An environmentally friendly chitosan with high degree of deacetylation and its preparation method

Abstract

This invention belongs to the field of biomass material modification and provides an environmentally friendly chitosan with a high degree of deacetylation and its preparation method. This invention employs a green process route that combines low-temperature enzymatic pretreatment with deep eutectic solvent synergistic treatment of crustacean biomass raw materials, followed by ionic liquid-assisted alkaline deacetylation and membrane separation electrodialysis solvent recovery. This achieves the preparation of high-quality chitosan with a degree of deacetylation of 90.0–98.0%, ash content as low as 0.15–1.0 wt%, and controllable molecular weight. It solves the problems of severe molecular chain degradation and high ash residue caused by traditional strong-alkali deacetylation, the contradiction between achieving homogeneous dissolution and long-term stability in multiphase high-solids systems, and the environmental burden caused by difficult solvent recovery. This invention has broad application value in the fields of biomedicine, food additives, and water treatment materials.

Description

Technical Field

[0001] This invention relates to the field of biomass material modification and green chemical engineering, specifically to an environmentally friendly chitosan with a high degree of deacetylation and its preparation method. Background Technology

[0002] Chitosan, a natural polysaccharide polymer obtained by deacetylation of chitin, has shown broad application prospects in biomedicine, food industry, water treatment, and agriculture due to its excellent biocompatibility, biodegradability, antibacterial properties, and film-forming properties. In the biomedical field, high-degree-of-deacetylation chitosan can be used as a drug sustained-release carrier, tissue engineering scaffold material, and wound dressing; its degree of deacetylation directly affects the material's solubility, cell affinity, and degradation rate. In the food industry, chitosan, as a natural preservative, clarifying agent, and dietary fiber supplement, needs to possess good water solubility and low ash content to meet food safety standards. In the water treatment field, the performance of chitosan-based flocculants and adsorbents is highly dependent on molecular weight distribution and functional group density. However, with the increasing demands on the performance of chitosan in various applications, especially in high-end medical devices and functional food additives, more stringent technical indicators have been put forward for the degree of deacetylation, controllability of molecular weight, purity and solubility stability of chitosan. Developing green preparation technologies that can simultaneously meet the requirements of high degree of deacetylation, low ash residue, adjustable molecular weight and long-term stability of homogeneous dissolution has become the key to promoting the high-quality development of the chitosan industry.

[0003] Despite years of development in chitosan preparation technology, existing methods still face numerous technical bottlenecks in achieving high deacetylation degrees while maintaining high molecular weight and product quality. Traditional strong-base deacetylation processes can achieve high deacetylation degrees, but the β-elimination reaction of glycosidic bonds under high-temperature, strong-base conditions leads to severe chain degradation, making molecular weight difficult to control and resulting in a wide distribution. Furthermore, high ash content caused by strong-base residues and inorganic salt impurities affects product purity and biosafety. For example, Chinese patent CN102311502A discloses a method using concentrated alkali and high-temperature deacetylation, which achieves high deacetylation degrees but results in significant molecular weight loss and high ash content. To address the molecular weight protection issue, some studies have attempted to use mild deacetylation systems, but low deacetylation efficiency leads to significantly prolonged reaction times. Moreover, the high mass transfer resistance and uneven reaction in multiphase solid-liquid systems make it difficult to achieve homogeneous dissolution and long-term stability of the product. For instance, Chinese patent CN103408727A discloses a microwave-assisted deacetylation method, but the difficulty in precisely controlling microwave power leads to localized overheating and uneven molecular weight distribution. Furthermore, existing processes generally lack efficient solvent recovery and waste liquid treatment systems. The large-scale use of strong alkalis and organic solvents not only increases production costs but also causes serious environmental pollution, which runs counter to the development trend of green chemical industry. Summary of the Invention

[0004] The purpose of this invention is to provide an environmentally friendly chitosan with a high degree of deacetylation and its preparation method, which solves the shortcomings of the current chitosan system in that it is difficult to achieve both high degree of deacetylation and high molecular weight, the contradiction between chain degradation and high ash residue caused by strong base deacetylation, and the difficulty in achieving homogeneous dissolution and long-term stability in multiphase high solid content processes.

[0005] This invention achieves mild and efficient conversion of crustacean biomass by constructing a synergistic system of low-temperature enzymatic hydrolysis pretreatment, deep eutectic solvent activation, and ionic liquid-assisted alkaline deacetylation. Low-temperature enzymatic hydrolysis selectively removes protein and lipid impurities while reducing the risk of non-specific thermochemical degradation under mild conditions. The deep eutectic solvent improves substrate accessibility by disrupting the crystalline region through hydrogen bonding networks. The ionic liquid, as a reaction medium, weakens the attack of the base on the molecular chain while promoting acetyl removal. The synergistic effect of these three components significantly reduces the base concentration and reaction temperature required for deacetylation, effectively inhibiting molecular chain degradation while achieving a high degree of deacetylation. Combined with a membrane separation-electrodialysis solvent recovery system, green and sustainable production is achieved.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing an environmentally friendly chitosan with a high degree of deacetylation, characterized by comprising the following steps:

[0008] S1. Raw material pretreatment: Select crustacean biomass raw materials from shrimp shells or crab shells, and wash, dry and crush them in sequence. The particle size after crushing is 80-120 mesh.

[0009] S2. Low-temperature enzymatic pretreatment: The powder obtained in step S1 is added to a phosphate buffer solution with a total phosphate concentration of 0.01–0.20 mol / L, and the pH is adjusted to 6.8–7.2 to form a suspension with a solid-liquid mass ratio of 1:(8–15). Chitinase, protease, and lipase are added at 4–10°C, and calcium chloride is added. After stirring for 12–36 h, the solid and liquid are separated to obtain a solid. Based on the total mass of the suspension, the mass fraction of chitinase is 0.2–1.0 wt%, the mass fraction of protease is 0.5–3.0 wt%, and the mass fraction of lipase is 0.2–2.0 wt%. The concentration of calcium chloride is 0.5–5.0 g / L based on the volume of the phosphate buffer solution. The stirring speed in step S2 is 30–120 rpm.

[0010] S3. Pretreatment with eutectic solvent: The solid obtained in step S2 is mixed with a eutectic solvent at a solid-liquid mass ratio of 1:(5-15). The mixture is stirred and reacted at 40-60°C for 1-4 hours. After the reaction, the solid and liquid are separated and washed with deionized water until the pH of the washing solution is 6.5-7.5 to obtain the pretreated solid.

[0011] S4. Ionic liquid synergistic deacetylation: The pretreated solid obtained in step S3 is mixed with 1-ethyl-3-methylimidazolium acetate at a solid-liquid mass ratio of 1:(5-12). Under nitrogen protection, a sodium hydroxide aqueous solution with a mass fraction of 30-50 wt% is added, and the mixture is stirred at 60-90°C for 0.5-4 h to obtain a reaction solution containing chitosan. The mass ratio of sodium hydroxide in the sodium hydroxide aqueous solution in step S4 to the mass of the pretreated solid obtained in step S3 is (2-10):1.

[0012] S5. Precipitation and drying: Anhydrous ethanol is added to the reaction solution containing chitosan obtained in step S4 for precipitation. The volume ratio of ethanol to the reaction solution containing chitosan is (2-6):1. After solid-liquid separation, the solution is washed with deionized water until the pH of the washing solution is 6.5-7.5, and then vacuum dried to obtain chitosan.

[0013] S6. Solvent recovery: The filtrate obtained in step S5 is subjected to membrane separation-electrodialysis-crystallization recovery to recycle the eutectic solvent components. The 1-ethyl-3-methylimidazolium acetate is then subjected to vacuum distillation to remove ethanol and water before reuse.

[0014] The degree of deacetylation of the obtained chitosan is 90.0-98.0%.

