CN106520854A - Chinese yew solid state fermentation medium, preparation method thereof and Chinese yew solid state fermentation method - Google Patents
Chinese yew solid state fermentation medium, preparation method thereof and Chinese yew solid state fermentation method Download PDFInfo
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- CN106520854A CN106520854A CN201610695714.8A CN201610695714A CN106520854A CN 106520854 A CN106520854 A CN 106520854A CN 201610695714 A CN201610695714 A CN 201610695714A CN 106520854 A CN106520854 A CN 106520854A
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- 238000010563 solid-state fermentation Methods 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 241001149649 Taxus wallichiana var. chinensis Species 0.000 title abstract 10
- 239000000843 powder Substances 0.000 claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 239000007787 solid Substances 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 69
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 9
- 239000012138 yeast extract Substances 0.000 claims description 9
- 240000007594 Oryza sativa Species 0.000 claims description 8
- 235000007164 Oryza sativa Nutrition 0.000 claims description 8
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 8
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 8
- 235000012054 meals Nutrition 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 235000009566 rice Nutrition 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 235000015099 wheat brans Nutrition 0.000 claims description 8
- 238000003958 fumigation Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000012467 final product Substances 0.000 claims description 6
- 244000061456 Solanum tuberosum Species 0.000 claims description 5
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 2
- 235000001727 glucose Nutrition 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 235000002639 sodium chloride Nutrition 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 claims 1
- 229910052760 oxygen Inorganic materials 0.000 claims 1
- 239000001301 oxygen Substances 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 229930012538 Paclitaxel Natural products 0.000 abstract description 16
- 229960001592 paclitaxel Drugs 0.000 abstract description 16
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 abstract description 16
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 235000019764 Soybean Meal Nutrition 0.000 abstract 1
- -1 bran Substances 0.000 abstract 1
- 239000004455 soybean meal Substances 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000003808 methanol extraction Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 3
- 238000012807 shake-flask culturing Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000202349 Taxus brevifolia Species 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000004243 sweat Anatomy 0.000 description 2
- YWLXLRUDGLRYDR-ZHPRIASZSA-N 5beta,20-epoxy-1,7beta,10beta,13alpha-tetrahydroxy-9-oxotax-11-ene-2alpha,4alpha-diyl 4-acetate 2-benzoate Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](O)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 YWLXLRUDGLRYDR-ZHPRIASZSA-N 0.000 description 1
- ZVEGOBHUZTXSFK-TZIKQHFSSA-N 7-Xylosyltaxol Natural products O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)CO3)O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 ZVEGOBHUZTXSFK-TZIKQHFSSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- DBXFAPJCZABTDR-KUEXGRMWSA-N Cephalomannine Natural products O=C(O[C@@H]1C(C)=C2[C@@H](OC(=O)C)C(=O)[C@]3(C)[C@@H](O)C[C@@H]4[C@](OC(=O)C)([C@H]3[C@H](OC(=O)c3ccccc3)[C@@](O)(C2(C)C)C1)CO4)[C@@H](O)[C@H](NC(=O)/C(=C\C)/C)c1ccccc1 DBXFAPJCZABTDR-KUEXGRMWSA-N 0.000 description 1
- 241000218631 Coniferophyta Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 235000016408 Podocarpus macrophyllus Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- 244000162450 Taxus cuspidata Species 0.000 description 1
- 235000009065 Taxus cuspidata Nutrition 0.000 description 1
- 241001674343 Taxus x media Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- DBXFAPJCZABTDR-WBYYIXQISA-N cephalomannine Chemical compound O([C@@H]1[C@]2(O)C[C@@H](C(=C([C@@H](OC(C)=O)C(=O)[C@]3(C)[C@@H](O)C[C@H]4OC[C@]4([C@H]31)OC(C)=O)C2(C)C)C)OC(=O)[C@H](O)[C@@H](NC(=O)C(/C)=C/C)C=1C=CC=CC=1)C(=O)C1=CC=CC=C1 DBXFAPJCZABTDR-WBYYIXQISA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 1
- 229910001948 sodium oxide Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a Chinese yew solid state fermentation medium, a preparation method thereof and a Chinese yew solid state fermentation method, and provides a Chinese yew solid state fermentation medium for increasing the Chinese yew taxol yield and reducing production cost, a preparation method of the medium and the Chinese yew solid state fermentation method. The medium is formed by mixing Chinese yew branch and leaf powder, bran, soybean meal and the like, and solid fermentation is conducted on taxol so as to increase the taxol yield. The content of taxol in a fermentation and cultured product can be effectively increased by fermenting Chinese yew with the medium. In the method, solid fermentation is conducted on Chinese yew for the first time, and the method plays an important role in the taxol production industry.
