CN101307112B - Process for abstracting mycelium polysaccharide by cordyceps fermentation - Google Patents
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Abstract
The invention discloses a method for extracting and purifying polysaccharide from a fermented mycelium of Cordyceps sinensis. The method comprises the following steps of: using the fermented mycelium of Cordyceps sinensis as a raw material to produce crude polysaccharide of the Cordyceps sinensis via degreasing, aqueous extraction, concentration and alcohol precipitation; then, redissolving the crude polysaccharide into distilled water, deproteinizing the crude polysaccharide by Sevag, decoloring the deproteinized crude polysaccharide by H2O2, dialyzing the decolored crude polysaccharide, andprecipitating the dialyzed crude polysaccharide by alcohol to produce primarily purified polysaccharide; finally, purifying the primarily purified polysaccharide by ion-exchange chromatography, collecting different produced compositions stepwisely, dialyzing the compositions, precipitating the dialyzed compositions by alcohol, drying the precipitated compositions to produce the polysaccharide of the Cordyceps sinensis after the precipitated compositions are orderly washed by absolute ethyl alcohol, acetone, aether and so on. In the produced polysaccharide of the Cordyceps sinensis, MPS2 is identified to be homogeneous polysaccharide by ultraviolet absorption spectrum and gel chromatography, with high content of total sugar. The method has the advantages of simple and convenient extractionsteps and low cost.
Description
Technical field
The present invention relates to the method that a kind of Base in Mycelia of Cordyceps polysaccharide extracts.
Background technology
Polysaccharide is the macromole active compound that is polymerized by a plurality of monose or derivatives thereofs, confirms to have biologic activity such as antitumor, anti-inflammatory, anticoagulation, antiviral, radioprotective, hypoglycemic, reducing blood-fat through pharmacological research and clinical application.Polysaccharide becomes one of important directions of current new drug development just gradually, and lentinan, polyporusum bellatus, lycium barbarum polysaccharide etc. have been applied to clinical both at home and abroad at present.
Cordyceps sinensis [Cordyceps sinensis (Berk.) Sacc.] is that Cordyceps fungus colonizes in the especially last entomogenous fungi complex body that forms of Chinese caterpillar fungus bat [Thitarodes armoricanus (Oberth ü r)] of lepidopteran (Lepidoptera) Hepialidae (Hepialidae) larva, is a kind of rare Chinese medicine and the high tonic of China's special product.Cordyceps sinensis contains abundant physiologically active substance, has unique pharmaceutical use and pharmacological action widely, is a mcroorganism treasure-house that is worth excavation.
As the distinctive fungus in Qinghai-Tibet Platean, Cordyceps sinensis distribution region limitation, natural output is very limited, and has been classified as second class protection species [" national key protected wild plants register (first) ", 1999] by national correlation department.At the present situation of natural cordyceps resource scarcity, the whole nation successively has a plurality of R﹠D institutions to carry out the submerged fermentation research of Cordyceps sinensis, and what have has entered suitability for industrialized production.
Yet relevant in the market Cordyceps sinensis product mostly is the mycelia raw product of disposable direct utilization, is unfavorable for integrating with the world market, and is unfavorable for participating in the cut-throat competition of world market.Therefore the research and development of higher level product are very urgent even for the secondary of Cordyceps sinensis.Polyose will be an important breakthrough mouth of Cordyceps sinensis development research as one of the abundantest most important monoid in the contained physiologically active substance of Cordyceps sinensis, have a good application prospect.
Summary of the invention
The extracting method that the purpose of this invention is to provide a kind of Base in Mycelia of Cordyceps polysaccharide.
