CN101838316B - Method for preprocessing Ramoplanin fermentation liquor - Google Patents
Method for preprocessing Ramoplanin fermentation liquor Download PDFInfo
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- CN101838316B CN101838316B CN2009100477255A CN200910047725A CN101838316B CN 101838316 B CN101838316 B CN 101838316B CN 2009100477255 A CN2009100477255 A CN 2009100477255A CN 200910047725 A CN200910047725 A CN 200910047725A CN 101838316 B CN101838316 B CN 101838316B
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- ramoplanin
- fermented liquid
- fermentation liquor
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Abstract
The invention provides a method for preprocessing Ramoplanin fermentation liquor, which is characterized by comprising the following steps: a) adjusting the pH value of the Ramoplanin fermentation liquor to be 8-10; b) adding flocculant in the fermentation liquor; c) filtering; and d) extracting Ramoplanin of filter cake. The method for preprocessing the Ramoplanin fermentation liquor has the advantages of simple steps, short operation time and high yield.
Description
Technical field
The invention belongs to the pharmaceutical chemistry field, be specifically related to a kind of pretreated method of Ramoplanin fermentation liquor.
Background technology
Along with the extensive application of microbiotic in anti-infective therapy, the clinical drug-resistant problem becomes increasingly conspicuous.Ramoplanin (RAMOPLANIN) mainly is to research and develop as external application and the gastrointestinal tract mucous surface sense metachromia disease medicament of treatment as one type of New-type wide-spectrum resisting gram-positive bacteria microbiotic at present.
Ramoplanin is made by a kind of actinoplanes bacterial classification (ATCC is preserved, code ATCC33076).Its traditional pretreatment process is that the fermented liquid that the ATCC33076 fermentation strain ferments is out transferred pH to 3.5 earlier, centrifugal mycelium and the fermented liquid of obtaining, and wherein mycelium extracts with organic solvent; The extraction final vacuum concentrates; Again with n-butanol extraction repeatedly, with sherwood oil it is precipitated repeatedly behind the combining extraction liquid again and separate, vacuum-drying obtains bullion; And fermented liquid is used n-butanol extraction, makes bullion with quadrat method.
The pretreatment process of the ramoplanin of reporting among the USP4328316 is through high speed centrifugation, and fermented liquid and mycelium are separated, and handles fermented liquid and mycelium more respectively.The shortcoming of this method is to use expensive large centrifugal equipment, and because the viscosity of Ramoplanin fermentation liquor is very big, the effect of solid-liquid separation is bad; In addition, owing to must handle complex operation step respectively to fermented liquid and mycelium; The solvent usage quantity is big, is unfavorable for very much suitability for industrialized production.
Summary of the invention
Technical problem to be solved by this invention provides that a kind of step is simple, the running time is short, and keeps the method for the pre-treatment Ramoplanin fermentation liquor of high yield simultaneously.
The present invention is based on throwing out is applied in the pre-treatment of Ramoplanin fermentation liquor, through screening various flocculation agents, groping its ratio that adds fermented liquid and accomplish.
The principle of throwing out is under the condition that flocculation agent exists, and between suspended particles, produces bridging action and makes the micelle material in the fermented liquid form thick flocculation group, thereby make that fermented liquid is easy to filter.For Ramoplanin fermentation liquor; Because the specificity of its mycelia form, viscosity is very big, is suction filtration or centrifugally all is difficult to obtain good effect; After adding flocculation agent of the present invention; Because the bridging action that produces makes the bigger protein colloid material aggregation of fermented liquid medium viscosity become piece, thereby can increase filtering speed greatly.
The present invention finds two kinds of extraordinary flocculation agents of flocculating effect through the various flocculation agents of screening, and optimizes best addition.After adding flocculation agent of the present invention, originally the unusual heavy-gravity fermented liquid easy filtration very that becomes directly can be opened solid-liquid separation fast through B, thereby avoid the super-magnum centrifuge treating processes loaded down with trivial details, the problem that centrifugation time is long.
In addition, the present invention is through to the investigation of fermented liquid pH, find that pH with fermented liquid is transferred to 8～10 after, add flocculation agent again, filtering the back ramoplanin will almost not have unit in the filtrating fully in mycelium.Can ramoplanin be extracted with organic solvent for mycelium.Compare with existing patent, this technology has been removed from from mycelium and has been filtrated and extract ramoplanin respectively, only needs handle mycelium, thereby has simplified technology.
