Deep eutectic solvent for dissolving collagen as well as preparation method and application of deep eutectic solvent

Abstract

The invention discloses a deep eutectic solvent for dissolving collagen as well as a preparation method and application of the deep eutectic solvent, and belongs to the technical field of collagen solvents. The eutectic solvent for dissolving collagen is formed by combining a hydrogen bond acceptor and a hydrogen bond donor through hydrogen bond acting force. According to the present invention, zinc chloride and other hydrogen bond receptors are combined with formic acid and other hydrogen bond donors to form the new hydrogen bond, such that the structure stability of the eutectic solvent can be ensured, the excellent dissolution effect on collagen can be provided, and compared with other collagen solvents, the collagen solubility is significantly improved, and the collagen dissolution rate is significantly improved under the optimal condition; the solubility of the collagen in the deep-eutectic solvent can reach 27.4%, and meanwhile, the collagen dissolved by the deep-eutectic solvent prepared by the invention can keep a triple-helical structure of collagen, so that the problems that high-concentration collagen dissolution is difficult to realize under a low-temperature condition (0-20 DEG C) and the natural structure of the collagen can not be kept are successfully solved; the method has great potential for expanding the dissolving processing and application field range of the collagen.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen solvents, and in particular to a low eutectic solvent for dissolving collagen, and a preparation method and application thereof. Background Art

[0002] Collagen is an important structural protein in the extracellular matrix, accounting for about one-third of all vertebrate body proteins. The relative molecular mass of collagen is 300,000, and its typical triple helix structure gives collagen many excellent biological properties, such as low antigenicity, biodegradability and good biocompatibility. However, due to the large number of hydrogen bonds, ionic bonds, van der Waals forces and hydrophobic bonds within and between collagen molecules, it is difficult to dissolve in water or general organic solvents, which greatly limits the dissolution processing of collagen and its application areas (such as collagen electrospinning and large-scale preparation and application of electrospun collagen fibers). Therefore, how to efficiently dissolve and prepare collagen solutions through suitable solvents is the premise and basis for the high-value transformation and utilization of collagen.

[0003] Acetic acid solution (pH 2-3) is a common "good solvent" for collagen, but the collagen solution gradually forms a high-molecular entanglement network structure as the collagen concentration increases. When the collagen concentration reaches more than 3%, it will solidify to form a partial gel or even become a complete gel, thereby losing fluidity and solution processing properties.

[0004] The use of ionic liquids to dissolve and prepare high-concentration (>6%) collagen solutions often requires higher temperature conditions (>45°C). As a result, the triple helix structure of collagen will be destroyed to a large extent during the dissolution process, making it difficult to ensure the integrity of the triple helix structure of collagen. In addition, ionic liquids also have problems such as complex synthesis process, long preparation cycle, and high cost.

[0005] Deep eutectic solvent (DES) is a new type of solvent developed on the basis of ionic liquids. The solvent is composed of hydrogen bond donors and hydrogen bond acceptors. The hydrogen bond donors are widely available, of various types, and relatively low in price, which makes the entire solvent cost-effective. In addition, the viscosity of the solvent is greatly reduced due to the addition of hydrogen bond donors. In addition to the advantages of ionic liquids such as non-volatility, safety and stability, deep eutectic solvents also have obvious advantages in viscosity, cost, and preparation methods. Therefore, it is of great significance to seek a DES solvent that can achieve high-concentration collagen dissolution under low temperature conditions and maintain the natural structure of collagen. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a low eutectic solvent for collagen dissolution and its preparation method and application, so as to solve the current problem that it is difficult to achieve high concentration collagen dissolution under low temperature conditions (0-20°C) and maintain the natural structure of collagen.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] A low eutectic solvent for dissolving collagen, wherein the low eutectic solvent is formed by hydrogen bond acceptors and hydrogen bond donors combined by hydrogen bond forces;

[0009] Further, the hydrogen bond acceptor includes any one of zinc chloride, magnesium chloride and copper chloride;

[0010] Further, the hydrogen bond donor includes any one of formic acid, acetic acid, propionic acid, oxalic acid and lactic acid.

