CN105506021A - Taxol-containing culture composition and preparation method thereof - Google Patents

Taxol-containing culture composition and preparation method thereof Download PDF

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CN105506021A
CN105506021A CN201511027604.6A CN201511027604A CN105506021A CN 105506021 A CN105506021 A CN 105506021A CN 201511027604 A CN201511027604 A CN 201511027604A CN 105506021 A CN105506021 A CN 105506021A
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thalline
taxol
liquid
fermentation
ramulus
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CN105506021B (en
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程俊文
贺亮
胡传久
魏海龙
方茹
邹景泉
李海波
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Zhejiang Academy of Forestry
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Abstract

The invention discloses a taxol-containing culture composition and a preparation method thereof. The preparation method comprises the following steps: inoculating a fig aspergillus seed liquid into a taxus chinensis solid-state fermentation medium containing taxus chinensis var mairei and rhizoma amorphophalli and culturing; culturing in an alternating magnetic field environment to obtain a taxus chinensis solid-state fermentation product; performing subcritical water extraction to obtain a taxus chinensis fermentation subcritical extract I; and inoculating a taxol-producing endophytic fungi seed liquid into an endophytic fungi liquid fermentation medium containing the extraction remainder II, and culturing to obtain an endophytic fungi fermentation product III, wherein the composition refers to the taxus chinensis fermentation subcritical extract I and the endophytic fungi fermentation product III and has a good liver protection function. By adopting the preparation method, biodegradation and transformation can be effectively performed on the nutritional ingredients and active ingredients in the raw materials, thereby facilitating the improvement of content and activity of the active ingredients in the final product.

Description

A kind of containing taxol cultivation composition and method of making the same
Technical field
The present invention relates to biological fermentation field, be specifically related to a kind of containing taxol cultivation composition and method of making the same.
Background technology
Taxol (Paclitaxel) is a kind of tetracyclic diterpene class natural anti-cancer drugs, and Americanized scholar's Giovannis (M.C.Wani) in 1963 and Wal (MonreE.Wall) have been separated to the crude extract of taxol first from peaceful red deal (PacificYew) bark of one growth appellation Western United States's fully stocked wood and timber.The preparation of taxol adopts traditional extracting and developing method more, generally comprises a, extraction, take Ramulus et folium taxi cuspidatae as the extract that raw material obtains containing taxol; B, removal colloid, the colloid impurity in removing extract; C, separation and purification.
Fermentable utilizes microorganism, under appropriate conditions, raw material is converted into the process of the product that necessary for human is wanted through specific pathways metabolism.The chemical extraction methods green safety that fermentable is general more traditional, is thus widely used in recent years.The fermentable cultural method of taxol is as a kind of in: Chinese patent ZL200810152499.2 discloses to be utilized Two Liquid Phases to ferment to improve the method for fungi taxol, it comprises cultivates endophytic fungus associated with taxol plan dish stey (Pestalotiopsismicrospora) NK17 in the medium, when thalli growth reaches plateau, add organic solvent to continue to cultivate, to make in described strain cell and produce more in described substratum and assemble taxol, and reclaim and purification of paclitaxel in described cell and substratum.The method taxol yield can reach 551.72 μ g/L.If develop new paclitaxel prodrugs and microbial fermentation processes thereof, provide new approach by being expected to for taxol or containing the preparation of the product of taxol.
Summary of the invention
The invention provides and a kind of cultivate composition containing taxol, in said composition the content of activeconstituents taxol and activity high, have and good protect liver function effect.
Present invention also offers a kind of preparation method cultivating composition containing taxol, utilize multiple fungi fermentation southerm yew and SHENLIUGU, contribute to the content and the activity that improve activeconstituents in the finished product, obtain the composition with good liver-protecting function.
The present invention finds, by the specific fungi fermentation of the present invention and specific technique, effectively the nutritive ingredient in Ramulus et folium taxi cuspidatae and effective constituent can be carried out Biodegradation and biotransformation, each raw material resources are fully utilized, contribute to the content and the activity that improve activeconstituents in the finished product.
The technical solution used in the present invention is:
Cultivate a preparation method for composition containing taxol, comprise step:
(1) get the taxol-producing endophytic fungi strain inoculation after activation and cultivate 4h-6h in liquid MRS substratum, collected by centrifugation first thalline; First thalline is suspended in the liquid MRS substratum containing cholate and hatches 3h-40h in 20 DEG C-30 DEG C, collected by centrifugation second thalline; By the second thalline, after PBS buffer solution for cleaning is clean, Eddy diffusion is in liquid MRS substratum, and concussion shakes up, and cultivates 2 days-10 days, obtain taxol-producing endophytic fungi seed liquor at 20 DEG C-30 DEG C;
(2) get the Aspergillus ficuum bacterium strain inoculation after activation and cultivate 4h-6h in liquid MRS substratum, collected by centrifugation the 3rd thalline, 3rd thalline is suspended in the liquid MRS substratum containing NaCl and hatches 0.5h-3h in 20 DEG C-28 DEG C, collected by centrifugation the 4th thalline; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, 4th thalline liquid is inoculated in the liquid MRS substratum containing NaCl and cultivates 5h-30h in 20 DEG C-28 DEG C, obtain Aspergillus ficuum bacterium seed liquor;
(3) by the Aspergillus ficuum bacterium seed liquor of step (2) access Ramulus et folium taxi cuspidatae solid-state fermentation culture medium, cultivate 3 days-30 days at 16 DEG C-30 DEG C, then be placed in alternating magnetic field environment and cultivate 5 days-20 days again, cultured products obtains Ramulus et folium taxi cuspidatae product by solid-state fermentation after vacuum lyophilization, pulverizing; Containing southerm yew and SHENLIUGU in described Ramulus et folium taxi cuspidatae solid-state fermentation culture medium;
(4) by the Ramulus et folium taxi cuspidatae product by solid-state fermentation of step (3) through Subcritical Water Extraction, obtain Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization;
(5) by the taxol-producing endophytic fungi seed liquor of step (1) access endogenetic fungus liquid fermentation medium, adjust pH is 3-7, cultivates 5 days-25 days at 15 DEG C-25 DEG C, centrifugal, obtains endogenetic fungus tunning III; Containing extraction residuum II in described endogenetic fungus liquid fermentation medium;
Described is Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I and endogenetic fungus tunning III containing taxol cultivation composition.
