CN102172172B - Method for culturing Schizophyllum commune - Google Patents
Method for culturing Schizophyllum commune Download PDFInfo
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- CN102172172B CN102172172B CN2011100465857A CN201110046585A CN102172172B CN 102172172 B CN102172172 B CN 102172172B CN 2011100465857 A CN2011100465857 A CN 2011100465857A CN 201110046585 A CN201110046585 A CN 201110046585A CN 102172172 B CN102172172 B CN 102172172B
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Abstract
The invention discloses a method for culturing Schizophyllum commune. The method comprises the steps of activating a Schizophyllum commune mother strain to obtain a first-level strain; and then fermenting and culturing the first-level strain in a liquid fermentation medium for 4-6 days at temperature of 24-28 DEG C and rotation speed of 200-260 rpm/min to obtain a fermentation mixed solution; filtering the fermentation mixed solution; collecting Schizophyllum commune hyphae; drying and crushing the Schizophyllum commune hyphae and the like. The Schizophyllum commune with high yield is fermented and cultured by using the method disclosed by the invention; mycelia with wet weight of 400-480 g or dry weight of 54-60 g can be obtained from each liter of fermentation strain solution; and compared with the prior art, the Schizophyllum commune has largely increased yield.
Description
The application is that application number is: 200810247561.6, the applying date the dividing an application for the patent application of " Schizophyllum commune protein extract " that be on December 30th, 2008, denomination of invention.
Technical field
The invention belongs to the control of plant disease field, relate in particular to Schizophyllum commune protein extract; In addition, the cultural method and the medium that also relate to schizophyllum commune.
Background technology
The anthropoid cancer of viroses of plant class does not still have the highly effective method of preventing and treating at present.(Tobacco mosaic virus is one of the virus of tool Economic Importance TMV) to tobacco mosaic virus, can infect 300 various plants such as Solanaceae, Curcurbitaceae, Cruciferae; And cause serious harm; Viroses of plant control relies on the method for breeding and chemical agent to solve usually, but effect is all undesirable, and the former is subject to the source of disease-resistant gene; The latter is weak effect then, can cause problems such as environmental pollution simultaneously.In recent years, Along with people's is to the attention of environment, and the application of chemical agent is restricted, and environmentally friendly biogenic Antiphytoviral medicament comes into one's own.
Schizophyllum commune (Schizophyllum communeFr) is claimed white ginseng, tree flower again, belongs to Eumycota, Basidiomycotina, Hymenomycetes, Aphyllophorales, schizophyllum commune section, Schizophyllum.Schizophyllum commune is widely distributed in all over the world, and China's most of areas all has distribution.In recent years, states such as Japan and America and Europe are mainly studied schizophyllum commune from the aspects such as seed selection of molecular biology, anti-cancer active matter and strain excellent, and the research of China then concentrates on fermentation condition, artificial domesticating cultivation and the medical value of schizophyllum commune.Contain multiple anti-inflammation, antitumor in the schizophyllum commune, radioresistance isoreactivity material, but its application study focuses mostly at medical domain, and the effect aspect Antiphytoviral does not relate to.
Patent " culture technology of schizophyllum commune and utilization thereof " (patent No. ZL85103492) discloses the method for the medium culture schizophyllum commune of forming with analysis for soybean powder, glucose or sucrose, yeast extract, magnesium sulfate, potassium dihydrogen phosphate etc.; Improve the yield of mycelium and schizophan, and antitumor, anti-the putting of extraction rises bletilla adjustment body's immunity, the Schizophyllum commune Fr polysaccharides of producing the antibiotic synergist and the amino acid of needed by human from schizophyllum commune.Under this liquid culture technology, every liter of zymocyte liquid contains 200-300g mycelium (weight in wet base), but its output is still lower.
Summary of the invention
The present invention's first purpose provides a kind of Schizophyllum commune protein extract that can prevent and treat tobacco mosaic virus.
The present invention's second purpose provides the preparation method of above-mentioned Schizophyllum commune protein extract.
The present invention's the 3rd purpose provides the cultural method of schizophyllum commune.
The present invention's the 4th purpose provides the medium that is used to cultivate schizophyllum commune.
