CN103882081A - Method for improving bacitracin valence through continuous flowing fed-batch fermentation - Google Patents
Method for improving bacitracin valence through continuous flowing fed-batch fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 67
- 230000004151 fermentation Effects 0.000 title claims abstract description 67
- 108010001478 Bacitracin Proteins 0.000 title claims abstract description 37
- 229960003071 bacitracin Drugs 0.000 title claims abstract description 37
- 229930184125 bacitracin Natural products 0.000 title claims abstract description 37
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 19
- 239000000725 suspension Substances 0.000 claims abstract description 18
- 241000894006 Bacteria Species 0.000 claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 238000011218 seed culture Methods 0.000 claims abstract description 9
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000006052 feed supplement Substances 0.000 claims description 25
- 239000000843 powder Substances 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 11
- 210000000582 semen Anatomy 0.000 claims description 9
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 8
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 8
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 238000012262 fermentative production Methods 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 2
- 229920001817 Agar Polymers 0.000 claims description 2
- 244000068988 Glycine max Species 0.000 claims description 2
- 235000010469 Glycine max Nutrition 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 230000001133 acceleration Effects 0.000 claims description 2
- 239000008272 agar Substances 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 230000001186 cumulative effect Effects 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 238000012258 culturing Methods 0.000 abstract 2
- 239000000463 material Substances 0.000 abstract 1
- 239000008223 sterile water Substances 0.000 abstract 1
- 230000009469 supplementation Effects 0.000 abstract 1
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 235000008935 nutritious Nutrition 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
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- 229910052799 carbon Inorganic materials 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- 230000029142 excretion Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention discloses a method for improving the bacitracin valence through continuous flowing fed-batch fermentation. The method comprises the following steps: a, performing slant culture: inoculating bacillus licheniformis into a slant culture medium, and culturing for 24 hours at 37 DEG C; b, preparing bacterium suspension: preparing the slant strain prepared in the step a into bacterium suspension by using sterile water; c, performing one-class seed culture: inoculating the bacterium suspension prepared in the step b into a one-class seed jog with a seed culture medium, and culturing for 16 hours under the conditions that the temperature is 37 DEG C, the rotation speed is 600rpm and the gas supply amount is 0.5vvm; d, performing fed-batch fermentation: inoculating the one-class seeds prepared in the step c into a fermentation tank with a fermentation culture medium according to a volume ratio of 10%, fermenting under the conditions that the temperature is 37 DEG C, the rotation speed is 600rpm and the gas supply amount is 1.0vvm, when the strain enters into an index growth period, starting supplementing materials at uniform speed, after the supplementation, controlling the pH value to be 7.0-7.5, and stopping fermentation after 40 hours; and e, detecting fermentation unit: detecting the fermentation unit of bacitracin, and comparing the fermentation unit of bacitracin with the fermentation unit of the bacitracin without fed-batch fermentation in production.
Description
Technical field
The invention belongs to microorganism fermentation field, be specifically related to a kind of Continuous Flow and add the method that fed-batch fermentation raising bacitracin is tired.
Background technology
Bacitracin belongs to polypeptide antibiotics, it is the unsettled polypeptide being combined into by multiple amino acids, gram-positive microorganism and part negative bacterium are had to strong restraining effect, it can anti-bacteria cell walls synthetic, affect its protein synthesis and cell membrane function, thereby reach the object of eliminating pathogenic bacteria.Bacitracin is made up of multiple amino acid, because it is efficient, have no side effect, noresidue, do not develop immunity to drugs and cross resistance, excretion be fast etc. that advantage is widely used in livestock and poultry breeding industry.
Bacitracin fermentative production is used Bacillus licheniformis, take W-Gum, bean cake powder as main carbon and nitrogen sources, culture temperature is 37 ℃, through 40h oxygen consumption fermentation culture biosynthesizing bacitracin, but fermentation level is not high, production energy consumption and production cost are high, are mainly the optimization of fermention medium at present to the research of Production by Microorganism Fermentation bacitracin, or the optimization of fermentation condition, does not obtain good effect; Also the bacterial strain of high yield bacitracin screens in the factory having by traditional selection by mutation, although fermentation unit improves, the vigor of bacterial strain is but in continuous decline.
Summary of the invention
The invention provides a kind of Continuous Flow and add the method that fed-batch fermentation raising bacitracin is tired, its object is to overcome the deficiencies in the prior art part, improves fermentation unit, reduces production energy consumption and production cost.
