CN104450823A - Method for improving fermentation yield of nemadectin - Google Patents
Method for improving fermentation yield of nemadectin Download PDFInfo
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- CN104450823A CN104450823A CN201410787858.7A CN201410787858A CN104450823A CN 104450823 A CN104450823 A CN 104450823A CN 201410787858 A CN201410787858 A CN 201410787858A CN 104450823 A CN104450823 A CN 104450823A
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Abstract
The invention discloses a method for improving fermentation yield of nemadectin. The method comprises the following steps: A. selecting an original strain; B. carrying out slant culture to the strain; C. bottling and cultivating the strain; D. cultivating the strain in a seeding tank; and E. cultivating the strain in a fermentation tank. By refilling sodium acetate for one or more times in the middle and later periods of fermentation, the method for improving the fermentation yield of the nemadectin disclosed by the invention can be used for greatly improving the yield of the nemadectin.
Description
Technical field
The present invention relates to the fermentative production of nimoctin, be specifically related to a kind of method improving nimoctin fermentation yield.
Background technology
Nimoctin is a kind of 16-membered ring macrolides microbiotic produced by streptomyces chromogenes or cyaneogriseus streptomyces fermentation, and it is mainly used in synthesizing the stronger mosictin of lipotropy.Nimoctin is mainly used in the stronger agricultural chemicals veterinary drug mosictin (Moxidectin) of composite reactive.Start to use as insect repellent for animals since 20th century of mosictin the mid-80s, nematode and ectozoa can be killed efficiently, have good security to animal, be widely used in the wide spectrum of veterinary clinic, efficient, Novel macrocyclic lactone expelling parasite microbiotic at present simultaneously.
The yielding poorly of nimoctin in prior art, can not meet industrial requirement far away.The method improving nimoctin in prior art has the bacterial strain choosing high yield, mend sugar, add amino acid or other nutritive ingredient etc., such as, application number is disclose " in the process of fermentative production nimoctin in the patent " a kind of method of fermentative production nimoctin " of 201110242857.0, when the total sugar concentration in fermented liquid is lower than 30g/L, by to fermented liquid interval or add glucide continuously, the total sugar concentration in fermented liquid is made to control to ferment at 10-40g/L ", the output of nimoctin is improved by gap or continuous benefit sugar, but its effect is still undesirable, there is glucose metabolism reptation behavior during the fermentation, and the output of glucose on nimoctin has critical impact, thus, although by mending sugar to improve nimoctin output, but there is certain limitation.
Summary of the invention
The present invention is the technical problem that output when solving the yielding poorly of nimoctin in prior art, fermentation ends has much room for improvement, and provides and utilizes a kind of method that can improve nimoctin fermentation yield.
The present invention is the technical scheme realizing the employing of its object:
Improve a method for nimoctin fermentation yield, it is characterized in that, comprise the following steps:
A, choose starting strain: choose a strain sodium acetate resistance streptomyces chromogenes mutant strain, the specific name of a described strain sodium acetate resistance streptomyces chromogenes mutant strain is streptomyces chromogenes (Streptomycescyanneorisens) DC18-01, be deposited in China Committee for Culture Collection of Microorganisms, deposit number is CGMCC No.6217, and preservation date is on June 14th, 2012;
B, bacterial strain slant culture: streptomyces chromogenes (Streptomyces cyanneorisens) DC18-01 is inoculated in slant medium, in 26-30 DEG C, cultivate 96-144h under 30%-60% humidity condition, obtain slant strains;
C, bacterial strain kind bottle are cultivated: take 2-5cm
2slant strains prepared by step B is inoculated in kind of bottle substratum, cultivates 24-30h in 26-30 DEG C, 220-240rpm concussion, obtains ripe kind liquid;
D, seed tank culture: the maturation kind liquid obtained by step C is seeded in seed tank culture base with volume ratio 0.05% inoculum size, control culture temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, and the dissolved oxygen in culturing process controls more than 25%, obtains kind of a bottle nutrient solution;
E, fermentor cultivation: the kind bottle nutrient solution obtained by step D is seeded in fermention medium with volume ratio 10% inoculum size, control fermentation jar temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, dissolved oxygen in fermented liquid controls more than 60%, glucose concn controls at 0.5-2.0g/mL, in fermented liquid, the sodium acetate solution that concentration is 10-20mg/L is added at least one times after fermentation 150h, add the sodium acetate of 1-2% volume ratio altogether, stop mending sugar at fermentation 260-280h, when glucose concn stops fermentation lower than during 0.2g/mL, the obtained fermented liquid containing nimoctin.
