepin crude product filter cake B with the crystallized cordycepin mother liquor filter cake, putting into a No.1 column feeding solution preparation tank, and dissolving with 30-60% ethanol by volume fraction to prepare column feeding solution; the solute concentration of the upper column liquid is 5-10 mg/L; the prepared upper column liquid pump is used for automatically controlling the adsorption of a macroporous resin column system, and the adsorption flow rate is 2-3 BV; eluting with 30-60% ethanol; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent; pumping the adsorption effluent and the eluate into a concentrator respectively for concentration, and recovering the solvent to obtain a cordycepin crude product concentrated solution; pumping the obtained cordycepin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing, and recovering ethanol solvent to obtain cordycepin crude product filter cake; mixing a crude pentostatin filter cake and a crystallized pentostatin mother solution dry filter cake A, B, putting into a No. 2 column feed preparation tank, and dissolving with ethanol with the volume fraction of 70-80% to prepare column feed; the solute concentration of the upper column liquid is 5-15 mg/L; pumping the prepared upper column liquid into a No. 2 macroporous resin column system for adsorption, wherein the flow rate of the upper column liquid is 2-3 BV; eluting with deionized water and 70-80% ethanol by volume; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent; adsorbing the effluent and the eluent by a No. 2 macroporous resin column system, pumping the effluent and the eluent into a concentrator for concentration, and recovering the solvent to prepare a crude pentostatin concentrated solution; pumping the obtained pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering an ethanol solvent to obtain a pentostatin crude product filter cake; feeding the obtained cordycepin filter cake into a No.1 crystallization kettle, dissolving with a solvent, recrystallizing at low temperature, and filtering to obtain cordycepin wet crystal and mother liquor; pumping the prepared cordycepin mother liquor into a concentrator for concentration, centrifuging for desolventizing, and recovering the solvent to prepare a cordycepin mother liquor dry filter cake; feeding the obtained crude pentostatin filter cake into a freezing crystallization kettle, dissolving the crude pentostatin filter cake by using a solvent, crystallizing at a low temperature, and filtering crystals to obtain pentostatin crude crystals and pentostatin mother liquor; pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a dry filter cake A of the pentostatin mother liquor; feeding the obtained crude pentostatin crystal into a No. 2 crystallization kettle, dissolving the crude pentostatin crystal by using a solvent, recrystallizing at low temperature, and filtering the crystal to obtain a wet pentostatin crystal and a mother solution; pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a pentostatin mother liquor dry filter cake B; combining the pentostatin mother solution dry filter cake B with the pentostatin crude product filter cake to prepare upper column solution; the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake are sent into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare cordycepin crystal powder with the content of more than or equal to 98 percent and cordycepin powder with low content; feeding the prepared pentostatin wet crystal into a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare pentostatin crystal powder with the content of more than or equal to 98 percent; and respectively feeding the prepared cordycepin crystal powder with the content of more than or equal to 98 percent and the prepared cordycepin powder with the low content into a batch V-shaped mixer for batch mixing to prepare a cordycepin crystal powder product with the content of more than or equal to 98 percent and a cordycepin powder product with the low content. And (3) feeding the prepared pentostatin crystal powder with the concentration of more than or equal to 98 percent into a batch V-shaped mixer for batch mixing to prepare a pentostatin crystal powder product with the concentration of more than or equal to 98 percent.

In the industrial preparation method for producing cordycepin and pentostatin by combined fermentation, the separation, mutagenesis and preservation method of the cordyceps militaris strain and the streptomyces antibioticus comprises the following steps:

a. culturing cordyceps militaris: selecting high-yield, high-quality and early-maturing cordyceps militaris sporocarp, carrying out surface disinfection by using 75% alcohol, cleaning surface liquid medicine by using sterile water, suspending the liquid medicine above a container filled with a comprehensive culture medium, carrying out static culture at the temperature of 28-30 ℃ for 5-7 days, and picking single or multiple colonies in an inoculation box to place the single or multiple colonies in a test tube slant culture medium for culture when starburst-shaped cordyceps militaris colonies appear on the surface of the culture medium. Purifying after the cordyceps militaris hyphae are covered on the inclined plane.

b. Culturing antibiotic streptomycete: taking a strain with a preservation number as follows: the antibiotic streptomyces CGMCC No.1349 is subjected to static culture at 28-30 ℃ for 5-7 days, and when the antibiotic streptomyces colonies appear on the surface of a culture medium, a single colony or a plurality of colonies are picked up in an inoculation box and placed in a test tube slant culture medium for culture. Purifying after the cordyceps militaris hyphae are covered on the inclined plane.

c. The culture medium of the cordyceps militaris and the culture medium of the streptomyces antibioticus are respectively put into a constant-temperature incubator at the temperature of 28-30 ℃ for culture. Observing at regular time, sequentially transferring the newly grown hyphae to a new culture medium by using an inoculating needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane to preserve two strains;

d. preparation of bacterial suspension: taking several inclined planes of the dominant strains which are cultured for one week respectively, adding physiological saline containing synergistic isonicotin respectively, scraping off thalli on the inclined planes by using an inoculating ring, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to resist and shake, and filtering the mixture by using a funnel with a layer of sterile mirror wiping paper after shaking to obtain a monospore suspension of the strains. Counting spores with a hemocytometer (hemocytometer) to adjust the spore concentration to about 10⁶Per ml;

e. ultraviolet mutagenesis: respectively taking 5ml of the prepared cordyceps militaris and streptomyces antibioticus monospore suspension into an aseptic plate with the diameter of 9 mm. The 15W UV lamp was turned on to preheat and stabilize the light. The dish containing the bacterial suspension is placed on a magnetic stirrer, is away from a lamp tube by a certain distance, the dish cover is opened, and the cell is stirred and irradiated at the same time, so that the cell can uniformly absorb ultraviolet light waves. The top was operated in a red background, avoiding photorepair. The thallus after ultraviolet mutagenesis is immediately transferred into a sterile test tube and immersed in ice water for 30min to inhibit enzyme activity and repair. According to the principle of delay phenomenon, adding the irradiated bacterial suspension subjected to ice bath into a PDA culture medium for post-culture so as to improve mutation rate;

f. ARTP (atmospheric room temperature plasma) mutagenesis: respectively taking 10 mu L of spore suspension with proper concentration of ultraviolet mutagenized cordyceps militaris and streptomyces antibioticus, uniformly coating the spore suspension on a sterilized mutagenesis cup, then mutagenizing under the optimal condition, putting the mutagenesis cup into a 2mL centrifuge tube after mutagenesis, adding 1mL of sterile ultrapure water, shaking for 1min, coating the mixture on an ISP2 culture medium, and culturing for 6-8 days at 30 ℃. And observing the colony with color, grain and character changed differently from the contrast when the colony grows to the diameter of about 0.5cm, and screening. The strain with the highest yield is selected for low-temperature preservation and used for subsequent culture.

