CN111587735A - Production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris - Google Patents
Production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris Download PDFInfo
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Abstract
The invention discloses a production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris, and belongs to the technical field of production of edible and medicinal fungus sporocarp. The method comprises the following steps: (1) pulverizing rice, wheat and soybean, mixing, adding nutrient solution, and preparing growth medium; (2) taking a cordyceps militaris mother strain which simultaneously has a tegular paecilomyces varioti structure and a head-gathered verticillium spore-forming structure on the same hypha as an effective mother strain, taking a streptomycete mother strain as an associated strain, and inoculating the streptomycete mother strain to a liquid culture medium to obtain an effective composite liquid strain; (3) diluting the effective composite liquid seed, inoculating to a growth medium, and culturing for 40-60 days; (4) collecting fruiting body, spraying the fruiting body surface with the white sprinkle soaked with dry folium Pini, and oven drying. The invention can promote the content of various active ingredients to be synergistically improved and stably produce, particularly obviously improve the content of anticancer substances, namely pentostatin, polysaccharide, cordycepic acid and SOD enzyme, and can be industrially popularized and applied.
Description
Technical Field
The invention belongs to the technical field of edible and medicinal dual-purpose fungus fruiting body production, relates to cordyceps militaris cultivation, and particularly relates to a production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris.
Background
The cordyceps militaris is a rare dual-purpose fungus traditional Chinese medicine for food therapy, and has wide medicine and health care values. Research shows that the cordyceps militaris contains various nutrient components necessary for human bodies, such as protein, amino acid, macroelement, microelement, multivitamin and the like, is higher than other fungus foods, and is also rich in unique active substances, such as adenosine, cordycepin, pentostatin, cordycepic acid, cordyceps polysaccharide, SOD enzyme, organic selenium and the like. The active substances integrate the cordyceps militaris into a whole to form a natural large formula, and the physiological function of a human body is adjusted by the integral matching effect, so that the human body functions in various aspects such as blood, body fluid, nerves, hormone and the like can be developed and normalized, and the immunity of the human body is improved on the whole, therefore, the cordyceps militaris is considered to be the best natural food for preventing and treating diseases by combining Chinese and western medicines. Modern medical research shows that the cordyceps militaris has multiple functions of benefiting lung and tonifying kidney, stopping bleeding and reducing phlegm, protecting liver and resisting cancer and the like, particularly can improve the immune function, and is an excellent food for preventing diseases.
With the development of the modern artificial cultivation technology, the research on the active substances of the cordyceps militaris focuses on the research on a single active substance, but the research on the synergistic stable improvement of a plurality of active substances of the cordyceps militaris lacks. Chinese patent CN201010186974.5 discloses research on important anticancer active substance pentostatin from Cordyceps militaris with various deletions.
Disclosure of Invention
The invention aims to provide a production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris.
The invention is realized by the following technical scheme:
a production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris comprises the following steps:
(1) preparing a growth medium: pulverizing rice, semen Tritici Aestivi, and semen glycines, mixing, adding nutrient solution, sealing, and sterilizing;
(2) preparing an effective compound liquid seed with harmonious cordyceps militaris isoniazoic coordination:
taking a cordyceps militaris mother strain which simultaneously has a tegular paecilomyces and a verticillium sporulation structure aggregated into a head on the same hypha as an effective mother strain, taking a streptomycete mother strain as an associated strain, inoculating the effective mother strain and the associated strain into a liquid culture medium, and performing shake-flask culture to obtain an effective composite liquid strain;
(3) inoculation and culture:
diluting the effective composite liquid seed, inoculating to a growth culture medium, and performing coordinated culture for 40-60 days;
(4) harvesting and processing:
collecting fruiting body, stopping light, spreading for cooling, spraying 60% (v/v) white spray soaked with dry folium Pini onto fruiting body surface, stacking under 500LX light for three days, and oven drying at 60 deg.C until water content is less than or equal to 12%.
Further, the cordyceps militaris mother strain is preserved in China general microbiological culture Collection center with the preservation number: CGMCC NO 5577, preservation time: 2011.12.12.
the Cordyceps militaris mother strain is an anti-degeneration Cordyceps militaris strain recorded in patent number ZL 201210248399.6.
The streptomycete mother strain is CPCC200004 antitumor streptomycete and is purchased from the pharmaceutical institute of Chinese medical academy of sciences.