In described step e fermentor cultivation process, after fermentation starts, 168h fills into sodium acetate solution.
In described step e fermentor cultivation process, after fermentation starts, 168h, 216h fill into sodium acetate solution.
In described step e fermentor cultivation process, after fermentation starts, 156h, 192h and 228h fill into sodium acetate solution.
Slant culture based component described in step B and consumption (g/100mL) are: glucose 0.3-0.5, maltose 0.1-0.15, yeast extract paste or yeast powder 0.3-0.5, starch 0.4-0.6, soybean cake powder 0.4-0.6, dextrin 0.4-0.6, dipotassium hydrogen phosphate 0.01-0.03, agar 1-2.
Kind bottle medium component described in step C and consumption (g/100mL) are: glucose 0.5-2, dextrin 1-3, soybean cake powder 1-2, yeast extract paste or yeast powder 1-2, magnesium sulfate 0.05-0.2, dipotassium hydrogen phosphate 0.05-0.2.
Seed tank culture based component described in step D and consumption (g/100mL) are: glucose 2-3, soybean cake powder 1-2, yeast extract paste or yeast powder 1-2, magnesium sulfate 0.05-0.2, dipotassium hydrogen phosphate 0.05-0.2.
The composition of the fermention medium described in step e and consumption (g/100mL) are: glucose 4-6, soybean cake powder 2.5-3.5, yeast powder 0.3-0.5, magnesium sulfate 0.8-1.2, calcium carbonate 0.2-0.4.
The invention has the beneficial effects as follows: a kind of method improving nimoctin fermentation yield of the present invention, by adding sodium acetate at fermentation middle and later periods single or multiple, achieving the significantly lifting of nimoctin output.In the process of research, high and produce at need inhibiting to the synthetase series of nimoctin in the face of how overcoming sodium acetate content in fermented liquid, puzzlement is wherein unthinkable, we have passed through long-term creative research, overcome all difficulties, this process is difficult but the development of research to nimoctin of its successful is significant.
From the composition principle of nimoctin, sodium acetate is as a kind of precursor substance of nimoctin, the output of its addition to nimoctin plays critical effect, sodium acetate produces NaOH after being utilized by mycelium, fermented liquid pH value is raised, and the too high growth not only bad for thalline of fermented liquid pH value, and suppress the synthesis of nimoctin, from Fig. 1 prior art sodium acetate to when showing sodium acetate addition from 0-0.2% the research of nimoctin yield effect rule, nimoctin sustained production rises, when sodium acetate addition is more than 0.2%, cell concentration declines, the output of nimoctin also sharply declines, reason is that in fermented liquid, sodium acetate concentration raises the synthetase series generation restraining effect to nimoctin.And we are by long-term research, break the normal procedure, increase the addition of sodium acetate, originally thinking can because the sodium acetate of high addition produces restraining effect to the synthetase series of nimoctin, unexpected, the output of nimoctin does not only decline, and has had on the contrary and has significantly promoted, and overcomes the technology prejudice that prior art sodium acetate addition sharply declines more than the output of 0.2% nimoctin.
Accompanying drawing explanation
Fig. 1 is the affecting laws of sodium acetate to nimoctin output
Embodiment
The present invention is the technical problem that output when solving the yielding poorly of nimoctin in prior art, fermentation ends has much room for improvement, and provides and utilizes a kind of method that can improve nimoctin fermentation yield.Below by specific embodiment, the present invention is further illustrated.
