CN110066843A - Kitasamycin fermentation medium, kitasamycin and its fermentation process - Google Patents
Kitasamycin fermentation medium, kitasamycin and its fermentation process Download PDFInfo
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Abstract
The invention discloses a kind of kitasamycin fermentation mediums, every 1000ml fermentation medium includes: glucose 3.0-10.0g, soybean cake powder 10-25g, starch 10.0-25.0g, ammonium chloride 1.0-5.0g, potassium dihydrogen phosphate 0.3-1.0g, magnesium sulfate 0.5-5.0g, zinc sulfate 0.03-0.1g, calcium carbonate 1.0-5.0g and soya-bean oil 10.0-40.0g, and the fermentation medium further includes methyl oleate and surfactant.Methyl oleate is used in fermentation medium of the present invention; it is easier hydrolyzed metabolism; generate short chain fatty acids; accelerate the synthesis of macrocyclic lactone, while generating more acetylcoenzyme, enhances the activity of acyltransferase in metabolic process; A5 content is improved to opposite; but also pH is more stable in kitasamycin fermentation process, to reduce A1 content, compensates for soya-bean oil and be metabolized slow short slab bring deficiency.
Description
Technical field
The present invention relates to technical field of microbial fermentation, mould more particularly to a kind of kitasamycin fermentation medium, guitar
Element and its fermentation process.
Background technique
Kitasamycin (Kitasamycin) also known as kitasamycin (Leucomycin, LM), belong to the big ring of sixteen-ring
Lactone antibiotic, nineteen fifty-three is by Japanese Qin Tengshu et al. isolated one in the big Kubo soil in Shinjuku, Tokyo, Japan
Strain S. hitasatoensis (Streptomyces kitasatoensis SK) fermented, extracting and developing obtains, and has nearly 50 so far
Many years history.The structure of kitasamycin is mould by ten hexa-atomic sugared former times (ageyeone), the mould nitrogen base of carbon sugared (mye-aminose), carbon
Three parts sugared (Myeaeose) form, and due to the difference of producing strains hereditary capacity and fermentation condition, it is different can to form content
Each component.
Kitasamycin structural formula:
Kitasamycin antimicrobial spectrum is similar to erythromycin, spiramvcin, tylosin, to gram-positive bacterium and negative ball
Bacterium, Richettsia, conveyor screw and large-scale virus have stronger inhibiting effect.Clinically, kitasamycin is mainly used for treating hammer
It is infected caused by bacterium, especially hemolytic streptococcus, pneumococcus, light, moderate infection of staphylococcus aureus, and to penicillin
The patient of allergy；To bronchitis, scarlet fever, suppurative tonsillitis and septicemia caused by streptococcus and pneumococcus etc. etc.
The effective percentage of illness up to 90%, and can be used for treating skin caused by gram-positive cocci, soft tissue, respiratory tract infection,
Other secondary infections and osteomyelitis, diphtheria and syphilis.
In recent years, since the residual quantity of kitasamycin in vivo is low, using wide in terms of veterinary drug and feed addictive
General, therefore, kitasamycin is mainly used for preventing and treating livestock and poultry as a kind of efficient, safe and stable medicated feed additive
Respiratory tract and disease of digestive tract, low dosage use have significant growth promoting function for livestock and poultry, belong to the feeding of Ministry of Agriculture's publication
The unique macrolide antibiotic for allowing to use for a long time in feed in material medicated premix operating specification.
Soya-bean oil is the grease extracted from soybean, is one of kitasamycin fermenting and producing key raw material.Soya-bean oil passes through
S. hitasatoensis catabolism, produces short chain fatty acids, and short chain fatty acids are to synthesize the important as precursors of kitasamycin.Modern molecular
Biological study shows that the synthesis of kitasamycin macrocyclic lactone is by 5 propionic acid units 1 butyric acid unit 1 of acetic acid unit 1
Hydroxyacetic acid unit is condensed by synthesizing similar polyketide (polyketide) approach with fatty acid, i.e. lower fatty acid
It is connected to form an aliphatic chain " end to end " under the action of enzyme, then forms ester ring under the action of cyclization enzyme and modification enzyme.This table
Its bright important as precursors is short chain fatty acids.In the actual production process, it since soya-bean oil is not soluble in water and specific gravity is smaller, tends not to
Sufficiently by microbial metabolism, remaining soya-bean oil affects fermentation condition and fermentation level, while to fermentation downstream extraction work
Bring very big burden.
