CN101497907A - Fermentation process of spiramycin - Google Patents
Fermentation process of spiramycin Download PDFInfo
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- CN101497907A CN101497907A CNA2009101189202A CN200910118920A CN101497907A CN 101497907 A CN101497907 A CN 101497907A CN A2009101189202 A CNA2009101189202 A CN A2009101189202A CN 200910118920 A CN200910118920 A CN 200910118920A CN 101497907 A CN101497907 A CN 101497907A
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Abstract
The invention relates to a fermentation process for improving the output of spiramycin alkali through adding metallic ions. The fermentation process comprises the following steps: a certain amount of manganese ion solution is added after fermentation medium is prepared according to the conventional method. The fermentation level of the spiramycin alkali carried out by the fermentation process obviously exceeds that of the prior process, and the invention has the advantages of high titer, simple operation, low cost and convenient mass production, effectively overcomes the deficiency of the prior fermentation process, and lays a foundation for the realization of industrial industrialization at the next stage.
Description
Technical field
The present invention relates to the fermentation technology optimization method of Spiramycin Base.Be by adding a kind of zymotechnique that metal ion improves Spiramycin Base output specifically.
Background technology
Spiramycin Base is by giving birth to the polycomponent macrolide antibiotics that two streptomycetes (Streptomyces ambofaciens) are produced.The process of spiramycin streptomyces biosynthesizing Spiramycin Base is subjected to the regulating and controlling effect of many enzymes.Acyl group kinases and acyl-CoA synthetase are Spiramycin Base the lactone ring synthetic important enzymes.Because synthetic enzyme is active less, infers that it may be to encircle synthetic " bottleneck " greatly, if improve its activity, just might increase substantially fermentation titer.Add incitant at earlier fermentation, E.C. 2.7.2.1 is in the elementary metabolism, the active raising not quite to the secondary metabolism influence, and tiring, it is not obvious to improve.Add incitant at ferment middle, improved the E.C. 2.7.2.1 of participation secondary metabolism and the activity of acetyl-CoA synthetic enzyme greatly, broken through big ring synthetic restriction, thereby obviously improved the fermentation titer of Spiramycin Base.
People such as Mao Quangui are published in Chinese microbiotic magazine volume o. 11th in November in 2005 the 30th (civilian rope numbering 1001-8689 (2005) 11-0647-02) metal ion the biosynthetic literary composition that influences of Spiramycin Base were also once reported to produce in dyadic streptomycete (Streptomycesambofaciens) fermenting process at spiramycin-producing strain and added Cu
2+, Ca
2+, Fe
2+And Mg
2+Deng inorganic metal ion, measure of the influence of different ions concentration to the ferment of spiramycin level.Studies show that Cu
2+Biosynthesizing has stronger restraining effect, Ca to Spiramycin Base
2+Little to Spiramycin Base biosynthesizing influence, Fe
2+, Mg
2+Obvious activation is all arranged, wherein Fe
2+To the biosynthetic promoter action maximum of Spiramycin Base, the potency ratio control group is high by 18.3% when interpolation concentration is 2.5 μ g/ml; Mg
2+Effect is best when concentration is 3.0 μ g/ml, and the potency ratio control group exceeds 11.4%.
The fermentative production of existing Spiramycin Base is some conventional fermentative production modes, does not add the production technique of metal ion that is:, and zymotechnique is that freeze pipe is inoculated in female bottle seed culture 40-48h, sub-flask culture 24h, and first order seed is cultivated 23-30h; Secondary seed is cultivated 20-32h; Fermentation culture 100-136h is put jar.Existing outstanding problem is that fermentation unit is lower, and wastes time and energy, and does not often reach the requirement of production.The mn ion that the application adds is not compared with respect to adding metal ion in the prior art, and fermentation unit can improve 25%.
Summary of the invention
The object of the present invention is to provide a kind of easy Spiramycin Base production technique, reach the purpose that improves the SPIRAMYCIN BASE unit output by adding metal ion during the fermentation.The present invention realizes in the following manner:
In one embodiment, the invention provides a kind of zymotechnique of production SPIRAMYCIN BASE, described technology comprises: spiramycin-producing strain is taked seed culture and fermenting process, it is characterized in that during the fermentation, add the aqueous solution of mn ion, make that the concentration of fermentor tank mn ion is 15-40mM.
