CN103882080A - Effective method for preparing avermectin - Google Patents
Effective method for preparing avermectin Download PDFInfo
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- CN103882080A CN103882080A CN201410098148.3A CN201410098148A CN103882080A CN 103882080 A CN103882080 A CN 103882080A CN 201410098148 A CN201410098148 A CN 201410098148A CN 103882080 A CN103882080 A CN 103882080A
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a method for preparing avermectin (Avermectins). The method comprises the following steps: cultivating a streptomyces avermitilis strain capable of generating avermectin and genetic improved bacteria thereof in a first stage of fluid nutrient medium, and then inoculating into a second stage of fluid nutrient medium as a seed solution; fermenting and cultivating after inoculating the seed solution into the second stage of fluid nutrient medium, and feeding a sodium propionate precursor to carry out feeding cultivation; collecting the avermectin B1a from fermentation broth after fermentation is finished. The effective method has the characteristics of being high in production efficiency and the like.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of effective ways of preparing Avrmectin.
Background technology
Avrmectin (Avermectin) is ten hexa-atomic macrolide antibiotics of one group of synthetic structural similitude of Avid kyowamycin (Streptomyces avermitilis) secondary metabolism, has wide spectrum, efficient desinsection, acaricidal activity, is made up of 8 components, it is avermectin A1a, A2a,, B2a, A1b, A2b, B1b, B2b, wherein, the activity of Avermectin B1a is the highest.
Because Avrmectin has, activity is high, insecticidal spectrum wide,, chemical structure unique the be easy to advantages such as transformation residual, environmentally safe without persistence, become the focus that biological pesticide develops.1985, Avrmectin was formally introduced to the market as agricultural insecticide, was worldwide widely used in the control of the plant insects such as vegetables, fruit tree, field crop, gardens, evil mite, had obtained the effect attracting people's attention.1999, Avrmectin has become the large situation of selling well agricultural chemicals in the world the 21st, and the 4th large situation of selling well sterilant is current topmost new bio Insecticidal and acaricidal agent, be known as the world's two mcroorganism agricultural chemicals with Bacillus thuringiensis Bt, become the agricultural and veterinary chemicals product of the high-efficiency low-toxicity of Ministry of Agriculture's recommendation.Research in recent years shows, Avrmectin can not only kill tubercule bacillus, comprise resistance tubercule bacillus, and there is antineoplastic effect, the resistance of tumour cell is also had to certain restraining effect, this is indicating Avrmectin or can be used for researching and developing new treating pulmonery tuberculosis and antitumor drug, so Avrmectin all has higher using value and wide market outlook aspect three of agricultures, poultry, doctor.
Up to the present, Avrmectin is generally considered the new antibiotic mankind since the penicillin that continues with major contribution, the huge market demand, and since nineteen ninety-five, the former medicine of Avrmectin is every year with the speed increase of 50% left and right, and production capacity is amplified year by year.In China, exploitation, development and the suitability for industrialized production of Avrmectin are listed in national key scientific and technological project always, and existing several drugmakers are in the production of carrying out Avrmectin, but the Avrmectin manufacturing enterprise of China, the productive rate of producing bacterial classification is lower, and zymotechnique is backward in technique.Therefore, need badly and improve bacterial classification yield level, optimize fermentation manufacturing technique, improve fermentation unit, further reduce production costs.
The inventor carrying out finding after genetic improvement to a strain Avid kyowamycin (Streptomyces avermitilis), and mutant strain Streptomyces avermitilis AW-H1 has the characteristic of high yield Avrmectin.
Subsequently, the inventor is studied the processing parameter and the technological method that utilize Avid kyowamycin (Streptomyces avermitilis) fermentation to prepare Avrmectin, obtain a kind of technological method that improves Avrmectin output by supplementing Sodium Propionate precursor, therefore completed the present invention.
Summary of the invention
Object of the present invention:
One of object of the present invention, is to provide a kind of new bacterial strain for the production of Avrmectin;
Two of object of the present invention, is to provide and a kind ofly adds the precursor substance of Sodium Propionate as the synthetic Avrmectin of bacterial strain, fermentative production Avrmectin by stream;
Three of object of the present invention, is to provide the substratum for the production of Avrmectin.
