Green preparation process of chitin with high deacetylation degree

Abstract

The invention belongs to the technical field of green processing of biomass materials, and particularly relates to a green preparation process of high-deacetylation-degree chitin. The preparation method aims at the problem of high energy consumption of existing high-deacetylation-degree chitin preparation. According to the method, raw materials are pretreated by utilizing a compound enzyme system under a low-temperature condition, chitin crystal lattices are preliminarily dissociated by combining a deep eutectic solvent at a medium temperature, high deacetylation conversion is realized through short-time dissolution of an ionic liquid, and meanwhile, a membrane separation-electrodialysis-gradient freezing crystallization three-stage recycling technology is matched, so that recycling of the solvent is realized; the method greatly reduces the reaction temperature and energy consumption, avoids the use of corrosive reagents, and has the advantages of environmental protection, low energy consumption, high deacetylation degree of the product, excellent quality and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of green processing of biomass materials, and specifically relates to a green preparation process of chitin with a high deacetylation rate, which realizes the preparation of chitin with a high deacetylation rate while reducing energy consumption and chemical pollution. Background Art

[0002] Chitin is a natural polysaccharide found in a wide range of biomass, including crustacean exoskeletons and fungal cell walls. By removing some of its acetyl groups, it can be converted into chitosan, which has important applications in medicine, food, and materials due to its excellent biocompatibility, biodegradability, and functional group reactivity. However, the preparation of highly deacetylated chitin has always been a challenge in the industry, with traditional processes suffering from high energy and reagent consumption and a heavy environmental burden.

[0003] Currently, the industrial production of highly deacetylated chitin typically uses a strong alkaline chemical method, which involves treating the chitin raw material with a high-concentration sodium hydroxide solution at high temperature for a long time to break the acetyl bonds and achieve deacetylation. Although this method can produce products with a high degree of deacetylation, it also has obvious drawbacks: first, the high temperature and high alkaline conditions lead to huge energy consumption; second, the strong alkali is highly corrosive to the reaction equipment, requiring high equipment materials and maintenance requirements, and it also produces a large amount of strong alkaline waste liquid, which can cause environmental pollution if improperly handled; third, under such harsh conditions, the polysaccharide backbone is easily degraded, resulting in a reduction in the molecular weight of the resulting chitosan and impaired physical and chemical properties. In addition, because chitin raw materials often contain impurities such as calcium carbonate and protein, traditional processes often require pretreatment steps using strong acids and strong bases for demineralization and deproteinization, respectively, which further increases chemical reagent consumption and waste liquid discharge.

[0004] In recent years, numerous researchers have explored green alternatives to traditional strong acid-base methods, driven by environmental friendliness and sustainable development. For example, chitin deacetylation can be directly catalyzed by microbial chitin deacetylase, or by the use of proteases and chitosanase to remove impurities and improve the reaction environment. Enzymatic treatment offers the advantages of mildness and high selectivity, but achieving high deacetylation degrees using enzymatic deacetylation alone is currently difficult, and the enzyme costs are high. Alternatively, two-step fermentation and bioextraction methods have been used to purify chitin, but their overall efficiency and industrial applicability still need to be improved.

[0005] In summary, the existing technology has not yet provided a chitosan preparation process that combines low energy consumption, high deacetylation rate, and green recycling characteristics. A method that combines bio-enzyme treatment, green solvents, and process integration optimization is urgently needed to overcome the shortcomings of traditional methods and achieve efficient and clean production of highly deacetylated chitosan. Summary of the Invention

[0006] The application provides a green preparation process of high-deacetylation degree chitin, aiming at overcoming the shortcomings of high energy consumption and serious environmental pollution in the preparation of high-deacetylation degree chitin in the prior art, and providing a green and efficient preparation process of high-deacetylation degree chitin. The application innovatively combines the synergistic effect of low-temperature enzymatic pretreatment, low-eutectic solvent and ionic liquid, and is matched with a multi-stage solvent recovery and recycling technology, so that efficient deacetylation conversion of chitin is realized under mild conditions, energy and chemical consumption is significantly reduced, and the application has a good industrial application prospect.

[0007] The specific technical scheme is as follows: A green preparation process of high-deacetylation degree chitin is as follows: S1: raw material processing and preparation.

[0008] S11: the crab shells are broken to 5-8mm particle size by physical crushing, then are ground at low temperature by liquid nitrogen, and are crushed to 80-100 meshes at-50 DEG C; residual meat and impurities are removed by using a hydrocyclone separation, and the crushed crab shells are obtained.

[0009] S12: chlorocholine is mixed with oxalic acid, and the DES solvent is prepared by stirring at 60-70 DEG C for 2-3h.