The present invention is that the technical scheme that realizes its object employing is:
Continuous Flow adds fed-batch fermentation and improves the method that bacitracin is tired, and comprises following operation steps:
A, slant culture: Bacillus licheniformis is inoculated on slant medium, at 37 ℃, cultivates 24h;
B, prepare bacteria suspension: wash down and make bacteria suspension by the slant strains that sterilized water is prepared step a;
C, first order seed are cultivated: the bacterial suspension inoculation of preparing in step b, in the first class seed pot of seed culture medium is housed, is cultivated to 16h under the condition of 37 ℃ of temperature, rotating speed 600rpm, air flow 0.5vvm;
D, fed-batch fermentation: the first order seed of preparing in step c is inoculated according to the inoculum size of volume ratio 10% in the fermentor tank that fermention medium is housed, under the condition of 37 ℃ of temperature, rotating speed 600rpm, air flow 1.0vvm, ferment, when thalline enters exponential growth after date, start at the uniform velocity flow feeding, feed supplement finishes rear control pH7.0-7.5, and 40h stops fermentation;
E, detect fermentation unit: after fermentation ends, detect the fermentation unit of bacitracin, and compare with the fermentation unit of the bacitracin of no-feed supplement fermentative production.
In step a, the composition of slant medium used (m/v) and consumption are: glucose 1%, peptone 1.5%, extractum carnis 1%, yeast extract powder 0.2%, agar 2.0%, the pH7.0 of substratum.
In step c, the raw material of seed culture medium used (m/v) and consumption are: soybean cake powder 3%, Semen Maydis powder 2%, W-Gum 1%, corn steep liquor 0.5%, ammonium sulfate 0.15%, calcium carbonate 0.3%, the pH6.8 of substratum.
In steps d, the composition of fermention medium used (m/v) and consumption are: bean cake powder 9%, Semen Maydis powder 2%, W-Gum 3%, ammonium sulfate 0.3%, magnesium sulfate 0.003%, calcium carbonate 0.5%, the pH7.2 of substratum, the volume of fermention medium is the 50-80% of fermentor tank volume.
Feed supplement (m/v) composition and consumption described in steps d are: bean cake powder 6-9%, Semen Maydis powder 0.5-1%, W-Gum 2-3%, corn steep liquor 0.3-0.5%, glucose 5-8%, feed supplement pH is 5.5-6.5, feed supplement flow acceleration is the 1-2% that stream per hour adds fermentating liquid volume, and stream is with the 10%-20% of fermented liquid cumulative volume altogether.
In steps d feed supplement finish after by adjusting rotary speed control pH7.0-7.5.
The invention has the beneficial effects as follows: method provided by the invention has improved fermentation level, and feed supplement composition is simple, cheap, when fermentation ends, detect nutritive substance substantially depleted, alleviated the contradiction of the quick-acting nutritious deficiency of logarithmic phase simultaneously, be conducive to the raising of biomass, reduce production energy consumption and production cost, bacitracin output has raising by a relatively large margin compared with no-feed supplement.
Embodiment
The invention provides a kind of Continuous Flow and add the method that fed-batch fermentation raising bacitracin is tired, its object is to overcome the deficiencies in the prior art part, improve fermentation unit, reduce production energy consumption and production cost, below in conjunction with specific embodiment, the invention will be further described.
Specific embodiment 1, a kind of Continuous Flow adds fed-batch fermentation and improves the method that bacitracin is tired, and comprises following operation steps:
A, slant culture: Bacillus licheniformis is inoculated on slant medium, at 37 ℃, cultivates 24h;
B, prepare bacteria suspension: wash down and make bacteria suspension by the slant strains that sterilized water is prepared step a;
C, first order seed are cultivated: the bacterial suspension inoculation of preparing in step b, in the 15L first class seed pot of 10L seed culture medium is housed, is cultivated to 16h under the condition of 37 ℃ of temperature, rotating speed 600rpm, air flow 0.5vvm;
D, fed-batch fermentation: the first order seed of preparing in step c is inoculated according to the inoculum size of volume ratio 10% in the 50L fermentor tank that 30L fermention medium is housed, 37 ℃ of temperature, rotating speed 600rpm, under the condition of air flow 1.0vvm, cultivate after 3h, start take the speed of 0.3L/h at the uniform velocity stream add composition and content as bean cake powder 6%, Semen Maydis powder 0.5%, W-Gum 3%, corn steep liquor 0.3%, the feed supplement (m/v) of glucose 7%, stream adds 3L feed supplement altogether, 13h stream adds end, then within pH being controlled to the scope of 7.0-7.5 by adjusting rotary speed, 40h stops fermentation,
E, detection fermentation unit: the fermentation unit that detects bacitracin after fermentation ends is 959U/ml, and the fermentation unit of the bacitracin of no-feed supplement fermentative production is 830U/ml.
