CN108812051B - Cordyceps militaris culture method - Google Patents
Cordyceps militaris culture method Download PDFInfo
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Abstract
The invention discloses a cordyceps militaris culture method, which selects a proper culture medium and culturesCondition, by mycelium on Cr3+Enrichment of Cr in the inorganic state3+Transform organic chromium to effectively avoid inorganic Cr3+Natural oxidation to Cr6+Reducing the direct absorption of metal Cr by human body3+Damage to human body. And researches show that chromium in the cordyceps militaris also has a promoting effect on the conversion of polysaccharide in the mycelia, and simultaneously, the organic chromium and the cordyceps polysaccharide have combined action, so that the pancreatic function of a diabetic patient can be improved, and the use of artificial insulin is reduced.
Description
Technical Field
The invention relates to the technical field of strain culture, in particular to a cordyceps militaris culture method.
Background
Cordyceps militaris l.link, also known as Cordyceps militaris, Cordyceps militaris etc., according to the latest classification system, Cordyceps militaris belongs to Ascomycota, hypocrea of hypocrea, cordycetales, cordycepin of cordycecaceae, cordycepin of cordycepin (Sung et al, 2007). Cordyceps militaris has similar active ingredients and pharmacological effects to those of Cordyceps sinensis (Kim and Yun, 2005; Li et al, 2006; Wangjian Fang and Yangchun, 2005), and Cordyceps fermentation products have various biological activities of bacteriostasis, oxidation resistance, anti-inflammation, anti-tumor, immunity enhancement and the like. The cordyceps polysaccharide has obvious blood sugar reducing effect on various diabetes model animals, and the mechanism of the cordyceps polysaccharide, which is possible to inhibit hepatic glucose output, promote the activity of hepatic glucose metabolic enzyme and reduce the content of glucose transport protein, has gradually become an ideal substitute of cordyceps.
The number of diabetic patients reaches 1.5 hundred million worldwide, and reaches 3 hundred million by 2025. The diabetes patients in China are about 3000-4000 ten thousand, and can exceed 5000 ten thousand by 2025 years, and is the third major country with diabetes mellitus in the world after India and the United states. Diabetes is a serious complication, such as uremia caused by diabetic nephropathy, cardiovascular and cerebrovascular diseases (cerebral infarction, coronary heart disease and myocardial infarction can occur), eye diseases (cataract, retinopathy, hemorrhage, glaucoma and the like), peripheral neuropathy (limb numbness, systemic pruritus and the like), peripheral angiopathy (necrosis of lower limb toe ends, diabetic foot and intermittent claudication), various infections (gingivitis, tooth decay, urinary tract infection and easier aggravation and difficult control after infection), and once stressed (wounds such as severe pneumonia, operation and trauma are not easy to heal). The above finally affects the quality of life, brings great pain and inconvenience to patients, and brings heavy burden to family and the whole society. At present, the total medical cost for urban treatment of type 2 diabetes and complications thereof every year in China is 208.60 billion yuan, which accounts for 4.38% of the total medical and health cost in China, wherein the total medical cost for type 2 diabetes with complications is 164.51 billion yuan. The clinical treatment of diabetes at present is to control blood sugar by injecting insulin, and long-term use of insulin can cause insulin dependence and irreversible damage to pancreas. At present, a safe blood sugar-reducing food without side effect is urgently needed, the pancreatic secretion function of a diabetic patient is improved through daily diet, and the dependence of the diabetic patient on insulin is relieved.
Type 2 diabetes (T2DM) is a common endocrine metabolic disease, the main pathologies of which are represented by a deficiency in insulin resistance and islet function, accompanied by severe hyperinsulinemia, wherein insulin resistance plays an important role in the development and progression of type 2 diabetes. The PCS promotes the insulin of fat cells of high-sugar and insulin-induced IR to stimulate the glucose uptake, and simultaneously shows that the hypoglycemic mechanism of the PCS may be related to the promotion of the glucose metabolism of peripheral tissues.
