CN1304995A - Process for culturing cordyceps with domestic silkworm - Google Patents
Process for culturing cordyceps with domestic silkworm Download PDFInfo
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- CN1304995A CN1304995A CN 01103275 CN01103275A CN1304995A CN 1304995 A CN1304995 A CN 1304995A CN 01103275 CN01103275 CN 01103275 CN 01103275 A CN01103275 A CN 01103275A CN 1304995 A CN1304995 A CN 1304995A
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- cordyceps sinensis
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Abstract
A process for culturing cordyceps with Bombyx mori includes phycical, chemical and biological treatment of cordyceps strain, inoculating in Bambyx mori bodies, and culturing it simulative natural environment to grow out stromata, Its advantages are easy operation, and low cost.
Description
The present invention relates to cultivate the method for producing Cordyceps sinensis with silkworm.
Cordyceps sinensis is that Cordyceps fungus (Cordyceps sinensis) parasitizes the formed bacterium worm mixture in back on the Chinese caterpillar fungus bat moth insect body such as (Hepialusarmoricanus), main product is in the alpine scrub and high mountain steppe of height above sea level 3500～5000m such as Sichuan of China, Qinghai, Tibet, Yunnan, Gansu, with genseng, pilose antler is equally celebrated for their achievements is the three big famous and precious nourishing diseases prevention Chinese medicinal materialss of China.But, because Chinese caterpillar fungus bat moth life cycle of the natural host insect of Cordyceps fungus is long, and the many natural enemies of many diseases, i.e. the worm source deficiency of natural propagation.Again it, environmental pollution in recent years, grassy marshland is desertified, artificial coyoting is excessively too early dug excessively and the chance that immature Chinese caterpillar fungus reduced infect the host etc., the Cordyceps sinensis quality and quantity is sharply descended, and its natural resources pursues exhausted, and the producing region that has has been dug less than Cordyceps sinensis, endangered state can not satisfy the growing demand in domestic and international market far away.
In order to remedy the huge breach between the Cordyceps sinensis supply and demand, take the method for artificial culture to produce the surrogate of Cordyceps sinensis at present.The method of its artificial culture mainly contains two classes: 1) adopt the artificial eye-liquid cultural method, obtain the mycelia and the tunning of Cordyceps fungus, make product-feed markets such as " zhiling capsuleses ".But there are the easily assorted problems such as pharmacology medicine instability that bacterium mixes, effective constituent is easily decomposed destruction, mycelia and tunning in these class methods, and this product did not both have " grass " not have " worm " yet, be not Cordyceps sinensis in essence, far apart with natural Cordyceps sinensis.2) artificial breeding Chinese caterpillar fungus bat moth larvae, the artificial inoculation Cordyceps fungus is to cultivate Cordyceps sinensis again.But these class methods exist the Chinese caterpillar fungus bat moth life generation cycle long, the life restraining factors are many, raise and depend on natural condition mostly, be difficult to finish well digenesis, expense cost height, problem such as of poor benefits indoor, do not see so far to be applied to production.
The objective of the invention is to substitute Chinese caterpillar fungus bat moth larvae with silkworm (Bombyx mori L.), after Cordyceps fungus made the physics and chemistry biological processes, be inoculated in the silkworm body, simulate natural surroundings again, make Cordyceps fungus infect parasitic silkworm body and grow the method for cultivate producing Cordyceps sinensis with silkworm of stroma.
In order to achieve the above object, the technological method of the present invention's employing is as follows:
A kind of is to gather superfine Cordyceps sinensis, after 0.1～0.2% mercuric chloride liquid surface sterilization, cuts tissue block and places on the PDA substratum and cultivate, and isolates Cordyceps fungus; Be in the tender mycelia of Cordyceps fungus children of logarithmic phase with 4～8mg/mL Driselase enzyme liquid oscillation treatment, isolate protoplastis; Place 15W ultraviolet lamp to shine 20～60sec down protoplastis, obtain that silkworm is had the mutant that infects parasitics at a distance of 10～30cm; With 30～40%PEG-Ca
++Be fusion-inducing agent, to have the protoplastis fusion of the protoplastis and the former Cordyceps fungus of the Cordyceps fungus mutant that infects parasitics to silkworm, with the cordycepin of mycelia, cordycepic acid, Cordyceps polysaccharide content with to the infection parasitics of silkworm is indication trait, therefrom cultivates to filter out not only to have kept the original proterties of Cordyceps fungus but also can infect parasitic silkworm body and the Cordyceps sinensis bacteria strain of growth well; The Cordyceps sinensis bacteria strain is contained in 1000mL distilled water be inoculated in the silkworm body after cultivating breeding on the substratum of 100～150g potato, 30～40g peptone, 30～40g glucose, 1g dipotassium hydrogen phosphate, 0.5g sal epsom, 10～15g extractum carnis, 20～30g fresh silkworm chrysalis powder, 17～20g agar, pH5.0; Under 10～30 ℃ of temperature, humidity 70～100%, the clean environment of illumination 6～24h/ day, protect, the Cordyceps sinensis bacteria strain is fully grown in the silkworm larva body or in the silkworm chrysalis body also grow stroma at last.Treat to gather in the crops when stroma grows to 5～7cm, dry under 50～60 ℃, be used for making into new drug.
