CN113155999A - Method for detecting cordycepin content in cordyceps militaris - Google Patents
Method for detecting cordycepin content in cordyceps militaris Download PDFInfo
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- CN113155999A CN113155999A CN202110338243.6A CN202110338243A CN113155999A CN 113155999 A CN113155999 A CN 113155999A CN 202110338243 A CN202110338243 A CN 202110338243A CN 113155999 A CN113155999 A CN 113155999A
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- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 title claims abstract description 70
- OFEZSBMBBKLLBJ-BAJZRUMYSA-N cordycepin Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)C[C@H]1O OFEZSBMBBKLLBJ-BAJZRUMYSA-N 0.000 title claims abstract description 70
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000000855 fermentation Methods 0.000 claims abstract description 48
- 230000004151 fermentation Effects 0.000 claims abstract description 48
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 19
- 239000000047 product Substances 0.000 claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 239000000126 substance Substances 0.000 claims description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 12
- 238000007865 diluting Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 10
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- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- 229930186147 Cephalosporin Natural products 0.000 claims description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
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- 150000001780 cephalosporins Chemical class 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 238000000825 ultraviolet detection Methods 0.000 claims description 3
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
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- G01N30/02—Column chromatography
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Abstract
The invention relates to a method for detecting cordycepin content in cordyceps militaris, which comprises the steps of (1) collecting mycelium and fermentation supernatant of cordyceps militaris to prepare a product to be detected; and respectively carrying out high performance liquid chromatography detection on the products to be detected 1 and 2 and the prepared cordycepin standard solution under the same chromatographic condition, and calculating the cordycepin content of the samples to be detected 1 and 2 according to the detection result. The detection result shows that the cordycepin has good linear relation in the range of 0.02-0.1mg/mL, and the regression equation is that Y is 27692919.69X-135695.5252(R is20.999728197); the content of the cordycepin in the sample 1 to be detected is 70.39mg/L, and the content of the cordycepin in the sample 2 to be detected is 1080.20 mg/L. The method has the advantages of high analysis speed, high sensitivity, selectivity,Convenient operation and the like.
Description
Technical Field
The invention relates to a method for detecting cordycepin content in cordyceps militaris, in particular to a method for detecting the content of intracellular cordycepin and extracellular cordycepin in fermentation liquor obtained after liquid fermentation of cordyceps militaris by using high performance liquid chromatography.
Background
Cordyceps militaris (Cordyceps militaris), also known as Cordyceps militaris, is a complex of fungi of the genus Cordyceps of the family Clavipitaceae, the order Hypocreales, the phylum Ascomycota, consisting of stroma and sclerotium. Cordycepin is one of important active ingredients in Cordyceps militaris, and has biological activities of resisting tumor, inflammation, bacteria, oxidation, and immunity. The Cordyceps militaris has similar efficacy to Cordyceps sinensis, and the content of cordycepin is higher than that of Cordyceps sinensis. In view of the important food and medicinal value and the huge application prospect of cordycepin, the cordycepin can be used as a good substitute of cordyceps sinensis.
Cordycepin mainly has three production methods, one is traditionally extracted from cordyceps militaris, cordyceps sinensis and the like, and the method is greatly influenced by regions and climate and has low yield; secondly, the compound is obtained by chemical total synthesis, the synthetic process of the method is complex, the cost is high, and organic reagents are released; thirdly, the compound is obtained by microbial fermentation by adopting a genetic engineering technology. At present, the technology for artificially cultivating cordyceps militaris is mature, and the cordyceps militaris is mainly obtained through solid fermentation and liquid fermentation.
Cordycepin is one of important indexes for quality evaluation of cordyceps militaris, and at present, methods for detecting cordycepin content comprise an ultraviolet spectrophotometer method, a high performance liquid chromatography method, a capillary electrophoresis method, a thin layer chromatography method and the like. Therefore, the rapid and effective detection of cordycepin content has important guiding significance for guiding the cordyceps militaris liquid fermentation process.
