CN111893154A - Method for producing sisomicin - Google Patents
Method for producing sisomicin Download PDFInfo
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- CN111893154A CN111893154A CN202010818274.7A CN202010818274A CN111893154A CN 111893154 A CN111893154 A CN 111893154A CN 202010818274 A CN202010818274 A CN 202010818274A CN 111893154 A CN111893154 A CN 111893154A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
- C12P19/48—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin the cyclohexyl radical being substituted by two or more nitrogen atoms, e.g. destomycin, neamin
- C12P19/485—Having two saccharide radicals bound through only oxygen to non-adjacent ring carbons of the cyclohexyl radical, e.g. gentamycin, kanamycin, sisomycin, verdamycin, mutamycin, tobramycin, nebramycin, antibiotics 66-40B, 66-40D, XK-62-2, 66-40, G-418, G-52
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a method for producing sisomicin. The method for producing sisomicin by using micromonospora inonotus through fermentation belongs to the technical field of fermentation engineering, and adopts deep layer, aeration stirring, multi-stage fermentation and batch feeding. The invention has the advantages of improving the fermentation liquor yield by 50-90% through fed-batch, shortening the fermentation period, reducing the production cost, improving the product profit and finally realizing the effect of high-efficiency and stable production of sisomicin.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a method for producing sisomicin.
Background
Sisomicin, the english name Sisomicin, the chinese name Sisomicin, perillamycin, the english name Baymicin, Extramycin, Mensiso, Rickamicin, Sisomicin Sulfate, Sisomicin Sulfate, and western medicine. The molecular formula is as follows: C19H37N5O7, molecular weight: 447.53, the structural formula is as follows (formula 1)
Sisomicin is an aminoglycoside antibiotic produced by micromonospora. The traditional Chinese medicine composition is mainly used for local or systemic infection (ineffective for central infection) caused by gram-negative bacteria such as escherichia coli, dysentery bacillus, klebsiella, proteus and the like, and has a particularly good effect on urinary tract infection.
At present, the yield of sisomicin produced by fermentation in China is low, which is extremely unfavorable for industrial production, so that the price of the medicine is too high, and the popularization of the medicine is difficult.
Therefore, a new method for producing sisomicin needs to be invented, so that the yield of sisomicin produced by fermentation is increased, the price of medicines is reduced, and the method is suitable for industrial production and sale.
Disclosure of Invention
According to one aspect of the present invention, there is provided a method of producing sisomicin, comprising the steps of:
s1, inoculating micromonospora inyoensis into a slant seed culture medium, and culturing for 9-15 days at the temperature of 32-36 ℃ under the condition of 200-300 rmp;
s2, inoculating the seeds on a seed culture medium of a first-level seed tank according to the inoculation amount of 10-15%, wherein the temperature is 32-36 ℃, the rotating speed is 200-300 rmp, the tank pressure is 0.03-0.07 Mpa, the air volume is 1: 1.0-1: 3.0vvm, and the culture time is 40-50 h;
s3, inoculating the mycelia on a fermentation medium of a fermentation tank according to the inoculation amount of 10-20%, wherein the temperature is 32-36 ℃, the temperature is properly adjusted when the mycelia grow too fast or too slow, and the culture time is 120-168 hours;
s4, when the fermentation is carried out for 40-45h, 60-65h and 80-90h, the supplementary culture medium is supplemented.
In some embodiments, the weight ratio (g/L) of the slant culture medium in step S1 is: 10-20 parts of soluble starch, 3-8 parts of sucrose, 1-3 parts of dry yeast, 1-3 parts of potassium nitrate, 1-3 parts of magnesium sulfate, 3-5 parts of precipitated calcium carbonate, 0.01-0.04 part of aspartyl, 0.01-0.03 part of ferrous sulfate heptahydrate, 0.3-0.5 part of monopotassium phosphate, 10-20 parts of agar, 30-40 parts of bran and 7.0-8.0 parts of pH.
In some embodiments, the weight ratio (g/L) of the seed culture medium in step S2 is: 20-30 parts of corn starch, 15-25 parts of soybean cake powder, 0.8-1.2 parts of magnesium sulfate, 3-5 parts of precipitated calcium carbonate, 0.8-1.0 part of corn steep liquor dry powder and pH 7.0-7.6.
