Chitosan purification method combining adsorption and desorption of magnetic nano material

Abstract

The invention provides a method for purifying chitosan by combining adsorption and desorption of a magnetic nano material. The method comprises the following steps: S1, preparing crude chitosan filtrate; s2, adding Fe3O4-coated PNIPAM / beta-CD composite particles into the crude chitosan filtrate, adjusting the pH value of the solution, and stirring for adsorption; s3, after adsorption is finished, a constant magnetic field is applied, standing is carried out, a compound of Fe3O4-coated PNIPAM / beta-CD and impurities is adsorbed by the magnetic field, and upper-layer clear liquid is a preliminarily purified chitosan solution; s4, adding hot water, raising the temperature to 35 DEG C, maintaining the temperature, continuously desorbing, removing the magnetic field, adding deionized water, uniformly stirring, standing, and separating out chitosan precipitate; and S5, filtering and collecting the chitosan precipitate, washing to be neutral, and performing vacuum drying to obtain the chitosan. According to the method, the rapid separation characteristic of a magnetic material, the solvent-free response characteristic of a temperature-sensitive polymer and the molecular recognition characteristic of beta-cyclodextrin are integrated by adopting the technology of Fe3O4-coated PNIPAM / beta-CD composite particles and temperature-sensitive adsorption-desorption, so that the effects of breaking through the limitation of a traditional purification process and synchronously realizing high-purity chitosan acquisition and environment-friendly production are achieved.

Description

Technical Field

[0001] This invention relates to the technical field of chitosan preparation, specifically to a chitosan purification method using magnetic nanomaterials for adsorption-desorption. Background Technology

[0002] Chitosan, a natural amino polysaccharide obtained from chitin through deacetylation, possesses irreplaceable application value in pharmaceuticals, food, environmental protection, and cosmetics due to its excellent biocompatibility, biodegradability, antibacterial properties, and film-forming properties. In the pharmaceutical field, it can serve as a drug carrier, medical dressing, and tissue engineering scaffold material. In the food field, it can be used as a preservative, thickener, and heavy metal ion chelating agent. In the environmental field, it can be used for the adsorption and treatment of dyes and heavy metals in water. However, crude chitosan (usually extracted from crustacean waste such as shrimp and crab shells) contains a large number of impurities, mainly including proteins, metal ions, endotoxins, pigments, and small molecule peptides. These impurities not only affect the physicochemical properties of chitosan (such as reducing viscosity stability and disrupting film-forming properties) but also limit its application in high-end fields—for example, in the pharmaceutical field, excessive endotoxin levels may cause adverse reactions such as fever and shock, while in the food field, protein residues can easily lead to product spoilage and flavor deterioration. Therefore, efficient purification of crude chitosan to remove impurities and improve purity is a core prerequisite for realizing its high-value-added applications.

[0003] Currently, commonly used industrial chitosan purification methods are represented by the "acid-dissolution-alkali precipitation method" and the "ethanol precipitation method." While these traditional processes are simple to operate and low in cost, they have insurmountable technical limitations, such as incomplete impurity removal, low product purity, reliance on chemical reagents, significant safety risks and environmental pressures, low separation efficiency, product loss, and high energy consumption. To address the pain points of traditional processes, some improvement technologies have been researched, but significant shortcomings remain, and a technological breakthrough has not yet been achieved. Therefore, developing a novel purification technology that enables efficient adsorption, precise separation, and solvent-free desorption is crucial to overcoming the current bottleneck in chitosan purification and promoting its application in high-end fields. Summary of the Invention

[0004] Technical problem to be solved: To address the above-mentioned technical problems, the purpose of this invention is to provide a chitosan purification method using magnetic nanomaterials for adsorption-desorption. By employing the technology of "Fe3O4@PNIPAM / β-CD composite particles + thermosensitive adsorption-desorption", the rapid separation characteristics of magnetic materials, the solvent-free response characteristics of thermosensitive polymers, and the molecular recognition characteristics of β-cyclodextrin are integrated to overcome the limitations of traditional purification processes and simultaneously achieve the effect of obtaining high-purity chitosan and environmentally friendly production.

