CN109022510A - A method of production taxol and 10-DAB III are co-cultured using southerm yew VA Mycorrhizal Fungi and living tissue cells - Google Patents
A method of production taxol and 10-DAB III are co-cultured using southerm yew VA Mycorrhizal Fungi and living tissue cells Download PDFInfo
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Abstract
A method of production taxol and 10-DAB III being co-cultured using southerm yew VA Mycorrhizal Fungi and its living tissue cells, comprising steps of the 1) induction of southerm yew mycorhiza and acquisition；2) configuration of VA Mycorrhizal Fungi isolation medium；3) VA Mycorrhizal Fungi is separately cultured bacterium (E20)；4) bacterial strain rejuvenation；5) strain cultivation；6) preparation southerm yew living tissue cells；7) E20 bacterial strain with the in vitro living tissue cells of southerm yew are closed is protected from light co-cultivation；8) extraction purification.The present invention is co-cultured by the E20 bacterial strain obtained from separation screening in principal and subordinate's southerm yew mycorhiza with southerm yew branches and leaves cell living, callus cell in vitro, it rebuilds symbiosis interaction process in vitro using the two and realizes and continue, steadily improve system taxol and III yield of 10-DAB, the content of 10-DAB III and taxol can be made to improve.It is easy to operate, environmentally friendly, high-efficient.
Description
Technical field
The invention belongs to bioengineering and field of biological pharmacy, and in particular to a kind of to utilize plant living tissue cells and VA Mycorrhizal Fungi
Co-culture the method for rebuilding the symbiosis interaction system production medicinal secondary metabolism ingredient of plant or its precursor.
Background technique
Chinese yew has 11 kinds and several mutation in the whole world, and temperate zone to torrid areas is distributed on the Northern Hemisphere.Hair at present
Existing main species have yewtree (Taxus. brevifoliaNutt.), Canadian Chinese yew (T. cannadlensisMarsh.), cone Chinese yew (T. globose), hybrid Japanese yew (T. mediaRehd.), European red bean
China fir (T. baccataL.), Florida Chinese yew (T. floridanaChapm.), taxus chinensis in northeast (T. cuspidata
Sieb. et Zucc.), taxusyunnanensis (T. yunnanensisCheng et L. K. Fu), Chinese Chinese yew (T. chinesisRehd. Chinese Yew), southerm yew (T. chinensis var. mairei Cheng et L.
K. Fu) and Xizang Taxus chinensis (T.wallichinana Zucc.) etc.；Wherein there are 4 kind of 1 mutation, i.e., southern red bean in China
China fir (mutation), taxusyunnanensis, taxus chinensis in northeast, Xizang Taxus chinensis, Chinese Chinese yew, are distributed in Central China, south China, East China, west
The ground such as south, Tibet, northeast, northwest, but the producing region relatively concentrated is southwest, northeast and South China.South involved in the present invention
Chinese yew is widely distributed in Southwestern China, South China, easily cultivate, the speed of growth is very fast, but its branches and leaves, taxol in bark and
The content of 10-DAB III is lower (0.08 ‰~0.2 ‰), is estimated according to extraction cost and market value, does not have business substantially
Extract value.
Plant cell culture mainly has 2 kinds of approach at present, i.e., taxol is directly extracted from Chinese yew genus plants, or extracts purple
The precursor 10- deacetylate Bakating III (10-DAB III) of China fir alcohol, then molecular design taxol or Docetaxel.It is purple
Structure is complicated for China fir alcohol, and the fully synthetic technique of chemistry is cumbersome, at high cost, no commercial application value.Reported taxol-producing endophytic fungi
And engineering bacteria is also reported without industrialized production since paclitaxel produced ability is lower and unstable.It is precursor with 10-DAB III
Though molecular design taxol has been applied to industrialized production, Chinese yew resource is still relied on, and is relied on substantially from foreign countries
Import european yew raw material, cost occupies height, and the raw material production cycle is long, is difficult to meet the market demand.Cell and tissue structrue is raw
Paclitaxel produced, due to stability, cost of amplification etc., technology is not mature enough.So seeking low cost, stable purple
China fir alcohol medicine source is always the emphasis and hot spot of this field research.
