CN1228349C - Extraction separation method of algae polysaccharide - Google Patents
Extraction separation method of algae polysaccharide Download PDFInfo
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- CN1228349C CN1228349C CN 02145386 CN02145386A CN1228349C CN 1228349 C CN1228349 C CN 1228349C CN 02145386 CN02145386 CN 02145386 CN 02145386 A CN02145386 A CN 02145386A CN 1228349 C CN1228349 C CN 1228349C
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 49
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 49
- 150000004676 glycans Chemical class 0.000 title claims abstract description 48
- 241000195493 Cryptophyta Species 0.000 title claims abstract description 21
- 238000000926 separation method Methods 0.000 title claims abstract description 13
- 238000000605 extraction Methods 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000001556 precipitation Methods 0.000 claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims description 22
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 5
- 229920005654 Sephadex Polymers 0.000 claims description 5
- 239000012507 Sephadex™ Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 125000001453 quaternary ammonium group Chemical group 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 238000002242 deionisation method Methods 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000012047 saturated solution Substances 0.000 claims description 2
- 238000002525 ultrasonication Methods 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 4
- 239000000284 extract Substances 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- -1 and finally Substances 0.000 abstract description 2
- 238000005349 anion exchange Methods 0.000 abstract description 2
- 230000004071 biological effect Effects 0.000 abstract description 2
- 238000002270 exclusion chromatography Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000003809 water extraction Methods 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 3
- 241000195474 Sargassum Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 235000016425 Arthrospira platensis Nutrition 0.000 description 1
- 240000002900 Arthrospira platensis Species 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 1
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 1
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 1
- 229960002733 gamolenic acid Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000004223 radioprotective effect Effects 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 229940082787 spirulina Drugs 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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- Polysaccharides And Polysaccharide Derivatives (AREA)
- Medicines Containing Plant Substances (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention discloses an extraction separation method of algae polysaccharides, which uses the characteristic that polysaccharides and glycosidoprotein can be dissolved in water to leach by a water extraction method and separate by a fractionated precipitation method of ethanol, and finally, polysaccharides are purified by an anion exchange column and a gel exclusion chromatography. The method of the present invention uses common alga as raw materials, extracts and purifies various polysaccharides inside and outside algal cells, and can realize the coextract of algae protein and algae polysaccharides and keep the biological activity of protein and protein polysaccharides. The present invention furthest increases the utilization rate of the raw materials and reduces production cost; the method of the present invention is suitable for large-scale production and can be universally used for the separation and the purification of various algae polysaccharides.
Description
Technical field
The present invention relates to the extraction and separation method of algal polysaccharides.
Background technology
Marine alga is the widest photosynthetic prokaryotic organism of occurring in nature distribution, has very high nutritive value, and Sargassum protein contains each seed amino acid of needed by human, VITAMIN, various trace elements, gamma-linolenic acid and some natural pigments etc.Polysaccharides has very high pharmaceutical use, is improving the human body non-specific immune function, and anti-hypoxia, antitumor, radioprotective, anti-ageing, hypoglycemic, reducing blood-fat aspect have significant effect.(seaweed polysaccharide, SP) active substance in mostly is water-soluble acidity, the neutral polysaccharide that monose such as D-glucose, D-seminose, D-semi-lactosi, D-glucuronic acid are formed to Sargassum polysaccharides.In the past polysaccharides extracts purification technique and mainly concentrates in the extraction of exocellular polysaccharide, and commonly used be the hot-water soak method, for example, Chinese patent publication number 1220999 described polysaccharide extractive techniques, the result often causes the loss of activity of polysaccharide protein.Chinese patent publication number 1280855 used technology, though mention the broken wall problem, this method inevitably causes the sex change of glycoprotein and the hydrolysis of some polysaccharide.Chinese patent publication number 1112128 described methods for another example though avoided the damage of the too high glycoprotein that temperature caused, might be destroyed some polysaccharide fraction.
Summary of the invention
The objective of the invention is provides a kind of extraction and separation method of algal polysaccharides for overcoming above-mentioned existing problems.The extraction and separation method of algal polysaccharides of the present invention is to utilize polysaccharide, the water-soluble characteristic of glycoprotein, leaches with water extraction, and separates with the method for ethanol precipitation, uses anion-exchange column and gel exclusion chromatography purified polysaccharide at last.Specifically may further comprise the steps:
1) gets fresh algae or algae powder adding distil water or phosphate buffer solution and mix, carry out ultrasonication;
2) above-mentioned broken liquid is centrifugal, abandon precipitation, get supernatant;
3) supernatant is sloughed albumen;
4) in sloughing proteic solution, add 2~3 times of ethanol and precipitate to this liquor capacity, centrifugal collecting precipitation, washing with acetone, drying gets Crude polysaccharides;
5) get Crude polysaccharides and be dissolved in distilled water or Tris-HCl damping fluid, make saturated solution, the centrifugal precipitation of abandoning is splined on DEAE-Sepharose Fast Flow post with supernatant, with Tris-HCl is elutriant, carry out the NaCl gradient elution, the phenolsulfuric acid method is followed the tracks of and is detected, and collects simple spike, be splined on sephadexG-100 or sephadex G-75 post behind the dialysis deionization, carry out wash-out with deionized water, collect simple spike, lyophilize gets the single polysaccharides of purifying.
