CN111394412B - Method for reducing neomycin C content in neomycin fermentation process - Google Patents
Method for reducing neomycin C content in neomycin fermentation process Download PDFInfo
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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
- C12P19/50—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 having two saccharide radicals bound through only oxygen to adjacent ring carbon atoms of the cyclohexyl radical, e.g. ambutyrosin, ribostamycin
- C12P19/52—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 having two saccharide radicals bound through only oxygen to adjacent ring carbon atoms of the cyclohexyl radical, e.g. ambutyrosin, ribostamycin containing three or more saccharide radicals, e.g. neomycin, lividomycin
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Abstract
The invention relates to a method for reducing neomycin C content in a neomycin fermentation process, which comprises fermentation culture and a method for reducing neomycin C content by controlling dissolved oxygen in stages in the fermentation culture process, so as to obtain fermentation liquor with high titer and low neomycin C content. The method is simple to operate, does not additionally increase production cost on the basis of the original fermentation process, reduces the treatment difficulty of the later extraction process, improves the product yield and simplifies the process.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a method for reducing neomycin C content in a neomycin fermentation process.
Background
Neomycin is an aminoglycoside antibiotic obtained by a biological fermentation method, and three main components thereof are neomycin A, neomycin B and neomycin C respectively, wherein neomycin B is taken as the main component. The content of each component of neomycin is required in pharmacopoeia of each country, wherein the content of neomycin A is not more than 2%, and the content of neomycin C is not more than 15%. In the products obtained by the prior fermentation process, neomycin A basically does not exceed the standard range, but because neomycin C and neomycin B belong to stereoisomers, and neomycin B is synthesized from neomycin C through neomycin C epimerase, neomycin C is an essential precursor for neomycin B synthesis, and a synthesis route cannot bypass neomycin C through a technical means.
The structures of neomycin B and neomycin C are as follows:
the biological activity detection shows that the antibacterial activity of the neomycin C is only one third of that of the neomycin B, and the toxic and side effects are large, so that the neomycin C becomes one of the controlled impurities in the quality standards of various countries. The method for reducing neomycin C adopted in the current production mainly comprises the steps of extracting, for example, patent CN 104558068 provides a method for extracting high-quality neomycin and a device used by the method, wherein neomycin analysis solutions in different stages are screened by the method for extracting, so that a part with lower neomycin C content is obtained, finished product preparation is carried out, and the rest neomycin C content analysis solutions with higher neomycin content only can be used for preparing low-quality products. The essence of the method is that the method of extracting and analyzing liquid and screening at the same time is used for obtaining the high-quality neomycin, and the result of the method necessarily causes the cost increase of the high-quality neomycin or improves the complexity of the process. Through continuous research and improvement, there are also methods for directly reducing the content of neomycin C through fermentation, for example, patent CN 107557413 provides a method for producing quality of neomycin sulfate, which mainly comprises the steps of adding yeast powder and/or cysteine and/or S-adenosyl-L-methionine and/or ammonium sulfate and/or ferrous sulfate and/or magnesium sulfate and/or manganese sulfate in the fermentation culture process to obtain a fermentation liquor with relatively low neomycin C content, but the method increases the production cost of neomycin sulfate intangibly.
Disclosure of Invention
The invention aims to solve the technical problem of higher neomycin C content in the neomycin production process, and provides a novel neomycin C content control method which is different from the conventional neomycin C content control method, and by controlling the fermentation process culture mode, the fermentation liquor with low neomycin C content is obtained, so that a product meeting the EP standard can be obtained, the subsequent extraction process is simplified, the production cost and the process complexity are reduced, and the method is simple, effective and convenient to implement.