Another kind is to gather superfine Cordyceps sinensis, after 0.1～0.2% mercuric chloride liquid surface sterilization, cuts tissue block and places on the PDA substratum and cultivate, and isolates Cordyceps fungus; Be in the tender mycelia of Cordyceps fungus children of logarithmic phase with 4～8mg/mL Driselase enzyme liquid oscillation treatment, isolate protoplastis; From the Bombyx Batryticatus body, isolate silkworm cause of disease muscardine, be in the tender mycelia of silkworm cause of disease muscardine children of logarithmic phase, isolate protoplastis with 4～8mg/ml Driselase enzyme liquid oscillation treatment; With 30～40%PEG-Ca
++Be fusion-inducing agent, the protoplastis of silkworm cause of disease muscardine and the protoplastis of Cordyceps fungus are merged, with the cordycepin of mycelia, cordycepic acid, Cordyceps polysaccharide content with to the infection parasitics of silkworm is indication trait, therefrom cultivates to filter out not only to have kept the original proterties of Cordyceps fungus but also can infect parasitic silkworm body and the Cordyceps sinensis bacteria strain of growth well; The Cordyceps sinensis bacteria strain is contained in 1000mL distilled water be inoculated in the silkworm body after cultivating breeding on the substratum of 100～150g potato, 30～40g peptone, 30～40g glucose, 1g dipotassium hydrogen phosphate, 0.5g sal epsom, 10～15g extractum carnis, 20～30g fresh silkworm chrysalis powder, 17～20g agar, pH5.0; Under 10～30 ℃ of temperature, humidity 70～100%, the clean environment of illumination 6～24h/ day, protect, the Cordyceps sinensis bacteria strain is fully grown in the silkworm larva body or in the silkworm chrysalis body also grow stroma at last; Treat to gather in the crops when stroma grows to 5～7cm, dry under 50～60 ℃, be used for making into new drug.
More than the inoculation method of two kinds of methods go in the aqua sterilisa for getting mycelia and conidium, making concentration is 10
6～10
8The bacterium liquid of/mL, the clean silkworm chrysalis surface that is sprayed on the clean silkworm larva surface of just casting off a skin for 2～5 ages or pupated the 1st～2 day; Or dip in inoculating needle and to get mycelia and conidium, wear out body skin direct inoculation in the clean silkworm larva body in 2～5 ages or in the clean silkworm chrysalis body of pupating the 1st～12 day.
The advantage that the present invention has is: it can make full use of the silkworm resource of China's abundant, cultivate existing " worm " and " grass " arranged and again similar in appearance to natural Cordyceps sinensis, come Reasonable Protection and utilize the rare precious Chinese medicine of this China of Cordyceps sinensis, satisfy the demand of domestic and international market, and develop the traditional sericulture of development China with the Cordyceps sinensis of high added value; It is easy to cultivate production Cordyceps sinensis with the present invention, and cost is low, can create huge social benefit and economic benefit.
Be embodiments of the invention below.Embodiment 1.
With the mycelia that 6mg/mL Driselase enzyme liquid is handled Cordyceps fungus, isolate protoplastis, place 15W ultraviolet lamp to shine 45sec down protoplastis at a distance of 20cm, obtain that silkworm is had the mutant that infects parasitics, with 35%PEG-Ca
++Be fusion-inducing agent, to have the protoplastis fusion of the protoplastis and the former Cordyceps fungus of the Cordyceps fungus mutant that infects parasitics to silkworm, with the cordycepin of mycelia, cordycepic acid, Cordyceps polysaccharide content with to the infection parasitics of silkworm is indication trait, therefrom cultivate to filter out and not only kept the original proterties of Cordyceps fungus but also can infect the Cordyceps sinensis bacteria strain that parasitic silkworm body is also grown well, after cultivating breeding, making concentration is 10
7The conidium liquid of/mL; be sprayed on the clean silkworm chrysalis surface of the producer goods kind cyanines pine that pupated the 1st day; slightly do the clean natural lighting environment protection down of 25 ℃ of humidity 100% of rearmounted temperature, treat that 20 ℃ of the hyperphoric temperature in the stiff back of silkworm chrysalis body, humidity 95%, 60W fluorescent lamp shine the clean environment protection down of 18h every day.The Cordyceps sinensis bacteria strain infects parasitic silkworm chrysalis body as a result, fully stretch out external formation stroma after the growth in vivo, the color and luster of its stroma, form are similar to natural cordyceps, and growing stroma and percentage that can fine growth is 95.8%, inoculates 20 batches and all obtains identical result.