Methyl oleate is a kind of unsaturated high-grade fatty acid ester, reacts to obtain by oleic acid and methanol, and oleic acid is with glyceride
Form in soya-bean oil content be up to 20-36%.Tween 80 and span 85 are nonionic surface active agent, can reduce liquid
Body interface tension makes mutually insoluble liquid form emulsification, tween and sapn and uses frequently as oil-in-water emulsifiers, can cream
Change, with molten and solubilized.
In 2015 editions pharmacopeia, kitasamycin A5, which should be 35%~70%, A4, should be 5%~25%, and A1, A13 should be 3%
~15%；The sum of kitasamycin major constituent A9, A8, A7, A6, A5, A4, A1, A3, A13 must not be less than 85%.Current existing guitar
In mycin production, part A1 component is higher than 15% and A5 component and is lower than 35%, and end product quality is caused to be unable to reach pharmacopeia mark
It is quasi-.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
The purpose of the present invention is to provide a kind of kitasamycin fermentation mediums, enable to kitasamycin fermentation process
Middle pH is more stable, and A5 content can be improved, and reduces A1 content, compensates for soya-bean oil and is metabolized slow short slab bring deficiency.
To achieve the above object, the present invention provides a kind of kitasamycin fermentation medium, the fermentations of every 1000ml
Culture medium includes: glucose 3.0-10.0g, soybean cake powder 10-25g, starch 10.0-25.0g, ammonium chloride 1.0-5.0g, phosphoric acid
Potassium dihydrogen 0.3-1.0g, magnesium sulfate 0.5-5.0g, zinc sulfate 0.03-0.1g, calcium carbonate 1.0-5.0g and soya-bean oil 10.0-40.0g,
The fermentation medium further includes methyl oleate and surfactant.
In above-mentioned culture medium use methyl oleate, compared with soya-bean oil be easier enter metabolism utilize, and decompose after be converted into compared with
More acetylcoenzymes makes up the acetylcoenzyme amount that primary ferment proportion soya-bean oil metabolism generates, to improve the content of A5 component.
In kitasamycin fermentation process, since methyl oleate molecular weight is smaller, it is easier to which hydrolyzed metabolism generates short chain
Fatty acid, accelerates the synthesis of macrocyclic lactone, while generating more acetylcoenzyme, enhances the work of acyltransferase in metabolic process
Property, A5 content is improved thus opposite, and pH is more stable because methyl oleate is added, in kitasamycin fermentation process, to reduce
A1 content.
In a preferred embodiment, the content of above-mentioned methyl oleate is 2.5-10.0ml.
In a preferred embodiment, above-mentioned surface-active contents are 0.5-2.5ml.
In a preferred embodiment, above-mentioned surfactant is made of Tween 80 and span 85.
In a preferred embodiment, above-mentioned Tween 80 and the volume ratio of span 85 are 1:1.
Surface active agent tween 80 and span 85 are used in kitasamycin fermentation medium, what is mainly utilized is it with molten
And solubilization, be conducive to the dissolution transmitting of short chain fatty acids.
In a preferred embodiment, above-mentioned surfactant and methyl oleate after mixing, then with other components
It is mixed.
Another object of the present invention is to provide a kind of fermentation process of kitasamycin, comprising: accesses seed culture fluid
The step of fermenting and producing kitasamycin is carried out in any one of the above kitasamycin fermentation medium.