The technical scheme adopted for solving the technical problems is as follows:
the technical method adopted by the invention is as follows: a method for reducing neomycin C content during fermentation of neomycin, the method comprising the steps of:
1) Seed culture: inoculating the spore liquid into a seed tank, culturing at 35+/-1 ℃ for 35-40h to obtain seed liquid;
2) Fermentation culture: according to the composition of fermentation culture medium, 65-75g/L of long-shaped rice powder, 0.25-0.35g/L of amylase, 26-30g/L of peanut cake powder, 4-6g/L of soybean cake powder, 8-12g/L of soybean peptone, 15-25g/L of glucose, 6-10g/L of yeast powder, 0.3-0.5g/L of potassium dihydrogen phosphate, 5-7g/L of ammonium sulfate, 3.5-4.5g/L of calcium carbonate and 0.4-0.6g/L of defoaming agent are respectively weighed. Adding water into a fermentation tank to prepare a fermentation culture medium, stirring uniformly, sterilizing at high temperature, cooling to room temperature, inoculating the seed liquid obtained in the step 1) into the fermentation tank, fermenting and culturing at 35+/-1 ℃ under the pressure of 0.02-0.06MPa, introducing sterile air in the process, controlling a certain dissolved oxygen level, and fermenting for 200h to obtain a neomycin fermentation product.
3) The dissolved oxygen level selected in step 2) is: 0-30h, controlling the dissolved oxygen level to be 30-40%, and controlling the dissolved oxygen level to be 5-15% after 30 h-tank discharge.
The defoaming agent is one of polyether defoaming agent or organic silicon defoaming agent.
The invention provides a method for reducing neomycin C content in a neomycin fermentation process, which comprises the following technical steps:
1) The treatment difficulty of the extraction section is reduced:
according to the invention, the problem of higher neomycin C content is solved in the fermentation stage by optimizing the fermentation process control process, the fermentation liquor can meet the quality standard requirement, the difficulty of extraction treatment of the fermentation liquor is reduced, the part of analysis liquor with qualified neomycin C content is not required to be particularly collected, the analysis liquor with qualified neomycin C content can be obtained by adopting a conventional extraction treatment method, and the extraction process is simplified.
2) The complexity of the process is reduced:
compared with the method with similar results obtained by the patent, the method has the advantages that the operation procedure is not increased on the basis of the existing fermentation process, and the implementation of production operators is facilitated.
3) The production cost is reduced:
the method adopted by the invention reduces the difficulty of the extraction process, improves the product yield, reduces the operation of the fermentation process, and has a certain reduction effect on the production cost.
According to the invention, a reasonable dissolved oxygen adjustment period and adjustment range are finally determined through research and process optimization, and a plurality of batches of researches are carried out to determine that 30 hours is the optimal transition time point from primary metabolism to secondary metabolism of neomycin producing bacteria, at the moment, the influence of reducing the dissolved oxygen control level on bacterial concentration increase and titer is minimum, the growth of bacterial amount can be influenced by reducing the dissolved oxygen level at an earlier time point, bacterial amount is relatively less, titer is lower, and the later-stage production capacity of the fermentation tank is obviously reduced; the reduction of dissolved oxygen level at a later time point can lead to excessive thallus quantity, excessive nutrient consumption of fermentation liquor, vigorous primary metabolism and increased neomycin C content. And researches show that the dissolved oxygen level is controlled to be in a proper range of 5-15% in the productive phase, and the too low dissolved oxygen level is easy to cause obvious weakening of the metabolic activity of thalli, and the thalli enters the decay phase earlier, so that the later-stage production of the antibiotics is not facilitated; the dissolved oxygen control level is too high, the bacterial activity is vigorous, the neomycin C accumulation amount is large, and the standard range is easily exceeded.
Detailed Description
Drawings
FIG. 1 is a comparative schematic of the results of examples 1-7 of the present invention;
FIG. 2 is a comparative schematic of the results of examples 6, 8, 9 of the present invention.
The present invention is further illustrated below with reference to examples, but the present invention is not limited thereto. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
1) Seed culture: inoculating the spore liquid into a seed tank, culturing at 35+/-1 ℃ for 35-40h to obtain seed liquid;
2) Fermentation culture: according to the composition of fermentation culture medium, 65-75g/L of long-shaped rice powder, 0.25-0.35g/L of amylase, 26-30g/L of peanut cake powder, 4-6g/L of soybean cake powder, 8-12g/L of soybean peptone, 15-25g/L of glucose, 6-10g/L of yeast powder, 0.3-0.5g/L of potassium dihydrogen phosphate, 5-7g/L of ammonium sulfate, 3.5-4.5g/L of calcium carbonate and 0.4-0.6g/L of defoaming agent are respectively weighed. Adding water into a fermentation tank to prepare a fermentation culture medium, stirring uniformly, sterilizing at high temperature, cooling to room temperature, inoculating the seed liquid obtained in the step 1) into the fermentation tank, fermenting and culturing at 35+/-1 ℃ under the pressure of 0.02-0.06MPa, introducing sterile air during the process, and controlling a certain dissolved oxygen level: and (3) controlling the dissolved oxygen level to be 30-40% in the whole process, fermenting to 200h, and placing in a tank to obtain a neomycin fermentation product.
