CN115812514A - Method for cultivating cordyceps sobolifera sporocarp - Google Patents
Method for cultivating cordyceps sobolifera sporocarp Download PDFInfo
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Abstract
The invention belongs to the technical field of artificial cultivation of edible fungi, and particularly relates to an artificial cultivation method for improving the content of cordyceps sobolifera polysaccharide and adenosine in cordyceps sobolifera fruiting bodies. The method for cultivating cordyceps sobolifera sporocarp comprises the steps of performing liquid strain cultivation in a liquid culture medium and performing sporocarp cultivation in a solid culture medium, separating edible fungus biochar and an edible fungus carbonization liquid by using edible fungus residues generated after conventional edible fungus cultivation as raw materials in a mode of performing hydrothermal carbonization on the edible fungus residues, and respectively using the edible fungus biochar and the edible fungus carbonization liquid as a solid matrix and a nutrient solution for sporocarp cultivation, so that cordyceps polysaccharide and adenosine are effectively accumulated in a solid fermentation process of cordyceps sobolifera sporocarp, the content of cordyceps polysaccharide and adenosine in the cordyceps sobolifera sporocarp is effectively improved, and the nutritional value of cordyceps sobolifera is improved.
Description
Technical Field
The invention belongs to the technical field of artificial cultivation of edible fungi, and particularly relates to an artificial cultivation method for improving the content of cordyceps sobolifera polysaccharide and adenosine in cordyceps sobolifera fruiting bodies.
Background
Cordyceps cicadae is one of traditional Chinese medicinal materials, also called golden cordyceps cicadae and cordyceps cicadae, and is a dry complex of fungus paecilomyces cicadae parasitizing on cicada nymphs in Cordyceps of Clavicepitaceae. The existing research results show that the cicada fungus is similar to the main ingredient amino acid in various cordyceps sinensis in type and has different degrees of tonifying effects. The biological active substances contained in cordyceps sobolifera reported at present comprise nucleosides, proteins, saccharides, alkaloids, sterols and the like.
Researches find that the cordyceps sobolifera polysaccharide has the pharmacological effects of resisting tumors, inhibiting immunity, enhancing immunity, resisting oxidation and aging, protecting liver, reducing blood pressure and the like. Because the polysaccharide is a high polymer formed by connecting a plurality of same monosaccharide groups by glycosidic bonds, the molecular structure of the polysaccharide is complex, the artificial synthesis is difficult, and the polysaccharide is mostly extracted and prepared from natural products. However, the content of cordyceps cicadae miq extracted from cordyceps cicadae miq is low at present, mainly because the content of cordyceps cicadae miq polysaccharide in artificially cultivated cordyceps cicadae miq is not ideal.
The cordyceps adenosine is one of the important components of cordyceps and is also one of the index substances of cordyceps. The Chinese pharmacopoeia 2005 has clear regulations, and the adenosine content of the cordyceps as a medicine is not less than 0.01%. The cordyceps sinensis adenosine has various biological activities such as protecting a cardiovascular system and a cerebrovascular system, protecting a central nervous system, regulating lipolysis and the like, and is widely applied to treatment of various common human diseases. However, the content of adenosine extracted from cordyceps cicadae miq is not ideal at present.
For example, the artificial cultivation method for increasing the content of cordyceps polysaccharide in cordyceps sobolifera fruiting bodies disclosed in chinese patent CN113330984A effectively increases the content of cordyceps polysaccharide in cordyceps sobolifera through optimization of a solid substrate, but is not ideal for accumulation of adenosine. Also, as the cultivation method for increasing the content of N6- (2-hydroxyethyl) adenosine in cordyceps sobolifera fruiting bodies disclosed in Chinese patent CN113396773A, the content of HEA in cordyceps sobolifera is effectively increased by optimizing the solid matrix, but the method is not ideal for accumulation of cordyceps polysaccharide. Therefore, the content of cordyceps polysaccharide and adenosine in cordyceps sobolifera fruiting bodies is improved by regulating and controlling the artificial cultivation process of cordyceps sobolifera, so that the quality of cordyceps sobolifera is effectively improved, and the method has positive significance for development and popularization of cordyceps sobolifera products.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a method for cultivating cordyceps sobolifera sporocarp, so as to solve the problem that the content of cordyceps polysaccharide and adenosine in cordyceps sobolifera sporocarp is not ideal in the prior art;
the second technical problem to be solved by the invention is to provide cordyceps sobolifera cordyceps sinensis sporocarp rich in cordyceps sinensis polysaccharide and adenosine.