Number of patent application: 201010560051.1, publication number: 102071230 A of CN, denomination of invention: a kind of to utilize micro- life
The method that object fermentation method extracts taxol from Chinese yew.This method is to impregnate Chinese yew using the good fermentation liquid of saccharomycetes to make fermentation
Branches and leaves powder a couple of days, then soak is concentrated, the taxol dry product of high-content can be obtained in spray drying.The technology
Feature is the taxol extracted in Chinese yew using biologic enzymolysis method, and extraction time is short, and technique simplifies, and extraction efficiency is high, is reduced
Cost, and pollution-free, but it is unrelated with biological interaction to improve Secondary metabolites content principle.
Number of patent application: 201010170989.2, publication number: 1018921162 A of CN, a kind of denomination of invention: taxol
Producing strains fermentation branches and leaves of yew produces taxol new method.This method is the Chinese yew for instantaneously being dewaxed, being disinfected with high pressure
Fresh branches and leaves are fermentation main material, by utilizing the paclitaxel precursor object in paclitaxel produced bacterial strain microbe conversion branches and leaves of yew,
Obtain paste taxol crude extract.This method carries out solid fermentation using Taxol-producing fungi single culture, to convert Chinese yew
Paclitaxel precursor object Taxol Biosynthesis in branches and leaves.Integrated artistic simplifies, easy to industrialized production.But this method and this method
The strain and its fermentation main material utilized is different, and fermentation main material is after 125 DEG C of disinfection with high pressure steam handle 1 min
Fresh branches and leaves of yew, therefore branches and leaves cell has inactivated, fermentation main material is non-living body.
Number of patent application: 200810152500.1, publication number: 101386819 A of CN, denomination of invention: one plant is paclitaxel produced
Saprophytic fungus and application the bacterial strain production taxol method.This method is different from this method, and this method is more using quasi- disk
Hair spore category saprophytic fungus carries out liquid fermentation to obtain taxol.This method fermentation period is short, easy to industrialized production, in patent
Do not provide taxol yield in tunning as a result, whether its yield has, to commercially produce value also unknowable.
Number of patent application: 200710144533.7, publication number: 101220384 A of CN, a kind of denomination of invention: efficiently induction
The method for extracting four kinds of bearing taxanes.This method is identical as many method raw materials, is renewable resource red bean
The fresh branches and leaves of China fir efficiently produce branch of Ramulus et folium taxi cuspidatae using enzyme, ultraviolet irradiation and oxygen as elicitor by fresh mill homogenization process
The method of 10-DAB, Bakating III, Cephalomannine and taxol in leaf.This method uses amylase and cellulase, does not break
Bad ecological resources, operation is simple, is suitable for industrial application, is of great significance.But the principle of this method be not live strain with
Symbiosis interaction between raw material active somatic cell, but external cause induces.
Pertinent literature report is disclosed, and the fermentation liquid taxol yield of the endophytic fungus associated with taxol of early stage research discovery is 50
ng·L-1.Stierle and Strobel etc. most early in yewtree (T.brevifolia) branch in be separated to one kind can be with
Paclitaxel produced endogenetic fungus, and be named as An Delieya it is mould (Taxomyces andreanae).Li et al. is from bald cypress
(Taxodium distichum) bark, be all separated to paclitaxel produced endogenetic fungus in bast and xylemPestalotiopsis microspora.Strobel GA etc. from a kind of pine tree (Wollemia nobilis) in be separated to production
Taxol endogenetic fungusPestalotiopsis guepini.Illustrate that paclitaxel produced endogenetic fungus has diversity, these grind
Study carefully host's scope of resource of further expansion taxol-producing endophytic fungi.Later people further from podocarpus, bald cypress,Wollemi Pine, Chinese torreya (Torreya grandifolia) etc. be separated to paclitaxel produced fungi in non-Chinese yew genus plants.