More particularly, the invention provides a kind of effective ways of preparing Avrmectin, comprise following steps:
The actinomycetes that can produce Avrmectin for example, are cultivated in level liquid substratum (, culture medium A hereinafter described), as seed liquor; Described actinomycetes are mainly Avid kyowamycin (Streptomyces avermitilis) and genetic improvement bacterial strain thereof.
Will be in first step liquid nutrient medium cultured seed liquor be for example inoculated into, in second stage liquid nutrient medium (, substratum B) hereinafter described and cultivate; After second stage liquid nutrient medium inoculation first step seed liquor, cultivate one suitable period, start stream and add the precursor substance of certain density Sodium Propionate as the synthetic Avrmectin of bacterial strain, improve the output of Avrmectin.
After fermentation ends, from above-mentioned fermentation culture, collect Avrmectin.
Specific embodiment of the invention scheme is, adopts to produce in the Avid kyowamycin bacterial classification of Avrmectin and genetic improvement bacterium liquid medium within thereof to carry out second order fermentation cultivation, selects different substratum at every grade of fermentation stage.In second stage fermentation, to be suitable for the liquid nutrient medium of second stage fermentation, for example substratum B hereinafter described, as fermention medium.After inoculation first step seed liquor, at applicable temperature, cultivate for some time, for example, at 25 ℃-35 ℃, after fermentation culture 5-30 hour, stream adds Sodium Propionate.
The fed-batch mode of Sodium Propionate can adopt continuously (at the uniform velocity or at the uniform velocity non-) stream to add and/or intermittent type fed-batch mode, and (at the uniform velocity or at the uniform velocity non-) fed-batch mode is preferred continuously.
Described (at the uniform velocity or at the uniform velocity non-) continuously fed-batch mode, is that by certain density Sodium Propionate, Continuous Flow adds in the fermentor tank of the second stage with certain stream rate of acceleration (at the uniform velocity or at the uniform velocity non-), within the 120th hour, stops stream adding to fermenting.
Described intermittent type fed-batch mode, is the mode that adds a defective material with certain interval of time, and intermittent type is by certain density Sodium Propionate, and stream adds in the fermentor tank of the second stage.Intermittent time is preferably every feed supplement in 5-20 hour 1-5 time, more preferably about interval feed supplement in 10 hours 1-2 time.Those skilled in the art also can be as required, adopts other applicable timed interval feed supplement.
Fermentation condition: 25 ℃-35 ℃ of temperature, PH:4-7
Fermentation time: 11-13 days
After fermentation ends, after organic solvent extractionprocess, silica gel column chromatography, the condensing crystal method for mycelium that retain etc. are extracted, obtain Avrmectin product after filtering fermentation liquor.
Adopt high performance liquid chromatography to detect Avrmectin, condition used: chromatographic column HC-C184.6 × 250mm; Moving phase is methyl alcohol: water=88:12(0.5% glacial acetic acid); Flow velocity 1mL/min; Detect wavelength 245nm.
Adopt technique of the present invention, while fermentation in the second stage, stream adds certain density Sodium Propionate, can supplement the synthetic Sodium Propionate precursor of Avrmectin, thereby improves the output of Avrmectin.
In preferred specific embodiments, the present invention also adopts the technical schemes such as for example new bacterial strain further to improve the output of Avrmectin.
In preferred the inventive method, the present invention provides the genetic improvement bacterial strain Streptomyces avermitilis AW-H1 of a strain for the production of the Avid kyowamycin of Avrmectin especially; It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 15th, 2013, and preserving number is CGMCC No.7319, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Culture medium A, B that second order fermentation of the present invention adopts, it is specifically composed as follows:
Culture medium A (g/mL): W-Gum 0.3%-5.0%, α-amylase 1u/100mL-10u/100mL, bean cake powder 0.1%-1.5%, yeast extract paste 0.05%-1.5%, groundnut meal 0.3%-2.0%, cobalt chloride 0.05%-0.5%.