The present invention's the 5th purpose provides Schizophyllum commune protein extract in the purposes of preventing and treating on the tobacco mosaic virus.
Realize that technical scheme of the present invention is following:
Schizophyllum commune protein extract prepares according to following method:
(1) be that 1: 10～20 ratio is mixed the schizophyllum commune hypha powder with the phosphate buffer (PBS) of pH 7.0～9.0 according to w/v; Then at 2～6 ℃ of lixiviate 1～3h; Centrifugal 15～25min under 4 ℃, 7000～10000rpm condition collects supernatant again, obtains the schizophyllum commune crude extract;
(2) in step (1) gained schizophyllum commune crude extract, adding ammonium sulfate to degree of saturation is 40%, places 6～12 hours at 2～6 ℃ then, and centrifugal 15～25min under 4 ℃, 7000～10000rpm condition collects supernatant again;
(3) in step (2) gained supernatant, adding ammonium sulfate to degree of saturation is 85%, placed 6～12 hours at 2～6 ℃ then, and centrifugal 15～25min under 4 ℃, 7000～10000rpm condition again, the gained deposition is Schizophyllum commune protein extract.
The preparation method of above-mentioned Schizophyllum commune protein extract comprises the steps:
(1) be that 1: 10～20 ratio is mixed the schizophyllum commune hypha powder with the phosphate buffer (PBS) of pH 7.0～9.0 according to w/v; Then at 2～6 ℃ of lixiviate 1～3h; Centrifugal 15～25min under 4 ℃, 7000～10000rpm condition collects supernatant again, obtains the schizophyllum commune crude extract;
(2) in step (1) gained schizophyllum commune crude extract, adding ammonium sulfate to degree of saturation is 40%, places 6～12 hours at 2～6 ℃ then, and centrifugal 15～25min under 4 ℃, 7000～10000rpm condition collects supernatant again;
(3) in step (2) gained supernatant, adding ammonium sulfate to degree of saturation is 85%, placed 6～12 hours at 2～6 ℃ then, and centrifugal 15～25min under 4 ℃, 7000～10000rpm condition again, the gained deposition is Schizophyllum commune protein extract.
The particle diameter of described schizophyllum commune hypha powder is≤60 orders.
The cultural method of the schizophyllum commune described in the above-mentioned Schizophyllum commune protein extract comprises the steps:
(1) the female kind of schizophyllum commune activation: the female kind of schizophyllum commune transferred on the PDA solid culture medium, cultivated 7～10 days at 24～28 ℃ then, get dull and stereotyped activation mycelia;
(2) first class inoculum preparation: the dull and stereotyped activation mycelia of step (1) gained is cut into the fritter less than 1mm; According to the inoculum concentration of 0.4～0.8g mycelia/100ml with the schizophyllum commune mycelium inoculation in liquid fermentation medium; Under 24～28 ℃, 200～260rpm/min condition, carry out fermented and cultured 4～6 days then, obtain first class inoculum; The composition of said liquid fermentation medium and percentage by weight thereof are: soluble starch 4～6%, glucose 4～5.5%, wheat flour 2～3%, corn flour 2～4%, yeast extract 0.3～0.6%, analysis for soybean powder 0.5～1%, peptone 0.2～0.6%, KH
2PO
41%, MgSO
40.5%, VB
10.01%, all the other are water;
(3) fermented and cultured: be that the first class inoculum that 5～15% inoculum concentration obtains step (2) is inoculated in the liquid fermentation medium (the same step of its composition and percentage by weight thereof (2)) according to percent by volume; Under 24～28 ℃, 200～260rpm/min condition, carried out fermented and cultured 4～6 days then, mixed liquor must ferment;
(4) with step (3) gained fermentation mixed liquor suction filtration, collection schizophyllum commune mycelia, oven dry, pulverizing get the schizophyllum commune hypha powder.
The time of the fermented and cultured described in above-mentioned cultural method step (2) or the step (3) is preferably 6 days.
The described PDA solid culture medium of above-mentioned cultural method step (1) prepares according to method well-known to those skilled in the art, and its composition and percentage by weight thereof can be for: potato starchs 20%, glucose 20%, agar 20%, all the other are water.