Specific embodiment 2, a kind of Continuous Flow adds fed-batch fermentation and improves the method that bacitracin is tired, and comprises following operation steps:
A, slant culture: Bacillus licheniformis is inoculated on slant medium, at 37 ℃, cultivates 24h;
B, prepare bacteria suspension: wash down and make bacteria suspension by the slant strains that sterilized water is prepared step a;
C, first order seed are cultivated: the bacterial suspension inoculation of preparing in step b, in the 15L first class seed pot of 10L seed culture medium is housed, is cultivated to 16h under the condition of 37 ℃ of temperature, rotating speed 600rpm, air flow 0.5vvm;
D, fed-batch fermentation: the first order seed of preparing in step c is inoculated according to the inoculum size of volume ratio 10% in the 50L fermentor tank that 30L fermention medium is housed, 37 ℃ of temperature, rotating speed 600rpm, under the condition of air flow 1.0vvm, cultivate after 3h, start take the speed of 0.6L/h at the uniform velocity stream add composition and content as bean cake powder 9%, Semen Maydis powder 0.6%, W-Gum 2.3%, corn steep liquor 0.5%, the feed supplement (m/v) of glucose 5%, stream adds 5L feed supplement altogether, 11.5h stream adds end, then within pH being controlled to the scope of 7.0-7.5 by adjusting rotary speed, 40h stops fermentation,
E, detection fermentation unit: the fermentation unit that detects bacitracin after fermentation ends is 1036U/ml, and the fermentation unit of the bacitracin of no-feed supplement fermentative production is 830U/ml.
Specific embodiment 3, a kind of Continuous Flow adds fed-batch fermentation and improves the method that bacitracin is tired, and comprises following operation steps:
A, slant culture: Bacillus licheniformis is inoculated on slant medium, at 37 ℃, cultivates 24h;
B, prepare bacteria suspension: wash down and make bacteria suspension by the slant strains that sterilized water is prepared step a;
C, first order seed are cultivated: the bacterial suspension inoculation of preparing in step b, in the 15L first class seed pot of 10L seed culture medium is housed, is cultivated to 16h under the condition of 37 ℃ of temperature, rotating speed 600rpm, air flow 0.5vvm;
D, fed-batch fermentation: the first order seed of preparing in step c is inoculated according to the inoculum size of volume ratio 10% in the 50L fermentor tank that 30L fermention medium is housed, 37 ℃ of temperature, rotating speed 600rpm, under the condition of air flow 1.0vvm, cultivate after 4h, start take the speed of 0.4L/h at the uniform velocity stream add composition and content as bean cake powder 8%, Semen Maydis powder 1%, W-Gum 2%, corn steep liquor 0.5%, the feed supplement (m/v) of glucose 6%, stream adds 4L feed supplement altogether, 14h stream adds end, then within pH being controlled to the scope of 7.0-7.5 by adjusting rotary speed, 40h stops fermentation,
E, detection fermentation unit: the fermentation unit that detects bacitracin after fermentation ends is 1100U/ml, and the fermentation unit of the bacitracin of no-feed supplement fermentative production is 830U/ml.
Method provided by the invention has improved fermentation level, and feed supplement composition is simple, cheap, when fermentation ends, detect nutritive substance substantially depleted, alleviated the contradiction of the quick-acting nutritious deficiency of logarithmic phase simultaneously, be conducive to the raising of biomass, reduce production energy consumption and production cost, bacitracin output has raising by a relatively large margin compared with no-feed supplement.
Claims (6)
1. Continuous Flow adds the method that fed-batch fermentation raising bacitracin is tired, and it is characterized in that comprising following operation steps:
A, slant culture: Bacillus licheniformis is inoculated on slant medium, at 37 ℃, cultivates 24h;
B, prepare bacteria suspension: wash down and make bacteria suspension by the slant strains that sterilized water is prepared step a;
C, first order seed are cultivated: the bacterial suspension inoculation of preparing in step b, in the first class seed pot of seed culture medium is housed, is cultivated to 16h under the condition of 37 ℃ of temperature, rotating speed 600rpm, air flow 0.5vvm;
D, fed-batch fermentation: the first order seed of preparing in step c is inoculated according to the inoculum size of volume ratio 10% in the fermentor tank that fermention medium is housed, under the condition of 37 ℃ of temperature, rotating speed 600rpm, air flow 1.0vvm, ferment, when thalline enters exponential growth after date, start at the uniform velocity flow feeding, feed supplement finishes rear control pH7.0-7.5, and 40h stops fermentation;
E, detect fermentation unit: after fermentation ends, detect the fermentation unit of bacitracin, and compare with the fermentation unit of the bacitracin of no-feed supplement fermentative production.
