Method for dissolving silk fibroin with mixed acid

Abstract

The application provides a method for dissolving silk fibroin by mixed acid, comprising the following steps: S1. degumming silk to obtain degummed silk; S2. mixing organic acid and inorganic acid to obtain a mixed acid solution, and then adding the degummed silk prepared in step S1 into the mixed acid solution to obtain a silk fibroin solution. The application provides a method for dissolving silk fibroin by using organic acid and inorganic acid, which can simply and quickly dissolve silk fibroin, and has the advantages of easy control of conditions, good solution stability, less environmental pollution, and easy recovery and recycling.

Description

Technical Field

[0001] This invention relates to the field of natural polymer materials, and more specifically to a method for dissolving silk fibroin using a neutral salt-mixed acid. Background Technology

[0002] Silk has a wide range of applications. It can be used not only in textiles and clothing but also as a non-textile material. For example, fibroin can be obtained through certain methods and processed into fibroin materials for use in the biomedical field. Mulberry silk is composed of fibroin and sericin. Fibroin contains essential amino acids and can be degraded into low-molecular-weight fibroin peptides and amino acids, which can be used in cosmetics, food, pharmaceuticals, and the medical field.

[0003] Camille et al., in their article "Method of Making Silk Products," disclosed the use of strong inorganic acids to dissolve fibroin. While this method can completely degrade fibroin, the reaction conditions are intense, causing severe damage to amino acids, making the degree of hydrolysis difficult to control, and posing a health hazard. Therefore, it is unsuitable for biomedical applications. Firstly, hydrochloric acid disrupts the hydrogen bonds and van der Waals forces between the polypeptide chains of silk proteins, loosening the silk structure and potentially increasing the breaking elongation. However, as the strength of the hydrochloric acid increases, more pores and gaps form within the silk fibers, increasing weak points and ultimately reducing the breaking elongation.

[0004] Secondly, hydrochloric acid undergoes a chemical reaction in water, producing hydrogen and chloride ions, which increases the polarity of the water. Hydrochloric acid can also act as a catalyst for the hydrolysis of silk. The chloride ions in hydrochloric acid can break the amide bonds connecting polypeptide chains in proteins, decomposing the amino acid sequences in large protein molecules into smaller free amino acids. Hydrochloric acid can also combine with free amino acids to form chelates; for example, the groups in hydrochloric acid react with the tryptophan groups in silk fibroin peptide chains, thus causing the color to gradually deepen when dissolving silk.

[0005] Therefore, using hydrochloric acid to dissolve silk fibroin in silkworm silk to prepare regenerated silk fibroin membranes results in poor performance and significant structural changes. The regenerated silk fibroin membranes are readily soluble in water, have low crystallinity, low initial modulus, and poor mechanical properties; they also crack easily after drying. This is because hydrochloric acid strongly degrades silk, disrupting the hydrogen bonds and van der Waals forces between silk fibers, breaking down the large protein molecules in silk into smaller amino acids. This results in the regenerated silk fibroin membrane lacking macromolecular or intermolecular bonding, leading to its easy solubility in water and its tendency to crack.

[0006] Silk fibroin does not undergo significant changes in formic acid, but it slowly degrades over time. Meanwhile, regenerated silk fibroin is readily soluble in formic acid, indicating that formic acid acts not only as a dispersant but also as a dissolving agent in the regenerated silk fibroin protein during the dissolution process. Simultaneously, Ca... 2+ Li + Strongly polar cations can disrupt the "surface" of silk fibroin fibers under acidic conditions. However, compared to traditional methods, this approach lacks the support of high-temperature energy and cannot break the van der Waals forces and hydrogen bonds of silk fibroin molecules, thus failing to yield nanofibers. Formic acid can slowly dissolve silk fibroin to a certain extent, but rapid dissolution requires the assistance of strongly polar cations. In this case, the presence of strongly polar cations in the solution limits further utilization, while strong acids completely destroy the van der Waals forces and hydrogen bonds of silk fibroin molecules, ensuring the mechanical properties of the recycled material. Summary of the Invention

[0007] The technical problem to be solved: The purpose of this invention is to provide a method for dissolving silk fibroin using organic and inorganic acids, which achieves and balances high solubility, mild dissolution conditions and low solvent consumption. This method can dissolve silk fibroin simply and quickly, and the conditions are easy to control. The solution has good stability, causes little environmental pollution, and is easy to recycle and reuse.

