CN109207536A - A method of improving mibemycin A3 content in mibemycin tunning - Google Patents
A method of improving mibemycin A3 content in mibemycin tunning Download PDFInfo
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Abstract
The invention discloses a kind of methods of mibemycin A3 content in raising mibemycin tunning, it include: slant strains culture, liquid seeds culture, liquid fermentation and culture and etc., during fermented and cultured into fermentation liquid flow feeding sucrose solution, especially after fermentation to 48h, pass through the sodium acetate solution for intermittently or serially filling into the precursor 0.05% of specificity daily into fermentation liquid, the content of A3 in tunning is significantly improved, while also improving the fermentation yield of mibemycin.
Description
Technical field
The present invention relates to a kind of zymotechniques of mibemycin, belong to biopharmaceutical technology, are related specifically to use
The method of microbial liquid submerged fermentation technology production mibemycin.
Background technique
Mibemycin (Milbemycin) is the mixture of a series of ten hexa-atomic macrolide antibiotics, by water suction chain
Mould (Streptomyces hygroscopicus) fermented generation.It is most of to agriculture in 20 various ingredients of mibemycin
Industry pest has wide spectrum prevention and treatment activity, such as aphid, mite, malacosoma neustria, Enterozoa and other damages to crops and domestic animal
Helminth, especially have higher control efficiency to various mite class.Mibemycin has, dosage strong to pest effect
It is few, it is free from environmental pollution to safety of human and livestock, worm is only murdered, is a kind of the characteristics of not killing pest natural enemy, be not easy to produce drug resistance
The biological insecticides of very promising wide spectrum, efficient, novel no cross tolerance.
In mibemycin medicine series, since the bioactivity of mibemycin A3, A4 are stronger, yield is higher, institute
It is all carried out around mibemycin A3, A4 with the commercialization of mibemycin product.In addition to mixing for mibemycin A3, A4
It closes object to be used as other than acaricide extensively, the semi-synthetic oximated product of mibemycin A3, A4, milbemycin oxime (Milbemycin
Oxime, milbemycin oxime, No. CAS: 129496-10-2), it is a kind of novel macrolide anthelmintic, to internal epizoite
Worm have it is good kill effect, have good effect to control and the most of common pet parasitic disease of prevention.Mir
Shellfish oxime especially with its efficient feature of low toxicity be acknowledged as a kind of wide spectrum, efficiently, the anthelmintic of safety, especially to some
It is only safer to the dog that ivermectin is sensitive.
Milbemycin oxime is mixed in a certain proportion and is formed by milbemycin oxime A3 and milbemycin oxime A4, it is desirable that milbemycin oxime A3+A4
Content be no less than 95%, wherein the ratio of A3 and A4 be (20 ± 5): (80 ± 5).The structural formula of milbemycin oxime A3, A4 are such as
Shown in following formula:
Milbemycin oxime A3 (Milbemycin Oxime A3): R=CH3
Milbemycin oxime A4 (Milbemycin Oxime A4): R=CH2CH3
It is industrial mainly to use streptomyces hygroscopicus (Streptomyces hygroscopicus) fermenting and producing mibemycin,
During the fermentation, mibemycin A3, A4 is by thallus while to generate, and is present in fermentation liquid as a mixture.
Since the structure and property of A3, A4 are all closely similar, only substituent group on C-25 is different, is methyl and ethyl respectively, from
It is extremely difficult that A3, A4 one-component are isolated in fermentation liquid, so A3, A4 are all generally industrially considered as same component at present,
It extracts and separates from fermentation liquid together, after a series of semi-synthetic step such as oxidation, oximate, finally by separation
Purifying obtains milbemycin oxime finished product.This requires from the beginning, the ratio of mibemycin A3 and A4 in fermentation liquid are with regard to necessary
Precisely in (20 ± 5): in (80 ± 5) range, the ratio of the milbemycin oxime A3 and A4 that finally obtain in this way can be conformed to
It asks.