In a preferred embodiment, the preparation method of above-mentioned seed culture fluid includes: using 500ml triangular flask, often
Bottle culture medium loading amount 50ml amounts to 10-15 bottles, and the seed culture based component includes (counting according to 1000ml): glucose 5.0-
15.0g, soybean cake powder 10-20g, starch 15.0-25.0g, peptone 2.0-5.0g, yeast powder 2.0-5.0g, sodium chloride 3.0-
8.0g, potassium dihydrogen phosphate 0.3-1.0g, magnesium sulfate 0.5-5.0g, ammonium sulfate 2.0-5.0g, calcium carbonate 1.0-5.0g；Eight layers of gauze
Wrap up bottleneck, sterilizing；Mature slant pore aseptic condition access shaking flask is cultivated；Uniform 10 bottles of getting wall after culture
It is incorporated into 2000ml aseptic inoculation bottle in desinfection chamber, for use；
Preferably, the sterilising temp is 121-123 DEG C, and sterilization time is 30 minutes；
Preferably, when the shaking flask is cultivated shaking speed be 220rpm, 28.0 ± 1.0 DEG C of temperature, humidity 30-
60%, incubation time 12-20h.
In a preferred embodiment, above-mentioned fermentation medium preparation method includes: that will remove surfactant, oleic acid first
Other raw materials outside ester, soya-bean oil and calcium carbonate are put into proportion container respectively, and suitable quantity of water is added and is sufficiently mixed, uses ammonium hydroxide tune
Save pH to 6.8-7.5, add soya-bean oil mixing, calcium carbonate is added and mixes constant volume, obtain except surfactant and methyl oleate it
Then the surfactant is uniformly mixed by outer fermentation medium with methyl oleate, be mixed into the prepared fermentation training
It supports in base, finally in fermentor constant volume, obtains fermentation liquid；
Preferably, prepared fermentation medium is sterilized, sterilising temp is 121-123 DEG C, keeps the temperature 30 minutes, cold
But to 28.0 DEG C；
Preferably, the time of the fermentation is 68-72h；
Preferably, fermentation liquid oxalic acid adjusts pH=3.0-4.5, filtering；Filtrate adjusts pH=8.5- using ammonium hydroxide
10.5, then heat up 30-45 DEG C；Butyl acetate extraction is added, extract liquor is stripped using potassium phosphate buffer；
Gained strip liquor adjusts pH=7.5-10.0 using ammonium hydroxide, is warming up to 50-65 DEG C and crystallizes 30 minutes；Centrifuge separation, drying, obtains
Kitasamycin product.
Another object of the present invention is to provide the kitasamycins obtained by any one of the above fermentation process.
Compared with prior art, it has the following beneficial effects: according to the present invention
(1) methyl oleate is used in fermentation medium of the present invention, in kitasamycin fermentation process, due to methyl oleate point
Son amount is smaller, it is easier to which hydrolyzed metabolism generates short chain fatty acids, accelerates the synthesis of macrocyclic lactone, while generating more second
Acyl coenzyme enhances the activity of acyltransferase in metabolic process, so that opposite improve A5 content, and because methyl oleate is added,
PH is more stable in kitasamycin fermentation process, to reduce A1 content, especially A1 component, maximum reduce 5.6%, completely
Meet requirement of 2015 editions pharmacopeia to kitasamycin, compensates for soya-bean oil and be metabolized slow short slab bring deficiency.
(2) methyl oleate surface active agent tween 80 and span 85 are used in fermentation medium of the present invention, have with molten and
Solubilization is conducive to the dissolution transmitting of short chain fatty acids.
(3) fibroin powder is not used in fermentation medium of the present invention, because fibroin powder is compound protein hydrolysate, material
Source will cause the aminoacid ingredient deviation of fibroin powder, and unstable influence is caused to kitasamycin fermentation process.
Specific embodiment
Specific embodiment is described in detail below, to more fully understand the present invention.It is to be understood that of the invention
Protection scope be not limited by the specific implementation.