In some embodiments, the fermentation medium of step S3 has a weight ratio (g/L) of: 8-10 parts of corn starch, 30-40 parts of dextrin, 30-40 parts of soybean cake powder, 1-2 parts of precipitated calcium carbonate, 0.3-0.5 part of magnesium chloride, 5-7 parts of corn starch powder, 0.01-0.03 part of cobalt chloride and 7.0-7.6 parts of pH value.
In some embodiments, the weight ratio (g/L) of the feed medium in step S4 is: 10-20 parts of maltose, 20-30 parts of corn starch, 20-26 parts of soybean cake powder, 0.6-1 part of methionine and 0.8-1 part of sodium chloride.
In some embodiments, in step S3, the inoculation amount is 10% to 20%.
In some embodiments, the pressure of the fermentation tank in step S3 is 0.03-0.07 Mpa, and the air volume is 1: 0.2-1: 3.0 vvm.
In some embodiments, the weight ratio (g/L) of the slant culture medium in step S1 is: 10-15 parts of soluble starch, 3-5 parts of sucrose, 1-2 parts of dry yeast, 1-2 parts of potassium nitrate, 1-2 parts of magnesium sulfate, 3-4 parts of precipitated calcium carbonate, 0.01-0.02 part of aspartyl, 0.01-0.02 part of ferrous sulfate heptahydrate, 0.3-0.4 part of monopotassium phosphate, 10-15 parts of agar, 30-35 parts of bran and 7.0-7.5 of pH.
The method for producing sisomicin has the advantages that the method for producing sisomicin by using the micromonospora inovora through fermentation belongs to the technical field of fermentation engineering, deep layer and aeration stirring are adopted, multi-stage fermentation is adopted, and batch feeding is carried out.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1A method for producing sisomicin
The method comprises the following steps:
s1, inoculating micromonospora inonotus into a slant culture medium, and culturing for 9 days at 32 ℃;
s2, selecting a mature inclined plane, inoculating the mature inclined plane to a seed liquid culture medium, culturing the mature inclined plane on a shaking table at 32 ℃, culturing for 40h under 200rmp, inoculating the mature inclined plane to a fermentation tank;
s3, inoculating according to the inoculation amount of 10%, and after the inoculation is completed, placing the fermentation tank at 32 ℃ for culture;
s4, fermenting until 40h, 60h and 80h are supplemented with the feed medium. In the whole fermentation process, the pressure of the fermentation tank is 0.03MPa, the ventilation quantity is 1.0VVM, the culture is carried out for 120h, and the tank is placed. And separating and extracting the obtained fermentation liquor to obtain the sisomicin.
The above slant medium (g/L): soluble starch 10, sucrose 3, dry yeast 1, potassium nitrate 1, magnesium sulfate 1, precipitated calcium carbonate 3, aspartyl 0.01, ferrous sulfate heptahydrate 0.01, monopotassium phosphate 0.3, agar 10, bran 30, and pH 7.0.
Seed medium (g/L): 20 parts of corn starch, 15 parts of soybean cake powder, 0.8 part of magnesium sulfate, 3 parts of precipitated calcium carbonate, 0.8 part of corn steep liquor dry powder and pH 7.0.
Fermentation medium (g/L): corn starch 8, dextrin 30, soybean cake powder 30, precipitated calcium carbonate 1, magnesium chloride 0.3, corn starch powder 5, cobalt chloride 0.01 and pH 7.0.
Feed medium (g/L): maltose 10, corn starch 20, soybean cake powder 20, methionine 0.6 and sodium chloride 0.8.
Example 2A method for producing sisomicin
The method comprises the following steps:
s1, inoculating micromonospora inonotus into a slant culture medium, and culturing at 34 ℃ for 12 days;
s2, selecting a mature inclined plane, inoculating the mature inclined plane to a seed liquid culture medium, culturing on a shaking table at 34 ℃, culturing for 45 hours under 250rmp, inoculating, and connecting to a fermentation tank;
s3, inoculating according to the inoculation amount of 15%, and after the inoculation is completed, placing the fermentation tank at 34 ℃ for culture;
s4, fermenting until 43h, 63h and 85h are supplemented with the feed medium. In the whole fermentation process, the pressure of the fermentation tank is 0.05MPa, the ventilation rate is 2.0VVM, the culture is carried out for 144h, and the tank is placed. And separating and extracting the obtained fermentation liquor to obtain the sisomicin.