Lou waits quietly being also separated to taxol/alkane producing strains in midget crabapple, coniferae.Result of study is shown at present, natural
In boundary, plant endogenesis epiphyte is the major microorganisms with paclitaxel produced ability, it has been found that has more than 20 category, including quasi- disk crinosity
Spore category (Pestalotiopsis microspora), treelike menu category (Noduzisporium sylviforme), sickle-like bacteria
Belong to (Fusari-um), rod method Pseudomonas (Alternaria), Phoma (Phomopsis), tumor seat Pseudomonas
(Tuberculariasp), aspergillus (Aspergillus nigerVar), nigrospora category (Periconia sp) etc..But it arrives
So far, there are no the reports about the pure fermented and cultured industrialized production taxol of utilization microorganism or 10-DAB III, because
The bacterial strain of the separated paclitaxel produced or 10-DAB III arrived, it is unstable to show industry characteristics.The Standard General of fermentation yield
It needs to reach 1 mgL-1, just there are industrial potentiality in left and right.Report highest reachable 1 mgL of yield at present-1, already close to the requirement of industrialization.The researcher of one Zhi Liu drugmaker of Xi'an isolates one from Chinese bark of Ramulus et folium taxi cuspidatae
Kind fungi can make the mass concentration of Baccatin III and taxol in culture solution equal through recombination mutagenesis and strain optimization culture
Reach 2 mgL-1, the potentiality of industrialization production are all had, but have no the open report of industrialization production.Be currently known report compared with
It is consistent the result is that the hereditary capacity of bacterial strain this aspect is unstable, be difficult continue, stable industrialization production.This project
Group early-stage study also there is similar problem, 2~3 generation of pure culture, yield just starts to decay, until disappear, post analysis recognize
It is mostly fungal component for this kind of bacterium, once separating with its host, the metabolic function that reason is born with altogether is possible will be due to separation
It loses.Therefore, sustainable to its paclitaxel produced or 10-DAB III ability, it constructs the syntaxial system in vitro again, restores it
Possessed metabolic function is the key problem in technology of present aspect when symbiosis.
In conclusion there are many research direction both at home and abroad about plant cell culture approach, but molding industrialized
The approach of production still only there are two types of, i.e., directly extract taxol from Chinese yew genus plants, or extract the precursor 10- of taxol
Deacetylate Bakating III (10-DAB III) molecular design taxol or Docetaxel afterwards.Chinese yew natural resources are insufficient,
Cultivating Chinese yew slow growth cannot provide enough raw materials in the short time.Structure is complicated for taxane molecule, is difficult to carry out chemical conjunction
At industrialized production.Cell culture processes are then because time-consuming, the genetic stability of squamous subculture is difficult repeatedly for high yielding cell sarain breeding
The reasons such as height are occupied with control, cost and fail to be applied by industrialization production.It is raw that domestic and foreign scholars were once turning to fungi fermentation method
Paclitaxel produced research, but paclitaxel produced bacterial strain is mostly endophyte of plant, and once being detached from ecosystem environment, bacterial strain secondary metabolism is lost
The sustainable and stability critical issue passed is relatively difficult to resolve certainly, and performance is unstable, at the technical bottleneck of industrialization production.
Summary of the invention
For lasting and the stability, southerm yew branches and leaves, callus for solving above-mentioned taxol bacterial strain industrialization production heredity
The problem of tissue paclitaxel low yield, is provided a kind of co-cultured using southerm yew VA Mycorrhizal Fungi and living tissue cells and produces Japanese yew
The method of alcohol and 10-DAB III.
To achieve the above object, the technical solution adopted by the present invention: a kind of to utilize southerm yew VA Mycorrhizal Fungi and living tissue
Cell co-cultures production taxol and the method for 10-DAB III, includes the following steps:
1) induction of southerm yew mycorhiza and acquisition: between spring and summer, shallow cut intertillage is carried out to Chinese yew forest land and is loosened the soil, is extracted miscellaneous
Grass is covered on forest land, about 5~10 cm of thickness, excavates mycorhiza October~November；
2) VA Mycorrhizal Fungi is separately cultured: using 26271 solid mediums, separation obtains a variety of mycorhiza from southerm yew mycorhiza
Bacterium, then 26271 fluid nutrient mediums are respectively adopted and carry out the pure fermented and cultured of single strain, using HPLC and HPLC/MS to fermentation liquid and
The content of taxol and 10-DAB III are detected in mycelium, and filtering out has compared with III ability of High Yield of Taxol and 10-DAB
Bacterial strain, being denoted as E20 bacterial strain, (fungi, code name are to have the ability compared with High Yield of Taxol and 10-DAB III, type waits reflecting from intending
It is fixed), it is stored refrigerated；
3) go out 26271 special solid culture mediums through orthogonal experiment research trial: a large amount of+MS(of 1/2 MS are micro, it is organic without hormone,
Molysite)+0.5 g L-1+ 1 g L of yeast extract-1+ 100 mL potato of caseinhydrolysate boils+30 g L of filtrate-1Sucrose+7
~14 g L-1Agar；Culture medium adjusts pH 5.8~7 through boiling, and through 121 DEG C of 30 min of high pressure steam sterilization, puts inclined-plane, to be solidified
It shelves at room temperature afterwards after-ripening 7 days or more, it is spare；
4) potato boils filtrate: cutting the potato peeled and is placed in tap water, with 30min is boiled with soft fire, then filters out juice；
5) 26271 fluid nutrient medium: a large amount of+MS (micro, molysite organic without hormone)+0.5 g L of 1/2 MS-1Yeast extract
+ 1 g L-1+ 100 mL potato of caseinhydrolysate boils+30 g L of filtrate-1Sucrose；Adjust 5.8~7,121 DEG C of high steams of pH
Sterilizing, it is spare.
