CN103882081B - A kind of Continuous Flow adds the method that fed-batch fermentation raising bacitracin is tired - Google Patents
A kind of Continuous Flow adds the method that fed-batch fermentation raising bacitracin is tired Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 17
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- 238000011218 seed culture Methods 0.000 claims abstract description 8
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 7
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- 238000001514 detection method Methods 0.000 claims abstract description 6
- 238000011081 inoculation Methods 0.000 claims abstract description 6
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
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- 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
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- 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
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- 244000068988 Glycine max Species 0.000 claims description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
Continuous Flow adds the method that fed-batch fermentation raising bacitracin is tired, and step is as follows: a, slant culture: be inoculated in by Bacillus licheniformis on slant medium, at 37 DEG C, cultivate 24h; B, prepare bacteria suspension: wash down by the slant strains that step a is prepared by sterilized water and make bacteria suspension; C, first order seed are cultivated: by the bacterial suspension inoculation prepared in step b in the first class seed pot that seed culture medium is housed, under the condition of temperature 37 DEG C, rotating speed 600rpm, air flow 0.5vvm, cultivate 16h; D, fed-batch fermentation: the first order seed prepared in step c is inoculated according to the inoculum size of volume ratio 10% and is equipped with in the fermentor tank of fermention medium, ferment under the condition of temperature 37 DEG C, rotating speed 600rpm, air flow 1.0vvm, when thalline enters exponential growth after date, start at the uniform velocity flow feeding, feed supplement terminates rear control pH7.0-7.5,40h and stops fermentation; E, detection fermentation unit: detect the fermentation unit of bacitracin and compare with the fermentation unit of the bacitracin of no-feed supplement fermentative production.
Description
Technical field
The invention belongs to field of microbial fermentation, be specifically related to a kind of Continuous Flow and add fed-batch fermentation and improve the bacitracin method of tiring.
Background technology
Bacitracin belongs to polypeptide antibiotics, it is the polypeptide of the instability be combined into by multiple amino acids, strong restraining effect is had to gram-positive microorganism and part negative bacterium, it can the synthesis of anti-bacteria cell walls, affect its protein synthesis and cell membrane function, thus reach the object eliminating pathogenic bacteria.Bacitracin is made up of multiple amino acid, because it is efficient, has no side effect, noresidue, not to 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 uses Bacillus licheniformis, be main carbon and nitrogen sources with W-Gum, bean cake powder, culture temperature is 37 DEG C, through 40h oxygen consumption fermentation culture biosynthesizing bacitracin, but fermentation level is not high, production energy consumption and production cost high, at present to the optimization of the research of Production by Microorganism Fermentation bacitracin mainly fermention medium, or the optimization of fermentation condition, does not obtain good effect; Also the bacterial strain of high yield bacitracin screens by traditional selection by mutation in the factory had, 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 to add fed-batch fermentation and improve the bacitracin method of tiring, its object is to overcome the deficiencies in the prior art part, improve fermentation unit, reduce production energy consumption and production cost.
The present invention is the technical scheme realizing the employing of its object:
Continuous Flow adds the method that fed-batch fermentation raising bacitracin is tired, and comprises following operation steps:
A, slant culture: be inoculated in by Bacillus licheniformis on slant medium, cultivate 24h at 37 DEG C;
B, prepare bacteria suspension: wash down by the slant strains that step a is prepared by sterilized water and make bacteria suspension;
C, first order seed are cultivated: by the bacterial suspension inoculation prepared in step b in the first class seed pot that seed culture medium is housed, under the condition of temperature 37 DEG C, rotating speed 600rpm, air flow 0.5vvm, cultivate 16h;
D, fed-batch fermentation: the first order seed prepared in step c is inoculated according to the inoculum size of volume ratio 10% and is equipped with in the fermentor tank of fermention medium, ferment under the condition of temperature 37 DEG C, rotating speed 600rpm, air flow 1.0vvm, when thalline enters exponential growth after date, start at the uniform velocity flow feeding, feed supplement terminates rear control pH7.0-7.5,40h and stops fermentation;
E, detection fermentation unit: the fermentation unit detecting bacitracin after fermentation ends, 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 fermenter volume.
Feed supplement (m/v) composition described in steps d and consumption 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 fed-batch cultivation liquid per hour amasss, altogether the 10%-20% of fed-batch cultivation liquid cumulative volume.
In steps d feed supplement terminate after by adjusting rotary speed control pH7.0-7.5.