[0008] Technical solution: A method for dissolving silk fibroin with mixed acids, comprising the following steps:

[0009] S1. Degumming is performed on the silk to obtain degummed silk.

[0010] S2. Mix organic acid and inorganic acid to obtain a mixed acid solution, and then add the degummed silk prepared in step S1 to the mixed acid solution to obtain a silk fibroin solution.

[0011] Preferably, the silk is any one or a combination of two or more of mulberry silk, tussah silk, or castor silk.

[0012] Preferably, the organic acid is formic acid, and the inorganic acid is phosphoric acid or hydrochloric acid.

[0013] Preferably, the concentration of the organic acid is 80-100 wt.%, the concentration of the inorganic acid is 0.01-5 wt.%, and the volume ratio of the organic acid to the inorganic acid is 1:1.

[0014] Preferably, the concentration of the silk fibroin solution is 0.1-50 wt.%.

[0015] The regenerated silk fibroin solution prepared by any of the above methods of dissolving silk fibroin with mixed acids.

[0016] The above-mentioned regenerated silk fibroin solution is used in the preparation of regenerated silk fibroin materials.

[0017] Preferably, the regenerated silk fibroin material includes films, filaments, nanofibers, porous scaffolds, and hydrogels.

[0018] Beneficial effects: The method of dissolving silk fibroin with mixed acids of the present invention has the following advantages:

[0019] 1. Under normal conditions, i.e. at room temperature, silk fibroin can be dissolved by mixing a small amount of inorganic acid and organic acid without the need for strong polar cations. This method can dissolve silk fibroin protein simply and quickly, and the conditions are easy to control. The solution has good stability, causes little environmental pollution, and is easy to recycle and reuse.

[0020] 2. This invention overcomes the problems of traditional methods that use hydrochloric acid to dissolve silk fibroin in the solution to prepare regenerated silk fibroin membranes, which result in water-insoluble membranes with low crystallinity, low initial modulus, poor mechanical properties, and easy cracking after drying. The regenerated silk fibroin membrane prepared by the method of this invention can improve the problem of poor mechanical properties caused by direct dissolution with hydrochloric acid. In this invention, formic acid can slowly dissolve silk fibroin to a certain extent. Rapid dissolution requires the help of strong polar cations. In this case, the presence of strong polar cations in the solution will limit further utilization, while strong acids will completely destroy the van der Waals forces and hydrogen bonds of silk fibroin molecules, thus ensuring the mechanical properties of the regenerated material. Attached Figure Description

[0021] Figure 1 Line graphs showing the solubility of MSF in different hydrochloric acids and the solubility of MSF in hydrochloric acid-assisted formic acid;

[0022] Figure 2 The images show the appearance of regenerated silk fibroin membranes after drying for a period of time. In the figures, a is a silk fibroin membrane with MSF dissolved in 2wt.% calcium chloride and 98wt.% formic acid; b is a silk fibroin membrane with MSF dissolved in 1wt.% hydrochloric acid and 98wt.% formic acid; c is a silk fibroin membrane with MSF dissolved in 2wt.% hydrochloric acid and 98wt.% formic acid; d is a silk fibroin membrane with MSF dissolved in 3wt.% hydrochloric acid and 98wt.% formic acid; e is a silk fibroin membrane with MSF dissolved in 4wt.% hydrochloric acid and 98wt.% formic acid; and f is a silk fibroin membrane with MSF dissolved in 5wt.% hydrochloric acid and 98wt.% formic acid.

[0023] Figure 3The figures show FTIR spectra of silk fibroin in different dissolution systems. Figure a represents 0.8 g MSF dissolved in 2 wt.% calcium chloride-98 wt.% formic acid; b represents 0.8 g MSF dissolved in 1 wt.% hydrochloric acid-98 wt.% formic acid; c represents 0.8 g MSF dissolved in 5 wt.% hydrochloric acid-98 wt.% formic acid; d represents 0.8 g MSF dissolved in 10 wt.% hydrochloric acid-98 wt.% formic acid; e represents 0.8 g MSF dissolved in 20 wt.% hydrochloric acid-98 wt.% formic acid; and f represents 0.8 g MSF dissolved in 30 wt.% hydrochloric acid-98 wt.% formic acid.

