CN114438153A - Process for improving extraction rate of cordycepin from Guni cordyceps sinensis - Google Patents
Process for improving extraction rate of cordycepin from Guni cordyceps sinensis Download PDFInfo
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- CN114438153A CN114438153A CN202210269099.XA CN202210269099A CN114438153A CN 114438153 A CN114438153 A CN 114438153A CN 202210269099 A CN202210269099 A CN 202210269099A CN 114438153 A CN114438153 A CN 114438153A
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- cordyceps sinensis
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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 56
- 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 56
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 52
- 230000008569 process Effects 0.000 title claims abstract description 45
- 238000000605 extraction Methods 0.000 title claims abstract description 39
- 241001248610 Ophiocordyceps sinensis Species 0.000 title claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 30
- 241000190633 Cordyceps Species 0.000 claims abstract description 29
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000001035 drying Methods 0.000 claims abstract description 27
- 229910001868 water Inorganic materials 0.000 claims abstract description 27
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- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 17
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- 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 abstract description 14
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- 235000001727 glucose Nutrition 0.000 claims abstract description 3
- 238000010298 pulverizing process Methods 0.000 claims abstract description 3
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 3
- 241001264174 Cordyceps militaris Species 0.000 claims description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 3
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- 108010046377 Whey Proteins Proteins 0.000 claims description 3
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- 235000021119 whey protein Nutrition 0.000 claims description 3
- 239000012045 crude solution Substances 0.000 abstract description 2
- 238000010828 elution Methods 0.000 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 10
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- 239000003814 drug Substances 0.000 description 4
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
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- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a process for improving the extraction rate of cordycepin from Guni cordyceps sinensis, which comprises the following steps: mixing succus Mali Pumilae, sweet potato juice, glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate, and water, sterilizing, adding activated GUNI Cordyceps mycelium, culturing at 20-30 deg.C for 5-15 days, and centrifuging to obtain precipitate; drying the precipitate with hot air, keeping ventilation during drying, pulverizing, sieving, reflux-extracting in a reflux device in boiling water bath for 4-6 hr, and performing ultrasonic treatment during reflux-extracting to obtain crude solution; adjusting the pH value of the crude liquid to 5-5.5, cooling from room temperature to-40-60 ℃, preserving heat for 1-2h, heating to 30-50 ℃, stirring for 1-2h, extracting with ethyl acetate, then passing through a 4.5 mu m filter membrane, loading onto a macroporous resin D1400 column, eluting with 15-20% methanol after balance of elution with distilled water, detecting by HPLC, collecting a sample receiving tube with the cordycepin peak area exceeding 85%, concentrating and drying.
Description
Technical Field
The invention relates to the technical field of cordycepin extraction, in particular to a process for improving the extraction rate of cordycepin from Guni cordyceps.
Background
Cordycepin (Cordycepin) is an analog of adenosine, of the formula: c10H13N5O3Molecular weight: 251.24, alkaline, needle-like or plate-like crystal, melting point 230-231 deg.C, maximum absorption wavelength 259.0nm, solubility in water, hot ethanol and methanol, and insolubility in benzene, ether and chloroform.
Cordycepin is a natural-source medicine, has various pharmacological effects of resisting tumor, resisting bacteria and viruses, regulating immunity, removing free radicals and the like, and has good clinical application prospect. The research on cordycepin is now becoming an extremely active area in medicinal chemistry. Its specific antibacterial and antiviral activity has attracted much attention from developed countries in science and technology. It can inhibit the synthesis of viral RNA; has inhibitory effect on Bacillus subtilis and Mycobacterium avium; it also has killing effect on HIV-I type virus; especially has strong inhibiting effect on various solid malignant tumors. Therefore, it is a necessary trend to develop it as a new drug with a wide range of uses.
The traditional medicine considers that the cordyceps sinensis is mild in medicine property, warm but not dry, and free of stagnation, can tonify deficiency and strengthen the body, can treat and prolong the life, is more stable in medicine property than ginseng and pilose antler, is not easily limited by physique, diseases, seasons and ages, and can be used for improving immunity and disease resistance of strong people, weak people, healthy people and patients. Moreover, the modern life and work rhythm is too fast, the pressure is increased, the function of an immune system is weakened, and diseases such as tumor, hepatitis and the like are easy to cause. Men often have accelerated hair loss and impaired sexual function due to reduced renal function, while women often have emaciation, insomnia, mental fatigue, poor appetite, irritable and anxious symptoms, and the onset of menopause is accelerated.