[0005] Technical solution: A chitosan purification method using magnetic nanomaterial adsorption-desorption coupling, comprising the following steps: S1: Take crude chitosan, slowly add 1% acetic acid solution, stir until completely dissolved, prepare 2% chitosan solution, sonicate at 250W for 15min, then filter through 0.45μm filter membrane to obtain crude chitosan filtrate; S2: Add Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH of the solution to 5.8, and continue to stir and adsorb for 70-80 min at 28℃ and a stirring rate of 180 r / min. S3: After adsorption is complete, a constant magnetic field of 0.5T is applied outside the reactor and allowed to stand for 15 minutes. The complex of Fe3O4@PNIPAM / β-CD and impurities is adsorbed by the magnetic field, and the clear liquid in the upper layer is the preliminarily purified chitosan solution. S4: Add 37℃ hot water to raise the temperature to 35℃ and maintain it. Adjust the stirring speed to 150r / min and continue desorption for 48-50min. Remove the magnetic field, add deionized water, stir evenly and let stand for 30min to precipitate chitosan. S5: Collect the chitosan precipitate by filtration through a 0.22μm filter membrane, wash the precipitate with deionized water until neutral, and vacuum dry to obtain high-purity chitosan.

[0006] Furthermore, the preparation method of the Fe3O4@PNIPAM / β-CD composite particles is as follows: S21: Take 5.0g of Fe3O4 particles, disperse them in 100mL of N,N-dimethylformamide, add 0.4g of γ-methacryloxypropyltrimethoxysilane, reflux at 60℃ for 4h, centrifuge at 8000r / min for 15min, wash 3 times with N,N-dimethylformamide to obtain Fe3O4 modified with γ-methacryloxypropyltrimethoxysilane; S22: γ-methacryloxypropyltrimethoxysilane-modified Fe3O4 was redispersed in 80 mL of N,N-dimethylformamide, 15.0 g of N-isopropylacrylamide monomer and 0.3 g of azobisisobutyronitrile were added, and polymerization was carried out at 70 °C for 6 h under nitrogen protection. After centrifugation at 10000 r / min for 20 min, Fe3O4@PNIPAM particles were collected, washed 3 times with the mixture, and vacuum dried to obtain Fe3O4@PNIPAM thermosensitive particles. S23: Take 10.0g of food-grade β-cyclodextrin, dissolve it in 100mL of deionized water, add 0.01g of EDTA-2Na, stir for 30min, filter through a 0.22μm filter membrane to obtain a 10% β-CD solution; S24: Take 4.0g of Fe3O4@PNIPAM temperature-sensitive particles, disperse them in 10% β-CD solution, add 0.15g of Tween-80, and stir the mixture at 40℃ and 150r / min for 60min. S25: Spray drying yields Fe3O4@PNIPAM / β-CD composite particles.

[0007] Furthermore, the mixture in S22 is water-ethanol with a volume ratio of 1:1.

[0008] Furthermore, the mass ratio of Fe3O4@PNIPAM thermosensitive particles to 10% β-CD solution in S24 is 8:2.

[0009] Furthermore, the spray drying conditions in S25 are: inlet air temperature 120°C, outlet air temperature 60°C, and feed rate 5 mL / min.

[0010] Furthermore, the amount of Fe3O4@PNIPAM / β-CD composite particles added in S2 is 5-7% of the mass of crude chitosan.

[0011] The above-mentioned chitosan purification method using magnetic nanomaterial adsorption-desorption coupling also includes the following steps: collecting the complex of Fe3O4@PNIPAM / β-CD and impurities adsorbed by the magnetic field, adding 50 mL of deionized water, stirring and washing at 200 r / min for 10 min, centrifuging at 8000 r / min for 10 min, and vacuum drying at 55 °C for 6 h to obtain regenerated Fe3O4@PNIPAM / β-CD composite particles.