[0024] Figure 4 XRD patterns of silk fibroin in different dissolution systems are shown. In the figure, a represents 8 g MSF dissolved in 2 wt.% calcium chloride-98 wt.% formic acid; b represents 0.8 g MSF dissolved in 1 wt.% hydrochloric acid-98 wt.% formic acid; c represents 0.8 g MSF dissolved in 5 wt.% hydrochloric acid-98 wt.% formic acid; d represents 0.8 g MSF dissolved in 10 wt.% hydrochloric acid-98 wt.% formic acid; e represents 0.8 g MSF dissolved in 20 wt.% hydrochloric acid-98 wt.% formic acid; and f represents 0.8 g MSF dissolved in 30 wt.% hydrochloric acid-98 wt.% formic acid.

[0025] Figure 5 Electrophoretic images of silk fibroin dissolved in MSF in different dissolution systems are shown. In the figure, a is 8g MSF dissolved in 2wt.% calcium chloride-98wt.% formic acid; b is 0.8g MSF dissolved in 1wt.% hydrochloric acid-98wt.% formic acid; c is 0.8g MSF dissolved in 5wt.% hydrochloric acid-98wt.% formic acid; d is 0.8g MSF dissolved in 10wt.% hydrochloric acid-98wt.% formic acid; e is 0.8g MSF dissolved in 20wt.% hydrochloric acid-98wt.% formic acid; and f is 0.8g MSF dissolved in 30wt.% hydrochloric acid-98wt.% formic acid. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0027] Confirmatory experiments:

[0028] Hydrochloric acid solubility of degummed mulberry silk

[0029] S1. Natural mulberry silk was boiled in a 0.1 wt% sodium carbonate solution for 30 minutes to degumme it. This process was repeated 3 times to obtain degummed mulberry silk (MSF).

[0030] S2. Prepare hydrochloric acid solutions with concentrations ranging from 1 to 6 mol / L. Dissolve 0.8 g of MSF in 10 mL of hydrochloric acid of different concentrations at a bath ratio of 1:50, at a temperature of 40℃, and in a water bath for 4 hours to hydrolyze the MSF. Wash the hydrolyzed products with deionized water, dry them, and determine the solubility.

[0031] Hydrochloric acid-assisted formic acid dissolution rate of degummed mulberry silk

[0032] S1. Degumming is achieved by boiling natural mulberry silk in a 0.1wt% sodium carbonate solution for 30 minutes. This process is repeated three times to obtain degummed mulberry silk.

[0033] S2. Prepare dissolving systems of different masses (1 wt.% hydrochloric acid and 98 wt.% formic acid, 2 wt.% hydrochloric acid and 98 wt.% formic acid, 3 wt.% hydrochloric acid and 98 wt.% formic acid, 4 wt.% hydrochloric acid and 98 wt.% formic acid, 5 wt.% hydrochloric acid and 98 wt.% formic acid), adding MSF in increments of 1 wt.% until dissolution saturation is reached, then stop adding MSF and obtain the amount of MSF dissolved.

[0034] from Figure 1 As can be seen, formic acid has a swelling effect on MSF, while hydrochloric acid catalyzes the hydrolysis of MSF. In the hydrochloric acid-formic acid dissolution system, the two combine. When hydrochloric acid reacts with water to produce ions, these ions dissolve in water, increasing the polarity of the water. This makes silk fibroin more easily swell and dissolve in water, resulting in complete dissolution of MSF and an increased amount of MSF dissolved. Figure 1 The increase in the solubility curve of MSF with different hydrochloric acid concentrations is relatively slow, while... Figure 1 The solubility curve of MSF in formic acid-assisted hydrochloric acid showed a relatively rapid increase. Therefore, it can be concluded that formic acid-assisted hydrochloric acid can better dissolve MSF.

[0035] Example 1

[0036] A method for dissolving silk fibroin with mixed acids includes the following steps:

[0037] S1. Degumming is achieved by boiling natural mulberry silk in a 0.1wt% sodium carbonate solution for 30 minutes. This process is repeated three times to obtain degummed mulberry silk.

[0038] S2. Mix 98 wt.% organic acid and 1 wt.% inorganic acid at a volume ratio of 1:1 to obtain a mixed acid solution. Then add the degummed silk prepared in step S1 to the mixed acid solution and stir at room temperature to dissolve it to obtain a silk fibroin solution with a concentration of 8 wt.%.

