Reduced natural eutectic solvent as well as preparation method and application thereof

Abstract

The invention relates to a reduced natural eutectic solvent as well as a preparation method and application thereof. The method comprises the following steps: mixing citric acid serving as a hydrogen bond donor, L-cysteine serving as a hydrogen bond acceptor and deionized water, and reacting to obtain a mixed solvent, namely the reduced natural eutectic solvent. The reduced natural eutectic solvent is used for extracting keratin from a keratin-based biomass raw material. Compared with the prior art, citric acid and L-cysteine with reducibility are respectively used as a hydrogen bond donor and a hydrogen bond acceptor to prepare the reduced natural deep-eutectic solvent, and efficient dissolution of natural keratin-based biomass can be realized under normal pressure and mild conditions; and water-insoluble keratin and water-soluble keratin can be respectively obtained through a method of combining centrifugal separation and freeze drying, so that the purposes of environmental friendliness and energy consumption reduction are achieved, and the obtained water-insoluble keratin and water-soluble keratin have good cytocompatibility.

Description

Technical Field

[0001] The invention belongs to the technical field of dissolution-regeneration of protein-based biomass, and in particular relates to a reduced natural low eutectic solvent and a preparation method and application thereof. Background Art

[0002] In the context of sustainable development, the development of natural biopolymer materials that can replace petroleum-based materials based on green solvent systems can effectively cope with the increasingly severe environmental pollution and energy crisis. Keratin is a protein-based natural polymer with excellent biocompatibility, degradability and self-assembly properties. It is present in large quantities in protein-based biomass such as hair, feathers and wool. Keratin is a fibrous structural protein with highly ordered secondary structures (such as α-helices and β-folds), which are stabilized by a large number of disulfide bonds, hydrogen bonds and hydrophobic interactions. Keratin materials usually have a high protein content (up to 90%) and a higher cystine content (7-13%) compared to other structural proteins. It is precisely because of the complex structure of keratin that it is difficult to dissolve in conventional solvents, thus limiting its high-value utilization. Currently, a variety of methods for extracting keratin from keratin-based biomass have been developed, which can be mainly summarized as acid-base method, reduction method, oxidation method and enzymatic method. These methods have obvious advantages, but the disadvantages such as high reaction temperature, toxic solvents, high cost and long reaction cycle are all unfavorable factors restricting the industrialization process of keratin extraction.

[0003] With the development of green chemistry, deep eutectic solvents (DES) have been gradually applied in the field of keratin processing as "designable" green solvents. DES is a low melting point mixture composed of hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) in a certain stoichiometric ratio. It has many unique properties, such as low vapor pressure, good thermal stability, biodegradability and designability. In recent years, researchers prefer to use natural and environmentally friendly components to prepare DES, also known as natural deep eutectic solvents (NADES), such as certain natural organic acid and organic base combinations. At present, different NADES systems (such as choline chloride / urea and choline chloride / oxalic acid) have been developed to extract keratin, but there is still a problem of high reaction temperature when extracting keratin based on these NADES systems.

[0004] Therefore, it is of great significance to design a new NADES system to achieve efficient extraction of keratin under simple, green and mild conditions.

[0005] Chinese patent CN114213683B discloses a method for preparing a high-concentration keratin eutectic system solution, which belongs to the field of fiber dissolution and regeneration technology. The solution disclosed in the patent believes that the lactic acid / L-cysteine ​​eutectic system is not effective in dissolving and extracting keratin from wool. Based on this, the patent uses a high-efficiency pretreatment liquid to infiltrate the reducing agent into the hair fiber in advance, and then uses a non-reducing eutectic system to dissolve the hair, so that a keratin eutectic system solution with a concentration of 10%-50% can be easily prepared. It has the characteristics of high dissolution efficiency, simple operation, economy and environmental protection, and greatly reduces the difficulty of preparing a high-concentration keratin eutectic system solution. However, the problem with this solution is that the process is complicated, special wool pretreatment steps are required, and ball milling and screening are required after pretreatment. In addition, the final DES treatment is wool powder below 200μm. At the same time, the process of this solution uses too many reagents and the overall process time is relatively long. Summary of the invention

[0006] In view of the current situation that natural deep eutectic solvent (NADES) in the prior art is difficult to efficiently extract keratin under simple, green and mild conditions, the present invention provides a reduced natural deep eutectic solvent and a preparation method and application thereof.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention first provides a method for preparing a reduced natural deep eutectic solvent (reduced NADES), comprising the following steps:

[0009] The citric acid as a hydrogen bond donor, L-cysteine ​​as a hydrogen bond acceptor, and deionized water are mixed and reacted to obtain a mixed solvent, which is a reduced natural deep eutectic solvent.

