CN102206694B - Slow-release composition containing capric acid - Google Patents
Slow-release composition containing capric acid Download PDFInfo
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- CN102206694B CN102206694B CN 201110085101 CN201110085101A CN102206694B CN 102206694 B CN102206694 B CN 102206694B CN 201110085101 CN201110085101 CN 201110085101 CN 201110085101 A CN201110085101 A CN 201110085101A CN 102206694 B CN102206694 B CN 102206694B
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- capric acid
- slow releasing
- releasing composition
- daptomycin
- fermentation
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Abstract
The invention belongs to the field of biomedicine, and relates to a slow-release composition containing capric acid, particularly application of a slow-release composition containing capric acid in preparation of Daptomycin by fermentation. A certain amount of the slow-release composition containing capric acid can be supplemented at a specific time in the Daptomycin fermentation and production process, thereby synthesizing the Daptomycin. The invention reduces the number of times of shake flask fermentation and material supplementation, and lowers the probability of microbiological contamination; the slow-release capric acid reduces the toxicity for bacteria, and ensures good growth conditions for bacteria; and compared with the traditional batch-by-batch material supplementation method,the unit of the shake flask fermentation is increased by 4.1-5.8 times.
Description
Technical field
The invention belongs to biomedicine field, related to a kind of slow releasing composition that contains capric acid, relate in particular to a kind of slow releasing composition that contains capric acid and prepare the application in the method for daptomycin in fermentation.
Background technology
Daptomycin (Daptomycin) (chemical formula one is the chemical formula of daptomycin) is to extract the cyclic ester peptide matters obtained in streptomycete (S.reseosporus) fermented liquid, it not only has novel chemical structure, and its binding mode is also from arbitrary to have got permission microbiotic different: it can destroy the bacterial cell membrane function in many aspects, kills rapidly thus gram positive organism.The daptomycin decapacitation acts on outside most of clinical relevant gram positive organisms, the more important thing is in vitro and still to have a strong active to being the resistance isolated strains such as X-1497 (Methicillin), vancomycin and Linezolid.
Chemical formula one
At present, daptomycin is mainly by fermentative Production, and needs in process of production to add the precursor substance capric acid, yet within the adding and need to be controlled at certain level of capric acid, when the capric acid excessive concentration, before high toxicity, know from experience and make thalli growth be subject to certain inhibition; And capric acid concentration is when low, under-supply, the daptomycin synthetic ratio is low.Lu Wenyu etc. are at " Fed-Batch Fermentative Production of Daptomycin by Decanoic Acid Resistance Mutant " (University Of Tianjin's journal, 2006, the 39th phase, the 20-24 page) disclose at the fermentor tank of 7.5L and produced, adopt the technique of continuous flow feeding to eliminate the restraining effect of precursor, the daptomycin for preparing gained is tired as 210mg/L.More than continue three grade in " Streptomyces roseosporus NRRL113789 produces the fermention medium optimization of daptomycin precursor thing A21978C " (Agriculture of Anhui science, 2008, the 36th the 19th phase of volume, the 7974-7976 page) disclose in a literary composition in shake flask fermentation and improved daptomycin output by optimizing fermention medium, the fermentation unit of final A21978C component is 60mg/mL, for the daptomycin after the A21978C bio-modification, does not disclose related data.
To sum up can learn, the pertinent literature that shake flask fermentation prepares daptomycin is not disclosed at present, and add capric acid in the shake flask fermentation process owing to can not realizing automatization trace flow feeding continuously, generally adopt repeatedly feed supplement, so not only easily cause the fermentation microbiological contamination, and because the capric acid excessive concentration suppresses thalli growth, thereby make fermentation unit lower.
Summary of the invention
The invention provides a kind of slow releasing composition that contains capric acid, percentage ratio meter by weight, the component that it contains following significant quantity: capric acid 3.0～10.0%, ethanol 3.0～8.0%, stearic acid 0.5～1.0%, polyoxyethylene glycol 1.0～2.0%, agar powder 1.0～2.0%, water 77.0～91.5%; More preferably capric acid 6%, ethanol 6%, stearic acid 0.7%, polyoxyethylene glycol (PEG) 1.1%, agar powder 1.2%, water 85%.
