CN103214533A - Method for continuously preparing cordycepin and cordyceps polysaccharide by using membrane separation technology - Google Patents
Method for continuously preparing cordycepin and cordyceps polysaccharide by using membrane separation technology Download PDFInfo
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Abstract
The invention discloses a method for continuously preparing cordycepin and cordyceps polysaccharide by using a membrane separation technology. The method is applicable to the fine/deep processing of active ingredients, namely cordycepin and cordyceps polysaccharide, in artificially-cultured cordyceps militaris, and is used for solving the problems in cordyceps polysaccharide and cordycepin separation that the process is complicated, products are single, organic solvents are used, and the purity and recovery ratio of the products are low. According to the method, cordyceps militaris fruiting bodies or solid culture media obtained after cordyceps militaris is cultured and is subjected to fruiting body harvesting are adopted as raw materials and are subjected to hot-water extraction, suspended substances and macromolecular impurities are removed from an extract through the microfiltration of a ceramic membrane, a hyperfiltration membrane with certain molecular weight cutoff is selected to carry out separation, cutoff liquid is collected and is subjected to alcohol precipitation so as to prepare cordyceps polysaccharide, through liquid is then subjected to hyperfiltration by a hyperfiltration membrane with the molecular weight cutoff of 500 Da, and through liquid is collected so as to prepare cordycepin. The content of the obtained cordyceps polysaccharide reaches over 35%, the content of the obtained cordycepin reaches over 20%, and the recovery ratio of each of the two products exceeds 90%. The method has the advantages that the process is simple, the separation efficiency is high, the purity of the obtained products is high, no phase change exists during separation, the energy consumption is low, organic solvents are not used, and the environment is not polluted, thereby being application to industrial production.
Description
Technical field
The present invention relates to a kind of membrane separation technique prepares cordycepin and Cordyceps polysaccharide continuously from the Cordyceps militaris (L.) Link. material method, especially relate to a kind of method that adopts microfiltration membrane from Cordyceps militaris (L.) Link. or its substratum, to prepare Cordyceps polysaccharide and cordycepin continuously in conjunction with ultrafiltration membrane technique.
Background technology
Cordyceps militaris (L.) Link. (Cordyceps militaris L.) has another name called Cordyceps militaris (L.) Link., belongs to mycota Ascomycota ergot Cordyceps, with China rare traditional Chinese medicine Cordyceps sinensis (C.sinensis) for belonging to xenogenesis together.Cordyceps militaris (L.) Link. is rich in activeconstituents Cordyceps polysaccharide and cordycepin, and content of cordycepin is more much higher than Cordyceps sinensis, Cordyceps militaris (L.) Link. is easy to artificial culture for Cordyceps sinensis simultaneously, because the shortage of Cordyceps sinensis resource, Cordyceps militaris (L.) Link. is expected to become the substitute of Cordyceps sinensis.Cordyceps polysaccharide can improve immune function of human body, leukocyte increasing, the clinical malignant tumour that has been used for the treatment of.In addition Cordyceps polysaccharide also can be used for controlling pulmonary tuberculosis, breathe hard breath with cough, impotence, nocturnal emission, spontaneous sweating, soreness of waist and knee joint, hypoglycemic effect is arranged simultaneously.Multiple pharmacological effect such as that cordycepin has is antitumor, anti-bacteria and anti-virus, immunomodulatory, removing free radical have good potential applicability in clinical practice.Activeconstituents cordycepin and Cordyceps polysaccharide have good economic worth and social benefit in the high efficiency separation Cordyceps militaris (L.) Link..The extraction of Cordyceps polysaccharide, cordycepin adopts purifying process researchs such as ion exchange resin, macroporous adsorbent resin, charcoal absorption to have more report, these methods all to exist the product that obtains single, wasting of resources phenomenon is serious, the energy consumption height with an organic solvent, pollutes environment.Membrane separation technique because have separations concurrently, concentrate, purifying and purified function, efficient, energy-saving and environmental protection are arranged again, molecular level filters and filtration procedure is simple, be easy to feature such as control, obtains application more and more widely in middle pharmaceutically active ingredient separation.Cordycepin and Cordyceps polysaccharide adopt membrane separation technique effectively the two to be separated because molecular weight varies in size.Present method utilizes membrane separation technique to separate Cordyceps militaris (L.) Link. polysaccharide and cordycepin simultaneously, utilizes ultra-filtration membrane effectively catching Cordyceps polysaccharide, has both realized concentrating fast of Cordyceps polysaccharide, is separated with small-molecule substance and most of pigment again, reaches the purpose of separation and purification.Quick, efficient, economic separation method when the present invention has set up a Cordyceps polysaccharide and cordycepin.
