CN103828599A - Method for inducing formation of coral fungus entity - Google Patents
Method for inducing formation of coral fungus entity Download PDFInfo
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- CN103828599A CN103828599A CN201410057952.7A CN201410057952A CN103828599A CN 103828599 A CN103828599 A CN 103828599A CN 201410057952 A CN201410057952 A CN 201410057952A CN 103828599 A CN103828599 A CN 103828599A
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- methyl alcohol
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Abstract
The invention relates to a method for inducing formation of a coral fungus entity. According to the technical scheme, the method comprises the steps that after coral fungus slope strains are activated, fungus blocks are taken to be inoculated into a PDA culture medium, constant temperature culture is carried out for 15-40 d at 25 DEG C to 28 DEG C, mycelia and a solid culture medium are cut into small blocks and are soaked into organic solvents, organic leach liquor is concentrated to obtain extracts, extraction is carried out through ethyl acetate and pure water with the ratio of 1:1, an ethyl acetate phase is dehydrated and evaporated to be dried, and coral fungus organic crude extracts are manufactured. The organic extracts are directly added to the coral fungus culture medium, or a certain number of components after further separation are added to the coral fungus culture medium, the formation of the coral fungus entity can be induced, growth is 7-10 days earlier than normal growth, and growth is even.
Description
Technical field
The present invention relates to a kind of method of inducing club fungi fruit-body formation, relate to the artificial cultivation field of edible medicinal macro fungi.
Background technology
Fruit body development is the macro fungi important stage of the history of life, after macro fungi mycelium is nourished and grown, and through the stimulation of the envirment factor such as illumination, temperature certain hour, mycelium knot Cheng Yuanji, the quantity of former base and quality directly affect the output of fruit body.Natural Macro-Fungi Resource is abundant, and sum approximately has 2000-5000 kind, and approximately there is 1500-2000 kind in China, report reach 938 kinds, be under the jurisdiction of 166 genus, at present can artificial cultivation less than 100 kinds, approximately 20 kinds of large-scale production cultivation.A large amount of wild edible and medical fungis (as club fungi, Chinese caterpillar fungus, russule, hickory chick, bolete etc.) are though have special medical value and abundant nutritive value, but more difficultly obtain fruit body by artificial cultivation, limited making full use of of its medicinal nutritive value because natural resources are rare.
Club fungi is to be worth higher wild edible and medical fungi, its fine and tender taste, fresh and sweet tasty and refreshing, property flat taste sweet, contain abundant high-quality protein and several amino acids, normal food can beauty treatment skin, improve body immunity, there is easing stomach-QI, dispel the wind, delay medium effect, among the peoplely be commonly used to cure the diseases such as stomachache, indigestion and wind pain, Clavicoronic acid is the medicinal lead compound separating from club fungi fruit body, has the effect that suppresses reverse transcriptase activity.The method that the present invention utilizes the metabolite of club fungi self to induce its fruit-body formation to grow have not been reported.The present invention is also significant to inquiring into the cometabolism effect of wild edible and medical fungi and the relation of fruit body development and artificial cultivation utilization.
Summary of the invention
The object of the present invention is to provide a kind of method of inducing club fungi fruit-body formation, started the new technology of utilizing self extract induction edible medicinal macro fungi fruit-body formation to grow.
The technical scheme that the present invention takes is as follows:
By club fungi (
clavicoronapyxidata, Clavicorolides A and B, sesquiterpenoids from the Fermentation Products of
clavicoronapyxidata.organic Letters, 2009,11 (1): 109-112.) after slant strains activation, get bacterium piece and be inoculated in potato glucose solid culture medium, in 25-28 ℃ of constant temperature culture 15-40 d, mycelium is cut into small pieces together with solid culture medium, is immersed in organic solvent, then organic leaching liquor is used after Rotary Evaporators evaporate to dryness, obtain medicinal extract, extract with ethyl acetate and pure water 1:1, evaporate to dryness after ethyl acetate phase dehydration, makes club fungi organic coarse extract.Component after organic extract directly or is further separated joins in club fungi medium with a certain amount of, can induce the formation of club fungi fruit body.