Substratum B(g/mL):
| Composition | General range | Preferable range |
| Semen Maydis powder | 0.01%-15.0% | 0.02%-10.0% |
| Zulkovsky starch | 0.001%-10.0% | 0.005%-5.0% |
| W-Gum | 5.0%-25.0% | 10.0%-20.0% |
| Analysis for soybean powder | 0.001%-3.0% | 0.005%-1.0% |
| Bean cake powder | 0.05%-10.0% | 0.5-5.0% |
| Groundnut meal | 0.001%-2.0% | 0.005%-1.5% |
| Yeast extract paste | 0.001%-2.0% | 0.005%-1.0% |
| Yeast powder | 0.05%-5.0% | 0.1%-3.0% |
| Manganous sulfate | 0.001%-0.05% | 0.002%-0.03% |
| Ammonium sulfate | 0.001%-2.0% | 0.005%-0.5% |
| Dipotassium hydrogen phosphate | 0.0001%-1.0% | 0.0005%-0.01% |
| Cobalt chloride | 0.0005%-0.01% | 0.001%-0.006% |
| Sodium-chlor | 0.0001%-1.0% | 0.0002%-0.05% |
| Sodium orthomolybdate | 0.001%-0.01% | 0.0015%-0.005% |
| α-amylase | 10u/100mL-100u/100mL | 20u/100mL-50u/100mL |
| Calcium carbonate | 0.005%-3.0% | 0.01%-1.5% |
the zymotechnique whole process of the preferred embodiment of the invention is:
Avid kyowamycin bacterial classification after activation is inoculated in culture medium A, and solid culture or be loaded in triangular flask 25 ℃ of-35 ℃ of shake-flask culture after 24-40 hours, is inoculated in the inoculum size of 5%--20% and in the fermentor tank that is added with sterilising medium B, carries out fermentative production.Bacterial classification is inoculated in after the fermentor tank of the second stage in 25 ℃-35 ℃, and preferably 27 ℃ of-32 ℃ of fermentation culture, after 5-30 hours, start stream and add certain density Sodium Propionate.
The flow feeding mode of Sodium Propionate has two kinds, and one is continuously that (at the uniform velocity or at the uniform velocity non-) stream adds, and one is that intermittent type stream adds, and Continuous Flow add mode is preferred.
While adopting Continuous Flow add mode, with the speed of 0.001-10.0L/h continuously (at the uniform velocity or at the uniform velocity non-) stream add Sodium Propionate (1mmol/L-100mmol/L), within the 120th hour, stop stream in fermentation culture and add.
While adopting intermittent type stream to add Sodium Propionate (1mmol/L-100mmol/L), intermittent time can be every feed supplement in 5-20 hour 1-5 time, be preferably about interval feed supplement in 10 hours 1-2 time, each feed supplement amount is 0.001%-0.6% of fermented liquid cumulative volume, preferably 0.01%-0.2%.
Fermentation condition: 25 ℃-35 ℃ of temperature, PH:4-7
Accompanying drawing 1 is shown in full technical process of the present invention in fermentation time: 11-13 days.
Adopt technique of the present invention, bacterial strain is produced Avrmectin with higher substrate conversion efficiency and product synthesis rate, more than the output of Avrmectin active principle B1a can reach 8200ug/mL.
After fermentation ends, after organic solvent extractionprocess, silica gel column chromatography, the condensing crystal method for mycelium that retain etc. are extracted, obtain Avrmectin product after filtering fermentation liquor.
Adopt high performance liquid chromatography to detect Avrmectin, condition used: chromatographic column HC-C184.6 × 250mm; Moving phase is methyl alcohol: water=88:12(0.5% glacial acetic acid); Flow velocity 1mL/min; Detect wavelength 245nm.
In preferred the inventive method, provide especially the genetic improvement bacterial strain Streptomyces avermitilis AW-H1 of a strain for the production of the Avid kyowamycin of Avrmectin; It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 15th, 2013, and preserving number is CGMCC No.7319.
The present invention has advantages of:
One, the ability of the genetic improvement bacterial strain Streptomyces avermitilis AW-H1 of a strain Avid kyowamycin provided by the invention production Avrmectin is strong, prepares Avrmectin with this bacterial strain, can improve the efficiency of producing Avrmectin.