In the step a, the ethanol accounts for 75% of the volume fraction;

in the step c, the ultraviolet irradiation time is respectively 0min, 0.5min, 1min, 1.5min, 2min, 2.5min and 5 min;

in the step c, the cordyceps militaris potato culture medium (modified by PDA) is as follows: 20-30 g of glucose, 5-8 g of peptone, 200mL of 20% potato extract, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 4-6 g of yeast powder, 1000mL of distilled water and 6.5-7 of pH. The streptomyces antibioticus culture medium (modified by PDA) is as follows: 4-6 g of glucose, 4-6 g of yeast powder, 8-10 g of malt extract powder, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 20g of agar, 1000mL of distilled water and 6.5-7 of PH.

In the step e, the culture time in the constant-temperature incubator is 3-7 days;

in the steps a to e, the cordyceps militaris is taken to northeast to produce a high-yield, high-quality and early-maturing cordyceps militaris sporocarp; the streptomyces antibioticus strain is preserved in China general microbiological culture collection center (CGMCC), and the preservation number is as follows: CGMCC No. 1349.

i. the liquid shake flask strain culture medium is PDA, PH7. The initial pH was 5.0. Preparing 250mL of the mixture, subpackaging the mixture into 250mL of triangular bottles, sterilizing the mixture for 30 minutes at 121 ℃ in each bottle, and cooling the mixture to room temperature for later use;

ii. Preparing liquid shake flask strains: separately packaging Cordyceps militaris or Streptomyces antibioticus liquid culture medium into 500ml triangular flasks, adding 200ml culture solution into each flask, and sealing with 12 layers of gauze and one layer of kraft paper. After 13-30 minutes of sterilization, the mother seeds are inoculated into a triangular flask by an inoculation hook under the aseptic condition, and each inclined plane can be connected with more than 10 bottles. After inoculation, carrying out shake cultivation at 28 ℃ for about 5-7 days, and after uniform small balls are formed, the inoculation can be carried out.

iv, primary seed culture: after the first-stage seed culture tank is sterilized, when the temperature of a culture medium in the tank is reduced to 28-30 ℃, respectively inoculating the triangular flask fermentation liquor into a No. 1-8 500L first-stage seed fermentation tank through an aseptic inoculation system, performing seed culture by using the seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 28-30 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7.2, the rotation speed of the seed tank is 100r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 2 days, and collecting seed liquid after the fermentation culture.

vi, secondary seed culture: after the second-stage seed culture tank is sterilized, when the temperature is reduced to 25-28 ℃, the first-stage seed fermentation liquor which is fermented for 5 days is respectively inoculated into a No. 1-8 1000L second-stage seed fermentation tank through an aseptic inoculation system, seed culture is carried out by using a seed culture medium, the inoculation amount is 1-5%, the fermentation temperature is 25-28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is carried out for 5-7 days under the condition that the pH is 7, the rotation speed of the seed tank is 120r/min and the ventilation rate is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 150r/min and the ventilation rate is 0.8-1.4 vvm/min after 2 days, and seed liquid is collected after the fermentation culture.

x, supplementing liquid in real time according to the standard of the culture medium amount in the fermentation period; when the culture medium supplement reaches the standard and the fermentation time reaches the standard, finishing the fermentation to prepare a fermentation mixed solution of the fruiting body, the cordycepin and the pentostatin;

in the step i, the formula of the cordyceps militaris liquid shake flask strain culture medium is as follows (unit is gram/liter): 20-30 g of glucose, 5-8 g of peptone, 200mL of 20% potato extract, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 4-6 g of yeast powder, 0.1mg of arachidonic acid, 0.05mg of cinnamic acid, 1000mL of distilled water and 6.5-7 of pH; the formula of the streptomyces antibioticus liquid shake flask strain culture medium is as follows (unit is gram/liter): 4-6 g of glucose, 4-6 g of yeast powder, 8-10 g of malt extract powder, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 20g of agar, 1000mL of distilled water and 6.5-7 of PH.

In the step ii, the shaking table culture temperature is 28 ℃, and the rotating speed is 120 r/min;

in step iii, the cordyceps militaris potato medium (modified by PDA): 20-30 g of glucose, 5-8 g of peptone, 200mL of 20% potato extract, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 4-6 g of yeast powder, 1000mL of distilled water and 6.5-7 of pH. The streptomyces antibioticus culture medium (modified by PDA) is as follows: 4-6 g of glucose, 4-6 g of yeast powder, 8-10 g of malt extract powder, 3-5 g of ammonium sulfate, 1-2 g of monopotassium phosphate, 1-2 g of magnesium sulfate, 20g of agar, 1000mL of distilled water and 6.5-7 of PH.

In steps iv and vi, the fermentation tank is a full-automatic control seed fermentation tank; the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the pH is 7-7.2, the fermentation culture is carried out for 18h, the rotating speed of the seeding tank is 120r/min for 0-9 h, the ventilation rate is 0.8-1.4 vvm/min, and after 9h, the rotating speed of the seeding tank is 180r/min, and the ventilation rate is 0.8-1.4 vvm/min;

in step vii, the optimized medium formula: 90g of cane sugar, 25-30 g of bean curd jelly, 10g of peptone, 18g of yeast extract powder, 0.3mg of L-phenylalanine, 0.05mg of sodium acetate, 3-5 g of sodium chloride, 0.5-1 g of magnesium sulfate, 0.02mg of sodium benzoate, 0.1mg of arachidonic acid, 0.05mg of cinnamic acid, 40g of zinc acetate, 1000mL of distilled water and 7 of PH; the materials are conveyed to a seed culture medium preparation tank by a vacuum material conveyor;

step ix, the fermentation tank is a full-automatic control fermentation tank; the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.03Mpa, and the pH value is 7-7.4; the stirring speed is 130r/min, the ventilation rate is 0.6-1.2 vvm/min, and after 16 hours, the fermentation tank speed is 150r/min, and the ventilation rate is 0.8-1.4 vvm/min;

in the step ix, the single-tank fermentation time is 240 hours;

in the step x, when the pH value of the fermentation liquor is lower than 6.2, starting to introduce ammonia, wherein the pH value is 6.3-6.5 before 100h, the pH value is 6.2-6.3 after 100h, and stopping introducing ammonia 8h before tank placing; the liquid supplementing standard is that total sugar is supplemented according to the residual sugar value of the fermentation liquid, the residual sugar value is controlled to be 8.0-9% before 100 hours of fermentation, the residual sugar value is controlled to be 7-8% after 100 hours-150 hours of fermentation, and the residual sugar value is controlled to be 5% after 150 hours and 6 hours before tank placing.

