CN100515437C - Application of medical fungi bidirection solid fermentation engineering technology in processing thunder godvine to detoxify and keep effect - Google Patents
Application of medical fungi bidirection solid fermentation engineering technology in processing thunder godvine to detoxify and keep effect Download PDFInfo
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Abstract
An application of the bidirectional solid fermenting engineer with medicinal fungus in detoxicating the threewingnut root and improving its curative effect includes such steps as pre-treating the threewingnut root, pre-treating the fermenting bacteria, inoculating, bidirectional solid fermenting, and collecting the medicinal bacterial substance.
Description
Technical field
The invention belongs to the Chinese traditional medicine biology technical field, particularly relate to the application of medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated.
Background technology
Radix Tripterygii Wilfordii (Triptreygium wilfordii Hook.f.) is Celastraceae (Celastraceae) Thunder God Calamus (Triptreygium Hook.f.) plant, and medicinal part is root and stem.Radix Tripterygii Wilfordii is evident in efficacy to diseases such as rheumatic arthritis, lupus erythematosus, nephritis, bronchitis, hepatitis gravis, leukemia, organ transplantation, be a kind of good immunosuppressant and antiinflammatory, but it is again famous poisonous plants, its main effective ingredient is a diterpene, what content was the highest (has another name called Radix Tripterygii Wilfordii lactone alcohol for triptolide, triptolide), effective metering of triptolide is very close with the metering of poisoning, its LD50 is 1.19+0.204mg/kg (oral), belongs to hypertoxic scope.Therefore, clinical application is absolutely unsafe, and the toxicity that how to reduce Radix Tripterygii Wilfordii keeps certain curative effect (being called for short detoxify imitates) again, and keeping safe medication is current problem extremely to be solved.
It is fermented bacterium with medicinal fungi " Auricularia Sophorae " that China bacteriology meeting Zhuan Yi professor plays research in the eighties in last century, with agricultural byproducts is to carry out solid fermentation on the nutrient matrix, its medicinal mycoplasma claims " Trametes robiniophila Murr. ", and make the medical material fermentation can cause that its component of organization changes according to fungus, the principle that causes nature,taste and action to change, in fermentation substrate, mix a certain amount of through elite Chinese crude drug as medicinal mycoplasma, under certain condition after the fermentation, acquisition is called the medicinal fungal substance of " Chinese scholartree stilbene mycoplasma ", its relevant every immune indexes all obviously is better than medicinal mycoplasma, thereby proposed the theory of the novel two-way solid fermentation engineering of medicinal fungi and method (referring to the research of PTS Auricularia Sophorae electuary. Chinese Pharmaceutical Journal .1998,33 (5), P273～275).
Fungus not of the same race goes fermentation with a kind of medical material respectively, the property of the composition of the mycoplasma that is produced, imitate different, and use ferment the consequence of different Chinese crude drugs respectively with a kind of fungus also can be different.Therefore, but different fungus forms in a large number " fermentation combination " with different medical material combined crosswise, can obtain " medicinal fungal substance " of a large amount of heterogeneitys, effectiveness.
The design of the theoretical basis of the novel amphicheirality's solid fermentation of medicinal fungi engineering, fermentation combination, the contents such as pharmacological screening of mycoplasma can be referring to document (Zhuan Yi, medicinal fungi novel (two-way type) solid fermentation engineering. edible fungi of china .2002,21 (4): 3～6).
The key of medicinal fungi novel (amphicheirality) solid fermentation engineering (being called for short two-way fermentation) is that fermented bacterium and fermentation substrate are constituted the interior substrate part of fermentation combination, by in the past only with being rich in carbon, the agricultural byproducts of nutrition such as nitrogen (bagasse, Testa Tritici etc.) as unidirectional nutrient matrix (its product claims medicinal mycoplasma), change into to adopt and have the Chinese crude drug of certain active component as medicinal mycoplasma, it can provide the effect of the enzyme that the conk desired nutritional can be subjected to fungus again and be changed component of organization, thereby produce new nature,taste and action, so have the amphicheirality, its product claims medicinal fungal substance, as dual-purpose nutrition, two kinds of substrate of the property of medicine then are called wholeness matrix, and product also claims medicinal fungal substance.Medicinal fungal substance, might be added with better drug effect simply mutually than this fungus or medicinal mycoplasma itself or both, mainly show potentiation, enlarge application, aspects such as detoxifcation (referring to the development of medicinal fungi novel solid fermentation engineering and Chinese scholartree stilbene mycoplasma. Chinese Pharmaceutical Journal, 2004,39 (03), P175～178).
