CN105087711B - A kind of streptomycete fermentation production mibemycin fermentation medium and fermentation culture method - Google Patents
A kind of streptomycete fermentation production mibemycin fermentation medium and fermentation culture method Download PDFInfo
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Abstract
The invention discloses a kind of streptomycete fermentations to generate mibemycin fermentation medium and fermentation culture method, and fermentation medium composition is：Sucrose 11.5 12.5%, cottonseed meal 0.8 1.2%, soybean cake powder 0.8 1.2%, skimmed milk power 0.8 1.2%, maltose 1.8 2.2%, dipotassium hydrogen phosphate 0.1 0.15%, ferrous sulfate 0.01 0.015%, zeolite powder 0.38 0.42%, sodium molybdate 0.004 0.006%, copper sulphate 0.004 0.006%, calcium carbonate 0.28 0.32%, defomaing agent GPE 0.03 0.05%, water surplus.The present invention improves the fermentation level of mibemycin by the improvement of culture medium and zymotechnique, significantly improves the yield of mibemycin, reduces production cost, simple and practicable, is suitble to industrialized production.
Description
Technical field
The present invention relates to mibemycin production technical field, more particularly to a kind of streptomycete fermentation production mibemycin
With fermentation medium and fermentation culture method.
Background technology
Mibemycin (Milbemycins) is the macrolide antibiotics generated by streptomycete, including a series of knots
Very similar sixteen-ring class component on structure.Mibemycin has strong desinsection, mite killing, expelling parasite and the biologies such as antitumor
Activity is learned, it is most of that there is wide spectrum prevention activity to agricultural pests, for preventing as aphid, mite, malacosoma neustria, enteron aisle are parasitic
Worm and the parasite of other damages to crops and domestic animal.Mibemycin has effect strong, and dosage is few, to people's safety nothing
Poison, it is free from environmental pollution, the features such as only murdering worm, not killing pest natural enemy.Can as prevent such drug generate resistance compounding and
Rotation medication is the biological insecticides of the current most promising new no cross tolerance of broad-spectrum high efficacy.
Mibemycin was found that later the said firm finds in succession again from 1967 by Japanese Sankyo companies for the first time
The components such as milbemycin A3, milbemycin A4, milbemycin.American Cyanamid companies of the U.S. find
Another component LL-28249- α (Nemadectin, nimoctin).Have multiple mibemycin similar drugs in the world at present
Commercialization is realized, veterinary medicine or crop protection pesticide is widely used as, there is scholarly forecast, it can substitute current use
In the organophosphor carbamate pyrethroid of crop protection and organochlorine class compound, wide application city is shown
.
Milbemycin oxime (milbemycinoxime) is the mibemycin A that streptomycete fermentation generates3 It is mould with Mir shellfish
Plain A4On the basis of semi-synthetic 9 oxime derivate, be a kind of new macrolides anthelmintic, it is equal to internal epizoa
With effect is killed, there is good effect to controlling and preventing most of common parasitic worm disease.Commonly used to prevent to dislike silk
Parasitosis, the trichuriasis of dog, cat disease and dog that control nematode, hookworm trigger.Milbemycin oxime is generally acknowledged with wide spectrum, height
The anthelmintic of effect, safety, it is particularly relatively more safe to the dog of some ivermectins sensitivity.Current rice commercially available at home
You are useful for the tablet " dog is felt at ease (milbemycin A) " of anti-dirofilariaimmitis disease and for anti-dog, cat by shellfish oxime Related product
Drops " ear mite is gone out (milbeMite) " of ear mite etc..These are all import drugs, by Novartis factory of Switzerland or Japan three
Co., Ltd.'s production altogether, the country not yet develop such product.
Although mibemycin series of products tool has been widely used and market, due to only having Japan to possess strain money
Source, the whole world only have Sankyo companies of Japan production milbemycin A always3And A4Active compound.Northeast Agricultural University of China wins to text
Deng the bacterial strain for being also isolated to production mibemycin, Harbin streptomycete (Streptomyces bingchenggensis), pass through
Strain mutagenesis breeding improvement and fermentation technology optimization, the yield of mibemycin is up to 1380 μ g/mL.Further utilize gene
Engineering technology carries out strain improvement, and the yield of mibemycin is greatly improved, up to 2312 μ g/mL（Number of patent application：
201310409240.2）.It is the effective way for improving mibemycin fermentation titer and production capacity by strain improvement.But
Such change approach technical difficulty is big, and of high cost, time-consuming, and success rate is low.
