CN114891050A - Method for separating cordycepin from fermentation liquor or extracting solution - Google Patents
Method for separating cordycepin from fermentation liquor or extracting solution Download PDFInfo
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- 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 114
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 title claims abstract description 114
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 title claims abstract description 113
- 238000000034 method Methods 0.000 title claims abstract description 52
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- 230000004151 fermentation Effects 0.000 title claims abstract description 31
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- 239000007788 liquid Substances 0.000 claims abstract description 41
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- 238000001471 micro-filtration Methods 0.000 claims abstract description 25
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- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000190633 Cordyceps Species 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 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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- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
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- 101800000263 Acidic protein Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001248610 Ophiocordyceps sinensis Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000011358 absorbing material Substances 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A20/00—Water conservation; Efficient water supply; Efficient water use
- Y02A20/124—Water desalination
Abstract
The invention discloses a method for separating cordycepin from fermentation liquor or extracting solution, which comprises the following steps: s1: performing solid-liquid separation on fermentation liquor or extract containing cordycepin to obtain clear liquid and concentrated solution; adding water into the obtained concentrated solution for percolation to obtain microfiltration permeating liquid; s2: carrying out nanofiltration on the microfiltration permeating liquid obtained in the step S1 and the obtained clear liquid to obtain a first nanofiltration concentrated liquid and a first nanofiltration permeating liquid; adding water into the first nanofiltration concentrated solution for percolation to obtain a second nanofiltration permeating solution; s3: adjusting the pH values of the first nanofiltration permeating liquid and the second nanofiltration permeating liquid obtained in the step S2, performing electrodialysis, and collecting the solution in a desalting chamber to obtain deionized materials; s4: adjusting the pH of the deionized material obtained in the step S3, performing electrodialysis, and collecting the solution in a concentration chamber to obtain a concentrated cordycepin solution; s5: and (4) crystallizing. The method has the advantages of short process, high cordycepin yield and purity, low wastewater discharge, and industrial production potential.
Description
Technical Field
The invention belongs to the technical field of separation and purification in biochemical engineering, and particularly relates to a method for separating cordycepin from fermentation liquor or extracting solution.
Background
Cordycepin, also called cordycepin and cordycepin, is the main active ingredient in cordyceps militaris, belongs to nucleoside substances and purine alkaloids, and is also the first nucleoside antibiotic separated from fungi. In 1951, Cunningham, German scientist, purified Cordyceps militaris to obtain a crystal named cordycepin (3' -deoxyadenosine), with molecular formula of C 10 H 13 N 5 O 3 The ultraviolet-absorbing material has a relative molecular mass of 251.24, a melting point of 230-231 ℃, alkaline property, needle-like or sheet-like crystal, solubility in water, hot ethanol and methanol, insolubility in benzene, ether and chloroform, a maximum absorption wavelength of ultraviolet light of 260nm, and a structural formula shown in formula 1.
As an analog of adenosine, cordycepin has various pharmacological actions of resisting tumor, resisting bacteria and viruses, regulating immunity, eliminating free radicals and the like, and has good clinical application prospect. The research on cordycepin is becoming an extremely active area in medicinal chemistry at present. The content of cordycepin in wild cordyceps sinensis is very low, and the cordycepin used in the current research is basically extracted from fermented cordyceps militaris.