(1) pressing the prepared mycelium and fruiting body, cordycepin and pentostatin fermentation mixed liquor into a No. 1-2 40000L fermentation liquor temporary storage tank;

(2) pressing the prepared fermentation liquor into coarse filtration equipment for filtration to prepare mycelium, a fruiting body filter cake and filtrate;

(3) pumping the filtrate into a membrane ultrafiltration unit for ultrafiltration, and filtering out impurities in the fermentation liquor to obtain a mixed crude solution of cordycepin and pentostatin;

(4) centrifuging and desolventizing the filtered concentrated solution by using an automatic scraper centrifuge to prepare wastes such as sporocarp and the like;

(5) pumping the prepared mixed crude solution of the cordycepin and the pentostatin into a membrane nanofiltration concentration unit for concentration to prepare a mixed concentrated solution of the cordycepin and the pentostatin and wastewater;

(6) pumping the prepared cordycepin and pentostatin mixed concentrated solution into an automatic scraper centrifuge for centrifugation and desolventizing to prepare a cordycepin and pentostatin mixed crude product filter cake A;

(7) feeding the prepared mixed crude product filter cake A of the cordycepin and the pentostatin into a No.1 extraction kettle, pumping an lipophilic solvent which is 3 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, pumping deionized water which is 6 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, extracting the mixture for 2 to 3 times, and collecting and combining extract liquor;

(8) performing liquid-liquid centrifugal extraction on the extract liquor, and separating an organic phase and a water phase to obtain an organic phase containing pentostatin and a water phase containing cordycepin;

(9) filtering the organic phase by a bag filter, concentrating the filtrate by a No.1 concentrator, centrifuging, desolventizing and recovering the solvent to prepare a crude pentostatin dry filter cake A;

(10) pumping the water phase into a 6000L flocculation precipitation tank, adding a flocculating agent, stirring for 10-30 min, precipitating for 4-6 h, filtering by a bag filter, concentrating the filtrate by a No. 2 concentrator, desolventizing by a concentrated solution centrifuge, and recovering the solvent to obtain a cordycepin crude product dry filter cake A;

(11) respectively feeding the cordycepin crude product dry filter cake A and the pentostatin crude product dry filter cake A into a No. 2 or No. 3 3000L extraction kettle, respectively using an acetone solvent with the weight 3 times of that of the dry filter cake, adding a decolorizing agent with the weight 0.03 time of that of the dry filter cake, carrying out extraction and decolorization for 2-3 times at the temperature of 40-60 ℃, filtering, combining extraction liquids, and respectively preparing cordycepin and pentostatin extraction liquids;

(12) pumping the cordycepin extract and the pentostatin extract into a concentrator for concentration, and recovering the solvent to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution;

(13) pumping the cordycepin crude product concentrated solution and the pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and solvent recovery to respectively prepare a cordycepin crude product filter cake B and a pentostatin crude product filter cake B;

(14) mixing the cordycepin crude product filter cake B with the crystallized cordycepin mother liquor filter cake, putting into a No.1 column feeding solution preparation tank, and dissolving with 30-60% ethanol by volume fraction to prepare column feeding solution; the solute concentration of the upper column liquid is 5-10 g/L;

(15) fully automatically controlling the adsorption of the prepared upper column liquid pump by a macroporous resin column system, wherein the adsorption flow rate is 2-3 BV; eluting with 30-60% ethanol; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

(16) pumping the adsorption effluent and the eluent into a concentrator respectively for concentration, and recovering the solvent to obtain a cordycepin crude product concentrated solution;

(17) pumping the prepared cordycepin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing, and recovering an ethanol solvent to prepare a cordycepin crude product filter cake;

(18) mixing a crude pentostatin filter cake and a crystallized pentostatin mother solution dry filter cake A, B, putting into a No. 2 column feed preparation tank, and dissolving with ethanol with the volume fraction of 70-80% to prepare column feed; the solute concentration of the upper column liquid is 5-15 g/L;

(19) pumping the prepared upper column liquid into a No. 2 macroporous resin column system for adsorption, wherein the flow rate of the upper column liquid is 2-3 BV; eluting with deionized water and 70-80% ethanol by volume; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

(20) adsorbing the effluent and the eluent by a No. 2 macroporous resin column system, pumping the effluent and the eluent into a concentrator for concentration, and recovering the solvent to prepare a crude pentostatin concentrated solution;

(21) pumping the obtained pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering an ethanol solvent to obtain a pentostatin crude product filter cake;

(22) feeding the prepared cordycepin filter cake into a No.1 crystallization kettle, dissolving the cordycepin filter cake with a solvent, recrystallizing at low temperature, and filtering crystals to obtain cordycepin wet crystals and a mother solution;

(23) pumping the prepared cordycepin mother liquor into a concentrator for concentration, centrifuging for desolventizing, and recovering the solvent to prepare a cordycepin mother liquor dry filter cake;

(24) feeding the prepared crude pentostatin filter cake into a freezing crystallization kettle, dissolving the crude pentostatin filter cake by using a solvent, crystallizing at a low temperature, and filtering crystals to prepare pentostatin crude crystals and pentostatin mother liquor;

(25) pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a pentostatin mother liquor dry filter cake A;

(26) feeding the obtained crude pentostatin crystal into a No. 2 crystallization kettle, dissolving the crude pentostatin crystal by using a solvent, recrystallizing at low temperature, and filtering the crystal to obtain a wet pentostatin crystal and a mother solution;

(27) pumping the prepared pentostatin mother liquor into a concentrator for concentration, centrifuging and desolventizing, and recovering the solvent to prepare a pentostatin mother liquor dry filter cake B;

(28) combining the dry filter cake B of the pentostatin mother liquor with the filter cake of the pentostatin crude product to prepare upper column liquor;

(29) feeding the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare cordycepin crystal powder with the content of more than or equal to 98 percent and cordycepin powder with low content;

(30) feeding the prepared pentostatin wet crystal into a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare pentostatin crystal powder with the content of more than or equal to 98 percent;

(31) and respectively feeding the prepared cordycepin crystal powder with the content of more than or equal to 98 percent and the prepared cordycepin powder with the low content into a batch V-shaped mixer for batch mixing to prepare a cordycepin crystal powder product with the content of more than or equal to 98 percent and a cordycepin powder product with the low content.

(32) And feeding the prepared pentostatin crystal powder with the concentration of more than or equal to 98 percent into a batch V-shaped mixer for batch mixing to prepare a pentostatin crystal powder product with the concentration of more than or equal to 98 percent.