Sodium acetate is not added in embodiment 1.1.5T ferment tank process
A, choose starting strain: choose a strain sodium acetate resistance streptomyces chromogenes mutant strain, the specific name of a described strain sodium acetate resistance streptomyces chromogenes mutant strain is streptomyces chromogenes (Streptomycescyanneorisens) DC18-01, be deposited in China Committee for Culture Collection of Microorganisms, deposit number is CGMCC No.6217, and preservation date is on June 14th, 2012;
B, bacterial strain slant culture: streptomyces chromogenes (Streptomyces cyanneorisens) DC18-01 is inoculated in slant medium, in 26-30 DEG C, cultivate 96-144h under 30%-60% humidity condition, obtain slant strains;
C, bacterial strain kind bottle are cultivated: take 2-5cm
2slant strains prepared by step B is inoculated in kind of bottle substratum, cultivates 24-30h in 26-30 DEG C, 220-240rpm concussion, obtains ripe kind liquid;
D, seed tank culture: the maturation kind liquid obtained by step C is seeded in seed tank culture base with volume ratio 0.05% inoculum size, control culture temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, and the dissolved oxygen in culturing process controls more than 25%, obtains kind of a bottle nutrient solution;
E, fermentor cultivation: configure 0.9T fermention medium in 1.5T fermentor tank, 27.5-28.5 DEG C is cooled to after 120 DEG C of sterilizing 20min, the kind bottle nutrient solution obtained by step D is seeded in fermention medium with volume ratio 10% inoculum size, control fermentation jar temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, in fermenting process, dissolved oxygen controls more than 60%, glucose concn controls at 0.5-2.0g/mL, stop mending sugar at fermentation 260h, when glucose concn stops fermentation lower than during 0.2g/mL, the obtained fermented liquid containing nimoctin, measuring last fermentation yield is 2560mg/L.
In embodiment 2.1.5T ferment tank process, fermentation starts rear 168h and adds sodium acetate
A, choose starting strain: choose a strain sodium acetate resistance streptomyces chromogenes mutant strain, the specific name of a described strain sodium acetate resistance streptomyces chromogenes mutant strain is streptomyces chromogenes (Streptomycescyanneorisens) DC18-01, be deposited in China Committee for Culture Collection of Microorganisms, deposit number is CGMCC No.6217, and preservation date is on June 14th, 2012;
B, bacterial strain slant culture: streptomyces chromogenes (Streptomyces cyanneorisens) DC18-01 is inoculated in slant medium, in 26-30 DEG C, cultivate 96-144h under 30%-60% humidity condition, obtain slant strains;
C, bacterial strain kind bottle are cultivated: take 2-5cm
2slant strains prepared by step B is inoculated in kind of bottle substratum, cultivates 24-30h in 26-30 DEG C, 220-240rpm concussion, obtains ripe kind liquid;
D, seed tank culture: the maturation kind liquid obtained by step C is seeded in seed tank culture base with volume ratio 0.05% inoculum size, control culture temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, and the dissolved oxygen in culturing process controls more than 25%, obtains kind of a bottle nutrient solution;
E, fermentor cultivation: configure 0.9T fermention medium in 1.5T fermentor tank, 27.5-28.5 DEG C is cooled to after 120 DEG C of sterilizing 20min, the kind bottle nutrient solution obtained by step D is seeded in fermention medium with volume ratio 10% inoculum size, control fermentation jar temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, in fermenting process, dissolved oxygen controls more than 60%, glucose concn controls at 0.5-2.0g/mL, after fermentation starts, 168h fills into the sodium acetate solution that 10L concentration is 10mg/L, stop mending sugar at fermentation 260h, when glucose concn stops fermentation lower than during 0.2g/mL, the obtained fermented liquid containing nimoctin, measuring last fermentation yield is 2980mg/L.