Description
Technical field
This belongs to field of microbial fermentation, more particularly to a kind of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium and preparation method thereof and
Ramulus et folium taxi cuspidatae process for solid state fermentation.
Background technology
Bearing taxanes are the class chemical combination for being extracted from the positions such as bark, root and the leaf of high gymnosperm Ramulus et folium taxi cuspidatae
Thing, mainly contains paclitaxel, Cephalomannine, 10-DAB III, 7- xylosyl taxols etc., has many to have been demonstrated with cell
Toxicity and anti-tumor activity, and it is used for extracting the natural Ramulus et folium taxi cuspidatae resource-constrained of paclitaxel etc., thus natural Japanese yew raw polyol medicine
Production and supply be restricted, the clinical application of paclitaxel far can not be met.
Solid fermentation (Solid-state fermentation, abbreviation SSF) to be referred to and do carbon source and energy using nature substrate
Source, or do solid support using inert substrate, its system are anhydrous or close to anhydrous any sweat, solid fermentation skill
Art develops for a long time, is widely used.China just has been applied in the aspects such as yeast production wine brewing, cure foods, fertilizer accumulation in ancient times.
Limited by development in science and technology, solid-fermented technique all rests on the original backwardness of comparison within past a very long time
State, or even still such fermentation technique is being continued to use in modern commercial production.Nearest two during the decade, send out with regard to solid
The research and application of ferment technology is developed rapidly, and people are in the research in solid fermentation field and its in resource environment, albumen
Application in matter feedstuff achieves progress, is mainly manifested in biological feedstuff, bio-fuel, biological pesticide, bioconversion, biological solution
The application of the aspect such as poison and biological restoration.There is presently no patent utilization microorganism and Ramulus et folium taxi cuspidatae adopts solid-fermented technique, increase
Plus the content of taxus brevifolia alcohol.
Solid fermentation is had the advantage that in itself：Culture medium is simple and wide material sources, mostly cheap natural substrates or work
The leftover bits and pieces of industry production；Small investment, energy consumption are low, and technology is simpler；The yield of product is higher；Water content of substrate is low, can subtract significantly
The volume of few bioreactor, it is not necessary to which wastewater treatment, environmental pollution are less, and post processing is easy to process；Sweat is general not
Need strict sterile working；Ventilation typically can be completed by gas diffusion or intermittent aeration, it is not necessary to which continuous ventilating, air are general
Also it is not required to strict filtrated air.
So for improving the yield and quality of paclitaxel, reducing the production cost of paclitaxel, solid-state can be carried out to which and is sent out
Ferment is researched and developed to meet clinical growing demand.
The content of the invention
The invention aims to provide a kind of Ramulus et folium taxi cuspidatae improved taxus brevifolia alcohol yield, reduce its production cost
Solid-state fermentation culture medium and preparation method thereof and Ramulus et folium taxi cuspidatae process for solid state fermentation.