Natural chromium is Cr to Cr6+Exist in various valence states, but are Cr and Cr2+、Cr3+、Cr6+Most commonly, among them, Cr6+Is toxic to the human body. Natural environment(s)Middle Cr2+And Cr3+Poor stability in the inorganic ion state, and is easily formed of Cr2+And Cr3+Conversion to Cr6+Inorganic ions, Cr3+Is a trace element necessary for human body, and the chromium in the human body is almost all Cr3+The recent research of authorities such as the nutritional center of the U.S. department of agriculture proves that Cr3+The inorganic chromium has little effect of enhancing the insulin activity, and has obvious effect of enhancing the insulin activity after being converted into the organic chromium. As an important component of glucose tolerance factor. The research shows that the organic chromium has strong stability, can directly act on histiocytes through cell membranes smoothly as fat-soluble non-electrolyte, enhances the activity of insulin, improves the glycometabolism of human bodies, has important effect on the treatment of diabetes, and is suitable for diabetes, people with abnormal glucose tolerance, high risk of diabetes and healthy people. The existing culture method of cordyceps militaris does not yet culture inorganic Cr by chromium-rich culture3+Reports on the conversion of organic states.
Disclosure of Invention
The invention aims to provide a novel cordyceps militaris culture method, which selects a proper culture medium and culture conditions and carries out Cr culture3+Enrichment of Cr in the inorganic state3+Transform organic state to effectively avoid inorganic Cr3+Natural oxidation to Cr6+Meanwhile, the organic chromium and the cordyceps polysaccharide act together to improve the pancreatic function of the diabetic, thereby reducing the use of artificial insulin.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a cordyceps militaris culture method comprises the following steps:
(1) VB is carried out on the cordyceps militaris strain preserved at the temperature of 0-4 DEG C1Slant culture of strain:
A. preparing a cordyceps militaris strain culture medium: cutting 200 parts by weight of potato into 0.8-1.2cm pieces, decocting in boiling water for 20min, filtering, collecting juice, adding glucose 20 parts, magnesium sulfate 0.5 part, potassium dihydrogen phosphate 1 part, and vitamin B10.01 portion of agar and 20 portions of agar, after melting, the volume is fixed1000mL, natural pH value;
B. subpackaging and sterilizing a cordyceps militaris strain culture medium:
subpackaging the prepared strain culture medium into test tubes of 18mm × 180mm, sealing, and sterilizing at 121 deg.C for 20 min;
C. cooling and inoculating:
taking out the culture medium of the cordyceps militaris strain after sterilization, naturally cooling to 20-30 ℃, and inoculating the cordyceps militaris strain under the aseptic condition;
D. culturing:
placing the inoculated cordyceps militaris strain culture medium in a constant-temperature incubator, and culturing at 22-25 ℃ for 4-8 days to ensure that hyphae fully grow;
E. selecting:
VB of cordyceps militaris with good growth vigor and no degeneration phenomenon1Selecting slant strains;
(2) inoculation:
mixing scarlet caterpiller fungus VB1Inoculating slant strains to a cordyceps militaris liquid strain culture medium under aseptic operation, culturing for 5d in a shaking table with the temperature of 20 ℃ and the rotating speed of 150-;
the cordyceps militaris liquid strain culture medium is a strain culture medium without agar, and comprises the following components in parts by weight: 200 parts of potato, 20 parts of glucose, 0.5 part of magnesium sulfate, 1 part of monopotassium phosphate and vitamin B10.01 part and 1000 parts of water.
The chromium-rich culture medium comprises the following components in parts by weight: 30 parts of oat and CrCl30.4-0.6 part, 1 part of monopotassium phosphate and 50 parts of water. The preparation method comprises the following steps: firstly, 1 part of monopotassium phosphate and CrCl30.4 to 0.6 portion of the oat powder is added into 50 portions of water to be stirred and dissolved, 30 portions of the weighed oat powder is put into a 500mL tissue culture bottle, and the dissolved monopotassium phosphate and CrCl are added3Covering the mixed solution with a cover, sterilizing at 121 deg.C for 60min, taking out, and cooling.
(3) And (3) chromium-rich culture:
inoculating the cooled chromium-rich culture medium into the Cordyceps militaris liquid, culturing at 16-20 deg.C for 8-12 days, mechanically sterilizing, transferring to 18-20 deg.C, relative humidity of 85-95%, and illumination intensity of 150-; collecting with forceps when the fruiting body grows to 8-12cm, and removing culture medium connected with the fruiting body for collecting fruiting body.