The female kind of schizophyllum commune described in the above-mentioned cultural method can obtain from Chinese agriculture microorganism fungus kind preservation administrative center or China Committee for Culture Collection of Microorganisms's common micro-organisms center purchase.
Liquid fermentation medium described in above-mentioned cultural method step (2) or the step (3), its composition and percentage by weight thereof are: soluble starch 4～6%, glucose 4～5.5%, wheat flour 2～3%, corn flour 2～4%, yeast extract 0.3～0.6%, analysis for soybean powder 0.5～1%, peptone 0.2～0.6%, KH
2PO
41%, MgSO
40.5%, VB
10.01%, all the other are water.
The compound method of aforesaid liquid fermentation medium comprises proportionally with soluble starch, glucose, wheat flour, corn flour, yeast extract, analysis for soybean powder, peptone, KH
2PO
4, MgSO
4And VB
1Add in the entry, mix then and get final product.
The application of Schizophyllum commune protein extract of the present invention on the control tobacco mosaic virus disease.Concrete grammar is for to be diluted to the solution that concentration is 40 μ g/ml with above-mentioned Schizophyllum commune protein extract, sprays on the blade of plants such as tobacco in 3～5 leaf phases, capsicum.
The advantage that the present invention has: (1) Schizophyllum commune protein extract of the present invention is good to the control efficiency of tobacco mosaic virus, is spraying tobacco after 72 hours, and the inoculation tobacco mosaic virus can reach more than 70% to the inhibiting rate of tobacco mosaic virus; (2) Schizophyllum commune protein extract of the present invention belongs to natural extract, to human and environmentally friendly, belongs to environmentally friendly agricultural chemicals; (3) Schizophyllum commune protein extract preparation method of the present invention is simple, and cost is low, is suitable for carrying out large-scale production.(4) utilize fermentation medium of the present invention to cultivate schizophyllum commune output height, every liter of zymocyte liquid can obtain 400～480g mycelium weight in wet base or 54～60g mycelium dry weight, and compared with prior art, output is greatly improved.
Embodiment
The preparation of embodiment 1 schizophyllum commune mycelia
1, actication of culture: plant switching on PDA solid culture medium (potato starch 20%, glucose 20%, agar 20%, all the other be water) with schizophyllum commune is female, 24 ℃ of cultivations 10 days, mycelia was covered with flat board.
2, the fermented and cultured of schizophyllum commune
(1) preparation of liquid fermentation medium: take by weighing following composition respectively, 60g soluble starch, 55g glucose, 30g wheat flour, 40g corn flour, 6g yeast extract, 10g analysis for soybean powder, 6g peptone, 1gKH
2PO
4, 0.5gMgSO
4, 10mg VB
1, these materials are added in the entry and are settled to 1000ml, mixing gets final product.
(2) first class inoculum preparation: the dull and stereotyped mycelia that step 1 obtains is cut into the fritter less than 1mm; Changing the schizophyllum commune mycelia over to 500ml that the 100ml fermentation medium is housed according to the inoculum concentration of 0.4g mycelia/100ml shakes in the bottle; Obtained first class inoculum in 6 days at 24 ℃, 200rpm shake-flask culture, its concentration is 3.9g/100ml).
(3) fermented and cultured: be that first class inoculum that 5% inoculum concentration obtains step 2 (2) is inoculated into the 500ml that the said liquid fermentation medium of 100ml step 2 (1) is housed and shakes in the bottle according to percent by volume; Shake bottle cultivation 6 days down at 24 ℃, 200rpm, mixed liquor must ferment;
(4) above-mentioned fermentation mixed liquor suction filtration, collection mycelia are dried in 40 ℃ of baking ovens, every 100ml zymocyte liquid obtains 5.8g mycelia (dry weight), pulverizes 60 mesh sieves, obtains the schizophyllum commune hypha powder.
The preparation of embodiment 2 schizophyllum commune mycelia
1, actication of culture: plant switching on PDA solid culture medium (potato starch 20%, glucose 20%, agar 20%, all the other be water) with schizophyllum commune is female, 26 ℃ of cultivations 10 days, mycelia was covered with flat board.
