CN102584941B - Paddy rice Ustiloxin A extracting and purifying method - Google Patents
Paddy rice Ustiloxin A extracting and purifying method Download PDFInfo
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- CN102584941B CN102584941B CN 201210056447 CN201210056447A CN102584941B CN 102584941 B CN102584941 B CN 102584941B CN 201210056447 CN201210056447 CN 201210056447 CN 201210056447 A CN201210056447 A CN 201210056447A CN 102584941 B CN102584941 B CN 102584941B
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Abstract
The invention relates to a paddy rice Ustiloxin A extracting and purifying method. The method for preparing and purifying Ustiloxin A comprises the following steps: extracting toxins from rice false smut balls or an Ustilaginoidea viren toxin culture by a 2% formic acid solution, and purifying the obtained extract by a cation exchange column; 2, separating by a semi-preparative high performance liquid chromatograph under conditions that the mobile phase is a mixed solution of methanol and water according to a volume ratio of 2:8, the flow rate is 1.5ml/min, and the detection wavelength is 254nm; and 3, further purifying the extract through adopting thin layer chromatography, wherein a mixed solution of dichloromethane and methanol according to a volume ratio of 7:3 is used as a developing agent. The method of the invention has the advantages of breakthrough of the toxin source restriction, simple step and high efficiency, and the obtained Ustiloxin A which has a purity of greater than90% can be used as a standard substance.
Description
Technical field
The present invention relates to a kind of also method of purifying ustilaginoidea virens toxin Ustiloxin A of extracting.
Background technology
False smut is a kind of worldwide rice fungus diseases evil, its pathogenic bacteria
Ustilaginoidea virensInfect paddy rice fringe portion and produce the rice curve.In recent years, along with the change of rice cropping pattern, the increase of rice field amount of application of nitrogen fertilizer adds that the big area of novel high yield dense cluster type hybridisation rice is promoted, being on the rise of false smut.False smut not only has a strong impact on the output of paddy rice, and more seriously ustilaginoidea virens can produce the ustilaginoidea virens toxin that people and animals are harmful to.
Ustilaginoidea virens toxin Ustiloxins is the mitotic heterocycle peptide of the anti-eukaryotic cell of a class, comprises 13 Yuans rings, and an ether key is arranged in the ring.1994, state such as Koisoetal etc. first separate from the rice curve and have obtained 6 kinds of toxin, be respectively Ustiloxin A, Ustiloxin B, Ustiloxin C, Ustiloxin D, Ustiloxin F and Ustiloxin E, and identified its molecular formula and chemical structure.Wherein the content of Ustiloxin A is the abundantest and activity is the strongest.
Suwa (1915) has reported that at first the water extract of rice curve is to the toxicity of rabbit, Nakamura reports such as (1992) has eaten by the feed of ustilaginoidea virens endotoxin contamination and can cause the serious liver and kidney disease of animal (metabolism disorder), 1993 can cause the mouse liver by Electronic Speculum histological chemistry proof ustilaginoidea virens toxin, kidney and urethra apoptosis and preceding stomach ulcer .Koiso etc. (1994) find, carry out disposable injection with Ustiloxin A, liver cell and kidney duct shape cell that mouse is exsomatized are sharply downright bad, and can contain organ cell's mitotic division or cause abnormal mitotic division that this is similar to the symptom that causes with the colchicine processing.Huang Shiwen (2002) report is fed rabbit, White Rock cockerel and local hen with false smut grain mix fodder, feeds through 35-84d and raises, and can cause dead and internal organs pathology; Cavings with the band ustilaginoidea virens feeds pigs, and stillborn foetus, corpse tire and monster etc. appear producing in sow.The experimental results shows subsequently, the ustilaginoidea virens toxin can stop the polyreaction of tubulin, the formation of interference cell skeleton, and existing tubulin had unzipping, then suppress cell mitotic division, but or it is because can not permeates cell membranes and do not possess cytotoxicity, thereby can not influence bacterium or fungi growth.Research at present thinks that Ustiloxin A and Ustiloxin B can also suppress multiple human tumor cell's mitotic division, and the plant and animal cell is had biological activity widely.
Along with the generation of false smut in recent years is more and more heavier, the ustilaginoidea virens toxin study is also extensively carried out in succession, scientific research field is also increasing to the demand of ustilaginoidea virens toxin standard substance, yet the standard substance of ustilaginoidea virens toxin are not sold in the market, and this has brought great inconvenience to scientific research.Consult pertinent literature, doctor Kobayashi who has only Tokyo Univ Japan at present from the rice curve separation and purification to the ustilaginoidea virens toxin, this method utilizes the rice curve as the source of toxin, adopts multisteps such as C18 pillar, silicagel column to purify means, and step is more loaded down with trivial details, consuming time.