Concrete
A kind of method of inducing club fungi fruit-body formation, it is characterized in that: by after the activation of club fungi slant strains, getting bacterium piece is inoculated in potato glucose solid culture medium, in 25-28 ℃ of constant temperature culture 15-40d, mycelium is cut into small pieces together with solid culture medium, be immersed in and in organic solvent, obtain organic leaching liquor, then organic leaching liquor is used after Rotary Evaporators evaporate to dryness, obtain medicinal extract, with ethyl acetate and pure water 1:1(v/v) extract, evaporate to dryness after ethyl acetate phase dehydration, makes club fungi organic coarse extract; The component that club fungi organic coarse extract directly or is further separated joins in potato glucose medium, and additional proportion is crude extract or the further component 1mg separating: potato glucose medium 20mL, can promote the growth of club fungi fruit body; The component of wherein said further separation is by after RP18 chromatographic column by club fungi organic coarse extract, carry out wash-out take water, 30% methyl alcohol, 50% methyl alcohol, 70% methyl alcohol and 100% methyl alcohol as eluant, eluent, collect the wash-out part of 100% methyl alcohol, concentrated by rotary evaporation is removed methyl alcohol, obtains component.
Described potato glucose solid culture medium is: 200 g/L murphy juice+glucose 20 g/L+ agar 10 g/L.
Described organic solvent is that volume ratio is ethyl acetate: the mixed solvent of methyl alcohol: acetic acid=80:15:5.
The present invention adopts club fungi self extract to induce its fruit-body formation, have the fruit-body formation time shorter, grow the advantages such as neat.Fruit body development is the macro fungi important stage of the history of life, after macro fungi mycelium is nourished and grown, in the stimulation of the suitable physicochemical environment such as illumination, temperature factor certain hour, mycelium knot Cheng Yuanji, the quantity of former base and quality directly affect the output of fruit body.Production practices show that the physical chemical factor of the environment such as strain quality, medium component, training method, illumination, temperature and ventilation can affect fruit body of edible fungi output, the beneficial microbes such as many bacteriums, actinomycetes and mould also can promote fruit body development, but the artificial cultivation to wild edible and medical fungi and to improving output, the effect of above conventional method is very limited.The present invention has disclosed the effect of its secondary metabolite of macro fungi (comprising edible and medical fungi) in fruit body development process, utilize the metabolic components induction of club fungi self to form about 41 days of the time of fruit body, improve 7-10 days than normal growth, and grown neat.The present invention is that the illustrating of problem in science of fruit body development mechanism lays the foundation from brand-new angle, provides new method in particular for utilization that in a large number can not tame wild edible and medical fungi.
Accompanying drawing explanation
The induction of Fig. 1 club fungi organic extract subfraction to fruit-body formation.
Embodiment
Embodiment 1
The induction step of club fungi fruit-body formation is as follows:
1) preparation solid culture medium.Described solid culture medium is potato glucose medium (formula: 200 g peeling potatos are boiled and soak juice, glucose 20 g, agar 10 g, water 1 L), 121 ℃ of high pressure steam sterilization 30 min.
2) club fungi solid culture.By club fungi (
clavicoronapyxidata, Clavicorolides A and B, sesquiterpenoids from the Fermentation Products of
clavicoronapyxidata.organic Letters, 2009,11 (1): 109-112.) slant strains activation, the slant strains of activation is chosen by 0.5～1.0cm
2bacterium piece is inoculated in the culture dish (diameter 9 cm) of the described solid culture medium that 20 ml are housed, and in 26 ℃, cultivates 25d.
3) preparation of club fungi organic extract.Cultured club fungi mycelium is cut into small pieces together with solid culture medium, be immersed in organic extract (ethyl acetate: methyl alcohol: acetic acid=80:15:5(v/v)) in, after 24 hours by eight layers of filtered through gauze, organic leaching liquor is used after Rotary Evaporators evaporate to dryness, obtain medicinal extract, ethyl acetate and pure water 1:1(v/v for medicinal extract) extract, evaporate to dryness after anhydrous sodium sulfate dehydration for ethyl acetate, makes club fungi organic coarse extract 2.25 g.
4) preparation of club fungi organic extract subfraction.Organic coarse extract is carried out to RP18 silica gel column chromatography, and (170 g), with water, 30% methyl alcohol, 50% methyl alcohol, 70% methyl alcohol and 100% methyl alcohol are eluant, eluent wash-out 1L respectively, 2L, 2L, 2L, 2L, be in charge of collection, every pipe is collected 250 ml, after concentrating respectively, obtain different component, the wash-out part of 100% methyl alcohol is merged into Fr.8, have 241.9 ㎎, other elution fraction is selected silica gel plate G25 according to TLC(, solvent is chloroform: methyl alcohol=10:1) analysis result, merge into respectively Fr.1(114 ㎎), Fr.2(319.8 ㎎), Fr.3(4.6 ㎎), Fr.4(240.6 ㎎), Fr.5(195.2 ㎎), Fr.6(156.1 ㎎) and Fr.7(80.7 ㎎).
