Method for extracting chitin based on temperature-responsive eutectic solvent-water extraction system

Abstract

The invention discloses a temperature-responsive eutectic solvent-water extraction system which is composed of a temperature-responsive eutectic solvent and water, the temperature-responsive eutectic solvent is composed of lidocaine, oleic acid and acetic acid, and the molar ratio of lidocaine to oleic acid to acetic acid is (4-6): (2.5-3.5): (1.5-2.5); and the content of water is 15-25% of the weight of the temperature-responsive eutectic solvent. The extraction system is applied to chitin extraction. The invention further discloses a method for extracting chitin based on the temperature-responsive deep-eutectic solvent-water extraction system and a method for recovering the temperature-responsive deep-eutectic solvent. The temperature-responsive deep-eutectic solvent-water extraction system disclosed by the invention has a very good extraction effect when being used for extracting chitin. The method has a good application prospect, and is of great significance in promoting the process of industrialization of deep eutectic solvent extraction of chitin.

Description

Technical Field

[0001] The invention relates to a method for extracting chitosan based on a temperature-responsive low eutectic solvent-water extraction system, and belongs to the technical field of extracting chitosan. Background Art

[0002] Chitin is a natural polymer composed of N-acetyl-D-glucosamine repeating units connected by β (1→4) bonds. It is the second most abundant biopolymer after cellulose and is mainly found in the shells of crustaceans and insects, the cell walls of fungi, and other substances such as the cartilage of mollusks. As the only natural polysaccharide containing nitrogen, chitin has biocompatibility, biodegradability and multiple activities, and has important applications in food, medicine, environmental protection and other fields.

[0003] Shrimp and crab shells are the main raw materials used in the production of chitosan in industry. In addition to chitosan, the main components of shrimp and crab shells also include calcium carbonate and protein. Protein wraps the chitosan molecular chain to form a protein-chitosan composite fiber bundle, and crystalline minerals are deposited on it to form a hard shell of shrimp and crab. Therefore, the essence of extracting chitosan is actually the removal of minerals and proteins from shrimp and crab shells. The traditional acid-base method is the basic method for extracting chitosan in industry, but this method requires a large amount of acid and alkali, and the waste liquid generated will cause serious environmental pollution, which is not in line with the concept of green and sustainable development.

[0004] In recent years, the emergence of deep eutectic solvents (DES) has opened up a new way for the efficient extraction of chitin. Deep eutectic solvents are a homogeneous mixture of hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) in a certain stoichiometric ratio, which self-associate through intermolecular hydrogen bond interactions to form a melting point lower than that of each individual component. They have the advantages of being environmentally friendly, simple to prepare, and having good biodegradability and biocompatibility, and are considered to be a new generation of green solvents. At present, various types of DES have been developed and applied to the extraction of chitin. Commonly used hydrogen bond acceptors are mainly choline chloride and betaine, and hydrogen bond donors include organic molecules such as glycerol, ethylene glycol and urea. Among them, the combination of choline chloride and organic acid has high demineralization and deproteinization capabilities. Therefore, the extraction of chitin based on deep eutectic solvents is considered to be a method with fast extraction speed and high extraction efficiency. At the same time, the impurities such as calcium carbonate and protein remaining in DES after extraction can be removed by the anti-solvent method, and the DES can be recovered and reused. However, the DES currently used has the characteristics of low vapor pressure and strong hygroscopicity. After adding the antisolvent-water to precipitate impurities, the added antisolvent needs to be removed by rotary evaporation, which consumes a lot of energy and is difficult to achieve large-scale production. Therefore, designing and synthesizing a new temperature-responsive low eutectic solvent, changing the hydrophilicity / hydrophobicity of DES by increasing or decreasing the temperature of the system, achieving the combination or rapid separation of the DES phase and the water phase, and improving the DES extraction and recovery process are of great significance to promote the further application of low eutectic solvents in the field of chitin extraction. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides an extraction system based on a temperature-responsive deep eutectic solvent-water (TRSDES-H2O), a method for extracting chitosan using the extraction system, and a method for recovering the temperature-responsive deep eutectic solvent.

