CN109618815B - Method for culturing cordyceps sinensis mycelia by using red yeast rice extraction residues - Google Patents
Method for culturing cordyceps sinensis mycelia by using red yeast rice extraction residues Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention discloses a method for culturing cordyceps sinensis mycelia by using red yeast rice extraction residues, which provides necessary trace elements for the growth of cordyceps sinensis mycelia and is easy for the mycelia to absorb assimilated nutrients by extracting the residues through naturally fermented red yeast rice; the production and accumulation of cordycepin in the cordyceps can be increased by adding vitamins; the addition of the compound amino acid can effectively increase the amount of amino in the cordyceps sinensis, and has a promoting effect on the synthesis of adenosine and cordycepin; the nutrient components in the fermentation liquor can be fully decomposed and degraded through fermentation, so that the aim of quickly converting cordyceps mycelia is fulfilled; the polysaccharide content in the cordyceps sinensis mycelia can be effectively improved through alternate irradiation culture of blue light and white light; the processing cost of agricultural waste is reduced by effectively utilizing the red yeast rice extraction residue.
Description
Technical Field
The invention relates to the technical field of artificial cultivation of cordyceps sinensis, in particular to a method for cultivating cordyceps sinensis mycelia by using red yeast rice extraction residues.
Background
With the continuous improvement of the living standard and the living quality of people, the continuous expansion of the cognition field and the increasing demand of functional health care products and foods. Cordyceps sinensis is a medicinal and edible fungus with remarkable health care curative effect, wherein the best known Cordyceps sinensis is Cordyceps sinensis, and related series products of the Cordyceps sinensis are dozens of products, including health care products, foods, Chinese patent medicines and the like, and have been widely reputed all over the world. However, the hosts of the cordyceps sinensis are single, the living environment is harsh, and the natural host hepialus has long life cycle, more natural enemies, more diseases and serious shortage of naturally propagated larva yield, and the environmental pollution in the production area of the cordyceps sinensis is serious in these years, so that the chance of infecting the host by immature cordyceps sinensis is greatly reduced due to artificial early and excessive mining and digging and meadow desertification, and the like, so that the quantity and quality of the cordyceps sinensis are greatly reduced, the source of the cordyceps sinensis is scarce, the price is high, and the increasing demand of the market can not be met.
Therefore, a large amount of artificially cultured cordyceps sinensis is coming to the market, but in the artificial culture, a culture medium for culturing cordyceps sinensis strains cannot reach the growth environment of natural wild cordyceps sinensis, so that sufficient trace elements, amino acids, vitamins and the like cannot be obtained in the growth of mycelia, so that active ingredients in the artificially cultured cordyceps sinensis are far smaller than those of the natural wild cordyceps sinensis, the natural wild effect cannot be achieved, and the effective active ingredients of the cordyceps sinensis are known as adenosine, cordycepin and polysaccharide at the present stage.
Disclosure of Invention
The invention aims to provide a method for culturing cordyceps sinensis mycelia by using red yeast rice extraction residues and provide an artificial culture method of cordyceps sinensis with high-efficiency activity, aiming at the defects of the prior art.
The invention provides a method for culturing cordyceps sinensis mycelia by using red yeast rice extraction residues, which comprises the following steps:
(1) drying and powdering the red yeast rice extraction residue to obtain red yeast rice powder;
(2) mixing 30-45 parts of red yeast rice powder, 10-15 parts of glucose, 7-10 parts of vitamin, 3-6 parts of compound amino acid, 0.5-1 part of monopotassium phosphate, 0.5-1 part of magnesium sulfate, 5-10 parts of yeast and 900 parts of water 800-containing material by mass, and fermenting for 2-3d to obtain fermentation liquor;
(3) filtering the fermentation liquor residues, collecting filtrate, pouring the filtrate into a container, sealing, placing into a steam sterilization cabinet for sterilization at 121 ℃ for 30min, and cooling to obtain a liquid culture medium;
(4) inoculating the cordyceps sinensis strain into a liquid culture medium, wherein the amount of the monomer inoculum is 3% -5% of the weight of the corresponding culture medium each time, and performing shake culture after inoculation, wherein the culture temperature is 15-20 ℃, and the culture time is 8-12d, so as to obtain the liquid strain containing cordyceps sinensis mycelia.
Further, the red yeast rice extraction residue is subjected to enzymolysis by adding protease, then subjected to inactivation treatment of the protease, and then subjected to enzymolysis by adding cellulase after the protease is inactivated.
Furthermore, 1-3 parts of fructose, 1-3 parts of starch and 1-2 parts of saccharifying enzyme are required to be added after the red yeast rice extraction residue is subjected to enzymolysis.