The cordyceps militaris is the only strain capable of forming a large amount of cordycepin in more than 300 cordyceps militaris strains reported at present, and is mainly extracted from cordyceps militaris sporocarp. However, the production period of the fruiting body is long, the yield is low, and the content of cordycepin is low, so that the production cost of cordycepin is extremely high, which has become a bottleneck restricting the further research, development and utilization of cordycepin. At present, large-scale submerged fermentation of cordyceps militaris is realized, but the unit yield of cordycepin is very low. The yield of cordycepin of an original strain in the nature is not high, and the mutation and screening reports of high-yield strains are not frequent. Experiments show that most of cordycepin is secreted into fermentation liquor. Therefore, cordycepin can be separated from both fruiting bodies and fermentation liquor. Many methods for separating cordycepin have been reported, including ultrafiltration membrane separation and adsorption resin separation, but the purity of cordycepin obtained by these methods is low, the operation is complicated, and improvement is needed. Such as ultrafiltration membrane separation: the Chinese patent application 201310127153.6 discloses an ultrafiltration membrane separation method for continuously preparing cordycepin and cordyceps polysaccharide by using a membrane separation technology, wherein the molecular weight cut-off is 500Da, and the purity of the obtained cordycepin is only over 20 percent; such as resin adsorption separation method: the Chinese patent application 201410322498.3 discloses a method for separating and purifying cordycepin by using mixed resin, which is characterized in that 20-40% of ethanol solution is adopted for elution, the content of cordycepin in an extract obtained by concentrating an eluent is only 20-40%, and the purity of cordycepin can reach more than 90% after recrystallization of the ethanol solution; such as resin adsorption separation method: the invention application 201510129431.2 in China, a method for rapidly separating cordycepin from fermentation liquor, adopts macroporous adsorption resin (such as AB-8) to separate cordycepin from the fermentation liquor with the concentration of only 40%, and continuously adopts silica gel C18 as chromatographic packing to obtain cordycepin with the concentration of only 90%, the process needs two-step chromatographic separation, needs to consume a large amount of ethanol solution, increases the complexity and cost of operation, and the silica gel C18 is mainly used for analytical chromatography or small-scale high-pressure chromatographic preparation and is not suitable for industrial scale-up production.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for separating cordycepin from fermentation liquor or extracting solution aiming at the defects of the prior art.
The invention idea is as follows: according to different dissociation states of cordycepin in the solution under different pH values, the cordycepin is separated in a targeted manner by adopting an electrodialysis method. Firstly, the cordycepin exists in a molecular state at the pH value of 6.0-7.0, is not acted by an electric field in a solution, and removes most inorganic salts and dissociable impurities in the solution by adopting an electrodialysis membrane with high crosslinking degree and small membrane pore diameter. And then adjusting the pH value of the solution to 2.5-3.0, wherein the cordycepin is positively charged, performing electrodialysis by adopting an ion exchange membrane with low crosslinking degree and large membrane pore diameter to separate the cordycepin from the original extracting solution to obtain a high-purity solution, and simultaneously concentrating the cordycepin in the solution by controlling the volume ratio of the solution in an electrodialysis desalination chamber and a concentration chamber to directly reach the crystallizable concentration. The method has the advantages that the adopted process flow is short, and after pretreatment (solid-liquid separation and nanofiltration for removing macromolecular impurities), according to different dissociation states of the cordycepin in the solution at different pH values, ion exchange membranes with different membrane pore diameters are adopted for separation to obtain the high-purity cordycepin product.
In order to solve the technical problems, the invention discloses a method for separating cordycepin from fermentation liquor or extracting solution (figure 1), which comprises the following steps:
s1: adjusting pH of fermentation liquid or extractive solution containing cordycepin to 6.0-7.0, precipitating acidic protein or impurities therein, removing thallus, suspended substances, etc., and performing solid-liquid separation to obtain clear liquid and concentrated solution; adding water into the obtained concentrated solution for percolation to obtain microfiltration permeating liquid;
s2: carrying out nanofiltration on the microfiltration permeating liquid obtained in the step S1 and the obtained clear liquid to obtain a first nanofiltration concentrated solution mainly containing macromolecular substances such as protein, cordyceps polysaccharide, pigment and the like and a first nanofiltration permeating liquid mainly containing cordycepin; adding water into the first nanofiltration concentrated solution for percolation when the volume of the first nanofiltration concentrated solution is reduced to 1-1.5 times of the minimum circulation volume so as to reduce the retention rate of the ultrafiltration membrane on cordycepin and obtain a second nanofiltration permeate;