The fermention medium weight proportion of Shi Yonging is in the present invention: starch 10～15%, soybean cake powder 1～2%, corn steep liquor 2～3%, potassium primary phosphate 0.01～0.02%, sodium-chlor 1～1.5%, lime carbonate 1～1.2%, soya-bean oil 0.5～0.8%.
In the present invention further in the technical scheme, zymotechnique comprises freeze pipe is inoculated in female flask culture base, cultivates 40-48h, shaking speed 220--240 rev/mins down at 27 ± 0.5 ℃; Inoculate sub-flask culture base by 6% inoculum size, cultivate 24h, rotating speed 220--240 rev/min down at 27 ± 0.5 ℃; When proceeding to about 40 hours, fermentation adds the solution that contains mn ion.The concentration of preferred mn ion is 15-40mM.Preferred mn ion source is a tetrahydrate manganese chloride.
In the further method of the present invention, relate to the method for suitability for industrialized production Spiramycin Base, it is characterized in that described method comprises: freeze pipe is inoculated in female flask culture base, cultivates 40-48h, shaking speed 220--240 rev/min down at 27 ± 0.5 ℃; Inoculate sub-flask culture base by 6% inoculum size, at 27 ± 0.5 ℃ of bottom fermentation 24h, rotating speed 220--240 rev/min; Be inoculated in the first class seed pot by 10% inoculum size and ferment, cultivate 23-30h down at 25-28.5 ℃; Be inoculated in the secondary seed jar by 10% culture transferring amount and ferment, cultivate 20-32h down at 20-28 ℃; At last, be inoculated in the fermentor tank by 10% culture transferring amount and ferment, cultivate 100-136h down at 24-28.5 ℃, about when fermentation proceeds to 40 hours, add the aqueous solution of mn ion, make that the manganese ion concentration in the fermented liquid is 15-40mM, finally obtain Spiramycin Base, its fermentation unit can improve about 25%.
In the above-described embodiment, preferred fermentation unit is 100m
3Stainless cylinder of steel, 120～140 rev/mins of the preferable range of rotating speed, the preferable range 0.01～0.04MPa of tank pressure, the preferable range 1500～2000m of air quantity
3/ h.
The present invention compared with prior art has following characteristics:
(1) fermentation level height obviously surpasses the level of former technology.
(2) to not influence of component.
(3) simple to operate.
(4) cost is low, is convenient to scale operation.
This as can be seen method has overcome the deficiency of domestic existing production SPIRAMYCIN BASE technology effectively, is fit to the needs of large-scale commercial production.
Embodiment
Further specify the present invention below by embodiment.The preparation method who it should be understood that the embodiment of the invention is only used for illustrating the present invention, rather than limitation of the present invention, and under design prerequisite of the present invention, similarly the preparation method belongs to the scope of protection of present invention.Except as otherwise noted, percentage ratio among the present invention is weight percentage, in addition, (W/W) in the specification sheets of the present invention is meant weight ratio or weight ratio concentration, (V/V) be meant volume ratio or volume by volume concentration, W/V is meant the ratio (for example, grams per milliliter, g/ml or kg/liter) of weight and volume.
Embodiment 1,2,3 is a shake flat experiment, is with inclined-plane seed switching shake-flask seed substratum enlarged culturing, ferments in shaking bottle; Embodiment 4,5 and 6 is a scale-up, and seed is through one-level, secondary seed jar enlarged culturing, at 100m
3Ferment in the fermentor tank.
Embodiment 1
Inclined-plane (being bacterial classification) is inoculated in the shake-flask seed substratum, cultivates 48h, shaking speed 220r/min down at 27 ℃; Insert in the shake flask fermentation substratum by 6% inoculum size, rotating speed is 220r/min, and under 27 ℃, rotary shaking table is cultivated 96h.
Fermention medium is prepared (fermention medium: starch, potassium primary phosphate, sodium-chlor, ammonium nitrate, lime carbonate, corn steep liquor, soya-bean oil according to a conventional method, its ratio is a starch 10～15%, soybean cake powder 1～2%, corn steep liquor 2～3%, potassium primary phosphate 0.01～0.02%, sodium-chlor 1～1.5%, lime carbonate 1～1.2%, soya-bean oil 0.5～0.8%), when fermentation proceeds to 35 hours, disposable input contains in the manganese tetrachloride aqueous solution and fermented liquid of a certain amount of mn ion, makes that the manganese ion concentration of putting in the fermentation shake flask is 30mM.
