CN113755548A - Method for improving fermentation level of polymyxin B - Google Patents
Method for improving fermentation level of polymyxin B Download PDFInfo
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- CN113755548A CN113755548A CN202111024467.6A CN202111024467A CN113755548A CN 113755548 A CN113755548 A CN 113755548A CN 202111024467 A CN202111024467 A CN 202111024467A CN 113755548 A CN113755548 A CN 113755548A
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- fermentation
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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
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
- C07K7/62—Polymyxins; Related peptides
Abstract
The invention provides a method for improving fermentation level of polymyxin B, which comprises a fermentation medium and fermentation process control, wherein the fermentation medium contains basic components consisting of a carbon source, a nitrogen source and inorganic salts and antibiotics. The fermentation process control comprises the following steps: the fermentation inoculum size of polymyxin B is 7%, the culture temperature is 30 ℃, the dissolved oxygen level is controlled to be more than 30%, the pH is controlled to be 6.0, and the tank pressure is 0.04-0.06 MPa; adding antibiotics when fermenting and culturing for 16-20 h. The streptomycin is supplemented in the normal fermentation process, amino acid substances in bacteria are inhibited from turning to the synthesis direction of common protein, more amino acid precursor substances are accumulated, and the amino acid precursor substances are promoted to be transferred to the synthesis direction of polymyxin B, so that the fermentation titer level is improved. The method adopted by the invention belongs to simple and easy-to-realize operation in the fermentation industry, and is easy to understand and fast master for production operators.
Description
Technical Field
The invention belongs to the technical field of biological fermentation engineering, and particularly relates to a method for improving fermentation level of polymyxin B.
Background
Polymyxin B is a polypeptide antibiotic which is firstly separated from Paenibacillus polymyxa in 1947, and due to the use of antibacterial drugs, bacteria have wide drug resistance to the antibacterial drugs, and even super bacteria which are resistant to almost all clinically available antibacterial drugs appear. With the increasing number of drug-resistant strains, the drug resistance degree is also continuously enhanced, and particularly gram-negative bacilli often show multiple drug resistance. However, in recent clinical research, common gram-negative bacilli still maintain high sensitivity to polymyxin B, and since the bacilli come into the market, the market demand is always rising and is strongly increased.
At present, only a few domestic polymyxin B can be produced, the yield is limited, domestic preparation manufacturers mainly rely on raw materials imported from foreign countries to meet the requirements, the domestic production cost is high, the market competitiveness is weak, and besides the quality problem, the low fermentation level is also an important reason, so that the polymyxin B fermentation process needs to be further optimized, the fermentation level is improved, the fermentation capacity is expanded, and the production cost is reduced to meet the market requirements.
At present, domestic research on fermentation processes of polymyxin B mainly focuses on screening and transformation of strains and adjustment and optimization of a fermentation basic formula, for example, patent CN111100823B and patent CN101235407B optimize the fermentation basic formula and improve the fermentation unit of polymyxin B, but the basic formula adjustment is mainly optimized for specific strains, certain differences exist among different strains, formula screening and optimization are needed to be carried out again for different strains, and the method is not suitable for large-scale popularization and rapid application.
It can be seen from the structural composition of polymyxin B that the polymyxin B mainly comprises amino acids, and the fatty acid chain part is evolved by taking the amino acids as precursors, so that the concentration of the amino acids in the bacteria cells generated by the polymyxin B is increased, and the polymyxin B is bound to play a certain role in synthesis and accumulation. The polymyxin B synthesis process does not need the participation of intracellular ribosome, and is different from the synthesis process of intracellular common protein. The streptomycin, the tetracycline and the like mainly inhibit the function of the intracellular ribosome, and the invention assumes that the antibiotics are added to generate a certain inhibiting effect on the intracellular ribosome generated by the polymyxin B, so that the flow of the intracellular amino acid precursor substances to a protein synthesis way is reduced, more amino acid precursor substances are accumulated, the amino acid precursor substances are promoted to be transferred to the synthesis direction of the polymyxin B, and the fermentation level of the polymyxin B is improved. According to the invention, through experimental research, the process of adding streptomycin in the fermentation process is finally determined to improve the fermentation level of polymyxin B.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for improving the fermentation level of polymyxin B, which has the advantages of simple process, easy operation, low production cost and contribution to industrial popularization.