For achieving the above object, the technical solution used in the present invention is：A kind of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium includes that A is trained
Foster base and B culture medium, wherein A culture medium include following weight portion：100 parts of Ramulus et folium taxi cuspidatae powder, wheat bran 40-50g parts, bean cake
10 parts of 4-5 parts, rice meal；
B culture medium includes following weight portion：2 parts of peptone, yeast extract 1-2 parts, 5 parts of Sodium Chloride, potassium dihydrogen phosphate 0.3
Part, 0.2 part of magnesium sulfate, glucose 8-10 parts, murphy juice, the murphy juice volume are 20mL with the ratio of the Sodium Chloride quality：
1g。
It is further preferred that the A culture medium includes following weight portion：100 parts of Ramulus et folium taxi cuspidatae powder, wheat bran 50g parts,
5 parts of bean cake, 10 parts of rice meal；
The B culture medium includes following weight portion：：2 parts of peptone, 1 part of yeast extract, 5 parts of Sodium Chloride, potassium dihydrogen phosphate
0.3 part, 0.2 part of magnesium sulfate, 10 parts of glucose, murphy juice, the ratio of the murphy juice volume and the Sodium Chloride quality is 20mL：
1g。
The preparation method of above-mentioned Ramulus et folium taxi cuspidatae solid-state fermentation culture medium comprises the steps：By Ramulus et folium taxi cuspidatae powder formaldehyde fumigation
Mix homogeneously with wheat bran, bean cake, rice meal after at least 12h and obtain final product A culture medium；
By fresh potato decortication stripping and slicing, with the decocting in water boiling at least 30min of 5 times of stripping and slicing Rhizoma Solani tuber osi quality, then with filtered through gauze,
The corresponding volume of water that filtrate is settled to 5 times of stripping and slicing Rhizoma Solani tuber osi quality with distilled water after naturally cooling to room temperature obtains murphy juice；
After peptone, yeast extract, Sodium Chloride, potassium dihydrogen phosphate, magnesium sulfate and glucose are dissolved with murphy juice, hydrogen is used
Sodium oxide adjusts pH to 7, obtains final product B culture medium.
It is further preferred that described naoh concentration is 3mol/L.
A kind of Ramulus et folium taxi cuspidatae process for solid state fermentation carried out using above-mentioned Ramulus et folium taxi cuspidatae solid-state fermentation culture medium is comprised the steps：
A. culture medium prepares：The B culture medium for preparing is mixed homogeneously with the wheat bran in A culture medium, bean cake and rice meal
After sterilize, the mixture after sterilizing is mixed homogeneously with the Ramulus et folium taxi cuspidatae powder after formaldehyde fumigation and obtains final product culture medium, and culture medium is loaded on
In container, thickness is 0.8-1.2cm；
B. the preparation of strain liquid：Slant strains are inoculated in the sterilized container equipped with PDB culture medium, in temperature are
28 DEG C, constant temperature culture 60-80h under the conditions of mixing speed 200rpm/min, are obtained seed liquor；
C. it is inoculated with and cultivates：Seed liquor is accessed in the culture medium for having prepared by the amount of mass fraction 1%-5%, in
Quiescent culture 6-10 days at a temperature of 26-32 DEG C；
The bacterium numbering is CPCC 800037, is stored in Chinese pharmacy Microbiological Culture Collection administrative center.
Further, in step A, sterilising conditions are：Temperature is 121 DEG C, pressure is that 0.1MPa autoclaves are interior sterilizes
20min。
It is further preferred that the constant temperature culture time is 72h in step B.
Further, after seed liquor accesses culture medium in step B, gauze is covered in culture medium and ties up solid.
It is further preferred that the thickness of culture medium is 1cm in step A.
The culture medium provided using the present invention is carried out fermentation energy to Ramulus et folium taxi cuspidatae and effectively improves paclitaxel in fermentation culture product
Content, and the method is for attempting first carrying out solid fermentation to Ramulus et folium taxi cuspidatae, significant to plant cell culture industry.
Specific embodiment
With reference to specific embodiment, the present invention will be further described.The embodiment is only being preferable to carry out for the present invention
Example, is not limited to the present invention, and for a person skilled in the art, the present invention can have various changes and change
Change.All any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., should be included in the present invention
Protection domain within.
The Ramulus et folium taxi cuspidatae powder for being used for preparing in embodiment the fungi fermentation culture medium of powder solid containing Ramulus et folium taxi cuspidatae is this public affairs
The Taxus media branch and leaf of department's plantation are obtained through air-dried crushing, and other every compositions can be obtained by commercial mode.
Embodiment one
Culture medium is configured：Ramulus et folium taxi cuspidatae powder 100g is standby with formaldehyde fumigation 12h.
Prepare B culture medium：Prepare murphy juice：By 200g fresh potato decortication stripping and slicing, 30min is boiled with the decocting in water of 1L, then
With 4 layers of filtered through gauze, filtrate is settled to distilled water after naturally cooling to room temperature that 1L is standby, and pH is nature.