Disclosure of Invention
The invention aims to provide a method for detecting cordycepin content in cordyceps militaris. Compared with the traditional detection method, the analysis method has the advantages of high sensitivity and good selectivity, and can accurately detect the cordycepin content in the cordyceps militaris.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for detecting cordycepin content in cordyceps militaris, which comprises the following steps:
(1) standard curve: preparing five cordycepin standard substances with different concentrations (preferably gradient concentration, more preferably 0.02, 0.04, 0.06, 0.08 and 0.10mg/mL) for high performance liquid chromatography detection, and performing linear fitting by taking the five different concentrations of the cordycepin standard substances as abscissas and the peak areas of the cordycepin standard substances as ordinates to obtain a standard curve; drawing a cordycepin standard curve;
(2) respectively collecting mycelia and fermentation supernatant of cordyceps militaris in cordyceps militaris fermentation liquor, drying the mycelia of the cordyceps militaris, adding deionized water, extracting at 75-85 ℃ for 2.5-3 h (extracting at 80 ℃ for 3h), centrifuging the obtained extracting solution, concentrating (distilling under reduced pressure) or diluting with water in the obtained supernatant, filtering with a 0.45-micrometer filter membrane, and detecting until the peak area is between the peak areas of the maximum concentration and the minimum concentration in the step (1), so as to obtain a product to be detected 1; diluting the fermentation supernatant with water, and filtering with a 0.45 μm microporous filter membrane to obtain a product 2 to be detected;
(3) and (3) respectively carrying out high performance liquid chromatography detection on the to-be-detected product 1 or the to-be-detected product 2 obtained in the step (2) under the same chromatographic condition as that of the step (1), calculating the concentration of the to-be-detected product 1 or the to-be-detected product 2 according to the standard curve in the step (1) and the peak areas of the to-be-detected samples 1 and 2, and further calculating the content of cordycepin in the mycelium of cordyceps militaris and the fermentation supernatant in unit volume of cordyceps militaris fermentation liquor according to the concentration or dilution times in the step (2).
Preferably, the preparation method of the cordycepin standard solution in the step (1) comprises the following steps: precisely weighing 2.5mg of cordycepin standard substance, dissolving with deionized water, diluting to a constant volume of 25mL to prepare a cordyceps standard substance stock solution with the solubility of 0.10mg/mL, storing at 4 ℃ for later use, taking 2.0, 4.0, 6.0, 8.0 and 10.0mL of the standard substance stock solution, diluting with deionized water to a constant volume of 10mL respectively, and filtering with a 0.45 mu m microporous filter membrane to obtain the cordycepin standard substance solution with the serial solubilities of 0.02, 0.04, 0.06, 0.08 and 0.10 mg/mL. Wherein the cordycepin standard product is a product of Aladdin company, and the product catalog number is C110081.
Further, the high performance liquid chromatography conditions of steps (1) and (3) are as follows: ultraviolet detection wavelength is 260nm, and mobile phase methanol is 25 mL: 75mL of water, the flow rate of 1.0mL/min and the sample volume of 5 mu L; the column was E clipse Plus C18(250 mm. times.4.6 mm 5 μm) available from Agilent; the reagent methanol (HPLC grade, bruddy).
Further, the mycelium and the fermentation supernatant of the cordyceps militaris in the step (2) are obtained by centrifuging the fermentation liquor of the cordyceps militaris at normal temperature of 10000r/min for 10min, obtaining the supernatant as the fermentation supernatant, washing the obtained precipitate with distilled water for three times, and drying the precipitate at 60 ℃ to constant weight.
Further, the cordyceps militaris fermentation broth in the step (2) is prepared by the following method: inoculating the cordyceps militaris seed liquid into a liquid fermentation culture medium containing 100 mu g/mL cephalosporin, culturing for 9 days at a constant temperature of 25 ℃ and under the condition of 150rpm to obtain cordyceps militaris fermentation liquid; wherein the final concentration of each component of the liquid fermentation medium is as follows: 25g/L of soluble starch, 20g/L of peptone and KH2PO4 1g/L，MgSO4·7H2O1 g/L, L-glycine 3g/L, distilled water as solvent and natural pH value; the inoculation volume of the cordyceps militaris seed liquid is 5% of that of the liquid fermentation culture medium.