[0006] The present invention is achieved through the following technical solutions: A temperature-responsive low eutectic solvent-water extraction system is composed of a temperature-responsive low eutectic solvent and water, wherein the temperature-responsive low eutectic solvent is composed of lidocaine, oleic acid and acetic acid, and the molar ratio of lidocaine, oleic acid and acetic acid is 4-6:2.5-3.5:1.5-2.5; the content of water is 15% to 25% of the weight of the temperature-responsive low eutectic solvent.

[0007] Preferably, the molar ratio of lidocaine, oleic acid and acetic acid is 5:3:2.

[0008] Preferably, the water content is 20% by weight of the temperature-responsive deep eutectic solvent.

[0009] The preparation method based on the temperature-responsive low eutectic solvent-water extraction system is as follows: lidocaine, oleic acid and acetic acid are mixed, heated to 80°C and stirred until transparent to obtain a slightly yellow liquid, which is the temperature-responsive low eutectic solvent; cooled to room temperature, water is added, and stirred evenly at room temperature to obtain the obtained liquid.

[0010] Application of the temperature-responsive low eutectic solvent-water extraction system in extracting chitin.

[0011] A method for extracting chitosan based on a temperature-responsive low eutectic solvent-water extraction system, comprising: mixing a crustacean shell with a temperature-responsive low eutectic solvent-water extraction system, and extracting at room temperature; centrifuging to obtain a precipitate, washing, decolorizing, and drying to obtain chitosan; wherein the weight ratio of the crustacean shell to the temperature-responsive low eutectic solvent is 1:(5-35); Or: mix crustacean shells, acetic acid and water, and pre-extract at room temperature; then add lidocaine and oleic acid, and extract at room temperature; centrifuge to obtain a precipitate, wash, decolorize, and dry to obtain chitin; wherein lidocaine, oleic acid and acetic acid constitute a temperature-responsive low eutectic solvent, the temperature-responsive low eutectic solvent and water constitute a temperature-responsive low eutectic solvent-water extraction system, and the molar ratio of lidocaine, oleic acid and acetic acid is 4-6:2.5-3.5:1.5-2.5; the amount of water used is 15%-25% of the weight of the temperature-responsive low eutectic solvent; the weight ratio of crustacean shells to the temperature-responsive low eutectic solvent is 1:(5-35).

[0012] Furthermore, the crustacean is a prawn.

[0013] Further, the crustacean shells are pretreated as follows: the crustacean shells are washed with water, dried, and crushed to obtain shell powder. Further, the drying temperature is 60° C., and the drying time is 1 to 2 days. Further, after crushing, the shells are sieved to obtain shell powder with a particle size of less than 0.30 mm.

[0014] Furthermore, the weight ratio of the crustacean shell to the temperature-responsive deep eutectic solvent is 1:(10-30), preferably 1:30.

[0015] Furthermore, the pre-extraction is stirring extraction, and the stirring rate is 400-600 rpm.

[0016] Furthermore, the pre-extraction time is 1 to 6 hours, preferably 2 hours.

[0017] Furthermore, the extraction is stirring extraction, and the stirring rate is 400-600 rpm.

[0018] Furthermore, the extraction time is 12 to 48 hours, preferably 24 to 36 hours.

[0019] Furthermore, the centrifugal conditions are: rotation speed 8000 rpm, time 10 min.

[0020] Furthermore, the washing is performed using deionized water at a temperature of 75 to 85°C.

[0021] Furthermore, the decolorization is carried out using a sodium hypochlorite solution with a concentration of 5% (weight percentage) and a solid-liquid ratio of 1:25 (w / v, unit: g / ml), which is a conventional method.

[0022] A method for recovering a temperature-responsive low eutectic solvent is as follows: after the above-mentioned extraction, the supernatant obtained after centrifugation is collected, water is added, the mixture is fully stirred, heated to 80°C and kept warm for 4 to 8 hours; the lower aqueous phase and the precipitate are removed, and the upper layer is the recovered temperature-responsive low eutectic solvent, which can be recycled and reused 1 to 3 times.

