CN105985150B - Cordyceps sobolifera sporostalk bundle liquid culture medium - Google Patents
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Abstract
The invention provides a liquid culture medium for artificially culturing cordyceps sobolifera sporophore bundles, which mainly comprises the following raw materials: 0.2-3% of yeast extract powder, 0.1-2% of soybean protein hydrolysate and the balance of water supplement to 100%; the culture medium can also be added with 1-8% of cane sugar, 0.005-0.1% of calcium chloride and 0.01-0.5% of monopotassium phosphate. The yield and the content of the effective components of the cordyceps sobolifera cultured by the culture medium are obviously improved, the product quality is stable, and the culture medium is suitable for large-scale industrial production.
Description
Technical Field
The invention relates to a liquid culture medium for artificially culturing cordyceps sobolifera bundles by cordyceps sobolifera, in particular to a novel process for artificially culturing cordyceps sobolifera.
Background
Cordyceps cicadae (Isaria cicadae Miquel) belongs to the phylum Ascomycota (Ascomycota), phylum Pezizomycotina (Pezizomycotina), class of Chaetomium (Sordariomycetes), order Hypocrea (Hypocrea), family of Cordyceps sinensis (Cordycipicaceae), genus Corynebacterium (Isaria) of the kingdom FUNGI (fungus).
Cordyceps sobolifera (Isaria cicadae Miquel) is a wide variety, and its strain can be isolated from the collected strain or purchased. The cicada fungus is widely distributed in 18 provinces, cities and regions in the south of Qinling mountain-Huaihe river in China. In subtropical and tropical regions in the south of the Yangtze river in China, and in valley regions such as the Jinshajiang river, the angjiang river, the lancang river and the Yalu Tinbjiang river in Yunnan Tibet plateau. It can be collected according to the collection method of common Chinese medicinal materials in the growing season of 6-8 months per year.
Cicada fungus is a rare Chinese medicinal material and is a cordyceps fungus parasitizing on cicadas (commonly known). The medicinal effect has more than 1000 years of history, is one of the traditional famous and precious medicinal materials in China, and has various medicinal values. The main components are as follows: adenosine, cordyceps polysaccharide, cordycepic acid (mannitol), cordycepin, uracil, sterol, alkaloid, vitamin, inorganic salt, mineral elements and the like.
The cordyceps sobolifera has the functions of regulating human body immunity, improving appetite, promoting sleep and enhancing self-disease resistance. Among the components, the cordyceps polysaccharide has unique biological activity, has the efficacies of resisting tumor, bacteria, viruses, radiation, aging and the like, and the immunoregulation function becomes a hotspot of research and development. Adenosine can inhibit central neuron excitability, dilate coronary and peripheral blood vessel, increase coronary blood flow, lower blood pressure, and slow heart rate. Adenosine also has platelet aggregation inhibiting, radioprotective, and antitumor effects. Moreover, the active ingredient ISP-1 in the cordyceps sobolifera has bidirectional immunoregulation activity, can greatly improve the success rate of organ transplantation operations, and has obvious positive effects on the rehabilitation of patients.
Since 1993 old Chenzhu' an Mr. studied artificial culture of Cordyceps cicadae, 20% of potato decoction, 3% of white granulated sugar and the balance of water were used for seed liquid and fermentation liquid of Cordyceps cicadae artificial culture of sporophyll bunch until the content of water reaches 100%.
The prior art has no related report and application of using the same culture medium formula for artificially culturing the coremium of cordyceps sobolifera.
Disclosure of Invention
The invention aims to provide a liquid culture medium for artificially culturing cordyceps sobolifera coremium.
The invention further provides a method for artificially culturing cordyceps sobolifera coremium.
A liquid culture medium for artificially culturing cordyceps sobolifera bundles mainly comprises the following raw materials: yeast extract powder, soybean protein hydrolysate and water.
Further, the raw material proportion of the culture medium is as follows (weight percentage): 0.2-3% of yeast extract powder, 0.1-2% of soybean protein hydrolysate and the balance of water to 100%.
Furthermore, the raw material ratio of the culture medium is (weight percentage): 0.3-1% of yeast extract powder, 0.2-1% of soybean protein hydrolysate and the balance of water to 100%.
Furthermore, the raw material ratio of the culture medium is (weight percentage): 0.4-0.8% of yeast extract powder, 0.3-0.8% of soybean protein hydrolysate and the balance of water supplement to 100%.
