CN1240723C - Method for extraction and purification of lipopolysaccharide from corn dipped in water - Google Patents
Method for extraction and purification of lipopolysaccharide from corn dipped in water Download PDFInfo
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- CN1240723C CN1240723C CN 200310109680 CN200310109680A CN1240723C CN 1240723 C CN1240723 C CN 1240723C CN 200310109680 CN200310109680 CN 200310109680 CN 200310109680 A CN200310109680 A CN 200310109680A CN 1240723 C CN1240723 C CN 1240723C
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Abstract
The present invention provides a method for extracting and purifying lipopolysaccharide from corn dipped in water. An isoelectric point method, an ultrafiltration method and a trichloroacetic acid method are used for separating protein dipped in water; concentration through a nanofiltration membrane is carried out; an ethanol precipitation method is used for further separating protein; lipopolysaccharide dipped in the water is extracted with active carbon; an active carbon chromatograph method and an ion exchange chromatograph method are used for purifying the lipopolysaccharide; the purity of a final obtained lipopolysaccharide product is more than 97.4%.
Description
Technical field
The present invention relates to the method that a kind of lipopolysaccharides extracts, purifies, particularly the method for extraction, purification lipopolysaccharides from soak water of maize
Background technology
In the prior art, adopt the ethanol distribution precipitator method to extract lipopolysaccharides, can make a large amount of protein with the lipopolysaccharides coprecipitation, the content of lipopolysaccharides very low (＜3%) in the product that obtains, on this basis, although adopted ion exchange chromatography to carry out purifying, product purity still undesirable (about 90%).
Summary of the invention
The objective of the invention is to overcome above deficiency, the method for a kind of extraction, purification lipopolysaccharides is provided, present method can simplify extraction, the purification operations step of lipopolysaccharides greatly, obviously improves the purity of the finished product simultaneously.
Purpose of the present invention realizes by following measure: adopt the protein in ultrafiltration process, isoelectric point method, three kinds of method combined separation of the trichloroacetic acid method soak water of maize, to soak water with nanofiltration membrane again concentrates, and further separate the protein that soaks in the water by ethanol precipitation, at last, with gac lipopolysaccharides is extracted from soak water:
1, isoelectric point method protein isolate and common polysaccharide:
Regulate the pH value to 7.3 that soaks in the water with saturated sodium hydroxide solution, soak solution is separated by separating machine, under per minute 3000 is walked around speed centrifugal 25 minutes, the precipitation separation supernatant liquor.
2, ultrafiltration process protein isolate and common polysaccharide:
Select the ultra-filtration membrane of molecular weight cut-off 100,000 for use, above-mentioned supernatant liquor is carried out ultrafiltration, get filtered liquid.
3, trichloroacetic acid method protein isolate and common polysaccharide:
In above-mentioned filtrate, add the trichoroacetic acid(TCA) account for its volume 0.5%, stirred 30 minutes, under per minute 3000 commentaries on classics conditions centrifugal 25 minutes, isolate supernatant liquor.
4, concentrate:
Adopt nanofiltration unit, the immersion water behind protein isolate and the common polysaccharide is concentrated 6-10 doubly, filter, get the immersion water after concentrating through nanofiltration membrane.Described nanofiltration membrane is the polymeric amide tubular membrane, membrane area 0, and 8-1.2 square metre, pure water flux is every square metre of 180-220 liter per hour.
5, separate again:
With further isolated protein of ethanol precipitation and general polysaccharides: add concentration and be 95% ethanol, make and soak that alcohol concn reaches 30% in the water, stirred 5 minutes, change to descend centrifugal 30 minutes, isolate supernatant liquor in per minute 2600.
6, extract:
Above-mentioned supernatant liquor pH value is adjusted into 6.50, adds the treated gac that accounts for its weight 1.5-2.5%, stirred 15 minutes, left standstill 10 minutes, filter at last, separate to take out and adsorbed the lipopolysaccharides gac.
Gac uses pre-treating process as follows: distilled water and nitrosonitric acid were mixed by 1: 1, active carbon powder is poured into wherein and soaked 5 hours, soaking temperature is 70～80 ℃.Release sour water after the immersion, wash to neutrality repeatedly with distilled water.
7, purify:
The gac that has adsorbed lipopolysaccharides filled in 1.6 * 90 centimetres the chromatographic column, carry out wash-out with every liter of Tutofusin tris-hydrochloric acid of 10 mmoles/every liter of sodium-chlor of 10 mmoles, eluent flow rate is per hour 8 milliliters, substep is collected elutriant, every pipe is collected 4 milliliters, collect 52 pipes altogether, the 27th～36 pipe elutriant is merged lyophilize.
DEAE-dextrane gel A-50 filled in 1.6 * 40 centimetres the chromatographic column.Get above-mentioned desciccate 0.25 gram, be dissolved in every liter of Tutofusin tris-hydrochloric acid of 10 mmoles/every liter of sodium-chlor of 10 mmoles of 6 milliliters, it is added chromatographic column.Tutofusin tris-hydrochloric acid/the sodium chloride solution that increases successively with concentration carries out wash-out, carries out substep simultaneously and collects.Every pipe is collected 6 milliliters, collects 60 pipes altogether, and the eluent flow velocity is per hour 8 milliliters.