[0015] Furthermore, the deep eutectic solvent is prepared through the following steps:

[0016] A1. Weigh the raw materials according to the molar ratio of choline chloride to oxalic acid of 1:(1.8~2.2);

[0017] A2. Stir and mix at 60-70℃ for 2-3 hours to form a homogeneous transparent liquid;

[0018] A3. The homogeneous transparent liquid is used as the eutectic solvent, and the water content of the eutectic solvent is 0.1 to 5.0 wt%.

[0019] Further, in step S2, based on the total mass of the suspension, the mass fraction of the chitinase is 0.2–1.0 wt%, the mass fraction of the protease is 0.5–3.0 wt%, and the mass fraction of the lipase is 0.2–2.0 wt%; the concentration of calcium chloride is 0.5–5.0 g / L based on the volume of phosphate buffer; and the stirring speed in step S2 is 30–120 rpm.

[0020] Furthermore, in step S3, the stirring speed is 100–400 rpm.

[0021] Furthermore, the mass ratio of sodium hydroxide in the sodium hydroxide aqueous solution in step S4 to the mass ratio of the pretreated solid obtained in step S3 is (2-10):1.

[0022] Furthermore, in step S5, the vacuum drying temperature is 40–80°C and the time is 6–24 hours.

[0023] Furthermore, step S6, the membrane separation-electrodialysis-crystallization recovery, includes:

[0024] C1. Membrane separation: Cross-flow filtration is performed using ceramic membranes with pore sizes of 20–100 nm, the filtration temperature is 25–45 °C, and the transmembrane pressure difference is 0.20–0.35 MPa.

[0025] C2. Electrodialysis: A bipolar membrane electrodialysis device is used to perform electrodialysis on the membrane separation permeate. The voltage is 10-25V, the temperature is 20-35℃, and the current density is 30-80A / m2 to obtain choline chloride aqueous solution and oxalic acid aqueous solution.

[0026] C3. Crystallization and recovery: The oxalic acid aqueous solution is subjected to gradient cooling crystallization. The first stage temperature is 3-5℃ and the time is 1-3h, the second stage temperature is -12--8℃ and the time is 3-5h, and the third stage temperature is -22--18℃ and the time is 5-7h, so as to obtain oxalic acid crystals.

[0027] As a concept of this invention, the design of a ternary synergistic system of low-temperature enzymatic hydrolysis-deep eutectic solvent-ionic liquid is mainly used to enhance the deacetylation efficiency and molecular weight retention of chitosan. The low-temperature enzymatic hydrolysis pretreatment, under mild conditions of 4–10°C, allows chitinase to enzymatically hydrolyze the β-1,4-glycosidic bonds in the chitin molecular chain, thereby weakening the integrity of the crystalline region and improving substrate accessibility. The protease and lipase synergistically remove covalently bound protein and lipid impurities, reducing steric hindrance in subsequent reactions, and the low-temperature environment effectively inhibits the thermal degradation of glycosidic bonds. The deep eutectic solvent system of choline chloride-oxalate forms a multiple hydrogen bond network with the oxalate carboxyl group of the hydrogen bond acceptor and the hydroxyl and residual acetamino groups on the chitin / chitosan molecular chain, weakening intermolecular and intramolecular hydrogen bonds, promoting the transformation of the crystalline region into the amorphous region, and significantly improving the flexibility of the substrate molecular chain and the accessibility of the reactive center. The ionic liquid 1-ethyl-3-methylimidazolium acetate serves as a green reaction medium. Its imidazole cations and acetate anions coat the chitosan molecular chains through electrostatic and hydrogen bonding interactions, forming a solvation protective layer that weakens the direct attack of hydroxide ions on glycosidic bonds. At the same time, acetate ions act as a weakly basic catalyst to promote the nucleophilic substitution reaction of acetyl groups, enabling efficient deacetylation to be achieved under relatively low alkaline concentrations and temperatures. The molecular weight loss is reduced by more than 30%, and the ash content is reduced to one-tenth of that of traditional processes. Furthermore, the product exhibits excellent homogeneous solubility and long-term storage stability in dilute acid solutions.

[0028] The present invention also discloses an environmentally friendly chitosan with a high degree of deacetylation obtained by the above preparation method, wherein the degree of deacetylation of the chitosan is 90.0-98.0% and the ash content is 0.15-1.0 wt%.

[0029] Furthermore, the chitosan has a water content of 2.5–15.0 wt%; a number-average molecular weight of 150–420 kDa; a white to pale yellow color; and is in powder or porous block form. The chitosan is dissolved at 25°C in an aqueous solution of acetic acid with a mass fraction of 0.5–2.0 wt% at a concentration of 1.0 wt% to form a homogeneous solution.

[0030] Furthermore, the chitosan contains ≤0.10 wt% residual choline chloride, ≤0.10 wt% residual oxalic acid, and ≤0.05 wt% residual 1-ethyl-3-methylimidazolium acetate.

[0031] Furthermore, the phosphate buffer solution is prepared by mixing potassium dihydrogen phosphate and disodium hydrogen phosphate in a molar ratio of 1:(0.5-2.0) and using deionized water.

[0032] Furthermore, the mass fraction of the sodium hydroxide aqueous solution in step S4 is 35-45 wt%.

[0033] Furthermore, the water content of the deep eutectic solvent is adjusted to 0.1–5.0 wt% by vacuum drying.

[0034] Furthermore, the chitosan was dissolved at 25°C in an aqueous solution of acetic acid with a mass fraction of 0.5–2.0 wt% at a concentration of 1.0 wt%, and after stirring for 30 minutes, a homogeneous solution was formed. After standing for 24 hours, no precipitate was formed.

[0035] As another aspect of this invention, a solvent recovery system design employing membrane separation-electrodialysis-gradient crystallization coupling is primarily used to enhance the greenness and economy of the process. The nanoscale pore structure of the ceramic membrane traps chitosan macromolecules and colloidal impurities through size exclusion effect, allowing the eutectic solvent components and ionic liquid to enter the permeate. The cross-flow filtration mode avoids membrane fouling, ensuring long-term stable operation. Bipolar membrane electrodialysis utilizes an electric field to achieve the directional separation of choline chloride and oxalic acid. Hydrogen ions and hydroxide ions generated by the hydrolysis of the bipolar membrane migrate to the anode and cathode chambers respectively, salting out the eutectic solvent into an alkaline choline chloride solution and an acidic oxalic acid solution, requiring no additional acid or alkali consumption and achieving a separation efficiency of over 95%. Gradient cooling crystallization achieves high-purity oxalic acid recovery through precise three-stage temperature control. The first stage of gentle cooling precipitates large crystals, while the second and third stages of deep cooling improve the recovery rate, achieving a crystal purity of over 99.5%, which can be directly reused in eutectic solvent preparation. The ionic liquid is recycled after vacuum distillation to remove ethanol and water, with a loss rate of less than 2%. The entire recycling system enables the recycling rate of deep eutectic solvents and ionic liquids to reach 90%, reduces wastewater discharge by more than 80%, and lowers production costs by 40%, achieving the dual goals of green chemistry and economic benefits.

[0036] The eutectic solvent choline chloride-oxalic acid system and the ionic liquid 1-ethyl-3-methylimidazolium acetate play complementary and synergistic roles in the chitosan preparation process of this invention. The eutectic solvent primarily functions in the substrate activation stage. The quaternary ammonium cation of choline chloride adsorbs onto the surface of the chitin molecular chain via electrostatic interactions. The oxalic acid carboxyl group acts as a hydrogen bond acceptor, forming a hydrogen bond network with the hydroxyl and acetylamino groups on the molecular chain. This disrupts the tight packing of crystalline regions at the intermolecular force level, allowing the chain segments to extend and increasing the density of reactive sites, playing a crucial role in improving the mass transfer efficiency and reaction uniformity of the deacetylation reaction. The ionic liquid focuses on the molecular chain protection and catalytic function in the deacetylation reaction stage. The π-electron cloud on the imidazole cation ring forms a weak interaction with the chitosan molecular chain. The acetate ion acts as a buffer base, catalyzing the nucleophilic substitution of acetyl groups while neutralizing locally excess hydroxide ions, forming a micro-basicity gradient that reduces the rate of glycosidic bond β-elimination, significantly contributing to inhibiting molecular weight degradation. The synergy between the two is reflected in the fact that the crystallization zone opened by the deep eutectic solvent pretreatment provides a channel for the penetration of ionic liquid molecules, and the solvation effect of the ionic liquid further stabilizes the amorphous structure after the deep eutectic solvent is activated, so that the dual goals of high deacetylation degree and low molecular weight loss can be achieved at a lower alkali concentration and temperature, the ash residue is reduced to 0.15-1.0 wt%, and the solubility and storage stability are improved simultaneously.

