CN1177925C - Fermenting culture process of produicng Cordyceps extract - Google Patents
Fermenting culture process of produicng Cordyceps extract Download PDFInfo
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- CN1177925C CN1177925C CNB011241098A CN01124109A CN1177925C CN 1177925 C CN1177925 C CN 1177925C CN B011241098 A CNB011241098 A CN B011241098A CN 01124109 A CN01124109 A CN 01124109A CN 1177925 C CN1177925 C CN 1177925C
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Abstract
The present invention provides a method adopting an airlift bioreactor for the large-scale culture of cordyceps militaris and separately extracting cordycepin from culture solution. The method cultures zymotic seeds on an MS medium, and then, the seeds are transmitted into a 20L bioreactor for large-scale culture. The culture solution in the bioreactor is separated and purified so as to obtain cordycepin of which the content reaches 46.25%, and the purity is 99%, so that the problem of raw material for producing cordycepin is solved. The present invention has the advantages of easy operation, low production cost and no environmental pollution, and reaches the level of industrial production.
Description
Technical field
The present invention relates to a kind of method of producing cordycepin by fermentation culture.Specifically be to be means with the plant cell culture technology, produce the method for cordycepin by the Cordyceps militaris (L.) Link. large scale culturing of in the cell biological reactor, carrying out.The invention belongs to field of biological pharmacy.
Background technology
Cordyceps militaris (L.) Link. (Corduceps militaris L; Sp, PL) give birth to for Ascomycotina (Ascomycotina) ergot Zoopagales (Clavicipitales) Clavicipitaceae (Cordycepps) fungi stroma list or have several to send from the insect head, sometimes send from joint, orange-yellow, scarcely branch is high 2～5 centimetres, the head clavate, long 1～2 centimetre, thick 0.3～0.5 centimetre.It is identical with Cordyceps sinensis to treat food.Monogony is that pupa is drawn up mould (Paecilmyces militarae), proves Cordyceps militaris (L.) Link. through modern science and not only has special nutritive value, and obvious medicinal value is arranged.Since ancient times, Cordyceps sinensis is used as the treasured in China's Chinese medicine treasure-house always, obtains paying attention to and application, and is called as China's three big tonics with genseng, pilose antler.Traditional Chinese medicine is thought that the Cordyceps sinensis nature and flavor are sweet, is equalled, goes into the lung kidney channel, and function benefit lung, kidney, cough-relieving are coughed, tonifying deficiency, beneficial vital essence.Liked by the human consumer.Cordyceps mycelium is cultivated in utilization biotechnological means submerged fermentation, finds to contain in the fermented liquid multiple essential trace elements such as multivitamin such as rich in protein, Va, Ve and selenium, zinc, manganese, chromium.Wherein remarkable with the pharmaceutical use of multiple biologically active substances such as cordycepic acid, cordycepin [Cordgxepin (i.e. 3 '-Desoxyadenosine)], Cordyceps polysaccharide and SOD especially.Particularly the natural content of cordycepin is apparently higher than natural cordyceps.The Cordyceps polysaccharide content that fermentation culture obtains is higher than the content in the natural cs far away.Relevant Cordyceps sinensis medicine and its raw material sources one of nutritious prod of domestic and international market sale so far are to take from natural Cordyceps sinensis, the 2nd, take from manual inseminating's Chinese caterpillar fungus, formulations such as making capsule and liquid oral is washed or washed to Cordyceps mycelium through traditional alcohol, the medicinal ingredients complexity, active constituent content is low.Because particular requirement that growth is geographical and strict parasitics cause the Cordyceps sinensis resource extremely rare, being difficult for of in addition gathering more makes the wild cordyceps resource extremely lack, thereby causes Cordyceps sinensis to cost an arm and a leg, and is worth to be higher than ginseng, young pilose antler.In the last few years, along with continuous expansion, to the demand of Chinese caterpillar fungus to Cordyceps sinensis understanding field, not only the domestic market enlarges day by day, and the demand of world markets such as South East Asia, Japan, the U.S. is subjected to the restriction of resource also increasing, rely on nature to gather very much, the phenomenon that supply falls short of demand usually occurs.In view of natural cordyceps has belonged to the class protection medicinal plant kind that country forbids plucking, raw material sources have become the key that can this kind further enlarge market.Just has great value so carry out research and the artificial culture of Chinese caterpillar fungus energetically.And its active constituent content of the Cordyceps sinensis of artificial culture or Cordyceps militaris (L.) Link. is extremely unstable, and the bacterial classification variation is bigger, wayward.So carry out energetically that the research of Chinese caterpillar fungus is particularly produced, the research of separation, extracting effective components just has great value.From the clinical efficacy and the nutritive value analysis of preserving the ecological environment and improving Cordyceps sinensis; both solved the raw material sources problem with biotechnology means exploitation Cordyceps sinensis; increased substantially the content of effective constituent cordycepin and Cordyceps polysaccharide etc. again, cost is low, and market potential is huge.In the document of having delivered and patent, do not see the report of utilization air lift type cell reactor submerged fermentation cultivation Cordyceps militaris technological development Cordyceps polysaccharide and cordycepin both at home and abroad.
