CN101475651A - Preparation of Gracilaria gigas Harvey polysaccharides - Google Patents
Preparation of Gracilaria gigas Harvey polysaccharides Download PDFInfo
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- CN101475651A CN101475651A CNA2009100012652A CN200910001265A CN101475651A CN 101475651 A CN101475651 A CN 101475651A CN A2009100012652 A CNA2009100012652 A CN A2009100012652A CN 200910001265 A CN200910001265 A CN 200910001265A CN 101475651 A CN101475651 A CN 101475651A
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 47
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 47
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 241001204911 Gracilaria gigas Species 0.000 title claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 53
- 239000000706 filtrate Substances 0.000 claims description 40
- 241000195493 Cryptophyta Species 0.000 claims description 28
- 238000001556 precipitation Methods 0.000 claims description 19
- 239000000843 powder Substances 0.000 claims description 18
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- 238000000605 extraction Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 108090000856 Lyases Proteins 0.000 claims description 11
- 102000004317 Lyases Human genes 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 230000007062 hydrolysis Effects 0.000 claims description 10
- 238000006460 hydrolysis reaction Methods 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 8
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 108010087558 pectate lyase Proteins 0.000 claims description 8
- 239000002953 phosphate buffered saline Substances 0.000 claims description 8
- 238000000108 ultra-filtration Methods 0.000 claims description 8
- 238000001291 vacuum drying Methods 0.000 claims description 8
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 7
- 108091005804 Peptidases Proteins 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 102000035195 Peptidases Human genes 0.000 claims description 6
- 230000001476 alcoholic effect Effects 0.000 claims description 6
- 238000013375 chromatographic separation Methods 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 108010059892 Cellulase Proteins 0.000 claims description 4
- 229920000615 alginic acid Polymers 0.000 claims description 4
- 235000010443 alginic acid Nutrition 0.000 claims description 4
- 229940106157 cellulase Drugs 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- AZHSSKPUVBVXLK-UHFFFAOYSA-N ethane-1,1-diol Chemical compound CC(O)O AZHSSKPUVBVXLK-UHFFFAOYSA-N 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 229940059442 hemicellulase Drugs 0.000 claims description 4
- 108010002430 hemicellulase Proteins 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 claims description 4
- 239000004744 fabric Substances 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000000967 suction filtration Methods 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 241000206581 Gracilaria Species 0.000 abstract description 6
- 241000206572 Rhodophyta Species 0.000 abstract description 4
- 241001134782 Gigartinales Species 0.000 abstract description 2
- 230000000259 anti-tumor effect Effects 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 241001428219 Gracilariaceae Species 0.000 abstract 1
- 230000001093 anti-cancer Effects 0.000 abstract 1
- 238000003912 environmental pollution Methods 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000007787 solid Substances 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
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- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 235000011837 pasties Nutrition 0.000 description 2
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 2
- CTYRPMDGLDAWRQ-UHFFFAOYSA-N phenyl hydrogen sulfate Chemical compound OS(=O)(=O)OC1=CC=CC=C1 CTYRPMDGLDAWRQ-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000195474 Sargassum Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
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- 238000005265 energy consumption Methods 0.000 description 1
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- 238000009776 industrial production Methods 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
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- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000004223 radioprotective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a preparation method for a Gracilaria polysaccharide. The Gracilaria polysaccharide is separated and extracted from common Gracilaria, Gracilaria, Gracilariaceae, Gigartinales, Florideae, and Rhodophyta. The molecular weight is between 500 and 50,000 Daltons. The Gracilaria polysaccharide solid is from faint yellow to white. The product can be developed into new anti-tumor and anticancer medicines. The preparation method is reliable and feasible and is suitable for the large-scale production. The preparation method has no environmental pollution and has low cost.
Description
Technical field
The present invention relates to a kind of preparation method of Gracilaria gigas Harvey polysaccharides.