The present invention also provides the preparation method of this slow releasing composition, and its step is as follows:
The present invention also provides this slow releasing composition to prepare the application in the method for daptomycin in fermentation, in the liquid nutrient medium that contains assimilable carbon and nitrogen sources and inorganic salt, carry out under the aerobic fermentation condition, in specified time adds nutrient solution by a certain amount of slow releasing composition that contains capric acid, thereby improve the daptomycin combined coefficient.The method that fermentation provided by the present invention prepares daptomycin comprises following steps:
A. Streptomyces roseosporus is inoculated on slant medium, in 30 ℃ of aerobic cultivations 10 days;
B. the bacterium of step a being cultivated is inoculated on the 20-50ml liquid seed culture medium in 28-32 ℃, 220rpm shaking culture 30-48h;
C. the bacterium of b being cultivated is inoculated in fermention medium by inoculum size 2-10%, and 30 ℃, 220rpm shaking culture 8 days;
D. cultivate 20-48h from inoculation fermentation and start to add the slow releasing composition that contains capric acid, every liter of fermented liquid is per hour added the slow releasing composition containing the 0.1-0.4ml capric acid, at interval of 24-72h, adds once, until fermentation 204h finishes;
E. adopt Shimadzu LC-20A liquid chromatograph to detect daptomycin, detect at interval of the 24h sampling after adding precursor;
F. 24h microscopy after feed supplement for the first time, during fermentation ends, the centrifugal survey relative biomass of 3000rpm.
The present invention has carried out preferably the slant culture based formulas in step a, and preferably, in above-mentioned steps a, slant medium consists of: yeast extract 4g/L, malt extract 10g/L, glucose 4g/L, agar 15g/L.
The present invention has carried out preferably the seed culture based formulas in step b, and preferably, in above-mentioned steps b, seed culture medium consists of: dextrin 20g/L, peptone 10g/L, yeast extract 3g/L, K
2hPO
40.1g/L, NaCl 0.2g/L.
The present invention has carried out preferably the fermentative medium formula in step c, and preferably, in above-mentioned steps c, fermention medium consists of: dextrin 30g/L, glucose 6g/L, soyflour 15g/L, yeast powder 4g/L, FeSO
47H
2o 0.5g/L, K
2hPO
40.2g/L, MgSO
47H
2o 0.1g/L.
The present invention has carried out preferably the time of origin of adding of slow releasing composition, preferably, above-mentionedly cultivates 20～36h from inoculation fermentation and starts to add the slow releasing composition that contains capric acid.
Carried out the interval time that the present invention adds slow releasing composition preferably, preferably, the above-mentioned slow releasing composition that once contains capric acid of adding at interval of 24～48h.
The present invention has carried out preferably the additional amount of slow releasing composition, and preferably, above-mentioned, every liter of fermented liquid is per hour added the slow releasing composition containing the 0.15-0.3ml capric acid.
The present invention compared with prior art has advantages of following outstanding:
(1) fermentation prepares in the process of daptomycin and adds the slow releasing composition that contains capric acid provided by the invention, reduces the number of times of adding of capric acid, greatly reduces the microbiological contamination probability;
(2) due to the slow releasing composition that contains capric acid provided by the invention, it is a kind of sustained-release materials, slow release by capric acid, alleviated the toxicity of capric acid to thalline in the fermenting process, guaranteed that thalline growth conditions in building-up process is good, 24h microscopy after feed supplement for the first time, visible mycelia is thicker, longer, color depth; During fermentation ends, the fermented liquid thickness;
(3) greatly improved the fermentation unit that shaking flask prepares daptomycin, the present invention's fermentation prepares tiring of daptomycin and is increased to 101.7mg/L, is up to 137.1mg/L, and the method for preparing daptomycin than traditional zymotic improves 4.1-5.8 doubly.
Embodiment
Further illustrate by the following examples the present invention, but these embodiment do not limit the present invention in any way.
Embodiment 1: the preparation method of the slow releasing composition that contains capric acid
| Capric acid (%) | Ethanol (%) | Stearic acid (%) | Polyoxyethylene glycol (%) | Agar powder (%) | Water (%) | |
| Composition 1 | 3 | 3 | 0.5 | 1.0 | 1.0 | 91.5 |
| Composition 2 | 10 | 8 | 1.0 | 2.0 | 2.0 | 77.0 |
| Composition 3 | 6 | 6 | 0.7 | 1.1 | 1.2 | 85.0 |
Embodiment 2: the preparation of fermention medium
Slant medium (g/L): yeast extract 4, malt extract 10, glucose 4, agar 15, become feed liquid by formulated, adjusts pH7.2,121 ℃ of sterilizing 30min;
Seed culture medium (g/L): dextrin 20, peptone 10, yeast extract 3, K
2hPO
40.1 NaCl 0.2, by formulated, becomes feed liquid, adjusts pH7.0,121 ℃ of sterilizing 30min;
Fermentative medium formula (g/L): dextrin 30, glucose 6, soyflour 15, yeast powder 4, FeSO
47H
2o 0.5, K
2hPO
40.2, MgSO
47H
2o 0.1, by formulated, becomes feed liquid, adjusts pH7.2,121 ℃ of sterilizing 30min.