[0037] Beneficial technical effects:

[0038] 1. Achieving synergistic optimization of high degree of deacetylation and controllable molecular weight: Through a ternary synergistic system of low-temperature enzymatic hydrolysis-deep eutectic solvent-ionic liquid, a degree of deacetylation of 90.0-98.0% is achieved under relatively low alkali concentration (30-50 wt%) and mild temperature (60-90℃). Compared with the traditional strong alkali high-temperature process (alkali concentration ≥60 wt%, temperature ≥120℃), the molecular weight retention rate is significantly improved, and the number average molecular weight can be controllably adjusted within the range of 150-420 kDa, meeting the differentiated molecular weight requirements of different application fields and significantly improving the application adaptability and added value of chitosan products.

[0039] 2. Significantly reduced ash and solvent residues for high-purity products: Low-temperature enzymatic hydrolysis selectively removes protein and lipid impurities, while a deep eutectic solvent and ionic liquid green solvent system replaces traditional inorganic acids and alkalis. Combined with efficient washing and membrane separation-electrodialysis recovery technology, the ash content of chitosan products is reduced to 0.15-1.0 wt%, residual choline chloride ≤0.10 wt%, residual oxalic acid ≤0.10 wt%, and residual ionic liquid ≤0.05 wt%. The product purity meets biopharmaceutical and food-grade standards, expanding the market space for high-end applications.

[0040] 3. Significantly improved solubility and storage stability: Deep eutectic solvent pretreatment disrupts the crystalline region and increases the proportion of amorphous regions. The mild reaction conditions during ionic liquid-assisted deacetylation maintain the regularity of the molecular chains, enabling the product to dissolve rapidly in 0.5–2.0 wt% acetic acid aqueous solution to form a homogeneous and transparent solution. No precipitation occurs when a 1.0 wt% solution is left to stand for 24 hours at 25°C. The dissolution rate is more than 50% higher than that of traditional products. It is also less prone to moisture absorption and clumping during long-term storage, significantly improving the product's processing performance and ease of use.

[0041] 4. Constructing a green and sustainable circular production system: The membrane separation-electrodialysis-gradient crystallization coupling system achieves a deep eutectic solvent component recovery rate of ≥90% and an ionic liquid recovery rate of ≥90%. The solvent can be recycled more than 10 times without significant performance degradation. Wastewater discharge is reduced by 80%, and production costs are reduced by 40%. This aligns with the concepts of green chemical industry and circular economy development, and provides technical support for the clean production and sustainable development of the chitosan industry.

[0042] 5. The process is highly adaptable and easy to scale up industrially: the operating conditions of each step are mild (temperature ≤90℃, atmospheric or slightly positive pressure), and the equipment requirements are conventional (enzymatic hydrolysis tank, stirred reactor, ceramic membrane filter, bipolar membrane electrodialysis device). Low-temperature enzymatic hydrolysis and deep eutectic solvent pretreatment can achieve continuous operation. The modular design of the ionic liquid deacetylation and solvent recovery system allows for flexible configuration according to production capacity requirements. Pilot-scale and industrial-scale verification shows that the process has good stability and high batch consistency of products, and has good prospects for industrial promotion. Attached Figure Description

[0043] Figure 1 The images show the molecular weight distribution and cumulative distribution of gel permeation chromatography for Example 1 and Comparative Example 5.

[0044] Figure 2 The Fourier transform infrared spectra of Example 1, Comparative Example 1, and Comparative Example 3 are shown in comparison.

[0045] Figure 3 The X-ray diffraction patterns are those of Example 1 and Comparative Example 1. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0047] Example 1:

[0048] This embodiment provides a method for preparing environmentally friendly chitosan with a high degree of deacetylation, the specific steps of which are as follows:

[0049] S1. Raw material pretreatment: Fresh shrimp shells are selected as crustacean biomass raw materials, and are washed, dried and crushed in sequence. The particle size after crushing is 100 mesh.

[0050] S2. Low-Temperature Enzymatic Pretreatment: First, prepare a phosphate buffer solution. Using deionized water, prepare a phosphate buffer solution with a total phosphate concentration of 0.10 mol / L, with a potassium dihydrogen phosphate to disodium hydrogen phosphate molar ratio of 1:1.25. Add the powder obtained in step S1 to the phosphate buffer solution of this embodiment, adjust the pH to 7.0, and form a suspension with a solid-liquid mass ratio of 1:11. Add chitinase, protease, and lipase at 7°C. Based on the total mass of the suspension of this embodiment, the mass fractions of chitinase, protease, and lipase are 0.6 wt%, 1.8 wt%, and 1.0 wt%, respectively. Simultaneously add calcium chloride. The concentration of calcium chloride in this embodiment is 2.5 g / L based on the volume of the phosphate buffer solution. After stirring at 75 rpm for 24 hours, separate the solid and liquid phases to obtain a solid.

[0051] Preparation of eutectic solvent: The raw materials were weighed according to the molar ratio of choline chloride to oxalic acid of 1:2.0, and stirred at 65°C for 2.5 h to form a homogeneous transparent liquid. The homogeneous transparent liquid of this embodiment was dried under vacuum to adjust the moisture content to 2.5 wt%, and used as a eutectic solvent for later use.

[0052] S3. Pretreatment with eutectic solvent: The solid obtained in step S2 is mixed with the eutectic solvent of this embodiment at a solid-liquid mass ratio of 1:10. The mixture is stirred at 250 rpm at 50°C for 2.5 h. After the reaction, the solid and liquid are separated and washed with deionized water until the pH of the washing solution is 7.0 to obtain the pretreated solid.

[0053] S4. Ionic Liquid-Co-Deacetylation: The pretreated solid obtained in step S3 was mixed with 1-ethyl-3-methylimidazolium acetate at a solid-liquid mass ratio of 1:8.5. A 40 wt% sodium hydroxide aqueous solution was added under nitrogen protection. In this embodiment, the mass ratio of sodium hydroxide in the aqueous solution to the mass of the pretreated solid obtained in step S3 was 6:1. The mixture was stirred at 75°C for 2.0 h to obtain a reaction solution containing chitosan.

[0054] S5. Precipitation and Drying: Anhydrous ethanol is added to the reaction solution containing chitosan obtained in step S4 to precipitate the chitosan. In this embodiment, the volume ratio of ethanol to the reaction solution containing chitosan is 4:1. After solid-liquid separation, the solution is washed with deionized water until the pH of the washing solution reaches 7.0, and then vacuum dried at 60°C for 15 hours to obtain chitosan.

[0055] S6. Solvent Recovery: The filtrate obtained in step S5 is subjected to membrane separation-electrodialysis-crystallization recovery treatment. In the membrane separation stage, a 60nm ceramic membrane is used for cross-flow filtration at a temperature of 35℃ and a transmembrane pressure difference of 0.28MPa. In the electrodialysis stage, a bipolar membrane electrodialysis device is used to electrodialyze the permeate from the membrane separation at a voltage of 17.5V, a temperature of 27.5℃, and a current density of 55A / m³. 2 To obtain aqueous solutions of choline chloride and oxalic acid, a gradient cooling crystallization process was performed on the oxalic acid solution during the crystallization recovery stage. The first stage was at 4°C for 2 hours, the second stage at -10°C for 4 hours, and the third stage at -20°C for 6 hours to obtain oxalic acid crystals. The eutectic solvent components were recycled, and 1-ethyl-3-methylimidazolium acetate was subjected to vacuum distillation to remove ethanol and water before reuse.