Furthermore, the raw material ratio of the culture medium is (weight percentage): 0.4 percent of yeast extract powder, 0.3 percent of soybean protein hydrolysate and the balance of water to 100 percent.
Furthermore, 1-8% of sucrose can be added into the raw material composition of the culture medium; furthermore, the adding amount of the sucrose is 2-5%; furthermore, the adding amount of sucrose is 3-4%; furthermore, the adding amount of the sucrose is 3.5 percent; further, the sucrose is white granulated sugar.
Furthermore, 0.005-0.1% of calcium chloride and 0.01-0.5% of monopotassium phosphate can be added into the raw materials of the culture medium; furthermore, the adding amount of the calcium chloride and the monopotassium phosphate is 0.008-0.05% of that of the calcium chloride and 0.02-0.2% of that of the monopotassium phosphate; furthermore, the addition amount of calcium chloride and potassium dihydrogen phosphate is 0.01-0.02% of calcium chloride and 0.05-0.1% of potassium dihydrogen phosphate; further, calcium chloride and potassium dihydrogen phosphate were added in an amount of 0.01% by weight and 0.05% by weight.
The culture medium is used for culturing cordyceps sobolifera strains through slant strain culture, liquid shake culture, seeding tank liquid culture, fermentation tank liquid fermentation culture and solid weed-producing culture, and the culture steps are carried out to obtain the sporophyte bundles, spore powder and mycoplasm; wherein the slant strain culture, liquid shake culture, seeding tank liquid culture, fermentation tank liquid fermentation culture and solid grass-growing culture can all adopt conventional methods.
The invention further provides a method for artificially culturing cordyceps sobolifera coremium, which comprises the steps of slant strain culture, liquid shake culture, seeding tank liquid culture, fermentation tank liquid fermentation culture and solid grass-emergence culture, wherein the liquid culture medium in the steps of liquid shake culture, seeding tank liquid culture, fermentation tank liquid fermentation culture and solid grass-emergence culture mainly comprises the following raw materials: yeast extract powder, soybean protein hydrolysate and water.
The culture method also comprises the steps of seed tank expansion culture.
Further, the raw material proportion of the culture medium is as follows (weight percentage): 0.2-3% of yeast extract powder, 0.1-2% of soybean protein hydrolysate and the balance of water to 100%.
Furthermore, the raw material ratio of the culture medium is (weight percentage): 0.3-1% of yeast extract powder, 0.2-1% of soybean protein hydrolysate and the balance of water to 100%.
Furthermore, the raw material ratio of the culture medium is (weight percentage): 0.4-0.8% of yeast extract powder, 0.3-0.8% of soybean protein hydrolysate and the balance of water supplement to 100%.
Furthermore, the raw material ratio of the culture medium is (weight percentage): 0.4 percent of yeast extract powder, 0.3 percent of soybean protein hydrolysate and the balance of water to 100 percent.
Furthermore, 1-8% of sucrose can be added into the raw material composition of the culture medium; furthermore, the adding amount of the sucrose is 2-5%; furthermore, the adding amount of sucrose is 3-4%; furthermore, the adding amount of the sucrose is 3.5 percent; further, the sucrose is white granulated sugar.
Furthermore, 0.005-0.1% of calcium chloride and 0.01-0.5% of monopotassium phosphate can be added into the raw materials of the culture medium; furthermore, the adding amount of the calcium chloride and the monopotassium phosphate is 0.008-0.05% of that of the calcium chloride and 0.02-0.2% of that of the monopotassium phosphate; furthermore, the addition amount of calcium chloride and potassium dihydrogen phosphate is 0.01-0.02% of calcium chloride and 0.05-0.1% of potassium dihydrogen phosphate; further, calcium chloride and potassium dihydrogen phosphate were added in an amount of 0.01% by weight and 0.05% by weight.