[0011] The beneficial effects of the present invention are as follows: zinc chloride and the like are used as hydrogen bond acceptors in the present invention, and can undergo molecular interactions such as hydrogen bonds and electrostatic interactions with active substances; formic acid and the like in the hydrogen bond donors contain carboxyl groups, and have a good dissolving effect on active substances of different polarities; new hydrogen bonds are formed by combining the hydrogen bond acceptors and the hydrogen bond donors, which can not only ensure the structural stability of the low eutectic solvent, but also have an excellent dissolving effect on collagen; compared with other collagen solvents, the solubility of collagen under low temperature conditions is significantly improved; and at the same time, the low eutectic solvent prepared by the present invention can maintain the triple helix structure of collagen, and has great potential for expanding the dissolution processing and application range of collagen.

[0012] Furthermore, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:3-6.

[0013] Preferably, the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:5.

[0014] The method for preparing the low eutectic solvent for dissolving collagen comprises the following steps: mixing and stirring a hydrogen bond acceptor and a hydrogen bond donor to form a transparent and uniform liquid.

[0015] Furthermore, the mixing and stirring is performed at a temperature of 40 to 60° C. and for a time of 30 to 60 minutes.

[0016] The above-mentioned low eutectic solvent for dissolving collagen is used as a collagen solvent.

[0017] A collagen solvent comprises the above-mentioned low eutectic solvent for dissolving collagen.

[0018] Furthermore, the above collagen solvent also includes water.

[0019] The beneficial effects of the present invention are as follows: the present invention combines a low eutectic solvent prepared with hydrogen bond acceptors such as zinc chloride and hydrogen bond donors such as formic acid as raw materials with water, thereby significantly improving the dissolution effect of the low eutectic solvent on collagen. When the mass fraction of water reaches 40%, the solubility of collagen under low temperature conditions (0-20°C) is increased from 11.5% to 27.4%.

[0020] Furthermore, the mass of water is 0 to 90% of the mass fraction of the collagen solvent.

[0021] Preferably, the mass of water is 30% to 50% of the mass fraction of the collagen solvent.

[0022] A method for preparing regenerated collagen comprises the following steps: firstly dissolving collagen in the above collagen solvent at 0-20°C, centrifugally separating the supernatant, then dialyzing the obtained solution, and finally freeze-drying the solution.

[0023] Furthermore, the collagen is any one of cowhide collagen, pig skin collagen, cow Achilles tendon collagen, pig Achilles tendon collagen, fish skin collagen, fish scale collagen, cow cartilage collagen and pig cartilage collagen.

[0024] Furthermore, the dialysis uses a 6-8 kDa dialysis bag, and the dialysis time is 3-7 days.

[0025] A regenerated collagen is prepared by adopting the above preparation method.

[0026] The present invention has the following beneficial effects:

[0027] 1. In the present invention, zinc chloride and the like are used as hydrogen bond acceptors, which can interact with active substances through hydrogen bonds and electrostatic interactions. Formic acid and the like in the hydrogen bond donors contain carboxyl groups, which have a good dissolving effect on active substances of different polarities.

[0028] 2. The present invention combines hydrogen bond acceptors and hydrogen bond donors to form new hydrogen bonds, which not only ensures the structural stability of the low eutectic solvent, but also has an excellent dissolving effect on collagen. Compared with other collagen solvents, the low eutectic solvent prepared by the present invention significantly improves the solubility of collagen. Under optimal conditions, the solubility of collagen in the low eutectic solvent can reach 27.4%.

[0029] 3. At the same time, the collagen dissolved in the low eutectic solvent prepared by the present invention can maintain the triple helix structure of collagen, successfully solving the problem of difficulty in achieving high-concentration collagen dissolution under low temperature conditions (0-20°C) and maintaining the natural structure of collagen, and has great potential for expanding the dissolution processing and application range of collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 FTIR spectra of the untreated collagen sample and the regenerated collagen in Experimental Example 1, wherein C is the untreated collagen sample, and 3-5 are the regenerated collagen obtained in Examples 5-7 respectively;

[0031] Figure 21 is the SDS-PAGE gel electrophoresis diagram of the untreated collagen sample and the regenerated collagen in Experimental Example 1, wherein M is the standard sample, C is the untreated collagen sample, and 3-5 are the regenerated collagens obtained in Examples 5-7 respectively;

[0032] Figure 3 1 is the XRD spectrum of the untreated collagen sample and the regenerated collagen in Experimental Example 1, wherein C is the untreated collagen sample, and 3-5 are the regenerated collagen obtained in Examples 5-7 respectively;