A problem is encountered in the fermenting and producing of country's milbemycin oxime at present, every batch of fermentation continues with course of fermentation,
Mibemycin totality fermentation unit sustainable growth, but the fermentation unit growth for the middle and later periods A3 that ferments is relatively slow, leads to A3/A4
Ratio also constantly declining, from the 1:3 or so of ferment middle, drop to 1:7 or so before tank to putting, and final products are wanted
Seeking Truth 1:4 or so, so often have to just puts tank in advance when fermentation unit is also lower.This encountered in production is difficult
Topic is just badly in need of finding a kind of method, is capable of the yield of the raising A3 of specificity, the yield without influencing A4 in this way can not only be from
The yield of mibemycin is improved on the whole, but also can substantially reduce the difficulty of subsequent semi-synthetic production milbemycin oxime,
Production cost is greatly lowered.
According to the parsing of the 400MHz 13C nuclear magnetic resoance spectrum to mibemycin α 2, α 4 and D component, glycocide skeleton
On carbon be to be derived from 7 acetates and 5 propionates, it is identical with the composition of avermectin glycocide skeleton.Institute
According to avermectin route of synthesis (H Ikeda, S Omura.Control of avermectin biosynthesis in
Streptomyces avermitilis for the selective production of a useful component.
" Cheminform ", 1996,27 (1): 549-62) infer, methyl substituents on the position C-25 of mibemycin A3 component
It is to be obtained by acetate metabolism derivative, the ethyl substituent on the position C-25 of A4 component is obtained by propionate metabolic derivatives.
Summary of the invention
The present invention is on the basis of existing fermenting and producing mibemycin technology, by adding in right amount during the fermentation
Specific precursor substance sodium acetate solves the slow technical problem of fermentation middle and later periods mibemycin A3 increase of production, provides
It is a kind of can specificity increase the process of mibemycin A3 fermentation yield, produced to improve mibemycin fermentation
The ratio of A3 in object.
Through research, the inventor has found that mibemycin is the secondary metabolite of streptomyces hygroscopicus, mibemycin
Specific ligands in A3 structure come from acetate, and opposite, and the specific ligands in mibemycin A4 structure come
From in propionate, illustrate that acetate is likely to be the specific precursor substance of mibemycin A3 biosynthesis, in fermentation liquid
The synthesis that acetate is likely to increase Mir shellfish A3 additionally is added, the synthesis without influencing A4 thus can be by fermenting
The method for adding sodium acetate in liquid in right amount, increases the yield of A3, without increasing A4 yield, so that reaching improves A3/A4 ratio
Purpose.The present inventor experiments prove that, mibemycin can be improved by a small margin by adding sodium acetate in right amount in fermentating formula
The yield of A3, while the yield of mibemycin A4 is not influenced substantially.
On the above Research foundation, thus it is speculated that adding acetate in right amount in the fermentation medium of mibemycin can save
Time and nutrition needed for thallus itself synthesis of acetic acid root, to improve the yield of A3, but are found through experiments that, using sending out
The measure of acetate is added in ferment basal medium, the effect for improving A3 yield is not obvious, and after fermentation period 48h, just
Grade metabolism is basically completed, and cometabolism adds acetate then when occupying main status have preferable effect, especially by several times
It adds daily or effect that continuous flow adds is more excellent.
On the basis of the above research, the aobvious of mibemycin A3 fermentation yield and ratio is realized by following technological means
It writes and improves:
(1) slant strains culture: streptomyces hygroscopicus (Streptomyces hygroscopicus) strain is inoculated in tiltedly
Face culture medium is cultivated 10-12 days under the conditions of 27-29 DEG C of temperature, humidity 20-60%, obtains mature slant strains.Inclined-plane training
Supporting based formulas is sucrose 0.4%, skimmed milk power 0.1%, yeast powder 0.2%, peptone 0.1%, agar 2.0%.
(2) liquid seeds culture: scraping appropriate spore inoculating in liquid seed culture medium from mature slant strains,
It 27-29 DEG C of temperature, cultivates 40-48 hours under the conditions of speed of agitator 100-500rpm, obtains mature seed liquor.Liquid seeds training
Supporting based formulas is sucrose 2%, skimmed milk power 0.1%, soybean cake powder 0.5%, peptone 0.5%, calcium carbonate 0.1%.