Further, the cordyceps militaris seed liquid is prepared by inoculating an original mother seed into a seed liquid culture medium containing 200 mug/mL of cephalosporin under an aseptic condition, and culturing for 5d at a constant temperature of 25 ℃ and under a condition of 150rpm to obtain a cordyceps militaris seed liquid; wherein the final concentration of each component of the seed liquid culture medium is as follows: glucose 20g/L, peptone 10g/L, KH2PO4 0.5g/L，K2HPO40.5g/L，MgSO4·7H20.5g/L of O, 1g/L of L-glycine, distilled water as a solvent and natural pH.
Further, the model of the high performance liquid chromatograph in the step (1) is LC-20A, and CAT.NO. 228-45003-38.
In the invention, the Cordyceps militaris is Cordyceps militaris AF 2021025; preserved in China center for type culture Collection, the preservation date is: year 2021, day 01, 04, address: china, wuhan university, 430072).
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for detecting cordycepin content in cordyceps militarisThe detection result of the method shows that the cordycepin has good linear relation in the concentration range of 0.02-0.1mg/mL, and the regression equation is that Y is 27692919.69X-135695.5252(R is20.999728197); the content of the cordycepin in the sample 1 to be detected is 70.39mg/L, and the content of the cordycepin in the sample 2 to be detected is 1080.20 mg/L. The method has the advantages of high analysis speed, high sensitivity, selectivity, convenience in operation and the like.
Drawings
FIG. 1 is an HPLC chromatogram of 0.08mg/mL cordycepin standard.
FIG. 2 is an HPLC chromatogram of sample 2.
FIG. 3 is an HPLC chromatogram of sample 1.
FIG. 4 is a standard curve of cordycepin, wherein the solubility on the abscissa is in mg/mL.
Detailed Description
The invention is further illustrated by the following examples:
example 1
The method for detecting the cordycepin content in the cordyceps militaris comprises the following steps:
1. preparing cordyceps militaris fermentation liquor: inoculating a small piece of original mother seed (Cordyceps militaris AF 2021025; preserved in China center for type culture Collection, preservation date: 2021, 01, 04, and address: China, Wuhan university, 430072) into 50mL of seed liquid culture medium containing 200 mug/mL of cephalosporin under aseptic condition, and culturing at constant temperature of 25 ℃ and 150rpm for 5d to obtain seed liquid of Cordyceps militaris; wherein the final concentration of each component of the formula of the seed liquid culture medium is as follows: glucose 20g/L, peptone 10g/L, KH2PO4 0.5g/L，K2HPO4 0.5g/L，MgSO4·7H20.5g/L of O, 1g/L of L-glycine, distilled water as a solvent and natural pH. Secondly, inoculating 5 percent of cordyceps militaris seed liquid (5 percent of the volume of the liquid fermentation culture medium is 5 percent) into 50mL of liquid fermentation culture medium containing 100 mu g/mL of cephalosporin, and culturing for 9d under the conditions of constant temperature of 25 ℃ and 150rpm to obtain cordyceps militaris fermentation liquid; wherein the final concentration of each component of the formula of the liquid fermentation medium is as follows: 25g/L of soluble starch, 20g/L of peptone and KH2PO4 1g/L，MgSO4·7H2O1 g/L and L-glycine 3g/L, and the solvent is distilled water with natural pH. A total of 3 bottles of 50mL liquid fermentation medium were prepared as described above.
