CN101130797A - Ubelin manufacturing technique - Google Patents
Ubelin manufacturing technique Download PDFInfo
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- CN101130797A CN101130797A CNA2007100257805A CN200710025780A CN101130797A CN 101130797 A CN101130797 A CN 101130797A CN A2007100257805 A CNA2007100257805 A CN A2007100257805A CN 200710025780 A CN200710025780 A CN 200710025780A CN 101130797 A CN101130797 A CN 101130797A
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Abstract
The present invention relates to a production process of citicoline. It is characterized by that said production process includes the following steps: firstly, adding water and glucose into a reaction tank, then adding yeast, making fermentation, after fermentation adding inorganic salt and choline phosphate, the adding cytidine monophosphate solution, an adding cane sugar, heating, then quickly-cooling by using water, pressing reaction liquid, washing by using water, removing salt from obtained clear liquor; then pressing said clear liquor and making said clear liquor be fed into a carbon column, rinsing said carbon column by using pure water, then using ethyl alcohol solution to make elution, collecting citicoline sodium; then vaporizing and concentrating eluent, diluting concentrate and making it be fed into a macroporous ion exchange resin column, then washing said column by using water and making elution, concentrating eluent, heating the collected citicoline sodium solution, removing impurity by utilizing ultrafiltration device, decolouring, adding ethyl alcohol and stirring them, storing the above-mentioned material in a refrigeration house and staying overnight; then making crystal solution undergo the processes of centrifugal separation, vacuum drying, bagging, weighing, sealing and dry-storage.
Description
Technical field:
The present invention relates to production process of chemical product, is a kind of brew-house to be produced the Ubelin manufacturing technique that the depleted yeast is handled.
Background technology:
Existing Ubelin manufacturing technique, the transformation efficiency and the recovery rate of product are low, and the depleted yeast causes very big pollution to environment in the production process, and the yeast resource can not be fully used, the production cost height.Product is behind biochemical reaction, and fermented liquid can not be removed impurity such as inorganic salt effectively, and the purity of product is low.Product color and albumen are difficult to remove, and the clarity of product is bad, has the living contaminants problem in the production process.Brewery produces the active low of depleted zymic bacterial classification, thereby makes the transformation efficiency of product and recovery rate all very low, is difficult to reach standard-required.
Summary of the invention:
The purpose of this invention is to provide a kind of product transformation efficiency and recovery rate height, reduce environmental pollution, reduce cost, improve the Ubelin manufacturing technique of product purity.
Technical solution of the present invention is: a kind of Ubelin manufacturing technique is characterized in that:
At first feed intake: in retort, add entry and glucose, be warmed up to 40～42 ℃ and drop into yeast, the fermentation back adds inorganic salt, phosphorylcholine, after waiting to heat up, drop into cytidylic acid solution, continue to rise wet 40 ± 1 ℃ of insulations, reaction again, add sucrose then, and every two hours add once, when transformation efficiency reaches 80% termination reaction when above; Be warmed up to 85 ℃, the water cool to room temperature send the squeezing machine squeezing reaction solution then rapidly; Squeezing finishes water top, back washes, and will regulate behind the PH gained clear liquid and send into and receive the film filter desalination;
Secondly, sample on the carbon post: carbon post on the squeezing clear liquid behind the carbon post end of the sample, with pure water rinsing carbon post, makes carbon post effluent liquid not have Cl
-With the alkali ethanolic soln citicoline sodium is eluted again, and when the uv-absorbing thing is arranged, begin to collect;
In the film vacuum concentration pot, vaporize elutriant concentrated;
Secondly be that ion exchange resin separates again: it is 2% that concentrated solution is diluted with water to concentration, last macroporous ion exchange resin post; Behind the end of the sample, the washing pillar; Be concentrated to concentration 75% again with the 1%NaCl wash-out for preparing, and with elutriant;
Make with extra care then: the cytidine diphosphate sodium solution of collecting is heated to about 40 degree delivers to 5000 molecular weight ultra-fine filter removal of impurities, decolouring; Again with destainer with 6000 molecular weight ultra-fine filter ultrafiltration, add ethanol then and stir, and place freezer and spend the night; Again crystal solution is carried out centrifugation, again the product of wetting are carried out vacuum-drying, oven dry back bagging and weighing hermetically drying is preserved.