) E20 bacterial strain rejuvenation: the oblique of 26271 solid mediums newly prepared is transferred to by stored refrigerated sterile point of strain
On face, the rejuvenation culture 5~7 days at 23 DEG C~28 DEG C.
) strain cultivation: the E20 bacterial strain after taking rejuvenation is transferred in the container for filling 29271 fluid nutrient mediums, and 120
rpm·min-1, under the conditions of 23 DEG C~28 DEG C, shaken cultivation 3~5 days, until growth animated period, is used as production inoculation；
8) it the acquisition of southerm yew branches and leaves and pretreatment: in autumn and winter clip southerm yew annual shoot leaf, smashs to pieces, becomes
The fragment of 2mm~5mm；
9) E20 bacterial strain and southerm yew are lived in vitro, and the progress of branches and leaves histocyte is closed to be protected from light fermentation: will be processed
Southerm yew is lived branches and leaves, and the E20 liquid spawn of above-mentioned culture is accessed in 10~20% ratio, after mixing thoroughly in 23 DEG C of temperature~
It is 28 DEG C, closed under humidity 50%~70%, it is protected from light, co-cultures 7~10 days；
10) extraction purification: the southerm yew branches and leaves that will state that treated through above-mentioned fermentation crush, mistake at once after 40 DEG C of drying
60 meshes, are then added the methanol of 10 times of volumes, and extracting solution is concentrated under reduced pressure by 30 min of ultrasound assisted extraction at 40 DEG C
Dry, the methanol that appropriate volume is then added redissolves, and adds isometric petroleum ether extraction, and extraction is collected methanol phase, subtracted in 40 DEG C
Pressure is concentrated to dryness, and adds appropriate volume methylene chloride to redissolve, and with Er Lv Jia Wan ︰ water=2 ︰ 1(V/V) it is extracted, water phase is discarded,
Methylene chloride phase is collected, 40 DEG C of reduced pressures sufficiently wash out concentration solid content with proper amount of methanol, and are diluted to debita spissitudo, adopt
It is separated with high performance liquid preparative chromatography, i.e., according to taxol and 10- when using high performance liquid chromatography detection under the same terms
The automatic collection mode of peak retention time setting sample of DAB III carries out sample collection, and then carrying out condensing crystallizing can be obtained
The relatively high sterling of III two kinds of target compounds of taxol and 10-DAB.
The present invention reconstructs symbiosis interaction using plant symbiosis bacterium and host plant histocyte living in vitro, and building is planted
Object secondary metabolites production system utilizes and separates the VA Mycorrhizal Fungi E20 bacterial strain obtained and southern red from southerm yew mycorhiza
The in vitro living tissue cells of beans China fir carry out co-culturing the technical side for rebuilding symbiosis interaction volume increase taxol and 10-DAB III in short-term
Method, it is characterised in that used E20 bacterial strain system inventor new separation screening from principal and subordinate's southerm yew mycorhiza obtains, and has
Relatively strong paclitaxel produced and 10-DAB III ability, it is further characterized in that the organ of southerm yew of the utilization with fresh and alive cell,
Tissue is also resided in when the bacterial strain after mostly no longer paclitaxel produced for subculture pure culture and 10-DAB III by having with southerm yew
Have that the organ of fresh and alive cell, tissue co-cultures that the content of 10-DAB III and taxol can be made in system compared with the natural branch of southerm yew
Leaf is respectively increased 3.2,6.38 times, can reach 1.132 ‰ (dry weights) and 0.512 ‰ (dry weights) respectively.