The invention has the beneficial effects as follows: method provided by the invention improves fermentation level, and feed component is simple, cheap, nutritive substance is detected substantially depleted during fermentation ends, alleviate 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 to add fed-batch fermentation and improve the bacitracin method of tiring, 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 the method that fed-batch fermentation raising bacitracin is tired, and comprises following operation steps:
A, slant culture: be inoculated in by Bacillus licheniformis on slant medium, cultivate 24h at 37 DEG C;
B, prepare bacteria suspension: wash down by the slant strains that step a is prepared by sterilized water and make bacteria suspension;
C, first order seed are cultivated: by the bacterial suspension inoculation prepared in step b in the 15L first class seed pot that 10L seed culture medium is housed, under the condition of temperature 37 DEG C, rotating speed 600rpm, air flow 0.5vvm, cultivate 16h;
D, fed-batch fermentation: the first order seed prepared in step c is inoculated according to the inoculum size of volume ratio 10% and is equipped with in the 50L fermentor tank of 30L fermention medium, temperature 37 DEG C, rotating speed 600rpm, after cultivating 3h under the condition of air flow 1.0vvm, start with the speed of 0.3L/h at the uniform velocity stream add composition and content for 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 by adjusting rotary speed, pH is controlled within the scope of 7.0-7.5, 40h stops fermentation,
E, detection fermentation unit: the fermentation unit detecting 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 the method that fed-batch fermentation raising bacitracin is tired, and comprises following operation steps:
A, slant culture: be inoculated in by Bacillus licheniformis on slant medium, cultivate 24h at 37 DEG C;
B, prepare bacteria suspension: wash down by the slant strains that step a is prepared by sterilized water and make bacteria suspension;
C, first order seed are cultivated: by the bacterial suspension inoculation prepared in step b in the 15L first class seed pot that 10L seed culture medium is housed, under the condition of temperature 37 DEG C, rotating speed 600rpm, air flow 0.5vvm, cultivate 16h;
D, fed-batch fermentation: the first order seed prepared in step c is inoculated according to the inoculum size of volume ratio 10% and is equipped with in the 50L fermentor tank of 30L fermention medium, temperature 37 DEG C, rotating speed 600rpm, after cultivating 3h under the condition of air flow 1.0vvm, start with the speed of 0.6L/h at the uniform velocity stream add composition and content for 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 by adjusting rotary speed, pH is controlled within the scope of 7.0-7.5, 40h stops fermentation,
E, detection fermentation unit: the fermentation unit detecting 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 the method that fed-batch fermentation raising bacitracin is tired, and comprises following operation steps:
A, slant culture: be inoculated in by Bacillus licheniformis on slant medium, cultivate 24h at 37 DEG C;
B, prepare bacteria suspension: wash down by the slant strains that step a is prepared by sterilized water and make bacteria suspension;
C, first order seed are cultivated: by the bacterial suspension inoculation prepared in step b in the 15L first class seed pot that 10L seed culture medium is housed, under the condition of temperature 37 DEG C, rotating speed 600rpm, air flow 0.5vvm, cultivate 16h;
D, fed-batch fermentation: the first order seed prepared in step c is inoculated according to the inoculum size of volume ratio 10% and is equipped with in the 50L fermentor tank of 30L fermention medium, temperature 37 DEG C, rotating speed 600rpm, after cultivating 4h under the condition of air flow 1.0vvm, start with the speed of 0.4L/h at the uniform velocity stream add composition and content for 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 by adjusting rotary speed, pH is controlled within the scope of 7.0-7.5, 40h stops fermentation,
E, detection fermentation unit: the fermentation unit detecting 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 improves fermentation level, and feed component is simple, cheap, nutritive substance is detected substantially depleted during fermentation ends, alleviate 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 (5)
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: be inoculated in by Bacillus licheniformis on slant medium, cultivate 24h at 37 DEG C;
B, prepare bacteria suspension: wash down by the slant strains that step a is prepared by sterilized water and make bacteria suspension;
C, first order seed are cultivated: by the bacterial suspension inoculation prepared in step b in the first class seed pot that seed culture medium is housed, under the condition of temperature 37 DEG C, rotating speed 600rpm, air flow 0.5vvm, cultivate 16h;
D, fed-batch fermentation: the first order seed prepared in step c is inoculated according to the inoculum size of volume ratio 10% and is equipped with in the fermentor tank of fermention medium, temperature 37 DEG C, rotating speed 600rpm, ferment under the condition of air flow 1.0vvm, when thalline enters exponential growth after date, start at the uniform velocity flow feeding, feed supplement terminates rear control pH7.0-7.5, 40h stops fermentation, described feed supplement (w/v) composition and consumption 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 fed-batch cultivation liquid per hour amasss, the 10%-20% of fed-batch cultivation liquid cumulative volume altogether,
E, detection fermentation unit: the fermentation unit detecting bacitracin after fermentation ends, 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 (w/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 (w/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 (w/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 fermenter volume.
5. a kind of Continuous Flow according to claim 1 adds fed-batch fermentation and improves the bacitracin method of tiring, and it is characterized in that, in steps d feed supplement terminate after by adjusting rotary speed control pH7.0-7.5.
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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 |
| 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 |
| CN110295213B (en) * | 2019-05-10 | 2020-12-08 | 衡阳师范学院 | Method for fermentation production of umami peptide based on index flow feeding principle |
| 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 |
| 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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| CN102899375A (en) * | 2012-09-20 | 2013-01-30 | 天津科技大学 | Method for increasing titer of bacitracin in fermentation liquid by using oxygen carrier |
| CN103146629A (en) * | 2013-03-05 | 2013-06-12 | 绿康生化股份有限公司 | Bacterial strain of bacillus licheniformis with vitreoscilla hemoglobin gene as well as construction method and application of bacterial strain |
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| CN102899375A (en) * | 2012-09-20 | 2013-01-30 | 天津科技大学 | Method for increasing titer of bacitracin in fermentation liquid by using oxygen carrier |
| CN103146629A (en) * | 2013-03-05 | 2013-06-12 | 绿康生化股份有限公司 | Bacterial strain of bacillus licheniformis with vitreoscilla hemoglobin gene as well as construction method and application of bacterial strain |
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Granted publication date: 20160210 |