[0039] Example 2

[0040] A method for dissolving silk fibroin with mixed acids includes the following steps:

[0041] S1. Degumming is achieved by boiling natural mulberry silk in a 0.1wt% sodium carbonate solution for 30 minutes. This process is repeated three times to obtain degummed mulberry silk.

[0042] S2. Mix 98 wt.% organic acid and 2 wt.% inorganic acid at a volume ratio of 1:1 to obtain a mixed acid solution. Then add the degummed silk prepared in step S1 to the mixed acid solution and stir at room temperature to dissolve it to obtain a silk fibroin solution with a concentration of 8 wt.%.

[0043] Example 3

[0044] A method for dissolving silk fibroin with mixed acids includes the following steps:

[0045] S1. Degumming is achieved by boiling natural mulberry silk in a 0.1wt% sodium carbonate solution for 30 minutes. This process is repeated three times to obtain degummed mulberry silk.

[0046] S2. Mix 98 wt.% organic acid and 3 wt.% inorganic acid at a volume ratio of 1:1 to obtain a mixed acid solution. Then add the degummed silk prepared in step S1 to the mixed acid solution and stir at room temperature to dissolve it to obtain a silk fibroin solution with a concentration of 8 wt.%.

[0047] Example 4

[0048] A method for dissolving silk fibroin with mixed acids includes the following steps:

[0049] S1. Degumming is achieved by boiling natural mulberry silk in a 0.1wt% sodium carbonate solution for 30 minutes. This process is repeated three times to obtain degummed mulberry silk.

[0050] S2. Mix 98 wt.% organic acid and 4 wt.% inorganic acid at a volume ratio of 1:1 to obtain a mixed acid solution. Then add the degummed silk prepared in step S1 to the mixed acid solution and stir at room temperature to dissolve it to obtain a silk fibroin solution with a concentration of 8 wt.%.

[0051] Example 5

[0052] A method for dissolving silk fibroin with mixed acids includes the following steps:

[0053] S1. Degumming is achieved by boiling natural mulberry silk in a 0.1wt% sodium carbonate solution for 30 minutes. This process is repeated three times to obtain degummed mulberry silk.

[0054] S2. Mix 98 wt.% organic acid and 5 wt.% inorganic acid at a volume ratio of 1:1 to obtain a mixed acid solution. Then add the degummed silk prepared in step S1 to the mixed acid solution and stir at room temperature to dissolve it to obtain a silk fibroin solution with a concentration of 8 wt.%.

[0055] Comparative Example 1

[0056] A method for dissolving silk fibroin using formic acid-CaCl2 includes the following steps:

[0057] S1. Degumming is achieved by boiling natural mulberry silk in a 0.1wt% sodium carbonate solution for 30 minutes. This process is repeated three times to obtain degummed mulberry silk.

[0058] S2. Add CaCl2 to a 98 wt.% formic acid solution, and then add the degummed silk prepared in step S1 to the mixed acid solution. The mass ratio of CaCl2 to degummed silk is 2:15. Stir and dissolve at room temperature to obtain a silk fibroin solution with a concentration of 8 wt.%.

[0059] Comparative Example 2

[0060] A method for dissolving silk fibroin with mixed acids includes the following steps:

[0061] S1. Degumming is achieved by boiling natural mulberry silk in a 0.1wt% sodium carbonate solution for 30 minutes. This process is repeated three times to obtain degummed mulberry silk.

[0062] S2. Mix 98 wt.% organic acid and 10 wt.% organic acid at a volume ratio of 1:1 to obtain a mixed acid solution. Then add the degummed silk prepared in step S1 to the mixed acid solution and stir at room temperature to dissolve it to obtain a silk fibroin solution with a concentration of 8 wt.%.

[0063] The silk fibroin solutions prepared in Examples 1-5 and Comparative Example 1 were spread evenly on flat petri dishes and placed in a dry, cool, and ventilated place until the membranes dried. The membrane containing calcium chloride in Comparative Example 1 was immersed in deionized water for 2 days, during which the deionized water needed to be changed. The deionized membranes were then air-dried.

