CN1821274A - Method for removing protein of Brazil mushroom crude polysaccharide - Google Patents
Method for removing protein of Brazil mushroom crude polysaccharide Download PDFInfo
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- CN1821274A CN1821274A CN 200610049894 CN200610049894A CN1821274A CN 1821274 A CN1821274 A CN 1821274A CN 200610049894 CN200610049894 CN 200610049894 CN 200610049894 A CN200610049894 A CN 200610049894A CN 1821274 A CN1821274 A CN 1821274A
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- removing protein
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 50
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 50
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 18
- 235000001674 Agaricus brunnescens Nutrition 0.000 title claims abstract description 15
- 239000011347 resin Substances 0.000 claims abstract description 44
- 229920005989 resin Polymers 0.000 claims abstract description 44
- 150000001450 anions Chemical class 0.000 claims abstract description 12
- 150000001768 cations Chemical class 0.000 claims abstract description 12
- 239000002253 acid Substances 0.000 claims abstract description 9
- 238000004440 column chromatography Methods 0.000 claims abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 150000002500 ions Chemical class 0.000 claims description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 230000007935 neutral effect Effects 0.000 claims description 12
- 241001327634 Agaricus blazei Species 0.000 claims description 10
- 238000002203 pretreatment Methods 0.000 claims description 7
- 229920002125 Sokalan® Polymers 0.000 claims description 6
- 239000004584 polyacrylic acid Substances 0.000 claims description 6
- 238000007667 floating Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 3
- 238000013375 chromatographic separation Methods 0.000 claims description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 claims description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 abstract description 9
- 239000003456 ion exchange resin Substances 0.000 abstract description 8
- 229920003303 ion-exchange polymer Polymers 0.000 abstract description 8
- 239000003960 organic solvent Substances 0.000 abstract description 6
- 238000003912 environmental pollution Methods 0.000 abstract description 4
- 239000003513 alkali Substances 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 description 8
- 238000004587 chromatography analysis Methods 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000945 filler Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000002572 peristaltic effect Effects 0.000 description 4
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000002585 base Substances 0.000 description 3
- 239000003729 cation exchange resin Substances 0.000 description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 239000003957 anion exchange resin Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229940088623 biologically active substance Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000003544 deproteinization Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the method of removing protein from crude Brazil mushroom polysaccharide. The crude Brazil mushroom polysaccharide is column chromatography separated with at least one kind of weak acid cation resin or weak alkali anion resin to separate free protein from polysaccharide. The method of the present invention has short operation time of 3-5 hr, high protein eliminating rate, less polysaccharide loss, no use of organic solvent. less environmental pollution, low cost and reusablility of ion exchange resin.
Description
(1) technical field
The present invention relates to a kind of method for removing protein of Brazil mushroom crude polysaccharide.
(2) background technology
Fungus polysaccharide is class biological activity goods, purer polysaccharide could play a role better generally to need to obtain relatively through separation and purification, especially in the time of it will being developed to the higher medicine of purity, remove requisite purification step of deproteinated technology of contained foreign protein in the Crude polysaccharides.The method that is applied to the polysaccharide deproteinization at present mainly contains to adopt and adds a certain proportion of organic solvent, as trichoroacetic acid(TCA), trifluoroacetic acid method, tannic acid, Sevag reagent (a certain proportion of mixed solution of trichoroacetic acid(TCA) and propyl carbinol), also can adopt enzyme process, enzyme-Sevag coupling method etc.Wherein the Sevag method is a classic methods all the time, is most widely used so far.
But when adopting the Sevag method to take off Agaricus Blazei Murrill coarse polysaccharide albumen, not only polysaccharide loss is serious, and deproteinated technology repeat could be more thorough more than 6 times, the organic solvent enormous amount that is consumed, cost height, environmental pollution and operator's injury are bigger, explosion-proof security is poor, operational cycle is long, generally takes more than 2 days.
(3) summary of the invention
Polysaccharide loss is serious in the existing removing protein of Brazil mushroom crude polysaccharide technology, the organic solvent quantity consumed is huge, cost is high in order to solve, environmental pollution and operator's injury are bigger, problems such as the operational cycle is long the invention provides a kind of method for removing protein of Brazil mushroom crude polysaccharide.
Method for removing protein of Brazil mushroom crude polysaccharide of the present invention adopts at least a acidulous cation resin or weak anion resin, and Agaricus Blazei Murrill polysaccharide is carried out column chromatography for separation floating preteins and polysaccharide.
