CN101240304A - Method for producing Huperzine by using fungi - Google Patents
Method for producing Huperzine by using fungi Download PDFInfo
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- CN101240304A CN101240304A CNA2007100035195A CN200710003519A CN101240304A CN 101240304 A CN101240304 A CN 101240304A CN A2007100035195 A CNA2007100035195 A CN A2007100035195A CN 200710003519 A CN200710003519 A CN 200710003519A CN 101240304 A CN101240304 A CN 101240304A
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Abstract
The invention relates to a new culture (cladospore mould CGMCC) for preparing tor alkalis and a method for preparing the same by utilizing new strain (cladospore mould CGMCC) comprising culturing the strain (cladospore mould CGMCC) in culture medium to prepare and amass tor alkalis in the strain cell and culture medium, and recovering tor alkalis from the cell and culture medium in high speed counterflow chromatograph separation. The invention is short periodic time, low cost and high production rate and can be used for industrialization production.
Description
1 one kinds of microorganisms
(1) PDA solid medium feature:
Utilize the PDA substratum to cultivate 4 days for 28 ℃, colony diameter is 2.0mm, and 7 days colony diameters are 3.7mm, and 7 days colony diameters are 6.0mm
(2) liquid culture feature:
Substratum PDA, 14 days shake-flask culture time, 28 ± 2 ℃ of culture temperature
The fermentation culture feature: cultivated the 3rd day, bacterial classification has odd white to cultivate the mycelia point; Cultivate and covered with white hypha in 4 days; It is intensive to cultivate 6 days fermented hyphas, and mycelia presents grey black; Cultivated the 10th day, the intensive one-tenth of fermented liquid mycelium is block.
(3) morphological specificity mycelia grey have every, conidiophore is branch not, the ultimate swelling stigma that grows thickly, its cochain living spore estranged.
(4) the DNA base sequence of 18sRNA
CTCTTGGTGATCATAATAACTTAACGAATCGCATGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTGCCCTATCAACT
TTCGATGGTAGGATAGTGGCCTACCATGGTATCAACGGGTAACGGGGAATTAGGGTTCGACTCCGGAGAGGGAGCCTGAG
AAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATAC
TGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGT
CTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAACC
TTGGGCCTGGCTGGCCGGTCCGCCTCACCGCGTGTACTGGTCCGGCCGGGCCTTTCCTTCTGGGGAACCTCATGCCCTTC
ACTGGGCGTGTTGGGGAACCGGAA
Its technological process is
------------fermentation does not have---high-speed counter-current chromatograph separating compound---effective constituent of extraction to fermentation culture to seed culture to bacterial classification in the test tube slant
Wherein, fermentation raw material is potato culture (PDA), and fermentation mode is the liquid culture fermentation; Strain inclined plane: the test tube slant spawn culture adopts potato glucose solid medium (solid PDA); Seed culture medium is potato culture (liquid PDA); Fermention medium is potato liquid nutrient medium (liquid PDA), the pH nature, and fermented incubation time 5 days, culture temperature: 28 ± 1 ℃ of test tube slant spawn culture temperature, the seed shaking table is cultivated 28 ± 1 ℃, and the fermentation culture temperature is 28 ± 2 ℃; Fermentation finishes, and collects fermented product, and fermentation dry powder is made in vacuum-drying.With following steps: carry out selagine and extract.
1. pre-treatment: adopt ordinary method that fermentation dry powder is fully soaked the back with 1.2% winestone acid solution and transfer the pH value of solution to weakly alkaline with ammoniacal liquor, extract repeatedly with chloroform, after extraction liquid concentrated evaporate to dryness, obtain the selagine crude product. with crude product with an amount of stationary phase solution dissolving afterwards after the sample introduction that promptly can be used for next step.
2. solvent system is chosen in the separation of countercurrent chromatography instrument, adopts the countercurrent chromatography system to separate.Solvent system is made of A, B, three components of C, and the A component can be selected from normal paraffins such as normal heptane, normal hexane, Skellysolve A, and the B component can be selected from Fatty Alcohol(C12-C14 and C12-C18) and aliphatic ketones such as methyl alcohol, ethanol, propyl carbinol, acetone, and the C component is a water.The volume ratio of three components is followed successively by: 1-10: 0.1-3: 1-10.Before using solvent system is placed separating funnel by above-mentioned volume ratio, shake up standing demix.Behind the ready to balance certain hour, upper and lower phase is separated, upward use, use down as moving phase as stationary phase.Before the sample introduction, be filled with whole pillar with stationary phase earlier, adjust engine speed, moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction, according to detector uv-spectrogram receiving target composition.