The technical scheme adopted by the invention is as follows: a method for improving fermentation level of polymyxin B comprises a fermentation medium and fermentation process control, wherein the fermentation medium contains basic components consisting of carbon sources, nitrogen sources and inorganic salts and antibiotics.
Further, the basic components of the fermentation medium are as follows: 100-140g/L of wheat flour, 25-30g/L of soybean meal, 30-40g/L of ammonium sulfate, 10-20g/L of corn steep liquor, 0.5-0.8g/L of monopotassium phosphate, 0.4-0.6g/L of magnesium sulfate and 5-7g/L of calcium carbonate.
Further, the fermentation process control comprises: the fermentation inoculum size of polymyxin B is 7%, the culture temperature is 30 ℃, the dissolved oxygen level is controlled to be more than 30% in the whole process, the pH is controlled to be 6.0 automatically, and the tank pressure is 0.04-0.06 MPa; adding antibiotics when fermenting and culturing for 16-20 h.
Further, antibiotics are added when fermentation culture is carried out for 18 hours in the fermentation process control.
Further, the antibiotic is one of streptomycin, erythromycin or tetracycline.
Further, the antibiotic is streptomycin.
Further, the concentration of the streptomycin is 0.04-0.06 mug/mL.
Further, the concentration of streptomycin was 0.05. mu.g/mL.
The beneficial effects obtained by the invention are as follows: 1) improving the fermentation level: by analyzing the synthesis mechanism of polymyxin B, the streptomycin is supplemented in a normal fermentation process, amino acid substances in bacteria are inhibited from being shifted to the direction of synthesizing common protein, more amino acid precursor substances are accumulated, and the amino acid precursor substances are promoted to be shifted to the direction of synthesizing polymyxin B, so that the fermentation titer level is improved. 2) The process is simple and easy to realize: the method adopted by the invention belongs to simple and easy-to-realize operation in the fermentation industry, and is easy to understand and fast master for production operators. 3) The production cost is reduced: by adopting the method, the yield of polymyxin B in the fermentation stage is obviously improved, and the relative product cost is further reduced.
The invention comprehensively analyzes the synthesis and metabolism paths of intracellular amino acid by analyzing the synthesis path of polymyxin B, determines that antibiotics such as streptomycin can generate inhibition effect on intracellular ribosome, accumulates more amino acid precursors for the synthesis of polymyxin B and promotes the improvement of fermentation level of polymyxin B. Combining with experimental results, the addition period and the addition concentration of the streptomycin are studied in multiple batches, and the fact that the synthesis of polymyxin B can be improved by adding the streptomycin with the final concentration of 0.05 mu g/mL for 18 hours is determined, and the growth and metabolism of bacteria are not obviously affected. The early or excessive addition of the polymyxin B can generate great influence on the growth and metabolism of the bacteria, directly influence the bacterial quantity or the activity of the bacteria and also generate influence on the accumulation of the polymyxin B; the effect of promoting polymyxin B synthesis cannot be generated at too late or in a small amount.