The content of cordycepin in the Guni cordyceps sinensis is low, a proper enrichment method is selected as a key for extracting cordycepin, the cordycepin is fat-soluble and is easy to damage by heating, 10 times of water is used for hot reflux extraction at 80 ℃ for 3 times for 90 minutes each time in the conventional extraction at present, but the extraction efficiency is not high, the purity of the cordycepin obtained by the method is relatively low, and although large-scale production can be realized, the process is relatively complex, so that the production cost is very high.
Therefore, the extraction process of cordyceps polysaccharide and cordycepin is further researched, so that the extraction rate is improved, and the method has important economic value and wide market application potential.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a process for improving the extraction rate of cordycepin from Guni cordyceps.
A process for improving the extraction rate of cordycepin from Guni cordyceps comprises the following steps:
(1) uniformly mixing apple juice, sweet potato juice, glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate and water, adding the activated Guni cordyceps sinensis mycelia after sterilization, fermenting and culturing for 5-15 days at 20-30 ℃, keeping the tank pressure at 0.02-0.04MPa and the stirring speed at 100-150r/min in the fermenting and culturing process, and centrifuging to obtain a precipitate;
(2) drying the precipitate with hot air, keeping ventilation in the drying process, pulverizing, sieving, adding into a reflux device, and adding 95% ethanol at a ratio of material to liquid of 1: reflux-extracting for 4-6h in a boiling water bath for 5-8 hours, and performing ultrasonic treatment during the reflux-extracting process to obtain a crude liquid;
(3) adjusting the pH value of the crude liquid to 5-5.5, cooling from room temperature to-40-60 ℃, preserving heat for 1-2h, heating to 30-50 ℃, stirring for 1-2h, extracting by ethyl acetate, then passing through a 4.5 mu m filter membrane, loading a macroporous resin D1400 column, eluting with 15-20% methanol after balance of rinsing by distilled water, detecting by HPLC, collecting a sample receiving tube with more than 85% of cordycepin peak area, concentrating, and drying to obtain the Guni cordyceps militaris cordycepin.
Preferably, in the step (1), the activated Guni cordyceps sinensis mycelia is subjected to sealing and sterilization of the first culture medium, and then the Acremonium terricola is inoculated under aseptic conditions and cultured at the constant temperature of 20-25 ℃ for 5-10 days; then inoculating 6-10% of the culture medium into the second sterilized culture medium, culturing at 15-25 deg.C for 2-4 days, and keeping ventilation of 0.01-0.03vvm during the culture process to obtain activated Guni Cordyceps mycelium.
Preferably, in the step (1), the first culture medium comprises the following components in parts by weight: 1-4 parts of maltose, 5-10 parts of egg albumen powder and 0.1-0.2 part of KH2PO40.1-0.2 part of magnesium sulfate and 80-100 parts of water;
the second culture medium comprises the following components in parts by weight: 15-25 parts of potato powder, 1-5 parts of glucose, 2-8 parts of whey protein concentrate and 0.1-0.2 part of KH2PO40.1-0.2 part of magnesium sulfate and 60-100 parts of water.
Preferably, in the step (1), the mass ratio of the apple juice, the sweet potato juice, the glucose, the peptone, the potassium dihydrogen phosphate, the magnesium sulfate and the activated Guni cordyceps mycelium is 1-5: 1-5: 1-3: 1-2: 0.01-0.05: 0.01-0.05: 5-10.
Preferably, in the step (1), the sterilization temperature is 115-125 ℃, and the sterilization time is 10-20 min.
Preferably, sterile air is continuously introduced during the fermentation culture process in the step (1), and the ventilation quantity is 0.02-0.03 vvm.
Preferably, in the step (2), the hot air drying temperature is 40-50 ℃, and the hot air drying time is 2-6 h.
Preferably, in the step (2), the ultrasonic frequency is 20-30kHz, and the ultrasonic power is 300-350W.
Preferably, in the step (3), the temperature is reduced from room temperature to-40 to-60 ℃ at a cooling rate of 5-10 ℃/min.
Preferably, in the step (3), the temperature rising speed from minus 40 to minus 60 ℃ to 30 to 50 ℃ is 1 to 3 ℃/min.
