CN1670179A - Method for preparing fruiting body and mycelium by artificial culture of inonotus obliquus - Google Patents
Method for preparing fruiting body and mycelium by artificial culture of inonotus obliquus Download PDFInfo
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- CN1670179A CN1670179A CN 200510038419 CN200510038419A CN1670179A CN 1670179 A CN1670179 A CN 1670179A CN 200510038419 CN200510038419 CN 200510038419 CN 200510038419 A CN200510038419 A CN 200510038419A CN 1670179 A CN1670179 A CN 1670179A
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Abstract
This invention discloses a method for acquiring Inonotus fruiting body and mycelium by fermenting in solid or liquid mediums. Wherein, in solid method, manually culturing Inonotus fruiting body and mycelium in solid medium; in liquid method, adopting shake fermenting culture, shake enlargement culture and fermenter culture. The invention supplies a new nutrition source and medicine source.
Description
Technical field:
The present invention relates to a kind of QINGGANJUN artificial culture and form sporophore, mycelial method.
Technical background:
QINGGANJUN Inonotus obliquus has another name called Chaga or Phaeopoms obliquus Fuscoporia obliqua, at Russia and the Eastern Europe Chaga that cries among the people, belong to Basidiomycotina, Hymenomycetes, non-brown Zoopagales, polyporaceae, the bark of being born in white birch, silvery birch, elm, alder etc. down or the bark of the standing tree of living down or on felling back trees dried-up.
16-17 is since century, and Russia, Poland, Finland etc. are among the people just extensively to utilize its wild sporophore and sclerotium to prevent various difficult and complicated cases, as various cancers (various digestion organs cancers such as cancer of the stomach, liver cancer, intestinal cancer) and heart trouble and diabetes etc.Poland just has been extensive use of QINGGANJUN and treats cancer since 1961.Just the researchist of Japan speaks highly of this medicine among the people of Russia, and thinks that this is a kind of peculiar present that God grants the suffering mankind, can be used to prevent and treat liver cancer, AIDS and O-157 intestinal bacteria and poisons.It is reported that the smart powder of the QINGGANJUN that the Muscovite Suo Molesiji of bearing (Komsomlski) drugmaker produces reaches 93% to the curative ratio of diabetes.(over yellow year, medicinal fungi-Phaeopoms obliquus among the people [J] that Russia is mysterious, edible fungi of china, 2002,21 (4): 7-8) the decocting Stewed Dish of QINGGANJUN has notable antitumor activity.The syrup of QINGGANJUN early is used for the treatment of cancer.QINGGANJUN contains multiple lanosterol type triterpene compound, lanosterol, the acid of bolt bacterium, folic acid derivatives, aromatic vanillic acid, syringic acid and polysaccharide etc.But the QINGGANJUN source is rare, and therefore, the present invention forms sporophore by the artificial culture QINGGANJUN and mycelium solves the rare problem in source.
Summary of the invention:
The purpose of this invention is to provide a kind of by artificial culture QINGGANJUN sporophore and mycelial method.
Realize that technical scheme of the present invention is: with the QINGGANJUN fungi is starting strain, adopts liquid and solid to send out this season and cultivates, with dry sporophore and the hypha powder of getting of the mycelia of liquid and solid fermentation cultivation.
QINGGANJUN artificial culture of the present invention forms sporophore and mycelial method may further comprise the steps:
1, the QINGGANJUN artificial culture forms sporophore, mycelial method steps:
(1) QINGGANJUN artificial culture formation sporophore and mycelial method are solid and liquid fermentation and culture method, and its key step is
1. strain separating: the fresh QINGGANJUN sclerotium of field acquisition is adopted tissue isolation after sterilizing, connect
1. strain separating: the fresh QINGGANJUN sclerotium of field acquisition is adopted tissue isolation after sterilizing, be inoculated on the bacterium culture medium, cultivated 6-7 days down, obtain pure strain I082669 at 20-28 ℃.
2. mycelium inoculation: with above-mentioned bacterial classification inoculation on the sporophore substratum that solid medium or liquid pH value through sterilization are 6.5-10.0.
3. mycelium culture: mycelial growth stage culture condition is a cultured continuously under the lucifuge condition, and temperature is 20-28 ℃, and relative humidity is 60-70%.
4. sporophore is cultivated: at sporophore cultivation stage culture condition is cultured continuously under the dark condition, and temperature is 2-28 ℃, and relative humidity is 70-85%, and solid medium was cultivated after 40 days, can grow faint yellow or the xanchromatic sporophore.
The 1. described spawn culture based formulas of step is: potato 200g/L, barley-sugar 6g/L, sucrose 5g/L, yeast extract paste 0.5g/L, agar 20g/L, distilled water 1L.