Background technology
Fragrant plant mentioned in ancient texts is perennial large-scale red algae, belongs to rhodophyta, true Rhodophyceae, and Gigartinales, fragrant plant mentioned in ancient texts section, Gracilaria, what world's fragrant plant mentioned in ancient texts algae kind had been reported has 100 kinds approximately, and the common fragrant plant mentioned in ancient texts algae of China mainly contains kind more than 10.The fragrant plant mentioned in ancient texts algae is one of China Hainan, Guangdong, Zhejiang, the coastal algal culture kind in Taiwan, mainly as fishery cultivating feed and production food collagen material, its research of carrying out the polysaccharide extraction and analysis is reported seldom.We constantly grope and combination some experiences in the past through fragrant plant mentioned in ancient texts polysaccharides extraction process, have invented the fragrant plant mentioned in ancient texts polysaccharides.
Gracilaria gigas Harvey polysaccharides is one of important biomolecule activeconstituents in the fragrant plant mentioned in ancient texts algae, polysaccharide is that a class of composition biological organism is to form a class important biomolecule macromole of biological organism, as physiological processs such as the mutual identification of informational molecule wide participation iuntercellular part and acceptor, intracellular signal transductions, with cell activation, propagation, differentiation and to produce cytokine etc. relevant.Along with deepening continuously of natural drug research, it is found that Sargassum polysaccharides has anti-infective, antitumor, anticoagulation, radioprotective, removing oxyradical and function in delaying senility.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of Gracilaria gigas Harvey polysaccharides, simple, safety, environmental protection, low cost.
For achieving the above object, technical scheme provided by the present invention is: a kind of preparation method of Gracilaria gigas Harvey polysaccharides may further comprise the steps:
(1), the fragrant plant mentioned in ancient texts algae of gathering is rinsed well, discard assorted algae and dirt, 50 ℃～100 ℃ oven dry, pulverizing algae powder; The algae powder adds the above water of 5 times of amounts and heats extraction, extracts more than 30 minutes more than 60 ℃, reduces to room temperature, filters, and gets filtrate 1.;
(2), in filtrate, add 10～20 million international units and carry out enzymolysis than the lyase of vigor by per kilogram fragrant plant mentioned in ancient texts algae powder, the pH value is 6.0～9.0, hydrolysis temperature is 30 ℃～60 ℃, enzymolysis time is 30～210 minutes; Enzymolysis finishes back adjust pH to 3.0～4.0, add 10～20 million international units by per kilogram fragrant plant mentioned in ancient texts algae powder and carry out enzymolysis than the proteolytic enzyme of vigor, after stirring evenly, keep the pH value 3.0～4.0,45 ℃～50 ℃ condition enzymolysis, enzymolysis time is 450～510 minutes, and enzymolysis finishes post-heating to 90 ℃～100 ℃ and carries out enzyme-deactivating, and the enzyme-deactivating time is 5～20 minutes;
(3), reduce to room temperature, filtration, collection filtrate; Filtrate is regulated pH value to 5.5～7.0; Ethanol to the alcoholic acid volumetric concentration of adding 95% is 10%～20%, and room temperature left standstill more than 3.0 hours;
(4), centrifugal, filter, collect filtrate, adding 95% ethanol alcohol, to be sink to the alcoholic acid volumetric concentration be 40%～95%, room temperature left standstill more than 3.0 hours;
(5), the solution behind the alcohol precipitation filters, taking precipitate, throw out are washed 2 times with 95% ethanol and acetone respectively, vacuum-drying promptly gets Gracilaria gigas Harvey polysaccharides.
This preparation method is further comprising the steps of:
(6), get the prepared Gracilaria gigas Harvey polysaccharides of step (5), add the above water of 10 times of amounts and stir and dissolve, centrifugal, filter, get filtered liquid;
(7), filtered liquid is carried out chromatographic separation with the DEAE chromatography column, with 10～30mmol/L, pH5.5～7.0 phosphate buffered saline buffers carry out wash-out;
(8), collect the target compound after the chromatographic separation, be that the ultra-filtration membrane of 5～100KD carries out ultrafiltration, the collection trapped fluid with molecular weight cut-off;
(9), trapped fluid adds 95% ethanol alcohol to be sink to the alcoholic acid volumetric concentration is 40%～95%, room temperature left standstill more than 3.0 hours;
(10), the solution behind the alcohol precipitation carries out centrifugally, filters, precipitation is washed 2 times with 95% ethanol and acetone respectively, carries out vacuum-drying, promptly gets Gracilaria gigas Harvey polysaccharides.