Summary of the invention
The present invention has initiated the semicontinuous cultivation of going down to posterity of employing seed, the air lift type submerged fermentation culture technique that the bacterial classification suspension culture combines according to the special mechanism of cordyceps militaris mycelium fermentation culture generation Cordyceps polysaccharide and cordycepin.Control the process of growth of bacterium ball well, and then shortened culture cycle, reduced cost, can effectively improve its output.
The culture process step of Cordyceps sinensis is as follows:
1, prepare seed earlier: liquid seed culture medium consists of: potato 200 grams, glucose 10 grams, KH
2PO
42 grams, MgSO
41 gram, peptone 5 grams, yeast extract paste 2 grams.
The culture condition of seed:
The Erlenmeyer flask that liquid seeds is housed is put on the rotary shaking table, and 200 rev/mins, 26 ℃ ± 1 cultivated 3-8 days, and temperature 23-28 ℃, scattered light, seed culture 3-8 days is one-period.
When industrialization is produced, adopt 2L air lift type cell reactor (the biochemical center of the Chinese Academy of Sciences is on sale) to make seeding tank, carry out the cultivation of seed.
2, fermentation culture:
After seed culture 3-8 days, it is limpid in sight to choose cultivation, bacterium ball uniformity, the seed liquor of about 3mm diameter, be seeded in the 20L fermentor tank by 10% inoculum size, under aseptic condition, cultivate, temperature 23-28 ℃, cultivated 3-8 days, air flow 200ml/min, fermentation culture 3-8 days is one-period.
The said seed of the present invention has carried out preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center, strain is called Coragyceps militaris bacterium, the Latin formal name used at school is Cordycepsmilitaris, and preserving number is CGMCC 0611, and preservation date is August 7 calendar year 2001.
It is identical with above-mentioned seed liquor substratum composition that the substratum of fermentation culture is formed.Cultivate as producing jar with 20L air lift type cell reactor.Culture condition is: temperature 23-28 ℃, cultivated air flow 200ml/min 3-8 days.Inoculum size 10%, scattered light.
3, through after the above-mentioned fermentation culture, carry out separation and purification, separation of the present invention and purge process are as follows:
Behind following jar of the cell culture fluid, cross post, obtain cordycepin crystal by HPLC.That is, the product of will cultivating are got dry thing 0.7g and are dissolved in 6ml H dry below 60 ℃
2Among the O,, get subsequent filtrate with the solution of gained 0.45um membrane filtration, repeat sample introduction 3 times, sample size is 10ul, and the peak area of gained is 2340604, calculating cordycepin concentration by the substitution typical curve is 7.1mg/l, and to account for the ratio of former dry product be 46.25% to cordycepin like this.The results are shown in and detect collection of illustrative plates (accompanying drawing 1).
The selection of high performance liquid phase (HPLC) chromatographic condition:
Utilize methyl alcohol: water (15: 85) reaches baseline separation as the cordycepin (CCS) in the dry thing of the mutual-assistance of flowing with other component, the retention time of cordycepin is less than 20 minutes, about about 12 minutes, go out peak (pharmacopeia regulation HPLC measures component should go out the peak with interior at 20 minutes) greatly, collect.
Experiment showed, and adopt air lift type submerged fermentation culture technique of the present invention to cultivate the nutrient solution that obtains, it is higher that its cordycepin content obtains cordycepin content than other cultural methods.
Following table is to adopt the result of the central cordycepin different output of nutrient solution of 2 kinds of training methods acquisitions.
2 kinds of different training methods are to the influence of cordycepin output
Training method bacterium ball surviving rate (%) cordycepin output (mg/l)
Shake-flask culture 80.7 0.65
Air lift type cultivates 100 7.10
Can see that from table the bacterium ball surviving rate of air lift type cell reactor and cordycepin output is all apparently higher than shake-flask culture, when cultivating, 0.08m
3The air flow of/h can make the bacterium ball grow preferably and produce cordycepin.Because shake-flask culture exists ventilation freely not cause the growth of bacterium ball unfavorable.The air lift type cell reactor does not then have above-mentioned situation.