When the present invention carries out solid state fermentation, the direct Aspergillus ficuum bacterium seed liquor adopted through hatching process containing liquid MRS substratum two step of NaCl, effectively the nutritive ingredient in Ramulus et folium taxi cuspidatae and effective constituent can be carried out Biodegradation and biotransformation, adopt composite southerm yew and SHENLIUGU simultaneously, contribute to the content and the activity that improve activeconstituents in the finished product.In step (4) and step (5), Ramulus et folium taxi cuspidatae product by solid-state fermentation is carried out Subcritical Water Extraction, make that the activity of effective constituent in extract is at utmost retained, content is greatly improved; Again using extraction after residue as endogenetic fungus liquid fermenting culture medium mainly, and adopt the taxol-producing endophytic fungi seed liquor through hatching process containing the liquid MRS substratum of cholate, by integrated recycle, each raw material resources are fully utilized, further increase content and the activity of activeconstituents in the finished product.
In order to reach better effect, preferably:
In step (1), described is 0.1%-0.5% containing the mass percentage of cholate in the liquid MRS substratum of cholate.
Described first thalline is 1:1-3 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:1-3 with the volume ratio for the liquid MRS substratum of second thalline that suspends.
In step (2), the mass percentage of the described liquid MRS NaCl in medium containing NaCl is 2%-6%.
Described 3rd thalline is 1:1-3 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, and the 4th thalline is 1:1-3 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends, and the inoculum size of the 4th thalline liquid is 1%-2%.
In step (3), described southerm yew can adopt the branches and leaves of southerm yew; Described SHENLIUGU can adopt the stem tuber of SHENLIUGU.
Containing 5.5g-9.0g southerm yew and 0.3g-3.0g SHENLIUGU in the every 1L of described Ramulus et folium taxi cuspidatae solid-state fermentation culture medium.Described Ramulus et folium taxi cuspidatae solid-state fermentation culture medium is made up of southerm yew, SHENLIUGU and fermentation basic medium, and described fermentation basic medium adopts the fermentation basic medium of this area routine, can adopt commercially available prod, and existing compound method also can be adopted to prepare.
The preparation method of described Ramulus et folium taxi cuspidatae solid-state fermentation culture medium, comprising: fresh southerm yew, SHENLIUGU are rinsed well under tap water, dry, chopping; Again 5.5g-9.0g southerm yew and 0.3g-3.0g SHENLIUGU fermentation basic medium are settled to 1L, mix, pH value nature, obtains Ramulus et folium taxi cuspidatae solid-state fermentation culture medium.
The access amount of described Aspergillus ficuum bacterium seed liquor is the 5%-25% of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium volume.
Current taxol-producing endophytic fungi fermentation technique ubiquity fermentation period is long, mycelial growth sprouts the problems such as slow, feature its secondary metabolites content is low.The present invention finds alternating magnetic field treatment technology to be applied in solid state fermentation, by extraneous factor effective stimuluss such as the suitable process of specific cultivation period, improve the growth metabolism of hypha,hyphae on southerm yew and SHENLIUGU culture base-material, shorten fermentation time, effectively promote the generation of secondary metabolite.In described alternating magnetic field environment, magneticstrength is 0.2mT-6mT, and field frequency is 3Hz-40Hz.
In step (4), the condition of described Subcritical Water Extraction is: water material mass ratio is 15-50:1, and extracting pressure is at 5MPa-20MPa, and extraction temperature is 100 DEG C-180 DEG C, and extraction time is 10min-60min.
In step (5), in the every 1L of described endogenetic fungus liquid fermentation medium, extract residuum II containing 0.2g-1g.Described endogenetic fungus liquid fermentation medium forms with fermentation basic medium by extracting residuum II, and described fermentation basic medium adopts the fermentation basic medium of this area routine, can adopt commercially available prod, and existing compound method also can be adopted to prepare.
General fermentation basic medium is made up of the component of following mass percent: glucose 1%-3%, K 2hPO 40.1%-0.2%, MgSO 4the water of 0.05%-0.1% and surplus.
The preparation method of described endogenetic fungus liquid fermentation medium, comprising: 0.2g-1g is extracted residuum II fermentation basic medium and be settled to 1L, mix, and pH value nature, obtains endogenetic fungus liquid fermentation medium.
The access amount of described taxol-producing endophytic fungi seed liquor is the 3%-20% of endogenetic fungus liquid fermentation medium volume.
Described taxol-producing endophytic fungi bacterial classification can adopt any one taxol-producing endophytic fungi bacterial classification existing, can adopt commercially available prod, such as: Fusarium mairei (Fusariummairei) bacterial classification, is purchased from Beijing North Na Chuanlian Bioteknologisk Institut.
Described Aspergillus ficuum bacterium bacterial classification can adopt any one Aspergillus ficuum bacterium bacterial classification existing, commercially available prod can be adopted, such as: Aspergillus ficuum bacterium (Aspergillusficuum) ACCC30360 bacterial classification, is purchased from Chinese agriculture Microbiological Culture Collection administrative center.
Described liquid MRS substratum adopts the liquid MRS substratum of this area routine, can adopt commercially available prod, and existing compound method also can be adopted to prepare.Consisting of of general liquid MRS substratum: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 20.0g, sodium acetate 5.0g, diammonium hydrogen citrate 2.0g, tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, manganous sulfate 0.05g and distilled water 1.0 liters.