The extracting method of Base in Mycelia of Cordyceps polysaccharide provided by the present invention may further comprise the steps:
(1) particle diameter is carried out skimming treatment less than the Cordyceps mycelium of 830 μ m (particle diameter that is equivalent to the Base in Mycelia of Cordyceps that can obtain by 20 mesh sieves), obtain the Cordyceps mycelium of degreasing;
(2) water extracts the degreasing Cordyceps mycelium that step (1) obtains, and collects extracting solution;
(3) extracting solution that step (2) is obtained precipitates with dehydrated alcohol, then described precipitation is used ether, acetone and absolute ethanol washing successively, obtains the Cordyceps mycelium Crude polysaccharides.
Described method also comprises the step of following purifying:
(a) deproteinated: adopt the Sevag method that the Cordyceps mycelium Crude polysaccharides that step (3) prepares is carried out deproteinated, obtain Deproteinated Cordyceps mycelium polysaccharide;
(b) decolouring: the Deproteinated Cordyceps mycelium polysaccharide that step (a) is obtained decolours with hydrogen peroxide, the Cordyceps mycelium polysaccharide that obtains decolouring;
(c) ion exchange chromatography: employing DEAE-52 ion exchange column separates the Cordyceps mycelium polysaccharide of the decolouring that step (b) obtains, be that 6-8,10mM Tris-HCl damping fluid carry out wash-out with the pH value earlier, be that the aqueous solution that 6-8 contains 0.1M-0.5M NaCl and 10mM Tris-HCl carries out wash-out with the pH value again, collect sugared peak, obtain the Cordyceps mycelium polysaccharide of purifying.
Wherein, the used reagent of the described skimming treatment of step (1) is that volumn concentration is the ethanol of 90-100%, and degreasing time is 12-24 hour.
Step is extracted as refluxing extraction described in (2).The temperature of described extraction is 70-90 ℃; In the described extraction, extract once at least, each time of extracting can be 1-3 hour.
The Deproteinated concrete grammar of step (a) is the Cordyceps mycelium Crude polysaccharides water dissolution that step (3) is obtained, form the aqueous solution of described Cordyceps mycelium polysaccharide, the described aqueous solution and Sevag reagent were mixed in 5: 1 by volume, extract, collect supernatant liquid, obtain Deproteinated Cordyceps Polysaccharide.
In the described deproteinated, take off albumen at least one time.
The concrete grammar of step (b) decolouring is the Deproteinated Cordyceps mycelium polysaccharide wiring solution-forming that described step (a) is obtained, the pH value of described solution is transferred to 7-8, be under 45-60 ℃ the condition in temperature, adding volume in described solution is the superoxol of 30% (volumn concentration) of described liquor capacity 20%, decolour the Cordyceps mycelium polysaccharide soln that obtains decolouring; The Cordyceps mycelium polysaccharide soln of described decolouring is precipitated with dehydrated alcohol, described precipitation is used ether, acetone and absolute ethanol washing successively, the Cordyceps mycelium polysaccharide that obtains decolouring.
The invention has the advantages that: by degreasing, water is carried, is concentrated, alcohol precipitation, deproteinated decolouring and chromatography obtain the Cordyceps sinensis purified polysaccharide, through ultra-violet absorption spectrum and gel chromatography identification of M PS2 is homogeneous polysaccharide, total sugar content reaches 98.26%, step is easy, with low cost, do not need specific installation.
Description of drawings
Fig. 1 is the extraction purifying schema of Cordyceps mycelium polysaccharide of the present invention.
Fig. 2 is the DEAE-52 column chromatography elution curve of the Cordyceps mycelium polysaccharide of embodiment 1.
Fig. 3 is the ultra-violet absorption spectrum of the Cordyceps mycelium polysaccharide of embodiment 1, (A) is the ultra-violet absorption spectrum of MPS1, (B) is the ultra-violet absorption spectrum of MPS2.
Fig. 4 is the gel chromatography wash-out collection of illustrative plates of the Cordyceps mycelium polysaccharide of embodiment 1, (A) is the wash-out collection of illustrative plates of MPS1, (B) is the wash-out collection of illustrative plates of MPS2.