The technical effects of the invention are as follows:
(1) in the step (2), after the precipitate is dried by hot air at the temperature of 40-50 ℃, the precipitate does not damage effective components, and is further matched with ethanol hot water bath for backflow, and ultrasonic treatment is assisted in the backflow extraction process, so that the usage amount of ethanol can be effectively reduced, and the extraction efficiency can be improved.
(2) The invention further adjusts the pH value of the crude liquid to 5-5.5, then carries out quick freezing and temperature reduction, does not generate any damage to components, and combines the quick temperature reduction with the slow temperature rise process, which can effectively cause the hydrophobic bond structure of the cell membrane to be broken, thereby leading the cell membrane and the cell wall to be broken, the acidic environment can also effectively enhance the permeability of the cell membrane and the cell wall, effectively promotes the quick diffusion of active components in the breaking process, and accelerates the separation of cordycepin components.
(3) Cordycepin is fat-soluble and easy to damage by heating, and the extraction efficiency is not high because the conventional extraction adopts thermal reflux extraction. The extraction process aims at solving the technical problem that after solid-liquid separation of fermentation, the Guni cordyceps mycelia are extracted by adopting improved low-temperature and other modes, so that not only can no damage be caused to components, but also the extraction rate of cordycepin is improved.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Example 1
A process for improving the extraction rate of cordycepin from Guni cordyceps comprises the following steps:
(1) uniformly mixing 1kg of apple juice, 1kg of sweet potato juice, 1kg of glucose, 1kg of peptone, 0.01kg of monopotassium phosphate, 0.01kg of magnesium sulfate and 500kg of water, sterilizing at 125 ℃ for 20min, adding 5kg of activated Guni cordyceps sinensis mycelia, fermenting and culturing at 30 ℃ for 15 days, keeping the tank pressure at 0.04MPa, the stirring speed at 150r/min and the ventilation volume of sterile air at 0.03vvm in the fermentation and culture process, and centrifuging to obtain a precipitate;
(2) drying the precipitate with hot air at 40 ℃ for 2h, keeping ventilation in the drying process, crushing, sieving with a 60-mesh sieve, adding into a reflux device, and adding 95% ethanol according to a material-liquid ratio of 1: 5, refluxing and extracting for 4 hours in a boiling water bath, wherein ultrasonic treatment is assisted in the refluxing and extracting process, the ultrasonic frequency is 20kHz, and the ultrasonic power is 300W, so as to obtain a crude liquid;
(3) adjusting the pH value of the crude liquid to 5-5.5 by using 0.1mol/L hydrochloric acid, cooling to-40 ℃ from room temperature at a speed of 5 ℃/min, preserving heat for 1h, heating to 30 ℃ at a speed of 1 ℃/min, stirring for 1h at a speed of 1000r/min, extracting by using ethyl acetate, then passing through a 4.5-micrometer filter membrane, loading a macroporous resin D1400 column, eluting and balancing by using distilled water, eluting by using 15-20% methanol, detecting by using HPLC, collecting a sample receiving tube with a cordycepin peak area exceeding 85%, concentrating, and drying to obtain the Guni cordycepin.
Example 2
A process for improving the extraction rate of cordycepin from Guni cordyceps comprises the following steps:
(1) uniformly mixing 5kg of apple juice, 5kg of sweet potato juice, 3kg of glucose, 2kg of peptone, 0.05kg of monopotassium phosphate, 0.05kg of magnesium sulfate and 500kg of water, sterilizing at 125 ℃ for 20min, adding 10kg of activated Guni cordyceps sinensis mycelia, fermenting and culturing at 30 ℃ for 15 days, keeping the tank pressure at 0.04MPa, stirring at 150r/min and the ventilation of sterile air at 0.03vvm in the fermentation and culture process, and centrifuging to obtain a precipitate;
(2) drying the precipitate with hot air at 50 ℃ for 6h, keeping ventilation in the drying process, crushing, sieving with a 60-mesh sieve, adding into a reflux device, and adding 95% ethanol according to a material-liquid ratio of 1: 8, refluxing and extracting for 6 hours in a boiling water bath, wherein ultrasonic treatment is assisted in the refluxing and extracting process, the ultrasonic frequency is 30kHz, and the ultrasonic power is 350W, so as to obtain crude liquid;
(3) adjusting the pH value of the crude liquid to 5-5.5 by using hydrochloric acid with the concentration of 1mol/L, cooling to-60 ℃ from room temperature at the speed of 10 ℃/min, preserving heat for 2h, heating to 50 ℃ at the speed of 3 ℃/min, stirring for 2h at the speed of 2000r/min, extracting by using ethyl acetate, then passing through a 4.5-micrometer filter membrane, loading a macroporous resin D1400 column, eluting with distilled water for balance, eluting with 15-20% methanol, detecting by HPLC, collecting a sample receiving tube with the cordycepin peak area exceeding 85%, concentrating, and drying to obtain the Guni cordyceps cordycepin.
