CN102283022A - Method for isolating Cordyceps sinensis Sacc. asexual-stage spawn - Google Patents
Method for isolating Cordyceps sinensis Sacc. asexual-stage spawn Download PDFInfo
- Publication number
- CN102283022A CN102283022A CN2011102063158A CN201110206315A CN102283022A CN 102283022 A CN102283022 A CN 102283022A CN 2011102063158 A CN2011102063158 A CN 2011102063158A CN 201110206315 A CN201110206315 A CN 201110206315A CN 102283022 A CN102283022 A CN 102283022A
- Authority
- CN
- China
- Prior art keywords
- sinensis
- cordyceps sinensis
- asexual
- moth larvae
- chinese
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention provides a method for isolating Hirsutslla sinensis asexual-stage spawns from the body fluid of a Hepialus armoricanus larva living body which is infected by Hirsutslla sinensis. When the Hirsutslla sinensis is isolated from the body fluid of the Hepialus armoricanus larva living body which is infected by the Hirsutslla sinensis, the Hepialus armoricanus larva living body which is infected by the Hirsutslla sinensis can be repetitively used for isolating the asexual-stage spawns which are used for culturing Cordyceps sinensis Sacc. Compared with a spore isolation method and a tissue isolation method, the method has the advantages that the operation is simpler, the microbial contamination rate is low, the biological material is saved and the Cordyceps sinensis Sacc. can still grow in the Hepialus armoricanus larva living body with the body fluid being extracted by continuous breeding. Therefore, the cyclic utilization of biological reproduction materials during the culture of the Cordyceps sinensis Sacc. is realized.
Description
Technical field
The present invention relates to a kind of strain separating method, relate to a kind of method of separating aweto from bat moth larvae live body in particular.
Technical background
Cordyceps sinensis is a kind of traditional famous and precious tonic Chinese herbal medicine material, is listed as three big invigorants with natural ginseng, pilose antler.Its property of medicine gentleness, equal edible throughout the year, the old or young, sick, weak, virtual person is all suitable, than the invigorant of other kinds medical value is widely arranged. and demand grows with each passing day both at home and abroad.Cordyceps sinensis is a kind of sac fungi, has conidium stage (phorozoon) and ascospore stage (epigamous) in its history of life.The aweto strain that uses in actual productions such as artificial culture, liquid fermentation is the asexual stage, and the correct evaluation of its bacterial classification is most important, so its phorozoon problem is noticeable especially.At present, the Cordyceps sinensis phorozoon bacterial classification of having reported has 22 formal names used at school, relates to 13 genus.By the regulation of international plant nomenclature, be divided into two classes in the literary composition: qualified novel species title of delivering (6 kinds) and defective deliver or as the title (9 kinds) of different name; By author's careful judgement, be divided into four classes again in the literary composition: can not be Cordyceps sinensis phorozoon title (3 kinds), may not be the title (2 kinds) of Cordyceps sinensis phorozoon, with the relation of Cordyceps sinensis title (12 kinds) still to be tested, can confirm as the title (6 kinds) of Cordyceps sinensis phorozoon at present.Molecular biology method repeatedly is applied to verifying the phorozoon of Cordyceps sinensis in recent years, on the bacterial classification verification method, that generally acknowledge in the whole world, the most reliable is according to Ke He rule (Koch's postulate), strain separated can form Cordyceps sinensis epigamous fruit body through artificial culture or after inoculating insect, produce ascospore, can determine that just it is the correct phorozoon of Cordyceps sinensis.Shen Nan English strain separated is on synthetic medium, uniquely can form Cordyceps sinensis epigamous stroma, produce ascospore, and (the patent No.: ZL85101971) can confirm the correct phorozoon bacterial classification of Cordyceps sinensis beyond doubt, afterwards this bacterial classification quilt hirsutella sinensis fungal really by name that patents.Hirsutella sinensis and Cordyceps sinensis DNA fingerprint collection of illustrative plates likelihood be up to 96 %, and the ITS sector sequence comparative analysis by rDNA, a plurality of independent studies results support that this bacterium is the asexual stage of Cordyceps sinensis.In addition, support also that by the microcycle conidiation experiment Hirsutella sinensis is the phorozoon of Cordyceps sinensis.