The kind of ustilaginoidea virens toxin is identified at present altogether 6 kinds, yet ustilaginoidea virens toxin Ustiloxin A is a kind of toxin that wherein content is maximum, activity is maximum, therefore, patent of the present invention is at this practical problems, set up a kind of new, can extract the method for purifying ustilaginoidea virens toxin Ustiloxin A on a large scale, safely, efficiently in the laboratory, have important Research Significance and using value.
Summary of the invention:
The objective of the invention is to: at there not being ustilaginoidea virens toxin Ustiloxin A standard substance to sell in the market, and a series of realistic problems such as existing ustilaginoidea virens toxin Ustiloxin A extracting method more complicated provide a kind of method that can extract purifying ustilaginoidea virens toxin Ustiloxin A in the laboratory on a large scale, safely, efficiently.
The object of the present invention is achieved like this: the extracting and purifying method of a kind of rice green smut verticillium toxin Ustiloxin A is characterized in that this method follows these steps to order and carries out:
A) get rice curve powder or ustilaginoidea virens toxin culture powder, add the PEG of its weight 1/5, add its weight 4-5 2% formic acid solution doubly again, place shaking table, the shaking table condition is room temperature, 200 rev/mins, takes out after 30 minutes, rotating speed be 8000 rev/mins centrifugal 10 minutes, get supernatant liquor;
B) add the isopyknic methyl alcohol of its weight in the filter residue, the same shaking table condition was taken out after 15 minutes, and centrifugal 10 minutes of the same condition is got supernatant liquor;
C) supernatant liquor of combining step a and step b, evaporation concentration obtains the crude extract 1 of toxin;
D) successively use the sodium hydroxide of methyl alcohol and 5N to wash the balance cation exchange column, when solvent liquid level arrives nh 2 column adsorption layer surface, the crude extract 1 that adds the toxin of step c acquisition immediately, the control flow velocity is at 1 droplet/second, successively distinguish the drip washing cationic exchange coloum with 5% ammoniacal liquor and the methyl alcohol of 1/5 sample volume again, last with target toxin under the meoh eluate wash-out of 1/5 sample volume, rotary evaporation concentrates and obtains thick toxin dry powder; Described meoh eluate is the methyl alcohol that contains 5% formic acid;
E) thick toxin dry powder is dissolved in the dry powder weight 10-20 methanol solvate doubly, by the separation of half preparative high performance liquid chromatography instrument, the retention time of contrast ustilaginoidea virens toxin Ustiloxin A standard specimen peak value is collected the crude extract 2 that obtains toxin; The volume ratio of methyl alcohol and water is 2:8 in the described methanol solvate;
F) crude extract 2 of the toxin that step e is obtained is evenly put at silica gel G F respectively with ustilaginoidea virens toxin Ustiloxin A standard specimen
254Chromatography cylinder is put into chromatoplate in the lower end of thin layer chromatography board, launches in developing agent, runs to apart from chromatoplate top 2 centimeters until developing agent, takes out chromatoplate, dries;
G) shine under ultraviolet lamp, the standard specimen of contrast ustilaginoidea virens toxin Ustiloxin A digs down and the band of standard specimen at same position;
H) silica gel is put into methyl alcohol and soak, shaking table took out after 15 minutes, and room temperature, 10000 rev/mins are centrifugal, get supernatant, and rotary evaporation concentrates, and namely got content greater than 90% ustilaginoidea virens toxin Ustiloxin A.
In the present invention, the rice curve powder described in the step a is to obtain like this: at the late growth stage of rice that false smut takes place, pluck globe blackish green or pale brown look from paddy rice fringe portion and be the rice curve, dry, clay into power, cross 20 mesh sieves, obtain rice curve powder, standby in-20 ℃ of preservations.
In the present invention, the ustilaginoidea virens toxin culture powder described in the step a is to obtain like this: at first get an amount of rice, soaked overnight, 121 ℃ of 20 minutes autoclavings; Insert the ustilaginoidea virens mycelia of activation then by 5% inoculum size, 28 ℃ of-30 ℃ of dark illumination were alternately cultivated 20-25 days, obtained ustilaginoidea virens toxin culture; With ustilaginoidea virens toxin culture oven drying at low temperature, clay into power again, cross 20 mesh sieves, obtain ustilaginoidea virens toxin culture powder, standby in-20 ℃ of preservations.
In the present invention, the separation condition of half preparative high performance liquid chromatography instrument described in the step e is: moving phase is that described methanol solvate, flow velocity are 1.5ml/min, and the detection wavelength is 254nm.