[0010] In one embodiment of the present invention, the molar ratio of citric acid to L-cysteine ​​is (1-5):1.

[0011] In one embodiment of the present invention, the amount of deionized water is 10% to 40% of the total mass of citric acid and L-cysteine.

[0012] In one embodiment of the present invention, the reaction conditions are: reaction at 70-90° C. for 30-120 min.

[0013] In one embodiment of the present invention, continuous stirring is performed during the reaction.

[0014] In one embodiment of the present invention, the stirring is magnetic stirring.

[0015] In one embodiment of the present invention, the reduced natural deep eutectic solvent is clear and transparent.

[0016] The present invention further provides a reduced natural deep eutectic solvent prepared based on the above method.

[0017] The present invention further provides the use of the reduced natural deep eutectic solvent prepared by the above method, wherein the reduced natural deep eutectic solvent is used to extract keratin from keratin-based biomass raw materials. This solvent system causes the keratin-based biomass to swell in the early stage of the reaction, causing its structure to be destroyed, thereby increasing the contact area between the solvent and the keratin-based biomass. In the further reaction, the role of L-cysteine ​​is mainly to destroy the disulfide bonds within and between keratin molecules, while the role of citric acid is mainly to break and destroy the peptide bonds, hydrogen bonds, salt bridges and hydrophobic interactions within and between keratin molecules (see Figure 1 b). In addition, the reducing and acidic properties of citric acid make L-cysteine ​​more stable and less likely to cause oxidation of L-cysteine ​​itself, thus maximizing the effect of L-cysteine.

[0018] In one embodiment of the present invention, the keratin-based biomass raw material is selected from natural keratin-based biomass such as hair, wool, waste wool fabrics, and nails.

[0019] In one embodiment of the present invention, the method for extracting keratin from keratin-based biomass raw materials using the reduced natural deep eutectic solvent comprises the following steps:

[0020] 1) Cleaning and degreasing of keratin-based biomass raw materials;

[0021] 2) using a reduced natural deep eutectic solvent to dissolve the keratin-based biomass to obtain a keratin solution;

[0022] 3) The keratin solution is dialyzed, and solid-liquid separation is performed after dialysis to obtain keratin from the solid and the supernatant respectively.

[0023] In one embodiment of the present invention, in step 1), the method for cleaning and degreasing the keratin-based biomass raw material is:

[0024] The keratin-based biomass raw material is washed with clean water, and after the raw material is dried, the dried raw material is extracted with a mixed reagent of n-hexane-dichloromethane for 16 to 24 hours to remove the oil in the keratin-based raw material.

[0025] In one embodiment of the present invention, in the n-hexane-dichloromethane mixed reagent, the volume ratio of n-hexane to dichloromethane is 1:1.

[0026] In one embodiment of the present invention, before performing step 2) of dissolving the keratin-based biomass using a reduced natural deep eutectic solvent, the keratin-based biomass raw material is subjected to a miniaturized treatment.

[0027] In one embodiment of the present invention, the miniaturization treatment of the keratin-based biomass raw material can be performed before or after the cleaning and degreasing treatments.

[0028] In one embodiment of the present invention, an optional operation method for miniaturizing the keratin-based biomass raw material is to crush the keratin-based biomass raw material (for example, by shearing) to make the length or particle size of the keratin-based biomass raw material 0.5 to 1.5 cm.

[0029] In one embodiment of the present invention, in step 2), when the keratin-based biomass is dissolved using a reduced natural deep eutectic solvent, the solid-to-liquid ratio of the keratin-based biomass in the reduced natural deep eutectic solvent is 1% to 20%.

[0030] In one embodiment of the present invention, in step 2), when the reduced natural deep eutectic solvent is used to dissolve the keratin-based biomass, the process is carried out under heating conditions, the heating conditions are 80-110° C., and the reaction time is 2h-12h.

[0031] In one embodiment of the present invention, in step 2), when the reduced natural deep eutectic solvent is used to dissolve the keratin-based biomass, continuous stirring is performed, and the stirring can be selected as magnetic stirring.