Embodiment 2
The induction step of club fungi fruit-body formation is as follows:
1) preparation solid culture medium.Described solid culture medium is potato glucose medium (formula: 200 g peeling potatos are boiled and soak juice, glucose 20 g, agar 10 g, water 1 L), 121 ℃ of high pressure steam sterilization 30 min.
2) club fungi solid culture.By club fungi (
clavicoronapyxidata, Clavicorolides A and B, sesquiterpenoids from the Fermentation Products of
clavicoronapyxidata.organic Letters, 2009,11 (1): 109-112.) slant strains activation, the slant strains of activation is chosen by 0.5～1.0cm
2bacterium piece is inoculated in the culture dish (diameter 9 cm) of the described solid culture medium that 20 ml are housed, and in 28 ℃, cultivates 15d.
3) preparation of club fungi organic extract.Cultured club fungi mycelium is cut into small pieces together with solid culture medium, be immersed in organic extract (ethyl acetate: methyl alcohol: acetic acid=80:15:5(v/v)) in, then use eight layers of filtered through gauze, organic leaching liquor is used after Rotary Evaporators evaporate to dryness, obtain medicinal extract, ethyl acetate and pure water 1:1(v/v for medicinal extract) extract, evaporate to dryness after anhydrous sodium sulfate dehydration for ethyl acetate, makes club fungi organic coarse extract.
4) preparation of club fungi organic extract subfraction.Organic coarse extract is carried out to RP18 silica gel column chromatography, and (170 g), difference wash-out 1L, 2L, 2L, 2L, 2L take water, 30% methyl alcohol, 50% methyl alcohol, 70% methyl alcohol and 100% methyl alcohol as eluant, eluent, be in charge of collection, every pipe is collected 250 ml, after concentrating respectively, obtains different component, and the wash-out part of 100% methyl alcohol is merged into Fr.8, other elution fraction is selected silica gel plate G25 according to TLC(, solvent is chloroform: methyl alcohol=10:1) analysis result, merge into respectively Fr.1～Fr.7,7 components.
Embodiment 3
The induction step of club fungi fruit-body formation is as follows:
1) preparation solid culture medium.Described solid culture medium is potato glucose medium (formula: 200 g peeling potatos are boiled and soak juice, glucose 20 g, agar 10 g, water 1 L), 121 ℃ of high pressure steam sterilization 30 min.
2) club fungi solid culture.By club fungi (
clavicoronapyxidata, Clavicorolides A and B, sesquiterpenoids from the Fermentation Products of
clavicoronapyxidata.organic Letters, 2009,11 (1): 109-112.) slant strains activation, the slant strains of activation is chosen by 0.5～1.0cm
2bacterium piece is inoculated in the culture dish (diameter 9 cm) of the described solid culture medium that 20 ml are housed, and in 25 ℃, cultivates 40d.
3) preparation of club fungi organic extract.Cultured club fungi mycelium is cut into small pieces together with solid culture medium, be immersed in organic extract (ethyl acetate: methyl alcohol: acetic acid=80:15:5(v/v)) in, after 24 hours by eight layers of filtered through gauze, organic leaching liquor is used after Rotary Evaporators evaporate to dryness, obtain medicinal extract, ethyl acetate and pure water 1:1(v/v for medicinal extract) extract, evaporate to dryness after anhydrous sodium sulfate dehydration for ethyl acetate, makes club fungi organic coarse extract.
4) preparation of club fungi organic extract subfraction.Organic coarse extract is carried out to RP18 silica gel column chromatography, and (170 g), difference wash-out 1L, 2L, 2L, 2L, 2L take water, 30% methyl alcohol, 50% methyl alcohol, 70% methyl alcohol and 100% methyl alcohol as eluant, eluent, be in charge of collection, every pipe is collected 250 ml, after concentrating respectively, obtains different component, and the wash-out part of 100% methyl alcohol is merged into Fr.8, other elution fraction is selected silica gel plate G25 according to TLC(, solvent is chloroform: methyl alcohol=10:1) analysis result, merge into respectively Fr.1 ~ Fr.7,7 components.