[0012] Chitosan prepared by the above method.

[0013] Furthermore, the purity of the chitosan is ≥99%.

[0014] Beneficial effects: 1. This invention integrates the rapid separation characteristics of magnetic materials, the solvent-free response characteristics of temperature-sensitive polymers, and the molecular recognition characteristics of β-cyclodextrin by adopting the technology of "Fe3O4@PNIPAM / β-CD composite particles + temperature-sensitive adsorption-desorption". This achieves the breakthrough of the limitations of traditional purification processes and simultaneously realizes the acquisition of high-purity chitosan and environmentally friendly production. 2. This invention replaces traditional centrifugation and filtration separation technologies with magnetic separation. By utilizing the strong magnetic responsiveness of the composite particles (saturation magnetic strength 52-55 emu / g), the adsorbent and solution can be quickly separated under the action of an external magnetic field, thereby significantly shortening the separation time, avoiding chitosan loss during the separation process, and improving the overall purification efficiency. 3. This invention uses temperature-sensitive desorption technology to replace chemical reagent desorption technology. Relying on the LCST characteristics of PNIPAM, the adsorption-desorption transition can be triggered by temperature adjustment alone, thus completely avoiding the risk of chemical desorbent residue and meeting the stringent safety requirements of pharmaceutical and food-grade chitosan. At the same time, it reduces solvent recovery costs and environmental treatment pressure. 4. This invention employs a composite modification technique of β-cyclodextrin and Fe3O4@PNIPAM. By utilizing the hydrophobic cavity structure of β-cyclodextrin, it achieves "host-guest" specific encapsulation of small molecule impurities (endotoxin fragments, pigment precursors) in chitosan. At the same time, its surface hydroxyl groups form stable hydrogen bonds with Fe3O4@PNIPAM, achieving the dual effect of enhancing the adsorption selectivity for small molecule impurities that are difficult to remove, while ensuring the structural stability of the composite material and avoiding the impact of material agglomeration on the adsorption effect. 5. This invention employs ultrasonic pretreatment and graded filtration technology for coarse chitosan to first remove insoluble large particulate impurities, and then adsorbs soluble impurities through composite particles, forming a process chain of "pretreatment-precise adsorption-deep purification". This achieves the effects of reducing ineffective consumption of adsorbent, improving the adsorption efficiency of adsorbent for target impurities, and extending the service life of adsorbent. Detailed Implementation

[0015] This invention proposes a chitosan purification method using a combination of adsorption and desorption with magnetic nanomaterials. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following examples will be used to further describe the invention in detail. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the invention.

[0016] Example 1 The preparation method of Fe3O4@PNIPAM / β-CD composite particles is as follows: S21: Take 5.0g of Fe3O4 particles, disperse them in 100mL of N,N-dimethylformamide, add 0.4g of γ-methacryloxypropyltrimethoxysilane, reflux at 60℃ for 4h, centrifuge at 8000r / min for 15min, wash 3 times with N,N-dimethylformamide to obtain Fe3O4 modified with γ-methacryloxypropyltrimethoxysilane; S22: γ-methacryloxypropyltrimethoxysilane-modified Fe3O4 was redispersed in 80 mL of N,N-dimethylformamide, 15.0 g of N-isopropylacrylamide monomer and 0.3 g of azobisisobutyronitrile were added, and polymerization was carried out at 70 °C for 6 h under nitrogen protection. After centrifugation at 10000 r / min for 20 min, Fe3O4@PNIPAM particles were collected, washed three times with a 1:1 water-ethanol mixture, and vacuum dried to obtain Fe3O4@PNIPAM thermosensitive particles. S23: Take 10.0g of food-grade β-cyclodextrin, dissolve it in 100mL of deionized water, add 0.01g of EDTA-2Na, stir for 30min, filter through a 0.22μm filter membrane to obtain a 10% β-CD solution; S24: Take 4.0g of Fe3O4@PNIPAM temperature-sensitive particles, disperse them in 10g of 10% β-CD solution, add 0.15g of Tween-80, and stir the mixture at 40℃ and 150r / min for 60min. S25: Spray drying, inlet air temperature 120℃, outlet air temperature 60℃, feed rate 5mL / min, to obtain Fe3O4@PNIPAM / β-CD composite particles.