Described PBS damping fluid and phosphate buffered saline(PBS), adopt the PBS damping fluid of this area routine, can adopt commercially available prod, existing compound method also can be adopted to prepare.General 1LPBS damping fluid: potassium primary phosphate 0.27g, Sodium phosphate dibasic 1.42g, sodium-chlor 8g and Repone K 0.2g, adds the abundant stirring and dissolving of appropriate amount of deionized water, and then add concentrated hydrochloric acid and adjust pH to 7.2, last constant volume is to 1L.
The temperature that Aspergillus ficuum bacterium bacterial classification after taxol-producing endophytic fungi bacterial classification after described activation, activation is cultivated in liquid MRS substratum is physical environment temperature, is preferably 20 DEG C-30 DEG C.1cm is accessed in general 1L liquid MRS substratum 2-4cm 2aspergillus ficuum bacterium bacterial classification bacterium block after taxol-producing endophytic fungi bacterial classification bacterium block after the activation of size, activation.The activation method of described taxol-producing endophytic fungi bacterial classification or Aspergillus ficuum bacterium bacterial classification adopts the actication of culture method of this area routine, comprise: by the taxol-producing endophytic fungi bacterial classification of slant preservation or Aspergillus ficuum bacterium strain inoculation on PDA plate culture medium, carry out activation culture, culture temperature 20 DEG C-30 DEG C, incubation time 3 days-25 days, obtains the Aspergillus ficuum bacterium bacterial classification after the taxol-producing endophytic fungi bacterial classification after activating or activation.The substratum that described PDA plate culture medium adopts this area seed culture conventional, can adopt commercially available prod.Further preferably, described PDA plate culture medium: potato 200g, glucose 20g and agar 15g-20g, be settled to 1000mL with water.
The percentage ratio of the volume of the thalline liquid that described inoculum size refers to an access and the ratio of culture volume.
Use after the present invention's substratum used all needs sterilizing, the condition of sterilizing adopts the normal condition of this area, such as can at 120 DEG C-125 DEG C sterilizing 20min-30min.
Preparation method containing taxol cultivation composition of the present invention prepares and cultivates composition containing taxol.Described cultivates composition containing taxol, is preferably made up of 40 weight part-70 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction things I and 10 weight part-35 weight part endogenetic fungus tunnings III.
The present invention cultivates composition containing taxol and has good liver-protecting function, can be used to prepare the healthcare products or medicine with liver-protecting function.Described healthcare products or medicine directly can be prepared by described composition and also can be prepared together with the auxiliary material of this area by described composition, product forms can be varied, such as comprise the soft capsule cultivating composition containing taxol, its content is made up of 40 weight part-70 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, 10 weight part-35 weight part endogenetic fungus tunnings III and 1 weight part-5 part by weight of vitamin E.The preparation of described soft capsule adopts the customary preparation methods of soft capsule.
Southerm yew (TaxusmaireiSYHu) aiphyllium, bark light gray, lobe strip thin slice; Blunt or the slightly point in perula top, comes off or part place is stored in sprig base portion.Leaf 2 arranges, nearly sickleshaped, long 1.5 ~ 4.5 centimetres, agalasisa brilliance matter projection on arteries and veins band in the back side, or sometimes have fragmentary distribution, or the Zhong Mai both sides contiguous with stomatal band have 1 to several corpora mammillaria cutin projections, color is different from stomatal band, light green, edge-band width and obviously.It is long avette, long 7 ~ 8 millimeters, usually upper wider that seed falls oval or column, is born in red meat cup-shaped arillus.Seed can extract oil; Bark is containing tannin; Timber can for effect material.There is cultivation in Nanjing and Shanghai; With seminal propagation, also can cuttage.Be distributed in each provinces and regions on the south the Yangtze valley, and Henan and Shaanxi.
SHENLIUGU: (AmorphophallusrivieriDurieu), also known as konjaku, amorphophallus rivieri Durieu, is the dry tuber of aroid konjaku or East China konjaku.Record according to " Chinese medicine voluminous dictionary ": this product taste cold in nature is pungent, bitter, for oral administration having effect of long-pending, detoxicating and resolving a mass, the pain relieving of the row stasis of blood of disappearing of reducing phlegm.
The present invention's raw material used all can adopt commercially available prod.
Beneficial effect of the present invention:
1, when the present invention carries out solid state fermentation, the direct Aspergillus ficuum bacterium seed liquor adopted through hatching process containing liquid MRS substratum two step of NaCl, effectively the nutritive ingredient in Ramulus et folium taxi cuspidatae and effective constituent can be carried out Biodegradation and biotransformation, adopt composite southerm yew and SHENLIUGU simultaneously, contribute to the content and the activity that improve activeconstituents in the finished product.In step (4) and step (5), Ramulus et folium taxi cuspidatae product by solid-state fermentation is carried out Subcritical Water Extraction, make that the activity of effective constituent in extract is at utmost retained, content is greatly improved; Again using extraction after residue as endogenetic fungus liquid fermenting culture medium mainly, and adopt the taxol-producing endophytic fungi seed liquor through hatching process containing the liquid MRS substratum of cholate, by integrated recycle, each raw material resources are fully utilized, further increase content and the activity of activeconstituents in the finished product.
2, the inventive method raw material sources are natural, quality controllable, and environmentally safe is suitable for suitability for industrialized production.
3, compared with common endogenetic fungus tunning, the present invention cultivates composition to CCl containing taxol 4hepatic tissue alanine aminotransferase (ALT) in liver injury mice serum, the reduction of aspartate aminotransferase (AST) content are more remarkable, to CCl 4the improvement effect of liver injury mouse liver histopathology indices is more remarkable, illustrate that the present invention cultivates composition and soft gel products thereof containing taxol, can significantly reduce ALT and AST content, and significantly improve liver injury mouse liver histopathology indices, there is significant liver-protecting function, can be used to prepare the healthcare products or medicine with liver-protecting function.
Embodiment
Below in conjunction with some embodiments, content of the present invention is illustrated further, but content of the present invention is not limited in the following examples.
