CN101818172A - Method for separating Wuyi rhzomorph antimicrobial active substance - Google Patents
Method for separating Wuyi rhzomorph antimicrobial active substance Download PDFInfo
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- CN101818172A CN101818172A CN201010156817A CN201010156817A CN101818172A CN 101818172 A CN101818172 A CN 101818172A CN 201010156817 A CN201010156817 A CN 201010156817A CN 201010156817 A CN201010156817 A CN 201010156817A CN 101818172 A CN101818172 A CN 101818172A
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- resin
- wuyiencin
- wuyi
- rhzomorph
- bacterium
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Abstract
The invention provides a method for separating a Wuyi rhzomorph antimicrobial active substance. The method has simple operation, little workload and low cost; a resin material can be recycled and is energy-saving and environment-friendly; and in the method, different polar parts of Wuyi rhzomorph can be obtained by rapidly separating, the bacteriostatic effect of the different polar parts on diseases caused by different pathogenic bacteria is determined, and technical basis is provided for developing and producing new breeds of Wuyi rhzomorph which aims at different crop diseases.
Description
Technical field
The invention belongs to field of biological pesticide, secondary metabolite-Wuyiencin in particular to a kind of superior strain (Streptomyces anhygroscopicus var.wuyiensis) F64 of not streptomyces hygroscopicus Wuyi mutation produces the invention still further relates to the method that the fermented liquid that utilizes this bacterial strain separates different antibacterial substances.
Background technology
Biological pesticide is the new direction of 21 century agricultural chemicals industry development, and wherein the antibiotics sterilant is one of the fastest product of biological pesticide industrialized development.Wuyiencin is the biological pesticide product with preventing disease production-increasing effect of Chinese Academy of Agricultural Sciences's development, it derives from a kind of microbiotic (microorganism journal that not streptomyces hygroscopicus Wuyi mutation (Streptomyces anhygroscopicus var.wuyiensis) produces, 24:401-402,1984), be mainly used in control farm crop fungus disease, have the characteristics of efficient, wide spectrum, low toxicity, environmental safety, therefore, in the ecological agriculture and pollution-free food production, have broad application prospects.The production of Wuyiencin is utilize to cultivate Wuyiencin to produce bacterium, the secondary metabolite of the microorganism that produces by biological submerged fermentation.
Separation method in the Wuyiencin production process mainly is to adopt high temperature concentrating under reduced pressure device and molecular film at present.High temperature concentrating under reduced pressure device is to evaporate moisture under 60-90 ℃ high temperature, obtains the concentrated solution of Wuyiencin effective constituent, and this method is destroyed effective constituent wherein easily, reduces sterilization effect.Adopt molecular film to separate Wuyiencin effective constituent and can only remove small-molecule substances such as sugar, inorganic salt, can not be separated to the effective constituent of different sites.The method of separation Wuyiencin provided by the invention, simple to operate, workload is few, and is with low cost; The opposed polarity position that can sharp separation obtains Wuyiencin, and definite opposed polarity position is to the fungistatic effect of the microbial disease of Different Kinds of Pathogens.
The highly efficient and productive production method of research Wuyiencin sterilant, the ability and the level of developing voluntarily, formulating for the agricultural antibiotic that improves China, improve new microbe agricultural chemicals industrialization level with independent intellectual property right, provide the basis not only directly for agriculture production provides the true tumor pesticide species, and for developing the microbial pesticide product.
Summary of the invention
The object of the present invention is to provide a kind of method of separating Wuyi rhzomorph antimicrobial active substance, thereby be that the utilization ratio that improves Wuyiencin, the new variety of exploitation Wuyiencin are put forward technical foundation.
The method of the separation Wuyi rhzomorph antimicrobial active substance that the contriver provides, critical step is: (1) preparation Wuyiencin fermented liquid; (2) the Wuyiencin fermented liquid separates through column chromatography, carries out gradient elution with the solvent of different concns such as the salts solution of aqueous methanol, ethanol etc. and different concns, obtains the wash-out position of opposed polarity, concentrating under reduced pressure, drying; (3) cultivate according to the culture condition of different bacterium or fungi.(4) cup-plate method or paper disk method are determined the bacteriostatic activity at opposed polarity position.
Anti-microbial activity of the present invention comprises has restraining effect to bacterium and fungi.
In above-mentioned second step, separatory column chromatography, used weighting agent is all models of different manufacturers production and the resin of specification, comprise polarity, low-pole and nonpolar macroporous adsorption resin, vinyl cyanide type and vinylbenzene macroporous adsorbent resin, polystyrene and acrylic acid series ion exchange resin, gel-type and macroreticular ion exchange resin, resin materials such as dextrane gel resin, polyamide resin, for example: AB-8 macroporous adsorbent resin, D113 ion exchange resin, SephadexLH-20 dextrane gel resin etc.