Generally be neutrality and the charged character of albumen according to the contained polysaccharide of Agaricus Blazei Murrill coarse polysaccharide, the present invention utilizes ion exchange resin and the mutual exchange adsorption property of biomolecules with opposite charges to realize removing protein of Brazil mushroom crude polysaccharide.Consider again simultaneously that biologically active substance should not adopt strong acid, strong base to adopt the ion exchange resin resin, so the present invention has adopted acidulous cation resin or weak anion resin.
Described acidulous cation resin can be selected from following resin: parent is a polyacrylic acid, and positively charged ion is a quaternary ammonium ion, specifically floats the D113 that Lai Te (China) company limited provides as Deqing.Described acidulous cation resin generally need carry out the pre-treatment of following step when just dispatching from the factory: place 2% hydrochloric acid soln to soak 2～4 hours resin, pure water washs 3～5 times to neutral, and then placing 2%NaOH to soak 2～4 hours the resin, pure water washing 3～5 times is to neutral; At last resin is placed 2% hydrochloric acid soln to soak 2～4 hours, pure water washs 3～5 times to neutral.
Described weak anion resin can be selected from following resin: parent is a polyacrylic acid, and negatively charged ion is an azochlorosulfonate acid ion, specifically floats the A830 that Lai Te (China) company limited provides as Deqing.Described weak anion resin generally need carry out the pre-treatment of following step when just dispatching from the factory: place 2%NaOH solution to soak 2～4 hours resin, pure water washs 3～5 times to neutral, and then placing 2% hydrochloric acid to soak 2～4 hours the resin, pure water washing 3～5 times is to neutral; At last resin is placed 2%NaOH solution to soak 2～4 hours, pure water washs 3～5 times to neutral.
The ion exchange resin description of product
| The resin model | Type | The ion pattern | The volume complete exchange capacity＞(eq/l) | Loading density (g/l) | Water content (%) | Wet true density (g/ml) |
| A830 D113 | The polyacrylic acid polyacrylic acid | The free alkali free acid | 2.7 4.2 | 690～725 720～800 | 45～52 47～53 | 1.10 1.14～1.20 |
Acidulous cation resin of the present invention or weak anion resin all can be regenerated.Weak base anion-exchange resin regeneration can be adopted: 2～4%NaOH solution dynamic 1 hour, replaces the NaOH wash-out to be washed till neutrality with pure water with 3 times of column volume wash-outs again.And the same weak base anion-exchange resin of the regeneration of weakly acidic cation-exchange resin, just with salt acid substitution NaOH.
When adopting the method for the invention, can the single-column chromatography, also can multicolumn series connection chromatography.When adopting two posts series connection chromatography, the step of recommendation is: adopt weak anion resin and acidulous cation resin successively, described Agaricus Blazei Murrill coarse polysaccharide is carried out post series connection chromatographic separation floating preteins and polysaccharide.
When adopting the method for the invention to carry out separating polyose and albumen, and the operating time weak point (general needs 3～5h), albumen decreasing ratio height, and polysaccharide loss is little; Operating process does not relate to organic solvent to be adopted, used acid, alkali cleaning take off at last can in and salify, environmental pollution is little; Owing to an organic solvent not adopting ion exchange resin, cost is low, and reusable.
(4) embodiment
The invention will be further described below in conjunction with embodiment, but protection scope of the present invention is not limited to this.
Embodiment 1 positively charged ion is crossed post 1 time
The ion exchange resin of handling well is filled in the chromatography column resin cation (R.C.) chromatography column specification: 2.6 * 55cm, the high 45cm of ion-exchanger filler;
Last sample: positively charged ion is crossed the post deproteinated, and applied sample amount is 10ml, polysaccharide content 13.00mg/ml in Agaricus Blazei Murrill coarse polysaccharide during last sample, and protein content 6.23mg/ml, the Crude polysaccharides material liquid pH is nature pH (=6.4) when extracting.
Wash-out: on the Zeo-karb behind the sample, deionized water peristaltic pump wash-out, flow velocity 1-4mL/min, the phenolsulfuric acid method detects effluent liquid sugar content, is eluted to sugar-free and separates out, and about 400～800ml regenerates then, reuses.Separating resulting sees Table 1.
Embodiment 2 negatively charged ion are crossed post 1 time
The ion exchange resin of handling well is filled in the chromatography column anionite-exchange resin specification: 2.6 * 55cm, the high 45cm of ion-exchanger filler.