Therefore, the present invention provides a kind of method for preprocessing ramoplanin fermentation liquor, and this method comprises the steps:
A) with the pH regulator of Ramoplanin fermentation liquor to 8-10;
B) in fermented liquid, add flocculation agent;
C) filter;
D) ramoplanin in the extraction filter cake.
Wherein, a) in the step pH of Ramoplanin fermentation liquor can regulate with NaOH solution; B) flocculation agent described in the step is preferably yellow prussiate of potash and zinc sulfate; The amount that said yellow prussiate of potash and zinc sulfate add is preferably 0.2～1% of said Ramoplanin fermentation liquor volume; Here the add-on of flocculation agent is minimum wants 0.2%; But do not limit maximum consumption, select 1% for the upper limit is the consideration from cost, 1% can be good at filtering Ramoplanin fermentation liquor fully; C) filter type in the step is preferably suction filtration; D) ramoplanin in the filter cake can be used the for example extractions such as methyl alcohol, ethanol or acetone of organic solvent of certain volume in the step.
Method for preprocessing ramoplanin fermentation liquor of the present invention is specifically operated according to following steps:
1) fermented liquid of ramoplanin is put jar back and transfer its pH to 8-10, leave standstill about 1h after the stirring with NaOH solution;
2) in fermented liquid, add flocculation agent (preferred yellow prussiate of potash and zinc sulfate), the amount of adding can leave standstill 0.5h for 0.2～1% (volume percent) after the stirring;
3) carry out suction filtration with B, the suction filtration time looks the volume of fermented liquid and decides, and is for the fermented liquid of several liters, fully can suction filtration in 20 minutes good;
4) suction filtration discards filtrate after finishing, and the organic solvent (particular methanol, ethanol, acetone etc.) of filter cake with certain volume extracted it.
Method for preprocessing ramoplanin fermentation liquor of the present invention has the advantage that step is simple, the running time is short and yield is high.
Embodiment
Ramoplanin fermentation liquor of the present invention is made by following method: with preserving number is that the fermentation strain of ATCC33076 ferments, wherein:
Slant culture condition: inclined-plane and plate culture medium (g/L): Carnis Bovis seu Bubali cream 3.0, casein 10.0, soluble starch 24.0, glucose 1.0, agar 22.0,7.0,121 ℃ of sterilizations of pH 30min before the sterilization.Bacterial classification inoculation was cultivated 5-9 days in 30 ℃ in slant medium.
The seed culture condition: second order fermentation seed culture condition is: Carnis Bovis seu Bubali cream 3.0, and casein 5.0, yeast powder 5.0, oatmeal 30.0, lime carbonate 4.0, the tap water preparation is with NaOH (perhaps HCl) the accent pH to 7.0 of 2mol/L, 121 ℃ of sterilization 30min.The seed culture medium loading amount is 100ml/750ml, and in 28 ℃, the rotary shaking table of 250rpm is cultivated 72h.
Fermentation culture conditions is: starch 50.0, and glucose 20.0, analysis for soybean powder 30.0, lime carbonate 10.0, L-Leu5.0, the tap water preparation is with NaOH (perhaps HCl) the accent pH to 7.0 of 2mol/L, 121 ℃ of sterilization 30min.Inoculum size 6%, loading amount 100ml/750ml, in 25 ℃, rotary shaking table 250rpm cultivated 5 days.
The used flocculation agent of the present invention:
1 yellow prussiate of potash
Chemical Reagent Co., Ltd., Sinopharm Group's analytical pure
2 zinc sulfate (Zinc vitriol)
Chemical Reagent Co., Ltd., Sinopharm Group's analytical pure
Embodiment 1～3 investigates the influence of the ratio of adding flocculation agent to the inventive method
Embodiment 1
The fermented liquid 3000ml of ramoplanin, unit is 1062ug/ml, put jar after, transfer its pH to 8.5 with NaOH solution; Leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate each 0.2%, leave standstill 0.5h after the stirring, leave standstill and then carry out suction filtration with B; Taking out the rate time is about 18min, and the intact back of suction filtration is detected with HPLC, and finding does not have unit in the filtrating; Discard filtrate, the methyl alcohol of filter cake with 1500ml is extracted it, surveying its unit is 882ug/ml.