[0017] Its lower critical solution temperature (LCST) was determined to be 32.2℃.

[0018] Example 2 A chitosan purification method using magnetic nanomaterials for adsorption-desorption coupling includes the following steps: S1: Take 20.0g of crude chitosan, slowly add 1000mL of 1% acetic acid solution, stir until completely dissolved, prepare a 2% chitosan solution, sonicate at 250W for 15min, and then filter through a 0.45μm filter membrane to obtain crude chitosan filtrate. S2: Add 1.0 g of Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH of the solution to 5.8, and continue to stir and adsorb for 70 min at 28℃ and a stirring rate of 180 r / min. S3: After adsorption is complete, a constant magnetic field of 0.5T is applied outside the reactor and allowed to stand for 15 minutes. The complex of Fe3O4@PNIPAM / β-CD and impurities is adsorbed by the magnetic field, and the clear liquid in the upper layer is the preliminarily purified chitosan solution. S4: Add 37℃ hot water to raise the temperature to 35℃ and maintain it. Adjust the stirring speed to 150r / min and continue desorption for 48min. Remove the magnetic field, add 1200mL of deionized water, stir evenly and let stand for 30min to precipitate chitosan. S5: Collect the chitosan precipitate by filtration through a 0.22μm filter membrane, wash the precipitate with deionized water until pH 7.0, and vacuum dry at 55℃ for 10h to obtain high-purity chitosan.

[0019] Example 3 A chitosan purification method using magnetic nanomaterials for adsorption-desorption coupling includes the following steps: S1: Take 20.0g of crude chitosan, slowly add 1000mL of 1% acetic acid solution, stir until completely dissolved, prepare a 2% chitosan solution, sonicate at 250W for 15min, and then filter through a 0.45μm filter membrane to obtain crude chitosan filtrate. S2: Add 1.2g of Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH of the solution to 5.8, and continue to stir and adsorb for 75min at 28℃ and a stirring rate of 180r / min. S3: After adsorption is complete, a constant magnetic field of 0.5T is applied outside the reactor and allowed to stand for 15 minutes. The complex of Fe3O4@PNIPAM / β-CD and impurities is adsorbed by the magnetic field, and the clear liquid in the upper layer is the preliminarily purified chitosan solution. S4: Add 37℃ hot water to raise the temperature to 35℃ and maintain it. Adjust the stirring speed to 150r / min and continue desorption for 49min. Remove the magnetic field, add 1200mL of deionized water, stir evenly and let stand for 30min to precipitate chitosan. S5: Collect the chitosan precipitate by filtration through a 0.22μm filter membrane, wash the precipitate with deionized water until pH 7.0, and vacuum dry at 55℃ for 10h to obtain high-purity chitosan.

[0020] Example 4 A chitosan purification method using magnetic nanomaterials for adsorption-desorption coupling includes the following steps: S1: Take 20.0g of crude chitosan, slowly add 1000mL of 1% acetic acid solution, stir until completely dissolved, prepare a 2% chitosan solution, sonicate at 250W for 15min, and then filter through a 0.45μm filter membrane to obtain crude chitosan filtrate. S2: Add 1.4g of Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH of the solution to 5.8, and continue to stir and adsorb for 80min at 28℃ and a stirring rate of 180r / min. S3: After adsorption is complete, a constant magnetic field of 0.5T is applied outside the reactor and allowed to stand for 15 minutes. The complex of Fe3O4@PNIPAM / β-CD and impurities is adsorbed by the magnetic field, and the clear liquid in the upper layer is the preliminarily purified chitosan solution. S4: Add 37℃ hot water to raise the temperature to 35℃ and maintain it. Adjust the stirring speed to 150r / min and continue desorption for 50min. Remove the magnetic field, add 1200mL of deionized water, stir evenly and let stand for 30min to precipitate chitosan. S5: Collect the chitosan precipitate by filtration through a 0.22μm filter membrane, wash the precipitate with deionized water until pH 7.0, and vacuum dry at 55℃ for 10h to obtain high-purity chitosan.