Last sample: negatively charged ion is crossed the post deproteinated, and applied sample amount is 10ml, polysaccharide content 13.00mg/ml in Agaricus Blazei Murrill coarse polysaccharide during last sample, and protein content 6.23mg/ml, the Crude polysaccharides material liquid pH is nature pH (=6.4) when extracting.
Wash-out: on the anionite-exchange resin behind the sample, deionized water peristaltic pump wash-out, flow velocity 1-4mL/min, the phenolsulfuric acid method detects effluent liquid sugar content, is eluted to sugar-free and separates out, about 400～800ml.Separating resulting sees Table 1.
Embodiment 3 negatively charged ion are crossed post 1 time
Use 4%NaOH solution with 3 times of column volume wash-outs in embodiment 2 described anionite-exchange resin, dynamic 1 hour, replace the NaOH wash-out to be washed till neutrality with pure water again.
Last sample: negatively charged ion is crossed the post deproteinated, and applied sample amount is 10ml, polysaccharide content 13.00mg/ml in Agaricus Blazei Murrill coarse polysaccharide during last sample, and protein content 6.23mg/ml, the Crude polysaccharides material liquid pH is nature pH (=6.4) when extracting.
Wash-out: on the anionite-exchange resin behind the sample, deionized water peristaltic pump wash-out, flow velocity 1-4mL/min, the phenolsulfuric acid method detects effluent liquid sugar content, is eluted to sugar-free and separates out, about 400～800ml.Separating resulting sees Table 1.
The series connection of embodiment 4 zwitterions is crossed post 1 time
The ion exchange resin of handling well is filled in the chromatography column resin anion(R.A) chromatography column specification: 2.6 * 55cm, the high 45cm of ion-exchanger filler; Zeo-karb specification: 2.6 * 55cm, the high 45cm of ion-exchanger filler.
Last sample: the zwitterion post deproteinated of connecting, applied sample amount is 10ml, polysaccharide content 13.00mg/ml in Agaricus Blazei Murrill coarse polysaccharide during last sample, protein content 6.23mg/ml, the Crude polysaccharides material liquid pH is nature pH (=6.4) when extracting.
Wash-out: on the anion-cation exchange resin behind the sample, deionized water peristaltic pump wash-out, flow velocity 1-4mL/min, the phenolsulfuric acid method detects effluent liquid sugar content, is eluted to sugar-free and separates out, and about 400～800ml regenerates then, reuses.Separating resulting sees Table 1.
Table 1 anion-cation exchange resin deproteinated effect
| Protein content (mg) | Protein removal rate (%) | Polysaccharide content (mg) | Polysaccharide recovery (%) | (polysaccharide content/protein content) value | |
| Positively charged ion (after the pre-treatment) is crossed post 1 time | 12.24 | 80.35 | 121.41 | 93.39 | 9.92 |
| Negatively charged ion (after the pre-treatment) is crossed post 1 time | 17.16 | 72.45 | 114.01 | 87.70 | 6.64 |
| Negatively charged ion (regenerate 1 time after) is crossed post 1 time | 18.34 | 70.56 | 115.96 | 89.2 | 6.32 |
| Zwitterion (after each pre-treatment) series connection is crossed post 1 time | 5.49 | 92.36 | 107.71 | 82.85 | 19.62 |
Claims (6)
1, a kind of method for removing protein of Brazil mushroom crude polysaccharide is characterized in that: adopt at least a acidulous cation resin or weak anion resin, Agaricus Blazei Murrill polysaccharide is carried out column chromatography for separation floating preteins and polysaccharide.
2, method for removing protein of Brazil mushroom crude polysaccharide as claimed in claim 1 is characterized in that: described acidulous cation resin parent is a polyacrylic acid, and positively charged ion is a quaternary ammonium ion.
3, method for removing protein of Brazil mushroom crude polysaccharide as claimed in claim 2, it is characterized in that: described acidulous cation resin carries out the pre-treatment of following step: place 2% hydrochloric acid soln to soak 2～4 hours resin, pure water washs 3～5 times to neutral, and then placing 2%NaOH to soak 2～4 hours the resin, pure water washing 3～5 times is to neutral; At last resin is placed 2% hydrochloric acid soln to soak 2～4 hours, pure water washs 3～5 times to neutral.