[0010] S13: 2-methylimidazole is reacted with 1,4-butane sulfone lactone at 60 DEG C for 24h to generate a sulfonic acid functionalized imidazole intermediate; then the sulfonic acid functionalized imidazole intermediate is reacted with chlorocholine under nitrogen protection at 80 DEG C for 48h, and then is refluxed with bis-trifluoromethanesulfonimide silver salt in acetonitrile for 12h; metal impurities are removed by active carbon column chromatography, and the solvent is removed by rotary evaporation to obtain a chlorocholine-imidazolium hybrid ionic liquid; the chlorocholine-imidazolium hybrid ionic liquid is mixed with the DES solvent prepared in S12 to prepare a zwitterionic liquid-low eutectic solvent.

[0011] S2: enzyme hydrolysis-ionic liquid synergy.

[0012] S21: the crushed crab shells are added into a phosphate buffer solution with pH=7.0 to form a suspension, wherein the solid-liquid ratio is 1:10; low-temperature composite enzymes are added at 4-10 DEG C, and the mass / volume ratio of CaCl2 is 0.2%, and the enzyme hydrolyzed slurry is obtained by stirring and dissolving.

[0013] S22: the enzyme hydrolyzed slurry in S21 is centrifuged and separated, the solid part is transferred to a medium temperature reaction chamber with a temperature of 40-50 DEG C, the DES solvent prepared in S12 is added, and the DES pretreated mixture is obtained by stirring and reacting.

[0014] S23: mixing the mixture prepared in S22 with the zwitterionic liquid-DES prepared in S13 to obtain a mixed solution, stirring and dissolving, and passing nitrogen gas through the whole reaction process to obtain a chitosan product solution with a deacetylation degree of ≥90%.

[0015] S24: adding the chitosan product solution prepared in S23 into 95% pure ethanol, stirring and then standing for precipitation, centrifugal separation, then washing the solid with deionized water until neutral, and finally vacuum freeze-drying to obtain high-deacetylation degree chitin.

[0016] S3: solvent recovery.

[0017] S31: subjecting the filtrate obtained by centrifugal separation in S24 to cross-flow filtration through a ceramic ultrafiltration membrane with a pore size of 50 nm to obtain a permeate with a molecular weight of less than 10 kDa.

[0018] S32: subjecting the filtrate treated in S31 to electro-osmosis in a bipolar membrane electrodialysis cell, obtaining a choline chloride solution in the cathode chamber and an oxalic acid solution in the anode chamber to realize the directional recombination of the DES decomposition products.

[0019] S33: subjecting the oxalic acid solution obtained in S32 to gradient cooling crystallization to obtain oxalic acid crystals.

[0020] Further, the choline chloride and oxalic acid in S12 have a molar ratio of choline chloride to oxalic acid of 1:1.8 to 1:2.2.

[0021] The 2-methylimidazole and 1,4-butane sulfone lactone in S13 have a molar ratio of 1:1.05 to 1:1.15.

[0022] The sulfonic acid functionalized imidazole intermediate and choline chloride in S13 have a molar ratio of 1:1.1 to 1:1.3.

[0023] The silver bistrifluoromethanesulfonimide salt in S13 has a molar ratio of 1:1.05 to the sulfonic acid functionalized imidazole intermediate.

[0024] The mass ratio of the choline-imidazolium hybrid ionic liquid to the DES solvent in the mixture in S13 is 3:7.

[0025] Further, the low-temperature complex enzyme in S21 comprises chitinase, protease, and lipase, the mass fraction of chitinase is 0.4 to 0.6%, the mass fraction of protease is 1.8 to 2.2%, and the mass fraction of lipase is 0.8 to 1.2%.

[0026] The stirring and dissolving in S21 is set with the following parameters: speed 50 rpm, and time 24 to 36 h.

[0027] The centrifugal separation of S22 has the parameter setting: rotation speed 5000 rpm, time length 10 min.

[0028] The solid of S22 has a solid-liquid ratio of 1:10 with the DES solvent.

[0029] The mixed solution of S23 has a solid mass to the solid-liquid ratio of the zwitterionic liquid-eutectic solvent of 1:5-1:7 in the mixture.

[0030] The stirring of S23 has the parameter setting: rate 200-400 rpm, temperature 60-70℃, time length 2-4 h.

[0031] The ethanol of S24 has a volume ratio of 3:1 with the chitosan solution.

[0032] The centrifugal separation of S24 has the parameter setting: rotation speed 4000 rpm, time length 15 min.

[0033] The vacuum freeze-drying of S24 has the parameter setting: temperature -50℃, pressure 0.1 mbar, time length 24 h.

[0034] Further, the filtration of S31 has the parameter setting: temperature 40℃, pressure difference 0.28-0.32 MPa.

[0035] The electro-osmosis of S32 has the parameter setting: voltage 15 V, temperature 25℃, current density 45-55 A / m 2 .

[0036] The gradient cooling crystallization of S33 has the parameter setting: first stage, temperature 3-5℃, time length 1-3 h; second stage, temperature -8--12℃, time length 3-5 h; third stage, temperature -18--22℃, time length 5-7 h.