[0033] Figure 4 3-5 are DSC graphs of the untreated collagen sample and the regenerated collagen in Experimental Example 1, wherein C is the untreated collagen sample, and 3-5 are the regenerated collagens obtained in Examples 5-7 respectively. DETAILED DESCRIPTION

[0034] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0035] Embodiment 1:

[0036] A method for preparing a low eutectic solvent for collagen dissolution comprises the following steps:

[0037] The hydrogen bond acceptor zinc chloride and the hydrogen bond donor formic acid were mixed in a molar ratio of 1:3, and then stirred at 50° C. for 35 minutes to form a uniform transparent liquid. The liquid was cooled at room temperature to obtain a low eutectic solvent DES, which was stored in a brown bottle for later use.

[0038] The process of dissolving collagen with the above-mentioned deep eutectic solvent comprises the following steps:

[0039] Bovine collagen was placed in DES and stirred in an ice-water bath until the collagen no longer dissolved, and then the mixture was centrifuged for 5 minutes using a low-temperature centrifuge to separate the undissolved collagen and the collagen-DES solution. The undissolved collagen was washed with anhydrous ethanol several times to remove the DES, and the undissolved collagen was freeze-dried and weighed, and the solubility was calculated to be 4.13%.

[0040] The collagen-DES solution was transferred to a 6-8 kDa dialysis bag, and then immersed in distilled water for dialysis. The distilled water was changed several times during the dialysis. After 3 days, the retained solution was poured into a polytetrafluoroethylene mold and vacuum freeze-dried using a freeze dryer to obtain regenerated collagen.

[0041] Embodiment 2:

[0042] A method for preparing a low eutectic solvent for collagen dissolution comprises the following steps:

[0043] The hydrogen bond acceptor magnesium chloride and the hydrogen bond donor acetic acid were mixed in a molar ratio of 1:4, and then stirred at 40°C for 60 minutes to form a uniform transparent liquid. The liquid was cooled at room temperature to obtain a low eutectic solvent DES, which was stored in a brown bottle for later use.

[0044] The process of dissolving collagen with the above-mentioned deep eutectic solvent comprises the following steps:

[0045] The pig skin collagen was placed in DES and stirred in an ice water bath until the collagen no longer dissolved, and then the mixture was centrifuged for 5 minutes using a low-temperature centrifuge to separate the undissolved collagen and the collagen-DES solution. The undissolved collagen was washed with anhydrous ethanol several times to remove the DES, and the undissolved collagen was freeze-dried and weighed, and the solubility was calculated to be 9.05%.

[0046] The collagen-DES solution was transferred to a 6-8 kDa dialysis bag, and then immersed in distilled water for dialysis. The distilled water was changed several times during the dialysis. After 7 days, the retained solution was poured into a polytetrafluoroethylene mold and vacuum freeze-dried using a freeze dryer to obtain regenerated collagen.

[0047] Embodiment 3:

[0048] A method for preparing a low eutectic solvent for collagen dissolution comprises the following steps:

[0049] The hydrogen bond acceptor copper chloride and the hydrogen bond donor oxalic acid were mixed in a molar ratio of 1:5, and then stirred at 60°C for 30 minutes to form a uniform transparent liquid. The liquid was cooled at room temperature to obtain a low eutectic solvent DES, which was stored in a brown bottle for later use.

[0050] The process of dissolving collagen with the above-mentioned deep eutectic solvent comprises the following steps:

[0051] Fish skin collagen was placed in DES and stirred in an ice water bath until the collagen no longer dissolved, and then the mixture was centrifuged for 5 minutes using a low-temperature centrifuge to separate the undissolved collagen and the collagen-DES solution. The undissolved collagen was washed with anhydrous ethanol for multiple times to remove the DES, and the undissolved collagen was freeze-dried and weighed, and the solubility was calculated to be 11.20%.

[0052] The collagen-DES solution was transferred to a 6-8 kDa dialysis bag, and then immersed in distilled water for dialysis. The distilled water was changed several times during the dialysis. After 5 days, the retained solution was poured into a polytetrafluoroethylene mold and vacuum freeze-dried using a freeze dryer to obtain regenerated collagen.