Traditional method is spore separation method and tissue isolation, but these two kinds of methods all have certain defective.The spore separation method will obtain the just ripe fruit body of ascospore in the open air and be not easy very much, and collection procedure is numerous and diverse, very easily pollution in its process, obtain single spore difficulty especially.Therefore be the pure bacterial strain that the method separation and Culture is difficult to obtain Cordyceps sinensis with this.Tissue isolation not only growth rate is slow, and assorted bacterial classification class is many in separation process, pollution rate is high, be difficult for obtaining the pure bacterial strain of Cordyceps sinensis, just because of this, the isolated separately strain name of domestic how tame unit differs, comes in every shape since the eighties, but all thinks and control oneself that what separate is real Cordyceps strain, and truth but is all non-real awetos of great majority report, one time, serious trouble waters have been formed to aweto asexual stage bacterial classification understanding.
Summary of the invention
By discovering, the hirsutella sinensis fungal poor growth, aerial hyphae is undeveloped, bacterium colony is hard, by cultivating, repeatedly form stroma on solid culture medium, form ascospore after the light treatment on stroma, it is just the same that ascocarp, the ascospore that these artificial culture produce is considered to ascocarp, the ascospore morphological feature natural with Cordyceps sinensis.Therefore can confirm that substantially Hirsutella sinensis is the phorozoon of Cordyceps sinensis.The invention provides a kind of method of aweto asexual stage strain separating, promptly separated hirsutella sinensis fungal asexual stage bacterial classification the body fluid of the bat moth larvae live body of hair spore bacterium from infection of Chinese.
Described from infection of Chinese by the bat moth larvae of hair spore bacterium, the concrete steps of separating hirsutella sinensis fungal in the live body are:
1) the screening infection of Chinese is by the bat moth larvae live body of hair spore bacterium, and body surface is sterilized;
2) Wicresoft bat moth larvae body surface, the body fluid that body surface is penetrated places on the plating medium to be cultivated;
3) under 8～18 ℃ of conditions, cultivated 9～16 days, during, eliminate the removal of impurities bacterium, stay Cordyceps Militaris and continue to cultivate, obtain hirsutella sinensis fungal.
Described step 2) prescription of described plating medium is: in 1000 milliliters in the water, fresh potato piece 200g, glucose 200g, agar 20 g, peptone 15 g, magnesium sulfate 0.5 g, potassium dihydrogen phosphate 0.5g, dusty yeast 5g, wherein the pH of plating medium is 6.5～7.
Useful technique effect of the present invention is: separated hirsutella sinensis fungal from infection of Chinese the body fluid of the bat moth larvae live body of hair spore bacterium, an infection of Chinese can repeatedly be used for the asexual stage bacterial classification of separation and Culture aweto by the bat moth larvae live body of hair spore bacterium.The operation of spore separation method and tissue isolation is simpler relatively, and the living contaminants rate is low, saves biomaterial, and the bat moth larvae that was extracted body fluid is raised by continuation, also can grow Cordyceps sinensis.Realized the recycling of biological self reproducing material in the Cordyceps sinensis cultivating process.
Description of drawings
Fig. 1: the Chinese caterpillar fungus of being unearthed 20 days of the bat moth larvae that the bacterial classification microbiological contamination that the present invention filters out is cultivated;
Fig. 2: the Chinese caterpillar fungus that wild sense bacterium larva was unearthed 20 days;
Fig. 3: the bat moth larvae that bacterial classification microbiological contamination that the present invention filters out is cultivated perithecium fully-developed Chinese caterpillar fungus;
Fig. 4: wild sense bacterium larva perithecium fully-developed Chinese caterpillar fungus.
Embodiment
Take by weighing fresh potato piece 200g, glucose 200g, agar 20 g, peptone 15 g, magnesium sulfate 0.5 g, potassium dihydrogen phosphate 0.5g, dusty yeast 5g; Earlier with the stripping and slicing of horse bell administration, add the ripe filtration of poach, getting its filtrate puts in people's conical flask, supply water to 1000 milliliter, add other batchings again, shake up, and heating boil fully dissolve to agar powder after, be sub-packed in the triangular flask, after sealing with tampon and brown paper, put into steam disinfecting apparatus, under 100 kPa pressure, 115 ℃ of high-temperature sterilization 30 min, take out after waiting to reduce to room temperature, on super quiet workbench, aseptic condition was filled in the empty plate of aseptic glass that diameter is 90mm in following minute, and cover lid is after cooling is solidified it naturally, obtain plating medium, preserve stand-by in 4 ℃ of refrigerators.