Example 3
A process for improving the extraction rate of cordycepin from Guni cordyceps comprises the following steps:
(1) uniformly mixing 2kg of apple juice, 4kg of sweet potato juice, 1.5kg of glucose, 1.7kg of peptone, 0.02kg of monopotassium phosphate, 0.04kg of magnesium sulfate and 300kg of water, sterilizing at 122 ℃ for 13min, adding 8kg of activated Guni cordyceps mycelium, fermenting and culturing at 22 ℃ for 13 days, keeping the tank pressure at 0.025MPa in the fermentation and culture process, stirring at 140r/min, and carrying out centrifugation to obtain a precipitate, wherein the ventilation capacity of sterile air is 0.022 vvm;
(2) drying the precipitate with hot air at 47 ℃ for 3h, keeping ventilation in the drying process, crushing, sieving with a 60-mesh sieve, adding into a reflux device, and adding 95% ethanol according to a material-liquid ratio of 1: 7, refluxing and extracting for 4.5 hours in a boiling water bath, wherein ultrasonic treatment is assisted in the refluxing and extracting process, the ultrasonic frequency is 27kHz, and the ultrasonic power is 320W, so as to obtain a crude liquid;
(3) adjusting the pH value of the crude solution to 5-5.5 by using 0.6mol/L hydrochloric acid, cooling to-55 ℃ from room temperature at the speed of 6 ℃/min, preserving heat for 1.3h, heating to 35 ℃ at the speed of 2.5 ℃/min, stirring at the speed of 1800r/min for 1.3h, extracting by using ethyl acetate, then passing through a 4.5-micrometer filter membrane, loading a macroporous resin D1400 column, eluting and balancing by using distilled water, eluting by using 15-20% methanol, detecting by using HPLC, collecting a sample receiving tube with the cordycepin peak area exceeding 85%, concentrating, and drying to obtain the Guni cordyceps cordycepin.
Example 4
A process for improving the extraction rate of cordycepin from Guni cordyceps comprises the following steps:
(1) uniformly mixing 4kg of apple juice, 2kg of sweet potato juice, 2.5kg of glucose, 1.3kg of peptone, 0.04kg of monopotassium phosphate, 0.02kg of magnesium sulfate and 400kg of water, sterilizing at 118 ℃ for 17min, adding 6kg of activated cordyceps gunnii mycelia, fermenting and culturing at 28 ℃ for 8 days, keeping the tank pressure at 0.035MPa in the fermentation and culture process, stirring at 110r/min, and carrying out centrifugation to obtain a precipitate, wherein the aeration quantity of sterile air is 0.028 vvm;
(2) drying the precipitate with hot air at 43 ℃ for 5h, keeping ventilation in the drying process, crushing, sieving with a 60-mesh sieve, adding into a reflux device, and adding 95% ethanol according to a material-liquid ratio of 1: carrying out reflux extraction in a boiling water bath for 5.5h, wherein ultrasonic treatment is assisted in the reflux extraction process, the ultrasonic frequency is 24kHz, and the ultrasonic power is 330W, so as to obtain a crude liquid;
(3) adjusting the pH value of the crude liquid to 5-5.5 by using hydrochloric acid with the concentration of 0.3mol/L, cooling to-45 ℃ from room temperature at the speed of 8 ℃/min, preserving heat for 1.7h, heating to 45 ℃ at the speed of 1.5 ℃/min, stirring at the speed of 1200r/min for 1.7h, extracting by using ethyl acetate, then passing through a 4.5-micron filter membrane, loading to a macroporous resin D1400 column, eluting and balancing by using distilled water, eluting by using 15-20% methanol, detecting by HPLC, collecting a sample receiving tube with the cordycepin peak area exceeding 85%, concentrating, and drying to obtain the Gunie cordycepin.