Embodiment 2
The fermented liquid 3000ml of ramoplanin, unit is 967ug/ml, put jar after, transfer its pH to 8.5 with NaOH solution; Leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate each 1%, leave standstill 0.5h after the stirring, leave standstill and then carry out suction filtration with B; The suction filtration time is about 12min, and the intact back of suction filtration is detected with HPLC, and finding does not have unit in the filtrating; Discard filtrate, the methyl alcohol of filter cake with 1500ml is extracted it, surveying its unit is 891ug/ml.
Embodiment 3
The fermented liquid 3000ml of 1 ramoplanin, unit is 1096ug/ml, put jar after, transfer its pH to 9.0 with NaOH solution; Leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate each 0.5%, leave standstill 0.5h after the stirring, leave standstill and then carry out suction filtration with B; The suction filtration time is about 15min, and the intact back of suction filtration is detected with HPLC, and finding does not almost have unit in the filtrating; Discard filtrate, the methyl alcohol of filter cake with 1500ml is extracted it, surveying its unit is 877ug/ml.
Embodiment 4～6 investigates the influence of pH value scope to the inventive method
Embodiment 4
The fermented liquid 3000ml of ramoplanin, unit is 957ug/ml, put jar after; Transfer its pH to 8.0 with NaOH solution, leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate respectively 0.5%; Leave standstill 0.5h after the stirring, leave standstill the back and carry out suction filtration with B, the suction filtration time is about 15min; The intact back of suction filtration is detected with HPLC, and finding has a spot of ramoplanin in the filtrating, and unit is 12ug/ml; Discard filtrate, the methyl alcohol of filter cake with 1500ml is extracted it, surveying its unit is 882ug/ml.
Embodiment 5
The fermented liquid 3000ml of ramoplanin, unit is 855ug/ml, put jar after, transfer its pH to 10.0 with NaOH solution; Leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate each 0.5%, leave standstill 0.5h after the stirring, leave standstill and then carry out suction filtration with B; The suction filtration time is about 15min, and the intact back of suction filtration is detected with HPLC, and finding does not almost have unit in the filtrating; Discard filtrate, the methyl alcohol of filter cake with 1500ml is extracted it, surveying its unit is 796ug/ml.
Embodiment 6
The fermented liquid 3000ml of ramoplanin, unit is 952ug/ml, put jar after, transfer its pH to 9.0 with NaOH solution; Leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate each 0.5%, leave standstill 0.5h after the stirring, leave standstill and then carry out suction filtration with B; The suction filtration time is about 15min, and the intact back of suction filtration is detected with HPLC, and finding does not almost have unit in the filtrating; Discard filtrate, the methyl alcohol of filter cake with 1500ml is extracted it, surveying its unit is 844ug/ml.
Embodiment 7～8 investigates the influence of different solvents to the inventive method
Embodiment 7
The fermented liquid 3000ml of 1 ramoplanin, unit is 1120ug/ml, put jar after, transfer its pH to 9.0 with NaOH solution; Leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate each 0.5%, leave standstill 0.5h after the stirring, leave standstill and then carry out suction filtration with B; The suction filtration time is about 15min, and the intact back of suction filtration is detected with HPLC, and finding does not almost have unit in the filtrating; Discard filtrate, the ethanol of filter cake with 1500ml is extracted it, surveying its unit is 921ug/ml.
Embodiment 8
The fermented liquid 3000ml of ramoplanin, unit is 1120ug/ml, put jar after, transfer its pH to 9.0 with NaOH solution; Leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate each 0.5%, leave standstill 0.5h after the stirring, leave standstill and then carry out suction filtration with B; The suction filtration time is about 15min, and the intact back of suction filtration is detected with HPLC, and finding does not almost have unit in the filtrating; Discard filtrate, the methyl alcohol of filter cake with 1500ml is extracted it, surveying its unit is 1033ug/ml.