s3: adjusting the pH values of the first nanofiltration permeate and the second nanofiltration permeate obtained in the step S2 to 6.0-8.0, performing electrodialysis through an electrodialysis device (pumping the electrodialysis permeate into a desalination chamber of an electrodialysis membrane stack by a pump, starting a circulating device of electrodialysis equipment to enable the desalination chamber solution, a concentration chamber solution and a polar chamber solution to respectively circulate, then starting a direct-current power supply, enabling inorganic ions and dissociable impurities in the desalination chamber solution to permeate through an ion exchange membrane and enter the concentration chamber solution), maintaining the pH value of the solution in the desalination chamber to be 6.0-7.0 in the electrodialysis process, stopping the electrodialysis when the conductivity of the solution in the desalination chamber is below 0.2ms/cm, keeping cordycepin without charges under the pH value and in the desalination chamber solution, thereby realizing the primary separation of cordycepin, inorganic salts and electrolyte impurities in fermentation liquor, collecting the solution in the desalination chamber, obtaining the material after ion removal;
s4: adjusting the pH of the deionized material obtained in the step S3 to 2.5-3.0, performing electrodialysis by an electrodialysis device (pumping the solution into a desalting chamber of a second electrodialysis membrane stack, and allowing cordycepin to enter the solution in a concentration chamber from the desalting chamber under the action of a direct current electric field), maintaining the pH of the solution in the desalting chamber to 2.8-3.0 during the electrodialysis, stopping the electrodialysis when the conductivity of the solution in the desalting chamber is below 0.2ms/cm, and retaining uncharged impurities such as monosaccharides and polysaccharides in the fermentation liquor in the desalting chamber; synchronously realizing the concentration of the cordycepin by adjusting the volume ratio of the solution in the desalting chamber to the solution in the concentrating chamber, and collecting the solution in the concentrating chamber to obtain the concentrated cordycepin solution;
s5: and (4) transferring the concentrated cordycepin solution obtained in the step (S4) to a crystallization tank, adjusting the pH of the solution to 4.0-9.0, cooling to 0-8 ℃, keeping the temperature for more than 6 hours for crystallization, and performing suction filtration, water washing and drying on the obtained crystals to obtain cordycepin crystals.
Wherein the cordyceps militaris fermentation broth is obtained by performing solid culture on mycelia. The preparation method of the fruit body extracting solution comprises the following steps: firstly, drying and crushing cordyceps militaris sporocarp, then respectively adding sporocarp powder and a dilute hydrochloric acid solution with the pH value of 2.8-3.0 according to the solid-to-liquid ratio of 1:10-1:5, uniformly stirring, then raising the temperature of the solution to 45 ℃ in a water bath mode, and maintaining the temperature for 6 hours to obtain the cordyceps militaris sporocarp.
In step S1, the initial pH value of the fermentation broth of Cordyceps militaris mycelia is about 5-6, and the pH value of the fermentation broth is adjusted to about 3.0 with dilute hydrochloric acid to precipitate acid-insoluble protein and some impurities with low solubility under acidic conditions, and remove part of the impurities in the solution.
In step S1, the solid-liquid separation mode is that microfiltration is carried out by a ceramic membrane to remove thalli and suspended matters in the solution; the aperture of the ceramic membrane is 50-200nm, the microfiltration pressure is below 0.4MPa, and the microfiltration temperature is below 35 ℃.
In step S1, the solid-liquid separation method is centrifugation; the rotating speed of the centrifugation is 6000-10000r/min, and the time of the centrifugation is 5-15 min.
In step S1, the water adding and percolating step is to add water 1-1.5 times of the volume of the concentrated solution for each time and percolating 3-5 times.
In step S2, in the nanofiltration, the cut-off molecular weight of the nanofiltration membrane is 300-500Da, the nanofiltration pressure is below 3.5MPa, and the nanofiltration temperature is below 35 ℃.
In step S2, the diafiltration with water is performed 3-5 times by adding water 1-1.5 times the volume of the first nanofiltration concentrate each time.
In steps S3 and S4, the cation exchange membrane in the electrodialysis device is a sulfonic acid type electrodialysis cation exchange membrane, and the anion exchange membrane is a quaternary ammonium type electrodialysis anion exchange membrane.
In step S3, a cation exchange membrane and an anion exchange membrane with high crosslinking degree and small pore diameter are adopted to improve the retention rate of the membrane on cordycepin. Then, electrodialysis is performed in a constant current mode (the current of the direct current power supply is constant during electrodialysis) or a constant voltage mode (the voltage of the direct current power supply is constant during electrodialysis). Under the direct current electric field, inorganic salt and some micromolecule organic matters which are in a dissociation state under a neutral condition in the solution of the desalting chamber enter the solution of the concentrating chamber.