Described in the present invention when dropping into yeast, stir, make its fermentation add inorganic salt, phosphorylcholine again in 3～5 hours, when treating that temperature is raised to 30 ℃, drop into cytidylic acid solution, continue to be warmed up to 40 ± 1 ℃ again, the insulation timing.Temperature of reaction is controlled at 40 ± 1 ℃, and sampling send the analyzer room to survey transformation efficiency after 3 hours, adds sucrose then, its addition is half when feeding intake, after, every two hours just need add sucrose once, get final product termination reaction when above when transformation efficiency reaches 80%, can prolong the reaction times if be lower than 70%.Squeezing finishes water top, back washes, and up to concentration＜10% of effluent liquid CDP-e o'clock, stops to collect.Pressed liquor is regulated PH, and squeezing is handled again, and gained clear liquid metering cumulative volume, and sampling and measuring content calculate total content.On the carbon post during sample,, suitably reduce flow velocity after 4 hours with mixing up carbon post on the squeezing clear liquid of PH.Flushing carbon post is the pure water with PH6.0, and carbon post effluent liquid is not have Cl with 1% Silver Nitrate inspection
-The wash-out citicoline sodium is with 0.05N alkali ethanol (25% ethanol) solution, and effluent liquid is observed under ultraviolet lamp with the slide sampling, sees that the uv-absorbing thing just begins to collect.Behind the end of the sample, the washing pillar checks that flow velocity can suitably be accelerated when no uv-absorbing logistics went out.When adding the ethanol stirring, treat to continue again to stir 2 hours after white precipitate is separated out, place freezer then and spend the night.
Beneficial effect of the present invention is: at first solve and activate yeast cell system, make phosphorylcholine-cytidyl-transferase fully dissociate out, improve the transformation efficiency and the recovery rate of product, the depleted yeast is to the pollution of environment in the minimizing production process, the yeast resource is fully used, and production cost obviously descends.Solve product behind biochemical reaction, how fermented liquid removes impurity such as inorganic salt effectively, improves the purity of product.Solve product color and albumen and remove, improve the clarity of product, and the living contaminants problem in the production process.Stronger glycolysis-ability and higher phosphorylcholine-cytidyl-transferase are arranged in the yeast cell.According to this characteristic, the present invention produces the depleted yeast by supplementary carbon source with brew-house, cultivates, and through Secondary Fermentation, adds an amount of inorganic salt and activates phosphorylcholine-cytidyl-transferase in the yeast cell, and citicoline sodium is synthesized in catalysis.Adopt this method, the activity of barms is improved, thereby produce higher phosphorylcholine-cytidyl-transferase, the transformation efficiency of product is brought up to more than 90% by 80%, the recovery rate of product brings up to 120% by 100%.Adopt ultra-filtration technique, remove albumen effectively, reduce the color and luster of product, solved the living contaminants in the production process effectively, the purity of product is reached more than 99%, and make clarity and thermal source all reach " 2005 editions standard-requireds of Chinese pharmacopoeia.The present invention makes full use of the yeast resource, and after producing the yeast separation of citicoline sodium, the precursor ergot steroid dry yeast of yeast extract or vitamin D2 is produced in regeneration, make production cost drop to 1500 yuan/Kg, realize cleaner production, reduced environmental pollution, embodied recycling economy value.
The present invention is described in further detail below in conjunction with embodiment:
Embodiment:
Batching (with 5 '-cytidylic acid is a radix): 5 '-cytidylic acid 1Kg, cereuisiae fermentum 30Kg, sucrose 3Kg, glucose 3Kg, water 120Kg, phosphorylcholine 5Kg.