Said among the present invention supernatant is sloughed proteic method, can adopt in the following several method any:
A) supernatant Sevage method deproteinated;
Add isopyknic chloroform-primary isoamyl alcohol in supernatant liquor, (volume ratio of chloroform and primary isoamyl alcohol is 5: 1) left standstill after the concussion 2 hours, centrifugal 15 minutes of 7000~8000rmp, and repeated multiple times sevage method deproteinated is until no albumen.
B) supernatant uses protease hydrolyzed, enzymolysis solution to use Sevage method deproteinated more earlier;
Adopt enzymolysis better in conjunction with Sevage method deproteinated effect, generally use papain enzymolysis, 40 ℃~45 ℃ enzymolysis 5 hours, enzyme agent consumption was a supernatant 0.5%.
C) supernatant is saltoutd with quaternary ammonium earlier, uses Sevage method deproteinated again;
D) supernatant is saltoutd with quaternary ammonium earlier, uses protease hydrolyzed then, and enzymolysis solution is used Sevage method deproteinated again.
Because algae protein has very high nutritive value, before with Sevage method deproteinated, saltout with quaternary ammonium (as ammonium sulfate) earlier, then can separate out most of albumen, the albumen of removing can be used, and this albumen can renaturation, can use it for anything else.
Among the present invention, if adopt fresh algae, then fresh algae and distilled water or phosphoric acid buffer are generally with 1: 2 mixed of weightmeasurement ratio, and the pH value of phosphoric acid buffer is 7.6, and concentration is 50~100mmol/L.If adopt the algae powder, then algae powder and distilled water or phosphoric acid buffer are generally with 1: 20 mixed of weightmeasurement ratio, and the pH value of phosphoric acid buffer is 7.6, and concentration is 50~100mmol/L.
Among the present invention, the pH value of the Tris-HCl damping fluid of dissolving Crude polysaccharides is generally 7~8, and concentration is 10~50mmol/L.The NaCl concentration of making gradient elution is 0.1-0.8mol/L.
Available phenol one sulfuric acid process of the detection of polysaccharide arrives 2ml with the 0.2ml sample with distilled water diluting, adds 6% phenol solution 1ml, adds the 5ml vitriol oil again, reacts after 20 minutes in 490nm place detection OD value.
The inventive method is a raw material with general algae, extracts, the inside and outside polysaccharide of born of the same parents of the various algae of purifying, and can realize the co-extracted of algae albumen and polysaccharides, keeps the biological activity of albumen and protein-polysaccharide.Increase utilization ratio of raw materials to greatest extent, reduce production costs.The inventive method is suitable for scale operation, and can be widely used in the separation and purification of various algal polysaccharides.
Embodiment
Further specify the present invention by the following examples, but the present invention is not limited thereto embodiment.
Embodiment 1:
Get fresh spirulina algae mud 500g, add 1000ml water, broken 15 minutes of room temperature.7500rmp. centrifugal 15 minutes, abandon precipitation.Add papoid 0.5g, 45 ℃ of temperature were bathed 5 hours.Sample adds isopyknic chloroform-primary isoamyl alcohol (volume ratio of chloroform and primary isoamyl alcohol is 5: 1), and thermal agitation left standstill 2 hours, and centrifugal 10 minutes of 7500rmp repeats above step, until there not being precipitation.Supernatant precipitates (resetting and adding ethanol on getting, until there not being precipitation) with 2~3 times of volume ethanol.Precipitate at last with washing with acetone for several times, dry that Crude polysaccharides 8 restrains, polysaccharide yield is 1.6%.
It is 7.6 that above-mentioned Crude polysaccharides is dissolved in 20ml pH value, and concentration is among the Tris-HCl of 10mmol/L, and 7500rmp abandoned precipitation in centrifugal 10 minutes.Supernatant is splined on DEAE-Sepharose Fast Flow post, is that 7.6 Tris-HCl makes elutriant with the pH value, is that the NaCl of 0.1-0.8mol/L makes gradient elution with concentration, and flow rate control is at 6ml/min.The product of collecting detects absorbance value with phenol one sulfuric acid process at the 490nm place, obtain three absorption peaks.Collect three absorption peaks (component I, II, III) respectively, and in flowing water dialysis desalting.Solution concentration after will dialysing is splined on sephadex G-75 post more respectively, makes elutriant with distilled water, and flow rate control is followed the tracks of with the phenol sulfuric acid process equally and detected at 1ml/min.The result shows that component I, II, III all are symmetrical peaks after crossing G-75, illustrates that this component is an one-component, will get the fluffy polysaccharides of 2g white after the component I lyophilize.