The content of the invention
It is an object of the invention to provide a kind of streptomycete fermentations to produce mibemycin fermentation medium, passes through culture
The fermentation level for improveing to improve mibemycin of base, significantly improves the yield of mibemycin, reduces production cost.
The present invention also provides a kind of fermentation culture methods of the streptomycete fermentation production mibemycin of improvement, significantly carry
The high yield of mibemycin, it is simple and practicable, it is suitble to industrialized production.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of streptomycete fermentation produces mibemycin fermentation medium, and the fermentation medium is by following quality percentage
Raw material than meter is mixed：Sucrose 11.5-12.5%, cottonseed meal 0.8-1.2%, soybean cake powder 0.8-1.2%, defatted milk
Powder 0.8-1.2%, maltose 1.8-2.2%, dipotassium hydrogen phosphate 0.1-0.15%, ferrous sulfate 0.01-0.015%, zeolite powder
0.38-0.42%, sodium molybdate 0.004-0.006%, copper sulphate 0.004-0.006%, calcium carbonate 0.28-0.32%, defomaing agent GPE
0.03-0.05%, water surplus.
The present invention is deeply divided using metabolic engineering principle and technology influencing mibemycin biosynthesis pathway
Analysis, to activate the relevant enzyme of mibemycin biosynthesis by increasing（Such as metal ion molybdic acid sodium, copper sulphate addition）With
Increase mibemycin precursor as far as possible（The precursor needed for mibemycin biosynthesis may be contained in the ingredients such as cottonseed meal
Substance）The methods of, the combination of optimal nutrient media components is had found, significantly improves the yield of mibemycin.Especially plus
Enter cottonseed meal, skimmed milk power, zeolite powder, the control of the ingredients such as sodium molybdate, copper sulphate and its content effectively increases Mir shellfish
The potency of mycin.
Preferably, the pH controls of the fermentation medium are 7.2-7.6.
A kind of fermentation culture method of streptomycete fermentation production mibemycin, the fermentation culture method include following step
Suddenly：
（1）Slant activation culture：It goes bail for the strain deposited, is inoculated into slant medium, connects the incubator that bacterium is placed on 28 DEG C
In, relative humidity 40-60% cultivates 8-9d, obtains activated spawn；
（2）Seed expands culture：Kind is taken from slant medium, with aseptic inoculation ring aseptically from slant medium
On scrape spore spore suspension be made, it is 10 to make its concentration7-108A spore/mL, then takes spore suspension to be inoculated into seed culture
In base, 28 DEG C, shaking speed 220r/min, incubation time 46-48h of cultivation temperature obtains seed liquor；
（3）Fermented and cultured：Gained seed liquor is seeded in fermentation medium, 28 DEG C of cultivation temperature, shaking speed 220r/
Fermented and cultured, incubation time 150-180h are carried out under min.
Preferably, the slant medium is mixed by the raw material of following mass percent meter：Sucrose 0.3% takes off
Fat milk powder 0.1%, yeast extract 0.15%, malt extract 0.4%, agar 2.5%, water surplus, pH 7.2 ± 0.1.
Preferably, the seed culture medium is mixed by the raw material of following mass percent meter：Sucrose 1.5%, egg
White peptone 0.35%, yeast extract 0.5%, skimmed milk power 0.2%, dipotassium hydrogen phosphate 0.05%, defomaing agent GPE 0.04%, more than water
Amount, pH 7.0-7.4.
Preferably, the fermentation medium is mixed by the raw material of following mass percent meter：Sucrose 11.5-
12.5%, cottonseed meal 0.8-1.2%, soybean cake powder 0.8-1.2%, skimmed milk power 0.8-1.2%, maltose 1.8-2.2%, phosphorus
Sour hydrogen dipotassium 0.1-0.15%, ferrous sulfate 0.01-0.015%, zeolite powder 0.38-0.42%, sodium molybdate 0.004-
0.006%, copper sulphate 0.004-0.006%, calcium carbonate 0.28-0.32%, defomaing agent GPE 0.03-0.05%, water surplus, pH
7.2-7.6。
Preferably, step（3）Middle seed liquor inoculum concentration is the 5-15% of fermentation medium volume.
The beneficial effects of the invention are as follows：The fermentation water of mibemycin is improved by the improvement of culture medium and zymotechnique
It is flat, the yield of mibemycin is significantly improved, reduces production cost, it is simple and practicable, it is suitble to industrialized production.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is the conventional method of this field unless otherwise instructed.