[0021] Example 5 A chitosan purification method using magnetic nanomaterials for adsorption-desorption coupling includes the following steps: S1: Take 30.0g of crude chitosan, slowly add 1500mL of 1% acetic acid solution, stir until completely dissolved, prepare a 2% chitosan solution, sonicate at 250W for 15min, and then filter through a 0.45μm filter membrane to obtain crude chitosan filtrate. S2: Add 1.5g of Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH of the solution to 5.8, and continue to stir and adsorb for 72min at 28℃ and a stirring rate of 180r / min. S3: After adsorption is complete, a constant magnetic field of 0.5T is applied outside the reactor and allowed to stand for 15 minutes. The complex of Fe3O4@PNIPAM / β-CD and impurities is adsorbed by the magnetic field, and the clear liquid in the upper layer is the preliminarily purified chitosan solution. S4: Add 37℃ hot water to raise the temperature to 35℃ and maintain it. Adjust the stirring speed to 150r / min and continue desorption for 48min. Remove the magnetic field, add 1800mL of deionized water, stir evenly and let stand for 30min to precipitate chitosan. S5: Collect the chitosan precipitate by filtration through a 0.22μm filter membrane, wash the precipitate with deionized water until pH 7.0, and vacuum dry at 55℃ for 10h to obtain high-purity chitosan.

[0022] Example 6 A chitosan purification method using magnetic nanomaterials for adsorption-desorption coupling includes the following steps: S1: Take 15.0g of crude chitosan, slowly add 750mL of 1% acetic acid solution, stir until completely dissolved, prepare a 2% chitosan solution, sonicate at 250W for 15min, and then filter through a 0.45μm filter membrane to obtain crude chitosan filtrate. S2: Add 0.9g of Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH of the solution to 5.8, and continue to stir and adsorb for 78min at 28℃ and a stirring rate of 180r / min. S3: After adsorption is complete, a constant magnetic field of 0.5T is applied outside the reactor and allowed to stand for 15 minutes. The complex of Fe3O4@PNIPAM / β-CD and impurities is adsorbed by the magnetic field, and the clear liquid in the upper layer is the preliminarily purified chitosan solution. S4: Add 37℃ hot water to raise the temperature to 35℃ and maintain it. Adjust the stirring speed to 150r / min and continue desorption for 49min. Remove the magnetic field, add 900mL of deionized water, stir evenly and let stand for 30min to precipitate chitosan. S5: Collect the chitosan precipitate by filtration through a 0.22μm filter membrane, wash the precipitate with deionized water until pH 7.0, and vacuum dry at 55℃ for 10h to obtain high-purity chitosan.