Preferably, the conditions of the chromium-rich culture are: inoculating the cooled chromium-rich culture medium into the Cordyceps militaris liquid, culturing at 20 deg.C for 10 days, mechanically sterilizing after mycelia fully grow, transferring to 18 deg.C, relative humidity of 90%, and illumination intensity of 200 lux for 12 hr per day, and culturing for 15 days; and taking out the sporocarp by using tweezers when the sporocarp grows to 10 cm.
Wherein, the preparation method of the liquid strain culture medium in the step (2) comprises the following steps: cutting 200 parts by weight of potato into 0.8-1.2cm pieces, decocting in boiling water for 20min, filtering, collecting juice, adding glucose 20 parts, magnesium sulfate 0.5 part, potassium dihydrogen phosphate 1 part, and vitamin B10.01 portion, after melting, 1000mL of water is added, and the pH value is natural.
More specifically, the cordyceps militaris liquid strain culture medium in the step (2) is filled into each bottle with the volume of 250mL, and then sterilized at 121 ℃ for 30min, cooled and inoculated, wherein each bottle is inoculated with 20mL of liquid strain. The chromium-rich culture medium is prepared and then is subpackaged in 500ml can bottles, a sealing film special for edible fungi is used for sealing, and the sterilization is carried out for 20 minutes at the temperature of 121 ℃; taking out the tin bottle containing the chromium-rich culture medium after sterilization, and naturally cooling to 20-30 ℃.
The invention has the following advantages:
the method selects proper culture medium and culture conditions, and carries out the treatment of Cr3+Enrichment of Cr in the inorganic state3+Transform organic chromium to effectively avoid inorganic Cr3+Natural oxidation to Cr6+Reducing the direct absorption of metal Cr by human body3+Damage to human body.
And researches also find that chromium in cordyceps militaris also has a promoting effect on the conversion of polysaccharide in mycelium of the cordyceps militaris, so that an unexpected technical effect is achieved, and meanwhile, the organic chromium and the cordyceps militaris polysaccharide act together, so that the pancreatic function of a diabetic patient can be improved, and the use of artificial insulin is reduced.
Drawings
FIG. 1 is CrCl in chromium-rich medium3Influence of additive amount on the yield of the subelement;
FIG. 2 CrCl concentrations3Influence of the addition amount on the chromium content in the subelement;
FIG. 3 is the effect of chromium concentration on polysaccharide in mycelium.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Various reagents and test materials (including cordyceps militaris strains) adopted by the invention can be purchased from the market.
Example 1
A cordyceps militaris culture method comprises the following steps:
(1) VB is carried out on the cordyceps militaris strain preserved at the temperature of 0-4 DEG C1Slant culture of strain:
A. preparing a cordyceps militaris strain culture medium: cutting 200 parts by weight of potato into 0.8-1.2cm pieces, decocting in boiling water for 20min, filtering, collecting juice, adding glucose 20 parts, magnesium sulfate 0.5 part, potassium dihydrogen phosphate 1 part, and vitamin B10.01 part of agar and 20 parts of agar, and after melting, the volume is fixed to 1000mL, and the pH value is natural;
B. subpackaging and sterilizing a cordyceps militaris strain culture medium:
subpackaging the prepared strain culture medium into test tubes of 18mm × 180mm, sealing, and sterilizing at 121 deg.C for 20 min;
C. cooling and inoculating:
taking out the culture medium of the cordyceps militaris strain after sterilization, naturally cooling to 30 ℃, and inoculating the cordyceps militaris strain under the aseptic condition;
D. culturing:
placing the inoculated cordyceps militaris strain culture medium in a constant-temperature incubator, and culturing at 25 ℃ for 4 days to ensure that hyphae fully grow;
E. selecting:
VB of cordyceps militaris with good growth vigor and no degeneration phenomenon1Selecting slant strains;
(2) inoculation:
firstly, preparing a cordyceps militaris liquid strain culture medium: cutting 200 parts by weight of potato into 0.8-1.2cm pieces, decocting in boiling water for 20min, filtering, collecting juice, adding glucose 20 parts, magnesium sulfate 0.5 part, potassium dihydrogen phosphate 1 part, and vitamin B10.01 portion, after melting, 1000mL of water is added, and the pH value is natural.