Summary of the invention
The invention provides the method that a kind of membrane separation technique prepares Cordyceps polysaccharide and cordycepin continuously.This method adopts the microfiltration membrane technology that Cordyccps-militaris-(L.)-link. Sporophore or Cordyceps militaris (L.) Link. solid medium hot water extraction liquid are carried out pre-separation, remove impurity such as suspended particle, macro-molecular protein in the extracting solution, reduced Protein content in the Cordyceps polysaccharide, reduced the isolating film of ultra-filtration membrane and polluted.This method adopts ultra-filtration membrane to separate the preparation Cordyceps polysaccharide, can be as required, the ultra-filtration membrane of selection differing molecular interception (MWCO) prepares the Cordyceps polysaccharide of different molecular weight magnitude range, this method is simple to operate, does not need to use any organic solvent, does not have phase transformation in the sepn process, energy consumption is low, trapped fluid can obtain Cordyceps polysaccharide, sees through liquid and can further separate the preparation cordycepin, and effective constituent maximizes the use.This method adopts ultra-filtration membrane to separate the preparation cordycepin, what obtain behind the employing membrane sepn Cordyceps polysaccharide is the feedstock production cordycepin through liquid, this method has reduced the content of impurity (macromole such as polysaccharide, protein, pigment) in the stock liquid, the cordycepin purity of preparation is higher, has reduced the subsequent step of cordycepin separation and purification.
The objective of the invention is to be achieved through the following technical solutions.
It comprises the steps: that (1) extract: take by weighing the solid medium after dry Cordyccps-militaris-(L.)-link. Sporophore or the Cordyceps militaris (L.) Link. cultivation and collection sporophore, pack into and make by oneself in the cloth bag, with the distillation water as solvent, solid-to-liquid ratio 1:10 ~ 1:20, boiling water bath extracts 2h in extractor, filter cloth filter extracting solution, residue repeats to extract once, united extraction liquid, high speed centrifugation, it is standby to get supernatant liquor.(2) microfiltration membrane is separated: supernatant liquor is packed in the micro-filtration liquid storing barrel, selects the ceramic membrane of 0.05 ~ 0.2 μ m to carry out micro-filtration, 10 ℃ ~ 50 ℃ of feed temperatures, and working pressure 0.1 ~ 0.3MPa, pH5 ~ 9, micro-filtration time 1 ~ 3h collects that to see through liquid stand-by.(3) ultra-filtration membrane separates Cordyceps polysaccharide: the liquid that sees through in the step (2) is packed in the ultra-filtration equipment liquid storing barrel, selecting MWCO according to the size of required polysaccharide molecular weight is the organic membrane ultrafiltration of 1000Da ~ 300000Da, the selection ultrafiltration pressure is 0.1 ~ 0.3MPa, pH5 ~ 9, the ultrafiltration time is 0.5 ~ 3h, separate Cordyceps polysaccharide, trapped fluid is used for the separation of Cordyceps polysaccharide, sees through the separation that liquid is used for cordycepin.(4) ultra-filtration membrane separates cordycepin: will separate packing in the ultrafiltration instrument liquid storing barrel through liquid of Cordyceps polysaccharide, selecting MWCO is organic ultra-filtration membrane of 500Da, and the selection ultrafiltration pressure is 0.1 ~ 0.3MPa, and the ultrafiltration time is that 0.5 ~ 3h carries out ultrafiltration, discard trapped fluid, collect and see through liquid.(5) concentrate the alcohol precipitation Cordyceps polysaccharide: the trapped fluid in the step (3) is evaporated to 1/4 ~ 1/6 of original volume, and the cooling back adds 95% ethanol of 4 times of volumes of concentrated solution, and places 2 ~ 10h, treat that precipitation fully after, centrifugal or filter the Cordyceps polysaccharide precipitation.(6) the concentrating of cordycepin, crystallization: the liquid that sees through that step (4) is obtained carries out concentrating under reduced pressure and gets medicinal extract, adds the ethanol heating for dissolving, carries out recrystallization, obtains cordycepin crystal.(7) drying: Cordyceps polysaccharide precipitation and cordycepin crystallization are carried out vacuum lyophilization, obtain Cordyceps polysaccharide product and cordycepin product respectively.