[0023] Furthermore, the amount of water added is 20% to 40% of the weight of the supernatant, preferably 30%.

[0024] Furthermore, the heat preservation time is 6 hours.

[0025] The temperature-responsive low eutectic solvent-water extraction system of the present invention is composed of lidocaine, oleic acid, acetic acid and water. When used to extract chitin, it can be extracted in a one-step method or a one-pot two-step method, and has a very good extraction effect (experimental results show that the one-pot two-step method has a better extraction effect). After extraction, the low eutectic solvent can be recovered by changing the hydrophobicity of the solvent by heating, and can be reused 1 to 3 times while still maintaining a high extraction effect. The temperature-responsive low eutectic solvent-water extraction system of the present invention has good application prospects and is of great significance to promoting the industrialization of chitin extraction with low eutectic solvents.

[0026] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0028] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0029] The crustacean shells used in the present invention are taken from fresh prawns and pretreated as follows: the prawn shells are repeatedly soaked and washed in water, dried (dried at 60° C. for 1 day), crushed, and passed through a 50-mesh sieve to obtain prawn shell powder with a particle size of less than 0.30 mm for later use.

[0030] The mineral content of the samples involved in the following examples was determined by ignition weight method, and the protein content was determined by Bradford method.

[0031] The method for determining the demineralization rate is as follows: Weigh 1.0 g of chitosan sample into a porcelain crucible, place the porcelain crucible in a muffle furnace, burn at 525°C for 5 h until constant weight, take out the muffle furnace when the temperature drops below 100°C, and then place the crucible in a desiccator to cool and weigh it. The demineralization rate of the sample is calculated according to the following formula: , .

[0032] Where: m0 is the mass of the crucible, g; m1 is the mass of the crucible and the mineral, g; m2 is the mass of the crucible and the sample, g. M0 is the mineral content in the crustacean shell powder; M is the ash content in the chitin sample.

[0033] The method for determining the deproteinization rate is as follows: Weigh 0.1 g chitosan sample in 1 ml 5% NaOH solution, heat and stir at 95°C for 1 h, then centrifuge the mixture, collect the supernatant for dilution, then take 0.5 ml of dilution and mix with 2.5 ml of staining solution, let stand for 2 to 10 min, and measure the absorbance at 595 nm; finally, calculate the protein content based on the absorbance of the sample combined with the standard curve equation. The deproteinization rate of the sample is calculated according to the following formula: .

[0034] Where: P0 is the protein content in crustacean shell powder; P is the protein content in chitin sample.

[0035] Example 1 Extraction of chitosan (1) Preparation of a temperature-responsive low eutectic solvent-water extraction system: lidocaine, oleic acid and acetic acid were mixed in a molar ratio of 5:3:2, heated to 80°C in a water bath and magnetically stirred until transparent to obtain a slightly yellowish liquid, which is the temperature-responsive low eutectic solvent; cooled to room temperature, 0%, 5%, 10%, 20%, and 30% (weight percentage) of water were added, respectively, and stirred evenly at room temperature to obtain temperature-responsive low eutectic solvent-water extraction systems with different water contents.

[0036] (2) Extraction of chitosan: The shrimp shell powder was mixed with a temperature-responsive low eutectic solvent-water extraction system (the weight ratio of shrimp shell powder to temperature-responsive low eutectic solvent was 1:30, i.e., the solid-liquid ratio), and the mixture was extracted under stirring (500 rpm) for 24 h at room temperature. The precipitate was separated by centrifugation (8000 rpm, 10 min), and the precipitate was placed on a 250-mesh sieve and washed with hot deionized water (80°C) until the residual DES was removed. The precipitate was then decolorized with a sodium hypochlorite solution, washed again, and dried in an oven at 60°C until constant weight was obtained to obtain a chitosan sample.

[0037] It was determined that the temperature-responsive low eutectic solvent-water extraction system with a water content of 20% had the best chitin extraction effect, with a demineralization rate of 80.39% and a deproteinization rate of 67.01%. Therefore, the temperature-responsive low eutectic solvent-water extraction system with a water content of 20% was selected for subsequent experiments.