3. aftertreatment is concentrated into the solution decompression of the selagine collected dried, obtains the off-white color solid, with the proper amount of acetone dissolving, in 0-10 ℃ of following crystallization, will obtain the white powder solid behind the crystallizing and drying then.Detect through HPLC, purity is more than 99%.
Embodiment
Embodiment: 1
Get bacterial classification E.J.Wu, under aseptic condition,, be inoculated in the solid medium test tube of sterilization, placed 28 ± 1 ℃ of activation culture of incubator 96 hours with a little bacterial classification of inoculating needle picking.
Take out the activatory bacterial classification, under aseptic condition, get involved the activatory bacterial classification in the same way in the seed liquid PDA substratum of having sterilized, cultivated 72 hours, be the mould seed of a spore at 28 ± 1 ℃ of following shaking tables.
500 milliliters of glass triangle bottles are adopted in fermentation, pack into 100 milliliters of the liquid PDA substratum that prepare, 15 pounds of sterilizations are after 30 minutes, take out to be cooled to 30 ℃ under aseptic condition, get the mould seed of a spore, be inoculated in 500 milliliters of glass triangle bottles that 100 milliliters of liquid PDA substratum are housed, the seed inoculum size is that 5%～10% (28 ± 2 ℃) forwarded aerated culture to 5 days.During fermentation, note the leavening temperature temperature variation, check to have or not the microbiological contamination phenomenon.
Fermentation finishes, and collects fermented product, and fermentation dry powder is made in vacuum-drying
Fermentation dry powder is fully soaked the back pH value of transferring solution with ammoniacal liquor to weakly alkaline with 1.2% tartrate, extract repeatedly, behind the concentrated evaporate to dryness of extraction liquid, obtain the selagine crude product with chloroform; Crude product promptly be can be used for sample introduction after with the dissolving of an amount of stationary phase solution, choose the appropriate solvent system, adopting column volume is that the high speed adverse current chromatogram system of 1000ml separates.Solvent system is made of A, B, three components of C, and the A component can be selected from normal paraffins such as normal heptane, normal hexane, Skellysolve A, and the B component can be selected from Fatty Alcohol(C12-C14 and C12-C18) and aliphatic ketones such as methyl alcohol, ethanol, propyl carbinol, acetone, and the C component is a water.The volume ratio of three components is followed successively by: 1-4: 0.2-2: 1-3.Before using solvent system is placed separating funnel by above-mentioned volume ratio, shake up standing demix.Behind the ready to balance certain hour, upper and lower phase is separated, upward use, use down as moving phase as stationary phase.Before the sample introduction, be filled with whole pillar with stationary phase earlier, adjust engine speed, moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction, according to detector uv-spectrogram receiving target composition: it is dried to place round-bottomed flask to be evaporated to the solution of the selagine collected, obtains the off-white color solid; Use acetone solution then,, the white powder pulverulent solids that obtains behind the crystallizing and drying is finished product in 0-10 ℃ of following crystallization.
Embodiment: 2
Get bacterial classification E.J.Wu, under aseptic condition,, be inoculated in the solid medium test tube of sterilization, placed 28 ± 1 ℃ of activation culture of incubator 96 hours with a little bacterial classification of inoculating needle picking.
Take out the activatory bacterial classification, under aseptic condition, get involved the activatory bacterial classification in the same way in the seed liquid PDA substratum of having sterilized, cultivated 72 hours, be the mould seed of a spore at 28 ± 1 ℃ of following shaking tables.
500 milliliters of glass triangle bottles are adopted in fermentation, pack into 100 milliliters of the liquid PDA substratum that prepare, 15 pounds of sterilizations are after 30 minutes, take out to be cooled to 30 ℃ under aseptic condition, get the mould seed of a spore, be inoculated in 500 milliliters of glass triangle bottles that 100 milliliters of liquid PDA substratum are housed, the seed inoculum size is that 5%～10% (28 ± 2 ℃) forwarded aerated culture to 5 days.During fermentation, note the leavening temperature temperature variation, check to have or not the microbiological contamination phenomenon.
Fermentation finishes, and collects fermented product, and fermentation dry powder is made in vacuum-drying.