In step S3, the electrodialysis membrane is soaked in a 5% sodium chloride solution for more than 20 hours before use, and is rinsed with pure water before use. During electrodialysis, the current was controlled to be <0.5A and the voltage to be < 60V. The step is mainly to allow the cordycepin solution to enter the concentration chamber from the desalting chamber, and preferably to select a cation exchange membrane and an anion exchange membrane which have low crosslinking degree, high water content and large pore diameter in order to improve the yield of cordycepin and reduce the residue of cordycepin in the desalting chamber.
In step S3, the volume ratio of the desalting chamber to the concentrating chamber is 1 (0.5-1.5), preferably 1: 1.
In step S4, the volume ratio of the desalting chamber to the concentrating chamber is (12-17): 1, preferably 15: 1.
In step S3, the degree of crosslinking of the cation exchange membrane and the anion exchange membrane in the electrodialysis device is 8% or more.
In step S4, the degrees of crosslinking of the cation exchange membrane and the anion exchange membrane in the electrodialysis device are both 6% or less.
In step S4, because the molecular weight of cordycepin is small (251), in order to increase the transmittance of cordycepin, preferably, a sulfonic acid type cation exchange membrane and a quaternary amine type strongly basic anion exchange membrane with low crosslinking degree and large pore diameter are selected, and preferably, a sulfonic acid type cation exchange membrane (CJMC-3, Na type) and a quaternary amine type strongly basic anion exchange membrane (CJMA-3, Cl type) of the compost family are selected, so that the retention rate of cordycepin by the ion exchange membrane can be reduced, and the cordycepin can enter the solution in the concentration chamber from the solution in the desalting chamber as much as possible.
In step S3 and step S4, the current is 0.5A or less and the voltage is 60V or less during the electrodialysis.
In step S5, according to the difference of solubility of cordycepin at different pH values and temperatures, the solubility is high under acidic conditions (pH <3.0), but when the solution is at pH 4.5-8, the solubility is low, the pH value of the solution is adjusted to 4.5 for crystallization, and then the temperature is reduced to 5 ℃ and maintained at the temperature for a certain time, so that cordycepin is sufficiently precipitated, and the yield is improved.
In step S5, the temperature of the water washing is 5 ℃ or lower.
In step S5, since cordycepin is easily decomposed at high temperature, the drying temperature is 55 ℃ or lower.
Has the beneficial effects that: compared with the prior art, the invention has the following advantages:
1. the invention adopts an electrodialysis method to replace a method for separating macroporous adsorption resin and ion exchange resin, the single-step yield can reach more than 96 percent, the acid and alkali consumption in the regeneration process of the ion exchange resin is reduced, and the wastewater discharge is reduced by more than 80 percent.
2. The method has short operation time, can finish the separation and crystallization of the cordycepin within about 30 hours, and greatly shortens the production period of the cordycepin.
3. The method has simple operation, high cordycepin separation purity, total yield of one-time separation of over 75 percent, cordycepin content of over 98 percent and ash content of less than 0.1 percent in the final product.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a flow chart of the present invention for separating cordycepin from fermentation broth or extraction liquid.
FIG. 2 is a HPLC chart of the starting material in example 1 of the present invention.
FIG. 3 is a HPLC chart of the crystallized product in example 1 of the present invention.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The preparation method of the fermentation broth containing the cordycepin mycelia in the following examples comprises the following steps: the cordyceps militaris fermentation liquor is fermented by adopting cordyceps militaris CICC14014 and using glucose and yeast extract as nitrogen sources, and finally the concentration of cordycepin in the fermentation liquor is about 3-6 g/L. The main components of the culture medium are as follows: yeast powder, peptone, glucose, magnesium sulfate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, wherein the solid culture time of the mycelium is 15-30 days.
HPLC detection is carried out on raw materials and finished products in the following examples, and the specific method comprises the following steps: a chromatographic column: c18, mobile phase: 1.36g/L potassium dihydrogen phosphate solution and methanol were mixed at a volume ratio of 90:10 (filtered with a 0.45um aqueous membrane and sonicated for 15 minutes), and the flow rates were: 1ml/min, detection time: 25min, detection method: ultraviolet detector, detection wavelength: 254 nm.