Medicinal fungi novel (amphicheirality) solid fermentation engineering is that Chinese medicine research has been opened up a new field, promptly the fungus medicine (referring to Zhuan Yi. a frontier of Chinese medicine. new Chinese medicine and clinical pharmacology .1992,3 (2) 49～51).Still do not have reported in literature at present medicinal fungi novel (amphicheirality) solid fermentation engineering is used for the Radix Tripterygii Wilfordii processing, imitate, thereby improve range of application and the DEVELOPMENT PROSPECT of Radix Tripterygii Wilfordii with the detoxify of realizing Radix Tripterygii Wilfordii.
Summary of the invention
The purpose of this invention is to provide the application of medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated.
Technical scheme of the present invention is that medicinal fungi amphicheirality solid fermentation engineering is applied in the Radix Tripterygii Wilfordii detoxify effect, promptly by selecting for use certain medicinal fungi to be inoculated on the tripterygium wilfordii (medicinal mycoplasma) as fermented bacterium, constitute the reasonable set combination of effectively fermenting, carry out amphicheirality's solid fermentation under certain condition, obtaining medicinal fungal substance is the Radix Tripterygii Wilfordii medicinal fungal substance that detoxify is imitated.Its theoretical basis is as being fermented under certain condition, go mouldy behind the fungal infections such as harmful assorted bacterium such as penicillium sp, Mucor, aspergillosis in the air according to Chinese crude drug, this is based on physiological activity that these funguses carry out, the component of organization of medical material is changed, thereby cause the change of its nature,taste and action.
The alleged amphicheirality's solid fermentation of the present invention is meant that fungus (fermented bacterium) grow when fermentation can accept on the one hand that special component exerts an influence to fungus itself in the Chinese crude drug on containing the solid medicinal mycoplasma of specific Chinese crude drug, also can the component of organization of specific Chinese crude drug be exerted an influence on the other hand, its effect shows as two-way.Be medicinal mycoplasma when providing nutrient substance such as the required carbon of fungus, nitrogen to promote thalli growths, fungus also can decompose the component of organization of medical material as enzyme, thereby produce new composition and have new flavor efficacy, therefore be called " two-way fermentation ".
The alleged medicinal fungal substance of the present invention is meant the mycoplasma that medicinal fungi (fermented bacterium) is grown and fermented and obtain in containing the medicinal mycoplasma of Chinese crude drug.
The alleged medicinal mycoplasma of the present invention is meant that Chinese crude drug is as fermentation substrate.
The alleged mycoplasma of the present invention is meant the product of fungus behind solid fermentation, comprise thalline and secondary metabolites, what these secondary metabolites had is present in the somatic cells, what have is secreted in the substrate after the extracellular is present in fermentation, a large amount of fungal myceliums of being grown with the fermentation after substrate between can't separate, so the mycoplasma of being referred to as.
The alleged substrate of the present invention be meant can provide conk the material of essential composition.The substrate that wherein only contains agricultural byproducts is called nutrient matrix, and the substrate that only contains Chinese crude drug is called medicinal mycoplasma, is called wholeness matrix when having nutrient matrix and medicinal mycoplasma simultaneously.
The objective of the invention is to realize by following measures:
The application of medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated.
The application of described medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated, the concrete grammar of this application is: the processing of fermentation substrate Radix Tripterygii Wilfordii; Fermented bacterium is handled; Fermented bacterium is inoculated in fermentation substrate, under certain fermentation condition, carries out two-way solid fermentation; Collect medicinal fungal substance.
The application of described medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated, wherein the fermentation substrate processing method is that fermentation substrate is pulverized, and adds suitable quantity of water, sterilization.