Two, stream adds certain density Sodium Propionate and carries out fed-batch fermentation, can supplement the synthetic required Sodium Propionate precursor of Avrmectin, significantly improves the output of Avrmectin.
Accompanying drawing explanation
Accompanying drawing is the schema of abamectin fermented production.
Embodiment
Embodiment 1
With 10 of 500mL triangular flasks, every bottled 150mL culture medium A (W-Gum 3.0%, α-amylase 6u/100mL, bean cake powder 0.8%, yeast extract paste 0.4%, groundnut meal 1.0%, cobalt chloride 0.3%), in 120 ℃ of sterilizings 30 minutes, Avid kyowamycin (Streptomyces avermitilis) the AW-H1 bacterium liquid after cooling rear inoculation activation, be placed at 28 ℃ of temperature shake-flask culture 32 hours.Cultured strain liquid is inoculated in by the inoculum size of 5%-10% in the 50L fermentor tank of in-built 20L sterilising medium B, (substratum B: Semen Maydis powder 0.04%, Zulkovsky starch 0.01%, W-Gum 14.0%, analysis for soybean powder 0.008%, bean cake powder 2.8%, groundnut meal 0.006%, yeast extract paste 0.008%, yeast powder 1.0%, manganous sulfate 0.0072%, ammonium sulfate 0.025%, dipotassium hydrogen phosphate 0.0008%, cobalt chloride 0.002%, sodium-chlor 0.0003%, Sodium orthomolybdate 0.023%, α-amylase 28u/100mL, calcium carbonate 0.08%), at 27 ℃ of-32 ℃ of temperature, aeration-agitation fermentation is after 8-20 hours, add Sodium Propionate (5mmol/L) with the speed Continuous Flow of 0.01-1.0L/h, within the 120th hour, stopping stream in fermentation culture adds.Fermentation PH4-7, fermentation period 12 days.
After fermentation ends, after organic solvent extractionprocess, silica gel column chromatography, the condensing crystal method for mycelium that retain etc. are extracted, obtain Avrmectin product after filtering fermentation liquor.
Adopt high performance liquid chromatography to detect Avrmectin, condition used: chromatographic column HC-C184.6 × 250mm; Moving phase is methyl alcohol: water=88:12(0.5% glacial acetic acid); Flow velocity 1mL/min; Detect wavelength 245nm.
After testing, through fermentation in 12 days, Avrmectin output can reach 8400ug/mL fermented liquid, and product recovery rate reaches more than 85%.
Embodiment 2
With 12 of 500mL triangular flasks, every bottled 150mL culture medium A (W-Gum 4.0%, α-amylase 8u/100mL, bean cake powder 1.0%, yeast extract paste 0.4%, groundnut meal 0.8%, cobalt chloride 0.2%), in 120 ℃ of sterilizings 30 minutes, Avid kyowamycin (Streptomyces avermitilis) the AW-H1 bacterium liquid after cooling rear inoculation activation, be placed at 28 ℃ of temperature shake-flask culture 32 hours.Cultured strain liquid is inoculated in by the inoculum size of 5%-10% in the 50L fermentor tank of in-built 20L sterilising medium B, (substratum B: Semen Maydis powder 0.08%, Zulkovsky starch 0.02%, W-Gum 12.0%, analysis for soybean powder 0.02%, bean cake powder 3.0%, groundnut meal 0.01%, yeast extract paste 0.01%, yeast powder 1.5%, manganous sulfate 0.0024%, ammonium sulfate 0.02%, dipotassium hydrogen phosphate 0.001%, cobalt chloride 0.0025%, sodium-chlor 0.001%, Sodium orthomolybdate 0.025%, α-amylase 30u/100mL, calcium carbonate 0.08%), at 27 ℃ of-32 ℃ of temperature, aeration-agitation fermentation is after 8-20 hours, intermittent type stream adds Sodium Propionate (10mmol/L), intermittent time is about interval feed supplement in 10 hours 1 time, each feed supplement amount is the 0.01%-0.1% of fermented liquid cumulative volume, within the 120th hour, stopping stream in fermentation culture adds.Fermentation PH4-7, fermentation period 12 days.
