CN102876766B - Fermentation process of cephalosporin C - Google Patents
Fermentation process of cephalosporin C Download PDFInfo
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- CN102876766B CN102876766B CN201210429362.3A CN201210429362A CN102876766B CN 102876766 B CN102876766 B CN 102876766B CN 201210429362 A CN201210429362 A CN 201210429362A CN 102876766 B CN102876766 B CN 102876766B
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Abstract
The invention relates to a fermentation process of cephalosporin C. At a later fermentation stage, the cephalosporin C is defoamed by supplementing a defoaming agent instead of soya-bean oil, so that cost of raw materials can be reduced by 15% under a condition that valence increasing and filtering velocity are not influenced.
Description
Technical field
The invention belongs to biotechnology and microbiology field; More specifically, the present invention relates to a kind of fermentation process of cephalosporin.
Background technology
Cephalosporin is produced by cephalosporium acremonium (Cephalosporium acremonuim), is primary metabolite continuation under given conditions and the product of development of cephalosporium sp, is another important β-lactam antibitics after penicillin.
In cephalosporin industrial fermentation process, use soya-bean oil as main carbon source at present, also play the effect of the foam that disappears simultaneously.To the fermentation later stage, liquid level easily produces foam, because soya-bean oil the effect of eliminating foam reagent is poor, more soya-bean oil need to be added and liquid level could be effectively controlled, simultaneously due to the thalline self-dissolving that senesces, the ability of utilizing to soya-bean oil declines, and remaining in soya-bean oil too much in fermented liquid can impact the extraction of cephalosporin.
Therefore, this area needs the Cephalosporin C fermentation method of Improvement, overcomes above-mentioned technical problem.
Summary of the invention
The object of the present invention is to provide a kind of fermentation process of cephalosporin.
In a first aspect of the present invention, a kind of fermentation process of cephalosporin is provided, described method comprises: in Cephalosporin C fermentation process, in the time that needs defoam (preferably, in the time that generation makes foam that liquid level increases), with the foam processing that disappears of polyethers foam killer or high-carbon alcohols foam killer.
In a preference, described polyethers foam killer is: polyoxyethylene oxypropylene glycerine.
In another preference, described polyoxyethylene oxypropylene glycerine H
2c-O-(C
3h
6o) m-(C
2h
4o) n-H; Molecular weight 8000-10000.
In another preference, described high-carbon alcohols foam killer is: polyoxyethylene glycol.
In another preference, described polyoxyethylene glycol is the polyoxyethylene glycol of polymerization degree 1000-4000; More preferably, described polyoxyethylene glycol is PEG2000.
In another preference, after fermentation initial 70-120 hour, foam processing disappears; More preferably, after fermentation initial 90-110 hour, foam processing disappears.
In another preference, the addition of polyethers foam killer or high-carbon alcohols foam killer is: 0.01-0.1g/L bubble fermentation liquid total amount; It is preferably 0.02-0.08g/L bubble fermentation liquid total amount; It is more preferably 0.03-0.06g/L bubble fermentation liquid total amount.
In another preference, the fermention medium of employing is:
Peanut powder: 25g/L, Semen Maydis powder: 17.5g/L, gluten powder: 30g/L, tuna cream: 20mL/L, corn steep liquor: 40mL/L, glucose: 5g/L, sucrose: 10g/L, methionine(Met): 9g/L, ammonium sulfate: 10g/L, calcium sulfate: 12g/L, calcium carbonate: 5g/L, soya-bean oil: 70mL/L, foam killer: 1mL/L;
The content of mentioned component in substratum can ± 10% (preferably, ± 5%).
In another preference, earlier fermentation (preferably first 50 hours) is added ammonium ion, and the ammonium concentration in controlled fermentation liquid is at 1.2-1.5g/L.
In another preference, ferment and rise for initial 40-60 hour, add soya-bean oil with the speed of 20-40g/hr, within initial 90-110 hour, stop adding soya-bean oil to fermenting.
In another preference, before fermenting initial 35-45 hour, culture temperature control is 28 ± 1 DEG C, and after fermenting initial 35-45 hour, controlling is 25 ± 1 DEG C.
In another aspect of this invention, provide the purposes of polyethers foam killer or high-carbon alcohols foam killer, for fermentative production of cephalosporin C.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Brief description of the drawings
Fig. 1, the impact of different sorts foam killer on Cephalosporin C fermentation.
Fig. 2, add foam killer to the biosynthetic impact of cephalosporin.
Fig. 3, add the impact of foam killer on cephalosporin fermented liquid filtering velocity.
Embodiment
In order to address the deficiencies of the prior art, the inventor, through a large amount of research experiments, finds in the later stage of Cephalosporin C fermentation, with the alternative a part of soya-bean oil of foam killer, can effectively control the foam of liquid level, and biosynthesizing and filtering velocity on cephalosporin do not affect.After adding foam killer, material cost reduces approximately 15% than only adding soya-bean oil.