In above-mentioned second step, described column chromatography eluent is moisture lower alcohol, for example, and methyl alcohol or ethanol.
In above-mentioned the 3rd step, described bacterium or fungus culture condition and cultural method are conventional bacterium or fungus culture method.
In above-mentioned the 4th step, it is characterized in that described anti-microbial activity comprises that bacterium and fungi are had anti-microbial effect.
A kind of method of separating Wuyi rhzomorph antimicrobial active substance provided by the invention, simple to operate, workload is few, and is with low cost; Resin material can be recycled, energy-conserving and environment-protective; The opposed polarity position that can sharp separation obtains Wuyiencin, and definite opposed polarity position is to the fungistatic effect of the microbial disease of Different Kinds of Pathogens is for exploitation and the new variety of producing at the Wuyiencin of different crops disease provide technical foundation.
Embodiment
Embodiment 1 AB-8 macroporous adsorbent resin separates the antibacterial substance of Wuyiencin
(1) preparation Wuyiencin fermented liquid;
(2) the Wuyiencin fermented liquid passes through the AB-8 macroporous adsorbent resin, and the water-methanol of usefulness different ratios (1: 0,1: 9,3: 7,5: 5,7: 3,0: 1) carry out gradient elution, obtain the wash-out position of six opposed polarities, concentrating under reduced pressure, drying;
(3) cultivate according to the culture condition of different bacterium or fungi.
(4) cup-plate method or paper disk method are measured the bacteriostatic activity at six opposed polarity positions.
Concentrated solution drying under reduced pressure with six opposed polarity positions, methanol aqueous solution dissolving with 25%, serial dilution to 2,1,0.5,0.25, six different concns of 0.125,0.0625 (mg/ml), measure bacteriostatic activity, indicator is rhodotorula (Rhodotorula rubra), subtilis (Bacillus subtilis), bacillus megaterium (Bacillusmegaterium) and intestinal bacteria (Escherichia coli).
The observation experiment result: the result shows that all there is bacteriostatic activity at six opposed polarity positions, there is very strong antibacterial effect at first position (washing position) to all indicators, the 4th position is to the good antimicrobial effect of rhodotorula, and there is the good restraining effect at the 6th position to subtilis, bacillus megaterium and intestinal bacteria.
Embodiment 2 Diaion HP-20 macroporous adsorbent resins separate the antibacterial substance of Wuyiencin
(1) preparation Wuyiencin fermented liquid;
(2) the Wuyiencin fermented liquid passes through Diaion HP-20 macroporous adsorbent resin, and the water-methanol of usefulness different ratios (1: 0,1: 9,3: 7,5: 5,7: 3,0: 1) carry out gradient elution, obtain the wash-out position of six opposed polarities, concentrating under reduced pressure;
(3) cultivate according to the culture condition of different bacterium or fungi.
(4) cup-plate method or paper disk method are measured the bacteriostatic activity at six opposed polarity positions.
Concentrated solution drying under reduced pressure with six opposed polarity positions, methanol aqueous solution dissolving with 25%, serial dilution to 2,1,0.5,0.25, six different concns of 0.125,0.0625 (mg/ml), measure bacteriostatic activity, indicator is rhodotorula (Rhodotorula rubra), subtilis (Bacillus subtilis), bacillus megaterium (Bacillusmega terium) and intestinal bacteria (Escherichia coli).
The observation experiment result: the result shows that all there is bacteriostatic activity at six opposed polarity positions, there is very strong fungistatic effect at first position (washing position) to all indicators, the 4th position is to the good antimicrobial effect of rhodotorula, and there is the good restraining effect at the 6th position to subtilis, bacillus megaterium and intestinal bacteria.
Embodiment 3 Sephadex LH-20 dextrane geles separate the antibacterial substance of Wuyiencin
(1) preparation Wuyiencin fermented liquid;
(2) the Wuyiencin fermented liquid passes through Sephadex LH-20 macroporous adsorbent resin, and the water-methanol of usefulness different ratios (1: 0,1: 9,3: 7,5: 5,7: 3,0: 1) carry out gradient elution, obtain the wash-out position of six opposed polarities, concentrating under reduced pressure;
(3) cultivate according to the culture condition of different bacterium or fungi.
(4) cup-plate method or paper disk method are measured the bacteriostatic activity at six opposed polarity positions.
Concentrated solution drying under reduced pressure with six opposed polarity positions, methanol aqueous solution dissolving with 25%, serial dilution to 2,1,0.5,0.25, six different concns of 0.125,0.0625 (mg/ml), measure bacteriostatic activity, indicator is rhodotorula (Rhodotorula rubra), subtilis (Bacillus subtilis), bacillus megaterium (Bacillusmega terium) and intestinal bacteria (Escherichia coli).