[0053] Embodiment 4:

[0054] A method for preparing a low eutectic solvent for collagen dissolution comprises the following steps:

[0055] The hydrogen bond acceptor zinc chloride and the hydrogen bond donor lactic acid were mixed in a molar ratio of 1:6, and then stirred at 50° C. for 40 minutes to form a uniform transparent liquid. The liquid was cooled at room temperature to obtain a low eutectic solvent DES, which was stored in a brown bottle for later use.

[0056] The process of dissolving collagen with the above-mentioned deep eutectic solvent comprises the following steps:

[0057] Bovine Achilles tendon collagen was placed in DES and stirred in an ice water bath until the collagen no longer dissolved, and then the mixture was centrifuged for 5 minutes using a low-temperature centrifuge to separate the undissolved collagen and the collagen-DES solution. The undissolved collagen was washed with anhydrous ethanol several times to remove the DES, and the undissolved collagen was freeze-dried and weighed, and the solubility was calculated to be 10.12%.

[0058] The collagen-DES solution was transferred to a 6-8 kDa dialysis bag, and then immersed in distilled water for dialysis. The distilled water was changed several times during the dialysis. After 3 days, the retained solution was poured into a polytetrafluoroethylene mold and vacuum freeze-dried using a freeze dryer to obtain regenerated collagen.

[0059] Embodiment 5:

[0060] A method for preparing a collagen solvent comprises the following steps:

[0061] The hydrogen bond acceptor magnesium chloride and the hydrogen bond donor propionic acid were mixed in a molar ratio of 1:6, and then distilled water with a mass fraction of 30% of the total collagen solvent was added, and then stirred at 50°C for 40 minutes to form a uniform transparent liquid. The collagen solvent was cooled at room temperature and stored in a brown bottle for later use.

[0062] The process of dissolving collagen with the collagen solvent comprises the following steps:

[0063] The cowhide collagen was placed in a collagen solvent and stirred in an ice-water bath until the collagen no longer dissolved, and then the mixture was centrifuged for 5 minutes using a low-temperature centrifuge to separate the undissolved collagen and the collagen solution. The undissolved collagen was washed with anhydrous ethanol several times to remove the collagen solvent, and the undissolved collagen was freeze-dried and weighed, and the solubility was calculated to be 23.28%.

[0064] The collagen solution was transferred to a 6-8 kDa dialysis bag and then immersed in distilled water for dialysis. The distilled water was changed several times during the dialysis. After 3 days, the retained solution was poured into a polytetrafluoroethylene mold and vacuum freeze-dried using a freeze dryer to obtain regenerated collagen.

[0065] Embodiment 6:

[0066] A method for preparing a collagen solvent comprises the following steps:

[0067] The hydrogen bond acceptor zinc chloride and the hydrogen bond donor formic acid were mixed in a molar ratio of 1:5, and then distilled water with a mass fraction of 40% of the total collagen solvent was added, and then stirred at 60°C for 30 minutes to form a uniform transparent liquid. The collagen solvent was cooled at room temperature and stored in a brown bottle for later use.

[0068] The process of dissolving collagen with the collagen solvent comprises the following steps:

[0069] Fish skin collagen was placed in a collagen solvent and stirred in an ice water bath until the collagen no longer dissolved, and then the mixture was centrifuged for 5 minutes using a low-temperature centrifuge to separate the undissolved collagen and the collagen solution. The undissolved collagen was washed with anhydrous ethanol for multiple times to remove the collagen solvent, and the undissolved collagen was freeze-dried and weighed, and the solubility was calculated to be 27.44%.

[0070] The collagen solution was transferred to a 6-8 kDa dialysis bag and then immersed in distilled water for dialysis. The distilled water was changed several times during the dialysis. After 5 days, the retained solution was poured into a polytetrafluoroethylene mold and vacuum freeze-dried using a freeze dryer to obtain regenerated collagen.

[0071] Embodiment 7:

[0072] A method for preparing a collagen solvent comprises the following steps:

[0073] The hydrogen bond acceptor copper chloride and the hydrogen bond donor oxalic acid were mixed in a molar ratio of 1:3, and then distilled water with a mass fraction of 10% of the total collagen solvent was added, and then stirred at 50°C for 50 minutes to form a uniform transparent liquid. The collagen solvent was cooled at room temperature and stored in a brown bottle for later use.