With fermentation dry powder with 1.2% tartrate fully soak transfer solution with ammoniacal liquor in the back pH value to weakly alkaline, extract repeatedly with chloroform, extraction liquid is concentrated obtains the selagine crude product after doing; Crude product promptly be can be used for sample introduction after with the dissolving of an amount of stationary phase solution; Choose solvent system, adopting column volume is the high speed adverse current chromatogram system separation of 5000ml.Solvent system is made of A, B, three components of C, the A component can be selected from normal paraffins such as normal heptane, normal hexane, Skellysolve A, the B component can be selected from Fatty Alcohol(C12-C14 and C12-C18) and aliphatic ketones such as methyl alcohol, ethanol, propyl carbinol, acetone, the C component is a water, and the volume ratio of three components is followed successively by: 1.2-3: 0.3-2: 1.2-3.Before using solvent system is placed separating funnel by above-mentioned volume ratio, shake up standing demix.Behind the ready to balance certain hour, upper and lower phase is separated, upward use, use down as moving phase as stationary phase.Before the sample introduction, be filled with whole pillar with stationary phase earlier, adjust engine speed, moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction, according to detector uv-spectrogram receiving target composition; It is dried to place round-bottomed flask to be evaporated to the solution of the selagine collected, obtains the off-white color solid; Then above-mentioned off-white color solid is dissolved with proper amount of acetone,, the white powder solid that obtains behind the crystallizing and drying is finished product in 0-10 ℃ of following crystallization.
Embodiment: 3
Get bacterial classification E.J.Wu, under aseptic condition,, be inoculated in the solid medium test tube of sterilization, placed 28 ± 1 ℃ of activation culture of incubator 96 hours with a little bacterial classification of inoculating needle picking.
Take out the activatory bacterial classification, under aseptic condition, get involved the activatory bacterial classification in the same way in the seed liquid PDA substratum of having sterilized, cultivated 72 hours, be the mould seed of a spore at 28 ± 1 ℃ of following shaking tables.
500 milliliters of glass triangle bottles are adopted in fermentation, pack into 100 milliliters of the liquid PDA substratum that prepare, 15 pounds of sterilizations are after 30 minutes, take out to be cooled to 30 ℃ under aseptic condition, get the mould seed of a spore, be inoculated in 500 milliliters of glass triangle bottles that 100 milliliters of liquid PDA substratum are housed, the seed inoculum size is that 5%～10% (28 ± 2 ℃) forwarded aerated culture to 5 days.During fermentation, note the leavening temperature temperature variation, check to have or not the microbiological contamination phenomenon.
Fermentation finishes, and collects fermented product, and fermentation dry powder is made in vacuum-drying.
Fermentation dry powder is fully soaked the back pH value of transferring solution with ammoniacal liquor to weakly alkaline with 1,2% tartrate, extract repeatedly, behind the concentrated evaporate to dryness of extraction liquid, obtain the selagine crude product with chloroform; Crude product promptly be can be used for sample introduction after with the dissolving of an amount of stationary phase solution; Adopting column volume is the high speed adverse current chromatogram system separation of 5000ml.Solvent system is made of normal hexane, methyl alcohol, three components of water, and volume ratio is followed successively by: 2: 1.5: 4, before using solvent system is placed separating funnel by above-mentioned volume ratio, and shake up standing demix.Behind the ready to balance certain hour, upper and lower phase is separated, upward use, use down as moving phase as stationary phase.Before the sample introduction, be filled with whole pillar with stationary phase earlier, adjust engine speed, moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction, according to detector uv-spectrogram receiving target composition: it is dried to place round-bottomed flask to be evaporated to the solution of the selagine collected, obtain the off-white color solid, dissolve with proper amount of acetone then, in 0-10 ℃ of following crystallization, the white powder solid that obtains behind the crystallizing and drying is finished product.
CTCTTGGTGATCATAATAACTTAACGAATCGCATGGCCTTGCGCC
GGCGATGGTTCATTCAAATTTCTGCCCTATCAACT
TTCGATGGTAGGATAGTGGCCTACCATGGTATCAACGGGTAACG
GGGAATTAGGGTTCGACTCCGGAGAGGGAGCCTGAG
AAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTA
CCCAATCCCGACACGGGGAGGTAGTGACAATAAATAC
TGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATGAGTACAATT
TAAATCCCTTAACGAGGAACAATTGGAGGGCAAGT
CTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATAT
TAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAACC
TTGGGCCTGGCTGGCCGGTCCGCCTCACCGCGTGTACTGGTCC
GGCCGGGCCTTTCCTTCTGGGGAACCTCATGCCCTTC
ACTGGGCGTGTTGGGGAACCGGAA
Claims (14)
1. microorganism and fermentation method thereof are produced high-purity huperzine A, the used PDA solid medium of organism of fermentation feature: utilize the PDA substratum to cultivate 4 days for 28 ℃, colony diameter is 2.0mm, and 7 days colony diameters are 3.7mm, and 7 days colony diameters are 6.0mm
2. the used liquid nutrient medium feature of organism of fermentation:
Substratum PDA, 14 days shake-flask culture time, 28 ± 2 ℃ of culture temperature
The fermentation culture feature: cultivated the 3rd day, bacterial classification has odd white to cultivate the mycelia point; Cultivate and covered with white hypha in 4 days; It is intensive to cultivate 6 days fermented hyphas, and mycelia presents grey black; Cultivated the 10th day, the intensive one-tenth of fermented liquid mycelium is block.