The application of described medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated, wherein fermented bacterium is Ganoderma, Auricularia Sophorae, Coriolous Dersicolor (Fr.) Quel, Punoporus cinnabarius, Laetiporus sulphureus (Bull. Ex Fr.) Bond. Et Singer. or Ganoderma applanatum (Pers. Ex Wallr) Pat..
The application of described medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated, wherein the processing of fermented bacterium is fermented bacterium is activated and to carry out amplification cultivation; The method of amplification cultivation is to adopt the liquid medium shake-flask culture or adopt solid medium to cultivate.
The application of described medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated, wherein fermentation condition is that temperature range is 26-28 ℃, and the fermentation substrate water content is for adding the water yield of 120-150% by the substrate dry weight, and the fermentation natural law is 30-60 days.
Ganderma lucidum [Ganoderma lucidum (Leyss.ex Fr.) Karst.], Coriolous Dersicolor (Fr.) Quel fungus [the little Trametes robiniphila Murr of Coriolus versicolor (L.exFr.) Quel [Trametes robiniophila Murr.], Punoporus cinnabarius [Pycnoporus cinnabarirus (jacq) karst], Laetiporus sulphureus (Bull. Ex Fr.) Bond. Et Singer. [Laetiporus sulphureus (Fr.) Murrill], Ganoderma applanatum (Pers. Ex Wallr) Pat. [Ganoderma applanatumm (Pers) Pat].
The present invention also can detoxify to other toxic herb materials.
Beneficial effect of the present invention:
The present invention can realize the detoxify of Radix Tripterygii Wilfordii is imitated, thereby improves range of application and the DEVELOPMENT PROSPECT of Radix Tripterygii Wilfordii.
Pharmacodynamic experiment result of the present invention:
The experiment medicine:
Radix Tripterygii Wilfordii crude drug clear paste and medicinal fungal substance clear paste, provide by China Medicine University, wherein Radix Tripterygii Wilfordii crude drug clear paste is numbered CK1, (CK1 is that the Radix Tripterygii Wilfordii crude drug is not sterilized to CK2, CK2 is Radix Tripterygii Wilfordii substrate sterilize (0 day), organize in contrast), Radix Tripterygii Wilfordii medicinal fungal substance clear paste is numbered G1, G2, G3, G4, T1, T2, (strain represented in the numbering Chinese and English to T3, T represents Trametes robiniphila Murr, and G represents Ganderma lucidum, and (1 is 30 days to digitized representation fermentation natural law, 2 is 40 days, 3 is 50 days, and 4 is 60 days), fermentation condition and embodiment 1, fermentation condition used in 2 is corresponding, except fermentation natural law difference, do not have other difference), be made into 20% distilled water suspension respectively.
Cyclophosphamide, Hualian Pharmaceutical Co., Ltd., Shanghai's product.
Dexamethasone acetate tablets, Tianjin pharmaceutcal corporation, Ltd product.
Picryl chloride (PC), Hualian Pharmaceutical Co., Ltd., Shanghai's product.
Dou Shi liquid (cyanmethemoglobin reagent), Nanjing is built up bio-engineering research and is provided.
Sheep red blood cell (SRBC) (SRBC), guinea pig serum, Jiangning health and epidemic prevention station provides.
Laboratory animal: ICR mice, male and female half and half, body weight 18-22g; Cavia porcellus, the male and female dual-purpose, body weight 300-350g is provided by China Medicine University's Experimental Animal Center.
One, immunization
1, the Radix Tripterygii Wilfordii medicinal fungal substance is to mouse humoral immune inhibitory action (to the influence of serum hemolysin)
The mice random packet, every group 10, male and female half and half, positive controls give cyclophosphamide 10mg/kg, and other administration groups give Radix Tripterygii Wilfordii respectively, dosage is its LD50/10, the blank group gives co-content water, ig, once a day, continuous 6 days, respectively organized all ip sheep red blood cell (SRBC) immunity behind the administration 30min on the 1st day.
After the immunity 6 days, mice is plucked eyeball and gets blood, places 30min serum is fully oozed out.With the centrifugal 10min of 2000rpm, get serum 25ul, add the 975ul normal saline again.