[0074] The process of dissolving collagen with the collagen solvent comprises the following steps:

[0075] The fish scale collagen was placed in a collagen solvent and stirred in an ice water bath until the collagen no longer dissolved, and then the mixture was centrifuged for 5 minutes using a low-temperature centrifuge to separate the undissolved collagen and the collagen solution. The undissolved collagen was washed with anhydrous ethanol several times to remove the collagen solvent, and the undissolved collagen was freeze-dried and weighed, and the solubility was calculated to be 22.46%.

[0076] The collagen solution was transferred to a 6-8 kDa dialysis bag and then immersed in distilled water for dialysis. The distilled water was changed several times during the dialysis. After 3 days, the retained solution was poured into a polytetrafluoroethylene mold and vacuum freeze-dried using a freeze dryer to obtain regenerated collagen.

[0077] Test Example 1: Determination of physical and chemical properties

[0078] 1. Experimental Methods

[0079] (1) Infrared spectroscopy (FTIR) test

[0080] The collagen and regenerated collagen samples were ground with potassium bromide in a ratio of 1:100 in an agate mortar until the samples and potassium bromide were evenly ground. The ground powder was placed in a mold and pressed into a sheet by strong pressure. The pressed sheet sample was placed in a Fourier transform infrared spectrometer and then measured at 400 cm -1 Up to 4000cm -1 The range is 4cm -1 The sample was scanned at a resolution of .

[0081] (2) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0082] The collagen and regenerated collagen samples were dissolved in 0.1 mol / L acetic acid and sample treatment solution was added to make the concentration reach 1 mg / mL, boiled for 5 min and used for later use. According to the SDS-PAGE analysis method, 4% stacking gel and 7.5% separation gel were used for electrophoresis at 12 mA current, and 0.25% Coomassie Brilliant Blue R250 was used for staining for 30 min, and then decolorized with 7.5% acetic acid and 5% methanol solution.

[0083] (3) X-ray diffraction (XRD) test

[0084] The collagen and regenerated collagen samples were pressed into thin films, and the XRD spectra of the samples were measured by X-ray diffractometer, with CuKα as the ray source, tube voltage of 40 kV, tube current of 30 mA, and angle scanning range of 5° to 50°.

[0085] (4) Differential Scanning Calorimetry (DSC)

[0086] DSC test was performed on collagen and regenerated collagen samples. The test conditions were: weigh 3-4 mg of sample, place in a 40 μL special crucible, use a blank crucible as a reference cell, heat up at a rate of 5°C / min, scan temperature in the range of 20-120°C, and record the absorption curve.

[0087] 2. Experimental Results

[0088] (1) Infrared spectroscopy (FTIR)

[0089] The experimental results are as follows Figure 1 And as shown in Table 1.

[0090] Table 1 A of different collagen samples 1450 / A 1240 Absorption ratio

[0091]

[0092] Figure 1 Medium 3300cm -1 、2938cm -1 、1550cm -1 、1240cm -1 Nearby are the stretching vibration of amide A with NH in collagen molecule, the stretching vibration of amide B with NH, the bending vibration of NH with Ⅱ, and the deformation vibration of NH with Ⅲ; while 1650cm -1 Nearby is the stretching vibration of amide I with C=O. Figure 1 It can be seen that compared with the collagen sample not treated with DES, the regenerated collagen has a corresponding absorption peak at the same wave number. Due to the strong hydrogen bonding effect of DES, the amide A band and amide I vibration peak of the regenerated collagen will move to a lower frequency compared with the collagen sample, indicating that the regenerated collagen will be affected by the strong hydrogen bonding effect of DES, and the regenerated collagen still has the same functional groups and the structure remains intact without functional group changes. Collagen 1450cm -1 The ratio of the absorbance of the amide III band (denoted as A 1450 / A 1240 ) can also be used to assess the integrity of the collagen triple helix structure. Untreated collagen samples have a complete triple helix structure, with A 1450 / A 1240 The ratio is 0.988, which is close to 1.0. 1450 / A 1240 The ratio is relatively the highest, very close to the untreated collagen sample, while the A of the regenerated collagen in Examples 5 and 7 is 1450 / A 1240 The ratio is slightly lower, indicating that the triple helix structure of the regenerated collagen sample is basically intact.