Embodiment 4
The induction of the organic coarse extract that embodiment 1-3 obtains to club fungi fruit-body formation.By sterile working, organic coarse extract is made an addition on circular filter paper sheet (diameter 6 ㎜), addition is 1mg, filter paper is attached to inoculation to be had on potato glucose solid culture medium (20 ml) flat board of club fungi, after 25 days left and right mycelium cover with in 25 ℃ of constant incubators, move on to room temperature and cultivate.Do un-added blank test, result shows simultaneously, and organic coarse extract has obvious inducing action to the formation of club fungi fruit body, and the time of fruit-body formation is all than blank test 10 days ahead of time.
Embodiment 5
The induction of the Fr.8 component that embodiment 1-3 obtains to club fungi fruit-body formation.By sterile working, eight components are made an addition to respectively on circular filter paper sheet (diameter 6 ㎜), addition is every component 1mg, filter paper is attached to inoculation to be had on potato glucose solid culture medium (20 ml) flat board of club fungi, after 25 days left and right mycelium cover with in 25 ℃ of constant incubators, move on to room temperature and cultivate.Result shows, the 8th component has obvious inducing action (Fig. 1) to the formation of club fungi fruit body, and the time of fruit-body formation is done sth. in advance 7-10 days than the component of other separation.
Embodiment 6
The induction of the Fr.8 component that embodiment 1-3 obtains to club fungi fruit-body formation.By sterile working, Fr.8 is made an addition to respectively on circular filter paper sheet (diameter 6 ㎜), addition is 1mg, filter paper is attached to inoculation to be had on potato glucose solid culture medium (20 ml) flat board of club fungi, after 25 days left and right mycelium cover with in 25 ℃ of constant incubators, move on to room temperature and cultivate.Do un-added blank test, result shows simultaneously, and Fr.8 component has obvious inducing action to the formation of club fungi fruit body, and the time of fruit-body formation was than blank test 10 days ahead of time.
Claims (3)
1. induce the method for club fungi fruit-body formation for one kind, it is characterized in that: by after the activation of club fungi slant strains, getting bacterium piece is inoculated in potato glucose solid culture medium, in 25-28 ℃ of constant temperature culture 15-40d, mycelium is cut into small pieces together with solid culture medium, be immersed in and in organic solvent, obtain organic leaching liquor, then organic leaching liquor is used after Rotary Evaporators evaporate to dryness, obtain medicinal extract, with ethyl acetate and pure water 1:1(v/v) extract, evaporate to dryness after ethyl acetate phase dehydration, makes club fungi organic coarse extract; The component that club fungi organic coarse extract directly or is further separated joins in potato glucose medium, and additional proportion is crude extract or the further component 1mg separating: potato glucose medium 20mL, can promote the growth of club fungi fruit body; The component of wherein said further separation is by after RP18 chromatographic column by club fungi organic coarse extract, carry out wash-out take water, 30% methyl alcohol, 50% methyl alcohol, 70% methyl alcohol and 100% methyl alcohol as eluant, eluent, collect the wash-out part of 100% methyl alcohol, concentrated by rotary evaporation is removed methyl alcohol, obtains component.
2. the method for claim 1, is characterized in that, described potato glucose solid culture medium is: 200 g/L murphy juice+glucose 20 g/L+ agar 10 g/L.
3. the method for claim 1, is characterized in that described organic solvent is that volume ratio is ethyl acetate: the mixed solvent of methyl alcohol: acetic acid=80:15:5.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105875193A (en) * | 2015-01-09 | 2016-08-24 | 北京中农绿源工程技术有限公司 | Method for inducing toadstool anlage to form through Led lamp |
| CN106332654A (en) * | 2016-08-23 | 2017-01-18 | 山东省科创食用菌产业技术研究院 | Cultivation method of red grape-like coral fungi |
| CN106718052A (en) * | 2016-12-15 | 2017-05-31 | 防城港市蓝瀚达科技有限公司 | The artificial cultivation method of club fungi |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105875193A (en) * | 2015-01-09 | 2016-08-24 | 北京中农绿源工程技术有限公司 | Method for inducing toadstool anlage to form through Led lamp |
| CN106332654A (en) * | 2016-08-23 | 2017-01-18 | 山东省科创食用菌产业技术研究院 | Cultivation method of red grape-like coral fungi |
| CN106718052A (en) * | 2016-12-15 | 2017-05-31 | 防城港市蓝瀚达科技有限公司 | The artificial cultivation method of club fungi |
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