2. a kind of Continuous Flow according to claim 1 adds the method that fed-batch fermentation raising bacitracin is tired, it is characterized in that, in step a, the composition of slant medium used (m/v) and consumption are: glucose 1%, peptone 1.5%, extractum carnis 1%, yeast extract powder 0.2%, agar 2.0%, the pH7.0 of substratum.
3. a kind of Continuous Flow according to claim 1 adds the method that fed-batch fermentation raising bacitracin is tired, it is characterized in that, in step c, the raw material of seed culture medium used (m/v) and consumption are: soybean cake powder 3%, Semen Maydis powder 2%, W-Gum 1%, corn steep liquor 0.5%, ammonium sulfate 0.15%, calcium carbonate 0.3%, the pH6.8 of substratum.
4. a kind of Continuous Flow according to claim 1 adds the method that fed-batch fermentation raising bacitracin is tired, it is characterized in that, in steps d, the composition of fermention medium used (m/v) and consumption are: bean cake powder 9%, Semen Maydis powder 2%, W-Gum 3%, ammonium sulfate 0.3%, magnesium sulfate 0.003%, calcium carbonate 0.5%, the pH7.2 of substratum, the volume of fermention medium is the 50-80% of fermentor tank volume.
5. a kind of Continuous Flow according to claim 1 adds the method that fed-batch fermentation raising bacitracin is tired, it is characterized in that, feed supplement (m/v) composition and consumption described in steps d are: bean cake powder 6-9%, Semen Maydis powder 0.5-1%, W-Gum 2-3%, corn steep liquor 0.3-0.5%, glucose 5-8%, feed supplement pH is 5.5-6.5, feed supplement flow acceleration is the 1-2% that stream per hour adds fermentating liquid volume, and stream is with the 10%-20% of fermented liquid cumulative volume altogether.
6. a kind of Continuous Flow according to claim 1 adds fed-batch fermentation and improves the bacitracin method of tiring, it is characterized in that, in steps d feed supplement finish after by adjusting rotary speed control pH7.0-7.5.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104357520A (en) * | 2014-12-04 | 2015-02-18 | 丽珠集团福州福兴医药有限公司 | Supplemented culture medium of teicoplanin and method for producing teicoplanin |
| CN104830933A (en) * | 2015-05-29 | 2015-08-12 | 河北圣雪大成制药有限责任公司 | Method for increasing valence of bacitracin by feeding mixed amino acid mother liquor |
| CN110295213A (en) * | 2019-05-10 | 2019-10-01 | 衡阳师范学院 | A kind of delicate flavour peptide fermentation method for producing based on exponential fed-batch feed supplement principle |
| CN113897410A (en) * | 2021-09-15 | 2022-01-07 | 南京工业大学 | Method for preparing bacitracin by integrated biological processing of trichoderma reesei and bacillus licheniformis |
| 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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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357520A (en) * | 2014-12-04 | 2015-02-18 | 丽珠集团福州福兴医药有限公司 | Supplemented culture medium of teicoplanin and method for producing teicoplanin |
| CN104830933A (en) * | 2015-05-29 | 2015-08-12 | 河北圣雪大成制药有限责任公司 | Method for increasing valence of bacitracin by feeding mixed amino acid mother liquor |
| CN104830933B (en) * | 2015-05-29 | 2018-12-25 | 河北圣雪大成制药有限责任公司 | A method of stream plus mixed amino acid mother liquor improve bacitracin potency |
| CN110295213A (en) * | 2019-05-10 | 2019-10-01 | 衡阳师范学院 | A kind of delicate flavour peptide fermentation method for producing based on exponential fed-batch feed supplement principle |
| CN113897410A (en) * | 2021-09-15 | 2022-01-07 | 南京工业大学 | Method for preparing bacitracin by integrated biological processing of trichoderma reesei and bacillus licheniformis |
| CN113897410B (en) * | 2021-09-15 | 2023-10-03 | 南京工业大学 | Method for preparing bacitracin by utilizing Trichoderma reesei and bacillus licheniformis integrated biological processing |
| 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 |
| CN114317648B (en) * | 2022-01-26 | 2024-04-30 | 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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| CN103882081B (en) | 2016-02-10 |
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