In order to solve the technical problems, the method for cultivating cordyceps sobolifera sporocarp comprises the steps of culturing liquid strains in a liquid culture medium and culturing the sporocarp in a solid culture medium;
the solid culture medium comprises the following components in a mass ratio of 1:1-1.5 of solid matrix and nutrient solution;
the solid matrix comprises the following components in parts by weight: 10-20 parts of wheat, 20-30 parts of corn grit, 12-20 parts of oat bran, 8-15 parts of kudzu root residue, 10-20 parts of cicada pupa powder and edible fungus residue biochar;
the nutrient solution comprises edible fungus residue carbonized liquid, and is added with 5-12g/L of carbon source, 3-8g/L of nitrogen source and 1-3g/L of inorganic salt, and water is added to the mixture until the volume is 1L and the pH value is natural;
wherein the edible fungus residue biochar and the edible fungus residue carbonization liquid are respectively solid and liquid products obtained by solid-liquid separation after hydrothermal carbonization reaction of 5-15 parts by weight of edible fungus residue.
Specifically, in the method for cultivating cordyceps sobolifera sporocarp, the edible fungi residues comprise agaric fungi residues and/or shiitake fungus residues.
Specifically, the method for cultivating cordyceps sobolifera sporocarp further comprises the step of preparing the edible fungus dreg biochar and the edible fungus dreg carbonization liquid, and comprises the following steps:
(1) Drying and pulverizing edible fungi residue;
(2) Adding deionized water into the treated edible fungus residue, mixing, and placing in a stainless steel high-pressure reaction kettle for hydrothermal carbonization reaction;
(3) Collecting reaction products, filtering, collecting solid parts, drying to obtain the edible fungus dreg biochar, and collecting liquid parts to obtain the required edible fungus dreg carbonization liquid.
Specifically, in the step (2), the mass ratio of the edible fungus residues to the deionized water is 1:0.5-0.8.
Specifically, in the step (2), the temperature of the hydrothermal carbonization reaction is 150-250 ℃, the pressure is 1.5-2.0MPa, and the reaction time is 5-12h.
Specifically, the cordyceps sobolifera sporocarp cultivation method comprises a sporocarp cultivation step, a color conversion step and a sporocarp management step;
the control conditions of the spawn running step comprise: controlling the culture temperature to 15-18 ℃ and the humidity to 60-80% for light-proof culture;
the control conditions of the color conversion step comprise: controlling the illumination condition to be 300-400lux, and controlling the photoperiod light-dark ratio to be L16: d8, culturing at 15-18 ℃ and with the humidity of 60-80%;
the control conditions of the sub-entity management step include: controlling the illumination condition to be 300-400lux, and controlling the photoperiod light-dark ratio to be L16: d8, culturing at 20-25 ℃ and humidity of 60-80%.
Specifically, the method for cultivating cordyceps sobolifera sporocarp comprises the following steps:
the carbon source comprises glucose;
the nitrogen source comprises peptone;
the inorganic salt comprises potassium dihydrogen phosphate and/or dipotassium hydrogen phosphate.
Specifically, the liquid culture medium comprises the following components in percentage by mass: glucose 1-2%, yeast extract 0.2-0.4%, peptone 0.3-0.5%, and K 2 HPO 4 0.01-0.03％、KH 2 PO 4 0.01-0.03%, natural pH.
Specifically, the method for cultivating cordyceps sobolifera sporocarp comprises the following steps: controlling the fermentation temperature to be 20-25 ℃, and the stirring speed to be 150-180rpm.
Specifically, the method for cultivating cordyceps sobolifera sporocarp further comprises the step of carrying out conventional activation on the strain before the step of cultivating the liquid strain.
The invention also discloses cordyceps sobolifera sporocarp obtained by the cultivation method.