2. described solid sporophore of step and mycelium culture base are:
Corn solids substratum: corn 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0; Or wheat solid medium: wheat 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0; Or birch or willow wood sawdust solid medium: birch or willow wood sawdust 98%, sucrose 1%, peptone 1%,
Water content 60-65%, pH6.5-10.0.Above-mentioned substratum all gets through the autoclaving modulation.
The bacterial classification of the 2. described inoculation usefulness of step is that the mother that 1. step makes plants mycelia or liquid spawn.
3. or 4. step is preferably under the 23-26 ℃ of condition and cultivates.
Described liquid fermentation process is to adopt the liquid shaking bottle fermentation culture, liquid shaking bottle enlarged culturing again, and fermentor cultivation is to growing mycelium again, and concrete steps are as follows:
(1) shake-flask culture: the shake-flask culture base is: potato 200g/L, barley-sugar 6g/L, sucrose 5g/L, yeast extract paste 0.5g/L, adding distil water is to 1l, the pH value is 6.5-10.0, culture condition is: culture temperature 23-28 ℃, rotating speed 130-190rpm, cultivated 110-150 hour, switching is gone into to shake bottle and is carried out enlarged culturing then.
(2) shake a bottle enlarged culturing: shaking a bottle enlarged culturing base is: potato 100g/L, Semen Maydis powder 20g/L, barley-sugar 2g/L, sucrose 5g/L, yeast extract paste 0.5g/L, adding distil water, pH6.5-10.0, culture condition is: culture temperature 23-28 ℃, tank pressure is more than 0.1bar, rotating speed 120-180rpm cultivated 96-150 hour, and the first order seed cultivation is gone in switching then.
(3) first order seed is cultivated: the first order seed substratum is formed: white sugar 20g/L, peptone 4g/L, yeast extract paste 3g/L, MgSO
40.5g/L, KH
2PO
40.66g/L, K
2HPO
41g/L pH6.5-10.0, adding distil water is settled to volume required, culture condition is: culture temperature is 23-28 ℃, tank pressure is more than 0.1bar, stir speed (S.S.) is 220rpm, dissolved oxygen DO maintains and (controls) fermentation time by changing air flow and stir speed (S.S.) more than 20% is 72-96 hour, and ferment tank is gone in switching then.
(4) ferment tank is cultivated, the ferment tank culture medium is formed as described in the step (3), the pH value is pH6.5-10.0, inoculum size is 12%, and culture condition is: culture temperature 23-28 ℃, tank pressure is more than 0.1bar, stir speed (S.S.) 300rpm, dissolved oxygen DO maintains more than 20% and (controls by changing air flow and stir speed (S.S.)), and fermentation time is 90-260 hour, gets mycelium.
Embodiment
Further set forth method of the present invention below in conjunction with embodiment:
1, isolating QINGGANJUN bacterial classification is connected on the bacterium culture medium, under 23-25 ℃ of temperature, cultivates a week, obtain female mycelium of planting.
The spawn culture based formulas is: potato 200g/L, barley-sugar 6g/L, sucrose 5g/L, yeast extract paste 0.5g/L, agar 20g/L, distilled water 1L.
2 QINGGANJUN artificial culture form sporophore and mycelial method
Be respectively charged into corn culture medium or wheat broth or birch or willow sawmilling substratum with plastics bacterium bag and tighten plastics bacterium bag, put into high-pressure sterilizing pot, 1.5kg/cm
2Autoclaving 3-6 hour, promptly obtain the solid medium that mycelium is cultivated.Mother following of aseptic worktable plants mycelia or liquid spawn (method making routinely) 2-3ml, is inoculated in the above-mentioned solid medium.Be placed on the cultivation indoor cultivation, temperature is 23-26 ℃, cultivates 40-60 days, can collect mycelium.The corn solids culture medium prescription is: corn 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0; Wheat solid culture based formulas is: wheat 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0; Birch or willow wood sawdust solid medium: birch or willow wood sawdust 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0.
Be respectively charged into corn solids substratum or wheat solid medium or birch or willow wood sawdust solid medium with plastics bacterium bag and tighten plastics bacterium bag, put into high-pressure sterilizing pot, 1.5kg/cm
2Autoclaving 3-6 hour, promptly obtain the solid medium that sporophore is cultivated.Mother at the following embodiment 1 of aseptic worktable plants mycelium or liquid spawn (method making routinely) 2-3ml, is inoculated in the above-mentioned solid medium.The bacterium bag is placed on the cultivation indoor cultivation, and temperature is 23-26 ℃, wait mycelia to cover with the bacterium bag after, culture condition is a cultured continuously under the dark condition, and temperature is 20-28 ℃, and relative humidity is 70-85%, solid medium was cultivated after 40 days, can grow faint yellow or the xanchromatic sporophore.Cultivated 50-60 days, and can collect sporophore.The corn solids culture medium prescription is: corn (or corn stalk) 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0; Wheat solid culture based formulas is: wheat 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0; Birch or willow sawmilling solid medium: birch or willow wood sawdust 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0.