Comparative Examples 1
The fermented liquid 3000ml of ramoplanin, unit is 1096ug/ml, put jar after; Transfer its pH to 3.5 with hydrochloric acid soln, leave standstill 1h after the stirring, with BECKMAN whizzer high speed centrifugation; Rotating speed is 4000 rev/mins, behind the centrifugal 0.5h, supernatant is detected with HPLC; Its unit is 233ug/ml, and the handy 1500ml methanol extraction of solid collection is come out, and surveying its unit is 564ug/ml.
Can find out that by Comparative Examples 1 ramoplanin all has unit in supernatant and deposition, must further handle respectively, therefore increase separation steps.
Comparative Examples 2
The fermented liquid 3000ml of ramoplanin, unit is 896ug/ml, put jar after, transfer its pH to 4.0 with hydrochloric acid soln; Leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash and zinc sulfate respectively 0.5%, leave standstill 0.5h after the stirring; Leave standstill the back and carry out suction filtration with B, the suction filtration time is about 15min, and the intact back of suction filtration is detected with HPLC; Find that ramoplanin unit is 426ug/ml in the filtrating, the methyl alcohol of filter cake with 1500ml extracted it that surveying its unit is 451ug/ml.
Can find out by Comparative Examples 2, all contain ramoplanin in filtrating and the mycelium, must further handle respectively, therefore increase separation steps.
Comparative Examples 3
The fermented liquid 3000ml of ramoplanin, unit is 952ug/ml, put jar after; Transfer its pH to 9.0 with NaOH solution, leave standstill 1h after the stirring, in fermented liquid, add yellow prussiate of potash 1%; Leave standstill 0.5h after the stirring; Find that fermented liquid still is gluey, be difficult to when filtering filter that the 2h rear filtrate is collected about 400ml with B.
Comparative Examples 4
The fermented liquid 3000ml of ramoplanin, unit is 952ug/ml, put jar after; Transfer its pH to 9.0 with NaOH solution, leave standstill 1h after the stirring, in fermented liquid, add zinc sulfate 1%; Leave standstill 0.5h after the stirring; Find that fermented liquid is gluey, be difficult to when filtering filter that the 2h rear filtrate is collected about 350ml with B.
Can find out that by Comparative Examples 3 and 4 separating effect that only adds a kind of flocculation agent acquisition is not good.
Claims (5)
1. method for preprocessing ramoplanin fermentation liquor is characterized in that, comprises the steps:
A) with the pH regulator of Ramoplanin fermentation liquor to 8-10;
B) yellow prussiate of potash and the zinc sulfate of the 0.2-1% of adding fermentating liquid volume in fermented liquid;
C) filter;
D) ramoplanin in the extraction filter cake.
2. method according to claim 1 is characterized in that, regulates the pH of Ramoplanin fermentation liquor with NaOH solution.
3. method according to claim 1 is characterized in that, c) is filtered into suction filtration described in the step.
4. method according to claim 1 is characterized in that, with the ramoplanin in the organic solvent extraction filter cake.
5. method according to claim 4 is characterized in that, said organic solvent is methyl alcohol, ethanol or acetone.
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| CN2009100477255A CN101838316B (en) | 2009-03-17 | 2009-03-17 | Method for preprocessing Ramoplanin fermentation liquor |
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| CN2009100477255A CN101838316B (en) | 2009-03-17 | 2009-03-17 | Method for preprocessing Ramoplanin fermentation liquor |
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| CN101838316A CN101838316A (en) | 2010-09-22 |
| CN101838316B true CN101838316B (en) | 2012-11-28 |
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| CN103130874B (en) * | 2011-12-02 | 2015-08-05 | 华北制药集团新药研究开发有限责任公司 | A kind of preparation method of high purity ramoplanin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101024662A (en) * | 2006-02-24 | 2007-08-29 | 上海医药工业研究院 | Method for purifying Ramoplanin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101024662A (en) * | 2006-02-24 | 2007-08-29 | 上海医药工业研究院 | Method for purifying Ramoplanin |
Non-Patent Citations (1)
| Title |
|---|
| Wu Xiangjin et al.Improvement of Ramoplanin Production by Mutation and Medium Optimization.《Journal of Northeast Agricultural University(English Edition)》.2008,第15卷(第01期),18-22. * |
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