In above-mentioned serum product 0.5ml, add 4 * 10
8Sheep red blood cell (SRBC) 0.25ml cools off test tube in the ice bath, add complement (guinea pig serum goods) 0.5ml in test tube.Test tube is moved in 37 ℃ of waters bath with thermostatic control, and insulation 15min inserts in the ice bath 5min immediately with cessation reaction.With the centrifugal 10min of 3000rpm, get its supernatant.Add Dou Shi liquid 1.875ml in supernatant 0.5ml, jolting is placed 10min, in the 540nm colorimetric.The results are shown in Table 2.
Table 2 Radix Tripterygii Wilfordii medicinal fungal substance is to the influence of serum hemolysin (mean ± SD)
| Group | Dosage (g/kg) | Mus number (only) | OD(540nm) |
| The blank group | - | 10 | 0.197±0.016 |
| The cyclophosphamide group | 0.01 | 10 | 0.182±0.010* |
| CK1 | 0.21 | 10 | 0.172±0.020** |
| CK2 | 0.5 | 10 | 0.189±0.021 |
| G1 | 0.5 | 10 | 0.181±0.011* |
| G2 | 0.5 | 10 | 0.188±0.012 |
| G3 | 0.5 | 10 | 0.182±0.015* |
| G4 | 0.5 | 10 | 0.199±0.013 |
| T1 | 0.5 | 10 | 0.208±0.031 |
| T2 | 0.5 | 10 | 0.205±0.021 |
| T3 | 0.5 | 10 | 0.223±0.032* |
Compare * P＜0.05, * * P＜0.01 with the blank group.
The result shows that cyclophosphamide, CK1, G1, G3 can suppress the mouse humoral immune function by significance, and T3 then significance strengthens the mouse humoral immune function.
2, the Radix Tripterygii Wilfordii medicinal fungal substance is to mouse cell immunosuppressive action (to the influence of the tardy paraphilia reaction of picryl chloride (PC) inducing mouse)
The mice random packet, 10 every group, male and female half and half.Each Mus abdominal part unhairing, scope 3 * 3cm
2About, get 1%PC ethanol solution 100ul with microsyringe, be evenly coated in unhairing district sensitization, behind the 30min, positive controls gives dexamethasone acetate 7.5mg/kg, other each administration groups are given Radix Tripterygii Wilfordii, dosage is LD50/10, and the blank group gives co-content water, ig, once a day, continuous 6 days.
Sensitization the 7th day, 1%PC olive oil solution 30ul evenly is applied in every Mus auris dextra (two sides) to be attacked, after antigen is attacked 24h, the mice dislocation is put to death, cut left and right sides auricular concha along the auricle baseline, taking off the auricle of diameter 8mm respectively in same position with card punch, is the swelling degree with the poor/left ear recast of left and right sides auricle weight.The results are shown in Table 3.
Table 3 Radix Tripterygii Wilfordii medicinal fungal substance is to mouse cell Immune Effects (mean ± SD)
| Group | Dosage (g/kg) | Mus number (only) | The swelling degree |
| The blank group | - | 10 | 0.321±0.168 |
| The dexamethasone acetate group | 0.0075 | 10 | 0.138±0.055* |
| CKl | 0.21 | 10 | 0.243±0.121 |
| CK2 | 0.5 | 10 | 0.169±0.083* |
| G1 | 0.5 | 10 | 0.236±0.105 |
| G2 | 0.5 | 10 | 0.113±0.077** |
| G3 | 0.5 | 10 | 0.247±0.116 |
| G4 | 0.5 | 10 | 0.145±0.190** |
| T1 | 0.5 | 10 | 0.152±0.099* |
| T2 | 0.5 | 10 | 0.227±0.116 |
| T3 | 0.5 | 10 | 0.154±0.072* |
Compare * P＜0.05, * * P＜0.01 with the blank group.
The result shows that dexamethasone acetate, CK2, G2, G4, T1, T3 can suppress the mouse cell immunologic function by significance.