Embodiment 2
As the method for embodiment 1, disposable input contains in the aqueous solution and fermented liquid of a certain amount of mn ion when fermentation proceeds to 40 hours, and the manganese ion concentration that put in the fermentation shake flask this moment is 35mM.
Embodiment 3
As the method for embodiment 1, the aqueous solution that disposable input contains a certain amount of mn ion when fermentation proceeds to 45 hours is in fermented liquid, and the manganese ion concentration in the fermentation shake flask is 40mM at this moment.
Table 1. mn ion adding technology is in the experimental result of shaking on the bottle
It is corresponding with the content of Spiramycin Base to tire, and the height of tiring is a sign of estimating fermentation level.Calculate by dry product: every 1mg Spiramycin Base is equivalent to the potency unit of 900 Spiramycin Bases.This shows, in above-mentioned fermenting process, add the content that mn ion has significantly improved Spiramycin Base afterwards.
Embodiment 4
As the method for embodiment 1, insert in the shake flask fermentation substratum by 6% inoculum size, rotating speed is 220r/min, under 27 ℃, rotary shaking table is cultivated 27h.Be inoculated in 1m by 10% inoculum size
3Ferment in the first class seed pot, cultivate 23-30h for 25-28.5 ℃; Be inoculated in 10m by 10% culture transferring amount
3Ferment in the secondary seed jar, cultivate 20-32h for 20-28 ℃; Be inoculated in 100m by 10% culture transferring amount
3Ferment in the fermentor tank, cultivate 100-136h for 24-28.5 ℃.Contain in the aqueous solution (the manganese tetrachloride aqueous solution) and fermented liquid of a certain amount of mn ion when fermentation proceeds to 35 hours disposable inputs, the manganese ion concentration that makes put in the fermentation shake flask this moment is 30mM.
Embodiment 5
As the method for embodiment 4, disposable input contains in the aqueous solution and fermented liquid of a certain amount of mn ion when fermentation proceeds to 40 hours, and the manganese ion concentration that put in the fermentation shake flask this moment is 35mM.
Embodiment 6
As the method for embodiment 4, disposable input contains in the aqueous solution and fermented liquid of a certain amount of mn ion when fermentation proceeds to 45 hours, and the manganese ion concentration that put in the fermentation shake flask this moment is 35mM.
Table 2. mn ion adding technology is at 100m
3Experimental result on the fermentor tank
As can be seen from the above table: at 100m
3Test on the fermentor tank, earlier fermentation (about 40 hours) adding manganese ion concentration is 15-40mM, and the fermentation unit comparison that adds mn ion is according to exceeding about 25.0%.
Claims (2)
1. one kind is added the zymotechnique that metal ion improves SPIRAMYCIN BASE output, and zymotechnique is cultivated 40-48h, rotating speed 220--240 rev/min for 27 ± 0.5 ℃ for freeze pipe is inoculated in female flask culture base; By the 6% inoculum size sub-flask culture base of transferring, cultivate 24h, rotating speed 220--240 rev/min for 27 ± 0.5 ℃; Be inoculated in the first class seed pot by 10% inoculum size and ferment, cultivate 23-30h for 25-28.5 ℃; Be inoculated in the secondary seed jar by 10% culture transferring amount and ferment, cultivate 20-32h for 20-28 ℃; Be inoculated in the fermentor tank by 10% culture transferring amount and ferment, cultivate 100-136h for 24-28.5 ℃; It is characterized in that in the process of fermentation, adding the aqueous solution of mn ion, make that the concentration of mn ion is 15-40mM in the fermented liquid.
2. zymotechnique as claimed in claim 1 is characterized in that, when fermentation proceeds to about 40 hours, adds the aqueous solution of mn ion, makes that the manganese ion concentration in the fermented liquid is 15-40mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009101189202A CN101497907A (en) | 2009-03-06 | 2009-03-06 | Fermentation process of spiramycin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009101189202A CN101497907A (en) | 2009-03-06 | 2009-03-06 | Fermentation process of spiramycin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062947A (en) * | 2015-08-25 | 2015-11-18 | 中农颖泰林州生物科园有限公司 | Production method for bacillus licheniformis with high sporation rate |
-
2009
- 2009-03-06 CN CNA2009101189202A patent/CN101497907A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062947A (en) * | 2015-08-25 | 2015-11-18 | 中农颖泰林州生物科园有限公司 | Production method for bacillus licheniformis with high sporation rate |
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Open date: 20090805 |