[0032] In one embodiment of the present invention, in step 3), before dialyzing the keratin solution, solid-liquid separation is first performed to remove undissolved keratin-based biomass raw materials. The solid-liquid separation method can be selected to filter using a filter, for example, a 800-mesh nylon filter.

[0033] In one embodiment of the present invention, in step 3), when the keratin solution is dialyzed, a dialysis bag with a molecular weight cutoff of 3500Da is selected for dialysis, and the dialysis is performed for 72 to 96 hours, and the deionized water is replaced every 12 hours.

[0034] In one embodiment of the present invention, in step 3), solid-liquid separation is performed by centrifugation after dialysis, and the solid and the supernatant are separated and freeze-dried respectively. After the solid is freeze-dried, water-insoluble keratin (WIK) is obtained, and after the supernatant is freeze-dried, water-soluble keratin (WSK) is obtained.

[0035] Since keratin macromolecules are mainly connected by disulfide bonds, they constitute insoluble proteins. The key to keratin extraction is to break the cross-linked disulfide bonds (RSSR) between molecules. Therefore, the present application scheme introduces reducing components when designing NADES, and uses reducing citric acid and L-cysteine ​​as hydrogen bond donors and hydrogen bond acceptors, respectively, to prepare reduced NADES, which is more conducive to breaking the disulfide bonds between keratin molecules, thereby achieving the purpose of efficient dissolution of keratin-based biomass.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] The reducing natural deep eutectic solvent of the present application is mainly composed of citric acid, L-cysteine ​​and deionized water, with simple raw materials and green and environmentally friendly.

[0038] The present application provides a reduced natural low eutectic solvent and a preparation method and application thereof, wherein reduced NADES is prepared by using reducing citric acid and L-cysteine ​​as hydrogen bond donors and hydrogen bond acceptors, respectively, and efficient dissolution of natural keratin-based biomass can be achieved under normal pressure and mild conditions, and WIK and WSK can be obtained respectively by a combination of centrifugal separation and freeze-drying, thereby achieving the purpose of being environmentally friendly and reducing energy consumption.

[0039] By using the reduced natural low eutectic solvent provided in the present application to extract keratin from keratin-based biomass raw materials, the yield of WIK from wool can reach 58%, and that of WSK can reach nearly 18%. The yield of WIK from hair can reach nearly 60%, and that of WSK can reach nearly 19%. High-yield keratin can be obtained, which can further broaden the application field of keratin. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 (a) The preparation process and conditions of reduced NADES prepared by citric acid (CA) and L-cysteine ​​(L-Cys) in different ratios and (b) the action mechanism between citric acid and L-cysteine ​​and keratin molecules.

[0041] Figure 2 The basic properties of reduced NADES and the interactions between its components. Figures (a) and (b) show the thermal properties of NADES and the viscosity at different temperatures, respectively. Figures (c), (d) and (e) show the hydrogen bonding interactions, non-bonding interactions and three-dimensional density distribution of solvents between the components of the system at different ratios obtained by molecular dynamics simulation.

[0042] Figure 3Cell compatibility of (a) water-soluble wool keratin (WSK) and (b) water-insoluble wool keratin (WIK) obtained under the condition of a CA to L-Cys molar ratio of 2:1.

[0043] Figure 4 These are pictures of the dissolved state of various keratin-based biomass (wool, sweaters, nails, duck feathers and hair) dissolved by reduced NADES, as well as the corresponding WIK and WSK pictures obtained under various dissolution conditions. DETAILED DESCRIPTION

[0044] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] The present invention is further described in detail below in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto.

[0046] The protein content in the raw materials was determined by the Kjeldahl method, and the calculation formula for WIK and WSK yields was:

[0047]

[0048] Example 1

[0049] A preparation and application of a reduced natural deep eutectic solvent (NADES) comprises the following steps:

[0050] 1) Preparation of NADES, the preparation process is referenced Figure 1 (a):

[0051] Weigh a certain amount of citric acid (hydrogen bond donor) and L-cysteine ​​(hydrogen bond acceptor) in a reaction bottle (the molar ratio of the two is 1:1), and then add deionized water of 20% of the total mass of the two to the reaction bottle, place the reaction bottle under 85°C heating conditions, react for 60 minutes, and continuously stir with a magnetic stirrer until a clear and transparent mixed solvent is obtained, namely NADES. The viscosity of the solvent obtained under this condition at 25°C is 12.68 Pa·s;