Embodiment
The present invention extracts the method for Base in Mycelia of Cordyceps polysaccharide, may further comprise the steps:
The Cordyceps mycelium that the used starting material of the present invention obtain for fermentation.The used substratum that ferments is semisynthetic medium [Yao Yijian, a kind of semi-synthetic culture medium of cordyceps sineusis of Dong Caihong (2004). patent No. ZL200410090875.1] or synthetic medium [Yao Yijian, Dong Caihong, Wang Bo, a kind of Cordyceps sinensis compound culture method of Xie Xueqin (2005) and special culture media thereof. patent No. ZL200510137457.8.].Used cultural method is seen patent: Yao Yijian, Dong Caihong, Wang Bo, a kind of Cordyceps sinensis compound culture method of Xie Xueqin (2005) and special culture media thereof. patent No. ZL200510137457.8.
(1) be the ethanol of 90-100% with particle diameter less than adding volumn concentration in the Base in Mycelia of Cordyceps of 830 μ m, room temperature degreasing 12-24 hour obtains the Cordyceps mycelium of degreasing; And with the air-dry ethanol of removing of the Cordyceps mycelium room temperature of described degreasing;
(2) in the mycelium that step (1) obtains, add the water of 10-20 times of volume, carry out refluxing extraction in 70-90 ℃, extract finish after, suction filtration obtains filter residue and filtrate; The distilled water that adds 10-20 times of volume in described filter residue again repeats the step of described 1-3 refluxing extraction and suction filtration, collects filtrate;
(3) merge described filtrate, filtrate is concentrated into 1/3rd of original volume, get concentrated solution; Add the long-pending dehydrated alcohol of triploid in described concentrated solution, 4 ℃ of placements are spent the night, centrifugal 30 minutes of 8000g, and collecting precipitation is used ether, acetone and absolute ethanol washing successively with described precipitation, gets the Cordyceps sinensis Crude polysaccharides.
Described method also comprises the step of following purifying:
(a) deproteinated: adopt Sevag method deproteinated, with step (3) Cordyceps mycelium Crude polysaccharides water dissolution, (room temperature is higher than 25 ℃ to form the aqueous solution of described Cordyceps Polysaccharide, need to add toluene in the aqueous solution of described Cordyceps Polysaccharide), the described aqueous solution and Sevag reagent were mixed in 5: 1 by volume, pack in the separating funnel, thermal agitation, leave standstill 30-60min, supernatant liquor as stated above repeatedly deproteinated until two alternate do not have tangible egg white layer till, obtain Deproteinated Cordyceps Polysaccharide;
(b) decolouring: the Deproteinated Cordyceps Polysaccharide wiring solution-forming that step (a) is obtained, the pH value of described solution is transferred to 7-8, at 45-60 ℃ of superoxol that in described solution, adds 30% (volumn concentration) by 20% volume ratio, oxidative decoloration to Cordyceps Polysaccharide solution by brown become light yellow till; To distill water dialysis 24h, every 3h changes water once with the Cordyceps Polysaccharide solution after the decolouring; With the solution concentration after the dialysis, add 3 times of volume dehydrated alcohol precipitation polysaccharide, 4 ℃ of standing over night, centrifugal, precipitate respectively with ether the Cordyceps mycelium polysaccharide that acetone and absolute ethanol washing drying must be decoloured;
(c) ion exchange chromatography: the Cordyceps Polysaccharide of the described decolouring of step (b) is carried out anion exchange chromatography, earlier with 10mM Tris-HCl buffer solution elution, be 7.4 the aqueous solution that contains 0.1M NaCl and 10mM Tris-HCl more successively with the pH value, collect with Fraction Collector, sulfuric acid-anthrone method detects, and obtains two sugared peaks, collects respectively, dialysis, concentrated after drying gets Cordyceps mycelium polysaccharide MPS1 and two components of MPS2.