Further, the vitamins are vitamin B1 and B6, and the mass ratio of the vitamins B1 and B6 is 1: 1.
Further, the compound amino acid comprises 0.5-1 part of arginine, 0.5-1 part of lysine, 0.5-1 part of histidine, 0.5-1 part of threonine, 0.5-1 part of aspartic acid and 0.5-1 part of alanine according to the mass parts.
Further, in both steps (3) and (4), sterile operation is carried out in a sterile operation box.
Further, the light culture is performed 4 days after the culture in the step (4).
Further, the illumination culture is blue light and white light alternate illumination culture, and the blue light and the white light alternate illumination culture once a day.
Further, the blue light and the white light are both cold light sources.
According to the method for culturing cordyceps sinensis mycelia by using the red yeast rice extraction residues, the natural fermented red yeast rice extraction residues provide necessary trace elements for the growth of cordyceps sinensis mycelia and are easy for the mycelia to absorb assimilated nutrients; the production and accumulation of cordycepin in the cordyceps can be increased by adding vitamins; the addition of the compound amino acid can effectively increase the amount of amino in the cordyceps sinensis, and has a promoting effect on the synthesis of adenosine and cordycepin; the polysaccharide content in the cordyceps sinensis mycelia can be effectively improved through alternate irradiation culture of blue light and white light; the fermentation can fully decompose and degrade the nutrient components in the fermentation liquor, so that the aim of quickly converting the cordyceps mycelia is fulfilled, and the processing cost of agricultural waste is reduced by effectively utilizing the red yeast rice extraction residue.
Detailed Description
The invention will be further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, various changes or modifications of the present invention may be made by those skilled in the art, and equivalents may fall within the scope of the claims of the present application. The proportions in the examples of the invention are by weight.
Example 1
(1) Drying and powdering the red yeast rice extraction residue to obtain red yeast rice powder;
(2) mixing 30 parts of red yeast rice powder, 10 parts of glucose, 7 parts of vitamin, 3 parts of compound amino acid, 0.5 part of monopotassium phosphate, 0.5 part of magnesium sulfate, 5 parts of yeast and 800 parts of water according to the parts by mass, and fermenting for 2d to obtain fermentation liquor;
the compound amino acid in this example contains, by mass, 0.5 part of arginine, 0.5 part of lysine, 0.5 part of histidine, 0.5 part of threonine, 0.5 part of aspartic acid, and 0.5 part of alanine.
(3) Filtering the fermentation liquor residues, collecting filtrate, pouring the filtrate into a container, sealing, placing into a steam sterilization cabinet for sterilization at 121 ℃ for 30min, and cooling to obtain a liquid culture medium;
(4) inoculating cordyceps sinensis strains into a liquid culture medium, wherein the inoculation amount of each monomer is 3% of the weight of the corresponding culture medium, performing shake culture after inoculation, and performing illumination culture after 4 days at the culture temperature of 15-20 ℃ for 8d to obtain liquid strains containing cordyceps sinensis mycelia;
(5) slowly injecting the liquid strains into the sterile and dried barley pests by an injector, wherein the injection points are 3-5, and stopping injection after the surfaces of the liquid strains are all wet;
(6) culturing the host in a culture flask at 15-20 deg.C in dark for 30 d; then illumination culture is carried out for 35d, the culture temperature is 26-30 ℃, and the artificial cordyceps is obtained.
Example 2
(1) Drying and powdering the red yeast rice extraction residue to obtain red yeast rice powder;
(2) mixing 37 parts of red yeast rice powder, 13 parts of glucose, 8 parts of vitamin, 6 parts of compound amino acid, 0.7 part of monopotassium phosphate, 0.7 part of magnesium sulfate, 8 parts of yeast and 850 parts of water according to parts by mass, and fermenting for 3d to obtain fermentation liquor;
the compound amino acid in this example contains, in parts by mass, 1 part of arginine, 1 part of lysine, 1 part of histidine, 1 part of threonine, 1 part of aspartic acid, and 1 part of alanine.
(3) Filtering the fermentation liquor residues, collecting filtrate, pouring the filtrate into a container, sealing, placing into a steam sterilization cabinet for sterilization at 121 ℃ for 30min, and cooling to obtain a liquid culture medium;
(4) inoculating cordyceps sinensis strains into a liquid culture medium, wherein the amount of the monomer inoculum is 4% of the weight of the corresponding culture medium each time, performing shake culture after inoculation, and performing illumination culture after 4 days at the culture temperature of 15-20 ℃ for 10 days to obtain liquid strains containing cordyceps sinensis mycelia;
(5) slowly injecting the liquid strains into the sterile and dried barley pests by an injector, wherein the injection points are 3-5, and stopping injection after the surfaces of the liquid strains are all wet;
(6) culturing the host in a culture flask at 15-20 deg.C in dark for 30 d; then illumination culture is carried out for 35d, the culture temperature is 26-30 ℃, and the artificial cordyceps is obtained.