In embodiment 3.12T ferment tank process, fermentation starts rear 168h, 216h and adds sodium acetate A, chooses starting strain: choose a strain sodium acetate resistance streptomyces chromogenes mutant strain, the specific name of a described strain sodium acetate resistance streptomyces chromogenes mutant strain is streptomyces chromogenes (Streptomycescyanneorisens) DC18-01, be deposited in China Committee for Culture Collection of Microorganisms, deposit number is CGMCC No.6217, and preservation date is on June 14th, 2012;
B, bacterial strain slant culture: streptomyces chromogenes (Streptomyces cyanneorisens) DC18-01 is inoculated in slant medium, in 26-30 DEG C, cultivate 96-144h under 30%-60% humidity condition, obtain slant strains;
C, bacterial strain kind bottle are cultivated: take 2-5cm
2slant strains prepared by step B is inoculated in kind of bottle substratum, cultivates 24-30h in 26-30 DEG C, 220-240rpm concussion, obtains ripe kind liquid;
D, seed tank culture: the maturation kind liquid obtained by step C is seeded in seed tank culture base with volume ratio 0.05% inoculum size, control culture temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, and the dissolved oxygen in culturing process controls more than 25%, obtains kind of a bottle nutrient solution;
E, fermentor cultivation: configure 7.5T fermention medium in 12T fermentor tank, 27.5-28.5 DEG C is cooled to after 120 DEG C of sterilizing 20min, the kind bottle nutrient solution obtained by step D is seeded in fermention medium with volume ratio 10% inoculum size, control fermentation jar temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, in fermenting process, dissolved oxygen controls more than 60%, glucose concn controls at 0.5-2.0g/mL, the sodium acetate solution that 50L and 70L concentration is 15mg/L is filled into respectively during 168h and 216h after fermentation starts, stop mending sugar at fermentation 276h, when glucose concn stops fermentation lower than during 0.2g/mL, the obtained fermented liquid containing nimoctin, measuring last fermentation yield is 3343mg/L.
In embodiment 4.12T ferment tank process, fermentation starts rear 156h, 192h and 228h and adds sodium acetate
A, choose starting strain: choose a strain sodium acetate resistance streptomyces chromogenes mutant strain, the specific name of a described strain sodium acetate resistance streptomyces chromogenes mutant strain is streptomyces chromogenes (Streptomycescyanneorisens) DC18-01, be deposited in China Committee for Culture Collection of Microorganisms, deposit number is CGMCC No.6217, and preservation date is on June 14th, 2012;
B, bacterial strain slant culture: streptomyces chromogenes (Streptomyces cyanneorisens) DC18-01 is inoculated in slant medium, in 26-30 DEG C, cultivate 96-144h under 30%-60% humidity condition, obtain slant strains;
C, bacterial strain kind bottle are cultivated: take 2-5cm
2slant strains prepared by step B is inoculated in kind of bottle substratum, cultivates 24-30h in 26-30 DEG C, 220-240rpm concussion, obtains ripe kind liquid;
D, seed tank culture: the maturation kind liquid obtained by step C is seeded in seed tank culture base with volume ratio 0.05% inoculum size, control culture temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, and the dissolved oxygen in culturing process controls more than 25%, obtains kind of a bottle nutrient solution;
E, fermentor cultivation: configure 7.5T fermention medium in 12T fermentor tank, 27.5-28.5 DEG C is cooled to after 120 DEG C of sterilizing 20min, the kind bottle nutrient solution obtained by step D is seeded in fermention medium with volume ratio 10% inoculum size, control fermentation jar temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, in fermenting process, dissolved oxygen controls more than 60%, glucose concn controls at 0.5-2.0g/mL, 156h after fermentation starts, 60L is filled into respectively during 192h and 228h, 40L and 60L concentration is the sodium acetate solution of 20mg/L, stop mending sugar at fermentation 264h, when glucose concn stops fermentation lower than during 0.2g/mL, the obtained fermented liquid containing nimoctin, measuring last fermentation yield is 3690mg/L.
Claims (8)
1. improve a method for nimoctin fermentation yield, it is characterized in that, comprise the following steps:
A, choose starting strain: choose a strain sodium acetate resistance streptomyces chromogenes mutant strain, the specific name of a described strain sodium acetate resistance streptomyces chromogenes mutant strain is streptomyces chromogenes (Streptomycescyanneorisens) DC18-01, be deposited in China Committee for Culture Collection of Microorganisms, deposit number is CGMCC No.6217, and preservation date is on June 14th, 2012;
B, bacterial strain slant culture: streptomyces chromogenes (Streptomyces cyanneorisens) DC18-01 is inoculated in slant medium, in 26-30 DEG C, cultivate 96-144h under 30%-60% humidity condition, obtain slant strains;
C, bacterial strain kind bottle are cultivated: take slant strains prepared by 2-5cm2 step B and be inoculated in kind of bottle substratum, cultivate 24-30h in 26-30 DEG C, 220-240rpm concussion, obtain ripely planting liquid;
D, seed tank culture: the maturation kind liquid obtained by step C is seeded in seed tank culture base with volume ratio 0.05% inoculum size, control culture temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, and the dissolved oxygen in culturing process controls more than 25%, obtains kind of a bottle nutrient solution;
E, fermentor cultivation: the kind bottle nutrient solution obtained by step D is seeded in fermention medium with volume ratio 10% inoculum size, control fermentation jar temperature is 27.5-28.5 DEG C, tank pressure is 0.03-0.05MPa, dissolved oxygen in fermented liquid controls more than 60%, glucose concn controls at 0.5-2.0g/mL, in fermented liquid, the sodium acetate solution that concentration is 10-20mg/L is added at least one times after fermentation 150h, add the sodium acetate of 1-2% volume ratio altogether, stop mending sugar at fermentation 260-280h, when glucose concn stops fermentation lower than during 0.2g/mL, the obtained fermented liquid containing nimoctin.