2. Preparing a cordycepin standard solution: precisely weighing 2.5mg of cordycepin standard substance, dissolving with deionized water, diluting to a constant volume of 25mL, preparing into a Cordyceps standard substance stock solution with a concentration of 0.10mg/mL, and storing at 4 deg.C for use. Taking 2.0, 4.0, 6.0, 8.0 and 10.0mL of standard substance stock solution, diluting with deionized water respectively to constant volume to 10mL, and filtering with 0.45 μm microporous membrane to obtain cordycepin standard substance solution with series concentration of 0.02, 0.04, 0.06, 0.08 and 0.10 mg/mL.
3. Analyzing the cordycepin standard solution by adopting a high performance liquid chromatography:
high performance liquid chromatography conditions: c18 column (250 mm. times.4.6 mm), UV detection wavelength 260nm, mobile phase methanol (25 mL): water (75mL), flow rate of 1.0mL/min, sample size of 5. mu.L.
The measuring method comprises the following steps: precisely sucking cordycepin standard solutions (0.02, 0.04, 0.06, 0.08 and 0.10mg/mL) with different concentrations respectively by 5 μ L, sequentially injecting into a liquid chromatograph, recording chromatogram and peak area, repeating the experiment for 3 times, and taking average values as shown in Table 1. The results show that: cordycepin has good linear relation in the concentration range of 0.02-0.10mg/mL, and the regression equation is that Y (mV. min) ═ 27692919.69X-135695.5252 (R)20.999728197), the unit of X is mg/mL.
4. Preparing a to-be-detected product: centrifuging the fermentation liquid of Cordyceps militaris at room temperature of 10000r/min for 10min, washing with distilled water for three times, drying at 60 deg.C to constant weight, and collecting mycelia and fermentation supernatant of Cordyceps militaris. Grinding the collected Cordyceps militaris hyphae, sieving with No. 6 sieve to obtain powder, and weighing hyphae powder of three bottles of fermentation liquor respectively, wherein the average value is 0.67 g. Accurately weighing mycelium powder 50mg, adding 40mL deionized water, and heating and extracting with water at 80 deg.C for 3 h; centrifuging at 5000r/min to obtain supernatant, distilling under reduced pressure, diluting to 1mL, filtering with 0.45 μm filter membrane to obtain extractive solution, and detecting. In addition, 1mL of the fermentation supernatant was passed through a 0.45 μm microporous membrane for detection. Performing preliminary experimental analysis on the extracting solution and the fermentation supernatant by adopting a high performance liquid chromatography, recording a chromatogram and a peak area under the high performance liquid chromatography condition and the analysis of a cordycepin standard solution, and diluting the extracting solution and the fermentation supernatant by finding that the peak areas are not in the peak area range of a linear relation and a flat peak appears on the chromatogram of the extracting solution and the fermentation supernatant, wherein the specific dilution step comprises the following steps: diluting and uniformly mixing 100 mu L of extracting solution passing through a 0.45 mu m microporous filter membrane with 300 mu L of sterile deionized water, and marking as a sample 1 to be detected; and adding 1mL of sterile deionized water into 100 mu L of fermentation supernatant which passes through a 0.45 mu m microporous filter membrane to dilute and mix uniformly, and marking as a sample 2 to be detected.
5. And (3) analyzing the samples 1 and 2 to be detected by adopting a high performance liquid chromatography, precisely absorbing 5 mu L of each of the samples 1 and 2 to be detected respectively under the high performance liquid chromatography condition and the analysis of the cordycepin standard solution, sequentially injecting the samples into a liquid chromatograph, recording a chromatogram and a peak area, repeating the experiment for 3 times, and taking an average value of results, wherein the average value is shown in table 1.