The method for cultivating cordyceps sobolifera sporocarp provided by the invention is based on the traditional cordyceps sobolifera sporocarp cultivation process, edible fungus residues generated after conventional edible fungi cultivation are used as raw materials, an edible fungus biochar and an edible fungus carbonization liquid are obtained by separation in a mode of carrying out hydrothermal carbonization on the edible fungus residues, and the edible fungus biochar and the edible fungus carbonization liquid are respectively used as a solid matrix and a nutrient solution for sporocarp cultivation. Compared with an application mode of directly taking the edible fungus residues as a substrate, the method can induce the cordyceps polysaccharide and adenosine to be effectively accumulated in the solid fermentation process of cordyceps sobolifera, effectively improves the content of the cordyceps polysaccharide and adenosine in cordyceps sobolifera fruiting bodies, and improves the nutritive value of the cordyceps sobolifera.
Detailed Description
Example 1
Liquid strain culture
Preparing a liquid culture medium which comprises the following components in percentage by mass: glucose 2%, yeast extract 0.3%, peptone 0.4%, K 2 HPO 4 0.02％、KH 2 PO 4 0.02%, natural pH, sterilizing at 121 deg.C for 20min, and naturally cooling to room temperature.
Inoculating the stored Cordyceps cicadae test tube mother strain (Paecilomyces cicadae mother strain) into the liquid culture medium under aseptic condition, controlling fermentation temperature at 22 deg.C, stirring at 160rpm, and shake-flask culturing for 48h to obtain seed solution.
Culture of fruiting bodies
Drying Auricularia auricula residues, crushing, sieving with a 40-mesh sieve, taking 10 parts by weight of the Auricularia auricula residues, adding 8 parts by weight of deionized water, fully mixing, placing in a stainless steel high-pressure reaction kettle, and performing hydrothermal carbonization reaction for 8 hours at the temperature of 200 ℃ and the pressure of 1.5 MPa; and after the reaction is finished, collecting and filtering a reaction product, collecting a solid part, drying to obtain the edible fungus residue biochar, and collecting a liquid part, namely the required edible fungus residue carbonization liquid.
Preparing a solid matrix: comprises the following components in parts by weight: 15 parts of wheat, 25 parts of corn grit, 16 parts of oat bran, 12 parts of kudzu root grit, 15 parts of cicada pupa powder and the collected edible fungus grit biochar.
Preparing a nutrient solution: and (3) taking the collected edible fungus residue carbonization liquid, adding 8g/L glucose, 5g/L peptone, 1g/L potassium dihydrogen phosphate and 1g/L dipotassium hydrogen phosphate, adding deionized water to a constant volume of 1L, and keeping the pH value natural.
According to the following steps of 1:1, uniformly mixing the solid matrix and the nutrient solution, sealing the prepared solid culture medium, placing the culture medium in a high-pressure moist heat sterilization pot, and performing conventional sterilization at 121 ℃ for 40min.
Inoculating the seed solution into the solid culture medium according to the inoculation amount of 8wt% under the room temperature and aseptic condition, then placing the inoculated culture medium into a cultivation room for cultivation, controlling the temperature to be 17 ℃ and the humidity to be 70%, and carrying out light-proof cultivation for 3-5 days until the spawn running is complete.
And then adjusting the illumination condition, controlling the illumination condition to be 300lux, and controlling the photoperiod light-dark ratio to be L16: d8, continuously controlling the culture temperature to be 17 ℃ and the humidity to be 70%, continuously culturing for 5-8 days to finish color conversion and form stroma buds.
And then maintaining the illumination condition, continuously controlling the illumination condition to be 300lux, and controlling the photoperiod light-dark ratio to be L16: d8, controlling the culture temperature to be 22 ℃ and the humidity to be 70%, continuously culturing for 15-20 days, harvesting mature sporocarp, and drying to obtain the fruit body.
Example 2
Liquid strain culture
Preparing a liquid culture medium which comprises the following components in percentage by mass: glucose 2%, yeast extract 0.3%, peptone 0.4%, K 2 HPO 4 0.02％、KH 2 PO 4 0.02%, natural pH, sterilizing at 121 deg.C for 20min, and naturally cooling to room temperature.