After fermentation ends, after organic solvent extractionprocess, silica gel column chromatography, the condensing crystal method for mycelium that retain etc. are extracted, obtain Avrmectin product after filtering fermentation liquor.
Adopt high performance liquid chromatography to detect Avrmectin, condition used: chromatographic column HC-C184.6 × 250mm; Moving phase is methyl alcohol: water=88:12(0.5% glacial acetic acid); Flow velocity 1mL/min; Detect wavelength 245nm.
After testing, through fermentation in 12 days, Avrmectin output can reach 8200ug/mL fermented liquid, and product recovery rate reaches more than 85%.
Embodiment 3
With 10 of 500mL triangular flasks, every bottled 150mL culture medium A (W-Gum 4.0%, α-amylase 8u/100mL, bean cake powder 1.0%, yeast extract paste 0.5%, groundnut meal 1.0%, cobalt chloride 0.2%), in 120 ℃ of sterilizings 30 minutes, preserved in this laboratory of Avid kyowamycin (Streptomyces avermitilis) AW-12(after cooling rear inoculation activation) bacterium liquid, be placed at 28 ℃ of temperature shake-flask culture 32 hours.Cultured strain liquid is inoculated in to (substratum B: Semen Maydis powder 0.06% in the 50L fermentor tank of in-built 20L sterilising medium B by the inoculum size of 5%-10%, Zulkovsky starch 0.008%, W-Gum 16.0%, analysis for soybean powder 0.01%, bean cake powder 3.0%, groundnut meal 0.02%, yeast extract paste 0.02%, yeast powder 1.5%, manganous sulfate 0.003%, ammonium sulfate 0.025%, dipotassium hydrogen phosphate 0.002%, cobalt chloride 0.002%, sodium-chlor 0.0008%, Sodium orthomolybdate 0.023%, α-amylase 40u/100mL, calcium carbonate 0.1%), at 27 ℃ of-32 ℃ of temperature, aeration-agitation fermentation is after 8-20 hours, add Sodium Propionate (10mmol/L) with the speed Continuous Flow of 0.01-1.0L/h, until stopping stream on the 120th hour, fermentation culture adds.Fermentation PH4-7, fermentation period 12 days.
After fermentation ends, after organic solvent extractionprocess, silica gel column chromatography, the condensing crystal method for mycelium that retain etc. are extracted, obtain Avrmectin product after filtering fermentation liquor.
Adopt high performance liquid chromatography to detect Avrmectin, condition used: chromatographic column HC-C184.6 × 250mm; Moving phase is methyl alcohol: water=88:12(0.5% glacial acetic acid); Flow velocity 1mL/min; Detect wavelength 245nm.
After testing, through fermentation in 12 days, Avrmectin output can reach 5000ug/mL fermented liquid, and product recovery rate reaches more than 80%.
Claims (6)
1. effective ways of preparing Avrmectin, comprise following steps:
The actinomycetes that can produce Avrmectin are cultivated in first step liquid nutrient medium, and described actinomycetes are genetic improvement bacterial strain Streptomyces avermitilis AW-H1 of Avid kyowamycin;
Will be in first step liquid nutrient medium cultured first step seed liquor be inoculated on the liquid nutrient medium of the second stage and carry out fermentation culture, under applicable condition, cultivate after for some time, stream adds certain density Sodium Propionate, carries out fed batch fermentation cultivation.After fermentation ends, from above-mentioned fermentation culture, collect Avermectin B1a.The first step is that seed liquor is cultivated, and used medium is liquid nutrient medium A, and the second stage is fermentation culture, and used medium is liquid nutrient medium B.
2. the method for preparing Avrmectin described in claim 1, is characterized in that: the genetic improvement bacterial strain Streptomyces avermitilis AW-H1 that described bacterial classification is Avid kyowamycin, CGMCC No.7319.
3. the method for preparing according to claim 1 Avrmectin, is characterized in that: substratum specifically composed as follows:
Culture medium A:
W-Gum 0.3%-5.0%, α-amylase 1u/100mL-10u/100mL, bean cake powder 0.1%-1.5%, yeast extract paste 0.05%-1.5%, groundnut meal 0.3%-2.0%, cobalt chloride 0.05%-0.5%.