[0064] After drying the films in Examples 1-5 and Comparative Example 1 for a period of time, the surface surfaces were compared. Figure 2As can be seen from the diagram, a is a regenerated silk fibroin membrane made by dissolving MSF with calcium chloride-formic acid. It exhibits low brittleness, high toughness, and high crystallinity. Silk fibroin is composed of crystalline and amorphous regions. The peptide chains in the crystalline region have strong hydrogen bonds and intermolecular forces, resulting in tight binding, orderly arrangement, and strong tensile strength. The peptide chains in the amorphous region have weaker intermolecular forces, are unstable, and are arranged irregularly, making them easily soluble in acids, enzymes, and salts. Therefore, the effect of calcium chloride on MSF is to destroy the amorphous region of the regenerated silk fibroin membrane. b, c, d, e, and f are regenerated silk fibroin membranes made by dissolving MSF with hydrochloric acid-formic acid (corresponding to Examples 1-5, respectively), showing a gradual increase in brittleness. Hydrochloric acid is a strong acid that can violently hydrolyze MSF, destroying the intermolecular forces and hydrogen bonds of peptide chains, as well as peptide bonds in polypeptides. This breaks down the large silk fibroin molecules into smaller amino acids, making the regenerated silk fibroin membrane easily cracked and readily soluble in water.

[0065] To verify the effects of mixed acid dissolution and formic acid-CaCl2 dissolution on degummed silk, infrared and XRD tests were performed on the regenerated silk fibroin membrane. Infrared spectroscopy revealed that the silk fibroin dissolved in hydrochloric acid-formic acid produced a distinct absorption peak with a sharp peak shape; while the absorption peak of the silk fibroin solution dissolved in calcium chloride-formic acid broadened and became less sharp. This is due to the difference in chloride and calcium ions in the hydrochloric acid-formic acid and calcium chloride-formic acid dissolution systems. These ions act on groups or hydrogen bonds in the protein, causing changes in the protein groups and thus altering the protein structure. XRD analysis showed that curves a and b both exhibited α-helical structures (Silk I structure) and β-sheet structures (Silk II structure), with the β-sheet structure being the predominant structure. Curve c showed the Silk I structure, while curves d, e, and f all showed random coil structures. This indicates that hydrochloric acid can alter the structure of silk fibroin, and with increasing hydrochloric acid mass fraction, the diffraction peaks related to the β-sheet structure gradually disappeared, while diffraction peaks related to the random coil structure appeared, and the peak shapes became increasingly distinct. Therefore, this paper speculates that the increased amount of hydrochloric acid is the reason why more random coil structures appear in silk fibroin.

[0066] Figure 5Electrophoresis images obtained by SDS-PAGE gel electrophoresis after dissolving MSF under different dissolution conditions to obtain silk fibroin solutions of different molecular weights. From left to right, M in Figure 1 represents the standard protein marker (17-180 kDa). Significant differences were observed in the SDS-PAGE results of silk fibroin solutions dissolved in MSF using different dissolution systems. Silk fibroin is generally considered to be composed of heavy chains (H chains), light chains (L chains), and P25 protein, with relative molecular weights of 390 kDa for the H chain, 28 kDa for the L chain, and 25 kDa for the P25 protein. The heavy chains (H chains) and light chains (L chains) are linked together by disulfide bonds, while the P25 protein is linked to them non-covalently. Figure a shows the most widespread molecular weight distribution, ranging from 17 to 180 kDa. This is attributed to the effect of calcium chloride on the silk fibroin macromolecules, leading to the degradation of the heavy chains in the silk fibroin.

[0067] As the mass fraction of hydrochloric acid increases, the colors of bands b, c, d, f, and g (g corresponds to correspondence 2) in the 35-180 kDa range gradually fade. The molecular weights of bands b, c, d, f, and g are as follows: band b has a molecular weight of 35 kDa; bands c, d, and f all have molecular weights of 28 kDa; and band g has a molecular weight below 17 kDa. This indicates that hydrochloric acid can reduce the molecular weight of silk fibroin. The greater the mass fraction of hydrochloric acid, the greater the reduction in silk fibroin molecular weight. This confirms that hydrochloric acid severely damages the large molecules of silk fibroin, and the degree of damage increases with increasing hydrochloric acid mass fraction. Compared to Figure a, bands b, c, d, f, and g show the greatest reduction in molecular weight. This indicates that the molecular weight and distribution of silk fibroin are related to hydrochloric acid and its mass fraction. Hydrochloric acid has a significant impact on the molecular weight of silk fibroin; as the mass fraction of hydrochloric acid increases, the molecular weight of silk fibroin gradually decreases.