The cordyceps sobolifera (Isaria cicadae Miquel) used in the invention is a widely distributed species, and can be made by oneself or purchased. The cicada fungus is widely distributed in 18 provinces, cities and regions in the south of Qinling mountain-Huaihe river in China. In subtropical and tropical regions in the south of the Yangtze river in China, and in valley regions such as the Jinshajiang river, the angjiang river, the lancang river and the Yalu Tinbjiang river in Yunnan Tibet plateau. It can be collected according to the collection method of common Chinese medicinal materials in the growing season of 6-8 months per year. The collected cicada fungus can be separated into cicada fungus strains according to the following method, and the specific steps are as follows:
the separation method of the strain comprises the following steps: cleaning fresh Cordyceps with sterile water on an ultrascope workbench under aseptic condition, placing in sterilized culture dish with diameter of 15cm, and wrapping the inner sclerotium part (polypide) of Cordyceps with wetted absorbent cotton for keeping moisture. A sterilized glass slide is placed below the fruiting body of Cordyceps, and the sclerotium part of Cordyceps is covered with a small glass slide, so that the part capable of being pregnant does not directly contact with the glass slide, and the distance is about 0.5 cm. Then placing the culture dish in an illumination incubator at 20 +/-0.5 ℃ for culture, when cordyceps sobolifera spores are ejected on a glass slide, dropwise adding a little of the PDA culture medium which is just melted around the spores, removing the glass slide, placing the glass slide in another sterilized culture dish for moisture culture, after the mycelia grow out, picking a little of the mycelia by using an inoculating needle, and transferring the mycelia to another flat dish (SDAY culture medium) for culture (the same as above) until single purified colonies grow out. And the strain is verified by a morphological or molecular biology method to be a cordyceps sobolifera (Isaria cicadae Miquel) anamorph.
The invention preferably selects the Paecilomyces cicadae strain [ Paecilomyces cicadae (Miq.) Samson ] disclosed in CN102851353A which is registered and preserved in China general microbiological culture Collection center (CGMCC for short) at 11-18 th of 2009, and the preservation number is CGMCC No. 3453.
Compared with the prior culture medium technology, the invention has the following advantages: the original complicated process of cooking the potato juice is eliminated, the labor force is saved, and the labor productivity is improved; the problem of different quality of potatoes in different varieties and different seasons is solved; the invention provides the proportion of the soybean protein hydrolysate and the yeast leaching powder for the first time, the mixture is used for artificial cordyceps sobolifera sporostalk bundle liquid culture, the yield of the cordyceps sobolifera sporostalk bundle and the content of the effective components thereof are obviously improved, the stable product quality is ensured, the production environment is improved, and the method is suitable for large-scale industrial production.
The specific implementation mode is as follows:
the invention will be further described with reference to specific embodiments:
example 1
The formula of the culture medium is as follows: 0.3 percent of soybean protein hydrolysate, 0.4 percent of yeast extract powder, 3.5 percent of white granulated sugar, 0.01 percent of calcium chloride, 0.05 percent of potassium dihydrogen phosphate and the balance of distilled water which is 100 percent.
Example 2
The formula of the culture medium is as follows: 0.3 percent of soybean protein hydrolysate, 1 percent of yeast extract powder, 2 percent of white granulated sugar, 0.05 percent of calcium chloride, 0.02 percent of potassium dihydrogen phosphate and the balance of distilled water which is 100 percent.
Example 3
The formula of the culture medium is as follows: 1% of soybean protein hydrolysate, 0.2% of yeast extract powder, 5% of white granulated sugar, 0.008% of calcium chloride, 0.2% of potassium dihydrogen phosphate and the balance of distilled water, wherein the balance is 100%.
Example 4
The formula of the culture medium is as follows: 0.1 percent of soybean protein hydrolysate, 3 percent of yeast extract powder, 1 percent of white granulated sugar, 0.1 percent of calcium chloride, 0.01 percent of potassium dihydrogen phosphate and the balance of distilled water which is 100 percent.
Example 5
The formula of the culture medium is as follows: 2 percent of soybean protein hydrolysate, 0.2 percent of yeast extract powder, 6 percent of white granulated sugar, 0.005 percent of calcium chloride, 0.5 percent of potassium dihydrogen phosphate and the balance of distilled water which is up to 100 percent.
Example 6
The formula of the culture medium is as follows: 0.6 percent of soybean protein hydrolysate, 0.4 percent of yeast extract powder and 4 percent of white granulated sugar.
Example 7
The formula of the culture medium is as follows: 0.5 percent of soybean protein hydrolysate, 0.4 percent of yeast extract powder and 3.5 percent of white granulated sugar.
Example 8
The formula of the culture medium is as follows: 1.5 percent of soybean protein hydrolysate, 0.3 percent of yeast extract powder and 3.5 percent of white granulated sugar.
Example 9 screening experiment of Medium formulation
1. First order slant strain culture
The formula of the culture medium is as follows: 20% of potato cooking juice, 2% of cane sugar and 2% of agar, and the balance is water to 100%; pH 6.5.