Foam killer is conventionally also referred to as defoamer, and the foam killer existing in prior art has more kind, and conventional foam killer has the macromolecular compound of natural fats and oils (as Vegetable oil lipoprotein, animal grease) and some chemosynthesis etc.Chemistry foam killer kind is very many, be mainly: polyethers defoamer (containing GP type defoamer, GPE type defoamer, GPES type defoamer etc.), higher alcohols defoamer (phosphoric acid tri-n-butyl, the higher alcohols of C12～C22 etc.), silicon defoamer (containing polydimethylsiloxane etc.), polyether-modified silicon, self-emulsifying defoamer, polysiloxane defoamers etc.
At present in industrial fermentation, the conventional foam means that disappear comprise machinery foam and the chemistry foam that disappears that disappears.Although also can adopt some foam killers to eliminate the foam producing in fermenting process in industrial fermentation at present, apply which kind of foam killer, need to be depending on factors such as the bacterial classification physiological property of production, region situation, reaction conditionss.
The present invention relates to the fermentation of cephalosporin, in fermentation process in the past, conventionally, to double as foam killer as the soya-bean oil of main carbon source simultaneously, therefore generally no longer add chemical additive.But the inventor finds after further investigation, with the foam processing that disappears of polyethers foam killer or high-carbon alcohols foam killer.It is controlled fermentation liquid level better, and can greatly reduce fermentation costs.
Therefore, the invention provides a kind of fermentation process of cephalosporin, described method comprises: in Cephalosporin C fermentation process, in the time that needs defoam, particularly in the time that generation makes foam that liquid level increases, with the foam processing that disappears of polyethers foam killer or high-carbon alcohols foam killer.
As the particularly preferred mode of the present invention, foam killer used is polyoxyethylene oxypropylene glycerine or polyoxyethylene glycol.As the particularly preferred mode of the present invention, foam killer used is polyoxyethylene oxypropylene glycerine, and this foam killer is less on the impact of Cephalosporin C fermentation, even has synergism.
The inventor has also optimized fermenting process, comprises the interpolation opportunity of foam killer.Preferably, after fermentation initial 70-120 hour, foam processing disappears; More preferably, after fermentation initial 90-110 hour, foam processing disappears.The addition of foam killer is: 0.01-0.1g/L bubble fermentation liquid total amount; It is preferably 0.02-0.08g/L bubble fermentation liquid total amount; It is more preferably 0.03-0.06g/L bubble fermentation liquid total amount.Stop the interpolation of soya-bean oil and change with foam killer processing on this opportunity, being conducive to effectively reduce foam generation, and being conducive to the separating-purifying of the cephalosporin in downstream.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, the condition described in Science Press, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
I. materials and methods
1, bacterial classification
Cephalosporium acremonium (Cephalosporium acremonium) CA01 is provided by Weiqida Pharmaceutical Ind Co., Ltd..
2, substratum
Slant medium (weightmeasurement ratio)):
Wort 1.2, peptone 1.2, agar 2.2; Above composition is dissolved in deionized water to constant volume to 100 milliliter; Before disappearing, (before sterilization) pH is 7.0.
Seed culture medium:
Soybean cake powder: 30g/L, tuna cream: 20mL/L, corn steep liquor: 15g/L, sucrose: 25g/L, glucose: 10g/L, calcium carbonate: 5g/L, soya-bean oil: 10mL/L; Mentioned component is dissolved in deionized water.
Fermention medium:
Peanut powder: 25g/L, Semen Maydis powder: 17.5g/L, gluten powder: 30g/L, tuna cream: 20mL/L, corn steep liquor: 40mL/L, glucose: 5g/L, sucrose: 10g/L, methionine(Met): 9g/L, ammonium sulfate: 10g/L, calcium sulfate: 12g/L, calcium carbonate: 5g/L, soya-bean oil: 70mL/L, foam killer: 1mL/L.Mentioned component is dissolved in deionized water.
3, foam killer
The foam killer that is used for testing is as table 1.
Table 1
4, cultural method
Slant culture
The spore being stored in freezing glycerine pipe is received to Boiling tube inclined-plane, and 28 DEG C, relative humidity 40～65% is cultivated 12 days, 4 DEG C of refrigerator preservations.
Shake-flask seed is cultivated
Be equipped with in the 500mL triangular flask of 50mL seed culture medium by the access of freeze pipe spore, 28 DEG C, 220r/min rotary type shaking table (eccentricity 5cm) is cultivated 72h.