The liquid culture medium: 200g of potato, 30g of corn flour, 20g of white granulated sugar, 3g of peptone, 1.5g of monopotassium phosphate, 0.02g of sodium sulfite, 0.01g of indoleacetic acid and 1000ml of water, boiling and dissolving the materials, taking filtrate, supplementing 1000ml of water, subpackaging the obtained mixture in 500ml triangular bottles, filling the bottles with the amount of 120 ml/bottle, sealing, sterilizing at 120 ℃ for 30 minutes, and inoculating when the temperature is cooled to below 30 ℃.
And (3) inoculating the liquid culture medium, standing for 72-96 hours, controlling the temperature to be 25-28 ℃, entering a shake flask for culture after the liquid surface is fully dispersed with hyphae, and culturing at 23-25 ℃ at 120-.
The coordinated culture comprises spawn running culture, mycelium stimulation culture and color transfer bud forcing culture: promoting the growth and culture of grass, wherein the spawn running culture is 18-21 ℃, the humidity is 70-75%, the dark culture is 10-15 days, the spawn running culture is to dip lime water with the PH9 with a scrubbing brush, the fungus skin is brushed to break, the internal heterodyne of the PH value (the PH value in the culture medium is less than 6) is formed, the color transfer bud promoting culture temperature is 16-24 ℃, the humidity is 80-90%, the illumination is carried out for 24 hours, and the time is 7-10 days; the culture temperature for promoting the growth of grass is 18-22 ℃ and 28-35 days.
In the stage of promoting the growth and culture of grass, when the fruiting body grows to 3-5cm, the culture medium and the fruiting body are sprayed with magnetized water.
Firstly, regarding the selection of strains, because the cordyceps militaris is a typical heterotype matching, namely, one strain contains two genes of Paeilomyces and Vertcllium verticillium simultaneously, otherwise, the strains cannot seed, in addition, the proportion of the two genes is not coordinated, the fruiting bodies and the active substance content of the strains are different, the basic principle of the synergistic cultivation of multiple active substances of the cordyceps militaris from the aspect of seed production is adopted, the parent strain is inspected by a microscope, the parent strain of the cordyceps militaris, whether separated or introduced, is observed on the same hypha under a microscope, simultaneously has an imbricated paecilomyces type and a head-gathered verticillium spore type spore producing structure, the two types of configuration are basically balanced in quantity, namely, the two types of configuration are effective parent strains, only one type of the two types of configuration is an ineffective type, the invention preferably selects the cordyceps militaris and reserves the cordyceps militaris in the common microorganism center of China Committee for the preservation and management committee of microorganism strains, the preservation number is as follows: CGMCC NO 5577, preservation time: 2011.12.12, also available from patent nos.: ZL201210248399.6 is anti-degeneration Cordyceps militaris strain; the effective compound liquid needs to be accompanied by streptomycete, and the streptomycete is purchased from pharmaceutical institute of Chinese medical academy of sciences and numbered as CPCC200004 streptomycete for resisting tumors.
Secondly, preparing a nutrient-balanced liquid culture medium: 200g of potato, 30g of corn flour, 20g of white granulated sugar, 3g of peptone, 1.5g of monopotassium phosphate, 0.02g of sodium sulfite, 0.01g of indoleacetic acid and 1000ml of water, boiling and dissolving the materials, taking filtrate, supplementing 1000ml of water, subpackaging in 500ml triangular bottles, the filling amount is 120 ml/bottle, sealing, sterilizing at 120 ℃ for 30 minutes, inoculating when the temperature is cooled to be below 30 ℃, inoculating by aseptic operation, inoculating 2-3mm of cordyceps militaris effective mother seeds into each bottle22-3 fungus blocks with different sizes are inoculated into streptomycete 2mm2One, the fungus block should float on the liquid surface, and should not sink, and the light is closed, and the two kinds of fungus are fermented together; standing for 72-96 hours, controlling the temperature at 25-28 ℃, and performing shake culture after the liquid level is full of mycelia: at 23-25 ℃, 120-240 r/min and 72-120 hours, the mycelium pellets are filled in the liquid, and the liquid is clear, namely the effective composite liquid seed.