Embodiment one:
One, feed intake:
1, in retort, adds tap water and glucose, be warmed up to 41 ℃ then, drop into yeast, stir, make its fermentation add inorganic salt, phosphorylcholine again in 4 hours, when treating that temperature is raised to 30 ℃, drop into cytidylic acid solution, continue to be warmed up to 40 ℃ again, the insulation timing.
2, temperature of reaction is controlled at 40 ℃, and sampling send the analyzer room to survey transformation efficiency after 3 hours, adds sucrose then, its addition is half when feeding intake, after, every two hours just need add sucrose once, get final product termination reaction when above when transformation efficiency reaches 80%, can prolong the reaction times if be lower than 70%.
3, reaction ends, and is warmed up to 85 ℃, uses the water cycle cool to room temperature rapidly, send the squeezing machine squeezing reaction solution then.
4, squeezing finishes water top, back and washes, and up to concentration＜10% of effluent liquid CDP-e o'clock, stops to collect.
5, pressed liquor is regulated PH, squeezing is handled again, and gained clear liquid metering cumulative volume, and sampling and measuring content calculate total content.
6, above-mentioned clear liquid is sent into received the film filter desalination.
Two, sample on the carbon post
1, will mix up carbon post on the squeezing clear liquid of PH, suitably reduce flow velocity after 4 hours.
2, washing carbon post: behind the carbon post end of the sample,, wash carbon post effluent liquid always and do not have Cl with 1% Silver Nitrate inspection with the pure water rinsing carbon post of PH6.0
-
3, carbon post wash-out: behind the carbon post wash clean, with 0.05N alkali ethanol (25% ethanol) solution citicoline sodium is eluted, effluent liquid is observed under ultraviolet lamp with the slide sampling, sees that the uv-absorbing thing just begins to collect.
Three, concentrate
In the film vacuum concentration pot, vaporize elutriant concentrated.
Four, ion exchange resin separates
1, sample on the ion exchange resin: it is 2% that concentrated solution is diluted with water to concentration, last macroporous ion exchange resin post.
2, ion exchange resin washing: behind the end of the sample, the washing pillar checks that flow velocity can suitably be accelerated when no uv-absorbing logistics went out.
3, exchange resin elution: with the 1%NaCl wash-out for preparing, wash-out finishes, and calculates recovery rate.
4, elutriant is concentrated to concentration about 75%.
Five, refining
1, decolours: the cytidine diphosphate sodium solution of collecting is heated to about 40 degree delivers to 5000 molecular weight ultra-fine filter removal of impurities, decolouring.
2, crystallization: destainer is transferred PH,, add ethanol then and stir, treat to continue again to stir 2 hours after white precipitate is separated out, place freezer then and spend the night with 6000 molecular weight ultra-fine filter ultrafiltration.
3, centrifugal: that crystal solution is carried out centrifugation, centrifuge dripping.
4, drying: the product of wetting are put into Stainless Steel Disc, put into baking oven, vacuum-drying, oven dry back bagging and weighing hermetically drying is preserved.
Embodiment two:
One, feed intake:
1, in retort, adds tap water and glucose, be warmed up to 40 ℃ then, drop into yeast, stir, make its fermentation add inorganic salt, phosphorylcholine again in 3 hours, when treating that temperature is raised to 29 ℃, drop into cytidylic acid solution, continue to be warmed up to 39 ℃ again, the insulation timing.
2, temperature of reaction is controlled at 39 ℃, and sampling send the analyzer room to survey transformation efficiency after 2.5 hours, adds sucrose then, its addition is half when feeding intake, after, every two hours just need add sucrose once, get final product termination reaction when above when transformation efficiency reaches 80%, can prolong the reaction times if be lower than 70%.
3, reaction ends, and is warmed up to 80 ℃, uses the water cycle cool to room temperature rapidly, send the squeezing machine squeezing reaction solution then.
4, squeezing finishes water top, back and washes, and up to concentration＜10% of effluent liquid CDP-e o'clock, stops to collect.