The present invention is using fresh and alive leaf and green sprig, callus, through the mechanical fragment for suitably smashing into 2mm~5mm to pieces
It is inoculated with without sterilization afterwards, is co-cultured under room temperature (23 DEG C~28 DEG C) and can be used within 7~10 days product extraction separation.This
Technical matters is simple, and mild condition, processing time is short, high-efficient, can be by there are few the southerm yew branches and leaves of economic value
Be converted to high value resource, can greatly alleviate anti-cancer medicine paclitaxel raw materials for production supply problem and reduce taxol and
The production cost of 10-DAB III, industrial application value with higher.
Specific embodiment:
Embodiment one
1, reagent: taxol control product (HPLC detection reference substance, >=98.0%, the brilliant pure limited public affairs of biochemical technology share in Shanghai
Department), III reference substance of 10-DAB (HPLC detection reference substance, >=95.0%, Shanghai Jing Chun biochemical technology limited liability company), first
Alcohol (AR, Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd), (AR, the permanent emerging chemical reagent manufacture in Tianjin are limited for methylene chloride
Company), methanol (HPLC, Tianjin Kermel Chemical Reagent Co., Ltd.), acetonitrile (HPLC, Tianjin Ke Miou chemical reagent
Co., Ltd), redistilled water (self-control).
2, material: clip contains annual band leaf sprig in self-built southerm yew young growth:
3, pretreatment: diameter 2mm is pounded with machinery rapidly when the fresh band leaf sprig of the southerm yew of clip is multiplied fresh and alive
The fragment of~5mm；
4, it bottling inoculation: weighs through above-mentioned pretreated 1000 g of fresh and alive branches and leaves, with E20 bacterium of 200 mL after rejuvenation culture
Liquid is fitted into open mouth glass after mixing, and is sealed with brown paper.Totally 5 bottles.
5, co-culture: by it is above-mentioned be inoculated with install a bottle material be placed in 25 DEG C of temperature, humidity 60%, be protected from light under the conditions of cultivate 8
It；
6, tunning is harvested, the content of 10- deacetylate Bakating III and taxol in detection architecture carries out product extraction point
From.
7. conclusion
After being handled using above-mentioned process flow, 10- deacetylate Bakating III and content of taxol in the fresh branches and leaves of Chinese yew
The natural branches and leaves of more natural southerm yew improve 3.54 and 5.44 times respectively, have reached 1.078 ‰ and 0.397 ‰.
The results show co-cultures reconstruct symbiosis using southerm yew branches and leaves living and its VA Mycorrhizal Fungi E20 bacterial strain in short-term
Interaction system can get the yield of higher 10- deacetylate Bakating III and taxol, and working condition is mild, simple process,
At low cost, less energy consumption is pollution-free, and southerm yew branches and leaves resource can be made to be fully used, and has important industrialized production
Meaning.
Embodiment two
One, reagent:
Taxol control product (HPLC detection reference substance, >=98.0%, Shanghai Jing Chun biochemical technology limited liability company), 10-
III reference substance of DAB (HPLC detection reference substance, >=95.0%, Shanghai Jing Chun biochemical technology limited liability company), methanol (AR, day
Heng Xing chemical reagent Manufacturing Co., Ltd of Jinshi City), methylene chloride (AR, Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd), first
Alcohol (HPLC, Tianjin Kermel Chemical Reagent Co., Ltd.), acetonitrile (HPLC, the limited public affairs of Tianjin Ke Miou chemical reagent
Department), redistilled water (self-control).
Two, pretreatment:
1, the potato 100g to have peeled is cut, tap water 400ml is added, with 30min is boiled with soft fire, is squeezed later with four layers of cotton gauze
Filter out juice.
, 26271 solid mediums: a large amount of+MS(of 1/2 MS are micro, molysite organic without hormone)+0.5 g L-1Yeast leaching
+ 1 g L of cream-1+ 100 mL potato of caseinhydrolysate boils+30 g L of filtrate-1Sucrose+7~14 g L-1Agar.Culture medium warp
Boiling adjusts pH 5.8~7, through 121 DEG C of 30 min of high pressure steam sterilization after packing, inclined-plane is put, after shelving at room temperature after to be solidified
It is ripe 7 days or more, spare.