Example 2:
the difference from example 1 is that: in the step (2), the fermentation culture process controls a certain dissolved oxygen level as follows: 0-200h, 15-30%.
Example 3:
the difference from example 1 is that: in the step (2), the fermentation culture process controls a certain dissolved oxygen level as follows: 0-200h and 5-15%.
Example 4:
the difference from example 1 is that: in the step (2), the fermentation culture process controls a certain dissolved oxygen level as follows: 0-50h,30-40%,50h-200h and 5-15%.
Example 5:
the difference from example 1 is that: in the step (2), the fermentation culture process controls a certain dissolved oxygen level as follows: 0-40h,30-40%,40h-200h and 5-15%.
Example 6:
the difference from example 1 is that: in the step (2), the fermentation culture process controls a certain dissolved oxygen level as follows: 0-30h,30-40%,30h-200h and 5-15%.
Example 7:
the difference from example 1 is that: in the step (2), the fermentation culture process controls a certain dissolved oxygen level as follows: 0-20h,30-40%,20h-200h and 5-15%.
Example 8:
the difference from example 1 is that: in the step (2), the fermentation culture process controls a certain dissolved oxygen level as follows: 0-30h,30-40%,30h-200h and 0-5%.
Example 9
The difference from example 1 is that: in the step (2), the fermentation culture process controls a certain dissolved oxygen level as follows: 0-30h,30-40%,30h-200h and 15-25%.
The results of neomycin titers and neomycin B and neomycin C content obtained in examples 1-9 are shown in Table 1 and FIGS. 1-2
Table 1: influence of different dissolved oxygen control levels on neomycin fermentation units and component ratios in fermentation culture process
| Experimental group | Can release potency (U/mL) | Neomycin B content (%) | Neomycin C content (%) |
| Example 1 | 22850 | 71.25 | 25.28 |
| Example 2 | 20167 | 74.11 | 21.85 |
| Example 3 | 17323 | 88.79 | 8.13 |
| Example 4 | 22341 | 74.27 | 20.36 |
| Example 5 | 22311 | 78.19 | 18.03 |
| Example 6 | 21724 | 83.66 | 12.74 |
| Example 7 | 18357 | 85.79 | 9.94 |
| Example 8 | 18245 | 84.32 | 10.26 |
| Example 9 | 22018 | 78.36 | 16.68 |
From the data in Table 1 and the trend analysis in FIG. 1, it can be seen that, as the dissolved oxygen level is controlled to decrease in the fermentation process, the content of neomycin C gradually decreases, and the titer also decreases to some extent, wherein when the dissolved oxygen level decreases to 5-15%, the content of neomycin C is significantly reduced to a level below 10%. In order to balance the relation between the titer and the neomycin C content in the fermentation process, the influence of dissolved oxygen on the titer and the neomycin C content in different periods is examined by stages, the neomycin C content is in a decreasing trend along with the advance of a dissolved oxygen limiting time point, the titer is also decreased to a certain extent, especially after the dissolved oxygen level is limited for 20 hours, the titer is obviously decreased, analysis is considered to be mainly due to the premature limitation of the dissolved oxygen, the primary metabolism of thalli is weakened, the integral synthesis path of neomycin is obviously reduced, the accumulation amount of neomycin C is reduced, and the titer is obviously reduced. Examination of various limiting dissolved oxygen levels in FIG. 2 shows that increasing or decreasing the dissolved oxygen control level after 30 hours has an effect on potency or component C content, with a range of dissolved oxygen of 5-15% being most appropriate. And finally, analysis confirms that about 30 hours is proper to limit dissolved oxygen by 5-15%, at the moment, the concentration of the thalli reaches a certain level, the thalli starts to shift into secondary metabolism, no obvious influence is caused on the generation of potency, but the content of neomycin C can be controlled to a qualified level, so that the fermentation liquor reaches the product quality standard, the later extraction treatment difficulty is reduced, and the product yield and quality are improved.