[0037] Compared with the prior art, the present application has the following beneficial effects: 1. By means of the low-temperature composite enzyme system and the metal ion activator, the crystal structure of chitin is effectively destroyed, part of the protein and fat is decomposed, and the chitin molecular chain is cut off, thereby reducing the difficulty and energy consumption of subsequent chemical treatment.

[0038] 2. By means of the DES solvent pretreatment, the hydrogen bond network of chitin is effectively destroyed, the dense crystal lattice structure is preliminarily loosened and dissociated, and the amount of ionic liquid and the treatment intensity are reduced.

[0039] 3. By means of the zwitterionic liquid-eutectic solvent treatment, high deacetylation conversion is achieved.

[0040] 4. Through the construction of membrane separation-electrodialysis-frozen crystallization three-stage recovery process, the solvent is recycled and the by-products are resourceized, the cost is reduced and the environment is protected. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 A green preparation process flow chart of high deacetylation degree chitin.

[0042] Figure 2 A comparison chart of the deacetylation degree of chitin and the total energy consumption data of the final preparation of examples 1-4 and comparative examples 1-3.

[0043] Figure 3 A comparison chart of wastewater generation and DES solvent recovery rate in waste liquid data in the entire production process of examples 1-5 and comparative examples 1-3. DETAILED DESCRIPTION

[0044] The following examples further explain and illustrate the technical solutions of the present application. It is particularly pointed out that each specific embodiment is a specific embodiment and explanation of the technical solution, and should not be regarded as a limitation on the protection scope of the present application. Those skilled in the art still have the right to modify the technical solutions of these examples, to replace some or all of the technical features with equivalent ones, and these modifications or replacements do not change the essence of the corresponding technical solutions, and do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions described in the present application.

[0045] The present application proposes a green preparation process of high deacetylation degree chitin, which effectively solves the problems of high energy consumption and high pollution in traditional process through low temperature enzymatic hydrolysis-DES solvent pretreatment-ion liquid cooperative treatment combined with a green preparation system of three-stage recovery system. As shown in the accompanying drawings, Figure 1 The present application proposes a green preparation process of high deacetylation degree chitin, which effectively solves the problems of high energy consumption and high pollution in traditional process through low temperature enzymatic hydrolysis-DES solvent pretreatment-ion liquid cooperative treatment combined with a green preparation system of three-stage recovery system. As shown in the accompanying drawings, 1. Raw material processing and preparation.

[0046] 1.1 Raw material preparation; Physical crushing is used to crush the crab shells to 5-8mm particle size, increase the specific surface area of the raw material, and prepare for subsequent deep crushing and chemical reaction; then liquid nitrogen low temperature grinding is used to crush to 80-100 mesh at-50℃, the liquid nitrogen low temperature can make the material brittle, which is easier to crush, and can effectively inhibit the heat generated in the grinding process, thereby protecting the natural structure and molecular weight of biological macromolecules; water cyclone separation is used to remove residual meat and impurities, and obtain crushed crab shells.

[0047] 1.2 DES preparation; The DES solvent is prepared by stirring choline chloride as a hydrogen bond acceptor and oxalic acid as a hydrogen bond donor, wherein the molar ratio of choline chloride to oxalic acid is 1:1.8-1:2.2, which ensures that the two can form a stable, low-melting eutectic mixture.

[0048] 1.3 Preparation of zwitterionic liquid-DES; 2-methylimidazole is reacted with 1,4-butane sultone to generate a sulfonic acid functionalized imidazole intermediate, and a sulfonic acid group is introduced as a strong acidic functional group. Then, the sulfonic acid functionalized imidazole intermediate is reacted with choline chloride under nitrogen protection, and then refluxed with silver bistrifluoromethanesulfonimide in acetonitrile. The silver bistrifluoromethanesulfonimide is introduced to reduce the viscosity of the ionic liquid, improve its thermal stability and solubility for polymers. Then, silver chloride and possible residual silver ions are removed by activated carbon column chromatography, and the solvent is removed by rotary evaporation to obtain a choline-imidazolium hybrid ionic liquid. Finally, the zwitterionic liquid-DES is prepared by compounding with the above DES solvent. The compounding can reduce the viscosity of the system and utilize the oxalic acid in the DES to participate in the acidic catalytic process.

[0049] 2. Enzymatic hydrolysis-ion liquid cooperation.

[0050] 2.1 Low-temperature enzymatic hydrolysis; The crushed crab shells are added to a phosphate buffer solution with a pH of 7.0 to form a suspension. Low-temperature complex enzymes are added at 4-10°C to inhibit the growth of miscellaneous bacteria and prevent the deterioration of the raw materials. 0.2% (w / v) of CaCl2 is added to enhance the activity of the low-temperature complex enzymes. The solution is stirred at 50 rpm for 24-36 h to obtain an enzymatically hydrolyzed slurry. The low-temperature complex enzymes contain chitinase, protease and lipase. The chitinase partially dissolves the crystal structure, breaks the bonds between proteins and chitin, the protease hydrolyzes and removes proteins closely combined with chitin, and the lipase hydrolyzes and removes lipids.