(3) liquid fermentation and culture: mature seed liquor is seeded in fermentation medium according to the inoculum concentration of 4-12%,
27-29 DEG C of temperature, after fermentation to 120h, tank top fermentation is measured by sampling in fermented and cultured under conditions of speed of agitator 100-600rpm
Sucrose concentration in liquid starts to mend sucrose solution, maintains sucrose in tank top fermentation liquid when sucrose concentration is reduced to 2.0% or less
Concentration between 1.5-2.5%, put 12h before tank and stop mending sugar.Terminate fermentation after fermentation 240-288h, obtains containing Mir shellfish
The fermentation liquid of mycin.Liquid fermentation medium formula are as follows: sucrose 8%, skimmed milk power 1.0%, soybean cake powder 1.0%, peptone
1.0%, dipotassium hydrogen phosphate 0.5%, ferrous sulfate 0.1%, zinc sulfate 0.01%, copper sulphate 0.05%, sodium molybdate 0.05%, carbon
Sour calcium 0.5%.
(4) precursor is mended in fermentation: after fermentation to 48h, filling into 0.05% sodium acetate in fermentation liquid daily, sodium acetate is matched
At the solution of 5% concentration, after sterilizing, sodium acetate solution is filled into fermentor according to the 1% of fermentating liquid volume daily.
Technical solution of the present invention is as follows:
A method of mibemycin A3 content in mibemycin tunning being improved, including in fermentation process, to
A certain amount of acetate is filled into fermentation liquid.
Further, the acetate includes one or more of sodium acetate, potassium acetate, ammonium acetate, zinc acetate etc., excellent
It is selected as sodium acetate.
Further, the acetate adds as a solution；The concentration of acetate solution used is generally 0.5%-
20% (mass fraction)；The preferably sodium acetate solution of concentration 5%.
Further, when 48h is arrived in fermentation, it is molten that a certain amount of acetate (preferably sodium acetate) is filled into fermentation liquid daily
Liquid, until fermentation ends.
Further, the amount of the acetate filled into daily into fermentation liquid (refers to the acetate filled by 0.01-0.1%
Mass fraction in fermentation liquid), preferably 0.05%.
Further, the mode that acetate is filled into fermentation liquid is the side for intermittently filling into or being added once a day with continuous flow
Formula fills into.
Further, when total sugar concentration is reduced to 2.0% (mass fraction, similarly hereinafter) below in fermentation liquid, start to add
Sucrose solution maintains the concentration of total reducing sugar in fermentation liquid between 1.5-2.5%.
Further, after fermentation is to 120-150h, total sugar concentration in fermentation liquid is detected, when total sugar concentration drops in fermentation liquid
When as low as 2.0% or less, starts to add sucrose solution, maintain the concentration of total reducing sugar in fermentation liquid between 1.5-2.5%.
Further, sucrose is added in 12h stopping before putting tank (terminating fermentation).
Further, terminate fermentation after fermentation 240-288h.
Further, fermentation condition includes: 27-29 DEG C of temperature, speed of agitator 100-600rpm, ventilatory capacity 0.4-
1.2vvm controls oxygen dissolving value (DO) >=30% on fermentor.
Generally, the microorganism that this field routine production mibemycin can be used is fermented, such as streptomyces hygroscopicus
(Streptomyces hygroscopicus)。
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined with each other each preferably to get the present invention
Example.
Specifically, in above-mentioned raising mibemycin tunning mibemycin A3 ratio method, including walk as follows
It is rapid:
1) slant strains culture
Streptomyces hygroscopicus (Streptomyces hygroscopicus) strain is inoculated in slant medium, in temperature
It 27-29 DEG C, cultivates 10-12 days under the conditions of humidity 20-60%, obtains mature slant strains；
Inclined-plane culture based formulas are as follows: sucrose 0.4%, skimmed milk power 0.1%, yeast powder 0.2%, peptone 0.1%, agar
2.0%.
2) liquid seeds culture
Appropriate spore inoculating is scraped in liquid seed culture medium from mature slant strains, at 27-29 DEG C of temperature, stirring
Ventilation culture 40-48 hours, obtain mature seed liquor under the conditions of revolving speed 100-500rpm；
Alternatively, further mature seed liquor is carried out again once to expand culture by identical method；
Liquid seed culture medium formula are as follows: sucrose 2%, skimmed milk power 0.1%, soybean cake powder 0.5%, peptone 0.5%,
Calcium carbonate 0.1%；
Generally, the inoculum concentration of seed liquor is 1%-8%.