The specific method comprises the following steps: peeling and cooking potatoes, filtering to remove residues, preparing according to the proportion of 200 g to 1L, adding 2% of sucrose, fixing the volume, adding 2% of agar for melting, subpackaging into test tubes, sterilizing at 121 ℃ under the pressure of 0.1Mpa for 30min, discharging air, placing into an inclined plane, and naturally cooling. A cicada fungus strain CGMCC No.3453 is inoculated on a slope under the aseptic condition, and cultured for 15 days at 24 ℃ to obtain a production mother strain (the Paecilomyces cicadae (Miq.) Samson strain related by the invention is registered and preserved in China general microbiological culture Collection center (CGMCC for short) at 11-18 th of 2009, and the preservation number is CGMCC No. 3453).
2. Liquid shake flask seed culture
The formula of the culture medium is as follows: formulations 1-15, detailed in table 1.
The method comprises the following specific steps: the liquid loading of a 500ml triangular flask is 1/3, well-grown slant seeds of Cordyceps cicadae (Isaria cicadae Miquel) are taken, homogenized, inoculated into a shake flask according to the inoculation amount of 2 percent, and cultured for 3 days at the temperature of 140r/min on a constant temperature water bath shaker at 25 ℃. The cultured seeds are grayish black, a large number of fungus balls with the size of small rice grains are arranged in the seeds, the fungus balls are compacted, and the fungus liquid has mushroom fragrant sweet taste;
TABLE 1 comparison table of artificial culture of Cordyceps cicadae in different liquid culture media
3. Seed tank enlargement culture
The culture medium used in the seeding tank is the same as the liquid seed.
The method comprises the following specific steps: filling 20L of liquid culture medium into a 30L airlift fermentation tank, introducing steam to heat to 121 ℃ under the pressure of 14.7 multiplied by 104Pa, and maintaining the pressure for 30-45 minutes, wherein the temperature of the culture medium is higher than 95 ℃ and the pH value is 6.0-7.0; after sterilization, when the seeds are cooled to 20 ℃, 6 bottles of the cultured 500ml shake flask seeds are inoculated and cultured, the culture temperature is 25-26 ℃, and the ventilation volume is 1: 0.5V/V.min, maintaining the pressure for 4.5-7.0 multiplied by 104Pa, and performing aeration culture for 20-48h until the logarithmic growth phase is reached, wherein the final culture volume is 20L. When the amount of the bubbles is large, the edible antifoaming agent can be added, and the addition amount is 0.03%;
in the seeding tank, the time is 1-18 hours, the time is a lag phase, the time is 18-38 hours, the time is a rapid growth phase (logarithmic growth phase), and the time is 38-48 hours, the time is a rapid growth later phase. The culture time is preferably 48 hours.
4. Fermentation culture in fermentation tank
The culture medium used in the fermenter is the same as the liquid seed.
The method comprises the following specific steps: filling 200L of liquid culture medium into a 300L fermentation tank, wherein the pH value is 6.0-7.0, adding 0.03% of edible antifoaming agent, introducing steam, heating in situ to 121 ℃, and maintaining the pressure for 30-45 minutes under the pressure of 14.7 multiplied by 104 Pa; after sterilization, when the seed is cooled to 26 ℃, 50L of liquid seeds in the seed tank are all inoculated into a fermentation tank for culture, the culture temperature is 25-26 ℃, and the ventilation volume is 1: 0.5V/V.min, maintaining the pressure of 4.903-7.0845 multiplied by 104Pa, and performing aeration culture for 36-48h until the logarithmic growth phase is reached, wherein the final culture volume is 200L. When the amount of the bubbles is large, the edible antifoaming agent can be added, and the addition amount is 0.03%;
in the fermentation tank, 0-12 hours is a lag phase, 12-38 hours is a rapid growth phase, 38 hours later is a growth stabilization phase, 48 hours reaches a peak, and then enters a decay phase. Fermenting for 48 hours and transferring to solid culture.
In larger scale production, the fermentation tank can be used as a secondary seed tank, and then the fermented fermentation liquid is transferred into a fermentation tank with larger volume so as to obtain more culture liquid.