[0051] 2.2 Moderate-temperature reaction; The enzymatically hydrolyzed slurry is centrifuged to remove soluble proteins, peptides, amino acids and other impurities produced during enzymatic hydrolysis. The solid part is transferred to a moderate-temperature reaction chamber, and DES is added and stirred to react. The addition of DES is to gently remove minerals and initially remove acetyl groups.

[0052] 2.3 Ionic liquid dissolution; The DES pretreated mixture is added to the zwitterionic liquid- deep eutectic solvent (nitrogen is continuously introduced to prevent oxidative decomposition), and stirred and dissolved at 60-70°C. The zwitterionic liquid- deep eutectic solvent is added because of its acidic functional groups and strong hydrogen bond breaking ability, which can break the strong hydrogen bond network between and within chitin molecules, allowing it to dissolve and simultaneously undergo deep deacetylation. Nitrogen creates an inert atmosphere to protect the integrity of the chitosan molecular chain, and 60-70°C provides high reaction kinetic energy to accelerate the deacetylation reaction and dissolution process.

[0053] 2.4 post-treatment; The prepared chitosan product solution is added to 95% pure ethanol, stirred and then allowed to settle, centrifuged, and then the solid is washed with deionized water until neutral, and finally vacuum freeze-dried to obtain high deacetylation degree chitin. Ethanol is added because chitosan is insoluble in ethanol, and vacuum freeze-drying is used because it can maintain the porous sponge structure of chitosan, resulting in a very high specific surface area.

[0054] 3. solvent recovery.

[0055] 3.1 ceramic ultrafiltration membrane; The filtrate obtained by centrifugal separation is subjected to cross-flow filtration through a ceramic ultrafiltration membrane with a pore size of 50 nm, and a permeate with a molecular weight less than 10 kDa is obtained. Filtration is used to trap possible large molecular proteins, pigments or chitosan fragments that may leak.

[0056] 3.2 bipolar membrane electrodialysis; The permeate is placed in a bipolar membrane electrodialysis cell for electrodialysis, and a choline chloride solution is obtained in the cathode chamber, and an oxalic acid solution is obtained in the anode chamber, achieving the directional recombination of DES decomposition products. Electrodialysis is used because it has lower energy consumption.

[0057] 3.3 gradient cooling crystallization; The oxalic acid solution after electrodialysis is subjected to gradient cooling crystallization to obtain oxalic acid crystals. Gradient cooling crystallization results in oxalic acid crystals with regular crystal form, larger particles and higher purity, which is convenient for subsequent use.

[0058] Example 1: A green preparation process for high deacetylation degree chitin, as follows: Table 1: Main raw materials:

[0059] S1: Raw material processing and preparation.

[0060] S11: The crab shells are physically crushed to a particle size of 5-8 mm, then ground in liquid nitrogen at-50°C to a size of 80-100 mesh, and then the residual meat and impurities are removed by hydrocyclone separation to obtain crushed crab shells.

[0061] S12: Choline chloride was mixed with oxalic acid to prepare a DES solvent by stirring at 65℃ for 2.5h, wherein the molar ratio of choline chloride to oxalic acid was 1:2.

[0062] S13: 2-methylimidazole was reacted with 1,4-butane sultone at 60℃ for 24h to generate a sulfonic acid functionalized imidazole intermediate, wherein the molar ratio of 2-methylimidazole to 1,4-butane sultone was 1:1.1; then the sulfonic acid functionalized imidazole intermediate was reacted with choline chloride under nitrogen protection at 80℃ for 48h, and then refluxed with silver bistrifluoromethanesulfonimide in acetonitrile for 12h, wherein the molar ratio of the sulfonic acid functionalized imidazole intermediate to choline chloride was 1:1.2, and the molar ratio of silver bistrifluoromethanesulfonimide to the sulfonic acid functionalized imidazole intermediate was 1:1.05; metal impurities were removed by activated carbon column chromatography, and the solvent was removed by rotary evaporation to obtain a choline-imidazolium hybrid ionic liquid; the choline-imidazolium hybrid ionic liquid was mixed with the DES solvent prepared in S12, and the mass ratio was 3:7 to prepare a zwitterionic liquid-DES.

[0063] S2: Enzymatic hydrolysis-ion liquid synergy.

[0064] S21: The crushed crab shells were added to a phosphate buffer solution with a pH of 7.0 to form a suspension, wherein the solid-liquid ratio of the crab shells to the phosphate buffer solution was 1:10; low-temperature complex enzymes were added at 7℃, and 0.2% CaCl2 was added by mass volume ratio, and the solution was dissolved by stirring at 50rpm for 30h to obtain an enzymatically hydrolyzed slurry, wherein the low-temperature complex enzymes contained chitinase, protease and lipase, the mass fraction of chitinase was 0.5%, the mass fraction of protease was 2.0%, and the mass fraction of lipase was 1.0%.