[0093] (2) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0094] like Figure 2 As shown in the figure, the electrophoresis diagram of all collagen samples has two obvious bands around 100kD, which are α 1 Chain and α 2Chain, a band appeared near 250kD, which was the β chain formed by the aggregation of two α chains. Obviously, the electrophoresis band distribution of regenerated collagen was similar to that of untreated collagen samples, and no small molecule bands appeared, indicating that collagen was dissolved in DES without degradation, and the collagen subunit structure remained stable.

[0095] (3) X-ray diffraction (XRD)

[0096] Depend on Figure 3 It can be seen that the XRD spectrum of untreated collagen has two main diffraction peaks, both of which are characteristic peaks of natural collagen: one is at 2θ of 7°~8° (representing the triple helix conformation), which is the diffraction peak generated by the collagen crystal region; the other is a very wide peak near 2θ of 22°, representing the diffuse scattering caused by the internal structure of collagen. The characteristic peak intensity of regenerated collagen is weak, indicating that the strong hydrogen bonding effect of DES will have a certain effect on the spatial structure of collagen, which corresponds to the results of FTIR.

[0097] (4) Differential Scanning Calorimetry (DSC)

[0098] Depend on Figure 4 It can be seen that the thermal denaturation temperature of the untreated collagen sample is 73.0°C. Relatively speaking, the thermal denaturation temperature of the regenerated collagen samples has decreased, indicating that the spatial structure of the regenerated collagen has been damaged to a certain extent, which corresponds to the results of FTIR and XRD. Nevertheless, all the regenerated collagen samples have shown good thermal stability, indicating that the collagen dissolved by DES prepared by the present invention can basically maintain its triple helical structure. Among them, the collagen regenerated by DES with a molar ratio of zinc chloride and formic acid of 1:5 and a water content of 40% has the highest thermal denaturation temperature and the most intact structure.

[0099] In summary, the present invention has prepared a DES that has high solubility for collagen under low temperature conditions, and the addition of appropriate water can significantly improve the dissolution effect of DES on collagen. When the water content is 40% (mass fraction), the solubility of collagen is as high as 27.4%. Characterization tests were conducted on regenerated collagen, and the results showed that although its thermal stability decreased slightly, the test results showed that the regenerated collagen basically maintained its natural structure, which shows that the DES is a good collagen solvent and has great potential for broadening the processing and application range of collagen.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A deep eutectic solvent for dissolving collagen, characterized in that: The deep eutectic solvent is formed by combining a hydrogen bond acceptor and a hydrogen bond donor through hydrogen bonding forces; The hydrogen bond acceptor includes any one of zinc chloride, magnesium chloride and copper chloride; The hydrogen bond donor includes any one of formic acid, acetic acid, propionic acid, oxalic acid and lactic acid.

2. The deep eutectic solvent for dissolving collagen according to claim 1, characterized in that: The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:3-6.

3. The method for preparing the deep eutectic solvent for dissolving collagen according to claim 1 or 2, characterized in that: The method comprises the following steps: mixing and stirring a hydrogen bond acceptor and a hydrogen bond donor to form a transparent and uniform liquid.

4. The method for preparing a deep eutectic solvent for dissolving collagen according to claim 3, characterized in that: The mixing and stirring is performed at a temperature of 40 to 60° C. and for a time of 30 to 60 minutes.

5. Use of the low eutectic solvent for dissolving collagen according to claim 1 or 2 as a collagen solvent.

6. A collagen solvent, characterized in that The collagen solvent includes the low eutectic solvent for dissolving collagen according to claim 1 or 2.

7. The collagen solvent according to claim 6, characterized in that It also includes water; wherein the mass of the water is 0 to 90% of the mass fraction of the collagen solvent.

8. A method for preparing regenerated collagen, characterized in that: The following steps are involved: The collagen is first dissolved in the collagen solvent according to claim 6 or 7 at 0-20°C, the supernatant is separated by centrifugation, the obtained solution is then dialyzed, and finally freeze-dried to obtain the product.

9. The method for preparing regenerated collagen according to claim 8, characterized in that: The collagen is any one of cowhide collagen, pig skin collagen, cow Achilles tendon collagen, pig Achilles tendon collagen, fish skin collagen, fish scale collagen, cow cartilage collagen and pig cartilage collagen; the dialysis uses a 6-8kDa dialysis bag, and the dialysis time is 3-7 days.

10. A regenerated collagen, characterized in that: The method is prepared according to claim 8 or 9.

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