2, the fermented and cultured of schizophyllum commune
(1) preparation of fermentation medium: take by weighing following composition respectively, 40g soluble starch, 40g glucose, 20g wheat flour, 20g corn flour, 3g yeast extract, 5g analysis for soybean powder, 2g peptone, 0.1gKH
2PO
4, 0.5gMgSO
4, 10mg VB
1, mixing in these materials addings 1000ml water is got final product.
(2) first class inoculum preparation: the dull and stereotyped mycelia that step 1 obtains is cut into the fritter less than 1mm; Changing the schizophyllum commune mycelia over to 500ml that the 100ml fermentation medium is housed according to the inoculum concentration of 0.6g mycelia/100ml shakes in the bottle; In 26 ℃, 240rpm shake-flask culture 6 days, obtain first class inoculum (concentration is 4.2g/100ml).
(3) fermented and cultured: be that first class inoculum culture fluid that 10% inoculum concentration obtains step 2 (2) is inoculated into the 500ml that the said fermentation medium of 100ml step 2 (1) is housed and shakes in the bottle according to percent by volume; In 26 ℃, 240rpm shake-flask culture 6 days, mixed liquor must ferment.
(4) above-mentioned fermentation mixed liquor suction filtration is collected mycelia and in 40 ℃ of baking ovens, dry, every 100ml zymocyte liquid obtains 5.4g mycelia (dry weight), pulverizes 60 mesh sieves and obtains the schizophyllum commune hypha powder.
The preparation of embodiment 3 schizophyllum commune mycelia
1, actication of culture: plant switching on PDA solid culture medium (potato starch 20%, glucose 20%, agar 20%, all the other be water) with schizophyllum commune is female, 28 ℃ of cultivations 10 days, mycelia was covered with flat board.
2, the fermented and cultured of schizophyllum commune
(1) preparation of fermentation medium: take by weighing following composition respectively, 60g soluble starch, 55g glucose, 30g wheat flour, 40g corn flour, 6g yeast extract, 10g analysis for soybean powder, 6g peptone, 1gKH
2PO
4, 0.5gMgSO
4, 10mg VB
1, these materials are added entry are settled to the 1000ml mixing and get final product.
(2) first class inoculum preparation: the dull and stereotyped mycelia that step 1 obtains is cut into the fritter less than 1mm; Change the schizophyllum commune mycelia over to 500ml that the 100ml fermentation medium is housed according to the inoculum concentration of 0.8g mycelia/100ml and shake in the bottle, obtained first class inoculum (concentration is 4.1g/100ml) in 6 days in 28 ℃, 260rpm shake-flask culture.
(3) fermented and cultured: the first class inoculum culture fluid that step 1) is obtained according to percent by volume be 15% inoculum concentration be inoculated into 100ml step 2 is housed) 500ml of said fermentation medium shakes in the bottle, in 28 ℃, 260rpm shake-flask culture 6 days, mixed liquor must ferment;
(4) above-mentioned fermentation mixed liquor suction filtration is collected mycelia and in 40 ℃ of baking ovens, dry, every 100ml zymocyte liquid obtains 6g mycelia (dry weight), pulverizes 60 mesh sieves and obtains the schizophyllum commune hypha powder.
The preparation of embodiment 4 Schizophyllum commune protein extracts
Carry out according to following method:
(1) be that the ratio of 1g/10ml is mixed the schizophyllum commune hypha powder of gained among the 2g embodiment 1 with the phosphate buffer of 0.025mol/L pH7.0 according to w/v; After 1 hour, centrifugal 25min under 4 ℃ of 10000rpm collects supernatant, gets the schizophyllum commune crude extract 2 ℃ of lixiviates.
(2) in the schizophyllum commune crude extract that step (1) obtains, adding ammonium sulfate to degree of saturation is 40%, places 8 hours at 2 ℃, centrifugal under 4 ℃, 10000rpm condition, collects supernatant.
(3) in the supernatant that step (2) obtains, adding ammonium sulfate to degree of saturation is 85%, places 8 hours at 2 ℃, and centrifugal under 4 ℃, 10000rpm condition, collecting precipitation promptly obtains Schizophyllum commune protein extract.