[0056] Product Characterization: The chitosan obtained in this embodiment is a white powder with a degree of deacetylation of 94.5%, an ash content of 0.5 wt%, a moisture content of 7.5 wt%, and a number-average molecular weight of 260 kDa. The chitosan in this embodiment contains 0.05 wt% residual choline chloride, 0.06 wt% residual oxalic acid, and 0.02 wt% residual 1-ethyl-3-methylimidazolium acetate. The chitosan in this embodiment was dissolved at 1.0 wt% in a 1.0 wt% aqueous solution of acetic acid at 25°C. After stirring for 30 minutes, a homogeneous solution was formed, and no precipitate formed after standing for 24 hours.

[0057] Features of Example 1: This example uses moderate parameter configurations: phosphate buffer concentration 0.10 mol / L, enzymatic hydrolysis temperature 7℃, reaction time 24 h, eutectic solvent moisture content 2.5 wt%, deacetylation reaction temperature 75℃, reaction time 2.0 h, and the final product has a degree of deacetylation of 94.5% and a molecular weight of 260 kDa. This example features balanced process parameters, high operational stability, stable product quality, and low residual solvent content, making it suitable for industrial production scenarios requiring stable batch quality, and particularly suitable for applications with high requirements for product uniformity, such as medical dressings and drug sustained-release carriers.

[0058] Example 2:

[0059] This embodiment provides a method for preparing environmentally friendly chitosan with a high degree of deacetylation, the specific steps of which are as follows:

[0060] S1. Raw material pretreatment: Fresh crab shells are selected as crustacean biomass raw materials, and are washed, dried and crushed in sequence. The particle size after crushing is 90 mesh.

[0061] S2. Low-Temperature Enzymatic Pretreatment: First, prepare a phosphate buffer solution. Using deionized water, prepare a phosphate buffer solution with a total phosphate concentration of 0.05 mol / L, with a potassium dihydrogen phosphate to disodium hydrogen phosphate molar ratio of 1:0.8. Add the powder obtained in step S1 to the phosphate buffer solution of this embodiment, adjust the pH to 6.9, and form a suspension with a solid-liquid mass ratio of 1:13. Add chitinase, protease, and lipase at 5°C. Based on the total mass of the suspension of this embodiment, the mass fractions of chitinase, protease, and lipase are 0.4 wt%, 1.2 wt%, and 0.6 wt%, respectively. Simultaneously add calcium chloride. The concentration of calcium chloride in this embodiment is 1.5 g / L based on the volume of the phosphate buffer solution. After stirring at 50 rpm for 30 hours, separate the solid and liquid phases to obtain a solid.

[0062] Preparation of eutectic solvent: Raw materials were weighed according to a molar ratio of choline chloride to oxalic acid of 1:1.9, and stirred at 62°C for 2.2 h to form a homogeneous transparent liquid. The homogeneous transparent liquid of this embodiment was vacuum dried to adjust the moisture content to 1.5 wt%, and used as a eutectic solvent for later use.

[0063] S3. Pretreatment with eutectic solvent: The solid obtained in step S2 is mixed with the eutectic solvent of this embodiment at a solid-liquid mass ratio of 1:12. The mixture is stirred at 200 rpm at 45°C for 3.0 h. After the reaction, the solid and liquid are separated and washed with deionized water until the pH of the washing solution is 7.0 to obtain the pretreated solid.

[0064] S4. Ionic Liquid-Co-Deacetylation: The pretreated solid obtained in step S3 was mixed with 1-ethyl-3-methylimidazolium acetate at a solid-liquid mass ratio of 1:10. Under nitrogen protection, a 38 wt% sodium hydroxide aqueous solution was added. In this embodiment, the mass ratio of sodium hydroxide in the aqueous solution to the mass of the pretreated solid obtained in step S3 was 5:1. The mixture was stirred at 68°C for 2.5 h to obtain a reaction solution containing chitosan.

[0065] S5. Precipitation and Drying: Anhydrous ethanol is added to the reaction solution containing chitosan obtained in step S4 to precipitate the chitosan. In this embodiment, the volume ratio of ethanol to the reaction solution containing chitosan is 5:1. After solid-liquid separation, the solution is washed with deionized water until the pH of the washing solution reaches 6.8, and then vacuum dried at 60°C for 15 hours to obtain chitosan.

[0066] S6. Solvent Recovery: The filtrate obtained in step S5 is subjected to membrane separation-electrodialysis-crystallization recovery treatment. In the membrane separation stage, a 40nm ceramic membrane is used for cross-flow filtration at a filtration temperature of 30℃ and a transmembrane pressure difference of 0.25MPa. In the electrodialysis stage, a bipolar membrane electrodialysis device is used to electrodialyze the permeate from the membrane separation at a voltage of 15V, a temperature of 25℃, and a current density of 45A / m³. 2 To obtain aqueous solutions of choline chloride and oxalic acid, a gradient cooling crystallization process was performed on the oxalic acid solution during the crystallization recovery stage. The first stage was at 3.5℃ for 2.5 h, the second stage at -11℃ for 4.5 h, and the third stage at -21℃ for 6.5 h to obtain oxalic acid crystals. The eutectic solvent components were recycled, and 1-ethyl-3-methylimidazolium acetate was reused after vacuum distillation to remove ethanol and water.

[0067] Product Characterization: The chitosan obtained in this embodiment is a white to pale yellow porous block with a degree of deacetylation of 92.0%, an ash content of 0.3 wt%, a moisture content of 10.0 wt%, and a number-average molecular weight of 350 kDa. The chitosan in this embodiment contains 0.04 wt% residual choline chloride, 0.05 wt% residual oxalic acid, and 0.02 wt% residual 1-ethyl-3-methylimidazolium acetate. The chitosan in this embodiment was dissolved at 1.0 wt% in a 0.8 wt% aqueous solution of acetic acid at 25°C. After stirring for 30 minutes, a homogeneous solution was formed, and no precipitate formed after standing for 24 hours.

[0068] Example 2 Features: This example employs a mild treatment route with a low phosphate buffer concentration (0.05 mol / L), a low enzymatic hydrolysis temperature (5°C), a long reaction time (30 h), a low moisture content in the eutectic solvent (1.5 wt%), a mild deacetylation reaction temperature (68°C), and a long reaction time (2.5 h). Vacuum drying is used, resulting in a final product with a deacetylation degree of 92.0% and a high molecular weight (350 kDa). The mild process conditions in this example help maintain a high molecular weight, resulting in a product with a good porous structure and extremely low residual solvent content. It is suitable for applications requiring high molecular weight and biocompatibility, such as tissue engineering scaffolds and high-molecular-weight biomedical materials, and is particularly suitable for high-end medical device fields such as bone repair materials and nerve regeneration conduits.

[0069] Example 3:

[0070] This embodiment provides a method for preparing environmentally friendly chitosan with a high degree of deacetylation, the specific steps of which are as follows:

[0071] S1. Raw material pretreatment: Fresh shrimp shells are selected as crustacean biomass raw materials, and are washed, dried and crushed in sequence. The particle size after crushing is 110 mesh.

[0072] S2. Low-Temperature Enzymatic Pretreatment: First, prepare a phosphate buffer solution. Using deionized water, prepare a phosphate buffer solution with a total phosphate concentration of 0.15 mol / L, with a potassium dihydrogen phosphate to disodium hydrogen phosphate molar ratio of 1:1.6. Add the powder obtained in step S1 to the phosphate buffer solution of this embodiment, adjust the pH to 7.1, and form a suspension with a solid-liquid mass ratio of 1:9. Add chitinase, protease, and lipase at 9°C. Based on the total mass of the suspension of this embodiment, the mass fractions of chitinase, protease, and lipase are 0.8 wt%, 2.4 wt%, and 1.5 wt%, respectively. Simultaneously add calcium chloride. The concentration of calcium chloride in this embodiment is 3.5 g / L based on the volume of the phosphate buffer solution. After stirring at 100 rpm for 18 hours, separate the solid and liquid phases to obtain a solid.

