CN101440389A - Method for increasing cordycepin content in Cordceps militaris solid culture medium - Google Patents
Method for increasing cordycepin content in Cordceps militaris solid culture medium Download PDFInfo
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- CN101440389A CN101440389A CNA200810068949XA CN200810068949A CN101440389A CN 101440389 A CN101440389 A CN 101440389A CN A200810068949X A CNA200810068949X A CN A200810068949XA CN 200810068949 A CN200810068949 A CN 200810068949A CN 101440389 A CN101440389 A CN 101440389A
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Abstract
The invention provides a method for increasing cordycepin content in Chinese caterpillar fungus solid medium, which comprises three steps of activation of fungus, preparation of liquid fungus and culture of the Chinese caterpillar fungus. The method is characterized in that in the method, rice with the grain size of 20 meshes and 14 meshes is mixed according to the mass ratio of 1:2 to obtain culture medium; 3 percent of glucose, 3.5 percent of peptone, 0.05 percent of K2HPO4, 0.05 percent of MgSO4.7H2O, 2mg/L VB1 and 1,000mL water are used to prepare nutrient solution, and the Chinese caterpillar fungus is cultured according to the prior solid fermentation method; and after stroma of the Chinese caterpillar fungus grows out, illumination intensity and time are increased. The method has the advantages of greatly increasing the cordycepin content (more than 0.76 percent) in the culture medium and having important significance on improving yield and efficiency of producing the cordycepin by solid fermentation of the Chinese caterpillar fungus, along with simple and quick operation; therefore, the method is suitable for mass production of the cordycepin by the solid fermentation of the Chinese caterpillar fungus.
Description
Technical field
The present invention relates to the fungi fermentation engineering, in particular to Cordyceps militaris (L.) Link. solid culture high yield medical material cordycepin.
Background technology
The molecular formula of cordycepin is C
10H
13N
5O
3, molecular weight 251, alkalescence, needle-like or plate crystal, 230 ℃～231 ℃ of fusing points, water-soluble, hot ethanol and methyl alcohol are insoluble to benzene, EC, the anthrone reaction positive, the UV-light maximum absorption wavelength is 259nm.The structural formula of cordycepin is as follows:
The cordycepin that Cordyceps militaris (L.) Link. produces has the various biological activity, such as: bacteriostatic action, antitumor action, effect, antiobesity action, immunoregulation effect and obviously hypoglycemic, the reducing blood lipid of inhibition HIV (human immunodeficiency virus) HIVI C-type virus C.Wherein, the effect of reducing blood-fat aspect is particularly evident, with commercially available fenofibrate (production of method national power Bo Funi drugmaker) effect quite or be better than fenofibrate; Also entering clinical study (in November, 1997 american cancer institute, in July, 2008 U.S. OncoVista company) by FDA approval aspect the treatment leukemia in the U.S..
At present Cordyceps militaris (L.) Link. has been realized extensive solid culture, but cordycepin content accounts for the amount lower (about 30%) of cordycepin total in the culture in the Cordyceps sporophore.Make full use of this valuable resource and just must seek to improve the effective way of cordycepin output, also promptly improve the unit output of cordycepin in the substratum.It is reported that the content of cordycepin is on the low side in the artificial Chinese caterpillar fungus culture medium, only is about 0.2%.So improving the content of cordycepin in the substratum has great importance.
In recent years, people pay close attention to the technology that improves cordycepin content gradually, and the patent application that relates to cordycepin has No. 01124109.8 " fermentation culture is produced the method (liquid culture) of cordycepin ", No. 200810068734.8 " the adopting red yeast rice synergistic fermentation improves the method for Cordyccps-militaris-(L.)-link. Sporophore and cordycepin output ", No. 200310101650.7 " 3 '-application of Desoxyadenosine in the preparation blood lipid-lowering medicine " etc.Yet the patent about the Cordyceps militaris (L.) Link. solid fermentation mainly concentrates on sporophore process study aspect at present, seldom relates to the report of solid medium scale operation cordycepin.
Summary of the invention
The object of the present invention is to provide a kind of method that improves cordycepin content in the Cordyceps militaris (L.) Link. solid medium, thereby increase substantially the wherein content of cordycepin.