In the step (2), the coarse filtration equipment is an automatic scraper centrifuge, a bag filter or a plate and frame filter press;

in the step (3), the full-automatic control membrane ultrafiltration unit is membrane separation equipment; the productivity is 4000L/h;

in the step (4), the model of the automatic scraper centrifuge is PGZ-1000;

in the step (5), the full-automatic control membrane nanofiltration concentration unit is membrane concentration equipment; the productivity is 4000L/h;

in the step (6), the model of the automatic scraper centrifuge is PGZ-1000;

in the step (7), the lipophilic solvent is dichloromethane or ethyl acetate; the extraction temperature is 40-50 ℃;

in the step (10), the flocculating agent is a ZTC1+1 natural clarifying agent; the flocculation precipitation temperature is 60-80 ℃; the precipitation time is 4-6 h;

in the step (11), the extraction solvent is acetone; the decolorizing material is activated carbon or activated clay;

in the step (14), the upper column liquid is ethanol with the volume fraction of 30-60%; the solute concentration of the upper column liquid is 5-10 g/L;

in the step (15), the macroporous resin is D001, HD-8, 711 or ML-7; the macroporous resin has an adsorption flow rate of 2-3 BV; the elution solvent is ethanol with the volume fraction of 30-60%; the flow rate of the eluent is 3-5 BV;

in the step (18), the upper column solution is prepared by dissolving 70-80% ethanol by volume fraction; the solute concentration of the upper column liquid is 5-15 g/L;

in the step (19), the macroporous resin is HZ202 or D201 or D241 or D261; the adsorption flow rate is 3-4 BV/h; respectively using deionized water, PH 4-6 and 70-80% ethanol or methanol in volume fraction as eluent; the elution flow rate of the deionized water is 2-3 BV/h; eluting the ethanol or methanol at the speed of 3-5 BV/h;

in the steps (2), (4), (6), (9), (10), (13), (17), (21), (23), (25) and (27), the model of the full-automatic scraper centrifuge is PGZ-1000-1800;

in the steps (9), (10), (12), (16), (20), (23), (25) and (27), the concentrator is a fully-automatically controlled 1000L-4000L/h MVR evaporator or a full-electric evaporator or a membrane concentrator; the concentration temperature of the MVR evaporator or the full-electric evaporator is 60 ℃; the vacuum degree is 0.07-0.085 Mpa;

in the step (21), the No.1 crystallizing kettle is a full-automatic control 8000L crystallizing kettle; the crystallization solvent is ethanol or methanol with the volume fraction of 30-60%; stirring for 10-30 min each time; the crystallization temperature is 40-50 ℃ → 5 ℃; the crystallization time is 12-24 h each time;

in the step (23), the crystallization dissolving solvent is 80-95% ethanol, methanol or acetone by volume fraction; the freezing crystallization kettle is a full-automatic control 8000L crystallization kettle; the stirring time is 10-30 min each time; the crystallization temperature is 40-50 ℃→ -20 ℃; the crystallization time is 12-24 h each time;

in the step (25), the No. 2 crystallization kettle is a full-automatic control 8000L crystallization kettle; the crystallization solvent is ethanol or methanol with the volume fraction of 70-80%; stirring for 10-30 min each time; the crystallization temperature is 40-50 ℃ → 5 ℃; the crystallization time is 12-24 h each time;

in the steps (28) and (29), the supercritical drying device is a 200-400L double-extraction double-separation electric heating full-automatic control supercritical drying device; supercritical drying and residual solvent removal are carried out at the pressure of 20-30 Mpa, the temperature of 40-60 ℃ and CO₂The flow rate is 1500-4500L/h, and the drying time is 360-480 min;

in the step (30), the batch V-shaped mixer is V-1000; the batch mixing time is 30-60 min.

Example 1

iv, primary seed culture: after the first-stage seed culture tank is sterilized, when the temperature of a culture medium in the tank is reduced to 28-30 ℃, respectively inoculating the triangular flask fermentation liquor into a No. 1-8 500L first-stage seed fermentation tank through an aseptic inoculation system, performing seed culture by using the seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 28-30 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7.2, the rotation speed of the seed tank is 100r/min and the ventilation amount is 0.8-1.4 vvm/min for 0-2 days, after 2 days, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.5-1 vvm/min, and collecting seed liquid after the fermentation culture.

vi, secondary seed culture: after the second-stage seed culture tank is sterilized, when the temperature is reduced to 25-28 ℃, the first-stage seed fermentation liquor which is fermented for 5 days is respectively inoculated into a No. 1-8 1000L second-stage seed fermentation tank through an aseptic inoculation system, seed culture is carried out by using a seed culture medium, the inoculation amount is 1-5%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is carried out for 5-7 days under the condition that the pH is 7, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 150r/min and the ventilation amount is 0.5-1 vvm/min, and seed liquid is collected after the fermentation culture.

(2) pressing the prepared fermentation liquor into a bag type filter for filtering to obtain mycelium, a fruiting body filter cake and filtrate;

(3) pumping the filtrate into a 4000L/h full-automatic control membrane ultrafiltration unit for ultrafiltration, and filtering out impurities in the fermentation liquor to obtain a mixed crude solution of cordycepin and pentostatin;

(4) centrifuging the filtered concentrated solution with PZG-100 automatic scraper centrifuge to obtain waste such as fruiting body;

(5) pumping the prepared mixed crude solution of cordycepin and pentostatin into a 4000L/h full-automatic control membrane nanofiltration concentration unit for concentration to prepare a mixed concentrated solution of cordycepin and pentostatin and wastewater;

(6) pumping the prepared cordycepin and pentostatin mixed concentrated solution into an PZG-100 automatic scraper centrifuge for centrifugation and desolventizing to prepare a cordycepin and pentostatin mixed crude product filter cake A;

(7) feeding the prepared mixed crude product filter cake A of cordycepin and pentostatin into a No.1 extraction kettle, pumping a dichloromethane solvent with the weight of 3 times that of the mixed crude product filter cake A of cordycepin and pentostatin, pumping the mixed crude product filter cake A of cordycepin and pentostatin with deionized water with the weight of 6 times that of the mixed crude product filter cake A of cordycepin and pentostatin, extracting for 2-3 times, and collecting and combining the extract liquor;

(11) respectively feeding the cordycepin crude product dry filter cake A and the pentostatin crude product dry filter cake A into a No. 2 or No. 3 extraction kettle of 3000L, respectively using acetone solvent with the weight 3-6 times of the weight of the dry filter cake, adding activated clay with the volume 0.02-0.05 time of the dry filter cake, extracting and decoloring for 2-3 times at the temperature of 40-60 ℃, filtering, combining the extraction liquid, and respectively preparing cordycepin and pentostatin extraction liquid;

(12) pumping the cordycepin extract and the pentostatin extract into a 1000L/h full-automatic MVR evaporator for concentration and solvent recovery to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution;

(13) pumping the cordycepin crude product concentrated solution and the pentostatin crude product concentrated solution into an PZG-100 automatic scraper centrifuge for centrifugal desolventizing and solvent recovery to respectively prepare a cordycepin crude product filter cake B and a pentostatin crude product filter cake B;

(15) pumping the prepared upper column liquid into a No.1 full-automatic control D001 macroporous resin column system for adsorption, wherein the adsorption flow rate is 2-3 BV; eluting with 30-60% ethanol; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

(16) pumping the adsorption effluent liquid and the eluent into a 2000-4000L/h full-automatic MVR evaporator respectively for concentration, and recovering the solvent to prepare a cordycepin crude product concentrated solution;

(17) pumping the obtained cordycepin crude product concentrated solution into an PZG-100 automatic scraper centrifuge for centrifugal desolventizing, and recovering ethanol solvent to obtain cordycepin crude product filter cake;

(19) pumping the prepared upper column liquid into a No. 2 full-automatic control HD-8 macroporous resin column system for adsorption, wherein the flow rate of the upper column liquid is 2-3 BV; eluting with deionized water and 70-80% ethanol by volume; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