Taxol-producing endophytic fungi Fusarium mairei (Fusariummairei) bacterial classification, is purchased from Beijing North Na Chuanlian Bioteknologisk Institut.
Aspergillus ficuum bacterium (Aspergillusficuum) ACCC30360 bacterial classification, is purchased from Chinese agriculture Microbiological Culture Collection administrative center.
Cholate: No. 3 cholate, i.e. Sodium cholic acid, cholic acid-Sodium desoxycholate salt mixture (in the permanent industry in Beijing chemical industry far away).
Embodiment 1
One, material prepares
PDA plate culture medium: potato 200g, glucose 20g and agar 15g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
The Aspergillus ficuum bacterium bacterial classification of the taxol-producing endophytic fungi bacterial classification of slant preservation, slant preservation is inoculated on PDA plate culture medium respectively, carry out activation culture, culture temperature 22 DEG C, incubation time 10 days, obtains the Aspergillus ficuum bacterium bacterial classification after the taxol-producing endophytic fungi bacterial classification after activating, activation respectively.
Consisting of of liquid MRS substratum: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 20.0g, sodium acetate 5.0g, diammonium hydrogen citrate 2.0g, tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, manganous sulfate 0.05g and distilled water 1.0 liters.
Fermentation basic medium is made up of the component of following mass percent: glucose 1%, K 2hPO 40.1%, MgSO 40.05% and the water of surplus.
1LPBS damping fluid: potassium primary phosphate 0.27g, Sodium phosphate dibasic 1.42g, sodium-chlor 8g and Repone K 0.2g, adds the abundant stirring and dissolving of appropriate amount of deionized water, and then add concentrated hydrochloric acid and adjust pH to 7.2, last constant volume is to 1L.
Two, the preparation of endogenetic fungus fermented product
(1) 2cm is got 2taxol-producing endophytic fungi strain inoculation after size activation cultivates 5h in 25 DEG C in 1L liquid MRS substratum, and nutrient solution collects the first thalline through the centrifugal 10min of 6000rpm; First thalline is suspended in cholate mass percentage be 0.3% containing cholate liquid MRS substratum in hatch 25h in 25 DEG C, collect the second thalline through the centrifugal 10min of 6000rpm; By the second thalline, after PBS buffer solution for cleaning is clean, Eddy diffusion is in liquid MRS substratum, and concussion shakes up cultivates 6 days again at 25 DEG C, obtains taxol-producing endophytic fungi seed liquor;
Wherein, the first thalline is 1:1 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:1 with the volume ratio for the liquid MRS substratum of second thalline that suspends.
(2) 1cm is got 2aspergillus ficuum bacterium strain inoculation after size activation cultivates 5h in 25 DEG C in 1L liquid MRS substratum, nutrient solution collects the 3rd thalline through the centrifugal 10min of 8000 × g, 3rd thalline is suspended in NaCl mass percentage be 4% hatch 2h containing in the liquid MRS substratum of NaCl in 25 DEG C, collect the 4th thalline through the centrifugal 10min of 8000 × g; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, by the 4th thalline liquid by 1% inoculum size be inoculated into NaCl mass percentage be 4% containing NaCl liquid MRS substratum in 25 DEG C cultivate 15h, obtain Aspergillus ficuum bacterium seed liquor;
Wherein, the 3rd thalline is 1:1 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, and the 4th thalline is 1:1 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends.
(3) Aspergillus ficuum bacterium seed liquor is accessed in Ramulus et folium taxi cuspidatae solid-state fermentation culture medium by the access amount accounting for Ramulus et folium taxi cuspidatae solid-state fermentation culture medium volume 15%, cultivate 20 days at 22 DEG C, then be placed in the alternating magnetic field environment that magneticstrength is 3mT, field frequency is 25Hz and cultivate 12 days again, cultured products obtains Ramulus et folium taxi cuspidatae product by solid-state fermentation after vacuum lyophilization, pulverizing;
The preparation of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium comprises: the branches and leaves of fresh southerm yew, the stem tuber of SHENLIUGU are rinsed well under tap water, dry, chopping; Then 7g southerm yew and 1.6g SHENLIUGU fermentation basic medium are settled to 1L, mix, pH value nature, obtains Ramulus et folium taxi cuspidatae solid-state fermentation culture medium.
(4) Ramulus et folium taxi cuspidatae product by solid-state fermentation was pulverized 40 mesh sieves, through Subcritical Water Extraction, the condition of Subcritical Water Extraction is: water material mass ratio is 35:1, extracting pressure is at 15MPa, extraction temperature is 150 DEG C, extraction time is 35min, and obtain Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization.
(5) taxol-producing endophytic fungi seed liquor is accessed in endogenetic fungus liquid fermentation medium by the access amount accounting for endogenetic fungus liquid fermentation medium volume 10%, adjust pH is 5, cultivate 15 days at 20 DEG C, obtain endogenetic fungus tunning III through the centrifugal 10min of 6000rpm;
The preparation of endogenetic fungus liquid fermentation medium comprises: 0.6g is extracted residuum II fermentation basic medium and be settled to 1L, mix, and pH value nature, obtains endogenetic fungus liquid fermentation medium.
Cultivate composition containing taxol to be made up of 55 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction things I and 25 weight part endogenetic fungus tunnings III.
Three, the preparation of soft gel products: the weighting material of soft capsule is made up of 55 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, 25 weight part endogenetic fungus tunnings III and 3 part by weight of vitamin E.
Embodiment 2
One, material prepares
PDA plate culture medium: potato 200g, glucose 20g and agar 20g, be settled to 1000ml with water, natural pH, sterilizing 20min at 121 DEG C.
The Aspergillus ficuum bacterium bacterial classification of the taxol-producing endophytic fungi bacterial classification of slant preservation, slant preservation is inoculated on PDA plate culture medium respectively, carry out activation culture, culture temperature 20 DEG C, incubation time 25 days, obtains the Aspergillus ficuum bacterium bacterial classification after the taxol-producing endophytic fungi bacterial classification after activating, activation respectively.