8, desalination:
The elutriant of the 34th～52 pipe is merged, carry out desalination with sephadex G-15 chromatography column, with the ultrapure water wash-out, last freeze-drying obtains the lipopolysaccharides finished product.
Effect of the present invention is: when having simplified operation steps, significantly improved the purity of lipopolysaccharides product.Lipopolysaccharides content reaches more than 97.4% in the products obtained therefrom.
Embodiment
Embodiment 1: the method for corn soaking water extraction, purification lipopolysaccharides is as follows in the pharmacy procedure:
1, isoelectric point method protein isolate and common polysaccharide:
Regulate the pH value to 7.3 that soaks in the water with saturated sodium hydroxide solution, soak solution is separated by separating machine, under per minute 3000 is walked around speed centrifugal 25 minutes, the precipitation separation supernatant liquor.
2, ultrafiltration process protein isolate and common polysaccharide:
Select the ultra-filtration membrane of molecular weight cut-off 100,000 for use, above-mentioned supernatant liquor is carried out ultrafiltration, get filtered liquid.
3, trichloroacetic acid method protein isolate and common polysaccharide:
In above-mentioned filtrate, add the trichoroacetic acid(TCA) account for its volume 0.5%, stirred 30 minutes, under per minute 3000 commentaries on classics conditions centrifugal 25 minutes, isolate supernatant liquor.
4, concentrate:
Adopt nanofiltration unit, the immersion water behind protein isolate and the common polysaccharide is concentrated 8 times, filter, get the immersion water after concentrating through nanofiltration membrane.Described nanofiltration membrane is the polymeric amide tubular membrane, 1 square metre of membrane area, and pure water flux is per hour 200 liters every square metre.
5, separate again:
With further isolated protein of ethanol precipitation and general polysaccharides: add concentration and be 95% ethanol, make and soak that alcohol concn reaches 30% in the water, stirred 5 minutes, change to descend centrifugal 30 minutes, isolate supernatant liquor in per minute 2600.
6, extract:
Above-mentioned supernatant liquor pH value is adjusted into 6.50, adds the treated gac that accounts for its weight 2%, stirred 15 minutes, left standstill 10 minutes, filter at last, separate to take out and adsorbed the lipopolysaccharides gac.
Gac uses pre-treating process as follows: distilled water and nitrosonitric acid were mixed by 1: 1, active carbon powder is poured into wherein and soaked 5 hours, soaking temperature remains on 70～80 ℃.Release sour water after the immersion, wash to neutrality repeatedly with distilled water.
7, purify:
The gac that has adsorbed lipopolysaccharides filled in 1.6 * 90 centimetres the chromatographic column, carry out wash-out with every liter of Tutofusin tris-hydrochloric acid of 10 mmoles/every liter of sodium-chlor of 10 mmoles, eluent flow rate is per hour 8 milliliters, substep is collected elutriant, every pipe is collected 4 milliliters, collect 52 pipes altogether, the 27th～36 pipe elutriant is merged lyophilize.
DEAE-dextrane gel A-50 filled in 1.6 * 40 centimetres the chromatographic column.Get above-mentioned desciccate 0.25 gram, be dissolved in every liter of Tutofusin tris-hydrochloric acid of 10 mmoles/every liter of sodium-chlor of 10 mmoles of 6 milliliters, it is added chromatographic column.Tutofusin tris-hydrochloric acid/the sodium chloride solution that increases successively with concentration carries out wash-out, carries out substep simultaneously and collects.Every pipe is collected 6 milliliters, collects 60 pipes altogether, and the eluent flow velocity is per hour 8 milliliters.
8, desalination:
The elutriant of the 34th～52 pipe is merged, carry out desalination with sephadex G-15 chromatography column, with the ultrapure water wash-out, last freeze-drying obtains the lipopolysaccharides finished product.
