CN116253809A - Method for removing endotoxin in chitosan - Google Patents
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- CN116253809A CN116253809A CN202310421090.0A CN202310421090A CN116253809A CN 116253809 A CN116253809 A CN 116253809A CN 202310421090 A CN202310421090 A CN 202310421090A CN 116253809 A CN116253809 A CN 116253809A
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 142
- 239000002158 endotoxin Substances 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 51
- 239000004094 surface-active agent Substances 0.000 claims abstract description 70
- 239000006228 supernatant Substances 0.000 claims abstract description 22
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229920004929 Triton X-114 Polymers 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 16
- 239000000126 substance Substances 0.000 claims abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 229960000583 acetic acid Drugs 0.000 claims description 14
- 239000008367 deionised water Substances 0.000 claims description 14
- 229910021641 deionized water Inorganic materials 0.000 claims description 14
- 239000012362 glacial acetic acid Substances 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims 1
- 239000012567 medical material Substances 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 6
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 241000238557 Decapoda Species 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 230000006196 deacetylation Effects 0.000 description 2
- 238000003381 deacetylation reaction Methods 0.000 description 2
- 229940127554 medical product Drugs 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002510 pyrogen Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 208000037487 Endotoxemia Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 206010051379 Systemic Inflammatory Response Syndrome Diseases 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000002407 tissue scaffold Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides a method for removing endotoxin in chitosan, which relates to the field of medical materials, in particular to a method for removing endotoxin in chitosan, and the invention discloses a method for removing endotoxin in chitosan, which is characterized by comprising the following steps: (1) preparing chitosan solution; (2) Preparing a surfactant, wherein the surfactant contains triton X-114 and tween40; (3) Mixing surfactant solution with chitosan solution, and collecting supernatant; (4) Adding alkaline substance into the supernatant to precipitate chitosan. The method for removing the endotoxin in the chitosan can effectively remove the endotoxin in the chitosan and improve the purity of the chitosan raw material.
Description
Technical Field
The invention relates to the field of medical materials, in particular to a method for removing endotoxin in chitosan.
Background
Chitosan is a derivative obtained after deacetylation of chitin, mainly derived from shrimps and crabs, insoluble in water and alkali, and soluble in dilute hydrochloric acid, nitric acid and the like. The chitosan can be widely applied to medicines, medical materials, sewage treatment and the like, and can be prepared into dressing and tissue scaffold in the aspect of medical materials, however, the purity of the chitosan sold in the market is low, and the chitosan contains endotoxin and protein, so that the immune response of organisms can be caused, and the application of the chitosan on the medical materials is limited.
Endotoxins are lipopolysaccharides, also known as "pyrogens", on the surface of gram-negative bacteria. Endotoxins are highly stable, have molecular weights close to those of proteins, and are hydrophilic and hydrophobic, and are difficult to remove from materials. Humans are extremely sensitive to bacterial endotoxins. Very small amounts (1-5 ng/kg body weight) of endotoxin can cause the body temperature to rise and gradually subside after a heating response of about hours. it has been shown that endotoxin levels in blood are closely related to endotoxemia, systemic inflammatory response syndrome, multiple organ functional exhaustion, etc.
YY/T1699-2020, "tissue engineering medical products: the protein content required by the standard of chitosan is less than or equal to 0.2%, and the removal mode of endotoxin <0.05EU/mg bacterial endotoxin comprises an ion exchange method, a two-phase extraction method, an affinity chromatography method and an adsorption method. However, chitosan is a cation and is easily combined with the negative ions of pyrogen, so that the effect is not ideal by adopting the existing method for removing chitosan. The endotoxin content of the chitosan produced by the existing method for removing the endotoxin can not meet the requirements of chitosan for medical materials.
Therefore, a method for efficiently removing endotoxin in chitosan is highly demanded.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method for removing endotoxin in chitosan, which can efficiently remove the endotoxin in chitosan and improve the purity of chitosan.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
the invention provides a method for removing endotoxin in chitosan, which is characterized by comprising the following steps:
(1) Preparing chitosan solution;
(2) Preparing a surfactant, wherein the surfactant contains triton X-114 and tween40;
(3) Mixing surfactant solution with chitosan solution, and collecting supernatant;
(4) Adding alkaline substance into the supernatant to precipitate chitosan.