Example 5
A process for improving the extraction rate of cordycepin from Guni cordyceps comprises the following steps:
(1) uniformly mixing 3kg of apple juice, 3kg of sweet potato juice, 2kg of glucose, 1.5kg of peptone, 0.03kg of monopotassium phosphate, 0.03kg of magnesium sulfate and 350kg of water, sterilizing at 120 ℃ for 15min, adding 7kg of activated Guni cordyceps mycelium, fermenting and culturing at 25 ℃ for 10 days, keeping the tank pressure at 0.03MPa, stirring at 120r/min and the ventilation of sterile air at 0.025vvm in the fermentation and culture process, and centrifuging to obtain a precipitate;
sealing the first culture medium in the activated Guni cordyceps mycelia, sterilizing, inoculating acremonium terricola under aseptic conditions, and culturing at 23 deg.C for 6 days; then inoculating 8% of the inoculum size into a second sterilized culture medium, culturing at 25 ℃ for 3 days, and keeping ventilation for 0.02vvm in the culture process to obtain activated Guni cordyceps mycelium;
the first culture medium comprises the following components in parts by weight: 2 parts of maltose, 6 parts of egg albumen powder and 0.12 part of KH2PO40.12 part of magnesium sulfate and 100 parts of water; the second culture medium comprises the following components in parts by weight: 20 parts of potato powder, 2 parts of glucose, 4 parts of whey protein concentrate and 0.12 part of KH2PO40.12 part of magnesium sulfate and 100 parts of water;
(2) drying the precipitate with hot air at 45 ℃ for 4h, keeping ventilation in the drying process, crushing, sieving with a 60-mesh sieve, adding into a reflux device, and adding 95% ethanol according to a material-liquid ratio of 1: 6.5 refluxing and extracting for 5h in boiling water bath, wherein ultrasonic treatment is assisted in the refluxing and extracting process, the ultrasonic frequency is 26kHz, and the ultrasonic power is 325W, so as to obtain a crude liquid;
(3) adjusting the pH value of the crude liquid to 5-5.5 by using 0.45mol/L hydrochloric acid, cooling to-50 ℃ from room temperature at a speed of 7 ℃/min, preserving heat for 1.5h, heating to 40 ℃ at a speed of 2 ℃/min, stirring at a speed of 1500r/min for 1.5h, extracting by using ethyl acetate, then passing through a 4.5-micrometer filter membrane, loading to a macroporous resin D1400 column, eluting and balancing by using distilled water, eluting by using 15-20% methanol, detecting by HPLC, collecting a sample receiving tube with a cordycepin peak area exceeding 85%, concentrating, and drying to obtain the Guni cordyceps cordycepin.
Comparative example
A process for improving the extraction rate of cordycepin from Guni cordyceps comprises the following steps:
(1) uniformly mixing 3kg of apple juice, 3kg of sweet potato juice, 2kg of glucose, 1.5kg of peptone, 0.03kg of monopotassium phosphate, 0.03kg of magnesium sulfate and 350kg of water, sterilizing at 120 ℃ for 15min, adding 7kg of activated Guni cordyceps mycelium, fermenting and culturing at 25 ℃ for 10 days, keeping the tank pressure at 0.03MPa, stirring at 120r/min and the ventilation of sterile air at 0.025vvm in the fermentation and culture process, and centrifuging to obtain a precipitate; wherein the activated Guni cordyceps mycelia was the same as the activation method of example 5;
(2) drying the precipitate with hot air at 45 ℃ for 4h, keeping ventilation in the drying process, crushing, sieving with a 60-mesh sieve, adding into a reflux device, and adding 95% ethanol according to a material-liquid ratio of 1: 6.5 refluxing and extracting for 5h in boiling water bath, wherein ultrasonic treatment is assisted in the refluxing and extracting process, the ultrasonic frequency is 26kHz, and the ultrasonic power is 325W, so as to obtain a crude liquid;
(3) extracting the crude liquid with ethyl acetate, filtering with a 4.5 μm filter membrane, loading onto macroporous resin D1400 column, eluting with distilled water, eluting with 15-20% methanol, detecting by HPLC, collecting sample receiving tube with cordycepin peak area over 85%, concentrating, and drying to obtain Guni Cordyceps cordycepin.