4, method for removing protein of Brazil mushroom crude polysaccharide as claimed in claim 1 is characterized in that: described weak anion resin parent is a polyacrylic acid, and negatively charged ion is an azochlorosulfonate acid ion.
5, method for removing protein of Brazil mushroom crude polysaccharide as claimed in claim 4, it is characterized in that: described weak anion resin carries out the pre-treatment of following step: place 2%NaOH solution to soak 2～4 hours resin, pure water washs 3～5 times to neutral, and then placing 2% hydrochloric acid to soak 2～4 hours the resin, pure water washing 3～5 times is to neutral; At last resin is placed 2%NaOH solution to soak 2～4 hours, pure water washs 3～5 times to neutral.
6, as the described method for removing protein of Brazil mushroom crude polysaccharide of one of claim 1～5, it is characterized in that: adopt weak anion resin and acidulous cation resin successively, described Agaricus Blazei Murrill coarse polysaccharide is carried out post series connection chromatographic separation floating preteins and polysaccharide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610049894 CN1821274A (en) | 2006-03-17 | 2006-03-17 | Method for removing protein of Brazil mushroom crude polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610049894 CN1821274A (en) | 2006-03-17 | 2006-03-17 | Method for removing protein of Brazil mushroom crude polysaccharide |
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| Publication Number | Publication Date |
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| CN1821274A true CN1821274A (en) | 2006-08-23 |
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| CN 200610049894 Pending CN1821274A (en) | 2006-03-17 | 2006-03-17 | Method for removing protein of Brazil mushroom crude polysaccharide |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429254B (en) * | 2008-12-05 | 2011-05-04 | 云南植物药业有限公司 | Bletilla striata polysaccharide, preparation method and new uses thereof |
| CN103694363A (en) * | 2013-11-27 | 2014-04-02 | 广东药学院 | Preparation method of refined polysaccharide of isatis root |
| CN104497161A (en) * | 2015-01-04 | 2015-04-08 | 华东理工大学 | Purification method of grifolan |
| CN106349400A (en) * | 2016-08-29 | 2017-01-25 | 高其品 | Novel method for preparing hericium purified polysaccharides by combined application of macroporous resin |
| CN106883310A (en) * | 2017-04-17 | 2017-06-23 | 龙岩学院 | A kind of Deproteinated purification process of the very best beans polysaccharide decolouring |
| CN109535274A (en) * | 2018-11-30 | 2019-03-29 | 淮阴工学院 | A kind of method that common cattail polysaccharide takes off protein decolouring |
| CN111841080A (en) * | 2020-07-07 | 2020-10-30 | 江苏大学 | Method for improving acid-base elution effect in chromatographic decolorization of lycium barbarum polysaccharide by using ultrasonic waves |
-
2006
- 2006-03-17 CN CN 200610049894 patent/CN1821274A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429254B (en) * | 2008-12-05 | 2011-05-04 | 云南植物药业有限公司 | Bletilla striata polysaccharide, preparation method and new uses thereof |
| CN103694363A (en) * | 2013-11-27 | 2014-04-02 | 广东药学院 | Preparation method of refined polysaccharide of isatis root |
| CN103694363B (en) * | 2013-11-27 | 2016-01-27 | 广东药学院 | A kind of preparation method of Root of Indigowoad refined polysaccharide |
| CN104497161A (en) * | 2015-01-04 | 2015-04-08 | 华东理工大学 | Purification method of grifolan |
| CN104497161B (en) * | 2015-01-04 | 2016-09-14 | 华东理工大学 | A kind of method of purification of grifolan |
| CN106349400A (en) * | 2016-08-29 | 2017-01-25 | 高其品 | Novel method for preparing hericium purified polysaccharides by combined application of macroporous resin |
| CN106883310A (en) * | 2017-04-17 | 2017-06-23 | 龙岩学院 | A kind of Deproteinated purification process of the very best beans polysaccharide decolouring |
| CN109535274A (en) * | 2018-11-30 | 2019-03-29 | 淮阴工学院 | A kind of method that common cattail polysaccharide takes off protein decolouring |
| CN111841080A (en) * | 2020-07-07 | 2020-10-30 | 江苏大学 | Method for improving acid-base elution effect in chromatographic decolorization of lycium barbarum polysaccharide by using ultrasonic waves |
| CN111841080B (en) * | 2020-07-07 | 2022-03-25 | 江苏大学 | Method for improving acid-base elution effect in chromatographic decolorization of lycium barbarum polysaccharide by using ultrasonic waves |
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