Because cordycepin and Cordyceps polysaccharide are mainly based on exocytosis, the content of cordycepin that process is separated, purifying obtains and Cordyceps polysaccharide etc. all has raising extremely significantly than natural Cordyceps sinensis and tame Cordyceps militaris.After testing, cordycepin content is 7.1mg/L, than natural Cordyceps sinensis and tame Cordyceps militaris height nearly a hundred times.
The culture that obtains is cultivated in natural cordyceps, Cordyceps militaris (L.) Link., air lift type cell reactor submerged fermentation of the present invention, and its main pharmaceutical ingredient content Preliminary detection the results are shown in shown in the following table:
Natural cordyceps, Cordyceps militaris (L.) Link., air lift type cell are anti-
Answer the main pharmaceutical ingredient content table of device submerged fermentation culture
| Composition | Cordyceps militaris fruiting body | Natural cordyceps | Fermenting culture of the present invention | |
| Cordycepic acid % | 4.81 | 3.08 | 30.56 | |
| Cordycepin % | 0.603 | 0.0029 | 7.1 | |
| Cordyceps polysaccharide % | 12.8 | 7.0 | 75 | |
| The SOD enzyme activity compares vigor | 184.4u/ml 42.4u/mg | 149.4u/ml 10.3u/mg | --- | |
| Amino acid % | Aspartic acid | 2.55 | 2.02 | 1.63 |
| Threonine | 1.50 | 1.03 | 0.93 | |
| Serine | 1.57 | 1.16 | 0.91 | |
| L-glutamic acid | 3.97 | 3.81 | 1.74 | |
| Proline(Pro) | 0.90 | 1.26 | 0.76 | |
| Glycine | 1.64 | 1.17 | 1.40 | |
| L-Ala | 2.39 | 1.47 | 1.66 | |
| Gelucystine | 0.49 | 0.21 | 0.33 | |
| Xie Ansuan | 1.65 | 1.10 | 1.00 | |
| Methionine(Met) | 0.28 | 0.12 | 1.33 | |
| Isoleucine | 1.98 | 0.89 | 0.79 | |
| Leucine | 2.49 | 2.09 | 1.19 | |
| Tyrosine | 0.92 | 0.66 | 0.61 | |
| Phenylalanine | 1.89 | 0.71 | 0.68 | |
| Methionin | 1.09 | 1.23 | 0.89 | |
| Tryptophane | 0.30 | 0.32 | 0.94 | |
| Histidine | 0.46 | 0.66 | 0.70 | |
| Arginine | 1.26 | 1.76 | 0.69 | |
| Trace element (ppm) | K | 1.7％ | 3.5％ | 4.5％ |
| Ca | 249 | 634 | 476 | |
| Zn | 85 | 267 | 330 | |
| Fe | 210 | 543 | 588 | |
| Cr | 3.12 | 8.55 | 2.79 | |
| Cu | 8.05 | 21.1 | 13.5 | |
| Mn | 47.1 | 29.6 | 53.0 | |
| Ni | 2.18 | 7.24 | 1.42 | |
| Pb | 1.04 | --- | --- | |
| As | 0.30 | --- | --- | |
The Cordyceps mycelium preparation that domestic and international Cordyceps sinensis class healthcare products that gone on the market and medicine are natural extract.Because it has very high pharmaceutical use, liked by the patient.Yet because its complicated component, active constituent content is low, has influenced the clinical efficacy of this medicine and the further expansion in market greatly.Particularly natural cordyceps has been subjected to the focused protection of the world and the Chinese government as endangered plant species, forbids to pluck.Each country all drops into the substitute products of lot of manpower and material resources research Cordyceps sinensis for this reason.The substitute products of generally acknowledging in the world are artificial culture Cordyceps sinensis or cordyceps militaris mycelium at present, and the high bacterial classification of screening active constituent content carries out artificial culture, and the mycelium that obtains carries out some conventional processing and makes medicinal extract, capsule or oral liquid.This still belongs to the traditional industry processing mode of Chinese medicine.The content of cordycepin and Cordyceps polysaccharide etc. is then very low though priming cost is low, and it is about 75 days that the production cycle is about.
The present invention adopts air lift type cell reactor submerged fermentation culture technique, produces cordycepin and Cordyceps polysaccharide, unchangeability, and the purity height, cost is low, the cycle short (being about about 7 days).Has the market competitiveness.Particularly cordycepin content is, than natural Cordyceps sinensis and tame Cordyceps militaris height nearly a hundred times.
Description of drawings
Fig. 1 detects collection of illustrative plates from the cordycepin chromatogram that Chinese caterpillar fungus obtained that the present invention cultivates.