Example 3
(1) Drying and powdering the red yeast rice extraction residue to obtain red yeast rice powder;
(2) mixing 45 parts of red yeast rice powder, 15 parts of glucose, 10 parts of vitamin, 6 parts of compound amino acid, 1 part of monopotassium phosphate, 1 part of magnesium sulfate, 10 parts of yeast and 900 parts of water according to parts by mass, and fermenting for 3d to obtain fermentation liquor;
the compound amino acid in this example contains, in parts by mass, 1 part of arginine, 1 part of lysine, 1 part of histidine, 1 part of threonine, 1 part of aspartic acid, and 1 part of alanine.
(3) Filtering the fermentation liquor residues, collecting filtrate, pouring the filtrate into a container, sealing, placing into a steam sterilization cabinet for sterilization at 121 ℃ for 30min, and cooling to obtain a liquid culture medium;
(4) inoculating cordyceps sinensis strains into a liquid culture medium, wherein the amount of the monomer inoculum is 5% of the weight of the corresponding culture medium each time, performing shake culture after inoculation, and performing illumination culture after 4 days at the culture temperature of 15-20 ℃ for 12d to obtain liquid strains containing cordyceps sinensis mycelia;
(5) slowly injecting the liquid strains into the sterile and dried barley pests by an injector, wherein the injection points are 3-5, and stopping injection after the surfaces of the liquid strains are all wet;
(6) culturing the host in a culture flask at 15-20 deg.C in dark for 30 d; then illumination culture is carried out for 35d, the culture temperature is 26-30 ℃, and the artificial cordyceps is obtained.
Comparative example
(1) Mixing 45 parts of starch, 15 parts of glucose, 10 parts of vitamin, 6 parts of compound amino acid, 1 part of monopotassium phosphate, 1 part of magnesium sulfate, 10 parts of yeast and 900 parts of water according to the mass parts, and fermenting for 3d to obtain fermentation liquor;
the compound amino acid in this example contains, in parts by mass, 1 part of arginine, 1 part of lysine, 1 part of histidine, 1 part of threonine, 1 part of aspartic acid, and 1 part of alanine.
(2) Filtering the fermentation liquor residues, collecting filtrate, pouring the filtrate into a container, sealing, placing into a steam sterilization cabinet for sterilization at 121 ℃ for 30min, and cooling to obtain a liquid culture medium;
(3) inoculating cordyceps sinensis strains into a liquid culture medium, wherein the amount of the monomer inoculum is 5% of the weight of the corresponding culture medium each time, performing shake culture after inoculation, and performing illumination culture after 4 days at the culture temperature of 15-20 ℃ for 12d to obtain liquid strains containing cordyceps sinensis mycelia;
(4) slowly injecting the liquid strains into the sterile and dried barley pests by an injector, wherein the injection points are 3-5, and stopping injection after the surfaces of the liquid strains are all wet;
(5) culturing the host in a culture flask at 15-20 deg.C in dark for 30 d; then illumination culture is carried out for 35d, the culture temperature is 26-30 ℃, and the artificial cordyceps is obtained.
Evaluation:
the content detection of adenosine, cordycepin and polysaccharide is carried out on example 1, example 2, example 3, comparative example and wild cordyceps, and the detection results are shown in table 1.
The content of adenosine is determined according to the quality standard of cordyceps sinensis in Chinese pharmacopoeia, and the specific method comprises the following steps: octadecylsilane chemically bonded silica is used as a filler (C18), phosphate buffer (pH6.5, 0.01mol/L sodium dihydrogen phosphate 68.5mL is mixed with 0.01mol/L disodium hydrogen phosphate 315 mL) is used as a mobile phase, and methanol (85: 15) is used as a mobile phase; the detection wavelength is 260 nm; accurately weighing adenosine reference substance, and adding 90% methanol to obtain solution containing 20 μ g per 1mL to obtain reference substance solution; respectively taking 0.5g of each of example 1, example 2, example 3, comparative example and wild cordyceps, precisely weighing, placing in a conical flask with a plug, precisely adding 10mL of 90% methanol, sealing the plug, shaking up, weighing, heating and refluxing for 30min, cooling, supplementing with heavy materials, shaking up, filtering, and taking a subsequent filtrate to obtain each sample solution; respectively sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and calculating adenosine content of each sample by using the concentration ratio equal to the peak area ratio.