The extraction of embodiment 1, Base in Mycelia of Cordyceps polysaccharide, purifying
1) preparation of Base in Mycelia of Cordyceps
Will be from separation and purification on the natural cordyceps, and be that the bacterial strain of real Cordyceps strain inserts semi-synthetic culture medium of cordyceps sineusis through molecular biology identification, ferment according to a conventional method, collect mycelium.This semi-synthetic culture medium of cordyceps sineusis is prepared as follows: get sucrose 50.0 grams, and peptone 10.0 grams, yeast extract 3.0 grams, potassium primary phosphate 1.0 grams, sal epsom 0.5 gram is settled to 1000 milliliters with distilled water.
2) extraction of Cordyceps mycelium Crude polysaccharides
The extraction purifying schema of Cordyceps mycelium polysaccharide, as shown in Figure 1.
The Cordyceps mycelium that obtains with step 1) is a raw material, and extracting method comprises the steps:
Cordyceps sinensis mycelium powder is broken into particle diameter less than 830 μ m mycelium (particle diameter that is equivalent to the Cordyceps mycelium that can obtain by 20 mesh sieves).In the Cordyceps mycelium that 100g pulverizes, add 500ml dehydrated alcohol (volumn concentration is 100%), room temperature degreasing 12 hours, and the mycelium room temperature after the degreasing is air-dry.Accurately take by weighing mycelium and add the distilled water of 20 times of volumes, in 90 ℃ of water-bath refluxing extraction 2 hours, obtain filtrate and filter residue behind the suction filtration, the distilled water that adds 20 times of volumes in filter residue again repeats aforesaid operations 3 times.Merging filtrate concentrates, and 55 ℃ are concentrated into 1/3rd of original volume.In the concentrated solution that obtains, add the long-pending dehydrated alcohol of triploid while stirring, put 4 ℃ and spend the night, centrifugal (8000g) 30 minutes, precipitation with ether, acetone and absolute ethanol washing and dry, promptly gets 16.1g Cordyceps sinensis Crude polysaccharides respectively.
3) purifying of Cordyceps sinensis Crude polysaccharides
Get 10g step 2) the Cordyceps sinensis Crude polysaccharides add 500ml distilled water, the magnetic agitation dissolving is spent the night (when room temperature is higher than 25 ℃, it is anticorrosion to add several toluene), mixed in 5: 1 by volume with Sevag reagent (chloroform is the mixed solution that is made at 4: 1 with propyl carbinol by volume), pack in the separating funnel, thermal agitation leaves standstill 30min, supernatant liquor as stated above repeatedly deproteinated until two alternate do not have tangible egg white layer till, obtain Deproteinated Cordyceps Polysaccharide solution.
Get Deproteinated Cordyceps Polysaccharide solution the pH value is adjusted to 8, in the time of 50 ℃, add the hydrogen peroxide (H of 30% (volumn concentration) by 20% volume ratio
2O
2) solution, oxidative decoloration to sugar soln by brown become light yellow till.To distill water dialysis 24h, every 3h changes water once with the solution after the decolouring.Solution concentration after the dialysis to 1/3rd of original volume, is added 3 times of volume dehydrated alcohols precipitation polysaccharide, 4 ℃ of standing over night, centrifugal 15 minutes of 12000g, collecting precipitation will precipitate respectively then with ether the polysaccharide that acetone and absolute ethanol washing drying must be decoloured.