In the present invention, the developing agent among the step f is the mixed solution of methylene dichloride and methyl alcohol, and the volume ratio of methylene dichloride and methyl alcohol is 7:3.
The invention has the advantages that: except can from the rice curve that pluck in the field, extracting toxin, more can utilize the rice substratum to make ustilaginoidea virens under laboratory condition, produce poison, avoid the harvesting of rice curve to be subjected to restrictions such as time, occurring degree, thereby limited the source that toxin extracts.
The present invention be advantageous in that: adopt the C-18 post to purify with existing technological method different to be, the present invention adopts the cationic exchange coloum contratoxin to carry out purifying treatment, can access that purity is higher, impurity toxin crude extract still less, make things convenient for further purifying.
The present invention be advantageous in that: the purification process that adopts half preparative liquid chromatography and thin-layer chromatography to be used in conjunction mutually, make the toxin purity that obtains higher, and convenient concentrating, compared with prior art, have the efficient height, safe, purity is high, the purity of gained toxin detects through HPLC-UV/DAD and reaches more than 90%, can do standard model.
The present invention be advantageous in that: fill the domestic gaps, set up feasible method for extract purifying ustilaginoidea virens toxin Ustiloxin A on a large scale, safely, efficiently in the laboratory.
Description of drawings
The high-efficient liquid phase chromatogram of Fig. 1 ustilaginoidea virens toxin Ustiloxin A standard model;
Fig. 2 extracts the high-efficient liquid phase chromatogram of ustilaginoidea virens toxin Ustiloxin A from the rice curve;
Fig. 3 extracts the high-efficient liquid phase chromatogram of ustilaginoidea virens toxin Ustiloxin A from artificial inoculation rice culture.
Embodiment
The preparation method of standard specimen high-efficient liquid phase chromatogram
The used toxin Ustiloxin of the present invention A standard control is that doctor Kobayashi of Tokyo Univ Japan gives.
The standard substance of ustilaginoidea virens toxin Ustiloxin A are made into the standardized solution that final concentration is 2ppm with methanol solvate, utilize the separation of half preparative high performance liquid chromatography instrument, separation condition is: moving phase is that described methanol solvate (volume ratio of methyl alcohol and water is 2:8), flow velocity are 1.5ml/min, and the detection wavelength is 254nm.Target peak (Fig. 1) occurred at 8.98 minutes, the peak that the front occurred at 3.553 minutes is solvent peak, is the qualitative reference of following extraction toxin with the retention time of this normaltoxin.
The rice curve is to obtain like this:
At late growth stage of rice (the field piece that has false smut to take place) in rice terrace, plucking globe blackish green or pale brown look in paddy rice fringe portion is the rice curve, dries, and clays into power, and crosses 20 mesh sieves, obtains rice curve powder, standby in-20 ℃ of preservations.
From rice curve powder, extract the method for purifying ustilaginoidea virens toxin Ustiloxin A:
The configuration of solution
Meoh eluate: composite by methyl alcohol and formic acid, contain 5% formic acid in the elutriant, surplus is methyl alcohol.
Methanol solution: the methyl alcohol dilute with water, the volume ratio of methyl alcohol and water is 2:8 during dilution.
Developing agent: composite by methylene dichloride and methyl alcohol, the volume ratio of methylene dichloride and methyl alcohol is 7:3.
Extracting and purifying method:
A) get the PEG that rice curve powder that embodiment 2 obtains adds 10g, add 2% formic acid solution of 200ml again, place shaking table, the shaking table condition is room temperature, 200 rev/mins, take out after 30 minutes, rotating speed be 8000 rev/mins centrifugal 10 minutes, get supernatant liquor.
B) add the methyl alcohol of 50ml in the filter residue again, the same shaking table condition was taken out after 15 minutes, and centrifugal 10 minutes of the same condition is got supernatant liquor.
C) supernatant liquor of combining step 1 and step 2, evaporation concentration obtains the crude extract 1 of 50ml toxin.
D) successively use each 10ml of sodium hydroxide of methyl alcohol and 5N to wash balance cation exchange column, when solvent liquid level arrives nh 2 column adsorption layer surface, the crude extract 1 that adds the toxin of step C acquisition immediately, the control flow velocity is at 1 droplet/second, successively use 5% ammoniacal liquor and the methyl alcohol difference drip washing cationic exchange coloum of 10ml again, with target toxin under the 10ml meoh eluate wash-out, rotary evaporation concentrates and obtains thick toxin dry powder at last.