Preparing a chromium-rich culture medium: firstly, 1 part of monopotassium phosphate and CrCl3Adding 0.5 part of oat into 50 parts of water, stirring and dissolving, filling 30 parts of weighed oat into a 500mL tissue culture bottle, and adding dissolved potassium dihydrogen phosphate and CrCl3Covering the mixed solution with a cover, sterilizing at 121 deg.C for 60min, taking out, and cooling.
Then filling the cordyceps militaris liquid strain culture medium into each bottle with the volume of 250mL, then sterilizing at 121 ℃ for 30min, cooling and inoculating, wherein each bottle is inoculated with 20mL of liquid strain. The chromium-rich culture medium is prepared and then is subpackaged in 500ml can bottles, a sealing film special for edible fungi is used for sealing, and the sterilization is carried out for 20 minutes at the temperature of 121 ℃; taking out the tin bottle containing the chromium-rich culture medium after sterilization, and naturally cooling to 20-30 ℃.
Then mixing the Cordyceps militaris VB1Inoculating slant strains to Cordyceps militaris liquid strain culture medium under aseptic operation, culturing in shaking table at 20 deg.C and rotation speed of 160r/min for 5d, inoculating liquid fermented strains to chromium-rich culture medium, wherein the inoculation amount of each bottle is 20 mL;
(3) and (3) chromium-rich culture:
inoculating the cooled chromium-rich culture medium into the Cordyceps militaris liquid, culturing at 18 deg.C for 8 days, mechanically sterilizing, transferring to 20 deg.C, relative humidity of 95%, and illumination intensity of 250 lux, and culturing for 10 days; collecting fruiting body with tweezers when the fruiting body grows to about 12cm, and removing culture medium connected with the fruiting body to collect fruiting body.
Example 2
A cordyceps militaris culture method comprises the following steps:
(1) VB is carried out on the cordyceps militaris strain preserved at the temperature of 0-4 DEG C1Slant culture of strain:
A. preparing a cordyceps militaris strain culture medium: cutting 200 parts by weight of potato into 0.8-1.2cm pieces, decocting in boiling water for 20min, filtering, collecting juice, adding glucose 20 parts, magnesium sulfate 0.5 part, potassium dihydrogen phosphate 1 part, and vitamin B10.01 part of agar and 20 parts of agar, and after melting, the volume is fixed to 1000mL, and the pH value is natural;
B. subpackaging and sterilizing a cordyceps militaris strain culture medium:
subpackaging the prepared strain culture medium into test tubes of 18mm × 180mm, sealing, and sterilizing at 121 deg.C for 20 min;
C. cooling and inoculating:
taking out the culture medium of the cordyceps militaris strain after sterilization, naturally cooling to 25 ℃, and inoculating the cordyceps militaris strain under the aseptic condition;
D. culturing:
placing the inoculated cordyceps militaris strain culture medium in a constant-temperature incubator, and culturing at 24 ℃ for 6 days to ensure that hyphae fully grow;
E. selecting:
VB of cordyceps militaris with good growth vigor and no degeneration phenomenon1Selecting slant strains;
(2) inoculation:
firstly, preparing a cordyceps militaris liquid strain culture medium: cutting 200 parts by weight of potato into 0.8-1.2cm pieces, decocting in boiling water for 20min, filtering, collecting juice, adding glucose 20 parts, magnesium sulfate 0.5 part, potassium dihydrogen phosphate 1 part, and vitamin B10.01 portion, after melting, 1000mL of water is added, and the pH value is natural.
Preparing a chromium-rich culture medium: firstly, 1 part of monopotassium phosphate and CrCl3Adding 0.6 part of oat into 50 parts of water, stirring and dissolving, filling 30 parts of weighed oat into a 500mL tissue culture bottle, and adding dissolved potassium dihydrogen phosphate and CrCl3Covering the mixed solution, sterilizing at 121 deg.C for 60min, and cooling.
Then filling the cordyceps militaris liquid strain culture medium into each bottle with the volume of 250mL, then sterilizing at 121 ℃ for 30min, cooling and inoculating, wherein each bottle is inoculated with 20mL of liquid strain. The chromium-rich culture medium is prepared and then is subpackaged in 500ml can bottles, a sealing film special for edible fungi is used for sealing, and the sterilization is carried out for 20 minutes at the temperature of 121 ℃; taking out the tin bottle containing the chromium-rich culture medium after sterilization, and naturally cooling to 20-30 ℃.