Getting the decolouring polysaccharide dissolves with 10ml 10mM Tris-HCl damping fluid (pH value 7.4), equilibrate overnight, the presumable microprecipitation of centrifugal removal, the supernatant liquor of 1% column volume is added to equilibrated DEAE-52 anion-exchange chromatography post, and (column length of ion exchange column is 30cm, the internal diameter of post is 2.6cm) on, elder generation is with 10mM Tris-HCl (the pH value 7.4) buffer solution elution of 2 times of column volumes, use 0.1M NaCl-10mMTris-HCl damping fluid (the pH value 7.4) wash-out of 2 times of column volumes again, flow velocity is 16ml/h, collect with Fraction Collector, every pipe is collected 4 milliliters, sulfuric acid-anthrone method detects, and obtains two sugared peaks, collects the component of 8-21 pipe and the component of 61-73 pipe respectively, dialyse with distilled water water, concentrated after drying gets 2.8g MPSl and two components of 1.5g MPS2 (DEAE-52 column chromatography elution curve as shown in Figure 2).
Identify purity of polysaccharide with ultra-violet absorption spectrum and Sephadex G-100 gel chromatography at last.
The concrete grammar of gel chromatography:
Take by weighing 12g Sephadex G-100 behind the abundant swelling of distilled water, with 0.1M NaCl solution washing for several times, the dress post (1.6 * 80cm), with standby behind the 0.1M NaCl solution equilibria.Take by weighing intracellular polyse MPS1 and each 20mg of MPS2 that ion exchange chromatography obtains, carry out gel chromatography according to following method respectively: with 2ml 0.1MNaCl solution dissolving polysaccharide, upper prop, with 0.1M NaCl eluant solution, flow velocity is 9ml/h, and Fraction Collector is collected, every 40min one pipe.Sulfuric acid-anthrone method detects.
The result shows that the MPS1 component is a mixing polysaccharide, and the MPS2 component is a homogeneous polysaccharide.The ultraviolet absorpting spectrum of MPS1 and MPS2 and gel chromatography wash-out collection of illustrative plates are respectively as shown in Figure 3, Figure 4.
Measuring the MPS1 sugar degree with sulfuric acid-anthrone method (Zhang Weijie chief editor complex polysaccharide Biochemical Research technology, Shanghai science tech publishing house, 1987) is 94.30%, and the MPS2 total sugar content is 98.26%.
Claims (4)
1. the extracting method of a Base in Mycelia of Cordyceps polysaccharide may further comprise the steps:
(1) particle diameter is carried out skimming treatment less than the Cordyceps mycelium of 830 μ m, obtain the Cordyceps mycelium of degreasing; Used reagent is that volumn concentration is the ethanol of 90-100% in the described skimming treatment, and degreasing time is 12-24 hour;
(2) water extracts the degreasing Cordyceps mycelium that step (1) obtains, and collects extracting solution; The described refluxing extraction that is extracted as; The temperature of described extraction is 70-90 ℃; In the described extraction, extract once at least, each time of extracting is 1-3 hour;
(3) extracting solution that step (2) is obtained precipitates with dehydrated alcohol, and described precipitation is used ether, acetone and absolute ethanol washing successively, obtains the Cordyceps mycelium polysaccharide;
Described method also comprises the step of following purifying:
(a) deproteinated: adopt the Sevag method that the prepared Cordyceps mycelium polysaccharide of step (3) is carried out deproteinated, obtain Deproteinated Cordyceps mycelium polysaccharide;
(b) decolouring: the described Deproteinated Cordyceps mycelium polysaccharide of step (a) is decoloured the Cordyceps mycelium polysaccharide that obtains decolouring with hydrogen peroxide;
(c) ion exchange chromatography: adopt the DEAE-52 ion exchange column that the Cordyceps mycelium polysaccharide of the described decolouring of step (b) is separated, be that 6-8,10mMTris-HCl damping fluid carry out wash-out with the pH value earlier, be that the aqueous solution that 6-8 contains 0.1M-0.5M NaCl and 10mMTris-HCl carries out wash-out with the pH value again, collect sugared peak, obtain the Cordyceps mycelium polysaccharide of purifying.