Embodiment 2.
With the mycelia that 6mg/mL Driselase enzyme liquid is handled Cordyceps fungus, isolate protoplastis, place 15W ultraviolet lamp to shine 45sec down protoplastis at a distance of 20cm, obtain that silkworm is had the mutant that infects parasitics, with 35%PEG-Ca
++Be fusion-inducing agent; to have the protoplastis fusion of the protoplastis and the former Cordyceps fungus of the Cordyceps fungus mutant that infects parasitics to silkworm; cordycepin with mycelia; cordycepic acid; Cordyceps polysaccharide content and be indication trait to the infection parasitics of silkworm; therefrom cultivate to filter out and not only kept the original proterties of Cordyceps fungus but also can infect the Cordyceps sinensis bacteria strain that parasitic silkworm body is also grown well; after cultivating breeding; dip in inoculating needle and to get mycelia and conidium; wear out body skin direct inoculation in the clean silkworm chrysalis body of the rich 1 * 54A of producer goods kind of pupating the 3rd day; put the clean natural lighting environment protection down of 28 ℃ of humidity 100% of temperature, treat 18 ℃ of the hyperphoric temperature in the stiff back of silkworm chrysalis body; humidity 98%; the 60W fluorescent lamp shines the clean environment protection down of 20h every day.The Cordyceps sinensis bacteria strain infects parasitic silkworm chrysalis body as a result, fully stretch out external formation stroma after the growth in vivo, the color and luster of its stroma, form are similar to natural cordyceps, and growing stroma and percentage that can fine growth is 96.2%, inoculates 16 batches and all obtains identical result.
Embodiment 3.
Be the Driselase enzyme liquid processing silkworm cause of disease muscardine of 6mg/mL and the young tender mycelia of Cordyceps fungus with concentration respectively, isolate protoplastis; With 40%PEG-Ca
++Be fusion-inducing agent; the protoplastis of Cordyceps fungus and the protoplastis of silkworm cause of disease muscardine are merged; cordycepin with mycelia; cordycepic acid; Cordyceps polysaccharide content and silkworm is infected parasitics is indication trait; therefrom cultivate to filter out and not only kept the original proterties of Cordyceps fungus but also can infect the Cordyceps sinensis bacteria strain that parasitic silkworm body is also grown well; after cultivating breeding; dip in inoculating needle and to get mycelia and conidium; wear out body skin direct inoculation in the clean silkworm chrysalis body of the producer goods kind fragrant grass * stars at dawn of pupating the 5th day; put the clean natural lighting environment protection down of 26 ℃ of humidity 98% of temperature, treat 20 ℃ of the hyperphoric temperature in the stiff back of silkworm chrysalis body; humidity 100%; the 60W fluorescent lamp shines 22h protection down every day.The Cordyceps sinensis bacteria strain infects parasitic silkworm chrysalis body as a result, fully stretch out external formation stroma after the growth in vivo, the color and luster of its stroma, form are similar to natural cordyceps, and growing stroma and percentage that can fine growth is 95.6%, inoculates 23 batches and all obtains identical result.