5, pressed liquor is regulated PH, squeezing is handled again, and gained clear liquid metering cumulative volume, and sampling and measuring content calculate total content.
6, above-mentioned clear liquid is sent into received the film filter desalination.
Two, sample on the carbon post
1, will mix up carbon post on the squeezing clear liquid of PH, suitably reduce flow velocity after 3.5 hours.
2, washing carbon post: behind the carbon post end of the sample,, wash carbon post effluent liquid always and do not have Cl with 1% Silver Nitrate inspection with the pure water rinsing carbon post of PH6.0
-
3, carbon post wash-out: behind the carbon post wash clean, with 0.05N alkali ethanol (25% ethanol) solution citicoline sodium is eluted, effluent liquid is observed under ultraviolet lamp with the slide sampling, sees that the uv-absorbing thing just begins to collect.
Three, concentrate
In the film vacuum concentration pot, vaporize elutriant concentrated.
Four, ion exchange resin separates
1, sample on the ion exchange resin: it is 1.8% that concentrated solution is diluted with water to concentration, last macroporous ion exchange resin post.
2, ion exchange resin washing: behind the end of the sample, the washing pillar checks that flow velocity can suitably be accelerated when no uv-absorbing logistics went out.
3, exchange resin elution: with the 1%NaCl wash-out for preparing, wash-out finishes, and calculates recovery rate.
4, elutriant is concentrated to concentration about 70%.
Five, refining
1, decolours: the cytidine diphosphate sodium solution of collecting is heated to about 38 degree delivers to 5000 molecular weight ultra-fine filter removal of impurities, decolouring.
2, crystallization: destainer is transferred PH,, add ethanol then and stir, treat to continue again to stir 2 hours after white precipitate is separated out, place freezer then and spend the night with 6000 molecular weight ultra-fine filter ultrafiltration.
3, centrifugal: that crystal solution is carried out centrifugation, centrifuge dripping.
4, drying: the product of wetting are put into Stainless Steel Disc, put into baking oven, vacuum-drying, oven dry back bagging and weighing hermetically drying is preserved.
Embodiment three:
One, feed intake:
1, in retort, adds tap water and glucose, be warmed up to 42 ℃ then, drop into yeast, stir, make its fermentation add inorganic salt, phosphorylcholine again in 5 hours, when treating that temperature is raised to 31 ℃, drop into cytidylic acid solution, continue to be warmed up to 41 ℃ again, the insulation timing.
2, temperature of reaction is controlled at 41 ℃, and sampling send the analyzer room to survey transformation efficiency after 3.5 hours, adds sucrose then, its addition is half when feeding intake, after, every two hours just need add sucrose once, get final product termination reaction when above when transformation efficiency reaches 80%, can prolong the reaction times if be lower than 70%.
3, reaction ends, and is warmed up to 90 ℃, uses the water cycle cool to room temperature rapidly, send the squeezing machine squeezing reaction solution then.
4, squeezing finishes water top, back and washes, and up to concentration＜10% of effluent liquid CDP-e o'clock, stops to collect.
5, pressed liquor is regulated PH, squeezing is handled again, and gained clear liquid metering cumulative volume, and sampling and measuring content calculate total content.
6, above-mentioned clear liquid is sent into received the film filter desalination.
Two, sample on the carbon post
1, will mix up carbon post on the squeezing clear liquid of PH, suitably reduce flow velocity after 4.5 hours.
2, washing carbon post: behind the sample knot d bundle,, wash carbon post effluent liquid always and do not have Cl on the carbon post with 1% Silver Nitrate inspection with the pure water rinsing carbon post of PH6.0
-
3, carbon post wash-out: behind the carbon post wash clean, with 0.05N alkali ethanol (25% ethanol) solution citicoline sodium is eluted, effluent liquid is observed under ultraviolet lamp with the slide sampling, sees that the uv-absorbing thing just begins to collect.
Three, concentrate
In the film vacuum concentration pot, vaporize elutriant concentrated.
Four, ion exchange resin separates
1, sample on the ion exchange resin: it is 2.2% that concentrated solution is diluted with water to concentration, last macroporous ion exchange resin post.