By peptone 2g, yeast extract 1g, Sodium Chloride 5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate 0.2g, glucose 10g are molten
In 100ml murphy juices, pH is adjusted to 7 with the sodium hydroxide of 3mol/L, it is standby.
Remaining composition of the B culture medium for preparing with A culture medium in addition to Ramulus et folium taxi cuspidatae powder is mixed homogeneously, 121 DEG C,
0.1Mpa autoclaves were cooled down after 20 minutes, and the Ramulus et folium taxi cuspidatae powder 100g after fumigating is mixed homogeneously standby with the culture medium after sterilizing
With.
Inoculation：The strain liquid of shake-flask culture is accessed in the culture medium for having prepared, in 30 DEG C by the amount of mass fraction 3%
Under the conditions of cultivate 7 days, with methanol extraction, HPLC determines content of taxol.
Embodiment two
Culture medium is configured：Ramulus et folium taxi cuspidatae powder 100g is standby with formaldehyde fumigation 12h.
Prepare B culture medium：Prepare murphy juice：By 200g fresh potato decortication stripping and slicing, 30min is boiled with the decocting in water of 1L, then
With 4 layers of filtered through gauze, filtrate is settled to distilled water after naturally cooling to room temperature that 1L is standby, and pH is nature.
By peptone 2g, yeast extract 1g, Sodium Chloride 5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate 0.2g, glucose 10g are molten
In 100ml murphy juices, pH is adjusted to 7 with the sodium hydroxide of 3mol/L, it is standby.
Remaining composition of the B culture medium for preparing with A culture medium in addition to Ramulus et folium taxi cuspidatae powder is mixed homogeneously, 121 DEG C,
0.1Mpa autoclaves were cooled down after 20 minutes, and the Ramulus et folium taxi cuspidatae powder 100g after fumigating is mixed homogeneously standby with the culture medium after sterilizing
With.
It is prepared by strain liquid：Slant strains are inoculated in the sterilized triangular flask equipped with PDB culture medium, 28 DEG C,
200rpm/min, constant temperature culture 3 days are obtained seed liquor standby.
Inoculation：The strain liquid of shake-flask culture is accessed in the culture medium for having prepared, in 32 DEG C by the amount of mass fraction 1%
Under the conditions of cultivate 10 days, with methanol extraction, HPLC determines content of taxol.
Embodiment three
Culture medium is configured：Ramulus et folium taxi cuspidatae powder 100g is standby with formaldehyde fumigation 12h.
Prepare B culture medium：Prepare murphy juice：By 200g fresh potato decortication stripping and slicing, 30min is boiled with the decocting in water of 1L, then
With 4 layers of filtered through gauze, filtrate is settled to distilled water after naturally cooling to room temperature that 1L is standby, and pH is nature.
By peptone 2g, yeast extract 1g, Sodium Chloride 5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate 0.2g, glucose 10g are molten
In 100ml murphy juices, pH is adjusted to 7 with the sodium hydroxide of 3mol/L, it is standby.
Remaining composition of the B culture medium for preparing with A culture medium in addition to Ramulus et folium taxi cuspidatae powder is mixed homogeneously, 121 DEG C,
0.1Mpa autoclaves were cooled down after 20 minutes, and the Ramulus et folium taxi cuspidatae powder 100g after fumigating is mixed homogeneously standby with the culture medium after sterilizing
With.
It is prepared by strain liquid：Slant strains are inoculated in the sterilized triangular flask equipped with PDB culture medium, 28 DEG C,
200rpm/min, constant temperature culture 3 days are obtained seed liquor standby.
Inoculation：The strain liquid of shake-flask culture is accessed in the culture medium for having prepared, in 28 DEG C by the amount of mass fraction 5%
Under the conditions of cultivate 6 days, with methanol extraction, HPLC determines content of taxol.
With with the Ramulus et folium taxi cuspidatae that batch do not ferment, with methanol extraction, HPLC determines content of taxol as control, each embodiment
Contrasting data it is as shown in table 1.