Preferably, step (1) is to prepare a chitosan solution of 0.5% -2% (w/v) by dissolving chitosan powder in glacial acetic acid.
Preferably, the step (2) is to prepare 5% -12% (w/v) of surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 2:1-8:1.
Preferably, the step (3) is to mix the surfactant solution and the chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 50:1-1000:1, stir for 4-12h, and take the supernatant.
Preferably, the step (4) is to add 1% -3% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, wash the chitosan to neutrality with deionized water, and dry the chitosan.
Preferably, the method comprises the following steps:
(1) The chitosan powder is dissolved in glacial acetic acid to prepare 0.5% -2% (w/v) chitosan solution;
(2) Preparing 5% -12% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 2:1-8:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 50:1-1000:1, stirring for 4-12h, and taking an upper layer solution;
(4) Adding 1% -3% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
Preferably, the method comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 1% (w/v);
(2) Preparing 8% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 4:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 500:1, stirring for 8 hours, and taking an upper layer solution;
(4) Adding 2% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
Compared with the prior art, the method for removing endotoxin in chitosan has the following advantages:
(1) The surface active method adopted by the invention has low cost, and can obtain the chitosan raw material with purity conforming to the grade of medical materials;
(2) The surfactants triton X-114 and tween40 adopted by the invention can effectively remove endotoxin in chitosan.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention as long as they do not depart from the basic idea of the invention.
Chitosan was purchased from aladine, cas:9012-76-4; the deacetylation degree is more than or equal to 95%, the viscosity is 100-200 mpa.s, the product number is C105799-100, and the endotoxin content of chitosan is 28326.2EU/g by adopting a dynamic turbidity method;
triton X-114, tween40 was purchased from Alatine;
example 1 method for removing endotoxin from chitosan
The embodiment provides a method for removing endotoxin in chitosan, which comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare 0.5% (w/v) chitosan solution;
(2) Preparing 5% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 4:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 50:1, stirring for 4 hours, and taking an upper layer solution;
(4) Adding 1% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
The endotoxin content of chitosan was determined by dynamic turbidity method and was 32.4EU/g.
Example 2 method for removing endotoxin from chitosan
The embodiment provides a method for removing endotoxin in chitosan, which comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 2% (w/v);
(2) Preparing a 12% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 4:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 1000:1, stirring for 4 hours, and taking an upper layer solution;
(4) Adding 3% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
The endotoxin content of chitosan was determined by dynamic turbidity method and was 23.2EU/g.
Example 3A method for removing endotoxin from chitosan
The embodiment provides a method for removing endotoxin in chitosan, which comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 1% (w/v);
(2) Preparing 8% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 4:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 500:1, stirring for 8 hours, and taking an upper layer solution;
(4) Adding 2% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
The endotoxin content of chitosan was determined by dynamic turbidity method and was 36.4EU/g.
Example 4A method for removing endotoxin from chitosan
The embodiment provides a method for removing endotoxin in chitosan, which comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 1% (w/v);
(2) Preparing 8% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 2:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 500:1, stirring for 8 hours, and taking an upper layer solution;
(4) Adding 2% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
The endotoxin content of chitosan was determined by dynamic turbidity method and was 26.6EU/g.
Example 5A method for removing endotoxin from chitosan
The embodiment provides a method for removing endotoxin in chitosan, which comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 1% (w/v);
(2) Preparing 8% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 8:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 500:1, stirring for 8 hours, and taking an upper layer solution;
(4) Adding 2% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
The endotoxin content of chitosan was determined by dynamic turbidity method and was 24.3EU/g.