E) getting thick toxin dry powder 5mg is dissolved in the methanol solution of 50ml, separation by half preparative high performance liquid chromatography instrument, separation condition is: moving phase is that methanol solution, flow velocity are 1.5ml/min, the detection wavelength is 254nm, and the retention time of contrast ustilaginoidea virens toxin Ustiloxin A standard specimen peak value is collected the crude extract 2 that obtains toxin.
F) with the crude extract 2 of the toxin that obtains and ustilaginoidea virens toxin Ustiloxin A standard specimen respectively evenly point at silica gel G F
254The same straight line place of the lower end of thin layer chromatography board puts into chromatography cylinder with chromatoplate, launches in developing agent, runs to apart from chromatoplate top 2 centimeters until developing agent, takes out chromatoplate, dries.
G) shine under ultraviolet lamp, contrast ustilaginoidea virens toxin Ustiloxin A standard specimen digs down and the band of ustilaginoidea virens toxin Ustiloxin A standard specimen at same position.
H) silica gel is put into 30ml methyl alcohol and soaked, shaking table took out after 15 minutes, and room temperature, 10000 rev/mins are centrifugal, get supernatant, and rotary evaporation concentrates, and obtains ustilaginoidea virens toxin Ustiloxin A.
Utilize the identical method of embodiment 1, obtain from rice curve powder, to extract the high-efficient liquid phase chromatogram (see figure 2) of ustilaginoidea virens toxin Ustiloxin A.
Purity is through using high performance liquid chromatography tandem mass spectrum and nucleus magnetic resonance identification and analysis, and the content that records ustilaginoidea virens toxin Ustiloxin A is 94%.
Ustilaginoidea virens toxin culture powder is that this obtains like this:
Get the good rice 100g of soaked overnight, place triangular flask, 121 ℃ of autoclavings 20 minutes.Ustilaginoidea virens is transferred to the PDA culture medium flat plate from the refrigeration inclined-plane to be activated, 28 ℃ of-30 ℃ of dark illumination were alternately cultivated 7-10 days, because this germ aerial hyphae growth is not flouring, get in the triangular flask that bacterium colony 1/4 is inoculated in step 1,28 ℃ of-30 ℃ of dark illumination were alternately cultivated 20-25 days, obtained ustilaginoidea virens toxin culture.With the rice culture oven drying at low temperature that obtains, clay into power again, cross 20 mesh sieves, both got ustilaginoidea virens toxin culture powder, standby in-20 ℃ of preservations.
From ustilaginoidea virens toxin culture powder, extract the method for purifying ustilaginoidea virens toxin Ustiloxin A:
The difference of present embodiment and embodiment 3 only terminates in: step a is the rice curve powder of directly having replaced embodiment 2 acquisitions of adopting among the embodiment 3 with the ustilaginoidea virens toxin culture powder that embodiment 4 obtains.Whole extraction purge process is all identical with embodiment 3.
Utilize the identical method of embodiment 1, obtain from ustilaginoidea virens toxin culture powder, to extract the high-efficient liquid phase chromatogram (see figure 3) of ustilaginoidea virens toxin Ustiloxin A.
Present embodiment obtains ustilaginoidea virens toxin Ustiloxin A, and purity is through using high performance liquid chromatography tandem mass spectrum and nucleus magnetic resonance identification and analysis, and the content that records ustilaginoidea virens toxin Ustiloxin A is 94%.