Two, toxicity test
1, anxious poison experiment LD50
Trial test: the mice random packet, 10 every group, male and female half and half are dosage to try thing 5g/kg, every Mus is irritated the equal volume of stomach 0.5ml/20g.Observe once every day, record death toll, continuous 7 days.If none death of animal, and do not occur anyly, determine tentatively that then this LD50 value that is subjected to the reagent thing is greater than 5g/kg because of being tried the reaction that thing causes; If death occurs or drug reaction arranged, then carry out following formal test.
Formal test: get the mice random packet, every group 10, male and female half and half, be subjected to reagent trial test result to select to compare between suitable agent with this, each organizes the medicine of the variable concentrations of mouse stomach 0.5ml/20g body weight, and observe once every day, the record death toll, continuous 7 days, calculate the LD50 value that this is subjected to reagent at last.The results are shown in Table 4.
Each extract clear paste The acute toxicity tests of table 4 Radix Tripterygii Wilfordii
| The fermentation numbering | Triptolide content (1,/00 ten thousand) | LD 50(g/kg) |
| CK1 | 13.17 | 2.061 |
| CK2 | 9.64 | ＞5 |
| G1 | 7.97 | ＞5 |
| G2 | 3.24 | ＞5 |
| G3 | 3.51 | ＞5 |
| G4 | 2.88 | ＞5 |
| T1 | 2.25 | ＞5 |
| T2 | 1.07 | ＞5 |
| T3 | 0.96 | ＞5 |
Conclusion: the Radix Tripterygii Wilfordii medicinal fungal substance is compared with the Radix Tripterygii Wilfordii crude drug, and toxicity reduces, but has stronger humoral immunization and cellular immunization inhibitory action.
The specific embodiment
The invention will be further elaborated by the following examples.
Embodiment 1
The processing of fermentation substrate Radix Tripterygii Wilfordii (medicinal mycoplasma): the root of Radix Tripterygii Wilfordii belt leather dries or the 60-80 ℃ of oven dry section of being split into, be cut into thickness 1-2mm medical material sheet with microtome, pulverizer is ground into Semen setariae to the Semen phaseoli radiati bulky grain, be loaded on the solid fermentation bottle by every bottle of 180g, add amount of water (add by the substrate dry weight 130% the water yield), the lid bottle stopper was sterilized 2 hours for 121 ℃.
Fermented bacterium is prepared: get Ganderma lucidum inoculation PDA culture medium and carry out activation culture for 28 ℃, spend the night.Make bacteria culture fluid, composition is: glucose 30g, KH
2PO
42.25g, yeast powder 7.5g, MgSO
41.125g, CaCO
33.75g, vitamin B
10.15g, water 1500ml.Be respectively charged into after composition fully dissolves in the 500ml triangular flask, every bottle of 250ml sterilized 30 minutes for 121 ℃.Get the mycelium sterile working inoculated and cultured liquid after the activation, 130 rev/mins of shaking tables, 28 ℃ are shaken an about week of training, make it to produce a large amount of fungus ball, fungus ball liquid (promptly shaking the liquid spawn that the training back is obtained).
Solid fermentation: sterile working, inoculation fungus ball liquid 8-10ml in each solid fermentation bottle, ferment in 27 ± 1 ℃ of fermenting cellars, according to the different fermentations cycle of setting (set 30 days respectively, 40 days, 50 days, 60 days, the clear paste of the medicinal fungal substance that obtains through extracting preparation corresponds to G1, G2, G3, G4) ferment, form medicinal fungal substance.
Mycoplasma post processing: the wet medicinal fungal substance that the back forms that regularly ferments of medicinal mycoplasma Radix Tripterygii Wilfordii in the bottle is weighed,, preserve standby with 60 ℃ of electric heating drying.
Composition extracts: the medicinal fungal substance that is obtained is used 40% ethanol extraction respectively, be prepared into clear paste (being G1, G2, G3, G4) and carry out toxicity, the test of pesticide effectiveness (seeing beneficial effect part of the present invention for details).
Embodiment 2
The processing of fermentation substrate Radix Tripterygii Wilfordii (medicinal mycoplasma): the root of Radix Tripterygii Wilfordii belt leather dries or the 60-80 ℃ of oven dry section of being split into, be cut into thickness 1-2mm medical material sheet with microtome, pulverizer is ground into Semen setariae to the Semen phaseoli radiati bulky grain, be loaded on the solid fermentation bottle by every bottle of 180g, add amount of water (add by the substrate dry weight 130% the water yield), the lid bottle stopper was sterilized 2 hours for 121 ℃.