The above slant medium (g/L): soluble starch 15, sucrose 5, dry yeast 2, potassium nitrate 2, magnesium sulfate 2, precipitated calcium carbonate 4, aspartyl 0.02, ferrous sulfate heptahydrate 0.02, monopotassium phosphate 0.4, agar 15, bran 35, and pH 7.5.
Seed medium (g/L): 25 parts of corn starch, 20 parts of soybean cake powder, 0.9 part of magnesium sulfate, 4 parts of precipitated calcium carbonate, 0.9 part of corn steep liquor dry powder and pH 7.3.
Fermentation medium (g/L): 9 parts of corn starch, 35 parts of dextrin, 35 parts of soybean cake powder, 1.5 parts of precipitated calcium carbonate, 0.4 part of magnesium chloride, 6 parts of corn starch powder, 0.02 part of cobalt chloride and 7.3 parts of pH value.
Feed medium (g/L): maltose 15, corn starch 25, soybean cake powder 23, methionine 0.8 and sodium chloride 0.9.
Example 3A method for producing sisomicin
The method comprises the following steps:
s1, inoculating micromonospora inonotus into a slant culture medium, and culturing for 15 days at 36 ℃;
s2, selecting a mature inclined plane, inoculating the mature inclined plane to a seed liquid culture medium, culturing the mature inclined plane on a shaking table at 36 ℃, culturing for 50h at 300rmp, inoculating the mature inclined plane to a fermentation tank;
s3, inoculating according to the inoculation amount of 20%, and after the inoculation is completed, placing the fermentation tank at 36 ℃ for culture;
s4, fermenting until 45h, 65h and 90h are supplemented with the fed-batch culture medium. In the whole fermentation process, the pressure of the fermentation tank is 0.07MPa, the ventilation quantity is 3.0VVM, the culture is carried out for 168 hours, and the tank is placed. And separating and extracting the obtained fermentation liquor to obtain the sisomicin.
The above slant medium (g/L): cocoa soluble starch 20, sucrose 8, dry yeast 3, potassium nitrate 3, magnesium sulfate 3, precipitated calcium carbonate 5, aspartyl 0.04, ferrous sulfate heptahydrate 0.03, monopotassium phosphate 0.5, agar 20, bran 40, pH 8.0.
Seed medium (g/L): 30 parts of corn starch, 25 parts of soybean cake powder, 1.2 parts of magnesium sulfate, 5 parts of precipitated calcium carbonate, 1.0 part of corn steep liquor dry powder and 7.6 parts of pH value.
Fermentation medium (g/L): 10 parts of corn starch, 40 parts of dextrin, 40 parts of soybean cake powder, 2 parts of precipitated calcium carbonate, 0.5 part of magnesium chloride, 7 parts of corn pulp powder, 0.03 part of cobalt chloride and 7.6 parts of pH.
Feed medium (g/L): maltose 20, corn starch 30, soybean cake powder 26, methionine 1 and sodium chloride 1.
What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (8)
1. A method for producing sisomicin is characterized by comprising the following steps:
s1, inoculating micromonospora inyoensis into a slant seed culture medium, and culturing for 9-15 days at the temperature of 32-36 ℃ under the condition of 200-300 rmp;
s2, inoculating the seeds on a seed culture medium of a first-level seed tank according to the inoculation amount of 10-15%, wherein the temperature is 32-36 ℃, the rotating speed is 200-300 rmp, the tank pressure is 0.03-0.07 Mpa, the air volume is 1: 1.0-1: 3.0vvm, and the culture time is 40-50 h;
s3, inoculating the mycelia on a fermentation medium of a fermentation tank according to the inoculation amount of 10-20%, wherein the temperature is 32-36 ℃, the temperature is properly adjusted when the mycelia grow too fast or too slow, and the culture time is 120-168 hours;
s4, when the fermentation is carried out for 40-45h, 60-65h and 80-90h, the supplementary culture medium is supplemented.