Thirdly, regarding the preparation of the growth culture medium, according to the biological characteristics of the cordyceps militaris, the growth culture medium is rich in organic nutrients for a long timeThe growth medium of the invention is characterized in that active substances can be accumulated in a balanced manner, and the inorganic nutrient components are properly added to play a role in induction and stimulation, so that various active substances are increased in a synergistic manner, wherein the growth medium is prepared from rice: wheat: the soybean is crushed into coarse powder according to the weight ratio of 4:4:2 and mixed, and the nutrient solution is 40g of silkworm chrysalis powder, 20g of white granulated sugar, VC0.1g of white granulated sugar and VB10.2g, 0.1g of sodium sulfite and 1000ml of water are mixed, the growth culture medium is subpackaged, the amount of dissolved 1/40-1/16 is added, then nutrient solution is added, the amount of added nutrient solution is 1.2-1.4 times of that of the solid culture medium, after polypropylene is used for sealing, sterilization is carried out for 1 hour at 120 ℃, and the mixture is cooled to 30 ℃ for aseptic inoculation.
Fourthly, adding the effective compound liquid seeds into sterile water which is three times of the original seeds, diluting and uniformly mixing, inoculating the effective compound liquid seeds into a growth culture medium by using an inoculating gun under the aseptic condition, wherein the inoculating amount is 30-35% of the growth culture medium base material, sealing and then performing coordinated culture, wherein the operation is generally carried out for 40-60 days, and the method specifically comprises the following steps: the first step is spawn running culture, which requires light-tight, the temperature is 18-21 ℃, the humidity is 70-75%, ventilation is carried out once in the morning and at night every day, each time is not less than 1 hour, until a complete layer of mycoderm is formed on the surface, the time is 10-15 days, the second step is mycelium stimulation culture, lime water with PH9 is smeared by a scrubbing brush, the mycoderm is brushed to form internal heterodynia of the PH value (the PH value in a culture medium is less than 6), the balanced formation of sporocarp buds is facilitated, the third step is color-transferring bud-destroying culture, the temperature is 14-25 ℃, the temperature difference between day and night is not less than 8 ℃, the humidity is 80-90%, the illumination is 200-LX in 24 hours, ventilation is carried out once in the morning and at night every time is not less than 1 hour, the time is 7-10 days, the culture medium is totally turned into deep yellow, sporocarp buds grow uniformly on the surface, the fourth step is promotion of, wherein the illumination is 500-3500LX for not less than 12 hours every day, seven sections are pressed to irradiate 500-800-1500-2500-3000-3500 LX for not less than 1 hour in the morning and evening, the illumination is carried out by LED low-temperature illumination, the ventilation is carried out for 1 time in the morning and evening, the time is not less than 1 hour, meanwhile, plastic films with small holes are used for coating the plastic films to enhance the exchange of oxygen inside and outside the container, in addition, music can be played to achieve the purpose of stimulating the balanced accumulation of various active substances of the cordyceps sinensis, when the sporocarp grows to 3-5cm, the culture medium and the sporocarp are sprayed by magnetized water, when the sporocarp grows to be about to diffuse spores but not diffuse, the plastic films for sealing are removed, the sporocarp is irradiated by 40W ultraviolet lamps for 40-60 seconds at a distance of 30-40cm, the sealing is carried out, the light is closed, the culture is carried out for 3-5 days, the ventilation is kept at 18, collecting fruiting body in time when fruiting body has recession.
Fifthly, collecting the sporophores by using a disinfection clamp during collection, closing the room, spreading for cooling, drying surface water, spraying 20g of dry pine needles and 1000ml of 60-degree white spraying soak solution on the surfaces of the sporophores, slightly rubbing and softening, stacking for three days under 500LX light, turning over once every 12 hours to promote the stability of each active substance, drying at 60 ℃ until the water content is less than or equal to 12%, bagging, sealing and preserving.
