CN102276751A - Method for extracting glycosaminoglycan from bullacta exarata - Google Patents
Method for extracting glycosaminoglycan from bullacta exarata Download PDFInfo
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- CN102276751A CN102276751A CN 201110228545 CN201110228545A CN102276751A CN 102276751 A CN102276751 A CN 102276751A CN 201110228545 CN201110228545 CN 201110228545 CN 201110228545 A CN201110228545 A CN 201110228545A CN 102276751 A CN102276751 A CN 102276751A
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Abstract
The invention relates to a method for extracting glycosaminoglycan from bullacta exarata. The method has the main technical characteristics that: the bullacta exarata serving as a low-value shellfish and being widely distributed in the Bohai Bay is used as a raw material for extracting the glycosaminoglycan. The method for extracting the glycosaminoglycan comprises the following steps of: shelling and weighing the bullacta exarata; adding water to prepare homogenate; adding trypsin into the homogenate for enzymolysis; centrifuging enzymolysis liquid to remove precipitates; adding hydrogen peroxide into supernatant to decolorize; centrifuging; adjusting the pH value; centrifuging to remove acidic and neutral proteins; performing alcohol precipitation on the supernatant to obtain a glycosaminoglycan rough product with yield of 1.22 percent; adding ultra-pure water into the rough product to dissolve the rough product; ultra-filtering; performing alcohol precipitation; adding CTAB (Cetyl Trimethyl Ammonium Bromide); centrifuging; dissolving the precipitates in a NaCl solution to obtain dissociation liquid; then performing ultra-filtration and alcohol precipitation; and recovering the high-purity glycosaminoglycan. The method has mild reaction conditions and high yield and purity of the glycosaminoglycan and has the advantages that the process is simplified, the time is shortened and the purity of the obtained glycosaminoglycan is up to 90 percent compared with extraction of the glycosaminoglycan of other shellfishes.
Description
Technical field
The invention belongs to bioengineering field, relate to glycosaminoglycan, especially a kind of method of from mud snail, extracting glycosaminoglycan.
Background technology
At present the extracting method of glycosaminoglycan (GAG) mainly contains three kinds: the first non-edman degradation Edman refers to that water or salts solution are driven, extracts the method for glycosaminoglycan in the plant tissue; Second alkali extraction method based on the unstable of cardohydrata-peptide linkage in the protein-polysaccharide to alkali, makes sugar separate with protein with alkaline extraction; The 3rd enzyme liberating method, proteolytic enzyme make sugar chain obtain separating the degraded of the protein portion of proteoglycan, and be best with enzyme liberating method effect in three kinds of methods, because of its mild condition, and meets the needs of present energy-saving and emission-reduction.Yet even belong to the enzyme liberating method, owing to composition, content and the functionally active of the contained glycosaminoglycan of different extraction materials are different, extracting method has different, moreover also need consider applying in the production.The present invention chooses that the modal low value shellfish in Bohai Sea Gulf---mud snail is starting material, extracts, and separates, and the purifying glycosaminoglycan is in the hope of for medicine, health products trade provide high-quality raw material, for the precious animal cultivation in sea provides good immunostimulant.