Claims (3)
1. the extracting and purifying method of a rice green smut verticillium toxin Ustiloxin A is characterized in that, this method follows these steps to order and carries out:
A) get rice curve powder or ustilaginoidea virens toxin culture powder, add the PEG of its weight 1/5, add its weight 4-5 2% formic acid solution doubly again, place shaking table, the shaking table condition is room temperature, 200 rev/mins, takes out after 30 minutes, rotating speed be 8000 rev/mins centrifugal 10 minutes, get supernatant liquor;
B) add the isopyknic methyl alcohol of its weight in the filter residue, the same shaking table condition was taken out after 15 minutes, and centrifugal 10 minutes of the same condition is got supernatant liquor;
C) supernatant liquor of combining step a and step b, evaporation concentration obtains the crude extract 1 of toxin;
D) successively use the sodium hydroxide of methyl alcohol and 5N to wash the balance cation exchange column, when solvent liquid level arrives nh 2 column adsorption layer surface, the crude extract 1 that adds the toxin of step c acquisition immediately, the control flow velocity is at 1 droplet/second, successively distinguish the drip washing cationic exchange coloum with 5% ammoniacal liquor and the methyl alcohol of 1/5 sample volume again, last with target toxin under the meoh eluate wash-out of 1/5 sample volume, rotary evaporation concentrates and obtains thick toxin dry powder; Described meoh eluate is the methyl alcohol that contains 5% formic acid;
E) thick toxin dry powder is dissolved in the dry powder weight 10-20 methanol solvate doubly, by the separation of half preparative high performance liquid chromatography instrument, the retention time of contrast ustilaginoidea virens toxin Ustiloxin A standard specimen peak value is collected the crude extract 2 that obtains toxin; The volume ratio of methyl alcohol and water is 2:8 in the described methanol solvate, and the separation condition of described half preparative high performance liquid chromatography instrument is: moving phase is that described methanol solvate, flow velocity are 1.5ml/min, and the detection wavelength is 254nm;
F) crude extract 2 of the toxin that step e is obtained is evenly put at silica gel G F respectively with ustilaginoidea virens toxin Ustiloxin A standard specimen
254Chromatography cylinder is put into chromatoplate in the lower end of thin layer chromatography board, launches in developing agent, runs to apart from chromatoplate top 2 centimeters until developing agent, takes out chromatoplate, dries; Developing agent is the mixed solution of methylene dichloride and methyl alcohol, and the volume ratio of methylene dichloride and methyl alcohol is 7:3;
G) shine under ultraviolet lamp, the standard specimen of contrast ustilaginoidea virens toxin Ustiloxin A digs down and the band of standard specimen at same position;
H) silica gel is put into methyl alcohol and soak, shaking table took out after 15 minutes, and room temperature, 10000 rev/mins are centrifugal, get supernatant, and rotary evaporation concentrates, and namely got content greater than 90% ustilaginoidea virens toxin Ustiloxin A.
2. the extracting and purifying method of rice green smut verticillium toxin Ustiloxin A according to claim 1, it is characterized in that: the rice curve powder described in the step a is to obtain like this: at the late growth stage of rice that false smut takes place, pluck globe blackish green or pale brown look from paddy rice fringe portion and be the rice curve, dry, clay into power, cross 20 mesh sieves, obtain rice curve powder, standby in-20 ℃ of preservations.
3. the extracting and purifying method of rice green smut verticillium toxin Ustiloxin A according to claim 1, it is characterized in that: the ustilaginoidea virens toxin culture powder described in the step a is to obtain like this: at first get an amount of rice, soaked overnight, 121 ℃ of 20 minutes autoclavings; Insert the ustilaginoidea virens mycelia of activation then by 5% inoculum size, 28 ℃ of-30 ℃ of dark illumination were alternately cultivated 20-25 days, obtained ustilaginoidea virens toxin culture; With ustilaginoidea virens toxin culture oven drying at low temperature, clay into power again, cross 20 mesh sieves, obtain ustilaginoidea virens toxin culture powder, standby in-20 ℃ of preservations.
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| CN103760353B (en) * | 2014-02-08 | 2015-07-08 | 中国农业大学 | Method and special ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting ustiloxin A |
| US20170291921A1 (en) * | 2014-09-24 | 2017-10-12 | National University Corporation, Iwate University | Cyclic peptide derivative, method for preparing same and composition thereof |
| CN104569416B (en) * | 2015-01-22 | 2016-04-13 | 中国农业大学 | Detect method and the special ELISA reagent kit thereof of rice aspergin B |
| CN106279359A (en) * | 2016-08-11 | 2017-01-04 | 中国水稻研究所 | A kind of method preparing five kinds of ustilaginoidea virens toxin |
| CN106754621A (en) * | 2017-01-10 | 2017-05-31 | 广西大学 | The efficient botrytis cinerea for preventing from polluting is conidial to prepare cultural method |
| CN106867957A (en) * | 2017-01-10 | 2017-06-20 | 广西大学 | A kind of ustilaginoidea virens thin-walled of Pollution protection is conidial to prepare cultural method |
| CN106635948A (en) * | 2017-01-10 | 2017-05-10 | 广西大学 | Pollution-free preparing and culturing method for rice-false-smut-case thin-wall conidia |
| CN108982689A (en) * | 2018-07-04 | 2018-12-11 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of method of two kinds of rice song toxin in quick detection paddy |
| CN111763241A (en) * | 2020-06-11 | 2020-10-13 | 江苏省农业科学院 | Preparation method of pure ustiloxin A |
| CN112649523A (en) * | 2020-12-01 | 2021-04-13 | 华中农业大学 | Method for detecting ustilagin A or ustilagin B in food |
| CN114933632A (en) * | 2022-05-16 | 2022-08-23 | 杭州医学院 | Method for simultaneously separating, purifying and preparing 5 kinds of aspergillus oryzae toxins |
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