Embodiment 1：
The fermentation culture method of streptomycete fermentation production mibemycin includes the following steps：
（1）Slant activation culture：It goes bail for the strain deposited（Harbin streptomycete CGMCC NO. 1734）, it is inoculated into inclined-plane training
Base is supported, bacterium is connect and is placed in 28 DEG C of incubator, relative humidity 40% cultivates 9d, obtains activated spawn.
Slant medium is mixed by the raw material of following mass percent meter：Sucrose 0.3%, skimmed milk power 0.1%, ferment
Female extract（It is commercially available）0.15%, malt extract（It is commercially available）0.4%, agar 2.5%, water surplus, 7.2 ± 0.1,121 DEG C of pH
20 min of lower sterilizing, bevel.
（2）Seed expands culture：Kind is taken from slant medium, with aseptic inoculation ring aseptically from slant medium
On scrape spore spore suspension be made, it is 10 to make its concentration7A spore/mL, then takes 2mL spore suspensions to be inoculated into seed culture
In shaking flask, in seed culture shaking flask seed culture medium liquid amount be 1000 mL of 100mL/, 28 DEG C of cultivation temperature, shaking speed
220r/min, incubation time 48h, obtains seed liquor.
Seed culture medium is mixed by the raw material of following mass percent meter：Sucrose 1.5%, peptone 0.35%, ferment
Female extract 0.5%, skimmed milk power 0.2%, dipotassium hydrogen phosphate 0.05%, defomaing agent GPE（It is commercially available）0.04%, water surplus, pH
Sterilize 20 min at 7.0,121 DEG C.
（3）Fermented and cultured：Gained seed liquor is seeded in fermentation medium, seed liquor inoculum concentration is fermented and cultured matrix
Long-pending 5%, 28 DEG C of cultivation temperature carry out fermented and cultured, incubation time 180h under shaking speed 220r/min.
Fermentation medium is mixed by the raw material of following mass percent meter：Sucrose 11.5%, cottonseed meal 0.8%,
Soybean cake powder 1.2%, skimmed milk power 0.8%, maltose 2.2%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.015%, zeolite powder
0.38%, sodium molybdate 0.004%, copper sulphate 0.006%, calcium carbonate 0.28%, defomaing agent GPE 0.05%, water surplus, pH 7.2,
Sterilize 20 min at 121 DEG C.
After fermentation, zymotic fluid obtains mycelium filter cake through filter-cloth filtering, and filter cake uses industrial first again after being washed with deionized water
Alcohol soaked overnight filters to obtain methanol extract liquid.Supernatant 1mL is taken to centrifuge（12000r/min）3min, supernatant are micro- through 0.22 μm
High performance liquid chromatography measure is carried out after the membrane filtration of hole.High-efficient liquid phase chromatogram condition：HPLC, UV detector, wavelength 238nm,
Chromatographic column specification ODS C18 reversed-phase columns, mobile phase are：Methanol, water (V/V)=90/10；Flow velocity, 1mL/min；Column temperature, 25 DEG C；Into
Sample amount：1μL.
After testing, mibemycin yield is up to 2306 μ g/mL.
Embodiment 2：
The fermentation culture method of streptomycete fermentation production mibemycin includes the following steps：
（1）Slant activation culture：It goes bail for the strain deposited（Harbin streptomycete CGMCC NO. 1734）, it is inoculated into inclined-plane training
Base is supported, bacterium is connect and is placed in 28 DEG C of incubator, relative humidity 60% cultivates 8d, obtains activated spawn.
Slant medium is mixed by the raw material of following mass percent meter：Sucrose 0.3%, skimmed milk power 0.1%, ferment
Female extract（It is commercially available）0.15%, malt extract（It is commercially available）0.4%, agar 2.5%, water surplus, 7.2 ± 0.1,121 DEG C of pH
20 min of lower sterilizing, bevel.
（2）Seed expands culture：Kind is taken from slant medium, with aseptic inoculation ring aseptically from slant medium
On scrape spore spore suspension be made, it is 10 to make its concentration8A spore/mL, then takes 2mL spore suspensions to be inoculated into seed culture
In shaking flask, in seed culture shaking flask seed culture medium liquid amount be 1000 mL of 100mL/, 28 DEG C of cultivation temperature, shaking speed
220r/min, incubation time 46h, obtains seed liquor.