5. Solid grass-growing culture
Cleaning wheat in a cleaning tank, filtering water from the cleaned wheat, subpackaging into culture boxes, charging 330 g per box, adding 470ml of purified water, covering and sealing. Placing in a high pressure steam sterilizing pot, sterilizing with steam at 121 deg.C for 50min, and naturally cooling. The seed solution in the fermenter in 4 above was injected into the sterilized culture medium in Table 1 at a ratio of 5% by using an inoculator, and sufficiently shaken and mixed. And transferring the inoculated culture box to a culture room for culture. Adopting a three-dimensional culture frame for culture, wherein the temperature of a culture room is 22-24 ℃ in the initial stage, and performing dark culture for 3-5 days until mycelia grow to the culture material until the surface of the culture material is full; then the full-spectrum illumination culture is changed, the illumination intensity is 100-200 lux, the temperature of the culture room is kept at 20-22 ℃, the ventilation and the exhaust are carried out at regular time, and the air in the culture room is kept fresh. When the sporophore is mature (cultured for 23-25 days and biomass is maximum), harvesting in time. Taking out fruiting body from the culture box, separating the mycoplasm from the fruiting body, removing dampness, oven drying at 50 deg.C, and packaging.
Meanwhile, the yield (i.e. weed yield) of the coremium globosum bundle and the contents of adenosine, polysaccharide and protein which are main effective components are measured after the culture is finished. The results are shown in Table 2.
TABLE 2 comparison table of artificial culture of Cordyceps cicadae in different liquid culture media
| Numbering | The grass yield is% | Content of adenosine% | Content of polysaccharide% | Protein content% |
| 1 | 12.6 | 0.104 | 4.32 | 32.5 |
| 2 | 12.5 | 0.100 | 4.00 | 32.1 |
| 3 | 12.8 | 0.102 | 4.33 | 34.5 |
| 4 | 10.1 | 0.083 | 3.69 | 30.2 |
| 5 | 11.2 | 0.094 | 4.02 | 32.0 |
| 6 | 10.7 | 0.097 | 3.92 | 31.9 |
| 7 | 12.1 | 0.101 | 3.75 | 32.0 |
| 8 | 11.2 | 0.096 | 3.24 | 31.5 |
| 9 | 11.8 | 0.098 | 3.92 | 32.7 |
| 10 | 10.5 | 0.087 | 3.28 | 30.8 |
| 11 | 10.7 | 0.086 | 3.65 | 30.2 |
| 12 | 10.7 | 0.086 | 3.54 | 30.8 |
| 13 | 8.1 | 0.087 | 3.20 | 28.5 |
| 14 | 7.6 | 0.056 | 2.74 | 29.9 |
| 15 | 7.9 | 0.068 | 3.00 | 30.8 |
And (4) experimental conclusion:
as can be seen from the data in the table above, in the formula (formulas 1-12) added with the soybean hydrolyzed protein, the number of produced cordyceps sobolifera bundles is higher than that of the formula (formulas 13-15) without increasing the soybean hydrolyzed protein, which indicates that the soybean hydrolyzed protein is crucial in cordyceps sobolifera culture; except for the yield, the adenosine and polysaccharide which are main active ingredients of the cordyceps sobolifera are higher than those of the formula without the soybean protein hydrolysate. Therefore, the soybean protein hydrolysate is a breakthrough in the cordyceps fermentation process when being applied to the formula of the liquid fermentation culture medium of cordyceps sobolifera.
It can also be seen from the results of the test that the culture solutions with yeast powder added only (formulations 4, 10, 11, 12, 14) had significantly lower active ingredients than the culture solutions with yeast extract powder added (formulations 1-3).
Example 10 stability study
The culture was performed according to the method of example 9, and the yield and quality of the cordyceps sobolifera produced by different batches of the liquid culture medium were examined, and the results are shown in Table 3. (the formula of the culture medium is the formula of the embodiment 1, and the strain of the cordyceps sobolifera is CGMCC No. 3453).
TABLE 3 Change and stability of effective ingredients in liquid Medium Cork Stem bundle of the present invention
| Batch number | Percentage of weed yielded (%) | Polysaccharide (%) | Adenosine (%) |
| 20140516 | 12.3 | 5.173 | 0.106 |
| 20140520 | 12.1 | 5.200 | 0.101 |
| 20140523 | 11.8 | 5.205 | 0.098 |
| 20140527 | 12.0 | 5.179 | 0.099 |
| 20140530 | 12.3 | 5.132 | 0.091 |
The result shows that the yield of cordyceps sobolifera produced by the liquid culture medium is more than 10%, the polysaccharide content is higher than 5%, the adenosine content is higher than 0.09%, and the product quality stability is good.