Claims (2)
1. A method for reducing neomycin C content in a neomycin fermentation process, comprising a fermentation medium and a process culture method, the method comprising the steps of:
1) Seed culture: inoculating the spore liquid into a seed tank, culturing at 35+/-1 ℃ for 35-40h to obtain seed liquid;
2) Fermentation culture: according to the composition of a fermentation medium, 65-75g/L of long-shaped rice powder, 0.25-0.35g/L of amylase, 26-30g/L of peanut cake powder, 4-6g/L of soybean cake powder, 8-12g/L of soybean peptone, 15-25g/L of glucose, 6-10g/L of yeast powder, 0.3-0.5g/L of monopotassium phosphate, 5-7g/L of ammonium sulfate, 3.5-4.5g/L of calcium carbonate and 0.4-0.6g/L of defoamer are respectively weighed, put into a fermentation tank, added with water to prepare the fermentation medium, stirred evenly, sterilized at high temperature, after being cooled to room temperature, the seed liquid obtained in the step 1) is put into the fermentation tank for fermentation culture, the fermentation temperature is 35+/-1 ℃, the tank pressure is 0.02-0.06MPa, a certain dissolved oxygen level is controlled in the process, and the fermentation tank is put into a tank for 200h to obtain a neomycin fermentation product;
3) The dissolved oxygen level selected in step 2) is: 0-30h, controlling the dissolved oxygen level to be 30-40%, and controlling the dissolved oxygen level to be 5-15% after 30-200 h.
2. The method for reducing the content of neomycin C in a neomycin fermentation process according to claim 1, wherein the defoamer is one of a polyether defoamer GP type or a polyether defoamer GPE type or a polyether defoamer GPES type or a solid silicone defoamer or a liquid silicone defoamer.
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| CN112226475A (en) * | 2020-09-27 | 2021-01-15 | 山东齐发药业有限公司 | Production method for improving neomycin yield |
| CN112410390B (en) * | 2020-11-30 | 2022-08-23 | 浙江孚诺医药股份有限公司 | Fermentation process of neomycin sulfate |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1222440A (en) * | 1968-05-31 | 1971-02-10 | Corn Products Co | Production of neomycin, nutrient medium used therein and process for preparing medium |
| CN101210260A (en) * | 2007-12-25 | 2008-07-02 | 福州大学 | Cleaning fermentation technique for neomycin |
| CN104558068A (en) * | 2014-12-19 | 2015-04-29 | 宜昌三峡制药有限公司 | Method for extracting high-quality neomycin and device adopted thereby |
| CN107557413A (en) * | 2017-10-16 | 2018-01-09 | 河北圣雪大成制药有限责任公司 | A kind of method for improving the neomycinsulphate quality of production |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1222440A (en) * | 1968-05-31 | 1971-02-10 | Corn Products Co | Production of neomycin, nutrient medium used therein and process for preparing medium |
| CN101210260A (en) * | 2007-12-25 | 2008-07-02 | 福州大学 | Cleaning fermentation technique for neomycin |
| CN104558068A (en) * | 2014-12-19 | 2015-04-29 | 宜昌三峡制药有限公司 | Method for extracting high-quality neomycin and device adopted thereby |
| CN107557413A (en) * | 2017-10-16 | 2018-01-09 | 河北圣雪大成制药有限责任公司 | A kind of method for improving the neomycinsulphate quality of production |
Non-Patent Citations (1)
| Title |
|---|
| Characterization of a Radical S‑Adenosyl‑L‑methionine Epimerase, NeoN, in the Last Step of Neomycin B Biosynthesis;Fumitaka Kudo等;J. Am. Chem. Soc.;第136卷;第13909−13915页 * |
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