Drawings
FIG. 1 is a liquid phase diagram of the results of example 1 of the present invention;
FIG. 2 is a liquid phase diagram of the results of example 2 of the present invention;
FIG. 3 is a liquid phase diagram of the results of example 3 of the present invention;
FIG. 4 is a liquid phase diagram of the results of example 4 of the present invention;
FIG. 5 is a liquid phase diagram of the results of example 5 of the present invention;
FIG. 6 is a liquid phase diagram of the results of example 6 according to the present invention;
FIG. 7 is a liquid phase diagram of the results of example 7 according to the present invention;
FIG. 8 is a liquid phase diagram of the results of example 8 of the present invention;
FIG. 9 is a liquid phase diagram of the results of example 9 of the present invention;
FIG. 10 is a liquid phase diagram of the results of example 10 of the present invention;
FIG. 11 is a liquid phase diagram of the results of example 11 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1
Respectively weighing 140g/L of wheat flour, 25-30g/L of soybean meal, 30-40g/L of ammonium sulfate, 10-20g/L of corn steep liquor, 0.5-0.8g/L of potassium dihydrogen phosphate, 0.4-0.6g/L of magnesium sulfate and 5-7g/L of calcium carbonate according to the composition of a fermentation medium, putting the materials into a fermentation tank, adding water to prepare the fermentation medium, uniformly stirring, sterilizing at high temperature, cooling to room temperature, and inoculating the cultured seed liquid into the fermentation medium. Inoculating 7%, culturing at 30 deg.C, introducing sterile air, controlling dissolved oxygen level to be greater than 30%, controlling pH to 6.0, and tank pressure to be 0.04-0.06MPa, culturing for 48 hr, fermenting to obtain polymyxin B fermentation liquid, and detecting polymyxin B content in the fermentation liquid.
Example 2
The difference from the embodiment 1 is that: after the fermentation medium was sterilized, streptomycin was added to the medium at a sterilized final concentration of 0.05. mu.g/mL prior to inoculation.
Example 3
The difference from the embodiment 1 is that: at the 12h fermentation culture period, the sterilized streptomycin was added to a final concentration of 0.05. mu.g/mL.
Example 4
The difference from the embodiment 1 is that: at the 18h fermentation culture period, the sterilized streptomycin was added to a final concentration of 0.05. mu.g/mL.
Example 5
The difference from the embodiment 1 is that: at the time of the fermentation culture period of 24 hours, a sterilized streptomycin was added to a final concentration of 0.05. mu.g/mL.
Example 6
The difference from the embodiment 1 is that: at the 18h fermentation culture period, the sterilized streptomycin was added to a final concentration of 0.03. mu.g/mL.
Example 7
The difference from the embodiment 1 is that: at the 18h fermentation culture period, the sterilized streptomycin was added to a final concentration of 0.04. mu.g/mL.
Example 8
The difference from the embodiment 1 is that: at the 18h fermentation culture period, the sterilized streptomycin was added to a final concentration of 0.06. mu.g/mL.
Example 9
The difference from the embodiment 1 is that: at the 18h fermentation culture period, the sterilized streptomycin was added to a final concentration of 0.07. mu.g/mL.
Example 10
The difference from the embodiment 1 is that: after the fermentation medium was sterilized, tetracycline was added at a final sterilized concentration of 0.05. mu.g/mL prior to inoculation.
Example 11
The difference from the embodiment 1 is that: after the fermentation medium was sterilized, erythromycin at a final sterilized concentration of 0.05. mu.g/mL was added before inoculation.
The results of the polymyxin B contents obtained in examples 1 to 11 are shown in Table 1
TABLE 1 polymyxin B content results obtained in the different examples
| Experimental group | Content (× 10)4U/mL) | Increased proportion (%) |
| Example 1 | 2.23 | / |
| Example 2 | 0.57 | -74.44 |
| Example 3 | 2.41 | 8.07 |
| Example 4 | 2.95 | 32.29 |
| Example 5 | 2.3 | 3.14 |
| Example 6 | 2.46 | 10.31 |
| Example 7 | 2.82 | 26.46 |
| Example 8 | 2.56 | 14.80 |
| Example 9 | 1.84 | -17.49 |
| Example 10 | 2.59 | 16.14 |
| Example 11 | 2.63 | 17.94 |