, 26271 fluid nutrient mediums: a large amount of+MS (micro, molysite organic without hormone)+0.5 g L of 1/2 MS-1Yeast leaching
+ 1 g L of cream-1+ 100 mL potato of caseinhydrolysate boils+30 g L of filtrate-1Sucrose.PH 5.8~7 is adjusted, through 121 after packing
DEG C high pressure steam sterilization 30 min, it is spare.
Three, the step of co-culturing production taxol and 10-DAB III using southerm yew VA Mycorrhizal Fungi and living tissue cells:
1) between spring and summer, shallow cut intertillage is carried out to Chinese yew forest land and is loosened the soil, after extract weeds and be covered on forest land, thickness
About 5~10 cm can excavate mycorhiza October~November.
2) VA Mycorrhizal Fungi is separately cultured: using 26271 solid mediums of autonomous trial, being separated from southerm yew mycorhiza
Obtain more than 20 VA Mycorrhizal Fungis, then 26271 fluid nutrient mediums be respectively adopted and carry out the pure fermented and cultured of single strain, using HPLC and
HPLC/MS detects the content of taxol and 10-DAB III in fermentation liquid and mycelium, and filtering out has compared with high yield Japanese yew
(fungi, code name are to have the energy compared with High Yield of Taxol and 10-DAB III from intending to the bacterial strain E20 bacterial strain of III ability of alcohol and 10-DAB
Power, type are to be identified).
) strain in refrigerator will be stored in be transferred to 26271 solid mediums newly prepared for sterile point in superclean bench
Inclined-plane on, the rejuvenation culture 5~7 days at 23 DEG C~28 DEG C.
) use inoculation shovel 1 piece of quantitative (about 1.5x1.5 cm2) mycelia after rejuvenation is transferred to and fills 100 m L 29271
In 250 mL triangular flasks of fluid nutrient medium, 120 rpmmin-1, under the conditions of 23 DEG C~28 DEG C, shaken cultivation 3~5 days, until
Animated period is grown, is used as production inoculation.
) in autumn and winter clip southerm yew annual shoot leaf, it is suitably smashed to pieces with machinery, becomes the broken of 2mm~5mm
Piece；
6) by processed southerm yew branches and leaves living, the E20 liquid bacteria of above-mentioned culture is accessed in 10~20% ratio
Kind, it is closed after mixing thoroughly under 23 DEG C~28 DEG C of temperature, humidity 50%~70%, it is protected from light, co-cultures 7~10 days.
) the southerm yew branches and leaves that will state that treated through above-mentioned fermentation, powder at once after being dried rapidly in 40 DEG C of baking ovens
It is broken, cross 60 meshes, then be added 10 times of volumes methanol, 30 min of ultrasound assisted extraction, will extract 4 times after gained extracting solution in
It being concentrated to dryness at 40 DEG C, the methanol that appropriate volume is then added redissolves, and adds isometric petroleum ether extraction, it extracts 4 times,
Methanol phase is collected, is concentrated to dryness in 40 DEG C, adds appropriate volume methylene chloride to redissolve, and with Er Lv Jia Wan ︰ water=2 ︰ 1(V/
V it) is extracted, is repeated 3 times.Water phase is discarded, methylene chloride phase is collected, 40 DEG C of reduced pressures are sufficiently washed out dense with proper amount of methanol
Contracting solid content, and it is diluted to debita spissitudo, it is separated using high performance liquid preparative chromatography, i.e., it is high according to being used under the same terms
The automatic collection mode of the peak retention time of taxol and 10-DAB III setting sample carries out sample receipts when effect liquid phase chromatogram detects
Collection, then carrying out condensing crystallizing can be obtained the relatively high sterling of two kinds of target compounds.