Fermentation basic medium is made up of the component of following mass percent: glucose 3%, K 2hPO 40.2%, MgSO 40.1% and the water of surplus.
Liquid MRS substratum and PBS damping fluid are with embodiment 1.
Two, the preparation of endogenetic fungus fermented product
(1) 1cm is got 2taxol-producing endophytic fungi strain inoculation after size activation cultivates 6h in 20 DEG C in 1L liquid MRS substratum, and nutrient solution collects the first thalline through the centrifugal 10min of 6000rpm; First thalline is suspended in cholate mass percentage be 0.1% containing cholate liquid MRS substratum in hatch 40h in 20 DEG C, collect the second thalline through the centrifugal 10min of 6000rpm; By the second thalline, after PBS buffer solution for cleaning is clean, Eddy diffusion is in liquid MRS substratum, and concussion shakes up, and cultivates 10 days again at 20 DEG C, obtains taxol-producing endophytic fungi seed liquor;
Wherein, the first thalline is 1:2 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:2 with the volume ratio for the liquid MRS substratum of second thalline that suspends.
(2) 4cm is got 2aspergillus ficuum bacterium strain inoculation after size activation cultivates 4h in 30 DEG C in 1L liquid MRS substratum, nutrient solution collects the 3rd thalline through the centrifugal 10min of 8000 × g, 3rd thalline is suspended in NaCl mass percentage be 6% hatch 3h containing in the liquid MRS substratum of NaCl in 20 DEG C, collect the 4th thalline through the centrifugal 10min of 8000 × g; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, by the 4th thalline liquid by 2% inoculum size be inoculated into NaCl mass percentage be 6% containing NaCl liquid MRS substratum in 20 DEG C cultivate 30h, obtain Aspergillus ficuum bacterium seed liquor;
Wherein, the 3rd thalline is 1:3 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, and the 4th thalline is 1:3 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends.
(3) Aspergillus ficuum bacterium seed liquor is accessed in Ramulus et folium taxi cuspidatae solid-state fermentation culture medium by the access amount accounting for Ramulus et folium taxi cuspidatae solid-state fermentation culture medium volume 25%, cultivate 30 days at 16 DEG C, then be placed in the alternating magnetic field environment that magneticstrength is 0.2mT, field frequency is 3Hz and cultivate 20 days again, cultured products obtains Ramulus et folium taxi cuspidatae product by solid-state fermentation after vacuum lyophilization, pulverizing;
The preparation of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium comprises: the branches and leaves of fresh southerm yew, the stem tuber of SHENLIUGU are rinsed well under tap water, dry, chopping; Again 9.0g southerm yew and 3.0g SHENLIUGU fermentation basic medium are settled to 1L, mix, pH value nature, obtains Ramulus et folium taxi cuspidatae solid-state fermentation culture medium.
(4) Ramulus et folium taxi cuspidatae product by solid-state fermentation was pulverized 40 mesh sieves, through Subcritical Water Extraction, the condition of Subcritical Water Extraction is: water material mass ratio is 50:1, extracting pressure is at 20MPa, extraction temperature is 180 DEG C, extraction time is 10min, and obtain Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization.
(5) taxol-producing endophytic fungi seed liquor is accessed in endogenetic fungus liquid fermentation medium by the access amount accounting for endogenetic fungus liquid fermentation medium volume 20%, adjust pH is 7, cultivate 5 days at 25 DEG C, obtain endogenetic fungus tunning III through the centrifugal 10min of 6000rpm;
The preparation of endogenetic fungus liquid fermentation medium comprises: 1g is extracted residuum II fermentation basic medium and be settled to 1L, mix, and pH value nature, obtains endogenetic fungus liquid fermentation medium.
Cultivate composition containing taxol to be made up of 70 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction things I and 10 weight part endogenetic fungus tunnings III.
Three, the preparation of soft gel products: the weighting material of soft capsule is made up of 70 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, 10 weight part endogenetic fungus tunnings III and 5 part by weight of vitamin E.
Embodiment 3
One, material prepares
PDA plate culture medium, liquid MRS substratum and PBS damping fluid are with embodiment 1.
The Aspergillus ficuum bacterium bacterial classification of the taxol-producing endophytic fungi bacterial classification of slant preservation, slant preservation is inoculated on PDA plate culture medium respectively, carry out activation culture, culture temperature 30 DEG C, incubation time 3 days, obtains the Aspergillus ficuum bacterium bacterial classification after the taxol-producing endophytic fungi bacterial classification after activating, activation respectively.
Fermentation basic medium is made up of the component of following mass percent: glucose 2%, K 2hPO 40.15%, MgSO 40.08% and the water of surplus.
Two, the preparation of endogenetic fungus fermented product
(1) 4cm is got 2taxol-producing endophytic fungi strain inoculation after size activation cultivates 4h in 30 DEG C in 1L liquid MRS substratum, and nutrient solution collects the first thalline through the centrifugal 10min of 6000rpm; First thalline is suspended in cholate mass percentage be 0.5% containing cholate liquid MRS substratum in hatch 3h in 30 DEG C, collect the second thalline through the centrifugal 10min of 6000rpm; By the second thalline, after PBS buffer solution for cleaning is clean, Eddy diffusion is in liquid MRS substratum, and concussion shakes up, and cultivates 2 days again at 30 DEG C, obtains taxol-producing endophytic fungi seed liquor;
Wherein, the first thalline is 1:3 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:3 with the volume ratio for the liquid MRS substratum of second thalline that suspends.