Seed culture medium is mixed by the raw material of following mass percent meter：Sucrose 1.5%, peptone 0.35%, ferment
Female extract 0.5%, skimmed milk power 0.2%, dipotassium hydrogen phosphate 0.05%, defomaing agent GPE（It is commercially available）0.04%, water surplus, pH
Sterilize 20 min at 7.4,121 DEG C.
（3）Fermented and cultured：Gained seed liquor is seeded in fermentation medium, seed liquor inoculum concentration is fermented and cultured matrix
Long-pending 15%, 28 DEG C of cultivation temperature carry out fermented and cultured, incubation time 150h under shaking speed 220r/min.
Fermentation medium is mixed by the raw material of following mass percent meter：Sucrose 12.5%, cottonseed meal 1.2% are yellow
Beancake powder 0.8%, skimmed milk power 1.2%, maltose 1.8%, dipotassium hydrogen phosphate 0.15%, ferrous sulfate 0.01%, zeolite powder
0.42%, sodium molybdate 0.006%, copper sulphate 0.004%, calcium carbonate 0.32%, defomaing agent GPE 0.03%, water surplus, pH 7.6,121
Sterilize 20 min at DEG C.
After fermentation, zymotic fluid obtains mycelium filter cake through filter-cloth filtering, and filter cake uses industrial first again after being washed with deionized water
Alcohol soaked overnight filters to obtain methanol extract liquid.Supernatant 1mL is taken to centrifuge（12000r/min）3min, supernatant are micro- through 0.22 μm
High performance liquid chromatography measure is carried out after the membrane filtration of hole.High-efficient liquid phase chromatogram condition：HPLC, UV detector, wavelength 238nm,
Chromatographic column specification ODS C18 reversed-phase columns, mobile phase are：Methanol, water (V/V)=90/10；Flow velocity, 1mL/min；Column temperature, 25 DEG C；Into
Sample amount：1μL.
After testing, mibemycin yield is up to 2293 μ g/mL.
Embodiment 3：
The fermentation culture method of streptomycete fermentation production mibemycin includes the following steps：
（1）Slant activation culture：It goes bail for the strain deposited（Mir shellfish streptomycete（DSM41911）Purchased from DSMZ collections）,
Slant medium is inoculated into, bacterium is connect and is placed in 28 DEG C of incubator, relative humidity 40-60% cultivates 8-9d, obtains activation bacterium
Kind.
Slant medium is mixed by the raw material of following mass percent meter：Sucrose 0.3%, skimmed milk power 0.1%, ferment
Female extract（It is commercially available）0.15%, malt extract（It is commercially available）0.4%, agar 2.5%, water surplus, 7.2 ± 0.1,121 DEG C of pH
20 min of lower sterilizing, bevel.
（2）Seed expands culture：Kind is taken from slant medium, with aseptic inoculation ring aseptically from slant medium
On scrape spore spore suspension be made, it is 10 to make its concentration7A spore/mL, then takes 2mL spore suspensions to be inoculated into seed culture
In shaking flask, in seed culture shaking flask seed culture medium liquid amount be 1000 mL of 100mL/, 28 DEG C of cultivation temperature, shaking speed
220r/min, incubation time 47h, obtains seed liquor.
Seed culture medium is mixed by the raw material of following mass percent meter：Sucrose 1.5%, peptone 0.35%, ferment
Female extract 0.5%, skimmed milk power 0.2%, dipotassium hydrogen phosphate 0.05%, defomaing agent GPE（It is commercially available）0.04%, water surplus, pH
Sterilize 20 min at 7.2,121 DEG C.
（3）Fermented and cultured：Gained seed liquor is seeded in fermentation medium, seed liquor inoculum concentration is fermented and cultured matrix
Long-pending 10%, 28 DEG C of cultivation temperature carry out fermented and cultured, incubation time 160h under shaking speed 220r/min.
Fermentation medium is mixed by the raw material of following mass percent meter：Sucrose 12%, cottonseed meal 1%, soya bean
Cake powder 1%, skimmed milk power 1%, maltose 2%, dipotassium hydrogen phosphate 0.12%, ferrous sulfate 0.012%, zeolite powder 0.4%, molybdic acid
Sodium 0.005%, copper sulphate 0.005%, calcium carbonate 0.3%, defomaing agent GPE 0.04%, water surplus sterilize at 7.4,121 DEG C of pH
20 min。
After fermentation, zymotic fluid obtains mycelium filter cake through filter-cloth filtering, and filter cake uses industrial first again after being washed with deionized water
Alcohol soaked overnight filters to obtain methanol extract liquid.Supernatant 1mL is taken to centrifuge（12000r/min）3min, supernatant are micro- through 0.22 μm
High performance liquid chromatography measure is carried out after the membrane filtration of hole.High-efficient liquid phase chromatogram condition：HPLC, UV detector, wavelength 238nm,
Chromatographic column specification ODS C18 reversed-phase columns, mobile phase are：Methanol, water (V/V)=90/10；Flow velocity, 1mL/min；Column temperature, 25 DEG C；Into
Sample amount：1μL.