Example 1: method for separating cordycepin from fermentation liquor containing cordycepin mycelia
(1) Taking 30L fermentation broth containing cordycepin (cordycepin concentration is 4.5g/L), adjusting pH of the solution to 7.0 with dilute hydrochloric acid, and filtering with ceramic microfiltration membrane (membrane pore diameter is 200nm, and membrane area is 0.33 m) 2 ) Operating pressure<0.4MPa, temperature<Removing suspended substances and mycelia from the fermentation broth at 30 deg.C, and concentrating the fermentation broth to about 3L to obtain 27L of permeate. Adding pure water into the concentrated solution, percolating with 3.5L of water each time for 4 times, and collecting 41L of microfiltration permeate with cordycepin concentration of 3.26g/L for 3 hr.
(2) Then, nanofiltration is carried out on the microfiltration permeating liquid (the cut-off molecular weight of the nanofiltration membrane is 300-500Da), the operation pressure is less than 3MPa, and the temperature is less than 35 ℃ (the pH value is 5.5-7.0), so as to obtain the nanofiltration permeating liquid. The obtained nanofiltration permeate was reduced to about 3L, water was added for diafiltration (same microfiltration as for diafiltration, 4 times, 3.5L each time), and 52L of nanofiltration permeate was collected, the concentration of cordycepin was 2.52g/L, the single step yield was about 98.2%, and the process time was 4 hours.
(3) And then adjusting the pH value of the nanofiltration permeate to be between 6.0 and 8.0, and then performing electrodialysis, wherein ion exchange membranes in an electrodialysis membrane stack are respectively a strong acid type cation exchange membrane (Nippon Asian Stokes, CMX, Na type) and a strong base type anion exchange membrane (Nippon Asian Stokes, AMX, Cl type), a constant current mode is adopted, the current is less than 0.5A, the voltage is controlled to be less than 60V, and the volume ratio of the solution in a concentration chamber to the solution in a desalination chamber is 1:1, in the electrodialysis process, maintaining the pH value of the solution in the desalting chamber to be 6.0-7.0, stopping the electrodialysis when the conductivity of the desalting chamber is less than 0.2ms/cm, and finally collecting 51.8L of the solution (cordycepin solution) in the desalting chamber after impurity removal, wherein the concentration of cordycepin is 2.51g/L, and the time of the process is 3 hours.
(4) Adjusting the pH value of the cordycepin solution obtained in the previous step to 2.5-3.0, then adopting electrodialysis again, wherein ion exchange membranes in a membrane stack respectively adopt a sulfonic acid type cation exchange membrane (CJMC-3, Na type) and a quaternary amine type strong base anion exchange membrane (CJMA-3, Cl type), a constant current mode is adopted, the current is less than 0.5A, the voltage is less than 60V, and the volume ratio of the solution in a desalting chamber to that in a concentration chamber is 15:1, in the electrodialysis process, the pH value of the solution in the desalting chamber is maintained to be 2.8-3.0, the electrodialysis is stopped when the conductivity of the solution in the desalting chamber is less than 0.2ms/cm, 4.0L of solution in the concentration chamber is collected, and the concentration of cordycepin is 32.2 g/L. The time for this process was 3 hours.
(5) Putting the concentrated solution into a glass crystallization tank, adjusting the pH value of the concentrated solution to 4.5 by using a sodium hydroxide solution, reducing the temperature to 5 ℃ in a way of sleeve-removing heat exchange of the crystallization tank, maintaining the temperature for 10 hours, putting the concentrated solution into the tank, performing suction filtration, washing (10 ℃), drying (55 ℃), and obtaining 108g of cordycepin finished product with the content of 98%, wherein the total yield is 80%. The time of the crystallization process is 8 hours, the time of the suction filtration and the drying is 8 hours, and the time required by the whole separation process is 29 hours. HPLC of the starting material and the crystallized end product in this example are shown in FIG. 2 and FIG. 3, respectively.