Shake flask fermentation is cultivated
Seed liquor 8mL access is equipped with in the 500mL baffle flask of 46mL fermention medium, 25 DEG C of cultivations, 260r/min shaking table (eccentricity 5cm) is cultivated 3 days, ferment 72 hours every bottle add 1mL soya-bean oil, cultivate and within 5-6 days, put bottle.
50L fermentor cultivation
25L fermention medium is joined to fermentor tank (for adding foam killer group and not adding foam killer group; in initial fermention medium, soya-bean oil amount is identical); by the inoculum size of 15% (v/v), seed liquor is accessed in fermentor tank under flame protection from inoculation mouthful; cultivate 110-150hr left and right; 28 DEG C before of culture temperature 40h, after 40h, controlling is 25 DEG C.Fermenting process is added ammonium sulfate and soya-bean oil as required.
Ammonium sulfate: earlier fermentation, below ammonium concentration is reduced to 1.5g/L time, start to add ammoniumsulphate soln (20%W/V), the ammonium concentration in controlled fermentation liquid is at 1.2-1.5g/L.Ammonium sulfate stopped adding after 50 hours.
Soya-bean oil: beginning in 50 hours is added with the speed of 30g/hr.Add foam killer group and do not add foam killer group that to add soya-bean oil before 100 hours be all identical fermenting; Add foam killer group and stop adding soya-bean oil in 100 hours, start to add foam killer; Continue to add soya-bean oil until put tank, additional amount 30g/hr and do not add foam killer group.
5, experimental installation
FUS-50L (A) automatically controlled fermentor: fermentation system is except being equipped with pH, dissolved oxygen (DO), temperature, outside the Measurement and Control System of the routine such as rotating speed, overall LOAD CELLS and high precision feed supplement (matrix is also equipped with, soda acid, defoamer etc.) measurement and control, and be connected with exhaust analyzer, complete machine has 14 on-line parameters and detects or control function, and there is computer control and the data processing software bag BIOSTAR that can input the manual location parameter in laboratory, can further accurately obtain thus optimizing fermentation and amplify the necessary indirect parameter that comprises various metabolism stream features or engineering characteristics, as oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), breathe entropy (RQ), Volumetric Oxygen Transfer Coefficients (KLa) etc.
6, analytical procedure
The dense mensuration of bacterium: get 10mL fermented liquid in centrifuge tube, the centrifugal 20min of 4000r/min, calculating solid object volume accounts for the ratio of fermented liquid.
Cephalosporin titration: HPLC method, chromatographic column: Thermo Hypersil ODS C184.6 × 150mm, 5 μ m; Moving phase: 0.8g SODIUM PHOSPHATE, MONOBASIC is in the mixed solution of 1030mL methyl alcohol, acetonitrile and water (140:40:850), mixed dissolution, adjust pH to 7.3 0.45 μ m strainer to filter by TBAH, flow velocity: 1.0mL/min, ultraviolet detection wavelength: 254nm, room temperature, sample size 20 μ L.
II. result and discussion
Embodiment 1, the impact of different sorts foam killer on Cephalosporin C fermentation
In shake-flask culture level, (A, D belong to polyethers foam killer to select five kinds of foam killers, B, E belong to silicone foam killer, C belongs to high-carbon alcohols foam killer) add in fermention medium and (initially add soya-bean oil according to the ratio of 0.25% (v/v), the same control group of addition), control group does not add foam killer, cultivates 6 days according to the method for aforementioned " shake flask fermentation cultivation ", investigate the impact of different types of foam killer on Cephalosporin C fermentation, the results are shown in Figure 1.
In five kinds of foam killers, A, C, D tire (u/mL), higher than contrast, B, E tire lower than contrast.Wherein D tire the highlyest, tiring of E is minimum.Can find out that polyoxyethylene oxypropylene glycerine is on the impact of Cephalosporin C fermentation less (even having synergism), and silicone foam killer is larger on the impact of Cephalosporin C fermentation.
In the present embodiment, the adding proportion of foam killer higher (0.25% (v/v)), experimental result shows that bacterial classification has certain tolerance to foam killer, and depends on different foam killer kinds.Polyethers foam killer, high-carbon alcohols foam killer effect are better, wherein polyethers foam killer the best.
Therefore, utilizing foam killer to substitute soya-bean oil is a kind of feasible method, even can be higher than the contrast using soya-bean oil as main carbon source.
In embodiment 2, fermenting process, add the impact of foam killer on Cephalosporin C fermentation
In 50L fermentor tank, ferment according to aforementioned " 50L fermentor cultivation ".Select D foam killer to carry out the AD Experiment of foam killer, within 100 hours, start by peristaltic pump intermittent injecting foam killer, when fermented liquid foam rises to foam and fermented liquid total amount starts to add foam killer in the position of 35L (observing by fermentor tank visor), add 1.5g foam killer (being 0.043g/L bubble fermentation liquid total amount) at every turn.After ammonium sulfate to 50 hour, stop adding.Soya-bean oil starts to add with the speed of 30g/hr for 50 hours, within 100 hours, stops adding soya-bean oil, starts to add foam killer.Soya-bean oil additional amount reduces approximately 25% compared with control group.Controlled fermentation liquid level is in 35L left and right.