The method of example 5 and the comparative example are used for extracting the Guni cordyceps cordycepin, and the method comprises the following specific steps:
the formula for calculating the cordycepin extraction rate of the Guni cordyceps is as follows:
the purity of Coryni Cordyceps militaris cordycepin is calculated as follows:
from the above table, it can be seen that: the extraction rate of cordycepin from Cordyceps Guni by the method of the invention can reach 4.02 per mill, which is far beyond the comparative example (2.14 per mill), and the purity can reach 99.35%.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A process for improving the extraction rate of cordycepin from Guni cordyceps is characterized by comprising the following steps:
(1) uniformly mixing apple juice, sweet potato juice, glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate and water, sterilizing, adding activated Guni cordyceps sinensis mycelia, fermenting and culturing at 20-30 ℃ for 5-15 days, keeping the tank pressure at 0.02-0.04MPa in the fermentation and culture process, stirring at the speed of 100-;
(2) drying the precipitate with hot air, keeping ventilation in the drying process, pulverizing, sieving, adding into a reflux device, and adding 95% ethanol according to a material-liquid ratio of 1: reflux-extracting for 4-6h in a boiling water bath for 5-8 hours, and performing ultrasonic treatment during the reflux-extracting process to obtain a crude liquid;
(3) adjusting the pH value of the crude liquid to 5-5.5, cooling from room temperature to-40-60 ℃, preserving heat for 1-2h, heating to 30-50 ℃, stirring for 1-2h, extracting by ethyl acetate, then passing through a 4.5 mu m filter membrane, loading a macroporous resin D1400 column, eluting with 15-20% methanol after balance of rinsing by distilled water, detecting by HPLC, collecting a sample receiving tube with more than 85% of cordycepin peak area, concentrating, and drying to obtain the Guni cordyceps militaris cordycepin.
2. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis according to claim 1, wherein the first culture medium is sealed and sterilized in the activated Guni cordyceps sinensis mycelia in step (1), and Acremonium terricola is inoculated under aseptic conditions and cultured at a constant temperature of 20-25 ℃ for 5-10 days; then inoculating 6-10% of the culture medium into the second sterilized culture medium, culturing at 15-25 deg.C for 2-4 days, and keeping ventilation of 0.01-0.03vvm during the culture process to obtain activated Guni Cordyceps mycelium.
3. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis according to claim 1, wherein in the step (1), the first culture medium comprises the following components in parts by weight: 1-4 parts of maltose, 5-10 parts of egg albumen powder and 0.1-0.2 part of KH2PO40.1-0.2 part of magnesium sulfate and 80-100 parts of water;
the second culture medium comprises the following components in parts by weight: 15-25 parts of potato powder, 1-5 parts of glucose, 2-8 parts of whey protein concentrate and 0.1-0.2 part of KH2PO40.1-0.2 part of magnesium sulfate and 60-100 parts of water.
4. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis according to claim 1, wherein in the step (1), the mass ratio of the apple juice to the sweet potato juice to the glucose to the peptone to the potassium dihydrogen phosphate to the magnesium sulfate to the activated Guni cordyceps sinensis mycelia is 1-5: 1-5: 1-3: 1-2: 0.01-0.05: 0.01-0.05: 5-10.
5. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis as claimed in claim 1, wherein the sterilization temperature in step (1) is 115-125 ℃ and the sterilization time is 10-20 min.
6. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis according to claim 1, wherein in the fermentation culture process in the step (1), sterile air is continuously introduced, and the ventilation volume is 0.02-0.03 vvm.
7. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis according to claim 1, wherein the hot air drying temperature in the step (2) is 40-50 ℃ and the hot air drying time is 2-6 h.
8. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis as claimed in claim 1, wherein in the step (2), the ultrasonic frequency is 20-30kHz, and the ultrasonic power is 300-350W.
9. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis according to claim 1, wherein in the step (3), the temperature is reduced from room temperature to-40 to-60 ℃ at a rate of 5-10 ℃/min.
10. The process for improving the extraction rate of cordycepin from Guni cordyceps sinensis according to claim 1, wherein in the step (3), the temperature is raised from-40 to-60 ℃ to 30-50 ℃ at a temperature raising speed of 1-3 ℃/min.
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