(20) pumping the effluent and the eluent absorbed by a No. 2 macroporous resin column system into a 2000-4000L/h full-automatic MVR evaporator for concentration and solvent recovery to prepare a crude pentostatin concentrated solution;

(21) pumping the obtained crude pentostatin concentrated solution into an PZG-100 automatic scraper centrifuge for centrifugal desolventizing and recovering an ethanol solvent to obtain a crude pentostatin filter cake;

(22) feeding the prepared cordycepin filter cake into a No.1 crystallization kettle, dissolving the cordycepin filter cake in an ethanol solvent with the volume fraction of 30-60%, recrystallizing at low temperature, and filtering to obtain cordycepin wet crystals and mother liquor;

(23) pumping the prepared cordycepin mother liquor into a 1000L/h full-automatic MVR evaporator for concentration, centrifuging and desolventizing by an PZG-100 automatic scraper centrifuge, and recovering a solvent to prepare a cordycepin mother liquor dry filter cake;

(25) pumping the prepared pentostatin mother liquor into a 1000L/h full-automatic MVR evaporator for concentration, centrifuging and desolventizing by an PZG-100 automatic scraper centrifuge, and recovering a solvent to prepare a pentostatin mother liquor dry filter cake A;

(26) feeding the prepared crude pentostatin crystals into a No. 2 crystallization kettle, dissolving the crude pentostatin crystals in a methanol solvent with the volume fraction of 70-80%, recrystallizing at low temperature, and filtering the crystals to obtain wet pentostatin crystals and a mother solution;

(27) pumping the prepared pentostatin mother liquor into a 1000L/h full-automatic MVR evaporator for concentration, centrifuging and desolventizing by an PZG-100 automatic scraper centrifuge, and recovering a solvent to prepare a pentostatin mother liquor dry filter cake B;

(29) feeding the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare 641.08kg of cordycepin crystal powder with the content of more than or equal to 98 percent and 2112.06kg of low-grade cordycepin powder with the content of more than or equal to 50 percent;

(30) feeding the prepared pentostatin wet crystal into a No. 2 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain 19.86kg of pentostatin crystal powder with the content of more than or equal to 98 percent;

(32) And feeding the prepared pentostatin crystal powder with the concentration of more than or equal to 98 percent into a V-shaped mixer with the volume of 1000L for batch mixing to prepare a pentostatin crystal powder product with the concentration of more than or equal to 98 percent.

Example 2

d. preparation of bacterial suspension: respectively taking several dominant bacterial strain inclined planes which are cultured for one week, and respectively adding synergistic retained abnormal tobaccoThe unicellular suspension of the strain is obtained by scraping thalli on the inclined plane by using an inoculating loop, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to endure and shake, and filtering through a funnel with a layer of sterile mirror wiping paper after shaking. Counting spores with a hemocytometer (hemocytometer) to adjust the spore concentration to about 10⁶Per ml;

vi, secondary seed culture: after the second-stage seed culture tank is sterilized, when the temperature is reduced to 25-28 ℃, respectively inoculating first-stage seed fermentation liquor fermented for 5 days into a No. 1-8 1000L second-stage seed fermentation tank through an aseptic inoculation system, performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 150r/min and the ventilation amount is 0.8-1.4 vvm/min after 2 days, and collecting seed liquid after the fermentation culture.

(2) pressing the prepared fermentation liquor into a plate and frame filter press for filtering to obtain mycelium, a fruiting body filter cake and filtrate;

(7) feeding the prepared mixed crude product filter cake A of the cordycepin and the pentostatin into a No.1 extraction kettle, pumping an ethyl acetate solvent which is 3 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, pumping deionized water which is 6 times of the weight of the mixed crude product filter cake A of the cordycepin and the pentostatin into the extraction kettle, extracting the mixture for 2 to 3 times, and collecting and combining the extract liquor;

(11) respectively feeding the cordycepin crude product dry filter cake A and the pentostatin crude product dry filter cake A into a No. 2 or No. 3 extraction kettle of 3000L, respectively using acetone solvent with the weight 3-6 times of the weight of the dry filter cake, adding active carbon with the volume 0.02-0.05 time of the dry filter cake, extracting and decoloring for 2-3 times at the temperature of 40-60 ℃, filtering, combining the extraction liquid, and respectively preparing cordycepin and pentostatin extraction liquid;

(12) pumping the cordycepin extract and the pentostatin extract into a 1000L/h full-electric evaporator for concentration and solvent recovery to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution;

(15) pumping the prepared upper column liquid into a No.1 full-automatic control HD-8 macroporous resin column system for adsorption, wherein the adsorption flow rate is 2-3 BV; eluting with 30-60% ethanol; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

(16) pumping the adsorption effluent and the eluent into a 2000-4000L/h full-electric evaporator respectively for concentration and solvent recovery to prepare a cordycepin crude product concentrated solution;

(20) pumping the effluent and the eluent absorbed by a No. 2 macroporous resin column system into a 2000-4000L/h all-electric evaporator for concentration and solvent recovery to prepare a crude pentostatin concentrated solution;

(22) feeding the prepared cordycepin filter cake into a No.1 crystallization kettle, dissolving the cordycepin filter cake in a methanol solvent with the volume fraction of 30-60%, recrystallizing at low temperature, and filtering to obtain cordycepin wet crystals and mother liquor;

(23) pumping the prepared cordycepin mother liquor into a 1000L/h full-electric evaporator for concentration, centrifuging and desolventizing by an PZG-100 automatic scraper centrifuge, and recovering the solvent to prepare a cordycepin mother liquor dry filter cake;

(25) pumping the prepared pentostatin mother liquor into a full electric evaporator with the volume of 1000L/h for concentration, centrifuging and desolventizing by an PZG-100 automatic scraper centrifuge, and recovering a solvent to prepare a pentostatin mother liquor dry filter cake A;

(26) feeding the prepared crude pentostatin crystals into a No. 2 crystallization kettle, dissolving the crude pentostatin crystals in an acetone solvent with the volume fraction of 70-80%, recrystallizing at low temperature, and filtering the crystals to obtain wet pentostatin crystals and a mother solution;

(27) pumping the prepared pentostatin mother liquor into a 1000L/h full-electric evaporator for concentration, carrying out centrifugal desolventizing by an PZG-100 automatic scraper centrifuge, and recovering a solvent to prepare a pentostatin mother liquor dry filter cake B;

(29) feeding the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare 641.32kg of cordycepin crystal powder with the content of more than or equal to 98 percent and 2112.32kg of low-grade cordycepin powder with the content of more than or equal to 50 percent;

(30) feeding the prepared pentostatin wet crystal into a No. 2 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain 20.01kg of pentostatin crystal powder with the content of more than or equal to 98 percent;

Example 3

(2) pressing the obtained fermentation liquor into PZG-180 automatic scraper centrifuge for centrifuging to obtain mycelium, fruiting body filter cake and filtrate;