The contriver points out that the method for Cordyceps militaris (L.) Link. conventional solid fermentation is:
1. actication of culture
(1) join the PDA substratum: potato 200g, poach 30min with 4 layers of filtered through gauze, gets filtrate, glucose 20g, agar 15～20g adds water to 1000mL;
(2) the PDA substratum branch for preparing is installed to test tube, wrapping, 121 ℃ of sterilization 30mim;
(3) the gone out test tube culture medium solidifying forward swing inclined-plane of bacterium, cooling;
(4) original strain is taken out from 4 ℃ of refrigerators, under the room temperature on the ultra-clean operator's console aseptic inoculation;
(5) 26 ℃ of dark constant temperature culture are down put on the inclined-plane of inoculation Cordyceps militaris (L.) Link.;
After (6) 3～5 days, wait bacterial classification to cover with the inclined-plane, make spore suspension (aseptic technique on the ultra-clean operator's console) with aseptic washing inclined-plane.
2. liquid spawn is made
The Cordyceps militaris (L.) Link. spore suspension is diluted to suitable concn, then spore suspension is inserted in the liquid mother culture media, cultivate 4d with the rotating speed of 150r/min in 20 ℃～28 ℃ following shaking tables.
The prescription of aforesaid liquid mother culture media is a peptone 2%, sucrose 2%, MgSO
47H
2O 0.05%, KH
2PO
40.1%, water 1000mL, pH nature; Liquid amount is the 100mL/500mL triangular flask.
3. Cordyceps militaris (L.) Link. is cultivated
The 20g rice of packing in the 350ml flint glass Cans, the material-water ratio of pressing 1:1.6 adds the 32mL nutritive medium; Seal with high temperature resistant single-layer polypropylene plastic film, 121 ℃, autoclaving under the 30min condition, after the cooling, inoculation Cordyceps militaris (L.) Link. liquid spawn is put 25 ℃ of dark constant temperature culture 10d down; Be no less than 10h from scattered light illumination 11 day every day, temperature keeps 25 ℃, and relative humidity is increased to about 80%; After the Cordyceps militaris (L.) Link. stroma grew, every day, scattered light illumination was no less than 15h, and increased intensity of illumination, kept about 22 ℃ of temperature, atmospheric moisture about 85%; Treat that Cordyceps militaris (L.) Link. cultivates after 50 days, remove a small amount of orange-yellow sporophore, detect the content of cordycepin in the dried substratum.
The contriver points out that the detection method of cordycepin is as follows: cordycepin content detects by HPLC, and HPLC mensuration PC2000 type HPLC analyser (Scientific systems Inc., PA, USA); Chromatographic column adopting Kromasil C18 (4.6mm * 250mm, 5 μ m particle size, Scientificsystems Inc., PA, USA); Ultraviolet Detector (model:Laballiance 525, Scientificsystems Inc., and PA, USA); The detection wavelength is 254nm; 30 ℃ of column temperatures; Moving phase is 10mM KH
2PO
4Be dissolved in methyl alcohol/distilled water (15:85), flow velocity is 1mL/min; Sample size is 20 μ L.Elder generation's precision takes by weighing dry-eye disease 0.2g and places tool plug test tube, adds distilled water 10mL, supersound process 30min, and centrifugal 15min under 5000g then is after detect the wherein content of cordycepin after the 0.45 μ m membrane filtration removal of impurities.If sample then multiply by extension rate through dilution before measuring.
Method of the present invention is on the basis of Cordyceps militaris (L.) Link. conventional solid fermentation, realizes by change substratum granularity and nutritive medium process optimization.Specifically, to get the rice pellets degree be 20 orders and 14 orders cooperates as medium matrix according to 1: 2 mass ratio; Adopt glucose 3%, peptone 3.5%, K
2HPO
40.05%, MgSO
47H
2O 0.05%, VB
1(2mg/L), water 1000mL, the formulated nutritive medium of pH nature is cultivated Cordyceps militaris (L.) Link. according to aforesaid conventional solid fermentation process, after the Cordyceps militaris (L.) Link. stroma grows, increases intensity of illumination and time; The high yield that helps most cordycepin in the solid medium.
