CN102071230A - Method for extracting paclitaxel from taxus chinensis through microorganism fermentation - Google Patents
Method for extracting paclitaxel from taxus chinensis through microorganism fermentation Download PDFInfo
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- CN102071230A CN102071230A CN2010105600511A CN201010560051A CN102071230A CN 102071230 A CN102071230 A CN 102071230A CN 2010105600511 A CN2010105600511 A CN 2010105600511A CN 201010560051 A CN201010560051 A CN 201010560051A CN 102071230 A CN102071230 A CN 102071230A
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- ramulus
- folium taxi
- taxi cuspidatae
- pulverous
- taxol
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Abstract
The invention relates to a method for extracting paclitaxel from taxus chinensis through microorganism fermentation, which comprises the following steps of: crushing branches and leaves of the taxus chinensis into powder; and immersing the branch and leaf powder of the taxus chinensis in fermentation liquor fermented by yeast for several days, concentrating the soak solution and performing spray drying to obtain high-content dry paclitaxel. The method has the advantages of short paclitaxel extraction time, high purity and no environmental pollution.
Description
Technical field
The present invention relates to a kind of method of utilizing microbe fermentation method from Ramulus et folium taxi cuspidatae, to extract taxol.
Background technology
Taxol is to separate a kind of natural anti-cancer drugs that obtains from Ramulus et folium taxi cuspidatae, and it has the effect that suppresses tumor growth.Because the mechanism of action uniqueness of taxol, evident in efficacy, therefore be used for the clinical treatment of lung cancer, ovarian cancer, mammary cancer etc.Become the emphasis of medicine trade research for the high efficiency extraction of taxol.The taxol scarcity of resources costs an arm and a leg, and therefore, taking effective ways to improve its yield is the key factor that reduces the taxol cost.The technology of extracting taxus brevifolia alcohol at present mainly contains chemical methods such as acid degradation, alkaline degradation.The shortcoming of these class methods is often to cause the effective constituent sex change, and the hazard residue in the product is higher and be difficult to remove.Physics method method such as ultrasonic wave, roll, physical actions such as microwave, comminution by gas stream, shortcoming is that active substance is difficult to extract fully.Owing to have the hard cell walls of one deck in the yew cell tissue, adopt ordinary method to be difficult to make the abundant broken wall of its cell, and in a large number with an organic solvent, the ubiquity extracting cycle be long, yield is low, product contains shortcomings such as Organic pollutants.
Summary of the invention
The object of the present invention is to provide a kind of simple and convenient, the rate of recovery is high, with low cost extracts the method for taxol from artificial planted south yew, mainly is by the biologic enzymolysis method extraction process, this method is extracted fully taxol.This microbe fermentation method that utilizes extracts taxol, help destroying plant cell wall, help the leaching of taxol, extraction time is shortened, extraction process is simplified, and has improved extraction efficiency, reduced extraction cost and also can not produce the Organic pollutants that the class quasi-chemical method is brought.
The present invention adopts following technical scheme for achieving the above object:
2, a kind of method of utilizing microbe fermentation method to extract taxol from Ramulus et folium taxi cuspidatae is characterized in that utilizing microbe fermentation method to extract taxol in Ramulus et folium taxi cuspidatae, specifically may further comprise the steps:
(1) the Ramulus et folium taxi cuspidatae fragmentation is Powdered;
(2) LB culture medium preparation: take by weighing following material respectively: pulverous Ramulus et folium taxi cuspidatae 28-31g, peptone 0.5-1.5g, yeast extract paste 1.5-2.5g, agar powder 13-16g, NaCl18-12g by formula rate, put into beaker, add 1000ml distilled water and heating for dissolving, adjust pH value of solution to 7.0-7.5 with NaOH solution or HCl solution; Then high-temperature sterilization, shelve the inclined-plane, be under 28-32 ℃ in temperature, cultivate after 24-48 hour, change in the 3-4 ℃ of refrigerator preserve standby;
(3) yeast is inoculated in the slant medium, carries out activation treatment;
(4) seed culture medium: in 1000ml distilled water, add yeast extract paste 1.5-2.5g, MgSO47H
2O0.15-0.25g, citric acid 0.40-0.44g, KH
2PO42.5-3.5g, NH
4Cl 1.5-2.5g, CaCl
20.08-0.12g, pulverous Ramulus et folium taxi cuspidatae 28-32kg mixes as seed culture medium;
(5) fermention medium: in 1000ml distilled water, add yeast extract paste 0.5-1.5g, Mg8O
47H
2O0.15-0.25g, citric acid 0.40-0.44g, KH
2PO
40.5-1.5g, NH
4Cl 5.3-5.5g, CaCl
20.08-0.12g, pulverous Ramulus et folium taxi cuspidatae 38-42g, MnCl
20.01-0.012g the mixed fermention medium that gets;
(6) seed preparation: get yeast after step (3) activation and be linked in right amount and carry out shaking table in the seed culture medium that step (4) cultivates and cultivate, the temperature of nutrient solution is 30～40 ℃, rotating speed 80～110rpm, cultivated 24～48 hours, change in the two generation substratum, regulate pH and be 7.5～9.0,30～40 ℃ and cultivated 5～12 hours, the preparation secondary seed, standby;
(7) fermentation culture: fermented liquid is packed in the fermentor tank, loading capacity is 50-85%, the secondary seed of step (6) preparation is inserted wherein, inoculum size is 10～20%, pulverous Ramulus et folium taxi cuspidatae is packed into wherein, 30～40 ℃ of temperature are fermented under stir speed (S.S.) 80～110rpm condition again, the pH value of control fermented liquid is 6～8, ferments 4-7 days;
(8) with the fermented liquid that ferments by centrifugation, collect supernatant liquid;
(9) liquid of collecting is carried out cryoconcentration, spraying drying obtains high-load taxol dry product.