In order to verify the effect of the invention, the contents of seven ingredients of the cordyceps militaris sporocarp, such as adenosine, cordycepin, pentostatin, polysaccharide, cordycepic acid, SOD enzyme, organic selenium, and the like, are respectively detected, and the results are shown in Table 1, and the table 1 shows that the contents of various active substances of the cordyceps militaris produced by the method are synergistically high and stable, which are 3-30 times higher than the contents of conventional products and natural products, wherein the conventional products and the natural products are commercially available products of northeast China and Qinba.
| Adenosine (%) | Cordycepin (%) | Pentostatin (%) | Polysaccharide (%) | Cordycepic acid (%) | Selenium (mug/g) | SOD(µ/mg) | |
| Products of the invention | 0.55 | 0.37 | 0.37 | 50.41 | 28.22 | 0.96 | 1986 |
| Conventional cultivated product | 0.1-0.2 | 0.03-0.05 | 0 | 5-6.11 | 5-25.11 | 0.038 | 54 |
| Natural products | 0.11-0.48 | 0.31 | 0.003 | 2.5 | 4-8 | 0.038 | 691 |
| Detection method | High performance liquid chromatography | High performance liquid chromatography | High performance liquid chromatography | Phenol sulfate process | Sodium periodate colorimetric method | Atomic absorption spectrometry | Ultrasonic wall breaking method |
TABLE 1
In conclusion, the invention can promote the content of various active ingredients to be synergistically improved and stably produce, particularly obviously improve the content of anticancer substances, namely pentostatin, polysaccharide, cordycepic acid and SOD enzyme, and can be industrially popularized and applied.
Detailed Description
The following examples are merely preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent exchange, improvement and the like made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Example 1
A production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris comprises the following steps:
(1) preparing an effective compound liquid seed with harmonious cordyceps militaris isoniazoic coordination:
microscopic examination of mother seeds: taking separated cordyceps militaris mother strains, and observing under a microscope: on the same hypha, the two types have both a imbricate paecilomyces type and a head-gathered verticillium spore type spore-forming structure, the number of the two types is basically balanced, namely the two types are effective mother species, and only one or unbalance of the two types is abandoned;
determining one companion fungus as streptomyces species from institute of pharmacy of Chinese medical academy, with number CP20004, and streptomyces antibumor;
preparing a nutrient-balanced liquid culture medium: 200g of potato, 30g of corn flour, 20g of white granulated sugar, 3g of peptone, 1.5g of monopotassium phosphate, 0.02g of sodium sulfite, 0.01g of indoleacetic acid and 1000ml of water. Boiling the above materials, collecting filtrate, adding 1000ml of water, subpackaging in 500ml triangular bottles with a filling amount of 120 ml/bottle, sealing, sterilizing at 120 deg.C for 30 min, cooling to below 30 deg.C, and inoculating;
inoculation and culture: inoculating 2-3mm effective mother strain of Cordyceps militaris into each bottle according to aseptic operation requirement22-3 fungus blocks with different sizes are inoculated into streptomyces species with the diameter of 2mm2On one piece, the surely marked strain should not sink. And (3) stopping light, carrying out co-fermentation on the two bacteria, standing for 72-96 hours, controlling the temperature to be 25-28 ℃, and carrying out shake flask culture after the bacteria are developed: controlling the temperature at 23-25 ℃, culturing for 72-120 hours at 120-;
(2) preparing a growth culture medium and a nutrient solution with various coordinated nutrient components;
long culture medium (according to weight ratio): 40% of each of the rice and wheat and 20% of the soybean (coarse powder);
nutrient solution: 40g of silkworm chrysalis meal, 20g of white granulated sugar, 0.1g of VC and VB10.2g, 0.1g of sodium sulfite and 1000ml of water;
preparation: mixing growth culture medium, subpackaging in 500ml special culture bottle with a filling amount of 30 g/bottle, adding culture solution 36ml, sealing, sterilizing at 120 deg.C for 1 hr, cooling to below 30 deg.C, and inoculating;
(3) inoculation and culture:
adding sterile water which is three times as much as the original seeds into a triangular flask for effectively compounding the liquid seeds for dilution, uniformly mixing, inoculating 10 ml/bottle of diluted seeds in a growth medium bottle by a sterile operation method, sealing, performing coordinated culture for 40-60 days, and operating according to the following steps:
spawn running culture: stopping lighting, wherein the temperature is 18-21 ℃, the humidity is 70-75%, and the ventilation is performed once every morning and evening, each time is not less than 1 hour, until the hyphae grow to 4/5 of the culture medium, the surface of the hyphae is preferably a layer of complete mycoderm, the time is 10-15 days, and the time is not less than 10 days, and the temperature is controlled;
and (3) mycelium stimulation culture: brushing the mycoderm with lime water with pH =9 to form internal heterodynia of pH (pH in the culture medium is less than 6), which is favorable for formation of fruiting body bud;
color transformation and germination acceleration culture: controlling the temperature to be 16-24 ℃, forming day and night temperature difference not less than 8 ℃, humidity 80-90%, illuminating for 24 hours by 200-LX, ventilating for morning and evening once respectively, wherein each time is not less than 1 hour, the time is 7-10 days, the interior of the bottle is totally turned into yellow, and the surface of the bottle is good for uniformly growing stroma buds;
promoting the growth and culture of grass, taking 28-35 days totally, grasping the following points: controlling the temperature at 18-22 ℃ and illuminating 500-;
strengthening ventilation, ventilating every morning and evening for not less than 1 hour, sealing, pricking 7-10 small holes (using toothpick) with plastic film, and enhancing oxygen circulation inside and outside the container;
green potted plants are placed in the indoor corridor, one plant is placed in each 15 m, the oxygen increasing effect is generated under the irradiation of lamplight, and the growth of sporocarp and the accumulation of active ingredients are promoted;
1-2 crickets are placed on each potted plant, in the accompaniment of classical light music, the chirping sound of the crickets is added, the crickets are mixed into natural harmony music, the ecological environment like the nature is changed, and the balanced accumulation of various active substances is promoted;
watering with magnetized water, spraying the magnetized water onto the fruiting body and culture medium in the bottle when the fruiting body grows to 3-5cm, and completely wetting;
irradiating with ultraviolet ray until the sporophore grows to 3-8cm high, i.e. when spore is to be emitted but not emitted, removing the seal, irradiating with 40W ultraviolet lamp at a distance of 30-40cm perpendicular to the sporophore for 40-60 s, sealing, stopping light, culturing for 3-5 days, maintaining at 18-22 deg.C, ventilating for 4 hr per day, and collecting;
(4) harvesting and processing: collecting fruiting body, spreading to cool in room, stopping light, air drying surface water, mixing with 20g dried folium Pini and 1000ml600Spraying the liquor soak solution on the surface of the fruiting body, kneading, stacking under 500LX illumination for three days, turning over once every 12 hours, drying at 60 deg.C until the water content is less than 12%, sealing, preserving, and randomly sampling and detecting.
Embodiment 2 a production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris, which comprises the following steps:
(1) preparing an effective compound liquid seed with harmonious cordyceps militaris isoniazoic coordination:
taking a patent seed with the patent number of ZL201210248399.6, namely the anti-degeneration cordyceps militaris mother seed. Observation under a microscope: the same hypha has a imbricate paecilomyces type and a verticillium spore type spore-forming structure gathered into a head shape, and the two types are basically balanced, namely an effective mother strain; selecting a streptomycete, namely a pharmaceutical institute of Chinese medical academy, and numbering CP 200004; preparing a nutrient-balanced liquid culture medium: 200g of potato, 30g of corn flour, 20g of white granulated sugar, 3g of peptone, 1.5g of monopotassium phosphate, 0.02g of sodium sulfite, 0.01g of indoleacetic acid and 1000ml of water; boiling the materials, taking filtrate, adding 1000ml of water, subpackaging in 500ml triangular bottles with the filling amount of 120 ml/bottle, sealing, sterilizing at 120 ℃ for 30 minutes, cooling to below 30 ℃ and inoculating; according to the aseptic operation requirement, the effective mother seed of Cordyceps militaris is 2-3mm2Inoculating 2-3 pieces of large-sized fungus blocks into each bottle, and inoculating 2mm of streptomycete2One block, light-closed, co-fermentation. Standing for 72-96 hours, controlling the temperature to be 25-28 ℃, after the strains are developed, entering a shaking flask for culturing, controlling the temperature to be 23-25 ℃, controlling the temperature to be 120-;
(2) preparing a growth medium and a nutrient solution with coordinated nutrients:
the culture medium comprises the following raw materials in parts by weight: respectively 40% of rice and wheat and 20% of soybean coarse powder, mixing, placing into a special culture box with a loading of 200 g/box, simultaneously adding culture solution, adding 280ml into each box, sealing, sterilizing at 120 deg.C for 1 hr, cooling to below 30 deg.C, and inoculating. Preparing a nutrient solution: 40g of silkworm chrysalis meal, 20g of white granulated sugar, 0.1g of VC and VB10.2g, 0.1g of sodium sulfite and 1000ml of water are boiled and dissolved;
(3) inoculation and culture:
diluting the effective composite liquid seeds with three times of sterile water, inoculating 60ml of liquid seeds into each box by a sterile operation method, culturing according to the following steps,
spawn running culture: closing light, at 18-21 deg.C, humidity 70-75%, ventilating every morning and evening for no less than 1 hr, when mycelium grows to 4/5 of culture medium and surface forms a layer of complete bacterial skin, the time is 10-15 days;
and (3) mycelium stimulation culture: brushing the mycoderm with lime water with pH =9 to promote the uniform growth of sporophore bud;
color transformation and germination acceleration culture: controlling the temperature to be 16-24 ℃, controlling the humidity to be 80-90%, illuminating for 24 hours by 200 and 500LX, ventilating for morning and evening once respectively, wherein each time is not less than 1 hour, the time is 7-10 days, the interior of the bottle turns yellow, and the surface stroma is uniform;
promoting the growth and culture of grass, wherein 28-35 days are needed;
(4) harvesting and processing:
collecting fruiting body, spreading to cool in room, stopping light, air drying surface water, mixing with 20g dried folium Pini and 1000ml600Spraying the liquor soaking solution on the surface of the fruiting body, kneading, stacking under 500LX illumination for 3 days, turning over once every 12 hours, drying at 60 deg.C until the water content is less than 12%, sealing, preserving, and random sampling and detecting.
Embodiment 3 a production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris, comprising the following steps:
(1) preparing an effective compound liquid seed with harmonious cordyceps militaris isoniazoic coordination:
taking cordyceps militaris records, preserving the cordyceps militaris records in China general microbiological culture Collection center with a preservation number: CGMCC5577, preserving for 12 months and 12 days in 2011, and performing microscopic examination on the mother strain, wherein two balanced spore-producing structures of paecilomyces and verticillium are arranged on the same hypha of the mother strain as effective mother strains; taking streptomycete with the number of CP200004 as companion fungus; preparing a nutrient-balanced liquid culture medium: 200g of potato, 30g of corn flour, 20g of white granulated sugar, 3g of peptone, 1.5g of monopotassium phosphate, 0.02g of sodium sulfite, 0.01g of indoleacetic acid and 1000ml of water; boiling the materials, taking filtrate, adding 1000ml of water, subpackaging in 500ml triangular bottles with the filling amount of 120 ml/bottle, sealing, sterilizing at 120 ℃ for 30 minutes, cooling to below 30 ℃ and inoculating; inoculating effective Cordyceps militaris (L.) Link seed 2-3mm by aseptic method2Inoculating 2-3 pieces of large-sized fungus blocks into each bottle, and inoculating 2mm of streptomycete2One is put into a bottle, and the light is cut off and the fermentation is carried out together. Standing for 72-96 hours, controlling the temperature to be 25-28 ℃, after the strains are developed, entering a shaking flask for culturing, controlling the temperature to be 23-25 ℃, and controlling the temperature to be 120-240 r/min, culturing for 72-120 hours, forming bacterial balls, and obtaining clear liquid, namely the effective composite liquid.
(2) Preparing a growth medium and a nutrient solution with coordinated nutrients:
the culture medium comprises the following raw materials in parts by weight: 40% of rice and wheat and 20% of soybean coarse powder, uniformly mixing, filling into a 500ml can bottle, the filling amount is 30 g/bottle, adding 42ml of culture solution, and preparing nutrient solution: 40g of silkworm chrysalis meal, 20g of white granulated sugar, 0.1g of VC and VB10.2g, 0.1g of sodium sulfite and 1000ml of water are boiled and dissolved. Adding the mixture, sealing, sterilizing at 120 ℃ for 1 hour, cooling to below 30 ℃ and inoculating;
(3) inoculation and culture:
diluting the effective composite liquid with three times of sterile water, inoculating 10ml of liquid in each bottle, and culturing for 40-60 days;
(4) harvesting and processing: collecting fruiting body, spreading to cool in room, stopping light, air drying surface water, mixing with 20g dried folium Pini and 1000ml600Spraying the liquor soaking solution on the surface of the fruiting body, kneading, stacking under 500LX illumination for 3 days, turning over once every 12 hours, drying at 60 deg.C until the water content is less than or equal to 12%, sealing, preserving, and random sampling and detecting.