Bacterial strain used in the present invention is: the anamorph pupa of cordyceps militaris link bacterial strain (Cordyceps militaris) is drawn up mould (paecilomyces militaris) PM-71 bacterial strain, this bacterial strain on April 17th, 2008 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation numbering: the CGMCCNo.2459 that registers on the books; Address: Datun Road, Chaoyang District, Beijing City (Institute of Microorganism, Academia Sinica).
Above-mentioned rice pellets degree combination is to be medium matrix with whole rice, 10 order rice, 14 order rice, 20 order rice, 40 order rice and their combination in twos (according to different mass ratio combinations) respectively, and matrix: nutritive medium than proportioning, adds nutritive medium by 1: 1.6 quality; Inoculation Cordyceps militaris (L.) Link. in sterilization back is cultivated 50d, and the results substratum preferably comes out to the cordycepin assay.
The prescription of above-mentioned nutritive medium is optimum carbon source (glucose), nitrogenous source (peptone), the inorganic salt (K that the optimization of solid medium nutritive medium single-factor is obtained
2HPO
4And MgSO
47H
2O) and somatomedin (VB
1) carry out according to five factors, four levels that orthogonal experiment preferably comes out.
In the aforesaid method, after the Cordyceps militaris (L.) Link. stroma grew, every day, scattered light illumination was no less than 15h, increased intensity of illumination to 3500LX.
Verify through the optimised process of substratum being produced cordycepin, prove that this technology makes that the content of cordycepin reaches 0.76% in the substratum, has improved 192.31% than 0.26% before optimizing.
The method of cordycepin content in the raising of the present invention Cordyceps militaris (L.) Link. solid medium can increase substantially the content of cordycepin in the substratum, and to produce the efficient of cordycepin significant to improving the Cordyceps militaris (L.) Link. solid fermentation; The present invention has conveniently advantage, is applicable to Cordyceps militaris (L.) Link. solid fermentation scale operation cordycepin.
Embodiment
Embodiment 1: 10 volumes be 350ml, internal diameter be 6.2cm, highly for being respectively charged into 20 purpose rice and 14 purpose rice combination (according to 1: 2 quality than proportioning) in the cylindrical flint glass bottle of 15cm, add the 32mL nutritive medium by 1: 1.6 material-water ratio and (adopt glucose 3%, peptone 3.5%, K
2HPO
40.05%, MgSO
47H
2O 0.05%, VB
1(2mg/L), water 1000mL, the nutritive medium that the formulated of pH nature forms), seal back 121 ℃ with high temperature resistant single-layer polypropylene plastic film, the 30min autoclaving, cooling back inoculation Cordyceps militaris (L.) Link. (CGMCCNo.2459) liquid is female plants, and puts 25 ℃ of dark constant temperature culture 10 days down.Be no less than 10h from scattered light illumination every day in the 11st day, temperature keeps 25 ℃, and relative humidity is increased to about 80%; After the Cordyceps militaris (L.) Link. stroma grew, every day, scattered light illumination was no less than 15h, and increased intensity of illumination to 3500LX, and temperature keeps about 22 ℃, atmospheric moisture about 85%; Treat that Cordyceps militaris (L.) Link. cultivates after 50 days, remove a small amount of orange-yellow sporophore, detect the content of cordycepin in the dried substratum.
Embodiment 2: 10 volumes be 350ml, internal diameter be 6.2cm, highly for being respectively charged into 20 purpose rice and 14 purpose rice combination (according to 1: 2 quality than proportioning) in the cylindrical flint glass bottle of 15cm, add the 40mL nutritive medium by 1: 1.6 material-water ratio and (adopt glucose 3%, peptone 3.5%, K
2HPO
40.05%, MgSO
47H
2O 0.05%, VB
1(2mg/L), water 1000mL, the nutritive medium that the formulated of pH nature forms), seal back 121 ℃ with high temperature resistant single-layer polypropylene plastic film, the 30min autoclaving, cooling back inoculation Cordyceps militaris (L.) Link. (CGMCCNo.2459) liquid is female plants, and puts 25 ℃ of dark constant temperature culture 10 days down.Be no less than 10h from scattered light illumination every day in the 11st day, temperature keeps 25 ℃, and relative humidity is increased to about 80%; After the Cordyceps militaris (L.) Link. stroma grew, every day, scattered light illumination was no less than 15h, and increased intensity of illumination to 3500LX, and temperature keeps about 22 ℃, atmospheric moisture about 85%.Treat that Cordyceps militaris (L.) Link. cultivates after 50 days, remove a small amount of orange-yellow sporophore, detect the content of cordycepin in the dried substratum.