3) liquid fermentation and culture
Mature seed liquor is seeded in fermentation medium according to the inoculum concentration of 4-12%, at 27-29 DEG C of temperature, stirring
Aerobic fementation culture under the conditions of revolving speed 100-600rpm, ventilatory capacity 0.4-1.2vvm, control fermentor on oxygen dissolving value (DO) >=
30%；
Liquid fermentation medium formula are as follows: sucrose 8%, skimmed milk power 1.0%, soybean cake powder 1.0%, peptone 1.0%,
Dipotassium hydrogen phosphate 0.5%, ferrous sulfate 0.1%, zinc sulfate 0.01%, copper sulphate 0.05%, sodium molybdate 0.05%, calcium carbonate
0.5%；
4) when 48h is arrived in fermentation, the sodium acetate solution that concentration is 5% is filled into fermentation liquid daily, fills into sodium acetate daily
Amount be 0.01-0.1% (referring to mass fraction of the sodium acetate filled into fermentation liquid), preferably 0.05%, until fermentation knot
Beam；
The mode for filling into sodium acetate solution is the mode for intermittently filling into or being added once a day with continuous flow；
5) after fermentation to 120-150h, total sugar concentration in sample detection fermentation liquid, when total sugar concentration is reduced to 2.0% or less
When, start to mend sucrose solution, maintain the concentration of total reducing sugar in fermentation liquid between 1.5-2.5%, 12h stops mending sugar, hair before putting tank
Terminate fermentation after ferment 240-288h.
The invention also includes the tunnings obtained by the above method.
Beneficial effects of the present invention:
The present invention provides the methods that a species specificity improves mibemycin A3 fermentation yield, and to mibemycin A4
Yield substantially without influence, so improving ratio of the mibemycin A3 component in final product.Technique side of the invention
Method solves the problems, such as that traditional mibemycin fermentation middle and later periods mibemycin A3 fermentation yield increasess slowly, so that Mir shellfish
Mycin A3 and A4 can grow simultaneously, and the ratio of mibemycin A3 in fermentation later stage fermentation product be improved, to extend
Fermentation period significantly improves the fermentation yield of mibemycin.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..It is not specified in embodiment specific
Technology or conditions person, described technology or conditions according to the literature in the art, or carried out according to product description.It is used
Production firm person is not specified in reagent or instrument, is the conventional products that can be commercially available by regular distributor.
Slant strains culture used below: the strain of streptomyces hygroscopicus (Streptomyces hygroscopicus) is connect
Kind is cultivated 10-12 days under the conditions of 27-29 DEG C of temperature, humidity 20-60% in slant medium, obtains mature slant strains.
Inclined-plane culture based formulas are as follows: sucrose 0.4%, skimmed milk power 0.1%, yeast powder 0.2%, peptone 0.1%, agar 2.0%.
Liquid seed culture medium formula used below are as follows: sucrose 2%, skimmed milk power 0.1%, soybean cake powder 0.5%, albumen
Peptone 0.5%, calcium carbonate 0.1%.
Liquid fermentation medium formula used below are as follows: sucrose 8%, skimmed milk power 1.0%, soybean cake powder 1.0%, albumen
Peptone 1.0%, dipotassium hydrogen phosphate 0.5%, ferrous sulfate 0.1%, zinc sulfate 0.01%, copper sulphate 0.05%, sodium molybdate 0.05%,
Calcium carbonate 0.5%.
Embodiment 1
(1) strain inclined plane grown is taken, the spore inoculating scraped on the inclined-plane about 0.5cm × 0.5cm shakes to 500ml seed
In bottle, seed flask loading amount is 50ml, inoculated seed flask 28 DEG C of culture 48h on the shaking table that revolving speed is 180rpm.
(2) shake-flask seed will have been cultivated to be seeded on 5L glass fermentation tank according to 8% inoculum concentration, the loading amount of fermentor is
2.5L, fermentation tank culture condition are 28 DEG C of tank temperature, ventilatory capacity 0.8vvm, speed of agitator 200rpm, pass through raising in fermentation process
Speed of agitator controls oxygen dissolving value (DO) >=30% on fermentor.
(3) when fermented and cultured is to 48h, start the sodium acetate solution for mending 5% concentration sterilize, daily benefit once, every time
25ml is mended, i.e., respectively fills into a sodium acetate when fermentation is to 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h.
(4) when fermented and cultured is to 150h, starts stream plus mend sucrose solution, pass through to adjust and mend sugarcane in sugared speed control fermentation liquid
Sugared concentration stops when 228h mending sugar between 1.5-2.5%.