Described step (1) also includes: filter residue adds the above water of 4 times of weight and carries out enzymolysis and extraction, add 10～30 million international units by per kilogram fragrant plant mentioned in ancient texts algae powder and carry out enzymolysis than the prozyme of vigor, described prozyme contains cellulase and the hemicellulase that weight ratio is 2～1:1～2; Keep the pH value 3.0～5.5, hydrolysis temperature is 30 ℃～60 ℃, and the time is 60～250 minutes; Enzymolysis finishes post-heating and carries out enzyme-deactivating, and the enzyme-deactivating temperature is 90 ℃～100 ℃, and the enzyme-deactivating time is 5～20 minutes, and cooling is filtered, and gets filtrate 2.; In the described step (2) the lyase enzymolysis be filtrate 1. with filtrate merging filtrate 2..
Lyase in the described step (2) is algin catenase, alginate lyase, pectate lyase or pectate lyase.
Filter in the described step (3) and comprise, carry out coarse filtration with filter cloth earlier, carry out centrifugally again with high speed freezing centrifuge, carry out suction filtration again.
Filtration procedure in the described step (6) is: centrifugal with high speed freezing centrifuge, pour out supernatant liquor, and collecting precipitation is washed with 95% ethanol to container, drains with suction filter.
The advantage that the present invention has: the present invention is by carrying out enzymolysis processing to the fragrant plant mentioned in ancient texts algae, method through classification alcohol precipitation and alcohol alcohol precipitation, other impurity is removed in the first step separation and purification, carry out chromatographic separation through the DEAE chromatography column again, be that the ultra-filtration membrane of 5～10KD carries out ultrafiltration with molecular retention then, collect trapped fluid, pass through the alcohol alcohol precipitation again, other impurity is removed in the second step separation and purification.The advantage of this method is as follows:
1, the fragrant plant mentioned in ancient texts algae is directly extracted the fragrant plant mentioned in ancient texts polysaccharides through production technique such as enzymolysis, has advantages such as simple, safety, environmental protection, low cost.
2, the prepared fragrant plant mentioned in ancient texts polysaccharides purity height of method for preparing the fragrant plant mentioned in ancient texts polysaccharides of the present invention, the yield height, of good quality, but advantages such as mass production.
3, the used prozyme of the preparation method of fragrant plant mentioned in ancient texts polysaccharides of the present invention carries out the enzymolysis lixiviate second time to the fragrant plant mentioned in ancient texts algae-residue, makes fragrant plant mentioned in ancient texts frustule wall break, and promotes the stripping of polysaccharide, improves extraction yield in the hope of reaching, shortens extraction time, reduces purpose such as energy consumption.
4, the used lyase enzymolysis process of the preparation method of fragrant plant mentioned in ancient texts polysaccharides of the present invention can the cracking glycosidic link, obtains the lower oligose of molecular weight ratio, improves the biologic activity of fragrant plant mentioned in ancient texts polysaccharides.
5, the used protease hydrolyzed method of the preparation method of fragrant plant mentioned in ancient texts polysaccharides of the present invention can be decomposed glycoprotein, helps improving the final purity of polysaccharide.
6, preparation is simple, and raw material is easy to get, the reaction conditions gentleness, and the yield height, good stability, also very low to preparation equipment and place requirement, be suitable for large-scale industrial production.
Embodiment:
Below by specific embodiment the present invention is described in further detail.
Embodiment 1
The deionized water of getting 2000g fragrant plant mentioned in ancient texts algae powder adding 10L heats extraction, and the extraction temperature is 85 ℃, and extraction time is 4h, extracts end back temperature and reduces to room temperature, and filtration gets 1. 8.5L of filtrate.Filter residue adds the 10L deionized water, adds the salt acid for adjusting pH value to 4.8 of 0.5mol/L, and being heated to temperature is 50 ℃ ± 2 ℃.Advance enzymolysis in the ratio that every kg fragrant plant mentioned in ancient texts algae powder adds 20 million international units (than vigor) prozyme, add 40 million international units (than vigor) prozyme, prozyme contains cellulase and the hemicellulase that weight ratio is 2～1:1～2; After stirring evenly, keep the condition hydrolysis 120min of 50 ℃ ± 2 ℃ of pH4.8, temperature.Enzymolysis finishes post-heating and carries out enzyme-deactivating, and cooling is filtered, and gets 2. 9.0L of filtrate.