Experiment condition used in the examples and experimental method are as follows:
Seed culture condition and incubation: taking 500ml triangular flask, and every bottle of culture medium loading amount 50ml amounts to 10-15 bottles；
Eight layers of gauze wrap up bottleneck, and 121-123 DEG C of sterilising temp, sterilization time is 30 minutes；Mature slant pore aseptic condition access
Shaking flask is cultivated, shaking speed 220rpm, and 28.0 ± 1.0 DEG C of temperature, humidity 30-60%, incubation time 12-20h；Culture knot
Uniform 10 bottles of beam getting wall are incorporated into 2000ml aseptic inoculation bottle in desinfection chamber, for use；Wherein, seed culture based component
(being counted according to 1000ml): glucose 5.0-15.0g, soybean cake powder 10-20g, starch 15.0-25.0g, peptone 2.0-5.0g,
Yeast powder 2.0-5.0g, sodium chloride 3.0-8.0g, potassium dihydrogen phosphate 0.3-1.0g, magnesium sulfate 0.5-5.0g, ammonium sulfate 2.0-
5.0g, calcium carbonate 1.0-5.0g.
Fermentation culture conditions and incubation: 15000ml fermentor (the proud middle limited public affairs of bioengineering equipment in Shanghai are used
Department), fermentation medium meter Material product 9000ml, 30% or more dissolved oxygen control, and speed of agitator 0- is adjusted according to dissolved oxygen
800rpm, 28 ± 1.0 DEG C of temperature, intake 9L/ minutes, incubation time 60-80h；Wherein, fermentation medium except methyl oleate and
Ingredient (being counted according to 1000ml) except surfactant: glucose 3.0-10.0g, soybean cake powder 10-25g, starch 10.0-
25.0g, ammonium chloride 1.0-5.0g, potassium dihydrogen phosphate 0.3-1.0g, magnesium sulfate 0.5-5.0g, zinc sulfate 0.03-0.1g, calcium carbonate
1.0-5.0g with soya-bean oil 10.0-40.0g；The preparation method of fermentation medium: by other raw materials in addition to soya-bean oil and calcium carbonate point
Suitable quantity of water Tou Ru not be added to be sufficiently mixed in proportion container, adjust pH to 6.8-7.5 using ammonium hydroxide, soya-bean oil is then added and mixes, most
Calcium carbonate is added afterwards and mixes constant volume.
Broth extraction condition and extraction process: fermentation liquid oxalic acid adjusts pH=3.0-4.5, filtering；Filtrate uses ammonia
Water adjusts pH=8.5-10.5 and heats up 30-45 DEG C, and butyl acetate extraction is added；Extract liquor is stripped using potassium phosphate buffer
It takes；Strip liquor adjusts pH=7.5-10.0 using ammonium hydroxide, is warming up to 50-65 DEG C and crystallizes 30 minutes；Crystal solution is centrifugated lucky
His mycin product, is detected after drying.
Kitasamycin component and detection method of content are shown in " Chinese Pharmacopoeia " 2015 editions according to efficient liquid phase detection method.
Following embodiment is using identical seed culture condition, fermentation culture conditions and broth extraction condition.
Embodiment 1
It takes Tween 80 and each 3.0ml of span 85 to be added in 60ml methyl oleate and be uniformly mixed, is mixed into prepared above-mentioned hair
In ferment culture medium, pours into and add drinking water constant volume 9000ml in 15000ml fermentor；It is sterilized using steam reality tank, sterilising temp
121-123 DEG C, 30 minutes are kept the temperature, is cooled to 28.0 DEG C；By above-mentioned prepared seed culture fluid flame protection access fermentation training
It supports in base, ferment 70h.Efficient liquid phase detection is carried out after extraction, as a result such as the following table 1:
Table 1
| Component | A5% | A4% | A1% | A13% | The sum of major constituent % |
| Percentage composition | 50.8 | 10.0 | 11.4 | 8.6 | 91.5 |
Embodiment 2
It takes Tween 80 and each 4.5ml of span 85 to be added in 45ml methyl oleate and be uniformly mixed, is mixed into prepared above-mentioned hair
In ferment culture medium, pours into and add drinking water constant volume 9000ml in 15000ml fermentor；It is sterilized using steam reality tank, sterilising temp
121-123 DEG C, 30 minutes are kept the temperature, is cooled to 28.0 DEG C；By above-mentioned prepared seed culture fluid flame protection access fermentation training