[0052] 2) Cleaning and degreasing of wool raw materials:

[0053] Wool was selected as the raw material, and cleaned with water. After the raw material was dried, a mixed reagent of n-hexane and dichloromethane (volume ratio 1:1) was used to extract the dried raw material for 16 hours to remove the lanolin;

[0054] 3) Miniaturization of wool raw materials:

[0055] Use scissors to cut the degreased wool obtained by extraction in step 2) into pieces with a length of about 1 cm;

[0056] 4) NADES dissolving wool:

[0057] A certain amount of NADES prepared in step 1) is placed in a pressure bottle, and the shredded wool material in step 3) is added to the pressure bottle at a solid-liquid ratio of 2%, and the entire reaction system is heated at 90° C. for 3 hours with continuous magnetic stirring;

[0058] 5) Keratin regeneration:

[0059] The wool solution in the pressure bottle of step 4) was filtered through 800 mesh nylon to remove the undissolved wool, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed for 72 hours, and the deionized water was replaced every 12 hours. After the dialysis was completed, the solid-liquid two-phase separation was achieved by centrifugation, and the solid was separated from the supernatant and freeze-dried separately. After the solid was freeze-dried, water-insoluble keratin (WIK) was obtained, and after the supernatant was freeze-dried, water-soluble keratin (WSK) was obtained.

[0060] The yields of WIK and WSK based on wool raw material were 48.03%±0.92% and 16.20%±1.30%, respectively.

[0061] Example 2

[0062] A preparation and application of a reduced natural deep eutectic solvent (NADES) comprises the following steps:

[0063] 1) Preparation of NADES, the preparation process is referenced Figure 1 (a):

[0064] Weigh a certain amount of citric acid (hydrogen bond donor) and L-cysteine ​​(hydrogen bond acceptor) in a reaction bottle (the molar ratio of the two is 2:1), and then add 20% of the total mass of deionized water to the reaction bottle, place the reaction bottle under 85°C heating conditions, react for 60 minutes, and continuously stir with a magnetic stirrer until a clear and transparent mixed solvent is obtained, namely NADES. The viscosity of the solvent obtained under this condition at 25°C is 7.79 Pa·s;

[0065] 2) Cleaning and degreasing of wool raw materials:

[0066] Wool was selected as the raw material, and cleaned with water. After the raw material was dried, a mixed reagent of n-hexane and dichloromethane (volume ratio 1:1) was used to extract the dried raw material for 16 hours to remove the lanolin;

[0067] 3) Miniaturization of wool raw materials:

[0068] Use scissors to cut the degreased wool obtained by extraction in step 2) into pieces with a length of about 1 cm;

[0069] 4) NADES dissolving wool:

[0070] A certain amount of NADES prepared in step 1) is placed in a pressure bottle, and the shredded wool material in step 3) is added to the pressure bottle at a solid-liquid ratio of 2%, and the entire reaction system is heated at 90° C. for 3 hours with continuous magnetic stirring;

[0071] 5) Keratin regeneration:

[0072] The wool solution in the pressure bottle of step 4) was filtered through 800 mesh nylon to remove the undissolved wool, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed for 72 hours, and the deionized water was replaced every 12 hours. After the dialysis was completed, the solid-liquid two-phase separation was achieved by centrifugation, and the solid was separated from the supernatant and freeze-dried separately. After the solid was freeze-dried, water-insoluble keratin (WIK) was obtained, and after the supernatant was freeze-dried, water-soluble keratin (WSK) was obtained.

[0073] The yields of WIK and WSK based on wool raw material were 57.82%±1.25% and 17.61%±1.99%, respectively.

[0074] The WSK and WIK obtained in this example were cultured in fibroblasts (NIH3T3) to evaluate ( Figure 3 a) Water-soluble wool keratin (WSK) and ( Figure 3 b) Cytocompatibility of water-insoluble wool keratin (WIK).