Example 2: extraction and separation of cordycepin from extractive solution containing Cordyceps militaris fruiting body
(1) Collecting fruiting body powder (with cordycepin content of 2 wt%) 4kg, adding 30L water solution (pH of the solution is adjusted to 2.5 with hydrochloric acid), and extracting at 45 deg.C in water bath for 6 hr to obtain extractive solution. Firstly, primarily filtering the extracting solution by using gauze, adding pure water to wash filter residues, and combining the filtrate and washing liquid to obtain about 36L of extracting solution, wherein the concentration of cordycepin is about 2.1g/L, and the extraction rate of cordycepin is about 95%. Then filtering the extractive solution with microfiltration membrane (membrane pore diameter of 0.1 μm and membrane area of 0.1 m) 2 ) Operating pressure<0.4MPa, temperature<And (6) obtaining microfiltration permeating liquid at the temperature of 30 ℃. Adding pure water into the obtained microfiltration concentrate for percolation when the volume of the microfiltration concentrate is about 3L, and adding 3L of water each timeFor 4 times, 45L of the microfiltration permeate was collected.
(2) And (2) performing nanofiltration on the microfiltration permeating liquid obtained in the step (1) by adopting a nanofiltration membrane (the molecular weight cut-off is 300-500Da), wherein the operation pressure is less than 3MPa, the temperature is less than 35 ℃ (the pH value is 5.5-7.0), and the nanofiltration permeating liquid and the nanofiltration concentrated liquid are obtained. When the nanofiltration concentrate is about 3.5L, water is added for diafiltration, 3.5L each time for 4 times, and 55.5L nanofiltration permeate is collected.
(3) And then adjusting the pH value of the nanofiltration permeate to 7.0, performing electrodialysis, using a strong acid type cation exchange membrane (CMX, Na type) and a strong base type anion exchange membrane (AMX, Cl type) of Asiaston Japan as ion exchange membranes, adopting a constant current mode, controlling the current to be less than 0.4A and the voltage to be less than 60V, controlling the volume ratio of the solution in the concentration chamber to the solution in the desalination chamber to be 1:1, maintaining the pH value of the solution in the desalination chamber to be 6.0-7.0 in the electrodialysis process, stopping the electrodialysis when the conductivity of the desalination chamber is less than 0.2ms/cm, and collecting and obtaining the cordycepin solution in the desalination chamber.
(4) And then, adjusting the pH of the cordycepin solution obtained by separation to 2.5-3.0, then performing second electrodialysis (constant current mode, current: <0.5A, voltage <60V, operating temperature less than or equal to 30 ℃), wherein the ion exchange membranes in the membrane stack respectively adopt a sulfonic acid type cation exchange membrane (CJMC-3, Na type) and a quaternary amine type strong alkali type anion exchange membrane (CJMA-3, Cl type), and the volume ratio of the solution in the desalting chamber to the solution in the concentrating chamber is 15: 1. In the electrodialysis process, the pH value of the solution in the desalting chamber is maintained to be 2.8-3.0, the electrodialysis is stopped when the conductivity of the solution in the desalting chamber is less than 0.2ms/cm, 2L of solution is obtained by collection, and the concentration of cordycepin is 40 g/L.
(5) And (3) putting the concentrated solution into a glass crystallization tank for crystallization (adjusting the pH value to 4.5 by using sodium hydroxide, cooling to 5 ℃, and maintaining at the temperature for 10 hours), carrying out suction filtration on the precipitated cordycepin crystals, washing the cordycepin crystals at the temperature of <10 ℃, drying at the temperature of <55 ℃) to obtain 63.8g of cordycepin finished product with the content of more than 98%, wherein the total yield is about 79.8%.
Comparative example 1: changing the pH value of cordycepin solution in the nanofiltration process
Microfiltration is carried out on the cordyceps militaris fermentation liquor to obtain 30L of clear solution, the concentration of cordycepin is about 3.5g/L, the pH value is adjusted to 3.0, then nanofiltration (the molecular weight cut-off of a nanofiltration membrane is 300-500Da) is carried out, the operating pressure is less than 3MPa in the nanofiltration process, the temperature of the solution is controlled below 20 ℃, and finally 4L of concentrated solution is obtained, wherein the concentration of cordycepin is 8g/L, and 26L of nanofiltration permeate liquid is obtained, and the concentration of cordycepin in the permeate liquid is about 2.8 g/L. Adding water into 4L of the concentrated solution, percolating (same manner as microfiltration, 4 times, adding 4L of water each time), and collecting 42L of nanofiltration permeate again, wherein the concentration of cordycepin in the nanofiltration permeate is 1.73 g/L. If the mass of the cordycepin in the permeate liquid is calculated, the yield of the step is only 69.5 percent.