Substratum B:
Semen Maydis powder 0.02%-10.0%, Zulkovsky starch 0.005%-5.0%, W-Gum 10.0%-20.0%, analysis for soybean powder 0.005%-1.0%, bean cake powder 0.5%-5.0%, groundnut meal 0.005%-1.5%, yeast extract paste 0.005%-1.0%, yeast powder 0.1%-3.0%, manganous sulfate 0.002%-0.03%, ammonium sulfate 0.005%-0.5%, dipotassium hydrogen phosphate 0.0005%-0.01%, cobalt chloride 0.001%-0.006%, sodium-chlor 0.0002%-0.05%, Sodium orthomolybdate 0.0015%-0.005%, α-amylase 20u/100mL-50u/100mL, calcium carbonate 0.01%-1.5%.
4. the method for preparing according to claim 1 Avrmectin, is characterized in that: the mode that stream adds Sodium Propionate is Continuous Flow add mode and/or intermittent type fed-batch mode.
5. prepare according to claim 4 the method for Avrmectin, it is characterized in that: Continuous Flow add mode is at the uniform velocity or non-at the uniform velocity Continuous Flow add mode, add Sodium Propionate (1mmol/L-100mmol/L) with the speed Continuous Flow of 0.001-10.0L/h, within the 120th hour, stop stream to fermenting and add.
6. prepare according to claim 4 the method for Avrmectin, it is characterized in that: intermittent type stream adds Sodium Propionate, and (intermittent time of (1mmol/L-100mmol/L) can be every feed supplement in 5-20 hour 1-5 time, be preferably about interval feed supplement in 10 hours 1-2 time, each feed supplement amount is 0.001%-0.6% of fermented liquid cumulative volume, preferably 0.01%-0.2%.
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| CN114381386A (en) * | 2021-11-21 | 2022-04-22 | 河北兴柏农业科技有限公司 | Culture medium for producing abamectin by fermentation |
| CN114457134A (en) * | 2022-03-17 | 2022-05-10 | 石家庄华滋生物工程有限公司 | Abamectin fermentation method based on composite organic nitrogen source |
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| CN105803022A (en) * | 2014-12-30 | 2016-07-27 | 青岛海科生物技术有限公司 | Medium with slow release effects for avermectin and preparation method thereof |
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| CN106609288A (en) * | 2015-10-21 | 2017-05-03 | 上海国佳生化工程技术研究中心有限公司 | Method used for increasing industrial avermectin B<1a> yield via optimization of fermentation medium |
| CN106609288B (en) * | 2015-10-21 | 2020-11-13 | 上海国佳生化工程技术研究中心有限公司 | Method for improving industrial abamectin B by optimizing fermentation medium1aMethod of production of |
| CN106191177A (en) * | 2016-07-25 | 2016-12-07 | 宁夏泰益欣生物科技有限公司 | A kind of culture medium utilizing streptomyces griseus fermenting and producing avilamycin |
| CN106190928A (en) * | 2016-08-31 | 2016-12-07 | 华北制药集团爱诺有限公司 | A kind of avilamycin superior strain and screening technique thereof |
| CN110408668A (en) * | 2019-08-16 | 2019-11-05 | 齐鲁制药（内蒙古）有限公司 | A kind of fermentation process for improving abamectin fermented liquid and putting tank potency |
| CN110408668B (en) * | 2019-08-16 | 2023-01-03 | 齐鲁制药（内蒙古）有限公司 | Fermentation method for improving tank-discharging titer of abamectin fermentation liquor |
| CN114381386A (en) * | 2021-11-21 | 2022-04-22 | 河北兴柏农业科技有限公司 | Culture medium for producing abamectin by fermentation |
| CN114381386B (en) * | 2021-11-21 | 2023-12-15 | 河北兴柏农业科技股份有限公司 | Culture medium for producing avermectin through fermentation |
| CN114457134A (en) * | 2022-03-17 | 2022-05-10 | 石家庄华滋生物工程有限公司 | Abamectin fermentation method based on composite organic nitrogen source |
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Application publication date: 20140625 |