[0075] Test method: WIK was immersed in Dulbecco's Modified Eagle Medium (DMEM) for 24 hours at a concentration of 0.1 g / mL at 37°C to obtain an extract. NIH3T3 cells were inoculated in a 96-well plate at a density of 5×10 3 cells / well, 37°C, 5% CO 2 Culture. After the cells were cultured in 100 μL of DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin for 24 h, the medium was replaced with fresh medium containing different concentrations of WSK (0.1, 0.5, 1, 2.5 and 5 mg / mL) and fresh medium containing different dilutions of WIK extract (2X, 4 times, 10 times, 20 times and 100 times). The medium without WSK and WIK extract was used as the control group. Cell viability was determined by CCK-8 method after 1 day and 3 days of culture. The absorbance of the samples was recorded by a microplate reader (Thermo Fisher Scientific, Multiskan FC, USA).

[0076] Results Reference Figure 3After 3 days, the cell viability values ​​at each concentration were all above 70%. Therefore, the WSK ( Figure 3 a) and WIK( Figure 3 b) Both have good cell compatibility.

[0077] Example 3

[0078] A preparation and application of a reduced natural deep eutectic solvent (NADES) comprises the following steps:

[0079] 1) Preparation of NADES, the preparation process is referenced Figure 1 (a):

[0080] Weigh a certain amount of citric acid (hydrogen bond donor) and L-cysteine ​​(hydrogen bond acceptor) in a reaction bottle (the molar ratio of the two is 3:1), and then add deionized water of 20% of the total mass of the two to the reaction bottle, place the reaction bottle under 85°C heating conditions, react for 60 minutes, and continuously stir with a magnetic stirrer until a clear and transparent mixed solvent is obtained, namely NADES. The viscosity of the solvent obtained under this condition at 25°C is 6.72 Pa·s;

[0081] 2) Cleaning and degreasing of wool raw materials:

[0082] Wool was selected as the raw material, and cleaned with water. After the raw material was dried, a mixed reagent of n-hexane and dichloromethane (volume ratio 1:1) was used to extract the dried raw material for 16 hours to remove the lanolin;

[0083] 3) Miniaturization of wool raw materials:

[0084] Use scissors to cut the degreased wool obtained by extraction in step 2) into pieces with a length of about 1 cm;

[0085] 4) NADES dissolving wool:

[0086] A certain amount of NADES prepared in step 1) is placed in a pressure bottle, and the shredded wool material in step 3) is added to the pressure bottle at a solid-liquid ratio of 2%, and the entire reaction system is heated at 90° C. for 3 hours with continuous magnetic stirring;

[0087] 5) Keratin regeneration:

[0088] The wool solution in the pressure bottle of step 4) was filtered through 800 mesh nylon to remove the undissolved wool, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed for 72 hours, and the deionized water was replaced every 12 hours. After the dialysis was completed, the solid-liquid two-phase separation was achieved by centrifugation, and the solid was separated from the supernatant and freeze-dried separately. After the solid was freeze-dried, water-insoluble keratin (WIK) was obtained, and after the supernatant was freeze-dried, water-soluble keratin (WSK) was obtained.

[0089] The yields of WIK and WSK based on wool raw material were 56.28%±0.91% and 12.25%±0.50%, respectively.

[0090] Example 4

[0091] A preparation and application of a reduced natural deep eutectic solvent (NADES) comprises the following steps:

[0092] 1) Preparation of NADES, the preparation process is referenced Figure 1 (a):

[0093] Weigh a certain amount of citric acid (hydrogen bond donor) and L-cysteine ​​(hydrogen bond acceptor) in a reaction bottle (the molar ratio of the two is 3:1), and then add 20% of the total mass of deionized water to the reaction bottle, place the reaction bottle under 90°C heating conditions, react for 50 minutes, and continuously stir with a magnetic stirrer until a clear and transparent mixed solvent is obtained, which is NADES. The viscosity of the solvent obtained under this condition at 25°C is 6.72 Pa·s;

[0094] 2) Wool cleaning and degreasing:

[0095] Choose waste sweaters as raw materials, and wash the wool with clean water until the raw materials are dried;

[0096] 3) Miniaturization of wool yarn:

[0097] Use scissors to cut the wool yarn washed in step 2) into pieces of about 1 cm in length;

[0098] 4) NADES dissolving wool:

[0099] A certain amount of NADES prepared in step 1) was placed in a pressure bottle, and the chopped wool raw material in step 3) was added to the pressure bottle at a solid-liquid ratio of 2%. The entire reaction system was heated at 90° C. for 3.5 hours with continuous magnetic stirring.