2, ion exchange resin washing: behind the end of the sample, the washing pillar checks that flow velocity can suitably be accelerated when no uv-absorbing logistics went out.
3, exchange resin elution: with the 1%NaCl wash-out for preparing, wash-out finishes, and calculates recovery rate.
4, elutriant is concentrated to concentration about 80%.
Five, refining
1, decolours: the cytidine diphosphate sodium solution of collecting is heated to about 42 degree delivers to 5000 molecular weight ultra-fine filter removal of impurities, decolouring.
2, crystallization: destainer is transferred PH,, add ethanol then and stir, treat to continue again to stir 2.5 hours after white precipitate is separated out, place freezer then and spend the night with 6000 molecular weight ultra-fine filter ultrafiltration.
3, centrifugal: that crystal solution is carried out centrifugation, centrifuge dripping.
4, drying: the product of wetting are put into Stainless Steel Disc, put into baking oven, vacuum-drying, oven dry back bagging and weighing hermetically drying is preserved.
Claims (10)
1. Ubelin manufacturing technique is characterized in that:
At first feed intake: in retort, add entry and glucose, be warmed up to 40～42 ℃ and drop into yeast, the fermentation back adds inorganic salt, phosphorylcholine, after waiting to heat up, drop into cytidylic acid solution, continue to be warmed up to 40 ± 1 ℃ of insulations, reaction again, add sucrose then, and every two hours add once, when transformation efficiency reaches 80% termination reaction when above; Be warmed up to 85 ℃, the water cool to room temperature send the squeezing machine squeezing reaction solution then rapidly; Squeezing finishes back water prewashing, will regulate behind the PH gained clear liquid and send into and receive the film filter desalination;
Secondly, sample on the carbon post: carbon post on the squeezing clear liquid behind the carbon post end of the sample, with pure water rinsing carbon post, makes carbon post effluent liquid not have Cl
-With the alkali ethanolic soln citicoline sodium is eluted again, and when the uv-absorbing thing is arranged, begin to collect;
In the film vacuum concentration pot, vaporize elutriant concentrated;
Secondly be that ion exchange resin separates again: it is 2% that concentrated solution is diluted with water to concentration, last macroporous ion exchange resin post; Behind the end of the sample, the washing pillar; Be concentrated to concentration 75% again with the 1%NaCl wash-out for preparing, and with elutriant;
Make with extra care then: the cytidine diphosphate sodium solution of collecting is heated to about 40 degree delivers to 5000 molecular weight ultra-fine filter removal of impurities, decolouring; Again with destainer with 6000 molecular weight ultra-fine filter ultrafiltration, add ethanol then and stir, and place freezer and spend the night; Again crystal solution is carried out centrifugation, again the product of wetting are carried out vacuum-drying, oven dry back bagging and weighing hermetically drying is preserved.
2. Ubelin manufacturing technique according to claim 1 is characterized in that: when dropping into yeast, stir, make its fermentation add inorganic salt, phosphorylcholine again in 3～5 hours, when treating that temperature is raised to 30 ℃, drop into cytidylic acid solution, continue to be warmed up to 40 ± 1 ℃ again, the insulation timing.
3. Ubelin manufacturing technique according to claim 1 and 2, it is characterized in that: temperature of reaction is controlled at 40 ± 1 ℃, sampling send the analyzer room to survey transformation efficiency after 3 hours, add sucrose then, its addition is half when feeding intake, after, every two hours just need add sucrose once, get final product termination reaction when above when transformation efficiency reaches 80%, can prolong the reaction times if be lower than 70%.
4. Ubelin manufacturing technique according to claim 1 and 2 is characterized in that: squeezing finishes water top, back washes, and up to concentration＜10% of effluent liquid CDP-e o'clock, stops to collect.
5. Ubelin manufacturing technique according to claim 1 and 2 is characterized in that: pressed liquor is regulated PH, and squeezing is handled again, and gained clear liquid metering cumulative volume, and sampling and measuring content calculate total content.