[0038] Example 2 Extraction of chitosan The difference from Example 1 is that a temperature-responsive low eutectic solvent-water extraction system with a water content of 20% is selected for extraction, and the extraction time is 36 hours, and the rest is the same as Example 1. It is determined that the demineralization rate is 84.39% and the deproteinization rate is 76.40%.

[0039] Example 3 Extraction of chitosan The difference from Example 1 is that a temperature-responsive low eutectic solvent-water extraction system with a water content of 20% is selected for extraction, the solid-liquid ratio is 1:20, the extraction time is 36 hours, and the rest is the same as Example 1. It is determined that the demineralization rate is 83.27% and the deproteinization rate is 71.12%.

[0040] Example 4 Extraction of chitosan The difference from Example 1 is that a temperature-responsive low eutectic solvent-water extraction system with a water content of 20% is selected for extraction, the solid-liquid ratio is 1:10, the extraction time is 36 hours, and the rest is the same as Example 1. It is determined that the demineralization rate is 81.72% and the deproteinization rate is 65.99%.

[0041] Example 5 Extraction of chitosan Shrimp shell powder, acetic acid and water were mixed (the amounts of shrimp shell powder, acetic acid and water were the same as those in Example 2), and pre-extracted for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours under stirring (speed 500 rpm) at room temperature; the precipitate was separated by centrifugation (8000 rpm, 10 min), and the precipitate was placed in a 250-mesh sieve, washed with hot deionized water (80°C) until the residual DES was removed, and then decolorized with a sodium hypochlorite solution, washed again, and placed in a 60°C oven to dry until constant weight to obtain a chitosan sample.

[0042] It was determined that almost all calcium carbonate could be removed by pre-extraction for 2 hours, with a demineralization rate of 97.71%. Therefore, a pre-extraction time of 2 hours was selected for subsequent experiments.

[0043] Example 6 Extraction of chitosan Shrimp shell powder, acetic acid and water were mixed and pre-extracted at room temperature for 2 hours with stirring (speed 500 rpm); then lidocaine and oleic acid were added and extracted at room temperature with stirring (speed 500 rpm) for 24 hours; centrifugation (8000 rpm, 10 min) was performed to separate the precipitate, which was placed in a 250-mesh sieve and washed with hot deionized water (80°C) until the residual DES was removed, and then decolorized with sodium hypochlorite solution, washed again and placed in a 60°C oven to dry until constant weight, to obtain a chitosan sample. Among them, lidocaine, oleic acid and acetic acid constitute a temperature-responsive low eutectic solvent, and the temperature-responsive low eutectic solvent and water constitute a temperature-responsive low eutectic solvent-water extraction system, and the molar ratio of lidocaine, oleic acid and acetic acid is 5:3:2; the amount of water used is 20% of the weight of the temperature-responsive low eutectic solvent; the weight ratio of shrimp shell powder to the temperature-responsive low eutectic solvent (i.e., the material-liquid ratio) is 1:30.

[0044] The demineralization rate was determined to be 98.87% and the deproteinization rate was 87.35%.

[0045] Example 7 Extraction of chitosan This is a parallel test of Example 6. It was determined that the demineralization rate was 98.86% and the deproteinization rate was 87.69%.

[0046] Example 8 Extraction of chitosan Example 7 After extracting chitosan, the temperature-responsive low eutectic solvent is recovered by collecting the supernatant obtained after centrifugation, adding 30% (weight percentage) of water, stirring thoroughly, heating to 80° C. and keeping the temperature at room temperature for 6 hours; removing the lower aqueous phase and the precipitate, and the upper layer is the recovered temperature-responsive low eutectic solvent.

[0047] Extraction of chitosan: The method is the same as Example 7, except that "adding lidocaine and oleic acid" is replaced by "adding the recovered temperature-responsive low eutectic solvent". The demineralization rate is 98.70%, and the deproteinization rate is 87.70%.

[0048] Example 9 Extraction of chitosan After the chitosan is extracted in Example 8, the temperature-responsive low eutectic solvent is recovered in the same manner as in Example 8.