Embodiment 2:
Get 50 gram spiral algae powders, add the 1000ml phosphoric acid buffer, broken 20 minutes of room temperature.Centrifugal 15 minutes of 7500rmp abandons precipitation.Sample adds isopyknic chloroform-primary isoamyl alcohol (volume ratio of chloroform and primary isoamyl alcohol is 5: 1), and thermal agitation left standstill 2 hours, and centrifugal 10 minutes of 7500rmp repeats step more than 4 times, until there not being precipitation.Supernatant precipitates (resetting and adding ethanol on getting, until there not being precipitation) with 2~3 times of volume ethanol, the washing with acetone precipitation, and dry that Crude polysaccharides 4 restrains, productive rate is 8%.
It is 7.6 that above-mentioned Crude polysaccharides is dissolved in 20ml pH value, and concentration is among the Tris-HCl of 10mmol/L, and 7500rmp abandoned precipitation in centrifugal 10 minutes.Supernatant is splined on DEAE-Sepharose Fast Flow post, is that 7.6 Tris-HCl makes elutriant with the pH value, is that the NaCl of 0.1-0.8mol/L makes gradient elution with concentration, and flow rate control is at 6ml/min.The product of collecting detects absorbance value with the phenolsulfuric acid method at the 490nm place, obtain two absorption peaks.Collect two absorption peaks (component I, II) respectively, and in flowing water dialysis desalting.Solution concentration after will dialysing is splined on sephadex G-75 post, makes elutriant with distilled water, and flow rate control is followed the tracks of with the phenol sulfuric acid process equally and detected at 1ml/min.The result shows that component I is a symmetrical peak after crossing G75, will get the fluffy polysaccharides of 1.5g white after the component I lyophilize.
Claims (5)
1. the extraction and separation method of algal polysaccharides is characterized in that may further comprise the steps:
1) get fresh algae or algae powder adding distil water or phosphate buffer solution and mix, carry out ultrasonication, wherein the pH value of phosphate buffer solution is 7.6, and concentration is 50~100mmol/L;
2) above-mentioned broken liquid is centrifugal, abandon precipitation, get supernatant;
3) supernatant is sloughed albumen;
4) in sloughing proteic solution, add 2~3 times of ethanol and precipitate to this liquor capacity, centrifugal collecting precipitation, washing with acetone, drying gets Crude polysaccharides;
5) get Crude polysaccharides and be dissolved in distilled water or tris-HCI buffer, make saturated solution, the centrifugal precipitation of abandoning is splined on DEAE-Sepharose Fast Flow post with supernatant, with Tris-HCl is elutriant, carry out the NaCl gradient elution, the phenolsulfuric acid method is followed the tracks of and is detected, and collects simple spike, be splined on sephadex G-100 or sephadex G-75 post behind the dialysis deionization, carry out wash-out with deionized water, collect simple spike, lyophilize gets the single polysaccharides of purifying.
2. the extraction and separation method of algal polysaccharides according to claim 1 is characterized in that step 3) is said supernatant is sloughed proteic method to have following several;
A) supernatant Sevage method deproteinated;
B) supernatant uses protease hydrolyzed, enzymolysis solution to use Sevage method deproteinated more earlier;
C) supernatant is saltoutd with quaternary ammonium earlier, uses Sevage method deproteinated again;
D) supernatant is saltoutd with quaternary ammonium earlier, uses protease hydrolyzed then, and enzymolysis solution is used Sevage method deproteinated again.
3. the extraction and separation method of algal polysaccharides according to claim 1 is characterized in that fresh algae of step 1) and distilled water or phosphoric acid buffer with 1: 2 mixed of weightmeasurement ratio, and the pH value of phosphoric acid buffer is 7.6, and concentration is 50~100mmol/L.
4. the extraction and separation method of algal polysaccharides according to claim 1 is characterized in that step 1) algae powder and distilled water or phosphoric acid buffer with 1: 20 mixed of weightmeasurement ratio, and the pH value of phosphoric acid buffer is 7.6, and concentration is 50~100mmol/L.
5. the extraction and separation method of algal polysaccharides according to claim 1, the pH value that it is characterized in that dissolving the tris-HCI buffer of Crude polysaccharides is 7~8, concentration is 10~50mmol/L.
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Cited By (1)
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CN101724012B (en) * | 2008-10-10 | 2012-05-30 | 中国科学院大连化学物理研究所 | Deep processing method for cultivated gardon asparagus in gracilaria |
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CN101724012B (en) * | 2008-10-10 | 2012-05-30 | 中国科学院大连化学物理研究所 | Deep processing method for cultivated gardon asparagus in gracilaria |
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