CN1563400A - Method for separating and preparing prodigiosin - Google Patents
Method for separating and preparing prodigiosin Download PDFInfo
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- CN1563400A CN1563400A CN 200410017773 CN200410017773A CN1563400A CN 1563400 A CN1563400 A CN 1563400A CN 200410017773 CN200410017773 CN 200410017773 CN 200410017773 A CN200410017773 A CN 200410017773A CN 1563400 A CN1563400 A CN 1563400A
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- prodigiosin
- resin
- fermentation
- fermented liquid
- adsorption column
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Abstract
This invention discloses a method for preparing prodigiosin by separation utilizing big hole adsorption to adsorb prodigiosin directly in the fermented solution and realizing coupling of fermentation and separation to finish the process of concentration and initial impurities remove on the adsorption post. Then, according to the composition in the eluent, a suitable solvent system is applied to adsorb impurities instead of the products to get rather pure products.
Description
Technical field
The present invention relates to separate the method for preparing prodigiosin, be specifically related to directly from fermented liquid, separate the method for preparing prodigiosin.
Background technology
Prodigiosin is that its structural formula is by the red pigments of serratia marcescens at a kind of tripyrrole ring of fermenting process generation:
Molecular weight: 324
It has the biological activity that promotes cancer cell-apoptosis after deliberation, is a kind of compound candidate with cancer therapy drug potentiality.Existing general preparation method, as (Montaner, B.and P é rez-Tom á s, R., 2000.British Journal of Pharmacology 131,585-593.) the disclosed technology of document, be to be produced by fermentation by serratia marcescens, centrifugation obtains thalline after fermentation ends, uses methanol extraction then, after dividing the thalline of leaving away extracting solution is concentrated, use the chromatography method separation and purification at last.This explained hereafter link is many, and the organic solvent consumption is big, and the yield of product is lower, generally only is 50%.
Summary of the invention
The technical issues that need to address of the present invention are to disclose a kind of method for preparing prodigiosin of separating, to overcome the above-mentioned defective that prior art exists.
Technical conceive of the present invention is such:
The present invention uses macroporous adsorbent resin directly to adsorb prodigiosin in fermented liquid, and can realize fermentation-separation coupling, finishes the process that concentrates with preliminary removal of impurities on adsorption column.The situation of component in the elutriant that elutes according to adsorption column adopts proper solvent system, and adopts absorption impurity and the strategy of non-adsorbed product, and a step separates and can obtain purer product.This technology has been simplified separating step greatly, has reduced consumption of organic solvent, has improved product yield and production efficiency, is more suitable in large-scale production.
Method of the present invention comprises the steps:
(1) serratia marcescens is being equipped with the fermentation cylinder for fermentation of substratum, then fermented liquid is being sent into the adsorption column that is filled with resin by recycle pump from the bottom, flowing back to fermentor tank, circulating fermentation again through the fermented liquid behind the post bed.
The processing condition that fermentation is adopted are identical with conventional method, as document (High production ofprodigiosin by Serratia marcescens grown on ethanol., Biotechnol.Lett., 22 (22), 1761-1765 (English) 2000) disclosed technology, wherein the component of substratum and concentration are generally (g/L): glycerine 10; Peptone 10; Potassium primary phosphate 1; Sodium-chlor 0.5, culture temperature are 28 ℃, and dissolved oxygen is 40～60%%, cultivate to be 36h.
Said resin is a macroporous adsorbent resin, preferably adopts polystyrene-divinylbenzene type macroporous adsorbent resin, and the model of producing as (resin processing plant of Nankai University) is to be the polystyrene-divinylbenzene type macroporous adsorbent resin of X-5.
According to optimized technical scheme of the present invention, begin again fermented liquid is sent into the adsorption column that is filled with resin when fermenting to 10-20h;
Resin demand is the 5-10% of fermentating liquid volume;
The flow velocity of fermented liquid in adsorption column be 5 column volumes/hour-; Because the present invention is a kind of will the fermentation and the fractionation by adsorption method of carrying out synchronously, therefore, the linear velocity of fermented liquid in adsorption column can not be too high, too high will causing adsorbed more thalline, is unfavorable for fermentation, but can not be too low, too lowly can not adsorb prodigiosin fully, and make sepn process incomplete;
Should be noted that and simply fermentation and fractionation by adsorption to be made up that because carry out fractionation by adsorption in fermentation, the concentration of product is lower in the fermented liquid, therefore, should control certain flow velocity.