3. microbial morphology feature: the mycelia grey have every, conidiophore is branch not, the ultimate swelling stigma that grows thickly, its cochain living spore estranged.
4. the DNA base sequence of the 18sRNA of organism of fermentation
CTCTTGGTGATCATAATAACTTAACGAATCGCATGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTGCCCTATCAACT
TTCGATGGTAGGATAGTGGCCTACCATGGTATCAACGGGTAACGGGGAATTAGGGTTCGACTCCGGAGAGGGAGCCTGAG
AAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATAC
TGATACAGGGCTCTTTTGGGTCTTGTAATTGGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGT
CTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAACC
TTGGGCCTGGCTGGCCGGTCCGCCTCACCGCGTGTACTGGTCCGGCCGGGCCTTTCCTTCTGGGGAACCTCATGCCCTTC
ACTGGGCGTGTTGGGGAACCGGAA
5. method of producing selagine, it is characterized in that: in substratum, cultivate claim 1,2,3,4 described microorganisms so that produce in described microorganism cells and in the described substratum and assemble selagine, and reclaim and extract selagine in described cell and the substratum.
6. the method that requires according to right 1,2, the carbon source material that it is characterized in that containing in the described substratum in the conventional carbon source that glucose, sucrose, maltose, fructose, glycerine, starch, lactose, semi-lactosi and other help described microorganism growth one or more, and peanut powder, analysis for soybean powder, corn general, yeast powder, peptone, beef extract, yeast extract, nitrate, ammonium chloride and other help the conventional nitrogen source of described microorganism growth.
7. according to claim 1,2 described methods, it is characterized in that described substratum also contains phosphoric acid salt, magnesium salts, sodium salt and other help the conventional inorganic salt or the organic salt material of microorganism growth.
8. the production method of a high-purity huperzine A comprises the steps:
The a pre-treatment
The pH value of transferring solution with ammoniacal liquor after the employing ordinary method is fully soaked fermentation dry powder with 1.2% winestone acid solution (0.15～0.25% aqueous hydrochloric acid) extracts with chloroform repeatedly to weakly alkaline, behind the concentrated evaporate to dryness of extraction liquid, obtains the selagine crude product; Crude product promptly be can be used for sample introduction after with the dissolving of an amount of stationary phase solution;
B countercurrent chromatography instrument separates chooses solvent system, adopts the countercurrent chromatography system to separate;
The c aftertreatment
The solution decompression of the selagine collected is concentrated evaporate to dryness, obtains the off-white color solid,, with the proper amount of acetone dissolving,, the white powder solid that obtains behind the crystallizing and drying is finished product then in 0～10 ℃ of following crystallization.
9. the production method of a kind of high-purity huperzine A according to claim 8, it is characterized in that: described solvent system is made of A, B, three components of C, and the A component is a normal paraffin, and the B component is Fatty Alcohol(C12-C14 and C12-C18) or aliphatic ketone, and the C component is a water.The volume ratio of three components is followed successively by: 1-10: 0.1-3: 1-10.
10. the production method of a kind of high-purity huperzine A according to claim 8, it is characterized in that: described solvent system is made of A, B, three components of C, and the A component is a normal paraffin, and the B component is Fatty Alcohol(C12-C14 and C12-C18) or aliphatic ketone, and the C component is a water.The volume ratio of three components is followed successively by: 1-4: 0.2-2: 1-3.
11. the production method of a kind of high-purity huperzine A according to claim 8 is characterized in that: described solvent system is made of A, B, three components of C, and the A component is a normal paraffin, and the B component is Fatty Alcohol(C12-C14 and C12-C18) or aliphatic ketone, and the C component is a water.The volume ratio of three components is followed successively by: 1.2-3: 0.3-2: 1.2-3.
12. the production method of a kind of high-purity huperzine A according to claim 8 is characterized in that: described solvent system is made of A, B, three components of C, and the A component is a normal paraffin, and the B component is Fatty Alcohol(C12-C14 and C12-C18) or aliphatic ketone, and the C component is a water.The volume ratio of three components is followed successively by: 1-5: 0.1-2: 1-5.