By retrieval, find following three pieces of publication documents relevant with present patent application: 1, the invention provides the method for compositions (CN102124032A) that a kind of production comprises glycosaminoglycan, described method comprises utilizes film adsorber form chromatography substrate will comprise that the animal material homogenate of glycosaminoglycan carries out chromatography.The extraction preparation method (CN1746191) of 2, a kind of active substance---chlamys farreri glycosaminoglycan with vascular protection effect, its extracting and preparing technique is as follows: the chlamys farreri visceral mass is weighed, add water and make homogenate, add proteolytic enzyme in homogenate, reacting by heating gets enzymolysis solution; Enzymolysis solution is centrifugal in whizzer, discard throw out, centrifugate is added the secondary ethanolic soln, form precipitation; Throw out adding distil water after separating is made into aqueous solution post-heating and adds gac, and cooling is after centrifugal, and the collection supernatant liquor to the supernatant liquor suction filtration, gets clear liquid; With clear liquid filter membrane ultrafiltration, getting molecular weight is the filtrate of 5k～100k; The filtrate lyophilize is promptly made the chlamys farreri glycosaminoglycan.This method is simple to operate, reaction conditions is gentle, with low cost, yield and purity height, when extracting glycosaminoglycan, also can reclaim protein in a large number, help the comprehensive utilization of chlamys farreri visceral mass, the material of extraction has important value for control cardiovascular disease.3, a kind of extracting method of holothuria leucospilota glycosaminoglycan (CN101624426), method: one, the hojothuria leucospilota powder mixes with purified water, through enzymolysis and centrifugal after precipitate A; Two, precipitate A is mixed with purified water, through the heating and centrifugal after deposit B; Three, deposit B is mixed with purified water, and centrifuging and taking precipitation is with the purified water dissolving, through centrifugal and wash deposit C; Four, deposit C is mixed with damping fluid, through chromatography, washing, wash-out and centrifugal after deposit D; Five, deposit D is mixed with physiological saline, gets deposit E after centrifugal; Six, deposit E is mixed with water for injection, and dialysis or ultrafiltration are got solid product after damming, after the lyophilize promptly.The holothuria leucospilota glycosaminoglycan quality purity that the present invention extracts can be directly used in the preparation injection up to 93%～99%.
By comparison, the technical scheme of above-mentioned publication document and extraction object all have the different of essence with present patent application, and present patent application has novelty.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of method of extracting the high purity glycosaminoglycan from mud snail is provided, sufficient raw, the extraction cost of this method are low, for the widespread use of satisfying medicine, health products trade and extra large precious animal farming industry provides possibility.
The present invention is achieved through the following technical solutions:
A kind of method of from mud snail, extracting glycosaminoglycan, extraction may further comprise the steps:
(1) raw material is handled: mud snail is shelled, after software is partly cleaned, add the distilled water homogenate of 2-2.5 times of volume, form homogenate;
(2) enzymolysis: add trypsinase in above-mentioned homogenate, under 45 ℃ of water bath condition enzymolysis 7-8 hour, after enzymolysis solution boiled the enzyme that goes out, the centrifugal 10min of 4000r/min got supernatant liquor;
(3) decolouring: add the hydrogen peroxide decolouring;
(4) adjust pH goes precipitation: transfer pH to 2.0, the centrifugal precipitation of removing with HCl earlier; Transfer pH to 7.0, the centrifugal precipitation of removing with NaOH again;
(5) alcohol deposition method extracts raw sugar amine glycan: supernatant liquor slowly adds ethanol to pure content under constantly stirring be 60%, left standstill 24 hours under 4 ℃ of conditions, removes supernatant liquor, reclaims raw sugar amine glycan behind the centrifugal 10min of 4000r/min;
(6) ultrafiltration: raw sugar amine glycan is dissolved in distilled water, is made into 1% liquid glucose, the centrifugal 10min of 4000r/min carries out ultrafiltration after removing insolubles;
(7) dissociate after the solution complexing: add the 5%CTAB (cetyl trimethylammonium bromide) of 3 times of volumes, 40 ℃ precipitate 1-2 hour, the centrifugal 30min of 8000r/min down, collecting precipitation, under 40 ℃ precipitation is dissolved in the NaCl solution of 2mol/L, the GAG-CTAB complex compound is dissociated, form dissociation solution;
(8) alcohol precipitation, ultrafiltration, alcohol precipitation again: above-mentioned dissociation solution adds ethanol, make the alcohol amount reach 60%, the centrifugal 10min of 8000r/min, collecting precipitation is dissolved in distilled water with precipitation, use the tubular fibre membrane ultrafiltration, add ethanol-sodium acetate reagent in the solution after ultrafiltration, make the alcohol amount reach 60%, press alcohol deposition method and reclaim glycosaminoglycan, with the throw out oven dry, promptly make pure mud snail glycosaminoglycan.
And tryptic addition is the 0.8-1% of homogenate gross weight in the described step (2).
And the addition of hydrogen peroxide is the 6-8% of supernatant liquor weight in the described step (3).