(12) pumping the cordycepin extract and the pentostatin extract into a 1000L/h full-automatic membrane concentration unit for concentration and solvent recovery to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution;

(15) pumping the prepared upper column liquid into a No.1 full-automatic control ML-7 macroporous resin column system for adsorption, wherein the adsorption flow rate is 2-3 BV; eluting with 30-60% ethanol; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

(16) pumping the adsorption effluent and the eluent into a 2000-4000L/h full-automatic control membrane concentration unit respectively for concentration and solvent recovery to prepare a cordycepin crude product concentrated solution;

(19) pumping the prepared upper column liquid into a No. 2 full-automatic control D201 or D241 macroporous resin column system for adsorption, wherein the flow rate of the upper column liquid is 2-3 BV; eluting with deionized water and 70-80% ethanol by volume; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

(20) pumping the effluent and the eluent absorbed by a No. 2 macroporous resin column system into a 1000L/h full-automatic control membrane concentration unit for concentration and solvent recovery to prepare a crude pentostatin concentrated solution;

(23) pumping the prepared cordycepin mother liquor into a 1000L/h full-automatic control membrane concentration unit for concentration, centrifuging and desolventizing by an PZG-100 automatic scraper centrifuge, and recovering a solvent to prepare a cordycepin mother liquor dry filter cake;

(25) pumping the prepared pentostatin mother liquor into a 1000L/h full-automatic control membrane concentration unit for concentration, centrifuging and desolventizing by an PZG-100 automatic scraper centrifuge, and recovering a solvent to prepare a pentostatin mother liquor dry filter cake A;

(27) pumping the prepared pentostatin mother liquor into a 1000L/h full-automatic control membrane concentration unit for concentration, centrifuging and desolventizing by an PZG-100 automatic scraper centrifuge, and recovering a solvent to prepare a pentostatin mother liquor dry filter cake B;

(29) feeding the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to prepare 642.44kg of cordycepin crystal powder with the content of more than or equal to 98 percent and 2113.13kg of low-grade cordycepin powder with the content of more than or equal to 50 percent;

(30) feeding the prepared pentostatin wet crystal into a No. 2 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain 20.41kg of pentostatin crystal powder with the content of more than or equal to 98 percent;

Example 4

Detection and identification of cordycepin or pentostatin product

1. Method for evaluating mutagenesis results

First evaluation of TLC

Quantitatively absorbing 5 mul, 10 mul, 15 mul, 20 mul, 25 mul and 30 mul of 0.35mg/L cordycepin or pentostatin by a microsyringe respectively, spotting on an activated silica gel G plate, developing, taking out, airing, fumigating for 0.5h by using acetic anhydride vapor, taking out, baking for 0.5h at 160 ℃, inspecting by ultraviolet at 260nm or 280nm, taking a picture by a digital camera, photographing on a gel imager by a quantityone, scanning and analyzing by totallab to obtain a color spot light absorption value of cordycepin or pentostatin, observing the relation between the sample amount and the color spot light absorption value, and drawing a cordycepin or pentostatin standard curve. Quantitatively spotting the fermentation samples of all the mutant strains and cordycepin or pentostatin (high and low dosage) on the same silica gel plate, and calculating the content of the total cordycepin or pentostatin substances by measuring and calculating the content of the samples through totallab after the same method is developed.

Evaluation of HPLC-UV

HPLC-UV evaluation of Cordycepin

Establishing a cordycepin standard curve by using HPLC: weighing 0.85mg of cordycepin standard substance, adding methanol: dissolving with water (volume ratio: 25: 75) until the volume is 10 mL. High Performance Liquid Chromatography (HPLC) assay, C18 column (250 mm. times.4.6 mm), UV detection wavelength 260nm, mobile phase methanol (25 mL): water (75mL), flow rate of 1.0mL/min, sample size of 5. mu.L. Column temperature: room temperature; a single point draws a standard curve. The cordycepin sample and the cordycepin standard substance prepared in the first to third embodiments are detected by a high performance liquid chromatograph, and have the same peak-off time.

② HPLC-UV evaluation of pentostatin

Pentostatin standard curves were established using HPLC: pentostatin standard 0.85mg was weighed out, dissolved in methanol/acetonitrile/10 mmol/L ammonium acetate (pH7.6) (2.5/2.5/95, v/v/v) and adjusted to 10 mL. High Performance Liquid Chromatography (HPLC) determination, the chromatographic conditions are as follows: the chromatographic column is a Hyper-SilODS2 column (250 mm. times.4.6 mm,5 μm); the mobile phase is methanol/acetonitrile/10 mmol/L ammonium acetate (pH7.6) (2.5/2.5/95, v/v/v) and the flow rate is 1.0 mL/min; the detection wavelength is 280 nm; the column temperature is 40 ℃: the amount of sample was 10. mu.L. A single point draws a standard curve. The pentostatin samples and pentostatin standard prepared in the first to third examples were tested by HPLC and had the same peak discharge time.

A formula for calculating the content of cordycepin or pentostatin is as follows:

the content (mg/L) of cordycepin or pentostatin in the fermentation broth is equal to the volume (mL) of methanol used for dissolving the extract, i.e. the content (mg/mL) of cordycepin or pentostatin in methanol, 10⁶Volume of fermentation broth taken at extraction (m L).

The ultraviolet spectrum, infrared spectrum, mass spectrum and nuclear magnetic resonance analysis of cordycepin or pentostatin obtained in examples 1 to 3 were performed, and the results were the same as those of cordycepin or pentostatin standard samples, thereby showing that the cordycepin or pentostatin spectrum prepared by the present invention is highly consistent with the spectrum of the purchased standard sample.

The above-mentioned embodiments only show some embodiments of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the claims.

Claims

1. An industrial preparation method for producing cordycepin and pentostatin by combined fermentation is characterized by comprising the following steps:

s2: culturing antibiotic streptomycete: taking a strain with a preservation number as follows: the antibiotic streptomyces CGMCC No.1349 is subjected to static culture at 28-30 ℃ for 5-7 days, when the surface of a culture medium has antibiotic streptomyces colonies, a single or a plurality of colonies are picked in an inoculation box and put into a test tube slant culture medium for culture, and the cordyceps militaris mycelia are purified after being covered with a slant;

s7: the liquid shake flask strain culture medium is PDA with a pH of 7, the initial pH value is 5.0, 250mL is prepared, the mixture is subpackaged into 250mL triangular flasks with each flask containing 30mL, the mixture is sterilized at 121 ℃ for 30 minutes and then cooled to room temperature for later use;