Merging filtrate 1. with filtrate 2. altogether 17.5L filtrate, sodium hydroxide solution with 18% (weight percent) is adjusted to 6.8 with the pH value, temperature is 50 ℃ ± 2 ℃, the ratio that adds 18 million international units (than vigor) lyase (as: algin catenase, alginate lyase, pectate lyase or pectate lyase) in every kg fragrant plant mentioned in ancient texts algae powder is advanced enzymolysis, add 36 million international units (than vigor) lyase, after stirring evenly, keep the condition hydrolysis 90min of 50 ℃ ± 2 ℃ of pH6.8, temperature.Enzymolysis finishes the hydrochloric acid adjust pH to 3.2 of back with 0.5mol/L, temperature is 50 ℃ ± 2 ℃, the ratio that adds 15 million international units (than vigor) proteolytic enzyme in every kg fragrant plant mentioned in ancient texts algae powder, add 30 million international units (than vigor) proteolytic enzyme, after stirring evenly, keep pH3.2,50 ℃ ± 2 ℃ condition hydrolysis 480min.Enzymolysis finishes post-heating and carries out enzyme-deactivating;
Be cooled to room temperature, filter, collect filtrate 17.0L; Filtrate adds with the NaOH solution of 18% (weight percent) regulates pH value to 6.5; Transferring the ethanol of the filtrate adding 95% of pH value is 20% to determining alcohol, and room temperature leaves standstill; Centrifugal, filter, collect filtrate, adding 95% ethanol is 70% to the ethanol volumetric concentration, room temperature leaves standstill; Centrifugal, filter, get precipitation, precipitation is washed 2 times with 95% ethanol and acetone respectively, and vacuum-drying promptly gets fragrant plant mentioned in ancient texts polysaccharides crude product 243.16g;
Get fragrant plant mentioned in ancient texts polysaccharides crude product 200g, add the stirring of 8L water and dissolve, centrifugal, filter, get filtrate 7.5L.With DEAE furnishing pasty state, adopt slurry type method dress post with water for injection, dress column pressure 0.5Mpa.Clean with 400mmol/L pH6.5 phosphate buffered saline buffer earlier behind the dress post, use 20mmol/LpH6.5 phosphate buffered saline buffer balance again, above-mentioned filtrate is gone up the DEAE chromatography column adsorb, with 20mmol/LpH6.5 phosphate buffered saline buffer wash-out.Collect target compound and get 7.0kg, using molecular retention is that the filter membrane of 100K carries out ultrafiltration, collects trapped fluid and gets 6.0L.Centrifugal, filter, alcohol to the determining alcohol of adding 95% is 70%, standing over night.Centrifugal, filter, get precipitation, precipitation is washed 2 times with 95% ethanol and acetone respectively, drains, and puts drying in the vacuum drying oven, promptly gets fragrant plant mentioned in ancient texts polysaccharides elaboration 6.41g.Its molecular weight of sampling and measuring shows that the molecular weight of Gracilaria gigas Harvey polysaccharides is between 500～50000 dalton.Other detection is carried out in sampling in addition, and the result is as follows:
1. polysaccharide is differentiated: phenol-sulphate reagent reaction (positive)
2. polysaccharide content: 98.62% (measuring) with phenol-sulfuric acid process
Embodiment 2
The deionized water of getting 2000g fragrant plant mentioned in ancient texts algae powder adding 20L heats extraction, and the extraction temperature is 95 ℃, and extraction time is 6h, extracts end back temperature and reduces to room temperature, and filtration gets 1. 17.0L of filtrate.Filter residue adds the 15L deionized water, adds the salt acid for adjusting pH value to 3.5 of 0.5mol/L, and being heated to temperature is 55 ℃ ± 2 ℃.Advance enzymolysis in the ratio that every kg fragrant plant mentioned in ancient texts algae powder adds 15 million international units (than vigor) prozyme, add 30 million international units (than vigor) prozyme, prozyme contains cellulase and the hemicellulase that weight ratio is 2～1:1～2; After stirring evenly, keep the condition hydrolysis 150min of 45 ℃ ± 2 ℃ of pH3.5, temperature.Enzymolysis finishes post-heating and carries out enzyme-deactivating, and cooling is filtered, and gets 2. 13.0L of filtrate.