1 content of taxol comparing result of table
Claims (9)
1. a kind of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium, it is characterised in that：Including A culture medium and B culture medium, wherein A culture medium bag
Include following weight portion：100 parts of Ramulus et folium taxi cuspidatae powder, wheat bran 40-50g parts, bean cake 4-5 parts, 10 parts of rice meal；
B culture medium includes following weight portion：2 parts of peptone, yeast extract 1-2 parts, 5 parts of Sodium Chloride, 0.3 part of potassium dihydrogen phosphate,
0.2 part of magnesium sulfate, glucose 8-10 parts, murphy juice, the murphy juice volume are 20mL with the ratio of the Sodium Chloride quality：1g.
2. Ramulus et folium taxi cuspidatae solid-state fermentation culture medium according to claim 1, it is characterised in that：The A culture medium includes as follows
Weight portion：100 parts of Ramulus et folium taxi cuspidatae powder, wheat bran 50g parts, 5 parts of bean cake, 10 parts of rice meal；
The B culture medium includes following weight portion：：2 parts of peptone, 1 part of yeast extract, 5 parts of Sodium Chloride, potassium dihydrogen phosphate 0.3
Part, 0.2 part of magnesium sulfate, 10 parts of glucose, murphy juice, the murphy juice volume are 20mL with the ratio of the Sodium Chloride quality：1g.
3. the preparation method of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium according to claim 1 and 2, it is characterised in that：Including such as
Lower step：A culture medium is obtained final product by mixing homogeneously with wheat bran, bean cake, rice meal after Ramulus et folium taxi cuspidatae powder formaldehyde fumigation at least 12h；
By fresh potato decortication stripping and slicing, with the decocting in water boiling at least 30min of 5 times of stripping and slicing Rhizoma Solani tuber osi quality, then with filtered through gauze, filtrate
The corresponding volume of water that 5 times of stripping and slicing Rhizoma Solani tuber osi quality are settled to distilled water after naturally cooling to room temperature obtains murphy juice；
After peptone, yeast extract, Sodium Chloride, potassium dihydrogen phosphate, magnesium sulfate and glucose are dissolved with murphy juice, hydroxide is used
Sodium adjusts pH to 7, obtains final product B culture medium.
4. the preparation method of Ramulus et folium taxi cuspidatae solid-state fermentation culture medium according to claim 3, it is characterised in that：Described hydrogen-oxygen
Change na concn is 3mol/L.
5. a kind of Ramulus et folium taxi cuspidatae solid fermentation side carried out using Ramulus et folium taxi cuspidatae solid-state fermentation culture medium as claimed in claim 1 or 2
Method, it is characterised in that：Comprise the steps：
A. culture medium prepares：Go out after the B culture medium for preparing is mixed homogeneously with the wheat bran in A culture medium, bean cake and rice meal
Bacterium, the mixture after sterilizing are mixed homogeneously with the Ramulus et folium taxi cuspidatae powder after formaldehyde fumigation and obtain final product culture medium, and culture medium is loaded on container
In, thickness is 0.8-1.2cm；
B. the preparation of strain liquid：Slant strains are inoculated in the sterilized container equipped with PDB culture medium, are 28 in temperature
DEG C, constant temperature culture 60-80h under the conditions of mixing speed 200rpm/min, seed liquor is obtained；
C. it is inoculated with and cultivates：Seed liquor is accessed in the culture medium for having prepared, in 26-32 by the amount of mass fraction 1%-5%
Quiescent culture 6-10 days at a temperature of DEG C；
The bacterium numbering is CPCC 800037, is stored in Chinese pharmacy Microbiological Culture Collection administrative center.
6. Ramulus et folium taxi cuspidatae process for solid state fermentation according to claim 5, it is characterised in that：In step A, sterilising conditions are：
Temperature is 121 DEG C, pressure is sterilizing 20min in 0.1MPa autoclaves.
7. Ramulus et folium taxi cuspidatae process for solid state fermentation according to claim 5, it is characterised in that：In the step B during constant temperature culture
Between be 72h.
8. Ramulus et folium taxi cuspidatae process for solid state fermentation according to claim 5, it is characterised in that：In step B, seed liquor is accessed
After culture medium, gauze is covered in culture medium and ties up solid.
9. Ramulus et folium taxi cuspidatae process for solid state fermentation according to claim 5, it is characterised in that：The thickness of culture medium in step A
Spend for 1cm.
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