Detecting the content of cordycepin, using acetonitrile-water (7: 93) as a mobile phase and a C18 chromatographic column, wherein the flow rate is 1.0 mL/min, and the detection wavelength is 260 nm. Each sample was precisely weighed and placed in a 250 mL measuring flask, and 20 mL of 50% ethanol was added. Extracting with ultrasonic wave (power 100W, frequency 40 kHz) under oscillation for 20 min, cooling, and adding 50% ethanol to scale. Filtering with 0.5 μm microporous membrane to obtain sample solution. And calculating the content of the cordycepin in the sample by using an external standard method.
The polysaccharide content is detected by accurately sucking 1.0mL of each sample treatment solution, placing the sample treatment solution in a 10mL colorimetric tube, and adding distilled water to 2.0 mL. The blank control tube was filled with distilled water. Adding 1.0mL of 6% phenol solution into the sample tube and the blank tube, shaking up, then rapidly adding 5.0mL of concentrated sulfuric acid, shaking up, standing at room temperature for 5 min, heating in boiling water bath for 15 min, taking out, rapidly cooling to room temperature, and measuring absorbance at 490 nm.
As can be seen from the data in Table 1, adenosine, cordycepin and polysaccharide in the artificially cultured cordyceps sinensis are all obviously higher than those in the comparative examples, and although the effective active ingredients of the cordyceps sinensis are not high in wild cordyceps sinensis, the active ingredients are close to the wild cordyceps sinensis in content and have certain substituting capacity, so that the cordyceps sinensis provided by the invention has certain creativity.
The embodiments of the present invention have been described above by way of example, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the application of the present invention shall fall within the scope of the patent of the present invention.
Claims (7)
1. The method for culturing the cordyceps sinensis mycelia by using the red yeast rice extraction residues is characterized by comprising the following steps of: the method comprises the following steps:
(1) drying and powdering the red yeast rice extraction residue to obtain red yeast rice powder;
(2) mixing 30-45 parts of red yeast rice powder, 10-15 parts of glucose, 7-10 parts of vitamin, 3-6 parts of compound amino acid, 0.5-1 part of monopotassium phosphate, 0.5-1 part of magnesium sulfate, 5-10 parts of yeast and 900 parts of water 800-containing material by mass, and fermenting for 2-3d to obtain fermentation liquor;
(3) filtering the fermentation liquor residues, collecting filtrate, pouring the filtrate into a container, sealing, placing into a steam sterilization cabinet for sterilization at 121 ℃ for 30min, and cooling to obtain a liquid culture medium;
(4) inoculating cordyceps sinensis strains into a liquid culture medium, wherein the amount of the monomer inoculum is 3% -5% of the weight of the corresponding culture medium each time, and performing shake culture after inoculation, wherein the culture temperature is 15-20 ℃, and the culture time is 8-12d, so as to obtain liquid strains containing cordyceps sinensis mycelia; the method comprises the steps of firstly adding protease into the red yeast extraction residue for enzymolysis, then inactivating the protease, then adding cellulase for enzymolysis, and further adding 1-3 parts of fructose, 1-3 parts of starch and 1-2 parts of saccharifying enzyme into the red yeast extraction residue after the enzymolysis.
2. The method for culturing cordyceps sinensis mycelia by using the red yeast rice extraction residue as claimed in claim 1, wherein the method comprises the following steps: the vitamin is vitamin B1And B6Said vitamin B1And B6The mass ratio of (A) to (B) is 1: 1.
3. The method for culturing cordyceps sinensis mycelia by using the red yeast rice extraction residue as claimed in claim 1, wherein the method comprises the following steps: the compound amino acid comprises, by mass, 0.5-1 part of arginine, 0.5-1 part of lysine, 0.5-1 part of histidine, 0.5-1 part of threonine, 0.5-1 part of aspartic acid and 0.5-1 part of alanine.
4. The method for culturing cordyceps sinensis mycelia by using the red yeast rice extraction residue as claimed in claim 1, wherein the method comprises the following steps: in the steps (3) and (4), sterile operation is required to be carried out in a sterile operation box.
5. The method for culturing cordyceps sinensis mycelia by using the red yeast rice extraction residue as claimed in claim 1, wherein the method comprises the following steps: and (4) performing illumination culture 4 days after the culture in the step (4).
6. The method for culturing Chinese caterpillar fungus mycelia by using the red yeast rice extraction residue as claimed in claim 5, wherein: the illumination culture is blue light and white light alternate illumination culture, and the blue light and the white light alternate illumination culture once a day.
7. The method for culturing Chinese caterpillar fungus mycelia by using the red yeast rice extraction residue as claimed in claim 6, wherein: the blue light and the white light are both cold light sources.
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