2. The method for producing sisomicin according to claim 1, wherein the weight ratio (g/L) of the slant culture medium in step S1 is: 10-20 parts of soluble starch, 3-8 parts of sucrose, 1-3 parts of dry yeast, 1-3 parts of potassium nitrate, 1-3 parts of magnesium sulfate, 3-5 parts of precipitated calcium carbonate, 0.01-0.04 part of aspartyl, 0.01-0.03 part of ferrous sulfate heptahydrate, 0.3-0.5 part of monopotassium phosphate, 10-20 parts of agar, 30-40 parts of bran and 7.0-8.0 parts of pH.
3. The method for producing sisomicin according to claim 1, wherein the weight ratio (g/L) of the seed culture medium in the step S2 is: 20-30 parts of corn starch, 15-25 parts of soybean cake powder, 0.8-1.2 parts of magnesium sulfate, 3-5 parts of precipitated calcium carbonate, 0.8-1.0 part of corn steep liquor dry powder and pH 7.0-7.6.
4. The method for producing sisomicin according to claim 1, wherein the fermentation medium of step S3 has the following weight ratio (g/L): 8-10 parts of corn starch, 30-40 parts of dextrin, 30-40 parts of soybean cake powder, 1-2 parts of precipitated calcium carbonate, 0.3-0.5 part of magnesium chloride, 5-7 parts of corn starch powder, 0.01-0.03 part of cobalt chloride and 7.0-7.6 parts of pH value.
5. The method for producing sisomicin according to claim 1, wherein the weight ratio (g/L) of the feed medium in step S4 is: 10-20 parts of maltose, 20-30 parts of corn starch, 20-26 parts of soybean cake powder, 0.6-1 part of methionine and 0.8-1 part of sodium chloride.
6. The method for producing sisomicin as defined in claim 1, wherein the amount of the inoculated sisomicin is 10 to 20% in the step S3.
7. The method for producing sisomicin as defined in claim 1, wherein the fermentation tank in step S3 has a tank pressure of 0.03 to 0.07Mpa and an air flow of 1:0.2 to 1:3.0 vvm.
8. The method for producing sisomicin according to claim 1, wherein the weight ratio (g/L) of the slant culture medium in step S1 is: 10-15 parts of soluble starch, 3-5 parts of sucrose, 1-2 parts of dry yeast, 1-2 parts of potassium nitrate, 1-2 parts of magnesium sulfate, 3-4 parts of precipitated calcium carbonate, 0.01-0.02 part of aspartyl, 0.01-0.02 part of ferrous sulfate heptahydrate, 0.3-0.4 part of monopotassium phosphate, 10-15 parts of agar, 30-35 parts of bran and 7.0-7.5 of pH.
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| CN202010818274.7A CN111893154A (en) | 2020-08-14 | 2020-08-14 | Method for producing sisomicin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115181703A (en) * | 2022-07-18 | 2022-10-14 | 江南大学 | Solid culture medium for rapid growth of micromonospora for sisomicin production |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102505032A (en) * | 2011-11-03 | 2012-06-20 | 福建省建阳武夷味精有限公司 | Method for supplementing seed liquid in sisomicin fermentation process |
| CN102618473A (en) * | 2012-04-06 | 2012-08-01 | 福州大学 | Mutant strain for producing sisomicin |
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2020
- 2020-08-14 CN CN202010818274.7A patent/CN111893154A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102505032A (en) * | 2011-11-03 | 2012-06-20 | 福建省建阳武夷味精有限公司 | Method for supplementing seed liquid in sisomicin fermentation process |
| CN102618473A (en) * | 2012-04-06 | 2012-08-01 | 福州大学 | Mutant strain for producing sisomicin |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115181703A (en) * | 2022-07-18 | 2022-10-14 | 江南大学 | Solid culture medium for rapid growth of micromonospora for sisomicin production |
| CN115181703B (en) * | 2022-07-18 | 2023-10-27 | 江南大学 | Solid medium for fast growth of micromonospora for sisomicin production |
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Address after: 219 Furong Zhongsi Road, Xishan Economic and Technological Development Zone, Wuxi City, Jiangsu Province, 214000 Applicant after: Zhuohe Pharmaceutical Group Co.,Ltd. Address before: 219 Furong Zhongsi Road, Xishan Economic and Technological Development Zone, Wuxi City, Jiangsu Province, 214000 Applicant before: Zhuohe Pharmaceutical Group Co.,Ltd. |
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