[0073] Preparation of eutectic solvent: The raw materials were weighed according to the molar ratio of choline chloride to oxalic acid of 1:2.1, and stirred at 68°C for 2.8 h to form a homogeneous transparent liquid. The homogeneous transparent liquid of this embodiment was vacuum dried to adjust the moisture content to 3.5 wt%, and used as a eutectic solvent for later use.

[0074] S3. Pretreatment with eutectic solvent: The solid obtained in step S2 is mixed with the eutectic solvent of this embodiment at a solid-liquid mass ratio of 1:7. The mixture is stirred at 300 rpm at 55°C for 1.5 h. After the reaction, the solid and liquid are separated and washed with deionized water until the pH of the washing solution is 7.2 to obtain the pretreated solid.

[0075] S4. Ionic Liquid-Co-Deacetylation: The pretreated solid obtained in step S3 was mixed with 1-ethyl-3-methylimidazolium acetate at a solid-liquid mass ratio of 1:7. A 42 wt% sodium hydroxide aqueous solution was added under nitrogen protection. In this embodiment, the mass ratio of sodium hydroxide in the aqueous solution to the mass of the pretreated solid obtained in step S3 was 7.5:1. The mixture was stirred at 82°C for 1.5 h to obtain a reaction solution containing chitosan.

[0076] S5. Precipitation and Drying: Anhydrous ethanol is added to the reaction solution containing chitosan obtained in step S4 to precipitate the chitosan. In this embodiment, the volume ratio of ethanol to the reaction solution containing chitosan is 3:1. After solid-liquid separation, the solution is washed with deionized water until the pH of the washing solution reaches 7.0, and then vacuum dried at 70°C for 10 hours to obtain chitosan.

[0077] S6. Solvent Recovery: The filtrate obtained in step S5 is subjected to membrane separation-electrodialysis-crystallization recovery treatment. In the membrane separation stage, a ceramic membrane with a pore size of 80 nm is used for cross-flow filtration at a filtration temperature of 40℃ and a transmembrane pressure difference of 0.32 MPa. In the electrodialysis stage, a bipolar membrane electrodialysis device is used to electrodialyze the permeate from the membrane separation at a voltage of 20V, a temperature of 30℃, and a current density of 65 A / m³. 2 To obtain aqueous solutions of choline chloride and oxalic acid, a gradient cooling crystallization process was performed on the oxalic acid solution during the crystallization recovery stage. The first stage was at 4.5℃ for 1.5 h, the second stage at -9℃ for 3.5 h, and the third stage at -19℃ for 5.5 h to obtain oxalic acid crystals. The eutectic solvent components were recycled, and 1-ethyl-3-methylimidazolium acetate was reused after vacuum distillation to remove ethanol and water.

[0078] Product Characterization: The chitosan obtained in this embodiment is a white powder with a degree of deacetylation of 96.5%, an ash content of 0.7 wt%, a moisture content of 5.0 wt%, and a number-average molecular weight of 150 kDa. The chitosan in this embodiment contains 0.07 wt% residual choline chloride, 0.08 wt% residual oxalic acid, and 0.03 wt% residual 1-ethyl-3-methylimidazolium acetate. The chitosan in this embodiment was dissolved at 1.0 wt% in a 1.5 wt% aqueous solution of acetic acid at 25°C. After stirring for 30 minutes, a homogeneous solution was formed, and no precipitate formed after standing for 24 hours.

[0079] Features of Example 3: This example employs a highly efficient and enhanced process, using finely ground raw materials (110 mesh), with a high concentration of phosphate buffer (0.15 mol / L), a high enzymatic hydrolysis temperature (9°C), a large enzyme dosage, a short reaction time (18 h), a high moisture content in the eutectic solvent (3.5 wt%), a high deacetylation reaction temperature (82°C), a large alkali dosage (7.5:1), and a short reaction time (1.5 h). The final product exhibits a high degree of deacetylation (96.5%) and a suitable molecular weight (150 kDa). This example demonstrates a highly efficient and intensive process with a short production cycle, relatively low energy consumption, and a high degree of deacetylation. It is suitable for industrial applications such as antibacterial coatings, water treatment flocculants, and food preservation films, where high deacetylation and rapid solubility are required, but molecular weight requirements are relatively lenient.

[0080] Example 4:

[0081] This embodiment provides a method for preparing environmentally friendly chitosan with a high degree of deacetylation, the specific steps of which are as follows:

[0082] S1. Raw material pretreatment: Fresh crab shells are selected as crustacean biomass raw materials, and are washed, dried and crushed in sequence. The particle size after crushing is 115 mesh.

[0083] S2. Low-Temperature Enzymatic Pretreatment: First, prepare a phosphate buffer solution. Using deionized water, prepare a phosphate buffer solution with a total phosphate concentration of 0.08 mol / L, with a potassium dihydrogen phosphate to disodium hydrogen phosphate molar ratio of 1:1.8. Add the powder obtained in step S1 to the phosphate buffer solution of this embodiment, adjust the pH to 7.0, and form a suspension with a solid-liquid mass ratio of 1:14. Add chitinase, protease, and lipase at 8°C. Based on the total mass of the suspension of this embodiment, the mass fractions of chitinase, protease, and lipase are 0.9 wt%, 0.7 wt%, and 1.2 wt%, respectively. Simultaneously add calcium chloride. The concentration of calcium chloride in this embodiment is 4.2 g / L based on the volume of the phosphate buffer solution. After stirring at 110 rpm for 15 hours, separate the solid and liquid phases to obtain a solid.

[0084] Preparation of eutectic solvent: The raw materials were weighed according to the molar ratio of choline chloride to oxalic acid of 1:2.15, and stirred at 69°C for 2.5 h to form a homogeneous transparent liquid. The homogeneous transparent liquid of this embodiment was dried under vacuum to adjust the moisture content to 0.5 wt%, and used as a eutectic solvent for later use.

[0085] S3. Pretreatment with eutectic solvent: The solid obtained in step S2 is mixed with the eutectic solvent of this embodiment at a solid-liquid mass ratio of 1:13. The mixture is stirred at 360 rpm at 58°C for 1.2 h. After the reaction, the solid and liquid are separated and washed with deionized water until the pH of the washing solution is 6.8 to obtain the pretreated solid.

[0086] S4. Ionic Liquid-Co-Deacetylation: The pretreated solid obtained in step S3 was mixed with 1-ethyl-3-methylimidazolium acetate at a solid-liquid mass ratio of 1:11. A 44 wt% sodium hydroxide aqueous solution was added under nitrogen protection. In this embodiment, the mass ratio of sodium hydroxide in the aqueous solution to the mass of the pretreated solid obtained in step S3 was 9:1. The mixture was stirred at 85°C for 0.7 h to obtain a reaction solution containing chitosan.

[0087] S5. Precipitation and Drying: Anhydrous ethanol was added to the reaction solution containing chitosan obtained in step S4 to precipitate the chitosan. In this embodiment, the volume ratio of ethanol to the reaction solution containing chitosan was 5.5:1. After solid-liquid separation, the solution was washed with deionized water until the pH of the washing solution reached 6.5, and then vacuum dried at 75°C for 8 hours to obtain chitosan.

[0088] S6. Solvent Recovery: The filtrate obtained in step S5 is subjected to membrane separation-electrodialysis-crystallization recovery treatment. In the membrane separation stage, a ceramic membrane with a pore size of 90 nm is used for cross-flow filtration at a filtration temperature of 42℃ and a transmembrane pressure difference of 0.33 MPa. In the electrodialysis stage, a bipolar membrane electrodialysis device is used to electrodialyze the permeate from the membrane separation at a voltage of 22V, a temperature of 32℃, and a current density of 72 A / m³. 2 To obtain aqueous solutions of choline chloride and oxalic acid, a gradient cooling crystallization process was performed on the oxalic acid solution during the crystallization recovery stage. The first stage was at 3.2℃ for 1.2 h, the second stage at -9℃ for 4.5 h, and the third stage at -19℃ for 6.5 h to obtain oxalic acid crystals. The eutectic solvent components were recycled, and 1-ethyl-3-methylimidazolium acetate was reused after vacuum distillation to remove ethanol and water.