The preparation of embodiment 5 Schizophyllum commune protein extracts
Carry out according to following method:
(1) be that the ratio of 1g/20ml is mixed the schizophyllum commune hypha powder of gained among the 2g embodiment 2 with the phosphate buffer of 0.025mol/L pH8.0 according to w/v; 4 ℃ of lixiviates 2 hours, centrifugal 25min under 4 ℃ of 10000rpm collected supernatant, gets the schizophyllum commune crude extract.
(2) in the schizophyllum commune crude extract that step (1) obtains, adding ammonium sulfate to degree of saturation is 40%, places 8 hours at 4 ℃ then, centrifugal under 4 ℃, 10000rpm condition again, collects supernatant.
(3) in the supernatant that step (2) obtains, continue to add ammonium sulfate to degree of saturation 85%, placed 8 hours at 4 ℃, centrifugal under 4 ℃, 10000rpm condition again, collecting precipitation promptly obtains Schizophyllum commune protein extract.
The preparation of embodiment 6 Schizophyllum commune protein extracts
Carry out according to following method
(1) be that the ratio of 1g/20ml is mixed the schizophyllum commune hypha powder of gained among the 2g embodiment 3 with the phosphate buffer of 0.025mol/L pH9.0 according to w/v; 6 ℃ of lixiviates 3 hours, centrifugal 25min under 4 ℃ of 10000rpm collected supernatant, gets the schizophyllum commune crude extract then.
(2) in the schizophyllum commune crude extract that step (1) obtains, adding ammonium sulfate to degree of saturation is 40%, places 8 hours at 6 ℃ then, centrifugal under 4 ℃, 10000rpm condition again, collects supernatant.
(3) in the supernatant that step (2) obtains, add ammonium sulfate to degree of saturation 85%, placed 8 hours at 6 ℃ then, centrifugal under 4 ℃, 10000rpm condition again, collecting precipitation promptly obtains Schizophyllum commune protein extract.
The test of embodiment 7 Schizophyllum commune protein extract evoking tobacco activity of resisting tobacco mosaic virus
1, the mensuration of protein concentration
Detect the protein concentration of the Schizophyllum commune protein extract that extracts among the embodiment 4,5,6 with BCA protein concentration kit (U.S. Pierce company).
2, antiviral effect test
(1) the spraying control is handled: respectively the Schizophyllum commune protein extract dilute with water that obtains among the embodiment 4,5,6 is obtained the solution that total protein concentration is 40 μ g/ml; Respectively the withered spot three lives cigarette of three to four leaf phases is put in order the strain spraying; If three repetitions, each repeats three strains, three leaves of every strain; Simultaneously with the withered spot three lives cigarettes of in three to four leaf phases of fresh water spraying as contrast (CK), three repetitions are set, each repeats three strains, three leaves of every strain.
(2) preparation of virus inoculation liquid: viral source is that Beijing City Agriculture and Forestry Institute plant and Institute for Environmental Research plant disease and combine the common strain of the TMV that preserves anti-research department system (with common cigarette is the breeding host; Live body is preserved); The blade of the common cigarette that infects the common strain of tobacco mosaic virus TMV system is removed master pulse, mix by 1g/20ml with the 0.01mol/LPBS of pH8.0, every 20mlPBS adds 0.015g diamond dust; Blade is fully ground, obtain inoculation liquid.
(3) inoculation: the method for (1) spraying processing cigarette plant of the withered spot three lives is after 72 hours set by step; Inoculation liquid with step (2) obtains carries out using flushing with clean water immediately after juice frictional inoculation (planting disease research method (third edition), Fang Zhongda, the Chinese agriculture publishing house) inoculation; In the insect protected greenhouse, cultivate; Condition of culture is 28 ℃ of illumination 12 hours, and 18 ℃ of unglazed photographs 12 hours hocket.
(4) withered spot inhibiting rate calculates: inoculate the 72 hours withered spot three lives of " Invest, Then Investigate " tobacco leaf sheet and produce the withered spot number amount, be calculated as follows out withered spot inhibiting rate.
X=(CK-Y) * 100/CK (formula I)
Among the formula I, X is withered spot inhibiting rate, unit: %; CK is the average withered spot number of clear water contrast blade, unit: individual; Y is for inducing the average withered spot number of handling rear blade, unit: individual through Schizophyllum commune protein extract.