Example 11 investigation of the yield and quality of Cordyceps cicadae produced by liquid Medium formulation of different strains
Obtaining a strain: according to the methods disclosed in the publications "study of Pedioecilomyces cicadae (Chenzhu 'an, journal of fungi, 1991)" and "study of Artificial culture and pharmacological action of Cordyceps cicadae (Chenzhu' an, et al, journal of fungi, 1993, 12 (2)". The separation method of the strain comprises the following steps: cleaning fresh Cordyceps with sterile water on an ultrascope workbench under aseptic condition, placing in sterilized culture dish with diameter of 15cm, and wrapping the inner sclerotium part (polypide) of Cordyceps with wetted absorbent cotton for keeping moisture. A sterilized glass slide is placed below the fruiting body of Cordyceps, and the sclerotium part of Cordyceps is covered with a small glass slide, so that the part capable of being pregnant does not directly contact with the glass slide, and the distance is about 0.5 cm. Then placing the culture dish in an illumination incubator at 20 +/-0.5 ℃ for culture, when cordyceps sobolifera spores are ejected on a glass slide, dropwise adding a little of the PDA culture medium which is just melted around the spores, removing the glass slide, placing the glass slide in another sterilized culture dish for moisture culture, after hyphae grow out, picking a little hyphae by using an inoculating needle, transferring the hyphae to another dish (SDAY culture medium) for culture (the same as above), and obtaining five strains: BACC0019, BACC0033, BACC0036, BACC0041
BACC 0056. And the isolated strains are all cordyceps sobolifera (Isariacicadae Miquel) anamorph through the verification of morphological and molecular biological methods.
The culture method comprises the following steps: the culture was carried out in the same manner as in example 9.
The formula of the culture medium is as follows: the same formulation as in example 1.
TABLE 4 variation and stability of the coremium (encystment) cultured on different strains of the liquid medium of the invention
| Strain number | Percentage of weed yielded (%) | Polysaccharide (%) | Adenosine (%) |
| BACC0019 | 9.85 | 4.52 | 0.083 |
| BACC0033 | 9.47 | 5.10 | 0.082 |
| BACC0036 | 9.20 | 4.35 | 0.083 |
| BACC0041 | 9.43 | 4.08 | 0.075 |
| BACC0056 | 8.75 | 5.05 | 0.076 |
The result shows that the yield of the cordyceps militaris of different cordyceps sobolifera strains produced by the liquid culture medium is more than 8.75%, the polysaccharide content is higher than 4.08%, the adenosine content is higher than 0.075%, and the product quality stability is good.
Example 12 stability study
The liquid medium of the present invention, not only used in the slant medium and the mycelium medium specified in example 9, can obtain good results, but also when the slant medium is changed, or in the liquid culture of mycelia of Cordyceps cicadae, can obtain good results using the medium as well.
Change of slant medium: the medium in example 9 was changed to SDAY medium, i.e., peptone 10g/L, yeast extract 10g/L, glucose 40g/L, agar 20 g/L. The seed liquid and solid cultures were as in example 9, and the culture results were: the grass yield is 12.6 percent, the polysaccharide content is 5.23 percent, and the adenosine content is 0.105 percent.
Culturing mycelium in a fermentation tank: the mycelium cultured in the fermenter in the same manner as in the 1, 2, 3 and 4 steps of example 9 was centrifuged and dried to obtain a mycelium product, which was measured for the content of the main chemical components and compared with the original culturing method, and the results are shown in Table 5:
TABLE 5 changes in effective ingredients in mycelia after using liquid medium
| Batch number | The yield of mycelium is% | Adenosine% | Content of polysaccharide% |
| 20140423(CK) | 4.2 | 0.099 | 7.52 |
| 20140430 | 5.0 | 0.105 | 8.82 |
| 20140428 | 5.4 | 0.112 | 9.24 |
The results show that the quality and quantity of the product obtained by culturing the liquid culture medium of the invention and different slant culture media or mycelium culture combinations are obviously improved, and the product quality is stable.