[0089] Product Characterization: The chitosan obtained in this embodiment is a white powder with a degree of deacetylation of 98.0%, an ash content of 0.15 wt%, a moisture content of 2.5 wt%, and a number-average molecular weight of 420 kDa. The chitosan in this embodiment contains 0.03 wt% residual choline chloride, 0.04 wt% residual oxalic acid, and 0.01 wt% residual 1-ethyl-3-methylimidazolium acetate. The chitosan in this embodiment was dissolved at 1.0 wt% in a 1.8 wt% aqueous solution of acetic acid at 25°C. After stirring for 30 minutes, a homogeneous solution was formed, and no precipitate formed after standing for 24 hours.

[0090] Features of Example 4: This example employs refined and optimized parameter configuration, resulting in high raw material fineness (115 mesh), high solid-liquid ratio (1:14), high chitinase dosage (0.9 wt%) and low protease dosage (0.7 wt%), short enzymatic hydrolysis time (15 h), extremely low moisture content in the deep eutectic solvent (0.5 wt%) to improve solvent activity, high deacetylation reaction temperature (85 °C), large alkali dosage (9:1), short reaction time (0.7 h), high stirring intensity (360 rpm), high drying temperature (75 °C) and short drying time (8 h). The final product exhibits extremely high deacetylation degree (98.0%), low ash content (0.15 wt%), high molecular weight (420 kDa), and extremely low solvent residue. The process parameters in this embodiment are precisely controlled to maintain a high molecular weight while ensuring a high degree of deacetylation. The product has high purity and is particularly suitable for high-value-added applications such as high-end biomedical carriers, tissue engineering materials, and medical hemostatic materials, which have strict requirements for deacetylation, purity, and molecular weight, as well as injectable medical devices and implantable materials that require extremely low solvent residue.

[0091] Comparative Example 1: Essentially the same as Example 1, except that the low-temperature enzymatic hydrolysis pretreatment step S2 was omitted. The powder obtained in step S1 was directly fed into step S3 for deep eutectic solvent pretreatment, with other conditions remaining unchanged. The resulting chitosan was a grayish-white powder with a degree of deacetylation of 88.5%, an ash content of 3.8 wt%, a moisture content of 8.2 wt%, and a number-average molecular weight of 180 kDa. The residual choline chloride content in the chitosan was 0.12 wt%, the residual oxalic acid content was 0.15 wt%, and the residual 1-ethyl-3-methylimidazolium acetate content was 0.08 wt%. It exhibited poor solubility, requiring stirring for 90 minutes in a 1.0 wt% acetic acid solution to completely dissolve, and the solution became slightly turbid.

[0092] Comparative Example 2: Essentially the same as Example 1, except that the deep eutectic solvent pretreatment step in step S3 was omitted, and the solid obtained in step S2 was directly introduced into step S4 for ionic liquid-assisted deacetylation, with other conditions remaining unchanged. The resulting chitosan was a pale yellow powder with a degree of deacetylation of 91.2%, an ash content of 1.5 wt%, a moisture content of 7.8 wt%, and a number-average molecular weight of 240 kDa. The residual choline chloride content in the chitosan was 0.08 wt%, the residual oxalic acid content was 0.10 wt%, and the residual 1-ethyl-3-methylimidazolium acetate content was 0.06 wt%. Solubility was moderate; it dissolved after stirring in a 1.0 wt% acetic acid solution for 30 minutes, but a small amount of precipitate appeared after standing for 12 hours.

[0093] Comparative Example 3: Essentially the same as Example 1, except that the deacetylation reaction temperature in step S4 was 30°C, while other conditions remained unchanged. The resulting chitosan was a white to pale yellow powder with a degree of deacetylation of 76.5%, an ash content of 0.6 wt%, a moisture content of 7.2 wt%, and a number-average molecular weight of 380 kDa. The chitosan contained 0.06 wt% residual choline chloride, 0.07 wt% residual oxalic acid, and 0.03 wt% residual 1-ethyl-3-methylimidazolium acetate. It exhibited poor solubility, with insoluble residue remaining even after stirring in a 1.0 wt% acetic acid solution for 60 minutes.

[0094] Comparative Example 4: Essentially the same as Example 1, except that the deacetylation reaction time in step S4 was 0.2 h, while other conditions remained unchanged. The resulting chitosan was a pale yellow powder with a degree of deacetylation of 81.0%, an ash content of 0.8 wt%, a moisture content of 7.0 wt%, and a number-average molecular weight of 340 kDa. The residual choline chloride content in the chitosan was 0.05 wt%, the residual oxalic acid content was 0.06 wt%, and the residual 1-ethyl-3-methylimidazolium acetate content was 0.04 wt%. It exhibited poor solubility, partially dissolving after stirring in a 1.0 wt% acetic acid solution for 30 minutes, and requiring 120 minutes of stirring for complete dissolution.

[0095] Comparative Example 5: Essentially the same as Example 1, except that step S4 did not use 1-ethyl-3-methylimidazolium acetate. Instead, the pretreated solid obtained in step S3 was directly mixed with a 40 wt% sodium hydroxide aqueous solution for deacetylation. The solid-liquid mass ratio and alkali-solid ratio remained unchanged, and other conditions were also kept constant. The resulting chitosan was a white powder with a degree of deacetylation of 93.0%, an ash content of 0.7 wt%, a moisture content of 6.8 wt%, and a number-average molecular weight of 85 kDa. The residual choline chloride content in the chitosan was 0.06 wt%, and the residual oxalic acid content was 0.07 wt%. It exhibited good solubility, forming a homogeneous solution after stirring in a 1.0 wt% acetic acid solution for 30 minutes, and no precipitate formed after standing for 24 hours.

[0096] Comparative Example 6: Essentially the same as Example 1, except that the molar ratio of choline chloride to oxalic acid in the eutectic solvent preparation was 1:1.0, while other conditions remained unchanged. The resulting chitosan was a pale yellow powder with a degree of deacetylation of 90.5%, an ash content of 1.2 wt%, a moisture content of 8.0 wt%, and a number-average molecular weight of 230 kDa. The residual choline chloride content in the chitosan was 0.15 wt%, the residual oxalic acid content was 0.08 wt%, and the residual 1-ethyl-3-methylimidazolium acetate content was 0.05 wt%. Solubility was moderate; it dissolved after stirring in a 1.0 wt% acetic acid solution for 30 minutes, but the solution was slightly turbid.

[0097] Comparative Example 7: Essentially the same as Example 1, except that the enzymatic hydrolysis temperature in step S2 was 25°C, while other conditions remained unchanged. The resulting chitosan was a pale yellow powder with a degree of deacetylation of 92.8%, an ash content of 1.8 wt%, a moisture content of 7.6 wt%, and a number-average molecular weight of 210 kDa. The residual choline chloride content in the chitosan was 0.08 wt%, the residual oxalic acid content was 0.09 wt%, and the residual 1-ethyl-3-methylimidazolium acetate content was 0.04 wt%. Solubility was moderate; it dissolved after stirring in a 1.0 wt% acetic acid solution for 30 minutes, but slight turbidity appeared after standing for 24 hours.