(5) fermented and cultured to 240h when terminate to cultivate, take fermentation liquid to detect, the content of total mibemycin in fermentation liquid
For 1.88g/L, wherein mibemycin A3 content is 0.37g/L, A3:A4=1:4.08.
The fermentation liquid of sodium acetate is not added with same batch as control, Mir shellfish total in fermentation liquid is mould when cultivating to 240h
The content of element is 1.79g/L, and wherein the content of mibemycin A3 is 0.25g/L, A3:A4=1:6.16.
Illustrate that this batch mends the zymotechnique of sodium acetate and can be improved the yield about 48% of mibemycin A3, and by A3 with
The ratio of A4 improves 1:4 or so from 1:6 or so, has reached the requirement of final products milbemycin oxime, has greatly reduced milbemycin oxime
Production cost.
Embodiment 2
(1) strain inclined plane grown is taken, the spore inoculating scraped on the inclined-plane about 0.5cm × 0.5cm shakes to 500ml seed
In bottle, seed flask loading amount is 50ml, inoculated seed flask 28 DEG C of culture 48h on the shaking table that revolving speed is 180rpm.
(2) shake-flask seed will have been cultivated to be seeded on 10L stainless steel seeding tank according to 1% inoculum concentration, the dress of seeding tank
Amount is 5.0L, and seed tank culture condition is 28 DEG C of tank temperature, ventilatory capacity 1.0vvm, speed of agitator 100rpm, is passed through in incubation
Speed of agitator is improved, oxygen dissolving value (DO) >=30% on seeding tank is controlled, the seed tank culture time is for 24 hours.
(3) seeding tank seed liquor will have been cultivated to be seeded on 50L fermentor according to 8% inoculum concentration, the loading amount of fermentor
For 30L, fermentation tank culture condition is 28 DEG C of tank temperature, ventilatory capacity 0.8vvm, speed of agitator 100rpm, passes through raising in incubation
Speed of agitator controls oxygen dissolving value (DO) >=30% on fermentor.
(4) when fermented and cultured is to 48h, start the sodium acetate solution for mending 5% concentration to have sterilized, at the uniform velocity flow feeding
Mode, the amount of filling into are 300ml/ days, stop benefit sodium acetate when 288h.
(5) when fermented and cultured is to 150h, starts stream plus mend sucrose solution, pass through to adjust and mend sugarcane in sugared speed control fermentation liquid
Sugared concentration stops when 276h mending sugar between 1.5-2.5%.
(6) fermented and cultured to 288h when terminate to cultivate, take fermentation liquid to detect, the content of mibemycin is in fermentation liquid
2.17g/L, wherein mibemycin A3 content is 0.42g/L, A3:A4=1:4.17.
The fermentation liquid of sodium acetate is not added with same batch as control, Mir shellfish total in fermentation liquid is mould when cultivating to 288h
The content of element is 2.03g/L, and wherein the content of mibemycin A3 is 0.26g/L, A3:A4=1:6.81.
Show that this batch mends the zymotechnique of sodium acetate and can be improved the yield about 62% of mibemycin A3, and by A3 with
The ratio of A4 improves 1:4 or so from 1:7 or so, has also reached the requirement of final products milbemycin oxime, has no longer needed to isolate A3, A4
One-component, greatly reduce the production difficulty and production cost of milbemycin oxime.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. a kind of method for improving mibemycin A3 content in mibemycin tunning, including in fermentation process, Xiang Fa
A certain amount of acetate is filled into zymotic fluid.
2. the method according to claim 1, wherein the acetate include sodium acetate, potassium acetate, ammonium acetate,
One or more of zinc acetate；
Preferably, the acetate adds as a solution；It is preferred that the concentration of acetate solution used is 0.5%-20%, more
The preferably sodium acetate solution of concentration 5%.
3. method according to claim 1 or 2, which is characterized in that when 48h is arrived in fermentation, fill into one in fermentation liquid daily
Quantitative acetate, until fermentation ends；
And/or the amount of the acetate filled into daily into fermentation liquid be 0.01-0.1%, preferably 0.05%.
4. method according to claim 1-3, which is characterized in that the mode for filling into acetate into fermentation liquid is
It intermittently fills into or is filled into the mode that continuous flow adds once a day.