It supports in base, ferment 72h.Efficient liquid phase detection is carried out after extraction, as a result such as the following table 2:
Table 2
| Component | A5% | A4% | A1% | A13% | The sum of major constituent % |
| Percentage composition | 48.5 | 11.2 | 10.2 | 9.0 | 90.7 |
Embodiment 3
It takes Tween 80 and each 6.0ml of span 85 to be added in 60ml methyl oleate and be uniformly mixed, is mixed into prepared above-mentioned hair
In ferment culture medium, pours into and add drinking water constant volume 9000ml in 15000ml fermentor；It is sterilized using steam reality tank, sterilising temp
121-123 DEG C, 30 minutes are kept the temperature, is cooled to 28.0 DEG C；By above-mentioned prepared seed culture fluid flame protection access fermentation training
It supports in base, ferment 68h.Efficient liquid phase detection is carried out after extraction, as a result such as the following table 3:
Table 3
| Component | A5% | A4% | A1% | A13% | The sum of major constituent % |
| Percentage composition | 49.3 | 11.4 | 9.5 | 9.6 | 91.2 |
Embodiment 4
It takes Tween 80 and each 5.0ml of span 85 to be added in 45ml methyl oleate and be uniformly mixed, is mixed into prepared above-mentioned hair
In ferment culture medium, pours into and add drinking water constant volume 9000ml in 15000ml fermentor；It is sterilized using steam reality tank, sterilising temp
121-123 DEG C, 30 minutes are kept the temperature, is cooled to 28.0 DEG C；By above-mentioned prepared seed culture fluid flame protection access fermentation training
It supports in base and carries out fermentation 72h.Efficient liquid phase detection is carried out after extraction, as a result such as the following table 4:
Table 4
| Component | A5% | A4% | A1% | A13% | The sum of major constituent % |
| Percentage composition | 51.0 | 10.6 | 8.9 | 9.5 | 90.8 |
Comparative example (does not contain methyl oleate) in fermentation medium
It takes Tween 80 and each 6.0ml of span 85 to be uniformly mixed, is mixed into prepared above-mentioned fermentation medium, pours into
Add drinking water constant volume 9000ml in 15000ml fermentor；It is sterilized using steam reality tank, 121-123 DEG C of sterilising temp, keeps the temperature 30 points
Clock is cooled to 28.0 DEG C；By in above-mentioned prepared seed culture fluid flame protection access fermentation medium, ferment 72h.It extracts
Efficient liquid phase detection is carried out afterwards, as a result such as the following table 5:
Table 5
| Component | A5% | A4% | A1% | A13% | The sum of major constituent % |
| Percentage composition | 44.5 | 11.0 | 14.5 | 8.9 | 90.5 |
Data in table 5 are compared and analyzed with corresponding data in table 1- table 4, it is known that: A5% distinguishes in table 1- table 4
It is 50.8%, 48.5%, 49.3%, 51.0%, and A5% is 44.5% in table 5, lower than the percentage composition of A5 in table 1- table 4,
When that is not containing methyl oleate in fermentation medium, the content of A5 is reduced in kitasamycin product；In table 1- table 4
A1% is respectively 11.4%, 10.2%, 9.5%, 8.9%, and A1% is 14.5% in table 5, higher than the percentage of A1 in table 1- table 4
Content, that is to say, that when not containing methyl oleate in fermentation medium, the content of A1 is increased in kitasamycin product.
And in embodiment 1- embodiment 4, due to joined methyl oleate in fermentation medium, not with culture medium in comparative example
The case where adding methyl oleate, compares, it is found that the A5 constituent content in fermentation gained kitasamycin product improves, A1 component
Content reduces, and especially A1 component, maximum reduce 5.6%, fully meets requirement of 2015 editions pharmacopeia to kitasamycin.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (10)
1. a kind of kitasamycin fermentation medium, the fermentation medium of every 1000ml includes: glucose 3.0-10.0g, Huang
Beancake powder 10-25g, starch 10.0-25.0g, ammonium chloride 1.0-5.0g, potassium dihydrogen phosphate 0.3-1.0g, magnesium sulfate 0.5-5.0g,
Zinc sulfate 0.03-0.1g, calcium carbonate 1.0-5.0g and soya-bean oil 10.0-40.0g, which is characterized in that the fermentation medium also wraps
Include methyl oleate and surfactant.