Claims (3)
1, the method for extraction, purification lipopolysaccharides in a kind of soak water of maize, it is characterized in that present method passes through to adopt successively isoelectric point method, ultrafiltration process, three kinds of methods of trichloroacetic acid method to separate the protein in the soak water of maize, to soak water with nanofiltration membrane again concentrates, and further separate the protein that soaks in the water by ethanol precipitation, at last, with gac lipopolysaccharides is extracted from soak water:
(1) isoelectric point method protein isolate and common polysaccharide:
Regulate the pH value to 7.3 that soaks in the water with saturated sodium hydroxide solution, soak solution is separated by separating machine, under per minute 3000 is walked around speed centrifugal 25 minutes, the precipitation separation supernatant liquor;
(2) ultrafiltration process protein isolate and common polysaccharide:
Select the ultra-filtration membrane of molecular weight cut-off 100,000 for use, above-mentioned supernatant liquor is carried out ultrafiltration, get filtered liquid;
(3) trichloroacetic acid method protein isolate and common polysaccharide:
In above-mentioned filtrate, add the trichoroacetic acid(TCA) account for its volume 0.5%, stirred 30 minutes, under per minute 3000 commentaries on classics conditions centrifugal 25 minutes, isolate the supernatant liquor that contains albumen and common polysaccharide;
(4) concentrate:
Adopt nanofiltration unit, the immersion water behind protein isolate and the common polysaccharide is concentrated 6-10 doubly, filter, get the immersion water after concentrating through nanofiltration membrane;
(5) separate again:
In above-mentioned concentrated immersion water, add concentration and be 95% ethanol, make and soak that alcohol concn reaches 30% in the water, stirred 5 minutes, change to descend centrifugal 30 minutes, get supernatant liquor after the separation in per minute 2600;
(6) extract:
Above-mentioned supernatant liquor pH value is adjusted into 6.50, adds the treated gac that accounts for its weight 1.5-2.5%, stirred 15 minutes, left standstill 10 minutes, filter at last, separate to take out and adsorbed the lipopolysaccharides gac;
(7) purify:
The gac that has adsorbed lipopolysaccharides filled in 1.6 * 90 centimetres the chromatographic column, carry out wash-out with every liter of Tutofusin tris-hydrochloric acid of 10 mmoles/every liter of sodium-chlor of 10 mmoles, eluent flow rate is per hour 8 milliliters, substep is collected elutriant, every pipe is collected 4 milliliters, collect 52 pipes altogether, the 27th～36 pipe elutriant is merged lyophilize;
DEAE-dextrane gel A-50 filled in 1.6 * 40 centimetres the chromatographic column, get above-mentioned desciccate 0.25 gram, be dissolved in every liter of Tutofusin tris-hydrochloric acid of 10 mmoles/every liter of sodium-chlor of 10 mmoles of 6 milliliters, it is added chromatographic column, Tutofusin tris-hydrochloric acid/the sodium chloride solution that increases successively with concentration carries out wash-out, carries out substep simultaneously and collects, and every pipe is collected 6 milliliters, collect 60 pipes altogether, the eluent flow velocity is per hour 8 milliliters;
(8) desalination:
The elutriant of the 34th～52 pipe is merged, carry out desalination with sephadex G-15 chromatography column, with the ultrapure water wash-out, last freeze-drying obtains the lipopolysaccharides finished product.
2, the method for extraction, purification lipopolysaccharides in the soak water of maize according to claim 1, it is characterized in that described gac uses pre-treating process to be: distilled water and nitrosonitric acid were mixed by 1: 1, active carbon powder is poured into wherein and soaked 5 hours, soaking temperature is 70～80 ℃, release sour water after the immersion, wash to neutrality repeatedly with distilled water.
3, extract in the soak water of maize according to claim 1 and 2, the method for purification lipopolysaccharides, it is characterized in that described nanofiltration membrane is the polymeric amide tubular membrane, membrane area 0.8-1.2 square metre, pure water flux is every square metre of 180-220 liter per hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310109680 CN1240723C (en) | 2003-11-28 | 2003-11-28 | Method for extraction and purification of lipopolysaccharide from corn dipped in water |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310109680 CN1240723C (en) | 2003-11-28 | 2003-11-28 | Method for extraction and purification of lipopolysaccharide from corn dipped in water |
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| CN1546532A CN1546532A (en) | 2004-11-17 |
| CN1240723C true CN1240723C (en) | 2006-02-08 |
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| CN 200310109680 Expired - Fee Related CN1240723C (en) | 2003-11-28 | 2003-11-28 | Method for extraction and purification of lipopolysaccharide from corn dipped in water |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392032B (en) * | 2008-11-05 | 2011-04-20 | 中国林业科学研究院林产化学工业研究所 | Method for preparing bamboo leaf polysaccharide extract by member separation |
| CN101638436B (en) * | 2009-09-07 | 2011-11-02 | 山东龙力生物科技股份有限公司 | Extracted corn protein and production method thereof |
| CN102212595B (en) * | 2011-04-20 | 2013-04-17 | 江南大学 | Preparation method and application of water-soluble nano grain polysaccharide |
| CN102746414B (en) * | 2012-07-20 | 2014-04-16 | 成都生物制品研究所有限责任公司 | Sedimentation method of lipopolysaccharides |
| CN102796255B (en) * | 2012-09-07 | 2014-04-02 | 吉林中粮生化有限公司 | Method for separating and purifying gamma-polyglutamic acid produced by corn soaking water fermentation |
| CN102898537B (en) * | 2012-10-31 | 2014-09-10 | 云南沃森生物技术股份有限公司 | Purification method of lipopolysaccharide |
| CN103819577B (en) * | 2014-03-24 | 2016-05-04 | 福州大学 | A kind of preparation method of spirulina polysaccharide |
| JP6539400B1 (en) * | 2018-10-24 | 2019-07-03 | 株式会社アンチエイジング・プロ | Method for producing LPS-rich composition |
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