2. The industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 1, wherein the separation, mutagenesis and preservation method of the Cordyceps militaris strains and the Streptomyces antibioticus strains are as follows:

culturing cordyceps militaris: selecting high-yield, high-quality and early-maturing cordyceps militaris sporocarp, carrying out surface disinfection by using 75% alcohol, cleaning surface liquid medicine by using sterile water, suspending the liquid medicine above a container filled with a comprehensive culture medium, carrying out static culture at the temperature of 28-30 ℃ for 5-7 days, and picking single or multiple colonies in an inoculation box to place the single or multiple colonies in a test tube slant culture medium for culture when starburst-shaped cordyceps militaris colonies appear on the surface of the culture medium. Purifying after the cordyceps militaris hyphae are covered with the inclined plane; culturing antibiotic streptomycete: taking a strain with a preservation number as follows: the antibiotic streptomycete CGMCC No.1349 is subjected to static culture at 28-30 ℃ for 5-7 days, and when the antibiotic streptomycete colonies appear on the surface of a culture medium, a single colony or a plurality of colonies are picked up in an inoculation box and placed in a test tube slant culture medium for culture; purifying after the cordyceps militaris hyphae are covered with the inclined plane; putting the cordyceps militaris bacterium culture medium and the streptomyces antibioticus culture medium into a constant-temperature incubator at 28-30 ℃ for culture respectively; observing at regular time, sequentially transferring the newly grown hyphae to a new culture medium by using an inoculating needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane to preserve two strains; preparation of bacterial suspension: respectively taking several inclined planes of the dominant strains cultured for one week, respectively adding physiological saline containing synergistic isonicotin, scraping thalli on the inclined planes by using an inoculating loop, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to resist oscillation, and filtering the mixture by using a funnel with a layer of sterile mirror wiping paper after the oscillation is finished to obtain monospore suspension of the strains; counting spores with a hemocytometer (hemocytometer) to adjust the spore concentration to about 10⁶Per ml; ultraviolet mutagenesis: respectively taking 5ml of the prepared cordyceps militaris and streptomyces antibioticus monospore suspension into an aseptic plate with the diameter of 9 mm; a 15W ultraviolet lamp is turned on to preheat so as to stabilize light waves; placing the plate containing the bacterial suspension on a magnetic stirrer at a certain distance from the lamp tube, opening the dish cover whileStirring and irradiating to ensure that the cells uniformly absorb ultraviolet light waves; the top was operated in a red background, avoiding photorepair. The thallus after ultraviolet mutagenesis is immediately transferred into a sterile test tube and immersed in ice water for 30min to inhibit enzyme activity and repair. According to the principle of delay phenomenon, adding the irradiated bacterial suspension subjected to ice bath into a PDA culture medium for post-culture so as to improve mutation rate; carrying out normal-pressure room-temperature plasma mutagenesis: respectively taking 10 mu L of spore suspension with proper concentration of ultraviolet mutagenized cordyceps militaris and streptomyces antibioticus, uniformly coating the spore suspension on a sterilized mutagenesis cup, then mutagenizing under the optimal condition, putting the mutagenesis cup into a 2mL centrifuge tube after mutagenesis, adding 1mL of sterile ultrapure water, coating the mutagenesis cup on an ISP2 culture medium after shaking for 1min, culturing for 6-8 days at 30 ℃, observing colonies with color, texture and character changed in brightness difference compared with a control when the colonies grow to be about 0.5cm in diameter, and screening. The strain with the highest yield is selected for preservation and used for subsequent culture.

3. The industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 1, wherein the preparation and industrial fermentation methods of the strain are as follows:

the liquid shake flask strain culture medium is PDA with a pH of 7, the initial pH value is 5.0, 250mL is prepared, the mixture is subpackaged into 250mL triangular flasks with each flask containing 30mL, the mixture is sterilized at 121 ℃ for 30 minutes and then cooled to room temperature for later use; preparing liquid shake flask strains: respectively subpackaging cordyceps militaris or streptomyces antibioticus liquid culture medium into 500ml triangular flasks, adding 200ml of culture solution into each flask, sealing the flasks by adding a layer of kraft paper on 12 layers of gauze, sterilizing for 13-30 minutes, inoculating mother seeds into the triangular flasks by using an inoculating hook under an aseptic condition, connecting each inclined plane to more than 10 bottles, performing shake cultivation at 28 ℃ for about 5-7 days after inoculation, forming uniform pellets, and then using for inoculation, preparing a seed culture medium according to a formula standard, respectively pumping the seed culture medium into No. 1-8 500L first-stage seed culture tanks, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min; first-order seed culture: after the first-stage seed culture tank is sterilized, when the temperature of a culture medium in the tank is reduced to 28-30 ℃, respectively inoculating the triangular flask fermentation liquor into a first-stage seed fermentation tank of No. 1-8 500L through an aseptic inoculation system, performing seed culture by using the seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 28-30 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5-7 days under the condition that the pH is 7.2, the rotation speed of the seed tank is 100r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.5-1 vvm/min after 2 days, collecting seed liquid after the fermentation culture, preparing the seed culture medium according to a formula standard, respectively filling the seed culture medium into a second-stage seed fermentation tank of No. 1-8L, sterilizing by using a steam sterilization method in the seed tank, wherein the sterilization temperature is not lower than 121 ℃, and the sterilization time is not lower than 30 min; secondary seed culture: after the second-stage seed culture tank is sterilized, when the temperature is reduced to 25-28 ℃, respectively inoculating first-stage seed fermentation liquor fermented for 5-7 days into a 1000L second-stage seed fermentation tank with the number of 1-8 through an aseptic inoculation system, performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-5%, the fermentation temperature is 25-28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 5 days under the condition that the pH is 7, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.8-1.4 vvm/min after 0-2 days, the rotation speed of the seed tank is 150r/min and the ventilation amount is 0.8-1.4 vvm/min after 2 days, collecting seed liquid after the fermentation culture, accurately weighing the culture medium materials according to a formula, filling the culture medium materials into a 40000L multiplied by 3 culture medium preparation tank in batches, and stirring the purified water according to the formula for 30 min; pumping fermentation medium into No. 1-10 100000L fermentation single tank or No. 1-3 medium liquid supplementing tank in batches, filling the tank with coefficient of 0.7, sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min; after single-tank sterilization is finished, cooling culture medium in the tank to 28 ℃ by using chilled water, respectively inoculating the secondary seed fermentation liquor fermented for 18 hours from No.1 to No. 8 to a No.1 100000L fermentation tank through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 28 ℃, the tank pressure is 0.03MPa, the fermentation culture is carried out for 120 hours under the condition that the pH is 7, the stirring rotation speed is 130r/min, the ventilation amount is 0.8-1.4 vvm/min, and after 180 hours, the fermentation tank rotation speed is 100r/min and the ventilation amount is 1 vvm/min; supplementing liquid in real time according to the culture medium quantity standard in the fermentation period; and when the culture medium feeding reaches the standard and the fermentation time reaches the standard, ending the fermentation to prepare a fermentation mixed solution of the fruiting body, the cordycepin and the pentostatin.