Separate Cordyceps polysaccharide and the cordycepin that obtains through method of the present invention, cordycepin content can reach more than 20%, and Cordyceps polysaccharide content reaches more than 35%, and two kinds of product recovery rates all reach more than 90%.
In a word, advantage of the present invention is: can separate preparation cordycepin and Cordyceps polysaccharide continuously.Adopt microfiltration membrane that extracting solution is carried out pre-treatment; effectively separated macromolecular substance and the suspended particle in the extracting solution; the film that has reduced in the ultra-filtration membrane sepn process pollutes; the present invention adopts membrane separation technique to separate Cordyceps polysaccharide and cordycepin continuously; can play inspissated to Cordyceps polysaccharide solution; reduce the energy that is consumed in the follow-up concentration process of Cordyceps polysaccharide extracting solution, do not had phase transformation in the whole membrane sepn process, effectively protected the biological activity of Cordyceps polysaccharide and cordycepin.The present invention not with an organic solvent, for green sub from.The present invention separates Cordyceps polysaccharide and cordycepin continuously, has effectively utilized the Cordyceps militaris (L.) Link. resource, has saved resource and cost.
Embodiment
Embodiment 1
Take by weighing dry Cordyccps-militaris-(L.)-link. Sporophore 5kg, in the self-control cloth bag of packing into, place the extractor of 200L, add 100L distilled water, 100 ℃ are extracted 2h in extractor, filter cloth filter extracting solution, residue repeats to extract once, united extraction liquid, high speed centrifugation, it is standby to get supernatant liquor.The supernatant liquor ceramic membrane of packing into is separated in the liquid storing barrel, select the ceramic membrane of 0.1 μ m to carry out micro-filtration, 30 ℃ of feed temperatures, working pressure 0.2MPa, pH7, micro-filtration time 2h must see through liquid.To pack in the ultra-filtration equipment liquid storing barrel through liquid, selecting MWCO is the organic membrane ultrafiltration of 10KDa, and ultrafiltration pressure is 0.2MPa, pH7, and ultrafiltration time 2h, trapped fluid is used for the separation of Cordyceps polysaccharide, sees through the separation that liquid is used for cordycepin.The liquid that sees through that separates Cordyceps polysaccharide is packed in the ultra-filtration equipment liquid storing barrel, and selecting MWCO is the organic membrane of 500Da, and selections ultrafiltration pressure is 0.3MPa, and ultrafiltration 2h discards trapped fluid, collects through liquid, is used for the separation of cordycepin.Add 95% ethanol of 4 times of volumes in the trapped fluid that obtains toward the organic membrane ultrafiltration of 10KDa, place 8h, treat that precipitation fully after, centrifugal must the precipitation carried out vacuum lyophilization with precipitation, Cordyceps polysaccharide, polysaccharide content is about 35%.With molecular weight cut-off is that the liquid that sees through that the film of 500Da obtains is evaporated to medicinal extract, adds an amount of ethanol heating for dissolving, and carries out recrystallization, and crystal carries out vacuum lyophilization and gets cordycepin, and cordycepin purity is about 22%.