Advantage of the present invention and positively effect are:
1, present method reaction conditions gentleness, glycosaminoglycan yield and purity height, with the glycosaminoglycan extract phase ratio of other shellfishes, work simplification, the time shortens.
2, present method adopts mud snail as extracting object, and mud snail is the common low value shellfish in Bohai Sea Gulf, and material cost is low, and the glycosaminoglycan yield does not reduce, and greatly reduces extraction cost.
3, present method also can reclaim protein in a large number when extracting glycosaminoglycan, helps the comprehensive utilization of mud snail, the glycosaminoglycan of being extracted for remove free radical, anti-oxidant and anticoagulation has vital role; The glycosaminoglycan of gained also can be extra large precious animal farming industry good immunostimulant is provided.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The extraction object that present embodiment adopted is to be selected from low value shellfish-mud snail that Bohai Sea Gulf extensively distributes.
A kind of method of extracting glycosaminoglycan from mud snail may further comprise the steps:
(1) raw material is handled: mud snail is shelled, after software is partly cleaned, add the distilled water homogenate of 2-2.5 times of volume, form homogenate;
(2) enzymolysis: add trypsinase in above-mentioned homogenate, this tryptic addition is the 0.8-1% of homogenate gross weight; Under 45 ℃ of water bath condition enzymolysis 7-8 hour, after enzymolysis solution boiled the enzyme that goes out, the centrifugal 10min of 4000r/min got supernatant liquor;
(3) decolouring: add the hydrogen peroxide decolouring, the addition of hydrogen peroxide is the 6-8% of supernatant liquor weight;
(4) adjust pH goes precipitation: transfer pH to 2.0, the centrifugal precipitation of removing with HCl earlier; Transfer pH to 7.0, the centrifugal precipitation of removing with NaOH again;
(5) alcohol deposition method extracts raw sugar amine glycan: supernatant liquor slowly adds ethanol to pure content under constantly stirring be 60%, left standstill 24 hours under 4 ℃ of conditions, removes supernatant liquor, reclaims raw sugar amine glycan behind the centrifugal 10min of 4000r/min, and yield is 1.22%;
(6) ultrafiltration: raw sugar amine glycan is dissolved in distilled water, is made into 1% liquid glucose, the centrifugal 10min of 4000r/min carries out ultrafiltration after removing insolubles;
(7) dissociate after the solution complexing: the CTAB (cetyl trimethylammonium bromide) that adds 5% weight concentration of 3 times of volumes, 40 ℃ precipitate 1-2 hour down, the centrifugal 30min of 8000r/min, collecting precipitation, under 40 ℃ precipitation is dissolved in the NaCl solution of 2mol/L, the GAG-CTAB complex compound is dissociated, form dissociation solution;
(8) alcohol precipitation, ultrafiltration, alcohol precipitation again: above-mentioned dissociation solution adds ethanol, make the alcohol amount reach 60%, the centrifugal 10min of 8000r/min, collecting precipitation is dissolved in distilled water with precipitation, use the tubular fibre membrane ultrafiltration, add ethanol-sodium acetate reagent in the solution after ultrafiltration, make the alcohol amount reach 60%, press alcohol deposition method and reclaim glycosaminoglycan, with the throw out oven dry, promptly make pure mud snail glycosaminoglycan.By check, the glycosaminoglycan purity that obtains can reach 90%.