[0023] Example 7 A chitosan purification method using magnetic nanomaterials for adsorption-desorption coupling includes the following steps: S1: Take 25.0g of crude chitosan, slowly add 1250mL of 1% acetic acid solution, stir until completely dissolved, prepare a 2% chitosan solution, sonicate at 250W for 15min, and then filter through a 0.45μm filter membrane to obtain crude chitosan filtrate. S2: Add 1.75g ​​of Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH of the solution to 5.8, and continue to stir and adsorb for 76min at 28℃ and a stirring rate of 180r / min. S3: After adsorption is complete, a constant magnetic field of 0.5T is applied outside the reactor and allowed to stand for 15 minutes. The complex of Fe3O4@PNIPAM / β-CD and impurities is adsorbed by the magnetic field, and the clear liquid in the upper layer is the preliminarily purified chitosan solution. S4: Add 37℃ hot water to raise the temperature to 35℃ and maintain it. Adjust the stirring speed to 150r / min and continue desorption for 50min. Remove the magnetic field, add 1500mL of deionized water, stir evenly and let stand for 30min to precipitate chitosan. S5: Collect the chitosan precipitate by filtration through a 0.22μm filter membrane, wash the precipitate with deionized water until pH 7.0, and vacuum dry at 55℃ for 10h to obtain high-purity chitosan.

[0024] Comparative Example 1 (Traditional Acid Dissolution and Alkali Precipitation Method) S1: Take 20.0g of crude chitosan, slowly add 1000mL of 1% acetic acid solution, stir until completely dissolved, prepare a 2% chitosan solution, sonicate at 250W for 15min, and filter through a 0.45μm filter membrane; S2: Add 2 mol / L NaOH solution to the filtrate to adjust the pH to 9.0, precipitate chitosan, and let stand for 30 min; S3: Collect the precipitate by filtration through a 0.22 μm filter membrane, wash with deionized water until pH 7.0, and dry under vacuum at 55 °C for 10 h to obtain purified chitosan.

[0025] Comparative Example 2 (Composite particles lacking β-CD: Fe3O4@PNIPAM only) S1: Same as S1 in Example 2; S2: Add 1.2g Fe3O4@PNIPAM particles (without β-CD) to the crude chitosan filtrate, adjust the pH to 5.8, and stir at 28℃ and 180r / min for 75min to adsorb. S3-S5: Same as S3-S5 in Example 2.

[0026] Comparative Example 3 (Non-thermosensitive desorption: desorption using hydrochloric acid-ethanol) S1-S3: Same as S1-S3 in Example 2; S4: Remove the magnetic field, add 1800mL of 0.1mol / L hydrochloric acid-ethanol (1:1) mixture to the solution, stir at 40℃ and 150r / min for 49min to desorb, let stand for 30min, and chitosan precipitate will be formed. S5: Same as S5 in Example 2.

[0027] Comparative Example 4 (Adsorption temperature higher than LCST: 35℃) S1: Same as S1 in Example 2; S2: Add 1.2g Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH to 5.8, and stir at 35℃ and 180r / min for 75min to adsorb. S3-S5: Same as S3-S5 in Example 2.