[0049] Extraction of chitosan: The method is the same as Example 7, except that "adding lidocaine and oleic acid" is replaced by "adding the recovered temperature-responsive low eutectic solvent". The demineralization rate is 98.86% and the deproteinization rate is 83.63%.

[0050] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A temperature-responsive deep eutectic solvent-water extraction system, characterized in that: The invention is composed of a temperature-responsive low eutectic solvent and water, wherein the temperature-responsive low eutectic solvent is composed of lidocaine, oleic acid and acetic acid, and the molar ratio of lidocaine, oleic acid and acetic acid is 4-6:2.5-3.5:1.5-2.5; the content of water is 15%-25% of the weight of the temperature-responsive low eutectic solvent.

2. The temperature-responsive deep eutectic solvent-water extraction system according to claim 1, characterized in that: The molar ratio of lidocaine, oleic acid and acetic acid is 5:3:

2.

3. The temperature-responsive deep eutectic solvent-water extraction system according to claim 1, characterized in that: The water content is 20% by weight of the temperature-responsive deep eutectic solvent.

4. The preparation method based on the temperature-responsive deep eutectic solvent-water extraction system according to any one of claims 1 to 3, characterized in that: Mix lidocaine, oleic acid and acetic acid, heat to 80°C and stir until transparent to obtain a slightly yellow liquid, which is a temperature-responsive low eutectic solvent; cool to room temperature, add water, and stir evenly at room temperature to obtain the product.

5. Use of the temperature-responsive deep eutectic solvent-water extraction system according to any one of claims 1 to 3 in extracting chitosan.

6. A method for extracting chitosan based on a temperature-responsive deep eutectic solvent-water extraction system, characterized in that: Mixing the shell of a crustacean with the temperature-responsive low eutectic solvent-water extraction system according to any one of claims 1 to 3, extracting at room temperature; centrifuging to obtain a precipitate, washing, decolorizing, and drying to obtain chitosan; wherein the weight ratio of the shell of the crustacean to the temperature-responsive low eutectic solvent is 1:(5-35); Or: mix the shell of crustacean, acetic acid and water, and pre-extract at room temperature; then add lidocaine and oleic acid, and extract at room temperature; centrifuge to obtain a precipitate, wash, decolorize, and dry to obtain chitin; wherein lidocaine, oleic acid and acetic acid constitute a temperature-responsive low eutectic solvent, the temperature-responsive low eutectic solvent and water constitute a temperature-responsive low eutectic solvent-water extraction system, and the molar ratio of lidocaine, oleic acid and acetic acid is 4-6:2.5-3.5:1.5-2.5; the amount of water used is 15%-25% of the weight of the temperature-responsive low eutectic solvent; the weight ratio of crustacean shell to temperature-responsive low eutectic solvent is 1:(5-35).

7. The method for extracting chitosan based on a temperature-responsive deep eutectic solvent-water extraction system according to claim 6, characterized in that: The crustacean is a prawn; Or, the crustacean shells are pretreated as follows: the crustacean shells are taken, washed with water, dried, and crushed to obtain shell powder.

8. The method for extracting chitosan based on a temperature-responsive deep eutectic solvent-water extraction system according to claim 6, characterized in that: The weight ratio of the crustacean shell to the temperature-responsive low eutectic solvent is 1:(10-30).

9. The method for extracting chitosan based on a temperature-responsive deep eutectic solvent-water extraction system according to claim 6, characterized in that: The pre-extraction is a stirring extraction, the stirring rate is 400-600 rpm, and the time is 1-6 hours or 2 hours; Or: the extraction is stirring extraction, the stirring rate is 400-600 rpm, and the time is 12-48 hours or 24-36 hours.

10. A method for recovering a temperature-responsive deep eutectic solvent, characterized in that: After chitosan is extracted by the method of the claim, the supernatant obtained after centrifugation is collected, water is added, the mixture is fully stirred, heated to 80°C and kept warm for 4 to 8 hours; the lower aqueous phase and precipitate are removed, and the upper layer is the recovered temperature-responsive low eutectic solvent.