[0065] S22: The enzymatically hydrolyzed slurry of S21 was centrifuged, and the parameters were set as follows: rotation speed 5000rpm, time 10min; the solid part was transferred to a medium temperature reaction chamber with a temperature of 45℃, and the DES solvent prepared in S12 was added, wherein the solid-liquid ratio of the solid part to the DES solvent was 1:10, and the mixture was stirred to obtain a DES pretreated mixture, wherein the stirring parameters were set as follows: rotation speed 200rpm, time 3h.

[0066] S23: The mixture prepared in S22 was mixed with the zwitterionic liquid-DES prepared in S13 to obtain a mixed solution, which was stirred and dissolved, and nitrogen was introduced throughout the reaction to obtain a chitosan product solution with a degree of deacetylation of ≥90%, wherein the solid mass in the mixture and the zwitterionic liquid-DES had a solid-liquid ratio of 1:6, and the stirring parameters were set as follows: speed 300rpm, temperature 65℃, time 3h.

[0067] S24: The chitosan product solution prepared in S23 is added to 95% ethanol, stirred and then allowed to precipitate, centrifuged, and then the solid is washed with deionized water until neutral, and finally vacuum freeze-dried to obtain high deacetylation degree chitin, wherein the volume ratio of chitosan solution to 95% ethanol is 1:3; the centrifugation parameter setting is 4000 rpm for 15 min; the vacuum freeze-drying parameter setting is -50℃, 0.1 mbar, and 24 h.

[0068] S3: Solvent recovery.

[0069] S31: The filtrate obtained by centrifugation in S24 is subjected to cross-flow filtration through a ceramic ultrafiltration membrane with a pore size of 50 nm, to obtain a permeate with a molecular weight of less than 10 kDa, wherein the filtration temperature is 40℃ and the pressure difference is 0.3 MPa.

[0070] S32: The filtrate treated in S31 is placed in a bipolar membrane electrodialysis cell for electro-osmosis, with the following electro-osmosis parameter settings: voltage 15 V, temperature 25℃, current density 50 A / m 2 , and a choline solution is obtained in the cathode chamber and an oxalic acid solution is obtained in the anode chamber, realizing the directional recombination of the DES decomposition products.

[0071] S33: The oxalic acid solution obtained in S32 is subjected to gradient cooling crystallization to obtain oxalic acid crystals, wherein the gradient cooling crystallization parameter settings are as follows: first stage, temperature 4℃, time 2 h; second stage, temperature -10℃, time 4 h; third stage, temperature -20℃, time 6 h.

[0072] Example 2: Referring to the composition and preparation process of Example 1, the difference is that: In S12 of the preparation process, the molar ratio of choline chloride to oxalic acid is 1:2.2, and the other components are the same.

[0073] In S12 of the preparation process, the stirring parameter setting is temperature 70℃ and time 3 h, and the other steps are the same.

[0074] In S13 of the preparation process, the molar ratio of 2-methylimidazole to 1,4-butane sulfone lactone is 1:1.05, and the molar ratio of sulfonic acid functionalized imidazole intermediate to choline chloride is 1:1.1, and the other components are the same.

[0075] In the low-temperature complex enzyme in S21 of the preparation process, the mass fraction of chitinase is 0.4%, the mass fraction of protease is 1.8%, and the mass fraction of lipase is 0.8%, and the other components are the same.

[0076] In S21 of the preparation process, the low-temperature complex enzyme is added at 4℃, stirred and dissolved for 24 h, and the other steps are the same.

[0077] The temperature in S22 of the preparation process is controlled at 40℃, and other steps are the same.

[0078] In S23 of the preparation process, the solid mass in the mixture is 1:5 of the solid-liquid ratio of the zwitterionic liquid-eutectic solvent, and other compositions are the same.

[0079] In S23 of the preparation process, the stirring parameters are set as follows: speed 200 rpm, temperature 60℃, and time 2h, and other steps are the same.

[0080] In S31 of the preparation process, the pressure difference is 0.28 MPa, and other steps are the same.

[0081] In S32 of the preparation process, the current density in the electro-osmosis parameters is 45 A / m 2 , and other steps are the same.

[0082] In S33 of the preparation process, the gradient cooling crystallization parameters are set as follows: the first stage, temperature 3℃, time 1h; the second stage, temperature -8℃, time 3h; the third stage, temperature -18℃, time 5h, and other steps are the same.

[0083] Example 3: Referring to the composition and preparation process of Example 1, the difference is that: In S12 of the preparation process, the molar ratio of choline chloride to oxalic acid is 1:2.2, and other compositions are the same.

[0084] In S12 of the preparation process, the stirring parameters are set as follows: temperature 70℃, time 3h, and other steps are the same.

[0085] In S13 of the preparation process, the molar ratio of 2-methylimidazole to 1,4-butane sulfone lactone is 1:1.15, and the molar ratio of sulfonic acid functionalized imidazole intermediate to choline chloride is 1:1.3, and other compositions are the same.