Claims (3)
1. method of extracting glycosaminoglycan from mud snail is characterized in that: extract and may further comprise the steps:
(1) raw material is handled: mud snail is shelled, after software is partly cleaned, add the distilled water homogenate of 2-2.5 times of volume, form homogenate;
(2) enzymolysis: add trypsinase in above-mentioned homogenate, under 45 ℃ of water bath condition enzymolysis 7-8 hour, after enzymolysis solution boiled the enzyme that goes out, the centrifugal 10min of 4000r/min got supernatant liquor;
(3) decolouring: add the hydrogen peroxide decolouring;
(4) adjust pH goes precipitation: transfer pH to 2.0, the centrifugal precipitation of removing with HCl earlier; Transfer pH to 7.0, the centrifugal precipitation of removing with NaOH again;
(5) alcohol deposition method extracts raw sugar amine glycan: supernatant liquor slowly adds ethanol to pure content under constantly stirring be 60%, left standstill 24 hours under 4 ℃ of conditions, removes supernatant liquor, reclaims raw sugar amine glycan behind the centrifugal 10min of 4000r/min;
(6) ultrafiltration: raw sugar amine glycan is dissolved in distilled water, is made into 1% liquid glucose, the centrifugal 10min of 4000r/min carries out ultrafiltration after removing insolubles;
(7) dissociate after the solution complexing: add the 5%CTAB (cetyl trimethylammonium bromide) of 3 times of volumes, 40 ℃ precipitate 1-2 hour, the centrifugal 30min of 8000r/min down, collecting precipitation, under 40 ℃ precipitation is dissolved in the NaCl solution of 2mol/L, the GAG-CTAB complex compound is dissociated, form dissociation solution;
(8) alcohol precipitation, ultrafiltration, alcohol precipitation again: above-mentioned dissociation solution adds ethanol, make the alcohol amount reach 60%, the centrifugal 10min of 8000r/min, collecting precipitation is dissolved in distilled water with precipitation, use the tubular fibre membrane ultrafiltration, add ethanol-sodium acetate reagent in the solution after ultrafiltration, make the alcohol amount reach 60%, press alcohol deposition method and reclaim glycosaminoglycan, with the throw out oven dry, promptly make pure mud snail glycosaminoglycan.
2. the method for extracting glycosaminoglycan from mud snail according to claim 1, it is characterized in that: tryptic addition is the 0.8-1% of homogenate gross weight in the described step (2).
3. the method for extracting glycosaminoglycan from mud snail according to claim 1 is characterized in that: the addition of hydrogen peroxide is the 6-8% of supernatant liquor weight in the described step (3).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618614A (en) * | 2012-04-13 | 2012-08-01 | 天津高蜚生物科技发展有限公司 | Method for preparing urechis unicinctus glycosaminoglycan and antibacterial peptide simultaneously |
| CN103130904A (en) * | 2013-01-07 | 2013-06-05 | 天津科技大学 | High-valued utilization method for patinopecten yessoensis offal |
| CN103636913A (en) * | 2013-11-10 | 2014-03-19 | 陆思烨 | Mud snail healthcare ice cream and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1523041A (en) * | 2003-09-10 | 2004-08-25 | 吉林高科生态开发有限公司 | Bulgaria inquinans polysaccharide, method for preparing the same and pharmaceutical composition using the compound as active component |
| CN1749282A (en) * | 2005-10-11 | 2006-03-22 | 大连轻工业学院 | Belly bottom snail polysaccharide and its preparing method |
| CN101402982A (en) * | 2008-11-11 | 2009-04-08 | 华东理工大学 | Mud snail glue polysaccharide, preparation method and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1523041A (en) * | 2003-09-10 | 2004-08-25 | 吉林高科生态开发有限公司 | Bulgaria inquinans polysaccharide, method for preparing the same and pharmaceutical composition using the compound as active component |
| CN1749282A (en) * | 2005-10-11 | 2006-03-22 | 大连轻工业学院 | Belly bottom snail polysaccharide and its preparing method |
| CN101402982A (en) * | 2008-11-11 | 2009-04-08 | 华东理工大学 | Mud snail glue polysaccharide, preparation method and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618614A (en) * | 2012-04-13 | 2012-08-01 | 天津高蜚生物科技发展有限公司 | Method for preparing urechis unicinctus glycosaminoglycan and antibacterial peptide simultaneously |
| CN102618614B (en) * | 2012-04-13 | 2013-07-31 | 天津高蜚生物科技发展有限公司 | Method for preparing urechis unicinctus glycosaminoglycan and antibacterial peptide simultaneously |
| CN103130904A (en) * | 2013-01-07 | 2013-06-05 | 天津科技大学 | High-valued utilization method for patinopecten yessoensis offal |
| CN103636913A (en) * | 2013-11-10 | 2014-03-19 | 陆思烨 | Mud snail healthcare ice cream and preparation method thereof |
| CN103636913B (en) * | 2013-11-10 | 2014-11-05 | 陆思烨 | Mud snail healthcare ice cream and preparation method thereof |
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