TABLE 1 Peak areas of cordycepin standard and samples 1 and 2 to be tested at different concentrations
| Sample (I) | Peak area |
| 0.02mg/mL cordycepin standard substance | 423736.406 |
| 0.04mg/mL cordycepin standard substance | 952510.625 |
| 0.06mg/mL cordycepin standard substance | 1545935.375 |
| 0.08mg/mL cordycepin standard substance | 2075864.375 |
| 0.10mg/mL cordycepin standard substance | 2631351.500 |
| Item to be tested 1 | 1682675.350 |
| Product to be tested 2 | 2583692.125 |
The result shows that the peak area of the sample 1 to be detected is 1682675.350, and the calculated cordycepin concentration of the sample 1 to be detected is 0.0657mg/mL, so that the cordycepin concentration of the extracting solution is 0.2628mg/mL, namely the content of cordycepin in cells is 70.43mg/L (fermentation medium); the peak area of the sample 2 to be detected is 2583692.125, and the calculated cordycepin concentration of the sample 2 to be detected is 0.0981mg/L, so that the concentration of cordycepin in the fermentation supernatant is 1.0791mg/mL, namely the extracellular cordycepin content is 1079.10mg/L (fermentation medium).
Claims (8)
1. A method for detecting cordycepin content in Cordyceps militaris is characterized by comprising the following steps:
(1) standard curve: preparing five cordycepin standard substances with different concentrations, carrying out high performance liquid chromatography detection, taking the solubility of the five different concentrations as abscissa and the peak area of the cordycepin standard substance as ordinate, and carrying out linear fitting to obtain a standard curve; drawing a cordycepin standard curve;
(2) respectively collecting mycelia and fermentation supernatant of cordyceps militaris in cordyceps militaris fermentation liquor, drying the mycelia of the cordyceps militaris, adding deionized water, extracting for 2.5-3 h at 75-85 ℃, centrifuging the obtained extracting solution, concentrating or diluting the obtained supernatant with water, filtering with a 0.45-micrometer filter membrane, and detecting until the peak area is between the peak areas of the maximum concentration and the minimum concentration in the step (1), so as to obtain a product to be detected 1; diluting the fermentation supernatant with water, and filtering with a 0.45 μm microporous filter membrane to obtain a product 2 to be detected;
(3) and (3) respectively carrying out high performance liquid chromatography detection on the to-be-detected product 1 or the to-be-detected product 2 obtained in the step (2) under the same chromatographic condition as that of the step (1), calculating the concentration of the to-be-detected product 1 or the to-be-detected product 2 according to the standard curve in the step (1) and the peak areas of the to-be-detected samples 1 and 2, and further calculating the content of cordycepin in the mycelium of cordyceps militaris and the fermentation supernatant in unit volume of cordyceps militaris fermentation liquor according to the concentration or dilution times in the step (2).
2. The method for detecting the content of cordycepin in cordyceps militaris as claimed in claim 1, wherein: the five different concentrations in the step (1) are 0.02, 0.04, 0.06, 0.08 and 0.10mg/mL in sequence.
3. The method for detecting the content of cordycepin in cordyceps militaris as claimed in claim 1, wherein: the high performance liquid chromatography conditions of the steps (1) and (3) are as follows: ultraviolet detection wavelength is 260nm, and mobile phase methanol is 25 mL: 75mL of water, the flow rate of 1.0mL/min and the sample volume of 5 mu L; the column was E clipse Plus C18, available from Agilent.
4. The method for detecting the content of cordycepin in cordyceps militaris as claimed in claim 1, wherein: and (3) centrifuging the cordyceps militaris fermentation liquor at normal temperature of 10000r/min for 10min to obtain supernatant, washing the obtained precipitate with distilled water for three times, and drying at 60 ℃ to constant weight to obtain the mycelium.
5. The method for detecting the content of cordycepin in cordyceps militaris as claimed in claim 1, wherein: the cordyceps militaris fermentation liquor in the step (2) is prepared by the following method: inoculating the cordyceps militaris seed liquid into a liquid fermentation culture medium containing 100 mu g/mL cephalosporin, culturing for 9 days at a constant temperature of 25 ℃ and under the condition of 150rpm to obtain cordyceps militaris fermentation liquid; wherein the final concentration of each component of the liquid fermentation medium is as follows: 25g/L of soluble starch, 20g/L of peptone and KH2PO4 1g/L，MgSO4·7H2O1 g/L, L-glycine 3g/L, distilled water as solvent and natural pH value; the inoculation volume of the cordyceps militaris seed liquid is 5% of that of the liquid fermentation culture medium.