Fermentation period is 36-40h, and the prodigiosin adsorptive capacity can reach the 7mg/g resin;
(2) adopt conventional method wash-out from adsorption column to collect prodigiosin then.
Concrete wash-out is collected and is comprised the steps:
With volumetric concentration is 30～70% methanol aqueous solution washing adsorption column, to remove the impurity of absorption.Use methanol-eluted fractions then, obtain its crude product after the elutriant that will contain prodigiosin concentrates, content is greater than 40%.
Further, can the crude product that obtain is further refining with the method for silica gel column chromatography, its method is as follows:
Can remove insolubles with said dissolving crude product in non-polar solvent, go up silicagel column then, because in above-mentioned non-polar solvent, therefore a little less than the absorption of silica gel to prodigiosin, be not adsorbed and pass, thereby other impurity such as pigment are then reached the separation purpose by silica gel adsorption on post.Collection passes liquid, and concentrated, oven dry obtains the purified prodigiosin, and content can reach more than 90%.
Said non-polar solvent comprises ethyl acetate, its mixture of chloroform.
By above-mentioned disclosed technical scheme as seen, method explained hereafter link of the present invention is few, and the resin absorption amount of employing is big, and the organic solvent consumption is low, and the yield height of product is a kind of method of separating prodigiosin for preparing with suitability for industrialized production prospect.
Embodiment
Embodiment 1
(φ 60 * 300mm) to connect a resin column outside 5L fermentor tank (interior dress 2.5L fermented liquid), resin demand is 8% of a fermentating liquid volume, with pump is that power makes fermented liquid pass through resin column, the end, go out on advancing, fermented liquid through resin flows back in the jar again, the prodigiosin that fermenting process produces is adsorbed on the resin, and thalline can be got back to its effect of continuation in the fermented liquid by resin, ferment to 38h, fermentation ends, at this moment the prodigiosin content in the fermented liquid is very low, and major part is adsorbed on the resin.
The resin that absorption is saturated is removed impurity with deionized water and methanol (1: 1) washing, and consumption is each 5 column volume, use methanol-eluted fractions afterwards, after the whole wash-outs of prodigiosin to be adsorbed were intact, the methyl alcohol in the water flush away post used in order to next time, and resin does not need every batch all to regenerate.
The elutriant evaporation concentration that will contain prodigiosin is to smaller size smaller, mixed solvent dissolving with ethyl acetate/chloroform (2: 1), remove insolubles, dewater with anhydrous magnesium sulfate, go up silicagel column then, collect and pass liquid, concentrate the highly finished product that obtain prodigiosin after drying, content can reach 90%, and the yield of all processes can reach 70%.
Wherein the component of substratum and concentration are (g/L): glycerine 10; Peptone 10; Potassium primary phosphate 1; Sodium-chlor 0.5, culture temperature are 28 ℃, and dissolved oxygen is 40～60%%.
Claims (8)
1. one kind is separated the method for preparing prodigiosin, it is characterized in that, comprises the steps:
Serratia marcescens is being equipped with the fermentation cylinder for fermentation of substratum, then fermented liquid is being sent into the adsorption column that is filled with resin by recycle pump from the bottom, flowing back to fermentor tank, circulating fermentation again through the fermented liquid behind the post bed; Adopt conventional method wash-out from adsorption column to collect prodigiosin then, said resin is a polystyrene-divinylbenzene type macroporous adsorbent resin.
2. method according to claim 1 is characterized in that, begins fermented liquid is sent into the adsorption column that is filled with resin when fermenting to 10-20h again.
3. method according to claim 1 is characterized in that, resin demand is the 5-10% of fermentating liquid volume.
4. method according to claim 1 is characterized in that, the flow velocity of fermented liquid in adsorption column be 5 column volumes/hour.