Embodiment 3: add the capric acid fermentation and prepare daptomycin
A. Streptomyces roseosporus NRRL11379 is inoculated on slant medium, in 30 ℃ of aerobic cultivations 10 days;
B. the bacterium of step a being cultivated is inoculated on the 30m1 seed culture medium, in 30 ℃, 220rpm shaking culture 48h;
C. the bacterium of b being cultivated is inoculated in fermention medium by inoculum size 8%, 30 ℃, 220rpm shaking culture 8 days;
D. cultivate 36h from fermentation and start to add capric acid, every liter of fermented liquid is once added the mixed solution (contain capric acid and ethanol in this mixed solution, and its weight ratio being 1: 1) that contains the 3.6ml capric acid, at interval of 24h, adds once, until fermentation 204h finishes, add altogether 6 times;
E. adopt Shimadzu LC-20A liquid chromatograph to detect daptomycin, detect the 20.1mg/L of fermentation ends daptomycin unit after adding capric acid at interval of the 24h sampling;
F. 24h microscopy after feed supplement for the first time: mycelia is thin, and painted light, fracture is serious; During fermentation ends, fermented liquid is rare, and the centrifugal survey relative biomass of 3000rpm is 12%.
Embodiment 4: the method for preparing daptomycin
Steps d starts to add for from fermentation, cultivating 36h the slow releasing composition that contains capric acid, and every liter of fermented liquid is once added the slow releasing composition that contains the 7.2ml capric acid, at interval of 48h, adds once, until fermentation 204h finishes, adds altogether 3 times.
All the other steps are with embodiment 3.
Adopt Shimadzu LC-20A liquid chromatograph to detect daptomycin, detect the 133.6mg/L of fermentation ends daptomycin unit after adding slow releasing composition at interval of the 24h sampling; 24h microscopy after feed supplement for the first time: mycelia is thicker, longer, color depth; During fermentation ends, the fermented liquid thickness, the centrifugal survey relative biomass of 3000rpm is 21%.
Embodiment 5: the method for preparing daptomycin
Steps d starts to add for from fermentation, cultivating 36h the slow releasing composition that contains capric acid, and every liter of fermented liquid is once added the slow releasing composition that contains the 10.8ml capric acid, at interval of 72h, adds once, until fermentation 204h finishes, adds altogether 2 times.
All the other steps are with embodiment 3.
Adopt Shimadzu LC-20A liquid chromatograph to detect daptomycin, detect the 101.7mg/L of fermentation ends daptomycin unit after adding slow releasing composition at interval of the 24h sampling; 24h microscopy after feed supplement for the first time: mycelia is thin, longer, painted shallow, the mycelia fracture; During fermentation ends, fermented liquid is than thickness, and the centrifugal survey relative biomass of 3000rpm is 19%.
Embodiment 6: the method for preparing daptomycin
Steps d starts to add for from fermentation, cultivating 30h the slow releasing composition that contains capric acid, and every liter of fermented liquid is once added the slow releasing composition that contains the 5.4ml capric acid, at interval of 36h, adds once, until fermentation 204h finishes, adds altogether 4 times.
All the other steps are with embodiment 3.
Adopt Shimadzu LC-20A liquid chromatograph to detect daptomycin, detect the 137.1mg/L of fermentation ends daptomycin unit after adding slow releasing composition at interval of the 24h sampling; 24h microscopy after feed supplement for the first time: mycelia is thicker, longer, color depth; During fermentation ends, the fermented liquid thickness, the centrifugal survey relative biomass of 3000rpm is 22%.
Embodiment 7: the method for preparing daptomycin
Steps d starts to add for from fermentation, cultivating 20h the slow releasing composition that contains capric acid, and every liter of fermented liquid is once added the slow releasing composition that contains the 3.2ml capric acid, at interval of 32h, adds once, until fermentation 204h finishes, adds altogether 5 times.
All the other steps are with embodiment 3.
Adopt Shimadzu LC-20A liquid chromatograph to detect daptomycin, detect the 135.3mg/L of fermentation ends daptomycin unit after adding slow releasing composition at interval of the 24h sampling; 24h microscopy after feed supplement for the first time: mycelia is thicker, longer, color depth; During fermentation ends, the fermented liquid thickness, the centrifugal survey relative biomass of 3000rpm is 21%.