Beneficial effect of the present invention:
The present invention utilizes microbe fermentation method to extract taxol, help destroying plant cell wall, help the leaching of taxol, extraction time is shortened, extraction process is simplified, and has improved extraction efficiency, reduced extraction cost and also can not produce the Organic pollutants that the class quasi-chemical method is brought.
Embodiment
3, a kind of method of utilizing microbe fermentation method to extract taxol from Ramulus et folium taxi cuspidatae is characterized in that utilizing little
Biological fermentation process extracts taxol in Ramulus et folium taxi cuspidatae, specifically may further comprise the steps:
(1) the Ramulus et folium taxi cuspidatae fragmentation is Powdered;
(2) LB culture medium preparation: take by weighing following material respectively: pulverous Ramulus et folium taxi cuspidatae 28-31g, peptone 0.5-1.5g, yeast extract paste 1.5-2.5g, agar powder 13-16g, NaCl18-12g by formula rate, put into beaker, add 1000ml distilled water and heating for dissolving, adjust pH value of solution to 7.0-7.5 with NaOH solution or HCl solution; Then high-temperature sterilization, shelve the inclined-plane, be under 28-32 ℃ in temperature, cultivate after 24-48 hour, change in the 3-4 ℃ of refrigerator preserve standby;
(3) yeast is inoculated in the slant medium, carries out activation treatment;
(4) seed culture medium: in 1000ml distilled water, add yeast extract paste 1.5-2.5g, MgSO47H
2O0.15-0.25g, citric acid 0.40-0.44g, KH
2PO42.5-3.5g, NH
4Cl 1.5-2.5g, CaCl
20.08-0.12g, pulverous Ramulus et folium taxi cuspidatae 28-32kg mixes as seed culture medium;
(5) fermention medium: in 1000ml distilled water, add yeast extract paste 0.5-1.5g, MgSO
47H
2O0.15-0.25g, citric acid 0.40-0.44g, KH
2PO
40.5-1.5g, NH
4Cl5.3-5.5g, CaCl
20.08-0.12g, pulverous Ramulus et folium taxi cuspidatae 38-42g, MnCl
20.01-0.012g the mixed fermention medium that gets;
(6) seed preparation: get yeast after step (3) activation and be linked in right amount and carry out shaking table in the seed culture medium that step (4) cultivates and cultivate, the temperature of nutrient solution is 30～40 ℃, rotating speed 80～110rpm, cultivated 24～48 hours, change in the two generation substratum, regulate pH and be 7.5～9.0,30～40 ℃ and cultivated 5～12 hours, the preparation secondary seed, standby;
(7) fermentation culture: fermented liquid is packed in the fermentor tank, loading capacity is 50-85%, the secondary seed of step (6) preparation is inserted wherein, inoculum size is 10～20%, pulverous Ramulus et folium taxi cuspidatae is packed into wherein, 30～40 ℃ of temperature are fermented under stir speed (S.S.) 80～110rpm condition again, the pH value of control fermented liquid is 6～8, ferments 4-7 days;
(8) with the fermented liquid that ferments by centrifugation, collect supernatant liquid;
(9) liquid of collecting is carried out cryoconcentration, spraying drying obtains high-load taxol dry product.