5. method according to claim 1 is characterized in that, fermentation period is 36-40h.
6. according to each described method of claim 1～5, it is characterized in that wash-out collects to comprise the steps: with volumetric concentration being 30～70% methanol aqueous solution washing adsorption column, to remove the impurity that adsorbs.Use methanol-eluted fractions then, obtain its crude product after the elutriant that will contain prodigiosin concentrates.
7. method according to claim 6 is characterized in that, the crude product that obtains is further refining with the method for silica gel column chromatography, and its step is as follows:
Said dissolving crude product in non-polar solvent, is removed insolubles, go up silicagel column then, collect and pass liquid, concentrated, oven dry obtains the purified prodigiosin.
8. method according to claim 7 is characterized in that, said non-polar solvent is ethyl acetate, chloroform mixture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410017773 CN1563400A (en) | 2004-04-19 | 2004-04-19 | Method for separating and preparing prodigiosin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410017773 CN1563400A (en) | 2004-04-19 | 2004-04-19 | Method for separating and preparing prodigiosin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1563400A true CN1563400A (en) | 2005-01-12 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CN 200410017773 Pending CN1563400A (en) | 2004-04-19 | 2004-04-19 | Method for separating and preparing prodigiosin |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199366A (en) * | 2011-03-30 | 2011-09-28 | 嘉兴学院 | Biological dye containing prodigiosins, preparation method thereof and application thereof |
| CN102311981A (en) * | 2011-07-19 | 2012-01-11 | 东南大学 | Method for preparing and purifying prodigiosin |
| CN101717789B (en) * | 2009-12-14 | 2012-06-20 | 昆明理工大学 | Method for preparing culture medium for efficiently producing haematochrome |
| CN103436476A (en) * | 2013-09-17 | 2013-12-11 | 中国科学院烟台海岸带研究所 | Prodigiosin (PG) producing strain as well as preparation method and application thereof |
| CN103627650A (en) * | 2013-08-15 | 2014-03-12 | 上海理工大学 | Serratia marcescens strain and synchronous extraction and fermentation method thereof |
| CN103757069A (en) * | 2014-01-09 | 2014-04-30 | 嘉兴学院 | Method for producing prodigiosin by coupling resin adsorption in-situ separation with fermentation |
| CN104193665A (en) * | 2013-08-30 | 2014-12-10 | 西藏天虹科技股份有限责任公司 | Separating and purifying method for prodigiosin |
-
2004
- 2004-04-19 CN CN 200410017773 patent/CN1563400A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717789B (en) * | 2009-12-14 | 2012-06-20 | 昆明理工大学 | Method for preparing culture medium for efficiently producing haematochrome |
| CN102199366A (en) * | 2011-03-30 | 2011-09-28 | 嘉兴学院 | Biological dye containing prodigiosins, preparation method thereof and application thereof |
| CN102311981A (en) * | 2011-07-19 | 2012-01-11 | 东南大学 | Method for preparing and purifying prodigiosin |
| CN103627650A (en) * | 2013-08-15 | 2014-03-12 | 上海理工大学 | Serratia marcescens strain and synchronous extraction and fermentation method thereof |
| CN103627650B (en) * | 2013-08-15 | 2015-09-16 | 上海理工大学 | One strain Serratia bacteria strain and synchronous extractive fermentation method thereof |
| CN104193665A (en) * | 2013-08-30 | 2014-12-10 | 西藏天虹科技股份有限责任公司 | Separating and purifying method for prodigiosin |
| CN104193665B (en) * | 2013-08-30 | 2016-06-22 | 西藏天虹科技股份有限责任公司 | A kind of isolation and purification method of prodigiosin |
| CN103436476A (en) * | 2013-09-17 | 2013-12-11 | 中国科学院烟台海岸带研究所 | Prodigiosin (PG) producing strain as well as preparation method and application thereof |
| CN103757069A (en) * | 2014-01-09 | 2014-04-30 | 嘉兴学院 | Method for producing prodigiosin by coupling resin adsorption in-situ separation with fermentation |
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