Comparative example 1 method for removing endotoxin in chitosan
The embodiment provides a method for removing endotoxin in chitosan, which comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 1% (w/v);
(2) Preparing 8% (w/v) surfactant solution, wherein the surfactant is tritonX-114;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 500:1, stirring for 8 hours, and taking an upper layer solution;
(4) Adding 2% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
The endotoxin content of chitosan was determined by dynamic turbidity method and was 93.1EU/g.
Comparative example 2 method for removing endotoxin in chitosan
The embodiment provides a method for removing endotoxin in chitosan, which comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 1% (w/v);
(2) Preparing 8% (w/v) surfactant solution, wherein the surfactant is tween40;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 500:1, stirring for 8 hours, and taking an upper layer solution;
(4) Adding 2% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
The endotoxin content of chitosan was determined by dynamic turbidity method and was 189.2EU/g.
Comparative example 3 method for removing endotoxin in chitosan
The embodiment provides a method for removing endotoxin in chitosan, which comprises the following steps:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 1% (w/v);
(2) Preparing 8% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween20 in the surfactant is 4:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 500:1, stirring for 8 hours, and taking an upper layer solution;
(4) Adding 2% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
The endotoxin content of chitosan was determined by dynamic turbidity method and was 97.3EU/g.
In conclusion, the chitosan prepared by the invention accords with the medical industry standard YY/T1699-2020 tissue engineering medical product: the requirement that the content of bacterial endotoxins is less than 0.05EU/mg is specified in chitosan. The endotoxin content is 23.2-36.4EU/g, the surfactant of comparative example 1 adopts triton X-114, the endotoxin content is 93.1EU/g, the surfactant of comparative example 2 adopts tween40, the endotoxin content is 189.2EU/g, the surfactant of comparative example 3 adopts triton X-114 to be matched with tween20, and the endotoxin content is 97.3EU/g. The surfactant disclosed by the invention is prepared by mixing triton X-114 with tween40, so that the endotoxin content in chitosan can be effectively reduced.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (7)
1. A method for removing endotoxin from chitosan, comprising the steps of:
(1) Preparing chitosan solution;
(2) Preparing a surfactant, wherein the surfactant contains triton X-114 and tween40;
(3) Mixing surfactant solution with chitosan solution, and collecting supernatant;
(4) Adding alkaline substance into the supernatant to precipitate chitosan.
2. The method of claim 1, wherein step (1) is to prepare a chitosan solution of 0.5% -2% (w/v) chitosan powder dissolved in glacial acetic acid.
3. The method of claim 1, wherein step (2) comprises preparing a 5% -12% (w/v) surfactant solution, wherein the mass ratio of triton x-114 to tween40 in the surfactant is 2:1-8:1.
4. The method for removing endotoxin in chitosan as defined in claim 1, wherein the step (3) is to mix a surfactant solution with a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 50:1-1000:1, and stirring for 4-12 hours, and the supernatant is obtained.
5. The method for removing endotoxin in chitosan as claimed in claim 1, wherein step (4) is to add 1% -3% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, wash with deionized water to neutrality, and dry.
6. The method for removing endotoxin from chitosan as claimed in claim 1, comprising the steps of:
(1) The chitosan powder is dissolved in glacial acetic acid to prepare 0.5% -2% (w/v) chitosan solution;
(2) Preparing 5% -12% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 2:1-8:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 50:1-1000:1, stirring for 4-12h, and taking an upper layer solution;
(4) Adding 1% -3% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
7. The method for removing endotoxin from chitosan as claimed in claim 6, comprising the steps of:
(1) Dissolving chitosan powder in glacial acetic acid to prepare a chitosan solution with the concentration of 1% (w/v);
(2) Preparing 8% (w/v) surfactant solution, wherein the mass ratio of triton X-114 to tween40 in the surfactant is 4:1;
(3) Mixing a surfactant solution and a chitosan solution, wherein the volume ratio of the surfactant solution to the chitosan solution is 500:1, stirring for 8 hours, and taking an upper layer solution;
(4) Adding 2% (w/v) sodium hydroxide solution into the supernatant to precipitate chitosan, washing with deionized water to neutrality, and drying.
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