[0100] 5) Keratin regeneration:

[0101] The woolen solution in the pressure bottle of step 4) was filtered through 800 mesh nylon to remove the undissolved woolen yarn, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed for 72 hours, and deionized water was replaced every 12 hours. After the dialysis is completed, the solid-liquid two-phase separation is achieved by centrifugation, and the solid is separated from the supernatant and freeze-dried respectively. After the solid is freeze-dried, water-insoluble keratin (WIK) is obtained, and after the supernatant is freeze-dried, water-soluble keratin (WSK) is obtained.

[0102] The yields of WIK and WSK obtained based on waste sweater raw materials were 58.25%±1.85% and 1.58%±0.87%, respectively.

[0103] Example 5

[0104] A preparation and application of a reduced natural deep eutectic solvent (NADES) comprises the following steps:

[0105] 1) Preparation of NADES, the preparation process is referenced Figure 1 (a):

[0106] Weigh a certain amount of citric acid (hydrogen bond donor) and L-cysteine ​​(hydrogen bond acceptor) in a reaction bottle (the molar ratio of the two is 3:1), and then add 20% of the total mass of deionized water to the reaction bottle, place the reaction bottle under 90°C heating conditions, react for 50 minutes, and continuously stir with a magnetic stirrer until a clear and transparent mixed solvent is obtained, which is NADES. The viscosity of the solvent obtained under this condition at 25°C is 6.72 Pa·s;

[0107] 2) Nail cleaning and degreasing:

[0108] Nails were selected as raw materials, cleaned with clean water, and after drying, extracted with a mixture of n-hexane and dichloromethane (volume ratio 1:1) for 16 hours to remove the grease in the nails;

[0109] 3) Smaller nails:

[0110] Use scissors to cut the nails obtained by extraction in step 2) into pieces with a width of about 0.5 cm;

[0111] 4) NADES dissolves nails:

[0112] A certain amount of NADES prepared in step 1) is placed in a pressure bottle, and then the chopped nail material in step 3) is added to the pressure bottle at a solid-liquid ratio of 2%, and the entire reaction system is placed under heating conditions of 105° C. for 6 hours with continuous magnetic stirring;

[0113] 5) Keratin regeneration:

[0114] The nail dissolving solution in the pressure bottle of step 4) was filtered through 800 mesh nylon to remove the undissolved nails, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed for 96 hours, and the deionized water was replaced every 12 hours. After the dialysis is completed, the solid-liquid two-phase separation is achieved by centrifugation, and the solid and the supernatant are separated and freeze-dried respectively. After the solid is freeze-dried, water-insoluble keratin (WIK) is obtained, and after the supernatant is freeze-dried, water-soluble keratin (WSK) is obtained.

[0115] The yields of WIK and WSK based on nail raw materials were 26.10%±1.57% and 6.33%±0.70%, respectively.

[0116] Example 6

[0117] A preparation and application of a reduced natural deep eutectic solvent (NADES) comprises the following steps:

[0118] 1) Preparation of NADES, the preparation process is referenced Figure 1 (a):

[0119] Weigh a certain amount of citric acid (hydrogen bond donor) and L-cysteine ​​(hydrogen bond acceptor) in a reaction bottle (the molar ratio of the two is 3:1), and then add 20% of the total mass of deionized water to the reaction bottle, place the reaction bottle under 90°C heating conditions, react for 50 minutes, and continuously stir with a magnetic stirrer until a clear and transparent mixed solvent is obtained, which is NADES. The viscosity of the solvent obtained under this condition at 25°C is 6.72 Pa·s;

[0120] 2) Cleaning and degreasing of duck feathers:

[0121] Duck feathers were selected as raw materials, washed with clean water, dried, and then extracted with a mixed reagent of n-hexane-dichloromethane (volume ratio 1:1) for 18 hours to remove grease;

[0122] 3) Miniaturization of duck feathers:

[0123] Using scissors, cut the defatted duck feathers obtained by extraction in step 2) into pieces with a length of about 1 cm;

[0124] 4) NADES dissolves duck feathers:

[0125] A certain amount of NADES prepared in step 1) is placed in a pressure bottle, and then the chopped duck feather material in step 3) is added to the pressure bottle at a solid-liquid ratio of 2%, and the entire reaction system is placed under heating conditions of 100° C. for 5 hours with continuous magnetic stirring;

[0126] 5) Keratin regeneration:

[0127] The duck feather solution in the pressure bottle of step 4) was filtered through 800 mesh nylon to remove the undissolved duck feathers, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed for 72 hours, and deionized water was replaced every 12 hours. After the dialysis is completed, the solid-liquid two-phase separation is achieved by centrifugation, and the solids are separated from the supernatant and freeze-dried respectively. After the solids are freeze-dried, water-insoluble keratin (WIK) is obtained, and after the supernatant is freeze-dried, water-soluble keratin (WSK) is obtained.