Merging filtrate 1. with filtrate 2. altogether 30.0L filtrate, sodium hydroxide solution with 18% (weight percent) is adjusted to 8.0 with the pH value, temperature is 45 ℃ ± 2 ℃, advance enzymolysis in the ratio that every kg fragrant plant mentioned in ancient texts algae powder adds 10 million international units (than vigor) lyase, add 20 million international units (than vigor) lyase (as: algin catenase, alginate lyase, pectate lyase or pectate lyase), after stirring evenly, keep the condition hydrolysis 120min of 45 ℃ ± 2 ℃ of pH8.0, temperature.Enzymolysis finishes the hydrochloric acid adjust pH to 3.8 of back with 0.5mol/L, temperature is 45 ℃ ± 2 ℃, adds the ratio of 20 million international units than vigor proteolytic enzyme in every kg fragrant plant mentioned in ancient texts algae powder, adds the proteolytic enzyme of 40 million international units than vigor, after stirring evenly, keep pH3.8,45 ℃ ± 2 ℃ condition hydrolysis 450min.Enzymolysis finishes post-heating and carries out enzyme-deactivating;
Be cooled to room temperature, filter, collect filtrate 28.0L; Filtrate adds with the NaOH solution of 18% (weight percent) regulates pH value to 6.0; Transferring ethanol to the ethanol volumetric concentration of the filtrate adding 95% of pH value is 15%, and room temperature left standstill more than 3.0 hours; Centrifugal, filter, collect filtrate, adding 95% ethanol to ethanol volumetric concentration is 80%, room temperature left standstill more than 3.0 hours; Centrifugal, filter, get precipitation, precipitation is washed 2 times with 95% ethanol and acetone respectively, and vacuum-drying promptly gets fragrant plant mentioned in ancient texts polysaccharides crude product 268.33g;
Get fragrant plant mentioned in ancient texts polysaccharides crude product 200g, add the stirring of 10L water and dissolve, centrifugal, filter, get filtrate 9.5L.With DEAE furnishing pasty state, adopt slurry type method dress post with water for injection, dress column pressure 0.5Mpa.Clean with 400mmol/L pH6.0 phosphate buffered saline buffer earlier behind the dress post, use 20mmol/LpH6.0 phosphate buffered saline buffer balance again, above-mentioned filtrate is gone up the DEAE chromatography column adsorb, with 20mmol/LpH6.0 phosphate buffered saline buffer wash-out.Collect target compound and get 9.0kg, using molecular retention is that the filter membrane of 100K carries out ultrafiltration, collects trapped fluid and gets 8.0L.Centrifugal, to filter, alcohol to the ethanol volumetric concentration of adding 95% is 80%, standing over night.Centrifugal, filter, get precipitation, precipitation is washed 2 times with 95% ethanol and acetone respectively, drains, and puts drying in the vacuum drying oven, promptly gets fragrant plant mentioned in ancient texts polysaccharides elaboration 6.23g.Its molecular weight of sampling and measuring shows that the molecular weight of Gracilaria gigas Harvey polysaccharides is between 500～50000 dalton.Other detection is carried out in sampling in addition, and the result is as follows:
1. polysaccharide is differentiated: phenol-sulphate reagent reaction (positive)
2. polysaccharide content: 97.71% (measuring) with phenol-sulfuric acid process
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, though the present invention discloses as above with preferred embodiment, yet be not in order to limit the present invention, any those skilled in the art, in not breaking away from the technical solution of the present invention scope, when the technology contents that can utilize above-mentioned announcement is made a little change or is modified to the equivalent embodiment of equivalent variations, in every case be the content that does not break away from technical solution of the present invention, according to technical spirit of the present invention to any simple modification that above embodiment did, equivalent variations and modification all still belong in the scope of technical solution of the present invention.