Then mixing the Cordyceps militaris VB1Inoculating slant strains to Cordyceps militaris liquid strain culture medium under aseptic operation, culturing in shaking table at 20 deg.C and rotation speed of 160r/min for 5d, inoculating liquid fermented strains to chromium-rich culture medium, wherein the inoculation amount of each bottle is 20 mL;
(3) and (3) chromium-rich culture:
inoculating the cooled chromium-rich culture medium into the cordyceps militaris liquid, placing the cordyceps militaris liquid in a constant-temperature incubator, culturing for 10 days at 20 ℃, mechanically culturing for 15 days after mycelia fully grow, then, transferring the cordyceps militaris liquid to the condition of 18 ℃, relative humidity of 90% and illumination intensity of 200 lux for 12 hours every day, and collecting by using a pair of tweezers when the sporophores grow to 10cm, and removing the culture medium connected with the sporophores to collect the sporophores.
Example 3
A cordyceps militaris culture method comprises the following steps:
(1) VB is carried out on the cordyceps militaris strain preserved at the temperature of 0-4 DEG C1Slant culture of strain:
A. preparing a cordyceps militaris strain culture medium: cutting 200 parts by weight of potato into 0.8-1.2cm pieces, decocting in boiling water for 20min, filtering, collecting juice, adding glucose 20 parts, magnesium sulfate 0.5 part, potassium dihydrogen phosphate 1 part, and vitamin B10.01 part of agar and 20 parts of agar, and after melting, the volume is fixed to 1000mL, and the pH value is natural;
B. subpackaging and sterilizing a cordyceps militaris strain culture medium:
subpackaging the prepared strain culture medium into test tubes of 18mm × 180mm, sealing, and sterilizing at 121 deg.C for 20 min;
C. cooling and inoculating:
taking out the culture medium of the cordyceps militaris strain after sterilization, naturally cooling to 20 ℃, and inoculating the cordyceps militaris strain under the aseptic condition;
D. culturing:
placing the inoculated cordyceps militaris strain culture medium in a constant-temperature incubator, and culturing at 22 ℃ for 8 days to ensure that hyphae fully grow;
E. selecting:
VB of cordyceps militaris with good growth vigor and no degeneration phenomenon1Selecting slant strains;
(2) inoculation:
firstly, preparing a cordyceps militaris liquid strain culture medium: cutting 200 parts by weight of potato into 0.8-1.2cm pieces, decocting in boiling water for 20min, filtering, collecting juice, adding glucose 20 parts, magnesium sulfate 0.5 part, potassium dihydrogen phosphate 1 part, and vitamin B10.01 portion, after melting, 1000mL of water is added, and the pH value is natural.
Preparing a chromium-rich culture medium: firstly, 1 part of monopotassium phosphate and CrCl3Adding 0.4 part of oat into 50 parts of water, stirring and dissolving, filling 30 parts of weighed oat into a 500mL tissue culture bottle, and adding dissolved potassium dihydrogen phosphate and CrCl3Covering the mixed solution, sterilizing at 121 deg.C for 60min, taking out, and cooling;
then filling the cordyceps militaris liquid strain culture medium into each bottle with the volume of 250mL, then sterilizing at 121 ℃ for 30min, cooling and inoculating, wherein each bottle is inoculated with 20mL of liquid strain. The chromium-rich culture medium is prepared and then is subpackaged in 500ml can bottles, a sealing film special for edible fungi is used for sealing, and the sterilization is carried out for 20 minutes at the temperature of 121 ℃; taking out the tin bottle containing the chromium-rich culture medium after sterilization, and naturally cooling to 25 ℃.