[0086] In S21 of the preparation process, the mass fraction of chitinase in the low-temperature complex enzyme is 0.6%, the mass fraction of protease is 2.2%, and the mass fraction of lipase is 1.2%, and other compositions are the same.

[0087] In S21 of the preparation process, the low-temperature complex enzyme is added at 10℃, and stirred and dissolved for 36h, and other steps are the same.

[0088] In S22 of the preparation process, the temperature is controlled at 50℃, and other steps are the same.

[0089] In S23 of the preparation process, the solid mass in the mixture is 1:7 of the solid-liquid ratio of the zwitterionic liquid-eutectic solvent, and other compositions are the same.

[0090] In S23 of the preparation process, the stirring parameters are set as follows: speed 400 rpm, temperature 70℃, and time 4h, and other steps are the same.

[0091] The pressure difference in S31 of the preparation process is 0.32 MPa, and other steps are the same.

[0092] The current density in the electro-osmosis parameter in S32 of the preparation process is 55 A / m 2 , and other steps are the same.

[0093] In S33 of the preparation process, the gradient cooling crystallization parameter setting is: the first stage, the temperature is 5 ℃, and the time length is 3 h; the second stage, the temperature is -12 ℃, and the time length is 5 h; the third stage, the temperature is -22 ℃, and the time length is 7 h, and other steps are the same.

[0094] Example 4: The composition and preparation process of Example 1 are referred to, except that: The molar ratio of choline chloride to oxalic acid in S12 of the preparation process is 1:1.9, and other compositions are the same.

[0095] In S12 of the preparation process, the stirring parameter setting is: the temperature is 67 ℃, and the time length is 2.2 h, and other steps are the same.

[0096] In S13 of the preparation process, the molar ratio of 2-methylimidazole to 1,4-butane sulfolane is 1:1.12, and the molar ratio of the sulfonic acid functionalized imidazole intermediate to choline chloride is 1:1.18, and other compositions are the same.

[0097] In the low-temperature complex enzyme in S21 of the preparation process, the mass fraction of chitinase is 0.6%, the mass fraction of protease is 2.1%, and the mass fraction of lipase is 0.9%, and other compositions are the same.

[0098] In S21 of the preparation process, the low-temperature complex enzyme is added at 5 ℃, and stirred and dissolved for 30 h, and other steps are the same.

[0099] In S22 of the preparation process, the temperature is controlled at 42 ℃, and other steps are the same.

[0100] In S23 of the preparation process, the solid mass in the mixture and the solid-liquid ratio of the zwitterionic liquid-eutectic solvent are 1:5.5, and other compositions are the same.

[0101] In S23 of the preparation process, the stirring parameter setting is: the speed is 250 rpm, the temperature is 64 ℃, and the time length is 2.5 h, and other steps are the same.

[0102] The pressure difference in S31 of the preparation process is 0.31 MPa, and other steps are the same.

[0103] The current density in the electro-osmosis parameter in S32 of the preparation process is 52 A / m 2 , and other steps are the same.

[0104] The gradient cooling crystallization parameter settings in S33 of the preparation process are: the first stage, temperature 5°C, duration 2 h; the second stage, temperature -11°C, duration 4.5 h; the third stage, temperature -19°C, duration 5.5 h, and the other steps are the same.

[0105] Comparative Example 1: Referring to the composition and preparation process of Example 1, the difference is: In S21 of the preparation process, the low-temperature compound enzyme hydrolysis is deleted and the treatment is performed with conventional hydrochloric acid. The other steps are the same.

[0106] Comparative Example 2: Referring to the composition and preparation process of Example 1, the difference is: In S22 of the preparation process, the zwitterionic liquid-deep eutectic solvent treatment is deleted and the concentrated alkali method is used. The other steps are the same.

[0107] Comparative Example 3: Referring to the composition and preparation process of Example 1, the difference is: The gradient cooling crystallization in S33 of the preparation process is deleted and cooling crystallization is adopted. The other steps are the same.

[0108] Based on Examples 1-4 and Comparative Examples 1-3, the finally prepared chitosan was sampled and the degree of deacetylation was tested: the chitosan was hydrolyzed, the supernatant was taken, and filtered through a 0.45 μm microporous filter membrane. The concentration of acetate ions in the filtrate was then analyzed using an ion chromatograph equipped with a conductivity detector, and the degree of deacetylation was finally calculated.

[0109] Based on Examples 1-4 and Comparative Examples 1-3, the total energy consumption was calculated: all energy-consuming equipment in the preparation process was connected to an electric energy meter, the operating time and power of the equipment during the entire production process were recorded, and the energy consumption of each equipment was calculated using the following formula: , and the final total energy consumption is converted into the energy consumed in producing one kilogram of product.