Claims (6)
1, a kind of preparation method of Gracilaria gigas Harvey polysaccharides may further comprise the steps:
(1), the fragrant plant mentioned in ancient texts algae of gathering is rinsed well, discard assorted algae and dirt, 50 ℃～100 ℃ oven dry, pulverizing algae powder; The algae powder adds the above water of 5 times of amounts and heats extraction, extracts more than 30 minutes more than 60 ℃, reduces to room temperature, filters, and gets filtrate 1.;
(2), in filtrate, add 10～20 million international units and carry out enzymolysis than the lyase of vigor by per kilogram fragrant plant mentioned in ancient texts algae powder, the pH value is 6.0～9.0, hydrolysis temperature is 30 ℃～60 ℃, enzymolysis time is 30～210 minutes; Enzymolysis finishes back adjust pH to 3.0～4.0, add 10～20 million international units by per kilogram fragrant plant mentioned in ancient texts algae powder and carry out enzymolysis than the proteolytic enzyme of vigor, after stirring evenly, keep the pH value 3.0～4.0,45 ℃～50 ℃ condition enzymolysis, enzymolysis time is 450～510 minutes, and enzymolysis finishes post-heating to 90 ℃～100 ℃ and carries out enzyme-deactivating, and the enzyme-deactivating time is 5～20 minutes;
(3), reduce to room temperature, filtration, collection filtrate; Filtrate is regulated pH value to 5.5～7.0; Ethanol to the alcoholic acid volumetric concentration of adding 95% is 10%～20%, and room temperature left standstill more than 3.0 hours;
(4), centrifugal, filter, collect filtrate, adding 95% ethanol alcohol, to be sink to the alcoholic acid volumetric concentration be 40%～95%, room temperature left standstill more than 3.0 hours;
(5), the solution behind the alcohol precipitation filters, taking precipitate, throw out are washed 2 times with 95% ethanol and acetone respectively, vacuum-drying promptly gets Gracilaria gigas Harvey polysaccharides.
2, the preparation method of Gracilaria gigas Harvey polysaccharides according to claim 1 is characterized in that: this preparation method is further comprising the steps of:
(6), get the prepared Gracilaria gigas Harvey polysaccharides of step (5), add the above water of 10 times of amounts and stir and dissolve, centrifugal, filter, get filtered liquid;
(7), filtered liquid is carried out chromatographic separation with the DEAE chromatography column, with 10～30mmol/L, pH5.5～7.0 phosphate buffered saline buffers carry out wash-out;
(8), collect the target compound after the chromatographic separation, be that the ultra-filtration membrane of 5～100KD carries out ultrafiltration, the collection trapped fluid with molecular weight cut-off;
(9), trapped fluid adds 95% ethanol alcohol to be sink to the alcoholic acid volumetric concentration is 40%～95%, room temperature left standstill more than 3.0 hours;
(10), the solution behind the alcohol precipitation carries out centrifugally, filters, precipitation is washed 2 times with 95% ethanol and acetone respectively, carries out vacuum-drying, promptly gets Gracilaria gigas Harvey polysaccharides.
3, the preparation method of Gracilaria gigas Harvey polysaccharides according to claim 1, it is characterized in that: described step (1) also includes: filter residue adds the above water of 4 times of weight and carries out enzymolysis and extraction, add 10～30 million international units by per kilogram fragrant plant mentioned in ancient texts algae powder and carry out enzymolysis than the prozyme of vigor, it is 2～1: 1～2 cellulase and hemicellulase that described prozyme contains weight ratio; Keep the pH value 3.0～5.5, hydrolysis temperature is 30 ℃～60 ℃, and the time is 60～250 minutes; Enzymolysis finishes post-heating and carries out enzyme-deactivating, and the enzyme-deactivating temperature is 90 ℃～100 ℃, and the enzyme-deactivating time is 5～20 minutes, and cooling is filtered, and gets filtrate 2.; In the described step (2) the lyase enzymolysis be filtrate 1. with filtrate merging filtrate 2..
4, the preparation method of Gracilaria gigas Harvey polysaccharides according to claim 1 is characterized in that: the lyase in the described step (2) is algin catenase, alginate lyase, pectate lyase or pectate lyase.
5, the preparation method of Gracilaria gigas Harvey polysaccharides according to claim 1 is characterized in that: filter in the described step (3) and comprise, carry out coarse filtration with filter cloth earlier, carry out centrifugally again with high speed freezing centrifuge, carry out suction filtration again.
6, the preparation method of Gracilaria gigas Harvey polysaccharides according to claim 2, it is characterized in that: the filtration procedure in the described step (6) is: centrifugal with high speed freezing centrifuge, pour out supernatant liquor, and collecting precipitation is to container, ethanol with 95% is washed, and drains with suction filter.
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