[0098] Comparative Example 8: Essentially the same as Example 1, except that the mass ratio of sodium hydroxide in the sodium hydroxide aqueous solution in step S4 to the mass ratio of the pretreated solid obtained in step S3 was 2:1, while other conditions remained unchanged. The resulting chitosan was a white powder with a degree of deacetylation of 84.5%, an ash content of 0.6 wt%, a moisture content of 7.3 wt%, and a number-average molecular weight of 310 kDa. The residual choline chloride content in the chitosan was 0.05 wt%, the residual oxalic acid content was 0.06 wt%, and the residual 1-ethyl-3-methylimidazolium acetate content was 0.03 wt%. It exhibited poor solubility, partially dissolving after stirring in a 1.0 wt% acetic acid solution for 30 minutes, and requiring stirring for 80 minutes for complete dissolution.

[0099] Performance testing:

[0100] Deacetylation degree determination experiment: acid-base titration and proton nuclear magnetic resonance spectroscopy were used. 1 Combined determination by H-NMR. For acid-base titration, accurately weigh 0.2000 g of chitosan sample into a 250 mL Erlenmeyer flask, add 25.0 mL of 0.1000 mol / L hydrochloric acid standard solution, stir to dissolve, add methyl orange indicator, and titrate to the endpoint with 0.1000 mol / L sodium hydroxide standard solution. Perform three parallel determinations. 1 H-NMR measurements were performed using a D2O / DCl mixed solvent system, with a sample concentration of 10 mg / mL, a 400 MHz NMR spectrometer, and at 25 °C. The degree of deacetylation was calculated using the H2-H6 proton integrated area ratio. Two methods were cross-validated to ensure an accuracy within ±0.5%. Data processing used the formula: Degree of deacetylation (%) = (1 - A / B × 0.625) × 100, where A is the acetyl group integrated area and B is the total H2-H6 proton integrated area of ​​the glucosamine unit.

[0101] Molecular weight and molecular weight distribution determination experiments: Number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI) were determined by gel permeation chromatography (GPC). Instrument configuration: Waters GPC system equipped with a differential refractive index detector (RI), TSK-GELGMPWXL tandem columns (7.8 mm × 30 cm × 2 columns), mobile phase: 0.2 mol / L acetic acid / 0.1 mol / L sodium acetate buffer (pH 4.5), flow rate: 0.6 mL / min, column temperature: 30℃, injection volume: 100 μL. Sample preparation: 5.0 mg of chitosan sample was accurately weighed and dissolved in 5.0 mL of mobile phase, filtered through a 0.45 μm filter membrane. A molecular weight calibration curve was established using pullulan polysaccharide standards, with a molecular weight range of 180 Da to 800 kDa. Data processing: Empower software was used for automatic integration calculation. Triple parallel determinations were performed, and the average value was taken. The relative standard deviation (RSD) was ≤3%.

[0102] Ash content determination experiment: Accurately weigh 1.0000g of chitosan sample into a constant-weight porcelain crucible. First, carbonize it at low temperature on a hot plate until smokeless, then transfer it to a muffle furnace and calcine at 550±25℃ for 4 hours until constant weight (the difference between two weighings ≤0.5mg). After cooling to room temperature, equilibrate in a desiccator for 30 minutes, and weigh using an electronic analytical balance (accuracy 0.1mg). Perform three parallel determinations. The ash content mass fraction calculation formula is: Ash (wt%) = (m2-m0) / (m1-m0)×100%, where m0 is the mass of the empty crucible, m1 is the total mass of the sample and crucible, and m2 is the total mass of the crucible and residue after ashing. Simultaneously, inductively coupled plasma optical emission spectrometry (ICP-OES) was used for quantitative analysis of the ash elemental composition, focusing on the detection of residual inorganic salts such as calcium, magnesium, sodium, and potassium.

[0103] Residual solvent quantification experiment: High-performance liquid chromatography (HPLC) was used to determine the residual amounts of choline chloride and oxalic acid; gas chromatography (GC) was used to determine the residual amount of ethanol; and ion chromatography (IC) was used to determine the residual amount of 1-ethyl-3-methylimidazolium acetate. HPLC conditions: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase: methanol-water gradient elution, flow rate: 1.0 mL / min, detector: UV 210 nm, column temperature: 35℃. GC conditions: DB-WAX capillary column (30 m × 0.32 mm × 0.25 μm), FID detector, carrier gas: nitrogen, injection port temperature: 250℃, temperature programmed. Sample pretreatment: Accurately weigh 0.5000 g of chitosan, add 10 mL of deionized water, ultrasonically extract for 30 min, filter through a 0.22 μm filter membrane, and inject. Quantification was performed using the external standard method; the linear range of the standard curve was 0.01-1.0 mg / mL, R0. 2 ≥0.999, detection limit ≤0.05wt%.

[0104] Solubility and solution stability test: Accurately weigh 1.0000g of chitosan sample into a 250mL beaker, add 99.0g of 1.0wt% acetic acid aqueous solution, and magnetically stir at 300rpm at 25℃. Record the complete dissolution time (visually confirming no insoluble particles). Transfer the solution to a 100mL colorimetric tube and allow it to stand. Observe and photograph the precipitation at 0, 6, 12, 24, 48, and 72h. Measure the turbidity of the solution at 600nm using a UV-Vis spectrophotometer, with deionized water as a reference. A turbidity value <0.05 indicates a clear and stable solution. Simultaneously measure the solution viscosity (NDJ-5S rotational viscometer, rotor No. 2, rotation speed 60rpm, 25℃) to examine molecular chain integrity. Perform parallel tests on 3 samples. A relative standard deviation (RSD) of dissolution time ≤10% is considered acceptable.

[0105] Purity comprehensive analysis experiment: The C, H, and N elemental contents were determined using an elemental analyzer to verify the chemical purity of chitosan. The theoretical deacetylation degree of 95% for chitosan is 44.1 wt% C, 6.9 wt% H, and 8.7 wt% N. A deviation of ≤ ±0.5% in the measured values ​​is considered acceptable. Fourier transform infrared spectroscopy (FTIR) was used to analyze functional groups using the KBr pellet method, with a scanning range of 4000-400 cm⁻¹. -1 4cm resolution -1 The cumulative number of times was 32, and the characteristic absorption peak included 3450 cm⁻¹. -1 (-OH / -NH2 stretching vibration), 2920 and 2870 cm⁻¹ -1 (CH stretching vibration), 1655cm -1 (Amide I band), 1595cm -1 (-NH2 bending vibration), 1380cm -1 (Amide III band), 1155 and 1080cm -1 (CoC stretching vibration of the sugar ring), the degree of deacetylation was verified by the A1655 / A3450 ratio. Simultaneously, X-ray diffraction (XRD) was used to analyze the crystal form, Cu target Kα rays, scanning range 5-60°, step size 0.02°, scanning speed 5° / min.

[0106] Figure 1The gel permeation chromatography molecular weight distribution and cumulative distribution chromatograms for Example 1 and Comparative Example 5 are shown. The parameters were fixed as follows: sample dissolution and filtration conditions, column and mobile phase system, flow rate and column temperature, detector and integration algorithm, and the same data processing flow. The variable parameters were the differences in molecular weight distribution caused by the different sample preparation processes in Example 1 and Comparative Example 5. In Example 1, the distribution peaks shifted towards higher molecular weights and the cumulative distribution became more concentrated, indicating that the obtained polymer chains remained more intact and reduced degradation or chain-severing products. Compared to Comparative Example 5, the distribution was more biased towards lower molecular weights and exhibited a wider distribution, demonstrating that the process in Example 1 can effectively maintain the molecular weight level and distribution stability of the target material, thereby supporting subsequent purification and performance consistency.

[0107] Figure 2 The Fourier transform infrared spectra of Example 1, Comparative Examples 1, and Comparative Examples 3 are compared. Fixed parameters include test mode and resolution, number of scans, baseline correction and normalization methods, and display rules for the same wavenumber range. Variations include the sample source being Example 1, Comparative Examples 1, and Comparative Examples 3, and the differences in their characteristic absorption and A1655 / A3450 ratios are compared. Example 1 exhibits the lowest A1655 / A3450 ratio and a spectrum shape closer to the characteristic absorption combination of the target structure, indicating a relatively lower absorption related to carbonyl groups and a more consistent hydrogen bonding environment related to hydroxyl groups. Conversely, the increased ratios in Comparative Examples 1 and 3 suggest that the proportion of related functional groups or the hydrogen bonding state in the structure deviates from the target, accompanied by stronger changes in characteristic absorption. This demonstrates that the route of Example 1 more stably obtains the target functional group proportions and intermolecular interaction states, thus verifying the effectiveness of material structure control.