Example 13
Liquid culture medium: examples 2 to 8;
the culture method comprises the following steps: the same as in example 9;
the results are shown in Table 6:
| examples | Percentage of weed yielded (%) | Polysaccharide (%) | Adenosine (%) |
| 2 | 10.0 | 4.75 | 0.085 |
| 3 | 12.0 | 5.04 | 0.093 |
| 4 | 10.1 | 4.50 | 0.078 |
| 5 | 10.8 | 4.82 | 0.087 |
| 6 | 11.7 | 4.95 | 0.090 |
| 7 | 10.5 | 4.73 | 0.093 |
| 8 | 11.0 | 4.83 | 0.093 |
Claims (8)
1. A liquid culture medium for artificially culturing cordyceps sobolifera bundles is characterized by comprising the following raw materials in percentage by weight: 0.2-3% of yeast extract powder, 0.1-2% of soybean protein hydrolysate, 1-8% of sucrose and the balance of water supplement to 100%.
2. The liquid culture medium of claim 1, wherein the culture medium comprises the following raw materials in percentage by weight: 0.5 percent of soybean protein hydrolysate, 0.4 percent of yeast extract powder, 3.5 percent of white granulated sugar and the balance of distilled water.
3. The liquid culture medium of claim 1, wherein the culture medium comprises the following raw materials in percentage by weight: 1.5 percent of soybean protein hydrolysate, 0.4 percent of yeast extract powder, 3.5 percent of white granulated sugar and the balance of distilled water.
4. The liquid culture medium according to claim 1, wherein the medium comprises 0.005-0.1% of calcium chloride and 0.01-0.5% of potassium dihydrogen phosphate.
5. The liquid medium according to claim 4, wherein the calcium chloride and the potassium dihydrogen phosphate are added in an amount of 0.008 to 0.05% by weight and 0.02 to 0.2% by weight.
6. The liquid culture medium according to claim 5, wherein the culture medium comprises the following raw materials in percentage by weight: 0.3 percent of soybean protein hydrolysate, 0.4 percent of yeast extract powder, 3.5 percent of white granulated sugar, 0.01 percent of calcium chloride, 0.05 percent of potassium dihydrogen phosphate and the balance of distilled water.
7. A method for artificially culturing cordyceps sobolifera sporophores, which is characterized by comprising the steps of slant strain culture, liquid shake culture, seeding tank liquid culture, fermentation tank liquid fermentation culture and solid grass-emergence culture, wherein the liquid culture medium of any one of claims 1-6 is used in the steps of liquid shake culture, seeding tank liquid culture, fermentation tank liquid fermentation culture and solid grass-emergence culture.
8. The method of claim 7, wherein the culturing method further comprises a seeding tank scale-up step.
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| CN102199557A (en) * | 2010-03-24 | 2011-09-28 | 北京林业大学 | Method for high-density enrichment culture of lactic acid bacteria in yoghourt leavening agent |
| CN102242154A (en) * | 2010-05-14 | 2011-11-16 | 浙江泛亚生物医药股份有限公司 | Liquid fermentation method for producing paecilomyces cicadae mycelia and application of culture product |
| CN103460994A (en) * | 2013-09-05 | 2013-12-25 | 徐州工程学院 | Cordyceps militaris nano-selenium and preparation method thereof |
| CN103621308A (en) * | 2012-08-22 | 2014-03-12 | 上海泛亚生物医药集团有限公司 | Culture medium for producing isaria tenuipes entities and industrialized cultural method for isaria tenuipes entities |
| CN103655215A (en) * | 2013-11-19 | 2014-03-26 | 安徽农业大学 | Paecilomyces varioti extract with tyrosinase activity and scavenging free radical activity and application thereof |
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| CN102199557A (en) * | 2010-03-24 | 2011-09-28 | 北京林业大学 | Method for high-density enrichment culture of lactic acid bacteria in yoghourt leavening agent |
| CN102242154A (en) * | 2010-05-14 | 2011-11-16 | 浙江泛亚生物医药股份有限公司 | Liquid fermentation method for producing paecilomyces cicadae mycelia and application of culture product |
| CN103621308A (en) * | 2012-08-22 | 2014-03-12 | 上海泛亚生物医药集团有限公司 | Culture medium for producing isaria tenuipes entities and industrialized cultural method for isaria tenuipes entities |
| CN103460994A (en) * | 2013-09-05 | 2013-12-25 | 徐州工程学院 | Cordyceps militaris nano-selenium and preparation method thereof |
| CN103655215A (en) * | 2013-11-19 | 2014-03-26 | 安徽农业大学 | Paecilomyces varioti extract with tyrosinase activity and scavenging free radical activity and application thereof |
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