[0110] Based on Examples 1-4 and Comparative Examples 1-3, a DES solvent recovery test was conducted: the oxalic acid content prepared in S3 was measured by titration. Referring to the standard GB / T 1626-2008 "Industrial Oxalic Acid", the choline chloride aqueous solution prepared in S3 was measured by non-aqueous titration. The solution was titrated with a perchloric acid-glacial acetic acid standard solution. Two drops of crystal violet indicator were added, and the solution was titrated with a perchloric acid standard solution until the solution changed from purple to blue-green. Finally, the mass fraction of choline chloride was obtained by mass conversion.

[0111] Based on Examples 1-4 and Comparative Examples 1-3, a wastewater generation test was conducted: a collection device was set at all wastewater discharge ports, and various types of wastewater generated in a complete production process were collected and measured separately. The amount of wastewater from all sources was accumulated and calculated to obtain the amount of wastewater generated per kilogram of product prepared.

[0112] The above specific test results are shown in Table 2: Table 2: Comparison table of core performance of examples 1-4 and comparative examples 1-3:

[0113] Note: The total energy consumption is the total power consumption of each device for preparing each kilogram of high deacetylation degree chitin. The wastewater production is the cumulative volume of wastewater produced for preparing each kilogram of high deacetylation degree chitin.

[0114] From the above comparison results, it can be seen that the comprehensive performance of example 1 is the best, which adopts the enzyme hydrolysis-ion liquid synergistic process and high-efficiency solvent recovery system, realizes high deacetylation degree, low energy consumption, low wastewater production and high solvent recovery rate, and can best solve the problems of high energy consumption and environmental pollution; the comprehensive performance of examples 2 to 4 is slightly lower than that of example 1, which shows that low energy consumption and low pollution can still be achieved under a large range of parameter changes; comparative example 1 deletes enzyme hydrolysis and uses traditional hydrochloric acid treatment, resulting in low deacetylation degree, high energy consumption; comparative example 2 deletes ionic liquid and uses concentrated alkali method treatment, resulting in the lowest deacetylation degree and high pollution; comparative example 3 deletes gradient cooling crystallization and uses cooling crystallization, resulting in high energy consumption, large wastewater production and low solvent recovery rate.

[0115] In summary, through the above examples and comparative examples, it can be clearly seen that the green preparation process of high deacetylation degree chitin provided by the present application is superior to the traditional scheme in solving the problems of high energy consumption and environmental pollution, which is due to the innovative structure of the low-temperature enzyme hydrolysis-ion liquid synergistic treatment combined with the three-stage recovery system green preparation system, thereby solving the problems of high energy consumption and environmental pollution in preparing high deacetylation degree chitin.

Claims

1. A green preparation process for high-deacetylation chitin, characterized by: The green preparation process uses low-temperature composite enzymes for enzymatic hydrolysis, DES solvent pretreatment, and zwitterionic liquid-low eutectic solvent deep deacetylation for synergistic treatment, combined with a three-stage recycling technology of membrane separation-electrodialysis-gradient freezing crystallization, to achieve green preparation of high-deacetylation chitin under mild conditions.

2. A green preparation process for high-deacetylation chitin according to claim 1, characterized in that: The low-temperature complex enzyme comprises chitinase, protease and lipase, wherein the mass fraction of chitinase is 0.4-0.6%, the mass fraction of protease is 1.8-2.2%, and the mass fraction of lipase is 0.8-1.2%. The chitinase is used to partially hydrolyze chitin, the protease cuts the shell protein cross-linking, and the lipase hydrolyzes the lipid layer.

3. The green preparation process of high-deacetylation chitin according to claim 1, characterized in that: The DES solvent uses choline chloride as a hydrogen bond acceptor and oxalic acid as a hydrogen bond donor, wherein the molar ratio of choline chloride to oxalic acid is 1:1.8 to 1:2.

2. The DES solvent forms a hydrogen bond network with the chitin molecules, weakening the original strong hydrogen bond force between the chitin molecules and reducing the crystallinity.

4. The green preparation process of high-deacetylation chitin according to claim 1, characterized in that: The zwitterionic liquid-deep eutectic solvent is composed of a choline-imidazolium hybrid ionic liquid and a DES solvent, and the mass ratio of the choline-imidazolium hybrid ionic liquid to the DES solvent is 3:7; the choline-imidazolium hybrid ionic liquid is prepared by reacting a sulfonic acid functionalized imidazole intermediate with choline chloride and then reacting with bistrifluoromethanesulfonyl imide silver salt; the sulfonic acid functionalized imidazole intermediate is prepared from 2-methylimidazole and 1,4-butane sultone.

5. The green preparation process of high-deacetylation chitin according to claim 1, characterized in that: The membrane separation filters macromolecular substances through a ceramic ultrafiltration membrane, the electrodialysis is performed through a bipolar membrane electrodialysis cell to achieve the recycling of choline chloride solution, and the gradient freezing crystallization achieves the recycling of oxalic acid through three-stage stepped temperature control.