Claims (4)
1. cultivate the method for producing Cordyceps sinensis with silkworm, it is characterized in that:
1) gathers superfine Cordyceps sinensis, after 0.1～0.2% mercuric chloride liquid surface sterilization, cut tissue block and place on the PDA substratum and cultivate, isolate Cordyceps fungus; Be in the tender mycelia of Cordyceps fungus children of logarithmic phase with 4～8mg/mL Driselase enzyme liquid oscillation treatment, isolate protoplastis; Place 15W ultraviolet lamp to shine 20～60sec down protoplastis, obtain that silkworm is had the mutant that infects parasitics at a distance of 10～30cm; With 30～40%PEG-Ca
++Be fusion-inducing agent, to have the protoplastis fusion of the protoplastis and the former Cordyceps fungus of the Cordyceps fungus mutant that infects parasitics to silkworm, with the cordycepin of mycelia, cordycepic acid, Cordyceps polysaccharide content with to the infection parasitics of silkworm is indication trait, therefrom cultivates to filter out not only to have kept the original proterties of Cordyceps fungus but also can infect parasitic silkworm body and the Cordyceps sinensis bacteria strain of growth well; The Cordyceps sinensis bacteria strain is contained in 1000mL distilled water be inoculated in the silkworm body after cultivating breeding on the substratum of 100～150g potato, 30～40g peptone, 30～40g glucose, 1g dipotassium hydrogen phosphate, 0.5g sal epsom, 10～15g extractum carnis, 20～30g fresh silkworm chrysalis powder, 17～20g agar, pH5.0
2) under 10～30 ℃ of temperature, humidity 70～100%, the clean environment of 6～24 h/ days of illumination, protect, the Cordyceps sinensis bacteria strain is fully grown in the silkworm larva body or in the silkworm chrysalis body also grow stroma at last;
3) treat to gather in the crops when stroma grows to 5～7cm, dry under 50～60 ℃, be used for making into new drug.
2. method of cultivating production Cordyceps sinensis according to claim 1 with silkworm, it is characterized in that: inoculation method is gone in the aqua sterilisa for getting mycelia and conidium, and making concentration is 10
6～10
8The bacterium liquid of/mL, the clean silkworm chrysalis surface that is sprayed on the clean silkworm larva surface of just casting off a skin for 2～5 ages or pupated the 1st～2 day; Or dip in inoculating needle and to get mycelia and conidium, wear out body skin direct inoculation in the clean silkworm larva body in 2～5 ages or in the clean silkworm chrysalis body of pupating the 1st～12 day.
3. cultivate the method for producing Cordyceps sinensis with silkworm, it is characterized in that:
1) gathers superfine Cordyceps sinensis, after 0.1～0.2% mercuric chloride liquid surface sterilization, cut tissue block and place on the PDA substratum and cultivate, isolate Cordyceps fungus; Be in the tender mycelia of Cordyceps fungus children of logarithmic phase with 4～8mg/mL Driselase enzyme liquid oscillation treatment, isolate protoplastis; From the Bombyx Batryticatus body, isolate silkworm cause of disease muscardine, be in the tender mycelia of silkworm cause of disease muscardine children of logarithmic phase, isolate protoplastis with 4～8mg/mL Driselase enzyme liquid oscillation treatment; With 30～40% PEG-Ca
++Be fusion-inducing agent, the protoplastis of silkworm cause of disease muscardine and the protoplastis of Cordyceps fungus are merged, with the cordycepin of mycelia, cordycepic acid, Cordyceps polysaccharide content with to the infection parasitics of silkworm is indication trait, therefrom cultivates to filter out not only to have kept the original proterties of Cordyceps fungus but also can infect parasitic silkworm body and the Cordyceps sinensis bacteria strain of growth well; The Cordyceps sinensis bacteria strain is contained in 1000mL distilled water be inoculated in the silkworm body after cultivating breeding on the substratum of 100～150g potato, 30～40g peptone, 30～40g glucose, 1g dipotassium hydrogen phosphate, 0.5g sal epsom, 10～15g extractum carnis, 20～30g fresh silkworm chrysalis powder, 17～20g agar, pH5.0;
2) under 10～30 ℃ of temperature, humidity 70～100%, the clean environment of illumination 6～24h/ day, protect, the Cordyceps sinensis bacteria strain is fully grown in the silkworm larva body or in the silkworm chrysalis body also grow stroma at last;
3) treat to gather in the crops when stroma grows to 5～7cm, dry under 50～60 ℃, be used for making into new drug.
4. method of cultivating production Cordyceps sinensis according to claim 3 with silkworm, it is characterized in that: inoculation method is gone in the aqua sterilisa for getting mycelia and conidium, and making concentration is 10
6～10
8The bacterium liquid of/mL, the clean silkworm chrysalis surface that is sprayed on the clean silkworm larva surface of just casting off a skin for 2～5 ages or pupated the 1st～2 day; Or dip in inoculating needle and to get mycelia and conidium, wear out body skin direct inoculation in the clean silkworm larva body in 2～5 ages or in the clean silkworm chrysalis body of pupating the 1st～12 day.