6. Ubelin manufacturing technique according to claim 1 and 2 is characterized in that: on the carbon post during sample, with mixing up carbon post on the squeezing clear liquid of PH, suitably reduce flow velocity after 4 hours.
7. Ubelin manufacturing technique according to claim 1 and 2 is characterized in that: flushing carbon post is the pure water with PH6.0, and carbon post effluent liquid is not have Cl with 1% Silver Nitrate inspection
-
8. Ubelin manufacturing technique according to claim 1 and 2 is characterized in that: the wash-out citicoline sodium is with 0.05N alkali ethanol (25% ethanol) solution, and effluent liquid is observed under ultraviolet lamp with the slide sampling, sees that the uv-absorbing thing just begins to collect.
9. Ubelin manufacturing technique according to claim 1 and 2 is characterized in that: behind the end of the sample, the washing pillar checks that flow velocity can suitably be accelerated when no uv-absorbing logistics went out.
10. Ubelin manufacturing technique according to claim 1 and 2 is characterized in that: when adding the ethanol stirring, treat to continue to stir 2 hours after white precipitate is separated out again, place freezer then and spend the night.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100257805A CN101130797A (en) | 2007-08-01 | 2007-08-01 | Ubelin manufacturing technique |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100257805A CN101130797A (en) | 2007-08-01 | 2007-08-01 | Ubelin manufacturing technique |
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| CN101130797A true CN101130797A (en) | 2008-02-27 |
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| CNA2007100257805A Pending CN101130797A (en) | 2007-08-01 | 2007-08-01 | Ubelin manufacturing technique |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906126B (en) * | 2010-02-09 | 2012-04-25 | 南京工业大学 | Method for separating purified cytidine diphosphate choline by hydrophobic chromatography |
| CN102605025A (en) * | 2011-01-19 | 2012-07-25 | 中国科学院生物物理研究所 | Bioengineering method for synthesis of citicoline |
| CN103509073A (en) * | 2013-08-29 | 2014-01-15 | 洪军 | Citicoline sodium compound |
| CN109836468A (en) * | 2017-11-24 | 2019-06-04 | 苏州华赛生物工程技术有限公司 | A method of the purifying citicoline sodium from microbial fermentation solution |
| CN113831378A (en) * | 2021-09-24 | 2021-12-24 | 上海蔚之星生物科技有限公司 | Citicoline chromatographic separation method based on intelligent control |
| WO2023027178A1 (en) | 2021-08-26 | 2023-03-02 | 協和発酵バイオ株式会社 | Method for producing cytidine-5'-diphosphate compound |
-
2007
- 2007-08-01 CN CNA2007100257805A patent/CN101130797A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906126B (en) * | 2010-02-09 | 2012-04-25 | 南京工业大学 | Method for separating purified cytidine diphosphate choline by hydrophobic chromatography |
| CN102605025A (en) * | 2011-01-19 | 2012-07-25 | 中国科学院生物物理研究所 | Bioengineering method for synthesis of citicoline |
| CN102605025B (en) * | 2011-01-19 | 2014-07-02 | 中国科学院生物物理研究所 | Bioengineering method for synthesis of citicoline |
| CN103509073A (en) * | 2013-08-29 | 2014-01-15 | 洪军 | Citicoline sodium compound |
| CN103509073B (en) * | 2013-08-29 | 2016-01-06 | 洪军 | A kind of Citicoline sodium compound |
| CN109836468A (en) * | 2017-11-24 | 2019-06-04 | 苏州华赛生物工程技术有限公司 | A method of the purifying citicoline sodium from microbial fermentation solution |
| WO2023027178A1 (en) | 2021-08-26 | 2023-03-02 | 協和発酵バイオ株式会社 | Method for producing cytidine-5'-diphosphate compound |
| CN113831378A (en) * | 2021-09-24 | 2021-12-24 | 上海蔚之星生物科技有限公司 | Citicoline chromatographic separation method based on intelligent control |
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