Embodiment
Adopt 2L air lift type cell reactor (available from the biochemical center of the Chinese Academy of Sciences) to make seeding tank, liquid seeds was cultivated 24 ℃ of temperature, scattered light 96 hours at 26 ℃ ± 1 time.Liquid seed culture medium consists of: potato 200 grams, glucose 10 grams, KH
2PO
42 grams, MgSO
41 gram, peptone 5 grams, yeast extract paste 2 grams.
Liquid seeds was cultivated after 5 days, it is limpid in sight to choose cultivation, bacterium ball uniformity, the seed liquor of about 3mm diameter is seeded in the 20L airlift fermentor by 10% inoculum size, cultivates under aseptic condition, it is identical with above-mentioned seed liquor substratum composition that the substratum of fermentation culture is formed, 24 ℃ of culture temperature were cultivated air flow 200ml/min 5 days.
Through after the above-mentioned fermentation culture, cross post by HPLC, the product of will cultivating are got dry thing 0.7g and are dissolved in 6ml H dry below 60 ℃
2Among the O, with the solution of gained 0.45um membrane filtration, get subsequent filtrate, last chromatographic column repeats sample introduction 3 times, and sample size is 10ul, collects and separates the cordycepin that obtains.
Claims (7)
1, a kind of method of producing cordycepin by microbial fermentation, it is characterized in that this method is for carrying out the cultivation of seed culture and large scale fermentation on substratum, behind following jar of the cell culture fluid, cross post by HPLC, separate and obtain cordycepin crystal, said seed strain is called Cordyceps militaris (L.) Link. (the Latin formal name used at school is Cordycepsmilitaris), and preserving number is CGMCC 0611.
2, according to the method for claim 1, it is characterized in that seed culture 3-8 days be one-period, fermentation culture 3-8 days is one-period.
3, according to the method for claim 1, it is characterized in that the seed culture condition is put on the rotary shaking table for the Erlenmeyer flask that liquid seeds will be housed, 200 rev/mins, cultivated scattered light 3-8 days for 23-28 ℃.
4, according to the method for claim 1, it is characterized in that its said substratum consists of: potato 200 grams, glucose 10 grams, KH
2PO
42 grams, MgSO
41 gram, peptone 5 grams, yeast extract paste 2 grams.
5, according to the method for claim 1, the method that it is characterized in that fermentation culture is, after liquid seeds was cultivated 5 days, it was limpid in sight to choose cultivation, bacterium ball uniformity, the seed liquor of about 3mm diameter, be seeded in the 20L fermentor tank by 10% inoculum size, under aseptic condition, cultivate, 24 ℃ of temperature, cultivated air flow 200ml/min 5 days.
6,, it is characterized in that said mass cell cultivation employing air lift type cell reactor carries out submerged fermentation and cultivates according to the method for claim 1.
7, according to the method for claim 1, it is characterized in that high-efficient liquid phase chromatogram condition is: utilize methyl alcohol: cordycepin and other component that water=the mobile mutual-assistance of conduct in 15: 85 is cultivated in the product reach baseline separation, the retention time of cordycepin is less than 20 minutes, about about 12 minutes, go out the peak greatly, carry out separated and collected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011241098A CN1177925C (en) | 2001-08-13 | 2001-08-13 | Fermenting culture process of produicng Cordyceps extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011241098A CN1177925C (en) | 2001-08-13 | 2001-08-13 | Fermenting culture process of produicng Cordyceps extract |
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| CN1335389A CN1335389A (en) | 2002-02-13 |
| CN1177925C true CN1177925C (en) | 2004-12-01 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SG129276A1 (en) * | 2003-04-10 | 2007-02-26 | Auric Pacific Nutritech Pte Lt | Large-scale production method for cordyceps and ganoderma |
| CN101423835B (en) * | 2005-09-06 | 2011-01-12 | 中炬高新技术实业（集团）股份有限公司 | Gene order relating to cordycepin biological synthesis |
| CN101780118B (en) * | 2009-01-19 | 2013-04-24 | 华东理工大学 | Method for extracting active ingredients of artificially cultured cordceps militaris waste liquor by flocculation |
| CN103416223B (en) * | 2013-08-07 | 2015-05-13 | 中南林业科技大学 | Method for improving cordycepin output in cordyceps militaris fermentation broth |
| CN105670942A (en) * | 2016-03-15 | 2016-06-15 | 江苏神华药业有限公司 | Method for producing ophiocordyceps sinensis powder through rapid and deep liquid state fermentation |
| CN107586726A (en) * | 2017-10-13 | 2018-01-16 | 贵阳中医学院 | Improve Cordyceps militaris rhizoma Gastrodiae liquid fermentation method and the application of cordycepin content |
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