From a liquid chromatogram, the polymyxin B belongs to a multi-component product, the main components respectively peak at about 14min, 16.5min, 23min and 29min, and the sum of the four main components is taken as the potency result of the polymyxin B. The data result shows that the addition of streptomycin before inoculation has great influence on the accumulation of polymyxin B in the fermentation process, the total peak area of main components is obviously reduced by comparing the liquid chromatogram 2 with the liquid chromatogram 1, mainly because the early fermentation bacteria amount is less, the streptomycin has obvious inhibition effect on the normal metabolic growth of bacteria, and because the bacteria amount is less, the content of polymyxin B accumulated in the fermentation liquid is less until the culture is finished. In order to reduce the influence of streptomycin on the normal growth of thalli, a process supplementing mode is adopted, and as can be seen from the results of liquid chromatogram 3, 4 and 5, the titer is increased along with the delay of a supplementing period, but the effect of improving the result is not obvious after too late supplementing, wherein the streptomycin is supplemented most suitably when the fermentation culture is carried out for about 18 hours, the effect of promoting the accumulation of polymyxin B is best, the content of polymyxin B in the fermentation liquor is effectively improved by 32.29%, the total amount of the peak areas of four main components in the corresponding liquid chromatogram 5 is obviously improved, and particularly the peak areas of the components in about 29min are obviously improved. Under the condition, streptomycin with different concentrations also has different influences on the accumulation of polymyxin B in the fermentation process, the addition amount is small, the improvement range of the content of the polymyxin B is small, and liquid phase maps 6 and 7 respectively show the results of maps when the addition amount is lower than the optimal addition amount; when the liquid phase maps 8 and 9 respectively show that the addition amount is higher than the optimum addition amount, the peak areas of the four main components are obviously reduced relative to the optimum addition amount, which shows that the addition amount is too much, the normal metabolic function of thalli can be directly inhibited, the thalli are died prematurely, the polymyxin B content is too low, and therefore, the final concentration of 0.05 mu g/mL is most suitable. The liquid phase map figures 10 and 11 show that under the same conditions, the effect of streptomycin is the best, the effect of the polymyxin B accumulation in the fermentation liquor is the most obvious, the peak area sum of the four main components in the obtained fermentation liquor is the highest, the erythromycin is the next component, and the tetracycline is the last component.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. A method for improving fermentation level of polymyxin B comprises fermentation medium and fermentation process control, and is characterized in that: the fermentation medium contains basic components consisting of carbon sources, nitrogen sources and inorganic salts and antibiotics.
2. The method for improving fermentation level of polymyxin B according to claim 1, wherein: the basic components of the fermentation culture medium are as follows: 100-140g/L of wheat flour, 25-30g/L of soybean meal, 30-40g/L of ammonium sulfate, 10-20g/L of corn steep liquor, 0.5-0.8g/L of monopotassium phosphate, 0.4-0.6g/L of magnesium sulfate and 5-7g/L of calcium carbonate.
3. The method for improving fermentation level of polymyxin B according to claim 1, wherein: the fermentation process control comprises the following steps: the fermentation inoculum size of polymyxin B is 7%, the culture temperature is 30 ℃, the dissolved oxygen level is controlled to be more than 30% in the whole process, the pH is controlled to be 6.0 automatically, and the tank pressure is 0.04-0.06 MPa; adding antibiotics when fermenting and culturing for 16-20 h.
4. The method for increasing fermentation level of polymyxin B according to claim 3, wherein: and adding antibiotics when the fermentation culture is carried out for 18 hours in the control of the fermentation process.
5. The method for increasing fermentation level of polymyxin B according to any one of claims 1-4, wherein: the antibiotic is one of streptomycin, erythromycin or tetracycline.
6. The method for increasing fermentation level of polymyxin B according to any one of claims 1-4, wherein: the antibiotic is streptomycin.
7. The method for increasing fermentation level of polymyxin B according to claim 6, wherein: the concentration of the streptomycin is 0.04-0.06 mug/mL.
8. The method for increasing fermentation level of polymyxin B according to claim 7, wherein: the concentration of streptomycin is 0.05 mug/mL.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114317648A (en) * | 2022-01-26 | 2022-04-12 | 河北圣雪大成制药有限责任公司 | Method for improving fermentation level of polymyxin E |
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- 2021-09-02 CN CN202111024467.6A patent/CN113755548A/en active Pending
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317648A (en) * | 2022-01-26 | 2022-04-12 | 河北圣雪大成制药有限责任公司 | Method for improving fermentation level of polymyxin E |
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