2. kitasamycin fermentation medium according to claim 1, which is characterized in that the content of the methyl oleate is
2.5-10.0ml。
3. kitasamycin fermentation medium according to claim 1, which is characterized in that the surface-active contents are
0.5-2.5ml。
4. kitasamycin fermentation medium according to claim 1 or 3, which is characterized in that the surfactant is by spitting
Temperature 80 and span 85 composition.
5. kitasamycin fermentation medium according to claim 4, which is characterized in that the body of the Tween 80 and span 85
Product ratio is 1:1.
6. kitasamycin fermentation medium according to claim 1, which is characterized in that the surfactant and oleic acid first
Ester after mixing, then with other components is mixed.
7. a kind of fermentation process of kitasamycin, characterized in that it comprises: appoint seed culture fluid access claim 1-6
The step of fermenting and producing kitasamycin is carried out in one kitasamycin fermentation medium.
8. fermentation process according to claim 7, which is characterized in that the preparation method of the seed culture fluid includes: to adopt
With 500ml triangular flask, every bottle of culture medium loading amount 50ml amounts to 10-15 bottle, the seed culture based component including (according to
1000ml meter): glucose 5.0-15.0g, soybean cake powder 10-20g, starch 15.0-25.0g, peptone 2.0-5.0g, yeast powder
2.0-5.0g, sodium chloride 3.0-8.0g, potassium dihydrogen phosphate 0.3-1.0g, magnesium sulfate 0.5-5.0g, ammonium sulfate 2.0-5.0g, carbonic acid
Calcium 1.0-5.0g；Eight layers of gauze wrap up bottleneck, sterilizing；Mature slant pore aseptic condition access shaking flask is cultivated；Culture knot
It is incorporated into 2000ml aseptic inoculation bottle in desinfection chamber for uniform 10 bottles of getting wall after beam, for use；
Preferably, the sterilising temp is 121-123 DEG C, and sterilization time is 30 minutes；
Preferably, shaking speed is 220rpm when the shaking flask is cultivated, and 28.0 ± 1.0 DEG C of temperature, humidity 30-60%, is trained
Support time 12-20h.
9. fermentation process according to claim 7, which is characterized in that the fermentation medium preparation method includes: that will remove
Other raw materials outside surfactant, methyl oleate, soya-bean oil and calcium carbonate are put into proportion container respectively, and suitable quantity of water is added and fills
Divide mixing, adjust pH to 6.8-7.5 using ammonium hydroxide, add soya-bean oil mixing, calcium carbonate mixing constant volume is added, obtains except surface is living
Property agent and methyl oleate except fermentation medium, the surfactant is uniformly mixed with methyl oleate then, is mixed into and matches
In the fermentation medium made, finally in fermentor constant volume, fermentation liquid is obtained；
Preferably, prepared fermentation medium is sterilized, sterilising temp is 121-123 DEG C, keeps the temperature 30 minutes, is cooled to
28.0℃；
Preferably, the time of the fermentation is 68-72h；
Preferably, fermentation liquid oxalic acid adjusts pH=3.0-4.5, filtering；Filtrate adjusts pH=8.5-10.5 using ammonium hydroxide,
Then it heats up 30-45 DEG C；Butyl acetate extraction is added, extract liquor is stripped using potassium phosphate buffer；Gained
Strip liquor adjusts pH=7.5-10.0 using ammonium hydroxide, is warming up to 50-65 DEG C and crystallizes 30 minutes；Centrifuge separation, drying, obtains guitar
Mycin product.
10. the kitasamycin that the fermentation process according to any one of claim 7-9 obtains.
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| CN115385970A (en) * | 2022-03-24 | 2022-11-25 | 浙江普洛生物科技有限公司 | Extraction method of kitasamycin |
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| CN111961700A (en) | 2020-11-20 |
| CN110066843B (en) | 2020-10-09 |
| CN111961700B (en) | 2023-04-28 |
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