Inoculating the stored cordyceps sobolifera test tube mother strain into the liquid culture medium under the aseptic condition, controlling the fermentation temperature to be 22 ℃, and carrying out shake culture for 48 hours at the stirring speed of 160rpm to obtain a seed solution.
Fruit of seedIn vivo culture
Drying agaric fungus residues, crushing, sieving with a 40-mesh sieve, taking 5 parts by weight of agaric fungus residues, adding 4 parts by weight of deionized water, fully mixing, placing in a stainless steel high-pressure reaction kettle, and performing hydrothermal carbonization reaction for 8 hours at the temperature of 200 ℃ and the pressure of 1.5 MPa; and after the reaction is finished, collecting and filtering a reaction product, collecting a solid part, drying to obtain the edible fungus residue biochar, and collecting a liquid part, namely the required edible fungus residue carbonization liquid.
Preparing a solid matrix: comprises the following components in parts by weight: 10 parts of wheat, 20 parts of corn grit, 12 parts of oat bran, 15 parts of kudzu root grit, 20 parts of cicada pupa powder and the collected edible fungus grit biochar.
Preparing a nutrient solution: and (3) taking the collected edible fungus residue carbonization liquid, adding 5g/L of glucose, 8g/L of peptone, 0.5g/L of monopotassium phosphate and 0.5g/L of dipotassium phosphate, and adding deionized water to a constant volume of 1L and keeping the pH value natural.
According to the following steps: 1, uniformly mixing the solid matrix and the nutrient solution, sealing the prepared solid culture medium, placing the culture medium in a high-pressure moist heat sterilization pot, and performing conventional sterilization at 121 ℃ for 40min.
Inoculating the seed solution into the solid culture medium according to the inoculation amount of 8wt% under the room temperature and aseptic condition, then placing the inoculated culture medium into a cultivation room for cultivation, controlling the temperature to be 17 ℃ and the humidity to be 70%, and carrying out light-proof cultivation for 3-5 days until the spawn running is complete.
And then adjusting the illumination condition, controlling the illumination condition to be 300lux, and controlling the photoperiod light-dark ratio to be L16: d8, continuously controlling the culture temperature to be 17 ℃ and the humidity to be 70%, continuously culturing for 5-8 days to finish color conversion and form stroma buds.
And then maintaining the illumination condition, continuously controlling the illumination condition to be 300lux, and controlling the photoperiod light-dark ratio to be L16: d8, controlling the culture temperature to be 22 ℃ and the humidity to be 70%, continuously culturing for 15-20 days, harvesting mature sporocarp, and drying to obtain the fruit body.
Example 3
Liquid strain culture
Preparing a liquid culture medium which comprises the following components in percentage by mass: glucose 2%, yeast extract 0.3%, peptone 0.4%, K 2 HPO 4 0.02％、KH 2 PO 4 0.02%, natural pH, sterilizing at 121 deg.C for 20min, and naturally cooling to room temperature.
Inoculating the stored cordyceps sobolifera test tube mother strain into the liquid culture medium under the aseptic condition, controlling the fermentation temperature to be 22 ℃, and carrying out shake culture for 48 hours at the stirring speed of 160rpm to obtain a seed solution.
Culture of fruiting bodies
Taking mushroom residues, drying, crushing, sieving with a 40-mesh sieve, taking 15 parts by weight of the mushroom residues, adding 12 parts by weight of deionized water, fully mixing, placing in a stainless steel high-pressure reaction kettle, and performing hydrothermal carbonization reaction for 8 hours at the temperature of 200 ℃ and the pressure of 1.5 MPa; and after the reaction is finished, collecting and filtering a reaction product, collecting a solid part, drying to obtain the edible fungus residue biochar, and collecting a liquid part, namely the required edible fungus residue carbonization liquid.
Preparing a solid matrix: comprises the following components in parts by weight: 20 parts of wheat, 20 parts of corn grit, 20 parts of oat bran, 8 parts of kudzu root residue, 10 parts of cicada pupa powder and the collected edible fungus residue biochar.