Comparative example 2: electrodialysis desalting of cordycepin nanofiltration permeate at pH 3.0 (first electrodialysis)
The cordyceps militaris fermentation liquor is subjected to microfiltration and nanofiltration pretreatment to obtain nanofiltration permeate, 10L of the nanofiltration permeate is taken for an electrodialysis experiment, the pH of the solution is adjusted to 3.0, and the concentration of cordycepin is 2.4 g/L. In the electrodialysis membrane stack, a strong acid type cation exchange membrane (CMX, Na type) and a strong base type anion exchange membrane (AMX, Cl type) of Asiaston Japan are used as ion exchange membranes, a constant current mode is adopted, the current is less than 0.4A, the voltage is controlled to be less than 60V, and the volume ratio of a desalting chamber to a concentrating chamber is 1: 1. in the electrodialysis process, the pH of the solution in the desalting chamber is maintained at 3.0, the electrodialysis is stopped when the conductivity of the desalting chamber is less than 0.2ms/cm, and 10L of the cordycepin solution desalted by the desalting chamber is collected, wherein the concentration of the cordycepin is 0.8g/L, and the yield is only 33%. The concentration of cordycepin in the concentration chamber is about 1.6g/L, which accounts for about 67%. However, further separation is required due to the presence of large amounts of inorganic salts and dissociable impurities in the concentrating compartment.
Comparative example 3: the cordycepin solution was concentrated and purified at pH 7 (second electrodialysis)
Taking 10L of the cordycepin solution (initial pH 7.0) desalted by the first electrodialysis, wherein the concentration of the cordycepin is 2.1g/L, and performing electrodialysis purification and concentration again. The ion exchange membranes in the membrane stack are respectively sulfonic acid type cation exchange membrane (CJMC-3, Na type) and quaternary amine type strong alkali anion exchange membrane (CJMA-3, Cl type). The electrodialysis process adopts a constant current mode, and the current: the voltage is less than 0.5A, the voltage is less than 60V, the operation temperature is less than or equal to 30 ℃, and the ratio of the solution in the desalting chamber to the solution in the concentrating chamber is 10: 1. and in the electrodialysis process, the pH value of the solution in the desalting chamber is maintained to be 6.0-7.0, and the electrodialysis is stopped when the conductivity of the solution in the desalting chamber is less than 0.2 ms/cm. Finally obtaining 1L of solution in a concentration chamber, wherein the concentration of the cordycepin is 0.2g/L, and the yield is only 1%. Concentration and purification at this pH by electrodialysis is therefore not feasible.
The present invention provides a method and a method for separating cordycepin from fermentation broth or extract, and a plurality of methods and ways for implementing the technical scheme, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (10)
1. A method for separating cordycepin from fermentation liquor or extracting solution is characterized by comprising the following steps:
s1: adjusting pH of fermentation liquid or extractive solution containing cordycepin to 6.0-7.0, and performing solid-liquid separation to obtain clear liquid and concentrated solution; adding water into the obtained concentrated solution for percolation to obtain microfiltration permeating liquid;
s2: carrying out nanofiltration on the microfiltration permeating liquid obtained in the step S1 and the obtained clear liquid to obtain a first nanofiltration concentrated liquid and a first nanofiltration permeating liquid; adding water into the first nanofiltration concentrated solution for percolation when the volume of the first nanofiltration concentrated solution is reduced to 1-1.5 times of the minimum circulation volume to obtain a second nanofiltration permeating solution;
s3: adjusting the pH values of the first nanofiltration permeate and the second nanofiltration permeate obtained in the step S2 to 6.0-8.0, performing electrodialysis by using an electrodialysis device, maintaining the pH value of the solution in the desalting chamber to 6.0-7.0 in the electrodialysis process, stopping the electrodialysis when the conductivity of the solution in the desalting chamber is below 0.2ms/cm, and collecting the solution in the desalting chamber to obtain a material subjected to ion removal;
s4: adjusting the pH of the deionized material obtained in the step S3 to 2.5-3.0, performing electrodialysis by an electrodialysis device, maintaining the pH of the solution in the desalting chamber to 2.8-3.0 in the electrodialysis process, stopping the electrodialysis when the conductivity of the solution in the desalting chamber is below 0.2ms/cm, and collecting the solution in the concentration chamber to obtain the concentrated cordycepin solution;
s5: and (4) transferring the concentrated cordycepin solution obtained in the step (S4) to a crystallization tank, adjusting the pH of the solution to 4.0-9.0, cooling to 0-8 ℃, keeping the temperature for more than 6 hours for crystallization, and performing suction filtration, water washing and drying on the obtained crystals to obtain cordycepin crystals.