[0028] Performance testing: Chitosan purity detection method: High performance liquid chromatography (HPLC) was used. Calculation method: A standard curve was established using standard (chitosan with purity ≥ 99.8%), and the purity was calculated based on the peak area of ​​the sample. Protein residue detection method: Coomassie Brilliant Blue G-250 method. Take 0.1g of purified chitosan, dissolve it in 10mL of 1% acetic acid solution, add 5mL of Coomassie Brilliant Blue reagent, shake well and let stand for 5min, and measure the absorbance at 595nm wavelength; establish a standard curve with bovine serum albumin as standard, and calculate the protein content. Metal ion residue (Ca) 2+ Fe 3+ Detection method: Atomic absorption spectrophotometry: Take 1.0g of purified chitosan, digest it with a mixture of nitric acid and perchloric acid (volume ratio 4:1) until clear, and make up to 50mL; measure the absorbance at the characteristic wavelengths of Ca (wavelength 422.7nm) and Fe (wavelength 248.3nm) respectively, and calculate the metal ion concentration through the standard curve; Endotoxin detection method: Take 0.5g of purified chitosan, prepare a 0.1% solution with pyrogen-free water, and filter it through a 0.22μm pyrogen-free filter membrane; take 0.1mL of filtrate and mix it with 0.1mL of Limulus amebocyte lysate (LAL) reagent, incubate at 37℃ for 60min, and observe whether a gel forms to determine the endotoxin content; Ethanol residue detection method: Gas chromatography (GC); Calculation method: A standard curve was established using ethanol standards, and the ethanol residue was calculated based on the peak area of ​​the sample; the results are shown in Table 1 below: Table 1 Chitosan purity (%) Protein residue (%) Metal ion residue (mg / kg) Endotoxin (EU / mg) Ethanol residue (%) Example 1 99.2 0.28 28 0.24 Not detected Example 2 99.4 0.25 25 0.21 Not detected Example 3 99.3 0.26 26 0.22 Not detected Example 4 99.2 0.29 29 0.25 Not detected Example 5 99.3 0.27 27 0.23 Not detected Example 6 99.4 0.24 24 0.2 Not detected Comparative Example 1 92.5 4.8 120 4.5 Not detected Comparative Example 2 97.8 0.85 55 0.8 Not detected Comparative Example 3 99.1 0.3 27 0.23 0.15 Comparative Example 4 96.2 1.5 68 1.2 Not detected As shown in Table 1 above, the test results of the examples are highly consistent. The purity of chitosan is stable at 99.2%-99.4%, the protein residue is as low as 0.24%-0.29%, the metal ion residue is 24-29 mg / kg, the endotoxin is 0.20-0.25 EU / mg, and no ethanol residue is detected. It meets the standards for pharmaceutical grade (endotoxin <0.5 EU / mg) and food grade (no solvent residue). When the dosage of composite particles was increased from 5% to 7%, the impurity removal effect was slightly improved (e.g., in Example 1, with a dosage of 5%, the protein residue was 0.28%; in Example 3, with a dosage of 7%, the protein residue was 0.26%), but the improvement was small. Considering cost, a dosage of 6% (Examples 2 and 5) was the optimal choice, which could control material costs while ensuring purity. Extending the adsorption time from 70 min to 80 min had a limited impact on the results (the purity was 99.2%-99.3% in both Example 1 (adsorption for 70 min) and Example 3 (adsorption for 80 min)). This indicates that 70-75 min is sufficient for sufficient adsorption, and there is no need to extend the time excessively, which can improve production efficiency.

[0029] The purity of this invention is 6.7-6.9 percentage points higher than that of the traditional acid-dissolution-alkali precipitation method (92.5%) in Comparative Example 1, with a 94%-94.8% reduction in protein residue and a 94.4%-95.6% reduction in endotoxin, completely solving the problem of incomplete impurity removal in traditional processes. Comparative Example 2 lacks β-CD: the composite particles containing β-CD reduced endotoxin from 0.8 EU / mg to 0.20-0.25 EU / mg, and increased the removal rate of small molecule impurities by 68.7%-75%, demonstrating the crucial role of β-CD "molecular recognition adsorption". Comparative Example 3, with chemical desorption, has a purity and impurity residue similar to this invention, but this invention has no ethanol residue (Comparative Example 3 has 0.15% residue), avoiding solvent safety risks. In Comparative Example 4, when the adsorption temperature is higher than LCST (35℃), the purity decreases from 99.2%-99.4% to 96.2%, and the protein residue increases by 4.6 times, indicating that "adsorption below LCST" is the core prerequisite for the efficient operation of this invention.

Claims

1. A chitosan purification method using magnetic nanomaterials for adsorption-desorption coupling, characterized in that, Includes the following steps: S1: Take crude chitosan, slowly add 1% acetic acid solution, stir until completely dissolved, prepare 2% chitosan solution, sonicate at 250W for 15min, then filter through 0.45μm filter membrane to obtain crude chitosan filtrate; S2: Add Fe3O4@PNIPAM / β-CD composite particles to the crude chitosan filtrate, adjust the pH of the solution to 5.8, and continue to stir and adsorb for 70-80 min at 28℃ and a stirring rate of 180 r / min. S3: After adsorption is complete, a constant magnetic field of 0.5T is applied outside the reactor and allowed to stand for 15 minutes. The complex of Fe3O4@PNIPAM / β-CD and impurities is adsorbed by the magnetic field, and the clear liquid in the upper layer is the preliminarily purified chitosan solution. S4: Add 37℃ hot water to raise the temperature to 35℃ and maintain it. Adjust the stirring speed to 150r / min and continue desorption for 48-50min. Remove the magnetic field, add deionized water, stir evenly and let stand for 30min to precipitate chitosan. S5: Collect the chitosan precipitate by filtration through a 0.22μm filter membrane, wash the precipitate with deionized water until neutral, and vacuum dry to obtain high-purity chitosan.