Then mixing the Cordyceps militaris VB1Inoculating slant strains to Cordyceps militaris liquid strain culture medium under aseptic operation, culturing in shaking table at 20 deg.C and 150r/min for 5d, inoculating liquid fermented strains to chromium-rich culture medium, wherein the inoculation amount in each bottle is 20 mL;
(3) and (3) chromium-rich culture:
inoculating the cooled chromium-rich culture medium into the Cordyceps militaris liquid, culturing at 16 deg.C for 12 days, mechanically sterilizing, and culturing at 20 deg.C under relative humidity of 85% and illumination intensity of 150 lux for 13 hr per day for 15 days; collecting fruiting body with tweezers when the fruiting body grows to about 8cm, and removing culture medium connected with the fruiting body to collect fruiting body.
The inventors have also demonstrated the beneficial effects of the present invention by the following tests, in particular:
firstly, the inventor separates and measures trivalent organic chromium by a capillary electrophoresis method to determine that the existence form of chromium in cordyceps militaris is an organic state, and also performs the following tests:
one, CrCl in chromium-rich medium3Effect of addition on daughter yield
As can be seen from FIG. 1, when the concentration of chromium is below 0.6. mu.g/mL, the biomass of the fruiting body increases with the increase of the chromium concentration, the biomass of the fruiting body reaches the maximum at 0.6. mu.g/mL and is 21.3 g/bottle, the yield of the fruiting body is the highest at the concentration of chromium of 0.6. mu.g/mL, the biotransformation rate is 71%, and the biotransformation rate of the cordyceps militaris tested by the conventional cultivation method is about 60%.
Two, CrCl of different concentrations3Influence of the addition on the chromium content of the subelement
As can be seen from FIG. 2, the chromium content in the fruit body increased with the increase of the chromium concentration at a chromium concentration of 0.6. mu.g/mL or less, and the chromium content was in direct correlation with the chromium concentration, and reached a maximum at a chromium concentration of 0.6. mu.g/mL, the organic chromium was 314.33. mu.g/g, and exceeded 0.6. mu.g/mL, and the chromium content began to decrease.
Third, the Effect of chromium concentration on polysaccharides in mycelia
As can be seen from FIG. 3, the chromium concentration has a promoting effect on the transformation of polysaccharide in the mycelia, below 0.4. mu.g/mL, the polysaccharide content in the mycelia increases with the increase of the chromium concentration, the culture medium without chromium ions is added, the content of cordyceps polysaccharide is 0.23mg/g, 0.44mg/mL at 0.6. mu.g/mL, which is 2 times of that of the conventional cultivation method, and the optimum concentration for the transformation of polysaccharide content in mycelia at this time is more than 0.6. mu.g/g, the polysaccharide content begins to decrease.
Fourth, the influence of different culture methods on the blood sugar of cordyceps militaris
A mouse model of alloxan induced uropathy is adopted, and cordyceps militaris in a conventional culture mode and a chromium-rich culture mode are subjected to 2 dose groups (600mg/kg and 300mg/kg), a positive control group (diabetic mice with diabetes mellitus pills of 1000 mg/kg), 2 dose groups (6 mu g/kg and 10 mu g/kg) of chromium picolinate and a negative control group. The preparation is administered by gavage with a volume of 0.20ml/10g, 1 time daily for 7 days. Fasting is carried out 7h before the last administration, blood is collected from orbital venous plexus after 1h of administration, and fasting blood glucose is measured by glucose oxidase method from separated serum. Experiments show that the chromium-rich cordyceps militaris has a better treatment effect on a alloxan diabetes animal model when being administrated by gastric administration at 600mg/kg, has significant difference (P is less than 0.01 and P is less than 0.05) compared with a model control group, and has no obvious influence on the blood sugar of a normal animal under the dosage of 600mg/kg, so that the prompt that the chromium-rich cordyceps militaris has no obvious insulin release stimulation effect or insulin-like effect is provided, and the diabetes-eliminating pill 1000mg/kg has an influence on the blood sugar of the normal animal is provided.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. A cordyceps militaris culture method is characterized by comprising the following steps:
(1) VB Cordyceps militaris strain preserved at 0-4 deg.C1Slant culture of strain:
A. preparing a cordyceps militaris strain culture medium: cutting 200 parts by weight of potato into small pieces of 0.8-1.2cm, boiling in boiling water for 20min, filtering, collecting juice, adding 20 parts of glucose, 0.5 part of magnesium sulfate, 1 part of potassium dihydrogen phosphate, and vitamin B10.01 part of agar and 20 parts of agar, and after melting, the volume is fixed to 1000mL, thus obtaining the natural pH value;
B. subpackaging and sterilizing a cordyceps militaris strain culture medium:
subpackaging prepared Cordyceps militaris strain culture medium into test tubes of 18mm × 180mm, sealing, and sterilizing at 121 deg.C for 20 min;
C. cooling and inoculating:
taking out the culture medium of the cordyceps militaris strain after sterilization, naturally cooling to 20-30 ℃, and inoculating the cordyceps militaris strain under the aseptic condition;
D. culturing:
placing the inoculated cordyceps militaris strain culture medium in a constant-temperature incubator, and culturing at 22-25 ℃ for 4-8 days to ensure that hyphae fully grow;
E. selecting:
VB of cordyceps militaris with good growth vigor and no degeneration phenomenon1Selecting slant strains;
(2) inoculation:
mixing scarlet caterpiller fungus VB1Inoculating slant strains to a cordyceps militaris liquid strain culture medium under aseptic operation, culturing for 5d in a shaking table with the temperature of 20 ℃ and the rotating speed of 150-; the chromium-rich culture medium comprises the following components in parts by weight: 30 parts of oat and CrCl30.6 part, 1 part of monopotassium phosphate and 50 parts of water; CrCl in the chromium-rich medium3The concentration of (A) is 0.6 mug/mL;
(3) and (3) chromium-rich culture:
the conditions of the chromium-rich culture are as follows: inoculating the cooled chromium-rich culture medium into the cordyceps militaris liquid, placing the cordyceps militaris liquid in a constant-temperature incubator, culturing for 10 days at 20 ℃, mechanically culturing for 15 days after mycelia fully grow, then, transferring the cordyceps militaris liquid to the condition of 18 ℃, relative humidity of 90% and illumination intensity of 200 lux for 12 hours every day, and collecting by using a pair of tweezers when the sporophores grow to 10cm, and removing the culture medium connected with the sporophores to collect the sporophores.
2. The cordyceps militaris culture method according to claim 1, wherein the liquid strain culture medium in the step (2) comprises the following components in parts by weight: 200 parts of potato, 20 parts of glucose, 0.5 part of magnesium sulfate, 1 part of monopotassium phosphate and vitamin B10.01 part and 1000 parts of water.
3. The method for culturing Cordyceps militaris (L.) Link as claimed in claim 1, wherein the volume of the Cordyceps militaris liquid strain culture medium in step (2) is 250mL per bottle, and then the product is sterilized at 121 deg.C for 30min, cooled and inoculated, and 20mL of liquid strain is inoculated per bottle.
4. The method for culturing cordyceps militaris according to claim 1, wherein the chromium-rich medium in the step (2) is dispensed into 500ml tin bottles after being prepared, the sealing films special for edible fungi are sealed, and the mixture is sterilized for 20 minutes at 121 ℃; taking out the tin bottle containing the chromium-rich culture medium after sterilization, and naturally cooling to 20-30 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1297995A (en) * | 2001-01-12 | 2001-06-06 | 杨忠才 | Production process of chromium-rich cordyceps mycelium |
| CN103125275A (en) * | 2013-03-15 | 2013-06-05 | 熊艳 | Cultivation method of cordyceps militaris |
| CN104604522A (en) * | 2015-01-19 | 2015-05-13 | 河南省民兴茧丝绸有限责任公司 | Method for producing cordyceps militaris |
| CN105733957A (en) * | 2016-03-01 | 2016-07-06 | 鲁东大学 | Method for culturing cordyceps militaris with slant solid strains effectively instead of liquid strains |
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2018
- 2018-05-23 CN CN201810499060.0A patent/CN108812051B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297995A (en) * | 2001-01-12 | 2001-06-06 | 杨忠才 | Production process of chromium-rich cordyceps mycelium |
| CN103125275A (en) * | 2013-03-15 | 2013-06-05 | 熊艳 | Cultivation method of cordyceps militaris |
| CN104604522A (en) * | 2015-01-19 | 2015-05-13 | 河南省民兴茧丝绸有限责任公司 | Method for producing cordyceps militaris |
| CN105733957A (en) * | 2016-03-01 | 2016-07-06 | 鲁东大学 | Method for culturing cordyceps militaris with slant solid strains effectively instead of liquid strains |
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