2. a kind of method improving nimoctin fermentation yield according to claim 1, is characterized in that: in described step e fermentor cultivation process, after fermentation starts, 168h fills into sodium acetate solution.
3. a kind of method improving nimoctin fermentation yield according to claim 1, is characterized in that: in described step e fermentor cultivation process, after fermentation starts, 168h, 216h fill into sodium acetate solution.
4. a kind of method improving nimoctin fermentation yield according to claim 1, is characterized in that: in described step e fermentor cultivation process, after fermentation starts, 156h, 192h and 228h fill into sodium acetate solution.
5. a kind of method improving nimoctin fermentation yield according to claim 1, is characterized in that: the slant culture based component described in step B and consumption (g/100mL) are: glucose 0.3-0.5, maltose 0.1-0.15, yeast extract paste or yeast powder 0.3-0.5, starch 0.4-0.6, soybean cake powder 0.4-0.6, dextrin 0.4-0.6, dipotassium hydrogen phosphate 0.01-0.03, agar 1-2.
6. a kind of method improving nimoctin fermentation yield according to claim 1, is characterized in that: the kind bottle medium component described in step C and consumption (g/100mL) are: glucose 0.5-2, dextrin 1-3, soybean cake powder 1-2, yeast extract paste or yeast powder 1-2, magnesium sulfate 0.05-0.2, dipotassium hydrogen phosphate 0.05-0.2.
7. a kind of method improving nimoctin fermentation yield according to claim 1, is characterized in that: the seed tank culture based component described in step D and consumption (g/100mL) are: glucose 2-3, soybean cake powder 1-2, yeast extract paste or yeast powder 1-2, magnesium sulfate 0.05-0.2, dipotassium hydrogen phosphate 0.05-0.2.
8. a kind of method improving nimoctin fermentation yield according to claim 1, it is characterized in that: the composition of the fermention medium described in step e and consumption (g/100mL) are: glucose 4-6, soybean cake powder 2.5-3.5, yeast powder 0.3-0.5, magnesium sulfate 0.8-1.2, calcium carbonate 0.2-0.4.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108774626A (en) * | 2018-02-01 | 2018-11-09 | 上海莫息生物科技有限公司 | A kind of method and bacterial strain producing nimoctin |
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| CN101554121A (en) * | 2009-05-15 | 2009-10-14 | 江苏省农业科学院 | Fermentation method for high cordyceps militaris biomass and high cordycepin content |
| CN101899410A (en) * | 2010-07-13 | 2010-12-01 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces parvus and application thereof for preparing daptomycin |
| CN104109648A (en) * | 2014-07-10 | 2014-10-22 | 河北圣雪大成制药有限责任公司 | Streptomyces chromogenes of high-yield nemadectin and screening method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101554121A (en) * | 2009-05-15 | 2009-10-14 | 江苏省农业科学院 | Fermentation method for high cordyceps militaris biomass and high cordycepin content |
| CN101899410A (en) * | 2010-07-13 | 2010-12-01 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces parvus and application thereof for preparing daptomycin |
| CN104109648A (en) * | 2014-07-10 | 2014-10-22 | 河北圣雪大成制药有限责任公司 | Streptomyces chromogenes of high-yield nemadectin and screening method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108774626A (en) * | 2018-02-01 | 2018-11-09 | 上海莫息生物科技有限公司 | A kind of method and bacterial strain producing nimoctin |
| CN108774626B (en) * | 2018-02-01 | 2020-11-17 | 上海莫息生物科技有限公司 | Method and strain for producing nemadectin |
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