From the kangding of more than 3800～4300 meter of height above sea level, the Cordyceps sinensis artificial culture base on ground such as Shiqu, gather infection of Chinese by the bat moth of hair spore bacterium the larva that lives, as for the examination material.
From infection of Chinese by the back central authorities of the bat moth larvae live body of hair spore bacterium, after 75% alcohol degreasing cotton balls partly sterilised, puncture epidermis gently with acupuncture needle (traditional Chinese medical science acupuncture-moxibustion with), body fluid promptly oozes out, and the body fluid that will ooze out is stained with under aseptic condition with transfer needle smears (i.e. inoculation) on plating medium.
The plating medium of having smeared the larva body fluid alive that carries disease germs placed in 8～18 insulating boxs cultivate.After 3 days, observe once every day, in time eliminates the removal of impurities bacterium, 10-16 days rears inoculation place of plating medium form dark-grey, be transformed into the Hirsutella sinensis bacterium colony of pale brown and even black then.
With the hirsutella sinensis fungal of separating, change over to from flat plate (triangular flask is 1000 milliliters) in the triangular flask that fills the medium identical with plating medium prescription and preparation method, put under 18 ℃ of conditions and cultivate, move and grow 2 weeks of back, beginning sees that inoculum expands gradually and blackening gradually, and extremely undeveloped short and sparse mycelium is arranged on it.Be formed centrally the circle of diameter 6-10mm, light brown bacterium colony in after 2 months being, and have the dark brown pigment to infiltrate in the medium with inoculum.Behind 3 wheat harvesting periods, colony diameter reaches about 1.8cm.At this moment, the inoculum center is yellowish-brown, bacterium colony is hard, and sparse smooth micromycelial is arranged, and then forms the conidium of aweto.
With sterile water its spore is washed, is mixed with spore suspension, this bacterium liquid is sprayed body surface in (the promptly just decortication) larva that just entered for three length of times, spray 2 times (midfeather 2～3 hours) continuously after, larva is put into insect box raises; Raise after the inoculation about 1 year, larva can fall ill rigid again, forms bombys batryticatus, then germinates young shoot, grows into fruit body.
With the bacterial classification inoculation bat moth larvae that the present invention separates, sense bacterium, unearthed 20 days the Chinese caterpillar fungus of Chinese caterpillar fungus and wild sense bacterium larva of ossifing, being unearthed 20 days after the rudiment compare, as Fig. 1,2; With the bacterial classification inoculation bat moth larvae that the present invention separates, the sense bacterium, ossify, grow after the rudiment perithecium fully-developed Chinese caterpillar fungus and wild sense bacterium larva and grow perithecium fully-developed Chinese caterpillar fungus and compare, as Fig. 3,4.
Find that the morphological feature of bacterial classification inoculation that the present invention obtains, Cordyceps sinensis that microbiological contamination bat moth larvae obtains and wild cordyceps and ascocarp thereof, ascospore morphological feature are just the same.Can confirm that thus the bacterial classification that the present invention is separated to is the asexual stage Hirsutella sinensis of Cordyceps sinensis.
Take by weighing the bacterial classification microbiological contamination that the present invention of 5000/feather weight filters out respectively and cultivate Cordyceps sinensis and wild cordyceps 10g, pulverize respectively, the content of adenosine, 17 seed amino acids, mannitol and the ergosterol of Cordyceps sinensis is cultivated in the bacterial classification microbiological contamination that detection the present invention filters out.
Concrete outcome is as shown in table 1.The adenosine content that the bacterial classification microbiological contamination that the present invention filters out is cultivated in Cordyceps sinensis and the wild cordyceps is basic identical, the content of mannitol and ergosterol is a little less than wild Chinese caterpillar fungus, but the bacterial classification microbiological contamination that the present invention filters out cultivate in the Cordyceps sinensis seed amino acid in 17 all be higher than wild cordyceps.
Cordyceps sinensis and wild cordyceps sample are cultivated in the bacterial classification microbiological contamination that table 1 the present invention filters out
Active ingredient and content (g/mg)
Claims (3)
1. the method for aweto asexual stage strain separating is characterized in that: separated hirsutella sinensis fungal asexual stage bacterial classification the body fluid of the bat moth larvae live body of hair spore bacterium from infection of Chinese.