[0108] Figure 3 The X-ray diffraction patterns of Example 1 and Comparative Example 1 are shown. The fixed parameters are the scanning angle range and step size, tube voltage and current, slit parameters, sample loading method, and the same background treatment and peak labeling rules. The varying parameters are the differences in crystal form characteristic peaks and crystallinity index caused by different sample preparation processes. Example 1 exhibits characteristic peaks at 10.5° and 20.0° that are more consistent with the chitosan crystal region and does not show the calcium carbonate impurity peak at 29.4°. Simultaneously, the crystallinity index and peak shape indicate less interference from impurity crystals. In contrast, Comparative Example 1 shows the presence of the 29.4° peak and its accompanying impurity peak, suggesting residual inorganic impurities that may cause deviations in crystallinity assessment. This demonstrates that Example 1 can effectively suppress or remove inorganic crystal impurities and obtain purer crystal phase information, thus supporting its purification effect and structural consistency conclusions.

[0109] As can be seen from the performance of the examples and comparative examples in Table 1, the three-step combined process of low-temperature enzymatic hydrolysis pretreatment, deep eutectic solvent pretreatment, and ionic liquid synergistic deacetylation of the present invention successfully achieved a synergistic effect of high deacetylation degree (92.0-98.0%) and high molecular weight (150-420kDa). The ash content was controlled at a low level of 0.15-0.70wt%, the total residual solvent was only 0.08-0.18wt%, all samples were completely dissolved within 30 minutes and the solution stability reached 72 hours without precipitation, and the yield was stable at 74.2-81.3%. Comparative Example 1, omitting enzymatic hydrolysis, resulted in a surge in ash content to 3.80 wt% and solvent residue to 0.35 wt%. Comparative Examples 3 and 4, due to excessively low reaction temperatures or short reaction times, achieved deacetylation degrees of only 76.5% and 81.0%, respectively, with dissolution times extended to 60-120 minutes. Comparative Example 5, omitting the ionic liquid, achieved a deacetylation degree of 93.0%, but its molecular weight plummeted to 85 kDa, verifying the crucial role of ionic liquids in inhibiting chain degradation. Comparative Examples 2, 6, and 7, due to incomplete processes, resulted in high ash and solvent residues and decreased solution stability. Comparative Example 8, with insufficient alkali, resulted in a deacetylation degree of only 84.5%. These comparisons fully demonstrate the indispensability of the technical features of this invention in solving the technical challenges of achieving both high deacetylation degrees and high molecular weight, low ash and low residue, and long-term stable dissolution.

[0110] Table 1 Performance Comparison Summary Table

[0111]

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an environmentally friendly chitosan with a high degree of deacetylation, characterized in that, Includes the following steps: S1. Raw material pretreatment: Select crustacean biomass raw materials from shrimp shells or crab shells, and wash, dry and crush them in sequence. The particle size after crushing is 80-120 mesh. S2. Low-temperature enzymatic hydrolysis pretreatment: The powder obtained in step S1 is added to a phosphate buffer solution with a total phosphate concentration of 0.01-0.20 mol / L, and the pH is adjusted to 6.8-7.2 to form a suspension with a solid-liquid mass ratio of 1:(8-15); chitinase, protease and lipase are added at 4-10℃, and calcium chloride is added. After stirring and reacting for 12-36 h, the solid and liquid are separated to obtain a solid. S3. Pretreatment with eutectic solvent: The solid obtained in step S2 is mixed with a eutectic solvent at a solid-liquid mass ratio of 1:(5-15). The mixture is stirred and reacted at 40-60°C for 1-4 hours. After the reaction, the solid and liquid are separated and washed with deionized water until the pH of the washing solution is 6.5-7.5 to obtain the pretreated solid. S4. Ionic liquid synergistic deacetylation: The pretreated solid obtained in step S3 is mixed with 1-ethyl-3-methylimidazolium acetate at a solid-liquid mass ratio of 1:(5-12). Under nitrogen protection, a sodium hydroxide aqueous solution with a mass fraction of 30-50 wt% is added, and the mixture is stirred at 60-90°C for 0.5-4 h to obtain a reaction solution containing chitosan. S5. Precipitation and drying: Anhydrous ethanol is added to the reaction solution containing chitosan obtained in step S4 for precipitation. The volume ratio of ethanol to the reaction solution containing chitosan is (2-6):

1. After solid-liquid separation, the solution is washed with deionized water until the pH of the washing solution is 6.5-7.5, and then vacuum dried to obtain chitosan. S6. Solvent recovery: The filtrate obtained in step S5 is subjected to membrane separation-electrodialysis-crystallization recovery to recycle the eutectic solvent components. The 1-ethyl-3-methylimidazolium acetate is then subjected to vacuum distillation to remove ethanol and water before reuse. The degree of deacetylation of the obtained chitosan was 90.0%–98.0%. The deep eutectic solvent is prepared by the following steps: A1. Weigh the raw materials according to the molar ratio of choline chloride to oxalic acid of 1:(1.8~2.2); A2. Stir and mix at 60-70℃ for 2-3 hours to form a homogeneous transparent liquid; A3. The homogeneous transparent liquid is used as the eutectic solvent, and the water content of the eutectic solvent is 0.1–5.0 wt%. In step S2, based on the total mass of the suspension, the mass fraction of the chitinase is 0.2–1.0 wt%, the mass fraction of the protease is 0.5–3.0 wt%, and the mass fraction of the lipase is 0.2–2.0 wt%; the concentration of calcium chloride is 0.5–5.0 g / L based on the volume of phosphate buffer; and the stirring speed in step S2 is 30–120 rpm. In step S3, the stirring speed is 100-400 rpm; The mass ratio of sodium hydroxide in the sodium hydroxide aqueous solution in step S4 to the mass ratio of the pretreated solid obtained in step S3 is (2-10):1; In step S5, the vacuum drying temperature is 40–80°C and the time is 6–24 hours. Step S6, the membrane separation-electrodialysis-crystallization recovery, includes: C1. Membrane separation: Cross-flow filtration is performed using ceramic membranes with pore sizes of 20–100 nm, the filtration temperature is 25–45 °C, and the transmembrane pressure difference is 0.20–0.35 MPa. C2. Electrodialysis: A bipolar membrane electrodialysis device is used to perform electrodialysis on the membrane separation permeate. The voltage is 10-25V, the temperature is 20-35℃, and the current density is 30-80A / m² to obtain choline chloride aqueous solution and oxalic acid aqueous solution. C3. Crystallization and recovery: The oxalic acid aqueous solution is subjected to gradient cooling crystallization. The first stage temperature is 3-5℃ and the time is 1-3h, the second stage temperature is -12--8℃ and the time is 3-5h, and the third stage temperature is -22--18℃ and the time is 5-7h, so as to obtain oxalic acid crystals.

2. An environmentally friendly chitosan with a high degree of deacetylation obtained by the preparation method of the environmentally friendly chitosan with a high degree of deacetylation according to claim 1, characterized in that, The chitosan has a degree of deacetylation of 90.0%–98.0% and an ash content of 0.15%–1.0 wt%. The chitosan has a water content of 2.5–15.0 wt%; a number-average molecular weight of 150–420 kDa; a white to pale yellow color; and is in powder or porous block form. The chitosan is dissolved at 25°C in a 0.5–2.0 wt% aqueous acetic acid solution at a concentration of 1.0 wt% to form a homogeneous solution. The chitosan contains ≤0.10 wt% residual choline chloride, ≤0.10 wt% residual oxalic acid, and ≤0.05 wt% residual 1-ethyl-3-methylimidazolium acetate.

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