(2) 2cm is got 2aspergillus ficuum bacterium strain inoculation after size activation cultivates 6h in 20 DEG C in 1L liquid MRS substratum, nutrient solution collects the 3rd thalline through the centrifugal 10min of 8000 × g, 3rd thalline is suspended in NaCl mass percentage be 2% hatch 0.5h containing in the liquid MRS substratum of NaCl in 28 DEG C, collect the 4th thalline through the centrifugal 10min of 8000 × g; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, by the 4th thalline liquid by 1% inoculum size be inoculated into NaCl mass percentage be 2% containing NaCl liquid MRS substratum in 28 DEG C cultivate 5h, obtain Aspergillus ficuum bacterium seed liquor;
Wherein, the 3rd thalline is 1:2 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, and the 4th thalline is 1:2 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends.
(3) Aspergillus ficuum bacterium seed liquor is accessed in Ramulus et folium taxi cuspidatae solid-state fermentation culture medium by the access amount accounting for Ramulus et folium taxi cuspidatae solid-state fermentation culture medium volume 5%, cultivate 3 days at 30 DEG C, then be placed in the alternating magnetic field environment that magneticstrength is 6mT, field frequency is 40Hz and cultivate 5 days again, cultured products obtains Ramulus et folium taxi cuspidatae product by solid-state fermentation after vacuum lyophilization, pulverizing;
The preparation of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium comprises: the branches and leaves of fresh southerm yew, the stem tuber of SHENLIUGU are rinsed well under tap water, dry, chopping; Again 5.5g southerm yew and 0.3g SHENLIUGU fermentation basic medium are settled to 1L, mix, pH value nature, obtains Ramulus et folium taxi cuspidatae solid-state fermentation culture medium.
(4) Ramulus et folium taxi cuspidatae product by solid-state fermentation was pulverized 40 mesh sieves, through Subcritical Water Extraction, the condition of Subcritical Water Extraction is: water material mass ratio is 15:1, extracting pressure is at 5MPa, extraction temperature is 100 DEG C, extraction time is 60min, and obtain Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization.
(5) taxol-producing endophytic fungi seed liquor is accessed in endogenetic fungus liquid fermentation medium by the access amount accounting for endogenetic fungus liquid fermentation medium volume 3%, adjust pH is 3, cultivate 25 days at 15 DEG C, obtain endogenetic fungus tunning III through the centrifugal 10min of 6000rpm;
The preparation of endogenetic fungus liquid fermentation medium comprises: 0.2g is extracted residuum II fermentation basic medium and be settled to 1L, mix, and pH value nature, obtains endogenetic fungus liquid fermentation medium.
Cultivate composition containing taxol to be made up of 40 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction things I and 35 weight part endogenetic fungus tunnings III.
Three, the preparation of soft gel products: the weighting material of soft capsule is made up of 40 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, 35 weight part endogenetic fungus tunnings III and 1 part by weight of vitamin E.
Comparative example 1
One, material prepares
Taxol-producing endophytic fungi bacterial classification after PDA plate culture medium, activation, liquid MRS substratum and fermentation basic medium are all with embodiment 1.
Two, the preparation of endogenetic fungus fermented product
(1) 2cm is got 2taxol-producing endophytic fungi strain inoculation after size activation is cultivated 5 days in 25 DEG C in 1L liquid MRS substratum, obtains taxol-producing endophytic fungi liquid seeds liquid.
(2) taxol-producing endophytic fungi liquid seeds liquid is fermented in basic medium by the access amount access Ramulus et folium taxi cuspidatae accounting for Ramulus et folium taxi cuspidatae fermentation basic medium volume 15%, cultivate 10 days at 25 DEG C, cultured products obtains endogenetic fungus tunning after vacuum lyophilization, pulverizing;
The preparation of Ramulus et folium taxi cuspidatae fermentation basic medium comprises: the branches and leaves of fresh southerm yew, the stem tuber of SHENLIUGU are rinsed well under tap water, dry, chopping; Then 7g southerm yew and 1.6g SHENLIUGU fermentation basic medium are settled to 1L, mix, pH value nature, obtain Ramulus et folium taxi cuspidatae fermentation basic medium.
Three, the preparation of soft gel products: the weighting material of soft capsule is made up of 55 weight part endogenetic fungus tunnings and 3 part by weight of vitamin E.
Comparative example 2
Except " preparation of Ramulus et folium taxi cuspidatae fermentation basic medium comprises: rinsed well under tap water by the branches and leaves of fresh southerm yew, dry, chopping; Then 7g southerm yew fermentation basic medium is settled to 1L, mixes, pH value nature, obtain Ramulus et folium taxi cuspidatae fermentation basic medium." outside, all the other operations, with comparative example 1, obtain endogenetic fungus tunning and soft capsule thereof.
The mensuration of activeconstituents
(1) measurement of the polysaccharide content
Take appropriate amount of sample dry powder, cross 40 orders after pulverizing, be placed in beaker, add distilled water by solid-liquid ratio 1:10 (mass ratio), 90 DEG C of lixiviate 3h, extract twice, filtration under diminished pressure collects filtrate, concentrating under reduced pressure at 50 DEG C, concentrated solution adds 4 times of volume dehydrated alcohols, alcohol precipitation overnight, after 3000r/min, 20min are centrifugal, precipitation is Crude polysaccharides.Precipitation distilled water is settled to 50mL, and measure polysaccharide in fermentation liquid content with phend-sulphuric acid, replication is averaged for 3 times, i.e. polysaccharide content.
(2) mensuration of content of taxol
Mixing extract after tunning cell excusing from death fragmentation.HPLC chromatographic condition: chromatographic column is AgilentEclipseXDB-C18 post (4.6mm × 150mm), moving phase is methanol-water (volume ratio 65:35), and determined wavelength is 228nm, and flow velocity is 0.8mL/min, column temperature is 35 DEG C, and sample size is 20 μ L.
The each embodiment of table 1 and comparative example main component detected result
Note: compare with comparative example 1, comparative example 2, Δ Δ: P<0.01.
The data presentation of table 1, the present invention cultivates composition containing taxol, significantly improves the content of polysaccharide in product, taxol isoreactivity composition simultaneously.Compared with conventional culture methods, what adopt the inventive method to obtain improves 60.5%-75.2% containing polysaccharide content in taxol cultivation composition, and taxol yield improves 47.4%-54.3%.