2. the method for aweto asexual stage strain separating according to claim 1 is characterized in that: described concrete steps of being separated hirsutella sinensis fungal the bat moth larvae live body of hair spore bacterium from infection of Chinese are:
1) the screening infection of Chinese is by the bat moth larvae live body of hair spore bacterium, and body surface is sterilized;
2) Wicresoft bat moth larvae body surface, the body fluid that body surface is penetrated places on the plating medium to be cultivated;
3) under 8～18 ℃ of conditions, cultivated 10～16 days, during, eliminate the removal of impurities bacterium, stay Cordyceps Militaris and continue to cultivate, obtain hirsutella sinensis fungal.
3. the method for aweto asexual stage strain separating according to claim 2, it is characterized in that: described step 2) prescription of described plating medium is: 1000 milliliters in water, potato 200g, glucose 200g, agar 20 g, peptone 15 g, magnesium sulfate 0.5 g, potassium dihydrogen phosphate 0.5 g, dusty yeast 5 g, wherein the PH of plating medium is 6.5～7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102063158A CN102283022A (en) | 2011-07-22 | 2011-07-22 | Method for isolating Cordyceps sinensis Sacc. asexual-stage spawn |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102063158A CN102283022A (en) | 2011-07-22 | 2011-07-22 | Method for isolating Cordyceps sinensis Sacc. asexual-stage spawn |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102283022A true CN102283022A (en) | 2011-12-21 |
Family
ID=45330016
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011102063158A Pending CN102283022A (en) | 2011-07-22 | 2011-07-22 | Method for isolating Cordyceps sinensis Sacc. asexual-stage spawn |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102283022A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102696555A (en) * | 2012-06-13 | 2012-10-03 | 重庆市中药研究院 | Semi-wild manual Cordyceps-cultivating method |
| CN102845218A (en) * | 2012-08-23 | 2013-01-02 | 大化金玉草生物科技有限责任公司 | Original ecological separation method of cordyceps sinensis hirsutella sinensis |
| CN103535185A (en) * | 2013-09-30 | 2014-01-29 | 涂峰 | Method for separating liquid of hirsutella sinensis strains |
| CN103548780A (en) * | 2013-11-15 | 2014-02-05 | 重庆市中药研究院 | Method for preventing and controlling paecilomyces disease in artificially breeding ophiocordyceps sinensis larvae host process |
| CN104145719A (en) * | 2014-09-04 | 2014-11-19 | 重庆市中药研究院 | Cordyceps sinensis mycelium fermentation production method |
| CN106834279A (en) * | 2017-04-10 | 2017-06-13 | 成都图径生物科技有限公司 | The method of rapid extraction aweto genomic DNA |
| CN110305795A (en) * | 2019-03-15 | 2019-10-08 | 广东省生物资源应用研究所 | One plant of Hirsutella sinensis and its application |
| CN113994846A (en) * | 2021-09-17 | 2022-02-01 | 力源新资源开发(广东)有限公司 | Application of juvenile hormone analogue methoprene to promotion of stiff hepialus larvae |
| CN115024154A (en) * | 2022-04-26 | 2022-09-09 | 青海德爱智能科技有限公司 | Method for ecologically tending cordyceps sinensis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101843196A (en) * | 2010-04-19 | 2010-09-29 | 杨毅 | Method for culturing mycelia and sporocarps of aweto |
-
2011
- 2011-07-22 CN CN2011102063158A patent/CN102283022A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101843196A (en) * | 2010-04-19 | 2010-09-29 | 杨毅 | Method for culturing mycelia and sporocarps of aweto |
Non-Patent Citations (4)
| Title |
|---|
| 《中国中药杂志》 20010731 陈仕江 等 西藏那曲冬虫夏草无形型的研究 454-454页 1 第26卷, 第7期 * |
| 《中国食用菌》 20070915 朱斗锡 等 中国冬虫夏草人工栽培研究进展 11-13页 1-3 第26卷, 第5期 * |
| 《贵州农业科学》 20081015 杨晓峰 等 真菌诱导的昆虫免疫反应研究进展 73-76页 1-3 第36卷, 第5期 * |
| 谢放 等: "不同培养基对冬虫夏草菌丝生长的影响", 《中国微生态学杂志》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102696555A (en) * | 2012-06-13 | 2012-10-03 | 重庆市中药研究院 | Semi-wild manual Cordyceps-cultivating method |
| CN102845218A (en) * | 2012-08-23 | 2013-01-02 | 大化金玉草生物科技有限责任公司 | Original ecological separation method of cordyceps sinensis hirsutella sinensis |