Claims (1)
1. a kind of co-culture production taxol and the method for 10-DAB III using southerm yew VA Mycorrhizal Fungi and its living tissue cells,
It is characterized by comprising the following steps:
1) induction of southerm yew mycorhiza and acquisition: between spring and summer, shallow cut intertillage is carried out to Chinese yew forest land and is loosened the soil, is extracted miscellaneous
Grass is covered on forest land, about 5~10 cm of thickness, excavates mycorhiza October~November；
2) VA Mycorrhizal Fungi is separately cultured: using 26271 solid mediums, separation obtains a variety of mycorhiza from southerm yew mycorhiza
Bacterium, then 26271 fluid nutrient mediums are respectively adopted and carry out the pure fermented and cultured of single strain, using HPLC and HPLC/MS to fermentation liquid and
The content of taxol and 10-DAB III are detected in mycelium, and filtering out has compared with III ability of High Yield of Taxol and 10-DAB
Bacterial strain, being denoted as E20 bacterial strain, (fungi, code name are to have the ability compared with High Yield of Taxol and 10-DAB III, type waits reflecting from intending
It is fixed), it is stored refrigerated；
3) 26271 solid mediums are the special culture media that independent research trial goes out, composition and configuration process are as follows: 1/2 MS is big
Amount+MS(is micro, molysite organic without hormone)+0.5 g L-1+ 1 g L of yeast extract-1+ 100 mL potato of caseinhydrolysate
Boil+30 g L of filtrate-1Sucrose+7~14 g L-1Agar；Culture medium adjusts pH 5.8~7 through boiling, through 121 DEG C of high steams
Sterilize 30 min, puts inclined-plane, shelves at room temperature after to be solidified after-ripening 7 days or more, spare；
4) potato boils filtrate: cutting the potato peeled and is placed in tap water, with 30min is boiled with soft fire, then filters out juice；
5) 26271 fluid nutrient medium: a large amount of+MS (micro, molysite organic without hormone)+0.5 g L of 1/2 MS-1Yeast extract
+ 1 g L-1+ 100 mL potato of caseinhydrolysate boils+30 g L of filtrate-1Sucrose；Adjust 5.8~7,121 DEG C of high steams of pH
Sterilizing, it is spare；
6) E20 bacterial strain rejuvenation: stored refrigerated sterile point of strain is transferred to the inclined-plane for 26271 solid mediums newly prepared
On, the rejuvenation culture 5~7 days at 23 DEG C~28 DEG C；
7) strain cultivation: the E20 bacterial strain after taking rejuvenation is transferred in the container for filling 29271 fluid nutrient mediums, 120 rpm
min-1, under the conditions of 23 DEG C~28 DEG C, shaken cultivation 3~5 days, until growth animated period, is used as production inoculation；
8) it the acquisition of southerm yew branches and leaves and pretreatment: in autumn and winter clip southerm yew annual shoot leaf, smashs to pieces, becomes
The fragment of 2mm~5mm；
9) E20 bacterial strain and southerm yew are lived in vitro, and the progress of branches and leaves histocyte is closed to be protected from light culture: will be processed
Southerm yew is lived branches and leaves, and the E20 liquid spawn of above-mentioned culture is accessed in 10~20% ratio, after mixing thoroughly in 23 DEG C of temperature~
It is 28 DEG C, closed under humidity 50%~70%, it is protected from light, co-cultures 7~10 days；
10) it extraction purification: by the southerm yew branches and leaves after above-mentioned fermentation process, is crushed at once after 40 DEG C of drying, crosses 60 mesh
Sieve, is then added the methanol of 10 times of volumes, and extracting solution is concentrated to dryness, so by 30 min of ultrasound assisted extraction at 40 DEG C
The methanol that appropriate volume is added afterwards redissolves, and adds isometric petroleum ether extraction, and methanol phase is collected in extraction, is concentrated under reduced pressure in 40 DEG C
To doing, appropriate volume methylene chloride is added to redissolve, and with Er Lv Jia Wan ︰ water=2 ︰ 1(V/V) it is extracted, water phase is discarded, collects two
Chloromethanes phase, 40 DEG C of reduced pressures sufficiently wash out concentration solid content with proper amount of methanol, and are diluted to debita spissitudo, using efficient
Liquid phantom preparing chromatogram is separated, i.e., according to taxol when using high performance liquid chromatography detection under the same terms with 10-DAB's III
Peak retention time sets the automatic collection mode of sample and carries out sample collection, then carry out condensing crystallizing can be obtained taxol and
The relatively high sterling of III two kinds of target compounds of 10-DAB.
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