6. The green preparation process of high deacetylation degree chitin according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: Raw material processing and preparation; S11: crushing the crab shells into particles of 5 to 8 mm by physical crushing, then cryogenically grinding with liquid nitrogen and crushing them into 80 to 100 mesh at -50°C; removing residual meat and impurities by hydrocyclone separation to obtain crushed crab shells; S12: mixing choline chloride and oxalic acid, and stirring at 60-70° C. for 2-3 hours to prepare a DES solvent; S13: 2-Methylimidazole was reacted with 1,4-butane sultone at 60°C for 24 hours to generate a sulfonic acid functionalized imidazole intermediate; the sulfonic acid functionalized imidazole intermediate was reacted with choline chloride at 80°C under nitrogen protection for 48 hours, and then refluxed with bis(trifluoromethanesulfonyl)imide silver salt in acetonitrile for 12 hours; metal impurities were removed by activated carbon column chromatography, and the solvent was removed by rotary evaporation to obtain a choline-imidazolium hybrid ionic liquid; the choline-imidazolium hybrid ionic liquid was mixed with the DES solvent prepared in S12 to prepare a zwitterionic liquid-deep eutectic solvent; S2: enzymatic hydrolysis-ionic liquid synergy; S21: adding crushed crab shells to a phosphate buffer solution with a pH of 7.0 to form a suspension, wherein the solid-liquid ratio is 1:10; adding a low-temperature complex enzyme at 4-10°C, adding 0.2% w / v CaCl2, stirring and dissolving, to obtain an enzymatically hydrolyzed slurry; S22: The slurry after enzymatic hydrolysis in S21 is centrifuged and the solid portion is transferred to a medium temperature reaction chamber at a temperature of 40-50°C, the DES solvent prepared in S12 is added, and the mixture is stirred for reaction to obtain a DES pretreated mixture; S23: mixing the mixture prepared in S22 with the zwitterionic liquid-deep eutectic solvent prepared in S13 to obtain a mixed solution, stirring and dissolving the mixture, and introducing nitrogen gas throughout the reaction to obtain a chitosan product solution with a deacetylation degree of ≥90%; S24: adding the chitosan product solution prepared in S23 to 95% pure ethanol, stirring, allowing to settle, centrifuging, and then washing the solid with deionized water until neutral, and finally vacuum freeze-drying to obtain highly deacetylated chitin; S3: solvent recovery; S31: The filtrate obtained by centrifugation in S24 is filtered through a 50 nm pore size ceramic ultrafiltration membrane to obtain a permeate having a molecular weight of less than 10 kDa; S32: placing the filtrate treated in S31 into a bipolar membrane electrodialysis cell for electroosmosis, obtaining a choline chloride solution in the cathode chamber and an oxalic acid solution in the anode chamber, thereby achieving directed recombination of the DES decomposition products; S33: subjecting the oxalic acid solution obtained in S32 to gradient cooling crystallization to obtain oxalic acid crystals.

7. The green preparation process of high-deacetylation chitin according to claim 6, characterized in that: The molar ratio of 2-methylimidazole and 1,4-butane sultone described in S13 is 1:1.05 to 1:1.15; The sulfonic acid functionalized imidazole intermediate described in S13 and choline chloride have a molar ratio of 1:1.1 to 1:1.3; The molar ratio of the bistrifluoromethanesulfonyl imide silver salt described in S13 to the sulfonic acid functionalized imidazole intermediate is 1:1.

05.

8. The green preparation process of high-deacetylation chitin according to claim 6, characterized in that: The stirring dissolution in S21 has the following parameters: a speed of 50 rpm and a duration of 24 to 36 hours; The centrifugal separation described in S22 has the following parameters: a speed of 5000 rpm and a duration of 10 min; The solid part described in S22 has a solid-to-liquid ratio to the DES solvent of 1:

10.

9. The green preparation process of high-deacetylation chitin according to claim 6, characterized in that: The mixed solution described in S23, wherein the solid-to-liquid ratio of the solid mass in the mixture to the zwitterionic liquid-deep eutectic solvent is 1:5 to 1:7; The stirring described in S23 has the following parameters: speed 200-400 rpm, temperature 60-70° C., and duration 2-4 h; The volume ratio of ethanol described in S24 to chitosan solution is 3:1; The centrifugal separation described in S24 has the following parameters: speed 4000 rpm, duration 15 min; The vacuum freeze drying described in S24 has the following parameter settings: temperature -50°C, pressure 0.1 mbar, and duration 24 hours.

10. The green preparation process of high-deacetylation chitin according to claim 6, characterized in that: The filtration parameters of S31 are as follows: temperature 40°C, pressure difference 0.28-0.32 MPa; The electroosmosis described in S32 has the following parameters: voltage 15V, temperature 25°C, current density 45-55A / m 2 ; The gradient cooling crystallization described in S33 has the following parameter settings: first stage, temperature 3~5°C, duration 1~3h; second stage, temperature -8~-12°C, duration 3~5h; third stage, temperature -18~-22°C, duration 5~7h.

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