5. method according to claim 1-4, which is characterized in that when total sugar concentration is reduced in fermentation liquid
When 2.0% or less, starts to add sucrose solution, maintain the concentration of total reducing sugar in fermentation liquid between 1.5-2.5%；
Preferably, after fermentation is to 120-150h, after fermentation is to 120-150h, total sugar concentration in fermentation liquid is detected, fermentation is worked as
When total sugar concentration is reduced to 2.0% or less in liquid, starts to add sucrose solution, maintain the concentration of total reducing sugar in fermentation liquid in 1.5-
Between 2.5%；
It is highly preferred that terminating 12h stopping before fermenting adds sucrose.
6. method according to claim 1-5, which is characterized in that fermentation condition includes: 27-29 DEG C of temperature, is stirred
Revolving speed 100-600rpm, ventilatory capacity 0.4-1.2vvm are mixed, oxygen dissolving value (DO) >=30% on fermentor is controlled；And/or fermentation 240-
Terminate fermentation after 288h.
7. method according to claim 1-6, which is characterized in that fermenting microbe used is streptomyces hygroscopicus
(Streptomyces hygroscopicus)。
8. any one of -7 the method according to claim 1, which comprises the steps of:
1) slant strains culture
Streptomyces hygroscopicus (Streptomyces hygroscopicus) strain is inoculated in slant medium, in temperature 27-29
DEG C, it is cultivated 10-12 days under the conditions of humidity 20-60%, obtains mature slant strains；
Inclined-plane culture based formulas used are as follows: sucrose 0.4%, skimmed milk power 0.1%, yeast powder 0.2%, peptone 0.1%, agar
2.0%；
2) liquid seeds culture
Appropriate spore inoculating is scraped in liquid seed culture medium from mature slant strains, at 27-29 DEG C of temperature, speed of agitator
Ventilation culture 40-48 hours, obtain mature seed liquor under the conditions of 100-500rpm；Alternatively, further by mature seed liquor
It carries out once expanding culture again by identical method；
Liquid seed culture medium formula used are as follows: sucrose 2%, skimmed milk power 0.1%, soybean cake powder 0.5%, peptone 0.5%,
Calcium carbonate 0.1%；
3) liquid fermentation and culture
Mature seed liquor is seeded in fermentation medium according to the inoculum concentration of 4-12%, at 27-29 DEG C of temperature, speed of agitator
Aerobic fementation culture under the conditions of 100-600rpm, ventilatory capacity 0.4-1.2vvm control oxygen dissolving value >=30% on fermentor；
Liquid fermentation medium formula used are as follows: sucrose 8%, skimmed milk power 1.0%, soybean cake powder 1.0%, peptone 1.0%,
Dipotassium hydrogen phosphate 0.5%, ferrous sulfate 0.1%, zinc sulfate 0.01%, copper sulphate 0.05%, sodium molybdate 0.05%, calcium carbonate
0.5%；
4) when 48h is arrived in fermentation, the sodium acetate solution that concentration is 5% is filled into fermentation liquid daily, fills into the amount of sodium acetate daily
Mass fraction in fermentation liquid is 0.05%, until fermentation ends；
The mode for filling into sodium acetate solution is the mode for intermittently filling into or being added once a day with continuous flow；
5) after fermentation to 120-150h, total sugar concentration in sample detection fermentation liquid, when total sugar concentration is reduced to 2.0% or less,
Start to mend sucrose solution, maintain the concentration of total reducing sugar in fermentation liquid between 1.5-2.5%, 12h stops mending sugar, fermentation before putting tank
Terminate fermentation after 240-288h.
9. tunning obtained by any one of claim 1-8 the method.
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|---|---|---|---|---|
| CN112358979A (en) * | 2020-10-16 | 2021-02-12 | 内蒙古拜克生物有限公司 | Method for producing milbemycins through microbial fermentation |
| CN117568205A (en) * | 2023-10-12 | 2024-02-20 | 湖北宏中药业股份有限公司 | Milbemycin high-yield strain and application thereof |
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| CN112358979A (en) * | 2020-10-16 | 2021-02-12 | 内蒙古拜克生物有限公司 | Method for producing milbemycins through microbial fermentation |
| CN112358979B (en) * | 2020-10-16 | 2021-11-02 | 内蒙古拜克生物有限公司 | Method for producing milbemycins through microbial fermentation |
| CN117568205A (en) * | 2023-10-12 | 2024-02-20 | 湖北宏中药业股份有限公司 | Milbemycin high-yield strain and application thereof |
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