Preparing a nutrient solution: and (3) taking the collected edible fungus residue carbonization liquid, adding 12g/L of glucose, 3g/L of peptone, 1.5g/L of monopotassium phosphate and 1.5g/L of dipotassium phosphate, and adding deionized water to a constant volume of 1L and keeping the pH value natural.
According to the following steps of 1:1, uniformly mixing the solid matrix and the nutrient solution, sealing the prepared solid culture medium, placing the culture medium in a high-pressure moist heat sterilization pot, and conventionally sterilizing at 121 ℃ for 40min.
Inoculating the seed solution into the solid culture medium according to the inoculation amount of 8wt% under the room temperature and aseptic condition, then placing the inoculated culture medium into a cultivation room for cultivation, controlling the temperature to be 17 ℃ and the humidity to be 70%, and carrying out light-proof cultivation for 3-5 days until the spawn running is complete.
And then adjusting the illumination condition, controlling the illumination condition to be 300lux, and controlling the photoperiod light-dark ratio to be L16: d8, continuously controlling the culture temperature to be 17 ℃ and the humidity to be 70%, continuously culturing for 5-8 days to finish color conversion and form stroma buds.
And then maintaining the illumination condition, continuously controlling the illumination condition to be 300lux, and controlling the photoperiod light-dark ratio to be L16: d8, controlling the culture temperature to be 22 ℃ and the humidity to be 70%, continuously culturing for 15-20 days, harvesting mature sporocarp, and drying to obtain the fruit body.
Comparative example 1
The method for cultivating cordyceps sobolifera in the comparative example is the same as that in example 1, only the agaric fungus residues are directly added into the solid matrix, and the same amount of deionized water is added into the nutrient solution for preparation.
Comparative example 2
The method for cultivating cordyceps sobolifera in the comparative example is the same as that in example 1, and is only different in that the edible fungus residues are mixed with deionized water, then are soaked for 8 hours at normal temperature and normal pressure, and are filtered to collect solid and liquid parts respectively for preparing a solid matrix and a nutrient solution.
Comparative example 3
The method for cultivating cordyceps sobolifera in the comparative example is the same as that in example 1, only the difference is that the edible fungus residues are not added, and the deionized water is used for preparing the nutrient solution.
Examples of the experiments
The fruiting bodies harvested in the above example 1 and comparative examples 1-3 were collected respectively and subjected to determination of contents of Cordyceps sinensis polysaccharides and adenosine, and the results of the determination are shown in Table 1 below.
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 was used as a filler (C18), and a phosphate buffer (ph 6.5, 0.01mol/L sodium dihydrogenphosphate 68.5mL and 0.01mol/L disodium hydrogenphosphate 315mL mixed) was used as a mobile phase, methanol (85; the detection wavelength is 260nm; 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 sporocarp to be detected, precisely weighing, placing in a conical flask with a plug, precisely adding 10mL of 90% methanol, sealing the plug, uniformly shaking, weighing, heating and refluxing for 30min, cooling, re-supplementing, uniformly shaking, filtering, and taking 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.
The determination of the crude polysaccharide was performed by ultrasonic assisted-hot water extraction: accurately weighing 1g of the above fruiting body powder, diluting with distilled water to constant volume of 100ml, placing into a test tube with a plug, performing ultrasonic extraction for 1h, and repeatedly reversing and mixing uniformly every 20 min; transferring into constant temperature water bath, extracting at 80 deg.C for 4 hr, cooling to room temperature, centrifuging the extractive solution at 14000rpm for 5min, and collecting supernatant as crude polysaccharide solution. The content of crude polysaccharide is determined by concentrated sulfuric acid-phenol method (see the method described in Chinese patent CN113330984A for details).
TABLE 1 Cordyceps cicadae Miquel fruiting body polysaccharide and adenosine content mg/g
Therefore, the method can simultaneously improve the content of crude polysaccharide and adenosine in cordyceps sobolifera sporocarp by optimizing the sporocarp culture medium system.
It should be understood that the embodiments described herein are merely illustrative of the principles of embodiments of the present application. Other variations are also possible within the scope of the present application. Thus, by way of example, and not limitation, alternative configurations of the embodiments of the present application can be viewed as being consistent with the teachings of the present application. Accordingly, the embodiments of the present application are not limited to only those embodiments explicitly described and depicted herein.