2. method according to claim 1, it is characterized in that: the Deproteinated method of step (a) is the Cordyceps mycelium polysaccharide water dissolution that step (3) is prepared, form the aqueous solution of described Cordyceps mycelium polysaccharide, the described aqueous solution and Sevag reagent were mixed in 5: 1 by volume, extract, collect supernatant liquid and obtain Deproteinated Cordyceps Polysaccharide.
3. method according to claim 1 and 2 is characterized in that: in the described deproteinated, take off albumen at least one time.
4. method according to claim 1, it is characterized in that: the method for step (b) decolouring is the Deproteinated Cordyceps mycelium polysaccharide wiring solution-forming that described step (a) is obtained, the pH value of described solution is transferred to 7-8, be under 45-60 ℃ the condition in temperature, the adding volume is that the volumn concentration of described liquor capacity 20% is 30% superoxol in described solution, decolour the Cordyceps mycelium polysaccharide soln that obtains decolouring; The Cordyceps mycelium polysaccharide soln of described decolouring is precipitated with dehydrated alcohol, described precipitation is used ether, acetone and absolute ethanol washing successively, the Base in Mycelia of Cordyceps polysaccharide that obtains decolouring.
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| CN101760038B (en) * | 2008-12-24 | 2012-12-19 | 中国科学院微生物研究所 | Method for extracting melanin from cordyceps militeris fungus fermentation broth |
| CN101709092B (en) * | 2009-12-09 | 2011-10-26 | 福建农林大学 | Method for extracting rubus reflexus polysaccharide |
| CN101928353B (en) * | 2010-08-13 | 2012-07-18 | 内蒙古昆明卷烟有限责任公司 | Extraction of cordyceps polysaccharide and application of cordyceps polysaccharide in cigarettes |
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| CN103789212B (en) * | 2012-11-02 | 2015-10-28 | 中国科学院微生物研究所 | One plant height produces spore Cordyceps strain |
| CN103130907B (en) * | 2013-02-16 | 2014-12-03 | 浙江省林业科学研究院 | Chinese cordyceps sinensis protein polysaccharide and preparation and application thereof |
| CN103435712A (en) * | 2013-08-27 | 2013-12-11 | 王辉 | Extraction method of cordyceps sinensis intracellular polysaccharide |
| CN103431368A (en) * | 2013-09-05 | 2013-12-11 | 东方中科生命科学有限责任公司 | Preparation method and comprehensive utilization of submerged fermentation product of cordyceps sinensis liquid |
| CN103751224B (en) * | 2014-01-27 | 2015-12-02 | 正源堂（天津）生物科技有限公司 | Method and the application thereof of antitumor component is extracted from Cordyceps militaris (L.) Link. |
| CN103784481B (en) * | 2014-01-27 | 2016-06-01 | 正源堂（天津）生物科技有限公司 | A kind of method and application thereof extracting antitumor component from Cordyceps militaris (L.) Link. |
| CN103864945B (en) * | 2014-02-21 | 2016-01-20 | 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 strengthening polysaccharide in extraction Cordyceps militaris rice medium |
| CN104403012A (en) * | 2014-11-10 | 2015-03-11 | 苏州蔻美新材料有限公司 | Extraction technology of cordyceps militaris polysaccharide and polypeptide |
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| CN110713550B (en) * | 2018-07-12 | 2022-03-15 | 浙江省亚热带作物研究所 | Method for preparing refined polysaccharide with antibacterial activity by using cordyceps culture |
| CN110256591B (en) * | 2019-06-21 | 2022-02-15 | 东莞市东阳光冬虫夏草研发有限公司 | Cordyceps sinensis polysaccharide extract and preparation method and application thereof |
| CN110396138B (en) | 2019-08-29 | 2021-02-19 | 华南理工大学 | Method for decoloring and deproteinizing brown algae polysaccharide |
| CN111040044B (en) * | 2019-12-31 | 2022-03-25 | 华南师范大学 | Cordyceps militaris intracellular polysaccharide, preparation method and application thereof in regulating intestinal flora |
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