Fermented bacterium is prepared: get Trametes robiniphila Murr inoculation PDA culture medium and carry out activation culture for 28 ℃, spend the night.Make bacteria culture fluid, composition is: glucose 30g, KH
2PO
42.25g, yeast powder 7.5g, MgSO
41.125g, CaCO
33.75g, vitamin B
10.15g, water 1500ml.Be respectively charged into after composition fully dissolves in the 500ml triangular flask, every bottle of 250ml sterilized 30 minutes for 121 ℃.Get the mycelium sterile working inoculated and cultured liquid after the activation, 130 rev/mins of shaking tables, 28 ℃ are shaken an about week of training, make it to produce a large amount of fungus ball, fungus ball liquid (promptly shaking the liquid spawn that the training back is obtained).
Solid fermentation: sterile working, inoculation fungus ball liquid 8-10ml in each solid fermentation bottle, ferment in 27 ± 1 ℃ of fermenting cellars, according to the different fermentations cycle of setting (set 30 days respectively, 40 days, 50 days, the clear paste of the medicinal fungal substance that obtains through extracting preparation corresponds to T1, T2, T3) ferment, form medicinal fungal substance.
Mycoplasma post processing: the wet medicinal fungal substance that the back forms that regularly ferments of medicinal mycoplasma Radix Tripterygii Wilfordii in the bottle is weighed,, preserve standby with 60 ℃ of electric heating drying.
Composition extracts: the medicinal fungal substance that is obtained is used 40% ethanol extraction respectively, be prepared into clear paste (being T1, T2, T3) and carry out toxicity, the test of pesticide effectiveness (seeing beneficial effect part of the present invention for details).
Embodiment 3
The processing of fermentation substrate Radix Tripterygii Wilfordii (medicinal mycoplasma): the root of Radix Tripterygii Wilfordii belt leather dries or the 60-80 ℃ of oven dry section of being split into, be cut into thickness 1-2mm medical material sheet with microtome, pulverizer is ground into Semen setariae to the Semen phaseoli radiati bulky grain, be loaded on the solid fermentation bottle by every bottle of 180g, add amount of water (add by the substrate dry weight 120% the water yield), the lid bottle stopper was sterilized 2 hours for 121 ℃.
Fermented bacterium is prepared: get Coriolous Dersicolor (Fr.) Quel fungus inoculation PDA culture medium and carry out activation culture for 28 ℃, spend the night.Make bacteria culture fluid, composition is: glucose 30g, KH
2PO
42.25g, yeast powder 7.5g, MgSO
41.125g, CaCO
33.75g, vitamin B
10.15g, water 1500ml.Be respectively charged into after composition fully dissolves in the 500ml triangular flask, every bottle of 250ml sterilized 30 minutes for 121 ℃.Get the mycelium sterile working inoculated and cultured liquid after the activation, 130 rev/mins of shaking tables, 28 ℃ are shaken an about week of training, make it to produce a large amount of fungus ball, fungus ball liquid (promptly shaking the liquid spawn that the training back is obtained).
Solid fermentation: the sterile working, inoculation fungus ball liquid 8-10ml ferments in 27 ± 1 ℃ of fermenting cellars in each solid fermentation bottle, ferments according to the different fermentations cycle of setting (30-60d), forms medicinal fungal substance.
Mycoplasma post processing: the wet medicinal fungal substance that the back forms that regularly ferments of medicinal mycoplasma Radix Tripterygii Wilfordii in the bottle is weighed,, preserve standby with 60 ℃ of electric heating drying.
Composition extracts: use rare alcohol (40% ethanol) to extract respectively the medicinal fungal substance that obtains, be prepared into clear paste and carry out chemistry, toxicity, the test of pesticide effectiveness.