The observation experiment result: the result shows that all there is bacteriostatic activity at six opposed polarity positions, there is very strong fungistatic effect at first position (washing position) to all indicators, the 4th position is to the good antimicrobial effect of rhodotorula, and there is the good restraining effect at the 6th position to subtilis, bacillus megaterium and intestinal bacteria.
Claims (6)
1. a method of separating Wuyi rhzomorph antimicrobial active substance is characterized in that this method may further comprise the steps: (1) preparation Wuyiencin fermented liquid; (2) the Wuyiencin fermented liquid separates through column chromatography, carries out gradient elution with the solvent of different concns and the salts solution of different concns, obtains the wash-out position of opposed polarity, concentrating under reduced pressure, drying; (3) cultivate according to the culture condition of different bacterium or fungi; (4) cup-plate method or paper disk method are determined the bacteriostatic activity at opposed polarity position.
2. method according to claim 1, it is characterized in that the used weighting agent of separatory column chromatography is polarity, low-pole and nonpolar macroporous adsorption resin, vinyl cyanide type and vinylbenzene macroporous adsorbent resin, polystyrene and acrylic acid series ion exchange resin, gel-type and macroreticular ion exchange resin, dextrane gel resin, polyamide resin.
3. method according to claim 2, wherein said weighting agent are AB-8 macroporous adsorbent resin, D113 ion exchange resin or Sephadex LH-20 dextrane gel resin.
4. method according to claim 1, it is characterized in that separating used eluent is aqueous methanol or ethanol.
5. method according to claim 1 is characterized in that in the described step (3), and the culture condition of described bacterium or fungi is conventional bacterium or fungus culture method.
6. method according to claim 1 is characterized in that described bacteriostatic activity comprises that bacterium and fungi are had anti-microbial effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010156817A CN101818172A (en) | 2010-04-27 | 2010-04-27 | Method for separating Wuyi rhzomorph antimicrobial active substance |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201010156817A CN101818172A (en) | 2010-04-27 | 2010-04-27 | Method for separating Wuyi rhzomorph antimicrobial active substance |
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| CN101818172A true CN101818172A (en) | 2010-09-01 |
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| CN201010156817A Pending CN101818172A (en) | 2010-04-27 | 2010-04-27 | Method for separating Wuyi rhzomorph antimicrobial active substance |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103626855A (en) * | 2013-11-14 | 2014-03-12 | 中国农业科学院植物保护研究所 | Protein associated with biosynthesis of wuyiencin and encoding gene and application thereof |
| CN108659076A (en) * | 2018-04-16 | 2018-10-16 | 中国农业科学院植物保护研究所 | A method of detaching Wuyiencin from zymotic fluid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1620871A (en) * | 2004-10-22 | 2005-06-01 | 潍坊万胜生物农药有限公司 | Wuyiencin bactericide extraction and concentration method |
| CN101050441A (en) * | 2007-03-30 | 2007-10-10 | 中国农业科学院植物保护研究所 | Heavy producing bacterial strain F64 for Wuyi bacterin, and fermentation process |
-
2010
- 2010-04-27 CN CN201010156817A patent/CN101818172A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1620871A (en) * | 2004-10-22 | 2005-06-01 | 潍坊万胜生物农药有限公司 | Wuyiencin bactericide extraction and concentration method |
| CN101050441A (en) * | 2007-03-30 | 2007-10-10 | 中国农业科学院植物保护研究所 | Heavy producing bacterial strain F64 for Wuyi bacterin, and fermentation process |
Non-Patent Citations (1)
| Title |
|---|
| 张克诚等: "微生物农药武夷菌素的研究进展", 《中国重要会议论文全文数据库 农业生物灾害预防与控制研究》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103626855A (en) * | 2013-11-14 | 2014-03-12 | 中国农业科学院植物保护研究所 | Protein associated with biosynthesis of wuyiencin and encoding gene and application thereof |
| CN103626855B (en) * | 2013-11-14 | 2015-09-16 | 中国农业科学院植物保护研究所 | A kind of albumen relevant to Wuyiencin biosynthesizing and encoding gene thereof and application |
| CN108659076A (en) * | 2018-04-16 | 2018-10-16 | 中国农业科学院植物保护研究所 | A method of detaching Wuyiencin from zymotic fluid |
| CN108659076B (en) * | 2018-04-16 | 2020-05-05 | 中国农业科学院植物保护研究所 | Method for separating wuyiencin from fermentation liquor |
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Application publication date: 20100901 |