[0128] The yields of WIK and WSK based on duck feather raw materials were 35.80%±3.14% and 32.25%±0.13%, respectively.

[0129] Example 7

[0130] A preparation and application of a reduced natural deep eutectic solvent (NADES) comprises the following steps:

[0131] 1) Preparation of NADES, the preparation process is referenced Figure 1 (a):

[0132] Weigh a certain amount of citric acid (hydrogen bond donor) and L-cysteine ​​(hydrogen bond acceptor) in a reaction bottle (the molar ratio of the two is 3:1), and then add 20% of the total mass of deionized water to the reaction bottle, place the reaction bottle under 90°C heating conditions, react for 50 minutes, and continuously stir with a magnetic stirrer until a clear and transparent mixed solvent is obtained, which is NADES. The viscosity of the solvent obtained under this condition at 25°C is 6.72 Pa·s;

[0133] 2) Hair washing and degreasing:

[0134] Hair was selected as the raw material, and cleaned with water. After the raw material was dried, a mixed reagent of n-hexane-dichloromethane (volume ratio 1:1) was used to extract the dried raw material for 20 hours to remove the grease;

[0135] 3) Hair thinning:

[0136] Using scissors, cut the degreased hair obtained by extraction in step 2) into pieces with a length of about 1 cm;

[0137] 4) NADES dissolves hair:

[0138] A certain amount of NADES prepared in step 1) is placed in a pressure bottle, and the chopped hair raw material in step 3) is added to the pressure bottle at a solid-liquid ratio of 2%, and the entire reaction system is heated at 100° C. for 5 hours with continuous magnetic stirring;

[0139] 5) Keratin regeneration:

[0140] The hair solution in the pressure bottle in step 4) was filtered through 800 mesh nylon to remove undissolved hair, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed for 72 hours, and deionized water was replaced every 12 hours. After the dialysis was completed, the solid-liquid two-phase separation was achieved by centrifugation, and the solid and the supernatant were separated and freeze-dried respectively. After the solid was freeze-dried, water-insoluble keratin (WIK) was obtained, and after the supernatant was freeze-dried, water-soluble keratin (WSK) was obtained.

[0141] The yields of WIK and WSK based on hair raw materials were 59.51%±0.10% and 18.86%±0.42%, respectively.

[0142] The basic properties of NADES obtained in the above different embodiments and the interactions between the components are as follows Figure 2 As shown, Figure 2 (a) with Figure 2 (b) The thermal properties of NADES obtained from different examples and the viscosity at different temperatures. Figure 2 (c) Figure 2 (d) and Figure 2 (e) The three-dimensional density distribution diagrams of hydrogen bonding interactions, non-bonded interactions and solvents among the components of the system at different ratios obtained by molecular dynamics simulation.

[0143] in, Figure 2 The test method for the thermal properties and viscosity of the NADES solvents obtained in the different embodiments is as follows: the thermal properties and viscosity of the solvents are analyzed using a differential scanning calorimeter (DSC 3+ / 700, Mettler-Toledo, Switzerland) and a rheometer (ARES-G2, TA-Waters, USA).

[0144] The thermal performance analysis was carried out by heating NADES with different ratios from -80℃ to 120℃ at a heating rate of 10℃ / min. 2 The viscosity measurements were performed in a 30% CO atmosphere. The temperature range of the viscosity measurements was 293 K to 363 K, and for each CA to L-Cys molar ratio, the measurements were performed at 9 different temperatures (293 K, 298 K, 303 K, 313 K, 323 K, 333 K, 343 K, 353 K, and 363 K). Molecular dynamics simulations were performed by Gromacs.