Embodiment 4
The processing of fermentation substrate Radix Tripterygii Wilfordii (medicinal mycoplasma): the root of Radix Tripterygii Wilfordii belt leather dries or the 60-80 ℃ of oven dry section of being split into, be cut into thickness 1-2mm medical material sheet with microtome, pulverizer is ground into Semen setariae to the Semen phaseoli radiati bulky grain, be loaded on the solid fermentation bottle by every bottle of 180g, add amount of water (add by the substrate dry weight 150% the water yield), the lid bottle stopper was sterilized 2 hours for 121 ℃.
Fermented bacterium is prepared: get Ganoderma applanatum (Pers. Ex Wallr) Pat. bacterium inoculation PDA culture medium and carry out activation culture for 28 ℃, spend the night.Make bacteria culture fluid, composition is: glucose 30g, KH
2PO
42.25g, yeast powder 7.5g, MgSO
41.125g, CaCO
33.75g, vitamin B
10.15g, water 1500ml.Be respectively charged into after composition fully dissolves in the 500ml triangular flask, every bottle of 250ml sterilized 30 minutes for 121 ℃.Get the mycelium sterile working inoculated and cultured liquid after the activation, 130 rev/mins of shaking tables, 28 ℃ are shaken an about week of training, make it to produce a large amount of fungus ball, fungus ball liquid (promptly shaking the liquid spawn that the training back is obtained).
Solid fermentation: the sterile working, inoculation fungus ball liquid 8-10ml ferments in 27 ± 1 ℃ of fermenting cellars in each solid fermentation bottle, ferments according to the different fermentations cycle of setting (30-60d), forms medicinal fungal substance.
Mycoplasma post processing: the wet medicinal fungal substance that the back forms that regularly ferments of medicinal mycoplasma Radix Tripterygii Wilfordii in the bottle is weighed,, preserve standby with 60 ℃ of electric heating drying.
Composition extracts: use rare alcohol (40% ethanol) to extract respectively the medicinal fungal substance that obtains, be prepared into clear paste and carry out chemistry, toxicity, the test of pesticide effectiveness.
Claims (2)
1, the application of medicinal fungi amphicheirality solid fermentation engineering in the Radix Tripterygii Wilfordii detoxify is imitated;
Wherein the processing step of medicinal fungi amphicheirality solid fermentation engineering is as follows:
The processing of a, fermentation substrate Radix Tripterygii Wilfordii: fermentation substrate is pulverized, added suitable quantity of water, make the fermentation substrate water content for add the water yield of 120-150%, sterilization by the substrate dry weight;
B, fermented bacterium handle: fermented bacterium Ganoderma, Auricularia Sophorae, Coriolous Dersicolor (Fr.) Quel, Punoporus cinnabarius, Laetiporus sulphureus (Bull. Ex Fr.) Bond. Et Singer. or Ganoderma applanatum (Pers. Ex Wallr) Pat. are activated and carry out amplification cultivation;
C, fermented bacterium is inoculated in fermentation substrate, carries out two-way solid fermentation at 26-28 ℃, the fermentation natural law is 30-60 days;
D, collection medicinal fungal substance.
2, the application of medicinal fungi amphicheirality solid fermentation engineering according to claim 1 in the Radix Tripterygii Wilfordii detoxify is imitated, the method that it is characterized in that the fermented bacterium amplification cultivation are to adopt the liquid medium shake-flask culture or adopt solid medium to cultivate.
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| CN103860783B (en) * | 2014-03-17 | 2017-10-03 | 宜宾学院 | A kind of method for thizoma curculiginis of being fermented using food, medicinal fungus |
| CN104138417A (en) * | 2014-07-14 | 2014-11-12 | 黄丹民 | Tinospora crispa medicine composition with effect of blood sugar reduction and preparation method thereof |
| CN115957249B (en) * | 2022-12-30 | 2024-08-13 | 深圳市中医院 | Application of bi-directional solid fermentation product of tripterygium wilfordii in preparation of medicament for treating kidney diseases |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816689A (en) * | 2010-05-19 | 2010-09-01 | 陈心智 | Processing method of ginseng |
| CN101816689B (en) * | 2010-05-19 | 2012-02-01 | 陈心智 | Processing method of ginseng |
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| CN1911262A (en) | 2007-02-14 |
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