The tobacco that the Schizophyllum commune protein extract of result's (seeing table 1) embodiment 4,5,6 gained was handled all more than 70%, explains that Schizophyllum commune protein extract can produce resistance to tobacco mosaic virus by evoking tobacco to the inhibiting rate of tobacco mosaic virus.
Table 1. Schizophyllum commune protein extract evoking tobacco activity of resisting tobacco mosaic virus result of the test
Claims (4)
1. the cultural method of a schizophyllum commune comprises the steps:
(1) the female kind of schizophyllum commune transferred on the PDA solid culture medium, cultivated 7～10 days at 24～28 ℃ then, get dull and stereotyped activation mycelia;
(2) the dull and stereotyped activation mycelia of step (1) gained is cut into the fritter less than 1mm; According to the inoculum concentration of 0.4～0.8g mycelia/100ml with the schizophyllum commune mycelium inoculation in liquid fermentation medium; Under 24～28 ℃, 200～260rpm/min condition, carry out fermented and cultured 4～6 days then, obtain first class inoculum; The composition of described liquid fermentation medium and percentage by weight thereof are: soluble starch 4～6%, glucose 4～5.5%, wheat flour 2～3%, corn flour 2～4%, yeast extract 0.3～0.6%, analysis for soybean powder 0.5～1%, peptone 0.2～0.6%, KH
2PO
41%, MgSO
40.5%, VB
10.01%, all the other are water;
(3) be that 5～15% inoculum concentration is inoculated into the first class inoculum that step (2) obtains in the liquid fermentation medium according to percent by volume, under 24～28 ℃, 200～260rpm/min condition, carried out fermented and cultured 4～6 days then, mixed liquor must ferment; The same step of the composition of wherein said liquid fermentation medium and percentage by weight thereof (2);
(4) with step (3) gained fermentation mixed liquor suction filtration, collection schizophyllum commune mycelia, oven dry, pulverizing get the schizophyllum commune hypha powder.
2. according to the described cultural method of claim 1, the time that it is characterized in that the fermented and cultured described in its step (2) is 6 days.
3. according to the described cultural method of claim 1, the time that it is characterized in that the fermented and cultured described in its step (3) is 6 days.
4. according to claim 1,2 or 3 described cultural methods, it is characterized in that the composition of the PDA solid culture medium described in its step (1) and percentage by weight thereof are: potato starch 20%, glucose 20%, agar 20%, all the other are water.
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Families Citing this family (9)
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| CN103160550A (en) * | 2013-04-09 | 2013-06-19 | 徐宝军 | Preparation method of compound polysaccharide consisting of schizophyllum commune exopolysaccharide and oat polysaccharide |
| WO2015150964A1 (en) * | 2014-04-01 | 2015-10-08 | National Institute Of Plant Health Management (Niphm) | Novel media for growing fungi and bacteria and production method thereof |
| CN105567571B (en) * | 2015-12-23 | 2019-08-09 | 文山学院 | A kind of air-dried fructification strain separating method of white ginseng bacterium |
| CN107320541A (en) * | 2017-06-29 | 2017-11-07 | 李永跃 | A kind of schizophyllum commune spina date seed syrup and its manufacture craft |
| CN109156266A (en) * | 2018-08-03 | 2019-01-08 | 西北师范大学 | A kind of method schizophyllum commune cultivation culture medium and cultivate schizophyllum commune using it |
| CN109247189A (en) * | 2018-09-29 | 2019-01-22 | 云南菌视界生物科技有限公司 | A kind of white ginseng bacterial strain and its white ginseng low cost cultural method |
| CN109220524B (en) * | 2018-09-29 | 2020-10-09 | 云南菌视界生物科技有限公司 | Schizophyllum commune strain and ultrashort period cultivation method of fruiting body thereof |
| CN109355203A (en) * | 2018-11-27 | 2019-02-19 | 云南菌视界生物科技有限公司 | A kind of cultural method of schizophyllum commune liquid spawn |
| CN112691125B (en) * | 2021-01-05 | 2023-04-28 | 广东丸美生物技术股份有限公司 | Pharmaceutical composition for whitening or resisting aging, preparation method thereof and skin care product |
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