4. The industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 1, wherein the purification steps of cordycepin and pentostatin are as follows:

5. The industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 1, wherein the separation, mutagenesis and preservation method of the Cordyceps militaris strains and Streptomyces antibioticus comprises the following steps:

b. culturing antibiotic streptomycete: taking a strain with a preservation number as follows: the antibiotic streptomyces CGMCC No.1349 is subjected to static culture at 28-30 ℃ for 5-7 days, when the surface of a culture medium has antibiotic streptomyces colonies, a single or a plurality of colonies are picked in an inoculation box and put into a test tube slant culture medium for culture, and the cordyceps militaris mycelia are purified after being covered with a slant;

6. The industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 5, characterized in that:

in the step a, the ethanol accounts for 75% of the volume fraction;

in the step f, culturing the ISP2 culture medium for 6-8 days at 30 ℃;

7. The industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 1, characterized in that:

i. the liquid shake flask strain culture medium is PDA with a pH of 7, the initial pH value is 5.0, 250mL is prepared, the mixture is subpackaged into 250mL triangular flasks with each flask containing 30mL, the mixture is sterilized at 121 ℃ for 30 minutes and then cooled to room temperature for later use;

8. The industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 7, characterized in that:

in step vii, the optimized medium formula is as follows: 90g of cane sugar, 25-30 g of bean curd jelly, 10g of peptone, 18g of yeast extract powder, 0.3mg of L-phenylalanine, 0.05mg of sodium acetate, 3-5 g of sodium chloride, 0.5-1 g of magnesium sulfate, 0.02mg of sodium benzoate, 0.1mg of arachidonic acid, 0.05mg of cinnamic acid, 40g of zinc acetate, 1000mL of distilled water and pH 7;

in the step x, when the pH value of the fermentation liquor is lower than 6.2, starting to introduce ammonia, wherein the pH value is 6.3-6.5 before 100h, the pH value is 6.2-6.3 after 100h, and stopping introducing ammonia 8h before tank placing; the liquid supplementing standard is that total sugar is supplemented according to the residual sugar value of the fermentation liquid, the residual sugar value is controlled to be 8.0-9.0% before 100 hours of fermentation, the residual sugar value is controlled to be 7.0-8.0% after 100 hours-150 hours of fermentation, and the residual sugar value is controlled to be 5.0% after 150 hours before 6 hours of tank emptying;

9. the industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 7, characterized in that:

n2, pressing the obtained fermentation liquor into a coarse filtration device for filtration to obtain mycelium, a fruiting body filter cake and filtrate;

n7, feeding the prepared cordycepin and pentostatin mixed crude product filter cake A into a No.1 extraction kettle, pumping a lipophilic solvent which is 3 times of the weight of the cordycepin and pentostatin mixed crude product filter cake A, pumping deionized water which is 6 times of the weight of the cordycepin and pentostatin mixed crude product filter cake A into the extraction kettle, extracting for 2-3 times, and collecting and combining extract liquor;

n8, carrying out liquid-liquid centrifugal extraction on the extract, and separating an organic phase and a water phase to obtain an organic phase containing pentostatin and a water phase containing cordycepin;

n9, filtering the organic phase by a bag filter, concentrating the filtrate by a No.1 concentrator, centrifuging, desolventizing, and recovering the solvent to prepare a crude product of pentostatin dry filter cake A;

n11, respectively feeding the cordycepin crude product dry filter cake A and the pentostatin crude product dry filter cake A into an extraction kettle of No. 2 or No. 3 3000L, respectively using acetone solvent with the weight 3 times of that of the dry filter cake, adding decolorizing agent with the weight 0.03 time of that of the dry filter cake, extracting and decolorizing for 2-3 times at the temperature of 40-60 ℃, filtering, combining the extract solutions, and respectively preparing cordycepin and pentostatin extract solutions;

n12, pumping the cordycepin extract and the pentostatin extract into a concentrator for concentration and recovering the solvent to respectively prepare a cordycepin crude product concentrated solution and a pentostatin crude product concentrated solution;

n14, mixing the cordycepin crude product filter cake B with the crystallized cordycepin mother liquor filter cake, putting into a No.1 column feeding solution preparation tank, and dissolving with 30-60% ethanol by volume fraction to prepare column feeding solution; the solute concentration of the upper column liquid is 5-10 g/L;

n16, pumping the adsorption effluent and the eluent into a concentrator respectively for concentration, and recovering the solvent to obtain a cordycepin crude product concentrated solution;

n17, pumping the obtained cordycepin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing, and recovering an ethanol solvent to obtain a cordycepin crude product filter cake;

n18, mixing a crude pentostatin filter cake with a crystallized pentostatin mother solution dry filter cake A, B, putting into a No. 2 column feed preparation tank, and dissolving with ethanol with the volume fraction of 70-80% to prepare a column feed; the solute concentration of the upper column liquid is 5-15 g/L;

n19, pumping the prepared upper column liquid into a No. 2 macroporous resin column system for adsorption, wherein the flow rate of the upper column liquid is 2-3 BV; eluting with deionized water and 70-80% ethanol by volume; the flow rate of the eluent is 3-5 BV; respectively collecting adsorption effluent liquid and eluent;

n21, pumping the obtained pentostatin crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering an ethanol solvent to obtain a pentostatin crude product filter cake;

n22, feeding the prepared cordycepin filter cake into a No.1 crystallization kettle, dissolving the cordycepin filter cake with a solvent, recrystallizing at low temperature, and filtering to obtain cordycepin wet crystals and a mother solution;

n24, feeding the prepared crude pentostatin filter cake into a freezing crystallization kettle, dissolving the crude pentostatin filter cake by using a solvent, crystallizing at a low temperature, and filtering crystals to prepare coarse pentostatin crystals and a pentostatin mother solution;

n26, feeding the prepared crude pentostatin crystal into a No. 2 crystallization kettle, dissolving the crude pentostatin crystal by a solvent, recrystallizing at low temperature, and filtering the crystal to prepare wet pentostatin crystal and mother liquor;

n28, mixing the dry filter cake B of the pentostatin mother solution with the filter cake of the pentostatin crude product to prepare upper column solution;

n29, feeding the prepared cordycepin wet crystal and cordycepin mother liquor dry filter cake into a No.1 drying kettle of a supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain cordycepin crystal powder with the content of more than or equal to 98% and cordycepin powder with low content;

n31, respectively feeding the cordycepin crystal powder with the concentration of more than or equal to 98% and the cordycepin powder with low content into a batch V-shaped mixer for batch mixing to obtain cordycepin crystal powder products with the concentration of more than or equal to 98% and cordycepin powder products with low content,

n32, feeding the prepared pentostatin crystal powder with the concentration of more than or equal to 98 percent into a batch V-shaped mixer for batch mixing to prepare a pentostatin crystal powder product with the concentration of more than or equal to 98 percent.

10. The industrial preparation method of cordycepin and pentostatin by combined fermentation according to claim 9, characterized in that:

in step N4, the model of the automatic scraper centrifuge is PGZ-1000;

in step N6, the model of the automatic scraper centrifuge is PGZ-1000;

in step N11, the decolorant is activated clay or activated carbon;

in step N13, the model of the automatic scraper centrifuge is PGZ-1000;