| CN103535185A (en) * | 2013-09-30 | 2014-01-29 | 涂峰 | Method for separating liquid of hirsutella sinensis strains |
| CN103535185B (en) * | 2013-09-30 | 2015-11-25 | 涂峰 | A kind of liquid separation method of China pilose spore bacterial classification |
| CN103548780A (en) * | 2013-11-15 | 2014-02-05 | 重庆市中药研究院 | Method for preventing and controlling paecilomyces disease in artificially breeding ophiocordyceps sinensis larvae host process |
| CN103548780B (en) * | 2013-11-15 | 2016-01-13 | 重庆市中药研究院 | The host larva Paecilomyces varioti disease preventing control method of artificial feeding Cordyceps sinensis |
| CN104145719A (en) * | 2014-09-04 | 2014-11-19 | 重庆市中药研究院 | Cordyceps sinensis mycelium fermentation production method |
| CN106834279A (en) * | 2017-04-10 | 2017-06-13 | 成都图径生物科技有限公司 | The method of rapid extraction aweto genomic DNA |
| CN110305795A (en) * | 2019-03-15 | 2019-10-08 | 广东省生物资源应用研究所 | One plant of Hirsutella sinensis and its application |
| CN110305795B (en) * | 2019-03-15 | 2021-10-01 | 广东省生物资源应用研究所 | Hirsutella sinensis and application thereof |
| CN113994846A (en) * | 2021-09-17 | 2022-02-01 | 力源新资源开发(广东)有限公司 | Application of juvenile hormone analogue methoprene to promotion of stiff hepialus larvae |
| CN115024154A (en) * | 2022-04-26 | 2022-09-09 | 青海德爱智能科技有限公司 | Method for ecologically tending cordyceps sinensis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102283022A (en) | Method for isolating Cordyceps sinensis Sacc. asexual-stage spawn | |
| JP2007537737A (en) | Industrial Fermentative Production Method for Chinese Cordyceps Asexual Type Bacteria (HirsutellahepialiChen & Shen) | |
| CN102440149A (en) | Method for breeding cordyceps militaris by utilizing silkworm larvae | |
| CN101215527A (en) | Method for cultivating silkworm chrysalis Cordyceps sinensis | |
| CN104145719A (en) | Cordyceps sinensis mycelium fermentation production method | |
| CN103396956B (en) | A kind of saccharomyces cerevisiae and its screening and culturing method and the method for bean pulp fermentation | |
| CN107475130B (en) | Thin handle Isaria novel bacterial and its cultural method and purposes | |
| CN103103152B (en) | Corallococcus coralloides and application thereof | |
| CN105075667A (en) | Method used for artificially cultivating Chinese caterpillar fungus in low altitude region | |
| CN105925616A (en) | Fruit and vegetable culture and probiotics fermentation of medicinal and edible fungi | |
| CN103798058A (en) | Method for cultivating aseptic cordyceps militaris | |
| CN1224695C (en) | New technology of culturing cordyceps sinensis using insect | |
| CN109182151A (en) | The separating screening method of gingko endogenous fungus | |
| CN105557312A (en) | Method for cultivating Cordyceps products by taking Tenebrio molitor (L.) larvae as carriers | |
| CN106010978A (en) | Culture method of cordyceps sinensis anamorphic hirsutella sinensis pure strain | |
| CN106967669A (en) | A kind of quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria | |
| CN113481108B (en) | Nutritional matrix for stimulating growth of nematode-trapping fungi on trunk, and preparation method and application method thereof | |
| CN103843580A (en) | Paecilomyces hepialid mycelium and preparation method thereof | |
| CN102498947A (en) | Method for culturing cordyceps sinensis by performing endogenous pressurization and artificial infection of hirsutella sinensis on hepialus larvae | |
| CN114854604B (en) | Ganoderma lucidum, high-concentration oral liquid containing ganoderma lucidum and preparation method thereof | |
| CN103782794A (en) | Method for stimulating cordyceps sinensis to generate conidia | |
| KR102265286B1 (en) | Method for manufacturing cordyceps scorched rice using grain products | |
| CN109618811B (en) | Industrialized artificial cultivation method for cordyceps sinensis | |
| CN109055236B (en) | Isaria pinicola WSWM1171 and application thereof in prevention and control of potato corm moth pupae | |
| CN113046249A (en) | Verticillium lecanii LL-01 and biocontrol application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20111221 |