Liver-protecting function is tested
Laboratory animal: male mice in kunming, body weight 18 ± 2g.Sub-cage rearing, free diet, observes after 3 days for test.
Experimental design: carry out according to " protective foods inspection and assessment technical specifications " (2003 editions) judgement criteria, if basic, normal, high 3 dose of test groups and 1 negative Basal control group and 1 model control group, basic, normal, high 3 dosage component are not equivalent to people with 5 times, 10 times, 20 times of maximum recommended dosage, 0.15 namely, 0.30,0.60g/kgbw/d, basic, normal, high 3 dosage formulation concentration are respectively 15,30,60mg/mL.Basal control group and model control group all give distilled water, all edible full nutrition mixed feed of all laboratory animal, drinking-water of freely ingesting.
Per os gives the tested material of mouse corresponding dosage once a day, and mouse stomach amount is 10mL/kgbw, continuous gavage 30d, in experiment the 30th day by fasting 16h overnight for each treated animal.Model control group and tested group of gavage give 1%CCl 4vegetable oil solution (5mL/kgBW), Basal control group gives vegetables oil, and tested group is continued to give endogenetic fungus tunning in composition that embodiment obtains or comparative example to testing end (with CCl 4gavage interval 4h), give CCl 4after 24h, mouse is won eyeball and gets blood, measures Serum ALT, AST level, and gets liver and carry out histopathological examination.
Experimental result:
1. the composition that obtains of each embodiment is on the impact of Mouse Weight
As shown in Table 2, one-way analysis of variance is carried out to the average of the original body mass/end body weight (30d) of each group of mouse test, no significant difference (P>0.05), result shows that the body weight of endogenetic fungus tunning on animal in the composition that each embodiment obtains and comparative example has no obvious impact.
2. couple CCl 4the impact of liver injury mice serum ALT and AST
As shown in Table 3, ALT, AST content of model control group mouse is higher than negative Basal control group, statistical analysis difference has statistical significance (P < 0.05), composition that embodiment obtains 0.15,0.30, the ALT value of 0.60g/kgbw/d dosage group lower than model control group, statistical analysis difference has statistical significance (P < 0.05); The AST value of low middle high dose group is lower than model control group, and statistical analysis difference has statistical significance (P < 0.05).Result shows, the composition that embodiment 1-3 obtains is when being equivalent to 5 times, 10 times of human body recommended intake and 20 multiple dose, there is the effect reducing chemical damage mice serum ALT and AST, and the composition indices that under same dose, embodiment 1-3 is obtained has significant difference (P < 0.05) with comparing with comparative example 2 than comparative example 1, show to protect liver effect compared with the product that the present composition and conventional culture methods obtain more obvious.
3. the composition that embodiment is obtained is to CCl 4the histopathologic impact of liver injury mouse liver
As shown in Table 4, one-way analysis of variance is carried out to the average of each group of mouse liver histopathological indications quantized value, indices all with model control group comparing difference significance.Result shows, the composition that embodiment 1-3 obtains is when being equivalent to 5 times, 10 times of human body recommended intake and 20 multiple dose, experimentation on animals pathological examination is positive, show that it has the pathologic lesion effect alleviating chemical damage mouse liver, and the composition indices that under same dose, embodiment 1-3 is obtained has significant difference (P < 0.05) with comparing with comparative example 2 than comparative example 1, show to protect liver effect compared with the product that the present composition and conventional culture methods obtain more obvious.
Table 2 embodiment and comparative example product are on the impact of Mouse Weight
The product that table 3 embodiment and comparative example obtain is on the impact of mice serum ALT and AST
Group AST(U) ALT(U)
Basal control group 109.39±16.4 96.26±3.1
Model control group 405.38±18.0 320.24±4.1
Embodiment 1 low dose group 363.25±16.5 221.25±4.5
Dosage group in embodiment 1 311.25±17.9 189.65±4.9
Embodiment 1 high dose group 273.24±15.8 179.52±4.2
Embodiment 2 low dose group 356.27±18.4 219.59±5.7
Dosage group in embodiment 2 296.53±16.9 200.63±5.1
Embodiment 2 high dose group 257.26±18.1 173.54±4.2
Embodiment 3 low dose group 349.59±17.8 259.53±5.8
Dosage group in embodiment 3 286.37±15.9 223.19±4.7
Embodiment 3 high dose group 254.21±17.3 187.31±4.1
Comparative example 1 low dose group 379.54±17.1 292.67±5.3
Dosage group in comparative example 1 372.18±18.1 265.64±6.5
Comparative example 1 high dose group 371.12±18.3 259.65±6.7
Comparative example 2 low dose group 388.54±18.9 290.15±3.9
Dosage group in comparative example 2 375.33±17.5 273.25±4.9
Comparative example 2 high dose group 358.61±18.4 256.42±6.1
The product that table 4 embodiment obtains is to CCl 4the histopathologic impact of liver injury mouse liver
In the scope that formula of the present invention and preparation method limit, the change of each parameter does not affect the present invention and cultivates composition containing taxol and soft gel products protects liver function effect and preparation, and therefore in formula of the present invention and preparation method, the combination of arbitrary parameter all can obtain cultivating composition and soft gel products thereof containing taxol.Do not repeat them here.