2. The method according to claim 1, wherein in step S1, the solid-liquid separation is carried out by microfiltration through a ceramic membrane; the aperture of the ceramic membrane is 50-200nm, the microfiltration pressure is below 0.4MPa, and the microfiltration temperature is below 35 ℃.
3. The method according to claim 1, wherein in step S1, the solid-liquid separation method is centrifugation; the rotating speed of the centrifugation is 6000-10000r/min, and the time of the centrifugation is 5-15 min.
4. The method as claimed in claim 1, wherein in step S2, the nanofiltration membrane has a molecular weight cut-off of 300 and 500Da, a nanofiltration pressure of 3.5MPa or less, and a nanofiltration temperature of 35 ℃ or less.
5. The method of claim 1, wherein in steps S3 and S4, the cation exchange membrane in the electrodialysis device is a sulfonic acid type electrodialysis cation exchange membrane, and the anion exchange membrane is a quaternary ammonium type electrodialysis anion exchange membrane.
6. The method according to claim 1, wherein in step S3, the volume ratio of the desalting chamber to the concentrating chamber is 1 (0.5-1.5), preferably 1: 1; in step S4, the volume ratio of the desalting chamber to the concentrating chamber is (12-17): 1, preferably 15: 1.
7. The method according to claim 1, wherein in step S3, the degree of crosslinking of both the cation exchange membrane and the anion exchange membrane in the electrodialysis unit is 8% or more; in step S4, the degrees of crosslinking of the cation exchange membrane and the anion exchange membrane in the electrodialysis device are both 6% or less.
8. The method of claim 1, wherein in steps S3 and S4, the current is 0.5A or less and the voltage is 60V or less during the electrodialysis.
9. The method according to claim 1, wherein the temperature of the water washing in step S5 is 5 ℃ or lower.
10. The method according to claim 1, wherein the drying temperature is 55 ℃ or lower in step S5.
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| CN103214533A (en) * | 2013-04-19 | 2013-07-24 | 湖南农业大学 | Method for continuously preparing cordycepin and cordyceps polysaccharide by using membrane separation technology |
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| CN105859809A (en) * | 2016-04-09 | 2016-08-17 | 南昌大学 | Method for extracting beta-thymidine from fermentation liquid |
| CN111574372A (en) * | 2020-06-05 | 2020-08-25 | 精晶药业股份有限公司 | Method for extracting chlorogenic acid from Chinese herbal medicine by electrodialysis method |
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| CN103214533A (en) * | 2013-04-19 | 2013-07-24 | 湖南农业大学 | Method for continuously preparing cordycepin and cordyceps polysaccharide by using membrane separation technology |
| CN104072559A (en) * | 2014-05-08 | 2014-10-01 | 宁波广发文博生物科技有限公司 | Method for extracting cordycepin from cordyceps militaris |
| CN204455079U (en) * | 2015-02-10 | 2015-07-08 | 厦门昊源膜科技有限公司 | A kind of Cordyceps militaris (L.) Link. separation of fermentative broth is purified and concentrating unit |
| CN105624200A (en) * | 2016-02-05 | 2016-06-01 | 浙江工业大学 | Recycling method of hirsutella sinensis fermentation filtrate |
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