6. The method for detecting the content of cordycepin in cordyceps militaris as claimed in claim 4, wherein: the preparation method of the cordyceps militaris seed liquid comprises the steps of inoculating an original mother seed into a seed liquid culture medium containing 200 mu g/mL of cephalosporin under an aseptic condition, culturing for 5d at a constant temperature of 25 ℃ and under a condition of 150rpm to obtain a cordyceps militaris seed liquid; wherein the final concentration of each component of the seed liquid culture medium is as follows: glucose 20g/L, peptone 10g/L, KH2PO4 0.5g/L，K2HPO4 0.5g/L，MgSO4·7H20.5g/L of O, 1g/L of L-glycine, distilled water as a solvent and natural pH.
7. The method for detecting the content of cordycepin in cordyceps militaris as claimed in claim 1, wherein: and (3) concentrating in the step (2) in a vacuum distillation mode.
8. The method for detecting the content of cordycepin in cordyceps militaris as claimed in claim 1, wherein: the extraction in the step (2) is carried out for 3 hours at the temperature of 80 ℃.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492706A (en) * | 2008-11-25 | 2009-07-29 | 贵州大学 | Method for improving cordyceps sinensis bacterium native volume of production with cordyceps militaris link liquid fermentation |
| US20100021426A1 (en) * | 2003-10-03 | 2010-01-28 | Wang ya-chun | Methods for making and compositions comprising fermentation products of cordyceps sinensis |
| CN103215250A (en) * | 2013-03-30 | 2013-07-24 | 徐州鸿宇农业科技有限公司 | Protoplast mutation breeding method for improving cordycepin content of cordyceps militaris |
| CN107926482A (en) * | 2017-12-08 | 2018-04-20 | 江西师范大学 | A kind of method using fluid nutrient medium production fruiting bodies of cordyceps militaris |
| CN108220172A (en) * | 2018-02-06 | 2018-06-29 | 北京市辐射中心 | The Cordyceps militaris mutant strain of high yield cordycepin and application |
| AU2020102126A4 (en) * | 2020-09-03 | 2020-10-15 | Northwest Institute Of Plateau Biology, Chinese Academy Of Sciences | A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof |
-
2021
- 2021-03-30 CN CN202110338243.6A patent/CN113155999A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100021426A1 (en) * | 2003-10-03 | 2010-01-28 | Wang ya-chun | Methods for making and compositions comprising fermentation products of cordyceps sinensis |
| CN101492706A (en) * | 2008-11-25 | 2009-07-29 | 贵州大学 | Method for improving cordyceps sinensis bacterium native volume of production with cordyceps militaris link liquid fermentation |
| CN103215250A (en) * | 2013-03-30 | 2013-07-24 | 徐州鸿宇农业科技有限公司 | Protoplast mutation breeding method for improving cordycepin content of cordyceps militaris |
| CN107926482A (en) * | 2017-12-08 | 2018-04-20 | 江西师范大学 | A kind of method using fluid nutrient medium production fruiting bodies of cordyceps militaris |
| CN108220172A (en) * | 2018-02-06 | 2018-06-29 | 北京市辐射中心 | The Cordyceps militaris mutant strain of high yield cordycepin and application |
| AU2020102126A4 (en) * | 2020-09-03 | 2020-10-15 | Northwest Institute Of Plateau Biology, Chinese Academy Of Sciences | A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof |
Non-Patent Citations (3)
| Title |
|---|
| 张楠等: "蛹虫草深层发酵产虫草素培养基的优化", 《北方园艺》 * |
| 简利茹等: "不同碳源下蛹虫草的产量及虫草素和腺苷含量", 《贵州农业科学》 * |
| 羊悦: "利用啤酒糟发酵产虫草菌素及虫草多糖的研究", 《中国优秀博硕士学位论文全文数据库(硕士) 工程科技Ⅰ辑》 * |
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