3 QINGGANJUN artificial culture form sporophore and mycelial method
The triangular flask that the 500ml of 150ml corn liquid nutrient medium or wheat liquid nutrient medium or birch or willow wood sawdust liquid nutrient medium is housed is being put into high-pressure sterilizing pot, 1.5kg/cm
2Autoclaving 30 minutes, cooling is planted mycelia or liquid spawn (method making routinely) 2-3ml with bacterial classification the mother of the following embodiment 1 of aseptic worktable, is inoculated in the aforesaid liquid substratum.23-26 ℃ of shaking table cultivated 96 hours, and shaking speed is 130rpm/min.The shake-flask culture based formulas: potato 200g/L, barley-sugar 6g/L, sucrose 5g/L, yeast extract paste 0.5g/L, adding distil water is to 1L.The pH value is 6.5-10.0, and culture condition is: culture temperature 23-28 ℃, rotating speed 130-190rpm cultivated 110-150 hour, and switching is gone into to shake bottle and carried out enlarged culturing then.
The seeding tank seed culture: with shake-flask culture mycelia about one week, after inspection, in aseptic condition inoculation down, inoculum size is that 5-15% cultivates in jar.The first order seed substratum is formed: white sugar 20g/L, peptone 4g/L, yeast extract paste 3g/L, MgSO
40.5g/L, KH
2PO
40.66g/L, K
2HPO
41g/LpH6.5-10.0, adding distil water is settled to volume required, culture condition is: culture temperature is 23-28 ℃, tank pressure is more than 0.1bar, stir speed (S.S.) is 220rpm, dissolved oxygen DO maintains and (controls) fermentation time by changing air flow and stir speed (S.S.) more than 20% is 72-96 hour, and ferment tank is gone in switching then.Ferment tank is cultivated: inoculum size is 12%, and fermentation tank culture medium and seeding tank are together.Culture condition is: culture temperature 23-28 ℃, tank pressure is more than 0.1bar, and stir speed (S.S.) 300rpm, dissolved oxygen DO maintain more than 20% and (control by changing air flow and stir speed (S.S.)), and fermentation time is 90-260 hour.Mycelial collection is with dry: after the fermentation culture, using filtered through gauze, is the centrifugal 20-30 of 1500rpm minute with centrifuge speed, obtains mycelium.
Claims (6)
1, a kind of QINGGANJUN artificial culture forms sporophore and mycelial method, it is characterized in that with QINGGANJUN Inonotus obliquus be starting strain, adopt liquid and solid fermentation to cultivate, with dry sporophore and the mycelium of getting of the mycelia of liquid and solid fermentation cultivation, concrete grammar is as follows:
1. strain separating: the fresh QINGGANJUN sclerotium of field acquisition is adopted tissue isolation after sterilizing, be inoculated on the bacterium culture medium, cultivated 6-7 days down, obtain pure strain I082669 at 20-28 ℃;
2. mycelium inoculation: is on 6.5-10.0 solid medium or the liquid nutrient medium with above-mentioned bacterial classification inoculation in the pH value through sterilization;
3. mycelium culture: mycelial growth stage culture condition is for to carry out solid or liquid fermenting cultured continuously under the lucifuge condition, temperature is 23-28 ℃, and relative humidity is 60-70%, to growing mycelium;
4. sporophore is cultivated: in sporophore cultivation stage dark condition cultured continuously, temperature is 23-28 ℃, and relative humidity is 70-85%, and solid medium was cultivated after 40 days, grew faint yellow or the xanchromatic sporophore.
2, method according to claim 1 is characterized in that the 1. described spawn culture based formulas of step is: potato 200g/L, barley-sugar 6g/L, sucrose 5g/L, yeast extract paste 0.5g/L, agar 20g/L, distilled water 1L.
3, method according to claim 1 is characterized in that 2. described solid sporophore of step and mycelium culture base are:
Corn solids substratum: corn 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0 or wheat solid medium: wheat 98%, sucrose 1%, peptone 1%.Water content 60-65%, pH6.5-10.0 or birch or willow wood sawdust solid medium: birch or willow wood sawdust 98%, sucrose 1%, peptone 1%, water content 60-65%, pH6.5-10.0.Above-mentioned substratum all gets through the autoclaving modulation.