After testing, mibemycin yield is up to 2330 μ g/mL.
Embodiment described above is a kind of preferable scheme of the present invention, and not the present invention is made in any form
Limitation also has other variants and remodeling on the premise of without departing from the technical solution recorded in claim.
Claims (4)
1. a kind of streptomycete fermentation produces mibemycin fermentation medium, which is characterized in that the fermentation medium by with
The raw material of lower mass percent meter is mixed：Sucrose 11.5-12.5%, cottonseed meal 0.8-1.2%, soybean cake powder 0.8-
1.2%, skimmed milk power 0.8-1.2%, maltose 1.8-2.2%, dipotassium hydrogen phosphate 0.1-0.15%, ferrous sulfate 0.01-
0.015%, zeolite powder 0.38-0.42%, sodium molybdate 0.004-0.006%, copper sulphate 0.004-0.006%, calcium carbonate 0.28-
0.32%, defomaing agent GPE 0.03-0.05%, water surplus, medium pH control are 7.2-7.6.
2. a kind of streptomycete fermentation generates the fermentation culture method of mibemycin, which is characterized in that the fermentation culture method
Include the following steps：
（1）Slant activation culture：It goes bail for the strain deposited, is inoculated into slant medium, connects bacterium and be placed in 28 DEG C of incubator, phase
To humidity 40-60%, 8-9d is cultivated, obtains activated spawn；
（2）Seed expands culture：Kind is taken from slant medium, is aseptically scraped with aseptic inoculation ring from slant medium
Spore suspension is made in lower spore, and it is 10 to make its concentration7-108A spore/mL, then takes spore suspension to be inoculated into seed culture medium
In, 28 DEG C, shaking speed 220r/min, incubation time 46-48h of cultivation temperature obtains seed liquor；
（3）Fermented and cultured：Gained seed liquor is seeded in fermentation medium, inoculum concentration is the 5-15% of fermentation medium volume,
28 DEG C of cultivation temperature carries out fermented and cultured, incubation time 150-180h under shaking speed 220r/min；Wherein fermentation medium by
The raw material of following mass percent meter is mixed：Sucrose 11.5-12.5%, cottonseed meal 0.8-1.2%, soybean cake powder
0.8-1.2%, skimmed milk power 0.8-1.2%, maltose 1.8-2.2%, dipotassium hydrogen phosphate 0.1-0.15%, ferrous sulfate 0.01-
0.015%, zeolite powder 0.38-0.42%, sodium molybdate 0.004-0.006%, copper sulphate 0.004-0.006%, calcium carbonate 0.28-
0.32%, defomaing agent GPE 0.03-0.05%, water surplus, pH 7.2-7.6.
3. fermentation culture method according to claim 2, it is characterised in that：The slant medium is by following quality percentage
Raw material than meter is mixed：Sucrose 0.3%, skimmed milk power 0.1%, yeast extract 0.15%, malt extract 0.4%, fine jade
Fat 2.5%, water surplus, pH 7.2 ± 0.1.
4. fermentation culture method according to claim 2, it is characterised in that：The seed culture medium is by following quality percentage
Raw material than meter is mixed：Sucrose 1.5%, peptone 0.35%, yeast extract 0.5%, skimmed milk power 0.2%, phosphoric acid hydrogen
Dipotassium 0.05%, defomaing agent GPE 0.04%, water surplus, pH 7.0-7.4.
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| CN101037442A (en) * | 2007-01-17 | 2007-09-19 | 东北农业大学 | New compound prepared by ice city streptomyces and preparation method |
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Effective date of registration: 20220921 Address after: No. 26-1, Chunhui Middle Road, Fuying Street, Xianju County, Taizhou City, Zhejiang Province 318000 Patentee after: Xianju Liangshan Biotechnology Co.,Ltd. Address before: 311121 Room 531, 6 Blocks, Shunfan Science Park, Jingchang Road, Wenyi Road, Yuhang District, Hangzhou City, Zhejiang Province Patentee before: HANGZHOU CHENXI BIOTECHNOLOGY Co.,Ltd. |
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