[0145] DSC test results ( Figure 2 a) shows that NADES at different molar ratios all exhibit glass transition characteristics, and the glass transition temperature is around -45°C. Viscosity test results ( Figure 2b) shows that when the molar ratio of CA to L-Cys is 2:1 and 3:1, the viscosity of NADES is similar and lower than that of 1:1 mixture. In addition, the viscosity of all formulations decreases with increasing temperature, which is mainly due to the increase in molecular kinetic energy, which in turn promotes the free movement of molecules. Molecular dynamics simulation results show that the CA-water interaction contributes the most to the total number of hydrogen bonds, because CA can easily form hydrogen bonds with water through its carboxyl (-COOH) and hydroxyl (-OH) groups ( Figure 2 c). The total number of hydrogen bonds in the CA:L-Cys / 3:1 and CA:L-Cys / 2:1 systems is similar and slightly higher than that in the CA:L-Cys / 1:1 system. In addition, the non-bonded interaction energy ( Figure 2 d), including van der Waals interactions (E vdW ) and electrostatic interactions (E elec ). The results showed that E elec Significantly stronger than E in all systems vdW . E of CA:L-Cys / 3:1 system vdW The highest is close to -600 kJ / mol. The three-dimensional density distribution shows that ( Figure 2 e), compared with the CA:L-Cys / 1:1 system, the components in the CA:L-Cys / 2:1 and CA:L-Cys / 3:1 systems were more evenly distributed.

[0146] The pictures of the dissolved states of various keratin-based biomasses (wool, sweaters, nails, duck feathers and hair, etc.) using reduced NADES in the above different embodiments and the corresponding WIK and WSK pictures obtained under each dissolution condition are shown in Figure 2. Figure 4 As shown, it can be seen that the reduced NADES can basically dissolve various keratin-based biomass (wool, sweaters, nails, duck feathers and hair), and WIK and WSK can be obtained respectively.

[0147] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a reduced natural deep eutectic solvent, characterized in that: The following steps are involved: The citric acid as a hydrogen bond donor, L-cysteine ​​as a hydrogen bond acceptor, and deionized water are mixed and reacted to obtain a mixed solvent, which is a reduced natural deep eutectic solvent.

2. The method for preparing a reduced natural deep eutectic solvent according to claim 1, characterized in that: The molar ratio of citric acid to L-cysteine ​​is (1-5):1; The amount of the deionized water is 10% to 40% of the total mass of the citric acid and the L-cysteine.

3. The method for preparing a reduced natural deep eutectic solvent according to claim 1, characterized in that: The reaction conditions are: reacting at 70-90°C for 30-120 minutes; Continuous stirring was performed during the reaction.

4. A reduced natural deep eutectic solvent prepared by the method according to any one of claims 1 to 3.

5. Use of the reduced natural deep eutectic solvent prepared by the method according to any one of claims 1 to 3, characterized in that: The reducing natural deep eutectic solvent is used for extracting keratin from keratin-based biomass raw materials.

6. The use according to claim 5, characterized in that: The method for extracting keratin from keratin-based biomass raw materials using a reduced natural deep eutectic solvent comprises the following steps: 1) Cleaning and degreasing of keratin-based biomass raw materials; 2) using a reduced natural deep eutectic solvent to dissolve the keratin-based biomass to obtain a keratin solution; 3) The keratin solution is dialyzed, and solid-liquid separation is performed after dialysis to obtain keratin from the solid and the supernatant respectively.

7. The use according to claim 6, characterized in that: In step 2), when the keratin-based biomass is dissolved in the reduced natural deep eutectic solvent, the solid-liquid ratio of the keratin-based biomass in the reduced natural deep eutectic solvent is 1% to 20%.

8. The use according to claim 6, characterized in that: In step 2), when the reduced natural deep eutectic solvent is used to dissolve the keratin-based biomass, the process is carried out under heating conditions, wherein the heating conditions are 80 to 110° C. and the reaction time is 2 to 12 hours.

9. The use according to claim 6, characterized in that: In step 3), when the keratin solution is dialyzed, a dialysis bag with a molecular weight cutoff of 3500Da is selected for dialysis, and the dialysis is performed for 72 to 96 hours, and the deionized water is replaced every 12 hours.

10. The use according to claim 6, characterized in that: In step 3), after dialysis, solid-liquid separation is performed by centrifugation, and the solid and the supernatant are separated and freeze-dried respectively. After the solid is freeze-dried, water-insoluble keratin is obtained, and after the supernatant is freeze-dried, water-soluble keratin is obtained.

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