Claims (3)
1. improve the method for cordycepin content in the Cordyceps militaris (L.) Link. solid medium, this method comprises that actication of culture, liquid spawn are made, Cordyceps militaris (L.) Link. is cultivated 3 steps, it is characterized in that getting in this method the rice granularity and be 20 orders and 14 orders according to 1: 2 quality than proportioning as medium matrix; Adopt glucose 3%, peptone 3.5%, K
2HPO
40.05%, MgSO
47H
2O 0.05%, 2mg/L VB
1, water 1000mL, the formulated nutritive medium of pH nature is cultivated Cordyceps militaris (L.) Link. according to traditional solid fermentation method.
2. in accordance with the method for claim 1, it is characterized in that in the described Cordyceps militaris (L.) Link. culturing process that employed bacterial strain is that the anamorph pupa of Cordyceps militaris (L.) Link. is drawn up mould CGMCCNo.2459 bacterial strain.
3. in accordance with the method for claim 1, it is characterized in that in the described Cordyceps militaris (L.) Link. culturing process that after the Cordyceps militaris (L.) Link. stroma grew, every day, scattered light illumination was no less than 15h, used intensity of illumination is 3500LX.
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|---|---|---|---|
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| CNA200810068949XA CN101440389A (en) | 2008-10-23 | 2008-10-23 | Method for increasing cordycepin content in Cordceps militaris solid culture medium |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101791329A (en) * | 2010-03-30 | 2010-08-04 | 天津科技大学 | Astragalus waste cordyceps sinensis fermentation technique and application thereof |
| CN102084780A (en) * | 2010-05-31 | 2011-06-08 | 陈卫东 | Method for culturing cordyceps militaris sporocarp with high-content bioactive substances |
| CN102550300A (en) * | 2012-03-10 | 2012-07-11 | 何寒 | Method for cultivating cordyceps sinensis by taking unhusked rice as culture medium |
| CN102942422A (en) * | 2012-12-13 | 2013-02-27 | 重庆慈恩生物科技有限公司 | Culture medium for liquid state cultivation of cordyceps militaris sporocarp and culturing method thereof |
| CN103262797A (en) * | 2013-06-04 | 2013-08-28 | 神舟太空产品高科技成果推广中心集团有限公司 | Space-induced efficient cordyceps militaris and application thereof as well as preparation method of capsule preparation of space-induced efficient cordyceps militaris |
| CN103262798A (en) * | 2013-06-04 | 2013-08-28 | 神舟太空产品高科技成果推广中心集团有限公司 | Space-induced efficient cordyceps fungus and application thereof as well as preparation method of capsule preparation of space-induced efficient cordyceps fungus |
| CN104003796A (en) * | 2014-05-05 | 2014-08-27 | 安徽金豪生态农业科技有限公司 | Wheat grain medium for cultivation of cordyceps militaris |
| CN104641944A (en) * | 2015-03-19 | 2015-05-27 | 黄秀英 | Method for culturing cordyceps sinensis by taking unhulled wheat as culture medium |
| CN104774888A (en) * | 2015-05-08 | 2015-07-15 | 南通大学 | Cordycepin fermentation solid medium and preparation method and application thereof |
| CN105248150A (en) * | 2015-11-19 | 2016-01-20 | 江苏康能生物工程股份有限公司 | Method for increasing content of cordycepin in cordyceps militaris sporocarp |
| CN106171529A (en) * | 2016-08-05 | 2016-12-07 | 吴剑翔 | The Cordyceps cultural method of worm summer herb with high-content of cordycepin and the culture medium of use thereof |
| CN106282028A (en) * | 2015-05-29 | 2017-01-04 | 东海大学 | Method for culturing cordyceps sinensis |