2. The chitosan purification method using magnetic nanomaterial adsorption-desorption coupling according to claim 1, characterized in that, The preparation method of the Fe3O4@PNIPAM / β-CD composite particles is as follows: S21: Take 5.0g of Fe3O4 particles, disperse them in 100mL of N,N-dimethylformamide, add 0.4g of γ-methacryloxypropyltrimethoxysilane, reflux at 60℃ for 4h, centrifuge at 8000r / min for 15min, wash 3 times with N,N-dimethylformamide to obtain Fe3O4 modified with γ-methacryloxypropyltrimethoxysilane; S22: γ-methacryloxypropyltrimethoxysilane-modified Fe3O4 was redispersed in 80 mL of N,N-dimethylformamide, 15.0 g of N-isopropylacrylamide monomer and 0.3 g of azobisisobutyronitrile were added, and polymerization was carried out at 70 °C for 6 h under nitrogen protection. After centrifugation at 10000 r / min for 20 min, Fe3O4@PNIPAM particles were collected, washed 3 times with the mixture, and vacuum dried to obtain Fe3O4@PNIPAM thermosensitive particles. S23: Take 10.0g of food-grade β-cyclodextrin, dissolve it in 100mL of deionized water, add 0.01g of EDTA-2Na, stir for 30min, filter through a 0.22μm filter membrane to obtain a 10% β-CD solution; S24: Take 4.0g of Fe3O4@PNIPAM temperature-sensitive particles, disperse them in 10% β-CD solution, add 0.15g of Tween-80, and stir the mixture at 40℃ and 150r / min for 60min. S25: Spray drying yields Fe3O4@PNIPAM / β-CD composite particles.

3. The chitosan purification method using magnetic nanomaterial adsorption-desorption coupling according to claim 2, characterized in that, The mixture in S22 is water-ethanol with a volume ratio of 1:

1.

4. The chitosan purification method using magnetic nanomaterial adsorption-desorption coupling according to claim 2, characterized in that, The mass ratio of Fe3O4@PNIPAM thermosensitive particles to 10% β-CD solution in S24 is 8:

2.

5. The chitosan purification method using magnetic nanomaterial adsorption-desorption coupling according to claim 2, characterized in that, The spray drying conditions in S25 are: inlet air temperature 120℃, outlet air temperature 60℃, and feed rate 5mL / min.

6. The chitosan purification method using magnetic nanomaterial adsorption-desorption coupling according to claim 1, characterized in that, The amount of Fe3O4@PNIPAM / β-CD composite particles added in S2 is 5-7% of the mass of crude chitosan.

7. The chitosan purification method using magnetic nanomaterial adsorption-desorption coupling according to claim 1, characterized in that, The process also includes the following steps: collecting the complex of Fe3O4@PNIPAM / β-CD and impurities adsorbed by the magnetic field, adding 50 mL of deionized water, stirring and washing at 200 r / min for 10 min, centrifuging at 8000 r / min for 10 min, and vacuum drying at 55 °C for 6 h to obtain regenerated Fe3O4@PNIPAM / β-CD composite particles.

8. Chitosan prepared by the method according to any one of claims 1-7.

9. The high degree of deacetylation chitosan according to claim 8, characterized in that, The purity of the chitosan is ≥99%.