4, method according to claim 1, the bacterial classification that it is characterized in that the 2. described inoculation usefulness of step are female plant mycelia or liquid spawns.
5, method according to claim 1, after it is characterized in that inoculating, 3. or 4. step is preferably under the 23-26 ℃ of condition and cultivates.
6, method according to claim 1 is characterized in that described liquid fermentation process is to adopt the liquid shaking bottle fermentation culture, liquid shaking bottle enlarged culturing again, and fermentor cultivation is to growing mycelium again, and concrete steps are as follows:
(1) shake-flask culture: the shake-flask culture base is: potato 200g/L, barley-sugar 6g/L, sucrose 5g/L, yeast extract paste 0.5g/L, adding distil water is to 1L, the pH value is 6.5-10.0, culture condition is: culture temperature 23-28 ℃, rotating speed 130-190rpm, cultivated 110-150 hour, switching is gone into to shake bottle and is carried out enlarged culturing then.
(2) shake a bottle enlarged culturing: shaking a bottle enlarged culturing base is: potato 100g/L, Semen Maydis powder 20g/L, barley-sugar 2g/L, sucrose 5g/L, yeast extract paste 0.5g/L, adding distil water, pH6.5-10.0, culture condition is: culture temperature 23-28 ℃, tank pressure is more than 0.1bar, rotating speed 120-180rpm cultivated 96-150 hour, and the first order seed cultivation is gone in switching then.
(3) first order seed is cultivated: the first order seed substratum is formed: white sugar 20g/L, peptone 4g/L, yeast extract paste 3g/L, MgSO
40.5g/L, KH
2PO
40.66g/L, K
2HPO
41g/L pH6.5-10.0, adding distil water is settled to volume required, and culture condition is: culture temperature is 23-28 ℃, tank pressure is more than 0.1bar, and stir speed (S.S.) is 220rpm, and dissolved oxygen DO maintains more than 20%, fermentation time is 72-96 hour, and ferment tank is gone in switching then.
(4) ferment tank is cultivated, the substratum that ferment tank is cultivated is formed as described in the step (3), the pH value is pH6.5-10.0, inoculum size is 12%, and culture condition is: culture temperature 23-28 ℃, tank pressure is more than 0.1bar, stir speed (S.S.) 300rpm, dissolved oxygen DO maintains more than 20%, and fermentation time is 90-260 hour, gets mycelium.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502218B (en) * | 2009-03-11 | 2010-08-18 | 北京林业大学 | Artificial cultivation method for sclerotium and fruiting body of Inonotus obliquus |
| CN102523921A (en) * | 2012-01-11 | 2012-07-04 | 福建农林大学 | Preparation method and products of needle mushroom mycelium pellets |
| CN102523920A (en) * | 2012-01-11 | 2012-07-04 | 福建农林大学 | Preparation method of Grifola frondosa mycelial pellets and products thereof |
| JP2017176032A (en) * | 2016-03-30 | 2017-10-05 | 群馬県 | Cultivation method and cultivation apparatus for fruit body of mushroom |
| CN108795770A (en) * | 2017-04-27 | 2018-11-13 | 天明制药股份有限公司 | To improve the process for solid culture of polysaccharides content in inonotus obliquus |
-
2005
- 2005-03-11 CN CN 200510038419 patent/CN1670179A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502218B (en) * | 2009-03-11 | 2010-08-18 | 北京林业大学 | Artificial cultivation method for sclerotium and fruiting body of Inonotus obliquus |
| CN102523921A (en) * | 2012-01-11 | 2012-07-04 | 福建农林大学 | Preparation method and products of needle mushroom mycelium pellets |
| CN102523920A (en) * | 2012-01-11 | 2012-07-04 | 福建农林大学 | Preparation method of Grifola frondosa mycelial pellets and products thereof |
| CN102523921B (en) * | 2012-01-11 | 2013-12-04 | 福建农林大学 | Preparation method and products of needle mushroom mycelium pellets |
| CN102523920B (en) * | 2012-01-11 | 2013-12-04 | 福建农林大学 | Preparation method of Grifola frondosa mycelial pellets and products thereof |
| JP2017176032A (en) * | 2016-03-30 | 2017-10-05 | 群馬県 | Cultivation method and cultivation apparatus for fruit body of mushroom |
| CN108795770A (en) * | 2017-04-27 | 2018-11-13 | 天明制药股份有限公司 | To improve the process for solid culture of polysaccharides content in inonotus obliquus |
| CN108795770B (en) * | 2017-04-27 | 2021-09-03 | 天明制药股份有限公司 | Solid state culture method for increasing polysaccharide content in inonotus obliquus |
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