| CN107582604A (en) * | 2017-10-13 | 2018-01-16 | 贵阳中医学院 | A kind of Cordyceps militaris conversion bark of eucommia solid fermentation product and its preparation method and application |
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2008
- 2008-10-23 CN CNA200810068949XA patent/CN101440389A/en active Pending
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101791329A (en) * | 2010-03-30 | 2010-08-04 | 天津科技大学 | Astragalus waste cordyceps sinensis fermentation technique and application thereof |
| CN102084780A (en) * | 2010-05-31 | 2011-06-08 | 陈卫东 | Method for culturing cordyceps militaris sporocarp with high-content bioactive substances |
| CN102084780B (en) * | 2010-05-31 | 2013-02-13 | 陈卫东 | Method for culturing cordyceps militaris sporocarp with high-content bioactive substances |
| CN102550300A (en) * | 2012-03-10 | 2012-07-11 | 何寒 | Method for cultivating cordyceps sinensis by taking unhusked rice as culture medium |
| CN102942422A (en) * | 2012-12-13 | 2013-02-27 | 重庆慈恩生物科技有限公司 | Culture medium for liquid state cultivation of cordyceps militaris sporocarp and culturing method thereof |
| CN103262797B (en) * | 2013-06-04 | 2014-10-29 | 神舟太空产品高科技成果推广中心集团有限公司 | Space-induced efficient cordyceps militaris and application thereof as well as preparation method of capsule preparation of space-induced efficient cordyceps militaris |
| CN103262797A (en) * | 2013-06-04 | 2013-08-28 | 神舟太空产品高科技成果推广中心集团有限公司 | Space-induced efficient cordyceps militaris and application thereof as well as preparation method of capsule preparation of space-induced efficient cordyceps militaris |
| CN103262798A (en) * | 2013-06-04 | 2013-08-28 | 神舟太空产品高科技成果推广中心集团有限公司 | Space-induced efficient cordyceps fungus and application thereof as well as preparation method of capsule preparation of space-induced efficient cordyceps fungus |
| CN103262798B (en) * | 2013-06-04 | 2014-08-06 | 神舟太空产品高科技成果推广中心集团有限公司 | Space-induced efficient cordyceps fungus and application thereof as well as preparation method of capsule preparation of space-induced efficient cordyceps fungus |
| CN104003796A (en) * | 2014-05-05 | 2014-08-27 | 安徽金豪生态农业科技有限公司 | Wheat grain medium for cultivation of cordyceps militaris |
| CN104641944A (en) * | 2015-03-19 | 2015-05-27 | 黄秀英 | Method for culturing cordyceps sinensis by taking unhulled wheat as culture medium |
| CN104774888A (en) * | 2015-05-08 | 2015-07-15 | 南通大学 | Cordycepin fermentation solid medium and preparation method and application thereof |
| CN104774888B (en) * | 2015-05-08 | 2017-09-05 | 南通大学 | A kind of cordycepin fermentation solid culture medium and preparation method and application |
| CN106282028A (en) * | 2015-05-29 | 2017-01-04 | 东海大学 | Method for culturing cordyceps sinensis |
| CN106282028B (en) * | 2015-05-29 | 2020-02-18 | 东海大学 | Method for culturing cordyceps sinensis |
| CN105248150A (en) * | 2015-11-19 | 2016-01-20 | 江苏康能生物工程股份有限公司 | Method for increasing content of cordycepin in cordyceps militaris sporocarp |
| CN105248150B (en) * | 2015-11-19 | 2017-12-22 | 江苏康能生物工程股份有限公司 | A kind of method of cordycepin content in raising fruiting bodies of cordyceps militaris |
| CN106171529A (en) * | 2016-08-05 | 2016-12-07 | 吴剑翔 | The Cordyceps cultural method of worm summer herb with high-content of cordycepin and the culture medium of use thereof |
| CN107582604A (en) * | 2017-10-13 | 2018-01-16 | 贵阳中医学院 | A kind of Cordyceps militaris conversion bark of eucommia solid fermentation product and its preparation method and application |
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Open date: 20090527 |