The effective components of the above examples 1 to 3 were tested, and the results are shown in Table 2,
TABLE 2
As can be seen from table 2, the content of the seven active substances adenosine, cordycepin, pentostatin, polysaccharide, cordycepic acid, SOD enzyme, organic selenium, etc. in the cordyceps militaris fruiting bodies produced in examples 1-3 are significantly increased synergistically and the production is stable.
Claims (8)
1. A production method for synergistically improving and stabilizing multiple active substances of cordyceps militaris is characterized by comprising the following steps:
(1) preparing a growth medium: pulverizing rice, semen Tritici Aestivi, and semen glycines, mixing, adding nutrient solution, sealing, and sterilizing;
(2) preparing an effective compound liquid seed with harmonious cordyceps militaris isoniazoic coordination:
taking a cordyceps militaris mother strain which simultaneously has a tegular paecilomyces and a verticillium sporulation structure aggregated into a head on the same hypha as an effective mother strain, taking a streptomycete mother strain as an associated strain, inoculating the effective mother strain and the associated strain into a liquid culture medium, and performing shake-flask culture to obtain an effective composite liquid strain;
(3) inoculation and culture:
diluting the effective composite liquid seed, inoculating to a growth culture medium, and performing coordinated culture for 40-60 days;
(4) harvesting and processing:
collecting fruiting body, stopping light, spreading for cooling, spraying 60% (v/v) white spray soaked with dry folium Pini onto fruiting body surface, stacking under 500LX light for three days, and oven drying at 60 deg.C until water content is less than or equal to 12%.
2. The method for producing multiple active substances of cordyceps militaris in a synergistic manner, as claimed in claim 1, wherein the cordyceps militaris mother strain is deposited in the common microorganism center of the china committee for culture collection and management of microorganisms under the accession number: CGMCC NO 5577, preservation time: 2011.12.12.
3. the method for producing multiple active substances for synergistically improving and stabilizing cordyceps militaris according to claim 1, wherein the cordyceps militaris mother strain is an anti-degeneration cordyceps militaris strain described in patent No. ZL 201210248399.6.
4. The method for producing multiple active substances for synergistically improving and stabilizing cordyceps militaris according to claim 1 or 2, wherein the streptomyces parent strain is streptomyces CPCC200004 antitumor.
5. The method for producing multiple active substances for synergistically improving and stabilizing cordyceps militaris according to claim 1 or 2, wherein the liquid culture medium: 200g of potato, 30g of corn flour, 20g of white granulated sugar, 3g of peptone, 1.5g of monopotassium phosphate, 0.02g of sodium sulfite, 0.01g of indoleacetic acid and 1000ml of water, boiling and dissolving the materials, taking filtrate, supplementing 1000ml of water, subpackaging the obtained mixture in 500ml triangular bottles, filling the bottles with the amount of 120 ml/bottle, sealing, sterilizing at 120 ℃ for 30 minutes, and inoculating when the temperature is cooled to below 30 ℃.
6. The method for producing multiple active substances for synergistically improving and stabilizing cordyceps militaris as claimed in claim 1, wherein the liquid culture medium is inoculated, the standing is carried out for 72-96 hours, the temperature is controlled to be 25-28 ℃, the liquid surface is cultured in a shake flask after being filled with hyphae, the temperature is 23-25 ℃, the temperature is 120-.
7. The method for producing multiple active substances of cordyceps militaris in a synergistic manner according to claim 1, wherein the coordinated culture comprises spawn running culture, mycelium stimulation culture and chromogenic bud forcing culture: promoting the growth and culture of grass, wherein the spawn running culture is 18-21 ℃, the humidity is 70-75%, the dark culture is 10-15 days, the spawn running culture is to dip lime water with the PH9 with a scrubbing brush, the fungus skin is brushed to break, the internal heterodyne of the PH value (the PH value in the culture medium is less than 6) is formed, the color transfer bud promoting culture temperature is 16-24 ℃, the humidity is 80-90%, the illumination is carried out for 24 hours, and the time is 7-10 days; the culture temperature for promoting the growth of grass is 18-22 ℃ and 28-35 days.
8. The method for producing multiple active substances in cordyceps militaris for synergistically improving and stabilizing the activity of multiple active substances according to claim 7, wherein in the stage of promoting growth and culture of grass, when the sporophores grow to 3-5cm, the culture medium and the sporophores are sprayed with magnetized water.
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