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| CN 01103275 CN1304995A (en) | 2001-01-19 | 2001-01-19 | Process for culturing cordyceps with domestic silkworm |
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| CN 01103275 CN1304995A (en) | 2001-01-19 | 2001-01-19 | Process for culturing cordyceps with domestic silkworm |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102017866B (en) * | 2009-09-23 | 2012-08-15 | 北京农业生物技术研究中心 | Cordyceps militaris bacteria culturing method |
| CN101338278B (en) * | 2008-08-07 | 2012-11-21 | 王立臣 | Standard operation method for producing Cordycepsmilitaris of Chinese medicine |
| CN103262751A (en) * | 2013-05-15 | 2013-08-28 | 刘东波 | Method for cultivating cordyceps militaris by using cordyceps militaris strains to infect silkworm larvae |
| CN103270887A (en) * | 2013-05-13 | 2013-09-04 | 玄永男 | Cordyceps militaris factory-like cultivation technology |
| CN103283687A (en) * | 2013-06-21 | 2013-09-11 | 夏西超 | Method for preparing inactivated grubs through beauveria bassiana |
| CN103539498A (en) * | 2013-09-29 | 2014-01-29 | 台州康晶生物科技有限公司 | Cordyceps militaris culture medium and application thereof |
| CN103636408A (en) * | 2013-12-18 | 2014-03-19 | 钱国琛 | Factory-like production method of silkworm cordyceps |
| CN105660188A (en) * | 2016-02-23 | 2016-06-15 | 张家港顺泰元生物科技有限公司 | Cultivation method for cordyceps militaris |
| CN105724054A (en) * | 2016-02-23 | 2016-07-06 | 张家港顺泰元生物科技有限公司 | Liquid culture solution for dried cordyceps militaris and culture method for dried cordyceps militaris |
| CN108112423A (en) * | 2017-12-27 | 2018-06-05 | 湖南省蚕桑科学研究所 | A kind of method that emulated ecological environment earthing cultivates silkworm grass |
| CN108633615A (en) * | 2018-04-24 | 2018-10-12 | 王道富 | A method of cultivating cordyceps sinensis with silkworm larvae and pupa worm |
| CN108739044A (en) * | 2017-12-29 | 2018-11-06 | 谢远泰 | A kind of silkworm larva infection parent species bacterial strain and artificial cultivating method for north Chinese caterpillar fungus |
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2001
- 2001-01-19 CN CN 01103275 patent/CN1304995A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338278B (en) * | 2008-08-07 | 2012-11-21 | 王立臣 | Standard operation method for producing Cordycepsmilitaris of Chinese medicine |
| CN102017866B (en) * | 2009-09-23 | 2012-08-15 | 北京农业生物技术研究中心 | Cordyceps militaris bacteria culturing method |
| CN103270887A (en) * | 2013-05-13 | 2013-09-04 | 玄永男 | Cordyceps militaris factory-like cultivation technology |
| CN105359825A (en) * | 2013-05-15 | 2016-03-02 | 刘东波 | Silkworm insect grass cultivating method of infecting bombyxmori larva by cordyceps fungus strain |
| CN103262751A (en) * | 2013-05-15 | 2013-08-28 | 刘东波 | Method for cultivating cordyceps militaris by using cordyceps militaris strains to infect silkworm larvae |
| CN103262751B (en) * | 2013-05-15 | 2015-09-30 | 刘东波 | A kind of method utilizing Cordyceps militaris spawn infected silkworm larva to cultivate silkworm Chinese caterpillar fungus |
| CN103283687A (en) * | 2013-06-21 | 2013-09-11 | 夏西超 | Method for preparing inactivated grubs through beauveria bassiana |
| CN103539498A (en) * | 2013-09-29 | 2014-01-29 | 台州康晶生物科技有限公司 | Cordyceps militaris culture medium and application thereof |
| CN103636408A (en) * | 2013-12-18 | 2014-03-19 | 钱国琛 | Factory-like production method of silkworm cordyceps |
| CN105660188A (en) * | 2016-02-23 | 2016-06-15 | 张家港顺泰元生物科技有限公司 | Cultivation method for cordyceps militaris |
| CN105724054A (en) * | 2016-02-23 | 2016-07-06 | 张家港顺泰元生物科技有限公司 | Liquid culture solution for dried cordyceps militaris and culture method for dried cordyceps militaris |
| CN108112423A (en) * | 2017-12-27 | 2018-06-05 | 湖南省蚕桑科学研究所 | A kind of method that emulated ecological environment earthing cultivates silkworm grass |
| CN108739044A (en) * | 2017-12-29 | 2018-11-06 | 谢远泰 | A kind of silkworm larva infection parent species bacterial strain and artificial cultivating method for north Chinese caterpillar fungus |
| CN108633615A (en) * | 2018-04-24 | 2018-10-12 | 王道富 | A method of cultivating cordyceps sinensis with silkworm larvae and pupa worm |
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