Claims (1)
1. a method of utilizing microbe fermentation method to extract taxol from Ramulus et folium taxi cuspidatae is characterized in that utilizing microbe fermentation method to extract taxol in Ramulus et folium taxi cuspidatae, specifically may further comprise the steps:
(1) the Ramulus et folium taxi cuspidatae fragmentation is Powdered;
(2) LB culture medium preparation: take by weighing following material respectively: pulverous Ramulus et folium taxi cuspidatae 28-31g, peptone 0.5-1.5g, yeast extract paste 1.5-2.5g, agar powder 13-16g, NaCl18-12g by formula rate, put into beaker, add 1000ml distilled water and heating for dissolving, adjust pH value of solution to 7.0-7.5 with NaOH solution or HCl solution; Then high-temperature sterilization, shelve the inclined-plane, be under 28-32 ℃ in temperature, cultivate after 24-48 hour, change in the 3-4 ℃ of refrigerator preserve standby;
(3) yeast is inoculated in the slant medium, carries out activation treatment;
(4) seed culture medium: in 1000ml distilled water, add yeast extract paste 1.5-2.5g, MgSO47H
2O0.15-0.25g, citric acid 0.40-0.44g, KH
2PO42.5-3.5g, NH
4Cl 1.5-2.5g, CaCl
20.08-0.12g, pulverous Ramulus et folium taxi cuspidatae 28-32kg mixes as seed culture medium;
(5) fermention medium: in 1000ml distilled water, add yeast extract paste 0.5-1.5g, MgSO
47H
2O0.15-0.25g, citric acid 0.40-0.44g, KH
2PO
40.5-1.5g, NH
4Cl 5.3-5.5g, CaCl
20.08-0.12g, pulverous Ramulus et folium taxi cuspidatae 38-42g, MnCl
20.01-0.012g the mixed fermention medium that gets;
(6) seed preparation: get yeast after step (3) activation and be linked in right amount and carry out shaking table in the seed culture medium that step (4) cultivates and cultivate, the temperature of nutrient solution is 30～40 ℃, rotating speed 80～110rpm, cultivated 24～48 hours, change in the two generation substratum, regulate pH and be 7.5～9.0,30～40 ℃ and cultivated 5～12 hours, the preparation secondary seed, standby;
(7) fermentation culture: fermented liquid is packed in the fermentor tank, loading capacity is 50-85%, the secondary seed of step (6) preparation is inserted wherein, inoculum size is 10～20%, pulverous Ramulus et folium taxi cuspidatae is packed into wherein, 30～40 ℃ of temperature are fermented under stir speed (S.S.) 80～110rpm condition again, the pH value of control fermented liquid is 6～8, ferments 4-7 days;
(8) with the fermented liquid that ferments by centrifugation, collect supernatant liquid;
(9) liquid of collecting is carried out cryoconcentration, spraying drying obtains high-load taxol dry product.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010105600511A CN102071230A (en) | 2010-11-23 | 2010-11-23 | Method for extracting paclitaxel from taxus chinensis through microorganism fermentation |
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| CN2010105600511A CN102071230A (en) | 2010-11-23 | 2010-11-23 | Method for extracting paclitaxel from taxus chinensis through microorganism fermentation |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104605138A (en) * | 2013-11-01 | 2015-05-13 | 南京黑玉特种家禽养殖专业合作社 | Poultry feed additive containing fermented Chinese yew, and preparation method and applications thereof |
| CN104997018A (en) * | 2015-06-12 | 2015-10-28 | 江苏红豆杉药业有限公司 | Yew enzyme health-care product as well as preparation method and application thereof |
| CN105153077A (en) * | 2015-10-19 | 2015-12-16 | 丁玉琴 | Method for extracting paclitaxel from Taxus chinensis leaf |
| CN109329804A (en) * | 2018-10-23 | 2019-02-15 | 黛君本草生命科技（深圳）有限公司 | A kind of extracting method of jujube ingredient |
| CN113862325A (en) * | 2021-11-09 | 2021-12-31 | 四川贝美森生物科技有限公司 | Taxus chinensis fermentation liquor with high taxol content as well as preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101177666A (en) * | 2006-11-12 | 2008-05-14 | 黑龙江大学 | Novel bacterial producing paclitaxel and method for breeding and preparing paclitaxel |
| CN101245360A (en) * | 2008-03-24 | 2008-08-20 | 江西德宇集团 | Biological enzymatic hydrolysis extraction technique of paclitaxel from south yew |
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2010
- 2010-11-23 CN CN2010105600511A patent/CN102071230A/en active Pending
Patent Citations (2)
| 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 |
| CN101245360A (en) * | 2008-03-24 | 2008-08-20 | 江西德宇集团 | Biological enzymatic hydrolysis extraction technique of paclitaxel from south yew |
Non-Patent Citations (1)
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| 赵凯 等: "内生真菌发酵生产紫杉醇的研究现状与展望", 《微生物学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104605138A (en) * | 2013-11-01 | 2015-05-13 | 南京黑玉特种家禽养殖专业合作社 | Poultry feed additive containing fermented Chinese yew, and preparation method and applications thereof |
| CN104997018A (en) * | 2015-06-12 | 2015-10-28 | 江苏红豆杉药业有限公司 | Yew enzyme health-care product as well as preparation method and application thereof |
| CN105153077A (en) * | 2015-10-19 | 2015-12-16 | 丁玉琴 | Method for extracting paclitaxel from Taxus chinensis leaf |
| CN109329804A (en) * | 2018-10-23 | 2019-02-15 | 黛君本草生命科技（深圳）有限公司 | A kind of extracting method of jujube ingredient |
| CN113862325A (en) * | 2021-11-09 | 2021-12-31 | 四川贝美森生物科技有限公司 | Taxus chinensis fermentation liquor with high taxol content as well as preparation method and application thereof |
| CN113862325B (en) * | 2021-11-09 | 2024-04-16 | 四川贝美森生物科技有限公司 | Taxus chinensis fermentation liquor with high taxol content and preparation method and application thereof |
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Application publication date: 20110525 |