Claims (10)
1. A method for cultivating cordyceps cicadae miq sporocarp is characterized by comprising the steps of culturing liquid strains in a liquid culture medium and culturing the sporocarp in a solid culture medium;
the solid culture medium comprises the following components in a mass ratio of 1:1-1.5 of solid matrix and nutrient solution;
the solid matrix comprises the following components in parts by weight: 10-20 parts of wheat, 20-30 parts of corn grit, 12-20 parts of oat bran, 8-15 parts of kudzu root residue, 10-20 parts of cicada pupa powder and edible fungus residue biochar;
the nutrient solution comprises edible fungus residue carbonized liquid, and is added with 5-12g/L of carbon source, 3-8g/L of nitrogen source and 1-3g/L of inorganic salt, and water is added to the mixture until the volume is 1L and the pH value is natural;
wherein the edible fungus residue biochar and the edible fungus residue carbonization liquid are respectively solid and liquid products obtained by solid-liquid separation after hydrothermal carbonization reaction of 5-15 parts by weight of edible fungus residue.
2. The method for cultivating cordyceps sobolifera sporocarp according to claim 1, wherein the edible fungi residues comprise agaric fungi residues and/or shiitake fungus residues.
3. The method for cultivating cordyceps sobolifera sporocarp according to claim 1 or 2, wherein the method further comprises the step of preparing the edible fungus dreg biochar and the edible fungus dreg carbonization liquid, and the method comprises the following steps:
(1) Drying and pulverizing edible fungi residue;
(2) Adding deionized water into the treated edible fungus residue, mixing, and placing in a stainless steel high-pressure reaction kettle for hydrothermal carbonization reaction;
(3) Collecting reaction products, filtering, collecting solid parts, drying to obtain the edible fungus dreg biochar, and collecting liquid parts to obtain the required edible fungus dreg carbonization liquid.
4. The method for cultivating cordyceps sobolifera sporocarp according to claim 3, wherein in the step (2), the mass ratio of the edible fungus residues to the deionized water is 1:0.5-0.8.
5. The method for cultivating cordyceps sobolifera sporocarp according to claim 3 or 4, wherein in the step (2), the temperature of the hydrothermal carbonization reaction is 150-250 ℃, the pressure is 1.5-2.0MPa, and the reaction time is 5-12h.
6. The method for cultivating cordyceps sobolifera fruiting body according to any one of claims 1-5, wherein the fruiting body cultivation step includes a spawn running step, a color conversion step and a fruiting body management step;
the control conditions of the spawn running step comprise: controlling the culture temperature to 15-18 ℃ and the humidity to 60-80% for light-proof culture;
the control conditions of the color conversion step comprise: controlling the illumination condition to be 300-400lux, and controlling the photoperiod light-dark ratio to be L16: d8, culturing at 15-18 ℃ and with the humidity of 60-80%;
the control conditions of the sub-entity management step include: controlling the illumination condition to be 300-400lux, and controlling the photoperiod light-dark ratio to be L16: d8, culturing at 20-25 ℃ and humidity of 60-80%.
7. The method for cultivating cordyceps sobolifera sporocarp according to any one of claims 1 to 6, wherein in the nutrient solution:
the carbon source comprises glucose;
the nitrogen source comprises peptone;
the inorganic salt comprises potassium dihydrogen phosphate and/or dipotassium hydrogen phosphate.
8. The method for cultivating cordyceps sobolifera sporocarp according to any one of claims 1 to 7, wherein the liquid culture medium comprises the following components in mass content: glucose 1-2%, yeast extract 0.2-0.4%, peptone 0.3-0.5%, and K 2 HPO 4 0.01-0.03％、KH 2 PO 4 0.01-0.03%, natural pH.
9. The method of claim 8, wherein the conditions for culturing the liquid spawn comprise: controlling the fermentation temperature to be 20-25 ℃, and the stirring speed to be 150-180rpm.
10. The method for cultivating cordyceps sobolifera fruiting body according to any one of claims 1-9, wherein the method further comprises the step of subjecting the strain to conventional activation before the step of culturing the liquid seed culture.
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Application publication date: 20230321 |