[0068] This invention reveals that hydrochloric acid hydrolyzes MSF more vigorously, disrupting intermolecular forces, hydrogen bonds, and peptide bonds connecting polypeptides, breaking down large protein molecules into smaller amino acids. Furthermore, the solubility of MSF gradually increases with increasing hydrochloric acid concentration. Hydrochloric acid assists formic acid in dissolving MSF; formic acid swells MSF, while hydrochloric acid catalyzes its hydrolysis. In the hydrochloric acid-formic acid dissolution system, the two combine. When hydrochloric acid reacts in water to produce ions, these ions increase the polarity of water, making silk fibroin more easily swell and dissolve, resulting in complete MSF dissolution and increased solubility. Moreover, the solubility of MSF increases with increasing hydrochloric acid concentration compared to different concentrations of hydrochloric acid. Therefore, the hydrochloric acid-assisted formic acid solubility of MSF is better than that of MSF solubility by hydrochloric acid at different concentrations. Calcium chloride and hydrochloric acid both degrade MSF, reducing its molecular weight, but to different degrees. In the dissolution of MSF using calcium chloride-formic acid and hydrochloric acid-formic acid, calcium chloride has a smaller effect on reducing the molecular weight of MSF, while hydrochloric acid has a larger effect. Both methods result in a molecular weight distribution below 35 kDa, and the reduction in molecular weight decreases with increasing mass fraction of hydrochloric acid. Therefore, the method of this invention can be used for dissolving silk fibroin to obtain regenerated silk fibroin with a molecular weight requirement below 35 kDa.

[0069] To verify the spinnability of the silk fibroin solution prepared by the dissolution method of this invention, a mixed acid solution was prepared by mixing 98 wt.% organic acid and 0.5 wt.% inorganic acid at a volume ratio of 1:1. Degummed silk was then added to the mixed acid solution and stirred at room temperature to dissolve, resulting in a 12 wt.% silk fibroin solution. Regenerated silk fibroin filaments were prepared by wet spinning. The mechanical properties of the regenerated silk fibroin filaments prepared by this dissolution method were measured. The breaking strength was 117.2 ± 25.3 MPa, and the breaking productivity was 12.3 ± 3.9%. The breaking strength of the regenerated silk fibroin filaments prepared by wet spinning using the silk fibroin solution in the comparative example was 376.6 ± 44.5 MPa, and the breaking elongation was 19.2 ± 4.5%. These results show that although the mechanical properties of the regenerated silk fibroin filaments prepared by this invention are not as good as those in the comparative example, the data demonstrate that the silk fibroin solution prepared by the dissolution method of this invention also possesses spinnability and can be used to prepare regenerated silk fibroin filaments with certain mechanical properties. The regenerated silk fibroin solution of the present invention can be used to prepare regenerated silk fibroin materials.

[0070] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for dissolving silk fibroin with mixed acids, characterized in that, Includes the following steps: S1. Degumming is performed on the silk to obtain degummed silk. S2. Mix organic acid and inorganic acid to obtain a mixed acid solution, and then add the degummed silk prepared in step S1 to the mixed acid solution to obtain a silk fibroin solution; wherein, the organic acid is formic acid, the inorganic acid is hydrochloric acid, the concentration of the organic acid is 80-100 wt.%, the concentration of the inorganic acid is 1-5 wt.%, and the volume ratio of the organic acid to the inorganic acid is 1:

1.

2. The method for dissolving silk fibroin with mixed acids according to claim 1, characterized in that: The silk is any one or a combination of two or more of mulberry silk, tussah silk, or castor silk.

3. The method for dissolving silk fibroin with mixed acids according to claim 1, characterized in that: The concentration of the silk fibroin solution is 0.1-50 wt.%.

4. The regenerated silk fibroin solution prepared by any of the mixed acid dissolution methods according to claims 1-3.

5. The application of the regenerated silk fibroin solution according to claim 4 in the preparation of regenerated silk fibroin materials.

6. The application according to claim 5, wherein the regenerated silk fibroin material comprises a film, filament, nanofiber, porous scaffold, and hydrogel.

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