Embodiment 8: the method for preparing daptomycin
Steps d starts to add for from fermentation, cultivating 36h the slow releasing composition that contains capric acid, and every liter of fermented liquid is once added the slow releasing composition that contains the 14.4ml capric acid, at interval of 48h, adds once, until fermentation 204h finishes, adds altogether 3 times.
All the other steps are with embodiment 3.
Adopt Shimadzu LC-20A liquid chromatograph to detect daptomycin, detect the 135.2mg/L of fermentation ends daptomycin unit after adding slow releasing composition at interval of the 24h sampling; 24h microscopy after feed supplement for the first time: mycelia is thicker, longer, color depth; During fermentation ends, the fermented liquid thickness, the centrifugal survey relative biomass of 3000rpm is 22%.
Embodiment 9: the method for preparing daptomycin
Steps d starts to add for from fermentation, cultivating 48h the slow releasing composition that contains capric acid, and every liter of fermented liquid is once added the slow releasing composition that contains the 9.6ml capric acid, at interval of 24h, adds once, until fermentation 204h finishes, adds altogether 5 times.
All the other steps are with embodiment 3.
Adopt Shimadzu LC-20A liquid chromatograph to detect daptomycin, detect the 134.9mg/L of fermentation ends daptomycin unit after adding slow releasing composition at interval of the 24h sampling; 24h microscopy after feed supplement for the first time: mycelia is thicker, longer, color depth; During fermentation ends, the fermented liquid thickness, the centrifugal survey relative biomass of 3000rpm is 20%.
Claims (9)
1. a slow releasing composition that contains capric acid is characterized in that: percentage ratio meter by weight, and it is comprised of the component of following significant quantity: capric acid 3.0～10.0%, ethanol 3.0～8.0%, stearic acid 0.5～1.0%, polyoxyethylene glycol 1.0～2.0%, agar powder 1.0～2.0%, water 77.0～91.5%.
2. slow releasing composition according to claim 1 is characterized in that: percentage ratio meter by weight, it is comprised of the component of following significant quantity: capric acid 6%, ethanol 6%, stearic acid 0.7%, polyoxyethylene glycol (PEG) 1.1%, agar powder 1.2%, water 85%.
3. a method for preparing slow releasing composition as claimed in claim 1 or 2 is characterized in that it comprises the following steps:
4. a method for preparing daptomycin is characterized in that it comprises following steps:
A. Streptomyces roseosporus is inoculated on slant medium, in 30 ℃ of aerobic cultivation 8-10 days;
B. the bacterium of step a being cultivated is inoculated on the 20-50ml liquid seed culture medium in 28-32 ℃, 220rpm shaking culture 36-50h;
C. the bacterium of b being cultivated is inoculated in fermention medium by inoculum size 2-10%, and 30 ℃, 220rpm shaking culture 8-10 days;
D. cultivate 20-48h from inoculation fermentation and start to add slow releasing composition as claimed in claim 1 or 2, add once at interval of 24-72h, until fermentation ends.
5. method according to claim 4, is characterized in that in step a, slant medium consists of: yeast extract 4g/L, malt extract 10g/L, glucose 4g/L, agar 15g/L.
6. method according to claim 4, is characterized in that in step b, seed culture medium consists of: dextrin 20g/L, peptone 10g/L, yeast extract 3g/L, K
2hPO
40.1g/L, NaCl 0.2g/L.
7. method according to claim 4, is characterized in that in step c, fermention medium consists of: dextrin 30g/L, glucose 6g/L, soyflour 15g/L, yeast powder 4g/L, FeSO
4 .7H
2o 0.5g/L, K
2hPO
40.2g/L, MgSO
4 .7H2O 0.1g/L.
8. method according to claim 4, is characterized in that cultivating 20h～36h from inoculation fermentation in steps d starts to add slow releasing composition as claimed in claim 1 or 2.
9. method according to claim 4, is characterized in that adding once slow releasing composition as claimed in claim 1 or 2 at interval of 24h～48h in steps d.
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102703551A (en) * | 2012-06-27 | 2012-10-03 | 中国药科大学 | Novel method for adding decanoic acid in fermentation process of daptomycin |
| CN102796680B (en) * | 2012-07-04 | 2018-06-05 | 鲁南新时代生物技术有限公司 | A kind of Streptomyces roseosporus and its method for producing Daptomycin using precursor is combined |
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2011
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Effective date of registration: 20151218 Address after: Shandong province Linyi city Feixian County North Ring Road No. 1 Patentee after: Lunan New Era Biological Technology Co., Ltd. Address before: 273400 Shandong city of Linyi province Feixian County North Ring Road No. 1 Patentee before: Shandong Xinshidai Pharmaceutical Industry Co., Ltd. |