Embodiment 2
Take by weighing dry Cordyceps militaris (L.) Link. solid medium 10kg, in the self-control cloth bag of packing into after the pulverizing, place the extractor of 200L, add 150L distilled water, 100 ℃ are extracted 2h in extractor, filter cloth filter extracting solution, residue repeats to extract once, united extraction liquid, high speed centrifugation, it is standby to get supernatant liquor.Supernatant liquor is packed in the ceramic membrane tripping device liquid storing barrel, select the ceramic membrane of 0.1 μ m to carry out micro-filtration, 30 ℃ of feed temperatures, working pressure 0.2MPa, pH7, micro-filtration time 2h must see through liquid.To pack in the ultra-filtration equipment liquid storing barrel through liquid, selecting MWCO is the organic membrane ultrafiltration of 10KDa, and ultrafiltration pressure is 0.2MPa, pH7, and ultrafiltration time 2h, trapped fluid is used for the separation of Cordyceps polysaccharide, sees through the separation that liquid is used for cordycepin.The liquid that sees through that separates Cordyceps polysaccharide is packed in the ultra-filtration equipment liquid storing barrel, select the organic membrane of 500Da, selections ultrafiltration pressure is 0.3MPa, and ultrafiltration 2h discards trapped fluid, collects through liquid, is used for the separation of cordycepin.Add 95% ethanol of 4 times of volumes in the trapped fluid that obtains toward the organic membrane ultrafiltration of 10KDa, place 8h, treat that precipitation fully after, centrifugal must the precipitation carried out vacuum lyophilization with precipitation, Cordyceps polysaccharide, polysaccharide content is about 36%.With molecular weight cut-off is that the trapped fluid that the membrane ultrafiltration of 500Da is collected is evaporated to medicinal extract, and with dissolve with ethanol and carry out recrystallization, crystal carries out vacuum lyophilization and gets cordycepin, and cordycepin content is 21%.
Claims (6)
1. the membrane separation technique method for preparing cordycepin and Cordyceps polysaccharide continuously is characterized in that: adopt hot water lixiviate Cordyccps-militaris-(L.)-link. Sporophore or solid medium, obtain extracting solution, after being adopted as filter membrane separate out suspended impurity and macro-molecular protein, the size that obtains polysaccharide molecular weight as required selects the ultra-filtration membrane of certain molecular retention amount to carry out ultra-filtration and separation, and trapped fluid carries out alcohol precipitation, the dry polysaccharide that obtains the certain molecular weight scope; Seeing through liquid, to select the molecular retention amount again for use be that the ultra-filtration membrane of 500D carries out ultrafiltration, must see through liquid, sees through liquid and concentrate, alcohol crystal and recrystallization, dry acquisition cordycepin.
2. a membrane separation technique prepares the method for cordycepin and Cordyceps polysaccharide continuously, it is characterized in that, may further comprise the steps: (1) ultra-filtration membrane separates Cordyceps polysaccharide; (2) ultra-filtration membrane separates cordycepin.
3. claim 2 membrane separation technique prepares the method for cordycepin and Cordyceps polysaccharide continuously, it is characterized in that, described membrane sepn Cordyceps polysaccharide is: the organic membrane of selecting 1000Da ~ 300KDa, ultrafiltration pressure is 0.1 ~ 0.3MPa, pH 5 ~ 9, the ultrafiltration time is 0.5 ~ 3h, obtains trapped fluid and prepares Cordyceps polysaccharide; Described membrane sepn cordycepin is: is raw material with what separate Cordyceps polysaccharide through liquid, selects the ultra-filtration membrane of 500Da to carry out ultrafiltration, and ultrafiltration pressure is 0.1 ~ 0.3MPa, and pH 5 ~ 9, and the ultrafiltration time is 0.5 ~ 3h, and the ultrafiltration that obtains 500Da sees through liquid and prepares the cordycepin product.
4. the method for claim 2 comprises the steps: that also (3) concentrate the alcohol precipitation polysaccharide.
5. the method for right 4 is characterized in that, the polysaccharide trapped fluid is evaporated to 1/4 ~ 1/6 of original volume, and cooling back adds the ethanol of 4 times of volumes 95% of concentrated solution, and places 2 ~ 12h, treat that precipitation fully after, centrifugal or filter polysaccharide precipitation.
6. a membrane separation technique prepares the method for cordycepin and Cordyceps polysaccharide continuously, and its feature also is, this method is equally applicable to separating of cordycepin in Cordyceps militaris (L.) Link. fermented liquid that liquid fermentation and culture obtains and the mycelium and Cordyceps polysaccharide.