Claims (10)

1. cultivate a preparation method for composition containing taxol, it is characterized in that, comprise step:
(1) get the taxol-producing endophytic fungi strain inoculation after activation and cultivate 4h-6h in liquid MRS substratum, collected by centrifugation first thalline; First thalline is suspended in the liquid MRS substratum containing cholate and hatches 3h-40h in 20 DEG C-30 DEG C, collected by centrifugation second thalline; By the second thalline, after PBS buffer solution for cleaning is clean, Eddy diffusion is in liquid MRS substratum, and concussion shakes up, and cultivates 2 days-10 days, obtain taxol-producing endophytic fungi seed liquor at 20 DEG C-30 DEG C;
(2) get the Aspergillus ficuum bacterium strain inoculation after activation and cultivate 4h-6h in liquid MRS substratum, collected by centrifugation the 3rd thalline, 3rd thalline is suspended in the liquid MRS substratum containing NaCl and hatches 0.5h-3h in 20 DEG C-28 DEG C, collected by centrifugation the 4th thalline; By the 4th thalline after PBS buffer solution for cleaning is clean Eddy diffusion in liquid MRS substratum, concussion shakes up and obtains the 4th thalline liquid, 4th thalline liquid is inoculated in the liquid MRS substratum containing NaCl and cultivates 5h-30h in 20 DEG C-28 DEG C, obtain Aspergillus ficuum bacterium seed liquor;
(3) by the Aspergillus ficuum bacterium seed liquor of step (2) access Ramulus et folium taxi cuspidatae solid-state fermentation culture medium, cultivate 3 days-30 days at 16 DEG C-30 DEG C, then be placed in alternating magnetic field environment and cultivate 5 days-20 days again, cultured products obtains Ramulus et folium taxi cuspidatae product by solid-state fermentation after vacuum lyophilization, pulverizing; Containing southerm yew and SHENLIUGU in described Ramulus et folium taxi cuspidatae solid-state fermentation culture medium;
(4) by the Ramulus et folium taxi cuspidatae product by solid-state fermentation of step (3) through Subcritical Water Extraction, obtain Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I, after extraction, remaining residue is extracted residuum II after vacuum lyophilization;
(5) by the taxol-producing endophytic fungi seed liquor of step (1) access endogenetic fungus liquid fermentation medium, adjust pH is 3-7, cultivates 5 days-25 days at 15 DEG C-25 DEG C, centrifugal, obtains endogenetic fungus tunning III; Containing extraction residuum II in described endogenetic fungus liquid fermentation medium;
Described is Ramulus et folium taxi cuspidatae fermentation subcritical abstraction thing I and endogenetic fungus tunning III containing taxol cultivation composition.
2. preparation method according to claim 1, is characterized in that, in step (3), containing 5.5g-9.0g southerm yew and 0.3g-3.0g SHENLIUGU in the every 1L of described Ramulus et folium taxi cuspidatae solid-state fermentation culture medium.
3. preparation method according to claim 1, is characterized in that, in step (5), extracts residuum II in the every 1L of described endogenetic fungus liquid fermentation medium containing 0.2g-1g.
4. preparation method according to claim 1, is characterized in that, in step (1), described is 0.1%-0.5% containing the mass percentage of cholate in the liquid MRS substratum of cholate;
In step (2), the mass percentage of the described liquid MRS NaCl in medium containing NaCl is 2%-6%.
5. preparation method according to claim 1, it is characterized in that, in step (1), described first thalline is 1:1-3 with the volume ratio of the liquid MRS substratum containing cholate for first thalline that suspends, and the second thalline is 1:1-3 with the volume ratio for the liquid MRS substratum of second thalline that suspends;
In step (2), described 3rd thalline is 1:1-3 with the volume ratio of the liquid MRS substratum containing NaCl for the 3rd thalline that suspends, 4th thalline is 1:1-3 with the volume ratio for the liquid MRS substratum of the 4th thalline that suspends, and the inoculum size of the 4th thalline liquid is 1%-2%.
6. preparation method according to claim 1, is characterized in that, in step (3), in described alternating magnetic field environment, magneticstrength is 0.2mT-6mT, and field frequency is 3Hz-40Hz;
In step (4), the condition of described Subcritical Water Extraction is: water material mass ratio is 15-50:1, and extracting pressure is at 5MPa-20MPa, and extraction temperature is 100 DEG C-180 DEG C, and extraction time is 10min-60min.
7. preparation method according to claim 1, is characterized in that, in step (3), the access amount of described Aspergillus ficuum bacterium seed liquor is the 5%-25% of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium volume;
In step (5), the access amount of described taxol-producing endophytic fungi seed liquor is the 3%-20% of endogenetic fungus liquid fermentation medium volume.
8. cultivate a composition containing taxol, it is characterized in that, adopt the preparation method described in any one of claim 1-7 to prepare.
9. the taxol that contains according to claim 8 cultivates composition, it is characterized in that, described composition is made up of 40 weight part-70 weight part Ramulus et folium taxi cuspidatae fermentation subcritical abstraction things I and 10 weight part-35 weight part endogenetic fungus tunnings III.
10. cultivate an application for composition containing taxol, it is characterized in that, the taxol cultivation composition that contains according to claim 8 or claim 9 is preparing the application in the healthcare products or medicine protecting liver.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520854A (en) * 2016-08-19 2017-03-22 重庆市碚圣医药科技股份有限公司 Chinese yew solid state fermentation medium, preparation method thereof and Chinese yew solid state fermentation method

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101177666A (en) * 2006-11-12 2008-05-14 黑龙江大学 Novel bacterial producing paclitaxel and method for breeding and preparing paclitaxel
CN101386874A (en) * 2008-10-28 2009-03-18 南开大学 Two-liquid phase fermentation method for improving paclitaxel yield from fungi
CN104073529A (en) * 2014-06-18 2014-10-01 聊城大学 Method for producing taxol by utilizing Chinese yew seed endophyte

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101177666A (en) * 2006-11-12 2008-05-14 黑龙江大学 Novel bacterial producing paclitaxel and method for breeding and preparing paclitaxel
CN101386874A (en) * 2008-10-28 2009-03-18 南开大学 Two-liquid phase fermentation method for improving paclitaxel yield from fungi
CN104073529A (en) * 2014-06-18 2014-10-01 聊城大学 Method for producing taxol by utilizing Chinese yew seed endophyte

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106520854A (en) * 2016-08-19 2017-03-22 重庆市碚圣医药科技股份有限公司 Chinese yew solid state fermentation medium, preparation method thereof and Chinese yew solid state fermentation method

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