Control group is set simultaneously, in control group, within 50 hours, starts to add soya-bean oil until put tank, soya-bean oil is added according to the speed of 30g/hr until put tank, and ammonium sulfate stopped adding after 50 hours.
1, add liquid level situation after D foam killer
According to range estimation, to add after D foam killer, liquid level there is no foam, the effect of eliminating foam reagent is obviously better than soya-bean oil.
2, add D foam killer to the biosynthetic impact of cephalosporin
Add after D foam killer, growing way with add soya-bean oil and there is no obvious difference, see Fig. 2.
3, add the impact of D foam killer on fermented liquid filtering velocity
Within 115 hours, get the fermented liquid of experimental tank (adding D foam killer) and control canisters (not adding D foam killer) simultaneously and measure filtering velocity, the results are shown in Figure 3.
As can be seen from Figure 3, adding D foam killer does not affect fermented liquid filtering velocity.
Conclusion
About 100 hours, start to add foam killer at Cephalosporin C fermentation, can effectively control the foam of liquid level, on biosynthesizing and filtering velocity do not affect.By cost accounting, add foam killer after material cost reduce approximately 15% than only adding soya-bean oil.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (6)
1. a fermentation process for cephalosporin, is characterized in that, described method comprises: in Cephalosporin C fermentation process, ferment and rise for initial 40-60 hour, add soya-bean oil with the speed of 20-40g/hr, within initial 90-110 hour, stop adding soya-bean oil to fermenting; Fermentation after initial 90-110 hour with the foam processing that disappears of polyethers foam killer polyoxyethylene oxypropylene glycerine or high-carbon alcohols foam killer polyoxyethylene glycol, addition is 0.02-0.08g/L bubble fermentation liquid total amount;
The fermention medium adopting is: peanut powder: 25g/L, Semen Maydis powder: 17.5g/L, gluten powder: 30g/L, tuna cream: 20mL/L, corn steep liquor: 40mL/L, glucose: 5g/L, sucrose: 10g/L, methionine(Met): 9g/L, ammonium sulfate: 10g/L, calcium sulfate: 12g/L, calcium carbonate: 5g/L, soya-bean oil: 70mL/L, foam killer: 1mL/L;
In fermention medium, the content of each composition is ± 10%.
2. the method for claim 1, is characterized in that, after initial 100 hours of fermentation, foam processing disappears.
3. the method for claim 1, is characterized in that, the addition of polyethers foam killer or high-carbon alcohols foam killer is 0.03-0.06g/L bubble fermentation liquid total amount.
4. the method for claim 1, is characterized in that, in fermention medium, the content of described composition in substratum is ± 5%.
5. the method for claim 1, is characterized in that, earlier fermentation starts to add ammoniumsulphate soln below ammonium concentration is reduced to 1.5g/L time, and the ammonium concentration in controlled fermentation liquid is at 1.2-1.5g/L; Described earlier fermentation is first 50 hours of fermentation.
6. the method for claim 1, is characterized in that, before fermenting initial 35-45 hour, culture temperature control is 28 ± 1 DEG C, and controlling is afterwards 25 ± 1 DEG C.
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| CN103816699A (en) * | 2012-11-16 | 2014-05-28 | 季爱英 | Antifoaming agent for food fermentation |
| CN104263792B (en) * | 2014-09-30 | 2017-11-07 | 国药集团威奇达药业有限公司 | The fermentation medium used in the fermentation process and this method that prepare cephalosporin |
| CN106511281A (en) * | 2016-09-30 | 2017-03-22 | 华北制药河北华民药业有限责任公司 | Preparation method of Cefamandole Nafate powder injection for injection |
| CN112852907B (en) * | 2020-03-16 | 2021-11-30 | 中国科学院天津工业生物技术研究所 | Cephalosporin C producing strain and preparation method and application thereof |
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| EP0137365A2 (en) * | 1983-09-06 | 1985-04-17 | Takeda Chemical Industries, Ltd. | Cephalosporins and their production |
| CN102408462A (en) * | 2011-12-02 | 2012-04-11 | 伊犁川宁生物技术有限公司 | Preparation method of erythromycin thiocyanate |
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| EP0137365A2 (en) * | 1983-09-06 | 1985-04-17 | Takeda Chemical Industries, Ltd. | Cephalosporins and their production |
| CN102408462A (en) * | 2011-12-02 | 2012-04-11 | 伊犁川宁生物技术有限公司 | Preparation method of erythromycin thiocyanate |
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