13. the production method of a kind of high-purity huperzine A according to claim 8 is characterized in that described solvent system is made of A, B, three components of C, the A component is a normal paraffin, and the B component is Fatty Alcohol(C12-C14 and C12-C18) or aliphatic ketone, and the C component is a water.The volume ratio of three components is followed successively by: 2: 1.5: 4 or 2: 1: 1 or 5: 1: 5 or 4: 1: 3 or 2: 1: 4.
14. production method according to claim 9 or 10 or 11 or 12 or 13 described a kind of high-purity huperzine As, it is characterized in that described normal paraffin is normal heptane, normal hexane or Skellysolve A, described Fatty Alcohol(C12-C14 and C12-C18) is methyl alcohol, ethanol or propyl carbinol, and described aliphatic ketone is an acetone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100035195A CN101240304A (en) | 2007-02-07 | 2007-02-07 | Method for producing Huperzine by using fungi |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100035195A CN101240304A (en) | 2007-02-07 | 2007-02-07 | Method for producing Huperzine by using fungi |
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| CN101240304A true CN101240304A (en) | 2008-08-13 |
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| CNA2007100035195A Pending CN101240304A (en) | 2007-02-07 | 2007-02-07 | Method for producing Huperzine by using fungi |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914452A (en) * | 2010-07-14 | 2010-12-15 | 洪亚辉 | Huperzine A high yield strain TCM-01 |
| CN102168017A (en) * | 2010-09-29 | 2011-08-31 | 湖南农业大学 | Huperzine A high-producing strain and method for producing huperzine A by fermenting same |
| CN102907253A (en) * | 2012-07-02 | 2013-02-06 | 恩施清江生物工程有限公司 | Huperzia serrata endophyte and application thereof |
| CN103834577A (en) * | 2014-02-07 | 2014-06-04 | 福建中医药大学 | Phlegmariurus phlegmaria mingchegensis mycorrhizal fungi, method for production of huperzine A from the same, and application |
| CN104707358A (en) * | 2013-12-13 | 2015-06-17 | 中国科学院大连化学物理研究所 | Novel double water phase high-speed countercurrent chromatography |
| CN105085523A (en) * | 2015-09-02 | 2015-11-25 | 中南大学 | Method for separating and preparing high-purity huperzine C through HSCCC (high-speed countercurrent chromatography) |
| CN108384819A (en) * | 2017-02-03 | 2018-08-10 | 上海医药工业研究院 | A kind of culture medium and fermentation process for the tacrolimus that ferments |
-
2007
- 2007-02-07 CN CNA2007100035195A patent/CN101240304A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914452A (en) * | 2010-07-14 | 2010-12-15 | 洪亚辉 | Huperzine A high yield strain TCM-01 |
| CN102168017A (en) * | 2010-09-29 | 2011-08-31 | 湖南农业大学 | Huperzine A high-producing strain and method for producing huperzine A by fermenting same |
| CN102168017B (en) * | 2010-09-29 | 2012-07-11 | 湖南农业大学 | Huperzine A high-producing strain and method for producing huperzine A by fermenting same |
| CN102907253A (en) * | 2012-07-02 | 2013-02-06 | 恩施清江生物工程有限公司 | Huperzia serrata endophyte and application thereof |
| CN104707358A (en) * | 2013-12-13 | 2015-06-17 | 中国科学院大连化学物理研究所 | Novel double water phase high-speed countercurrent chromatography |
| CN103834577A (en) * | 2014-02-07 | 2014-06-04 | 福建中医药大学 | Phlegmariurus phlegmaria mingchegensis mycorrhizal fungi, method for production of huperzine A from the same, and application |
| CN103834577B (en) * | 2014-02-07 | 2016-01-06 | 福建中医药大学 | The methods and applications of phlegmariurus mycorrhizal fungi and product selagine thereof |
| CN105085523A (en) * | 2015-09-02 | 2015-11-25 | 中南大学 | Method for separating and preparing high-purity huperzine C through HSCCC (high-speed countercurrent chromatography) |
| CN108384819A (en) * | 2017-02-03 | 2018-08-10 | 上海医药工业研究院 | A kind of culture medium and fermentation process for the tacrolimus that ferments |
| CN108384819B (en) * | 2017-02-03 | 2021-06-25 | 上海医药工业研究院 | Culture medium for fermenting tacrolimus and fermentation method |
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Open date: 20080813 |