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| CN103772523A (en) * | 2014-01-14 | 2014-05-07 | 武汉新国峰科技开发有限公司 | New technology for preparing poria polysaccharide extract through membrane separation and purification technology |
| CN104352532A (en) * | 2014-10-15 | 2015-02-18 | 江西国药有限责任公司 | Purification process for fermentative cordycepic fungal powder |
| CN104447925A (en) * | 2014-11-26 | 2015-03-25 | 湖北省农业科学院农产品加工与核农技术研究所 | Method for extracting cordycepin from cordyceps militaris rice medium under ultrahigh pressure |
| CN104650255A (en) * | 2015-03-20 | 2015-05-27 | 海安县泓寿生物技术有限责任公司 | Method for separating and purifying cordyceps polysaccharide contained in cordyceps militaris solid culture medium |
| CN105294796A (en) * | 2015-11-11 | 2016-02-03 | 湖南炎帝生物工程有限公司 | Method for extracting cordycepin and cordyceps from cordyceps militaris solid medium residues |
| WO2016082327A1 (en) * | 2014-11-26 | 2016-06-02 | 南京中科药业有限公司 | Processing method of cordyceps sinensis mycelium |
| CN105801718A (en) * | 2016-04-21 | 2016-07-27 | 浙江工业大学 | Extraction method of cordyceps sinensis flower polysaccharide |
| CN106727733A (en) * | 2016-12-27 | 2017-05-31 | 广东东阳光药业有限公司 | A kind of combination medicine of fresh Cordyceps sinensis anti-tumor chemotherapeutic and its application |
| CN106810618A (en) * | 2017-01-06 | 2017-06-09 | 徐州鸿宇农业科技有限公司 | A kind of extraction from Chinese caterpillar fungus culture medium and the method for continuous polysaccharide enrichment |
| CN111363057A (en) * | 2020-03-23 | 2020-07-03 | 浙江工业大学 | Hierarchical preparation method of ganoderan based on multistage membrane separation technology |
| CN114456278A (en) * | 2022-03-14 | 2022-05-10 | 泸州品创科技有限公司 | Method for continuously extracting cordyceps militaris intracellular polysaccharide |
| CN114891050A (en) * | 2022-04-25 | 2022-08-12 | 南京高新工大生物技术研究院有限公司 | Method for separating cordycepin from fermentation liquor or extracting solution |
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| CN104447925B (en) * | 2014-11-26 | 2016-10-26 | 湖北省农业科学院农产品加工与核农技术研究所 | A kind of method of cordycepin in hyperpressure extraction Cordyceps militaris rice medium |
| CN104447925A (en) * | 2014-11-26 | 2015-03-25 | 湖北省农业科学院农产品加工与核农技术研究所 | Method for extracting cordycepin from cordyceps militaris rice medium under ultrahigh pressure |
| WO2016082327A1 (en) * | 2014-11-26 | 2016-06-02 | 南京中科药业有限公司 | Processing method of cordyceps sinensis mycelium |
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| CN105801718A (en) * | 2016-04-21 | 2016-07-27 | 浙江工业大学 | Extraction method of cordyceps sinensis flower polysaccharide |
| CN106727733A (en) * | 2016-12-27 | 2017-05-31 | 广东东阳光药业有限公司 | A kind of combination medicine of fresh Cordyceps sinensis anti-tumor chemotherapeutic and its application |
| CN106727733B (en) * | 2016-12-27 | 2020-03-24 | 广东东阳光药业有限公司 | Combined medicine for fresh cordyceps sinensis antitumor chemotherapy and application thereof |
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| CN111363057A (en) * | 2020-03-23 | 2020-07-03 | 浙江工业大学 | Hierarchical preparation method of ganoderan based on multistage membrane separation technology |
| CN114456278A (en) * | 2022-03-14 | 2022-05-10 | 泸州品创科技有限公司 | Method for continuously extracting cordyceps militaris intracellular polysaccharide |
| CN114891050A (en) * | 2022-04-25 | 2022-08-12 | 南京高新工大生物技术研究院有限公司 | Method for separating cordycepin from fermentation liquor or extracting solution |
| CN114891050B (en) * | 2022-04-25 | 2023-12-15 | 南京高新工大生物技术研究院有限公司 | Method for separating cordycepin from fermentation liquid or extracting liquid |
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Application publication date: 20130724 |