Dissolution solution for dissolving tussah silk, dissolution method, and tussah silk protein solution

Abstract

The present application belongs to the technical field of biological materials, and relates to a dissolving solution for dissolving tussah silk, a dissolving method and a tussah silk protein solution. The tussah silk is a natural protein fiber of medium and short types, and the main amino acid composition of the silk fibroin is alanine (about 43.1%), glycine (about 27.27%) and serine (about 11.25%), and the characteristic amino acid sequence is (-Ala-) n . The alanine repeat sequence forms a crystalline region of the tussah silk, in which nonpolar groups are dense, the peptide chains are regularly arranged, and the hydrogen bonds between the peptide chains and the intermolecular forces are strong, so that the tussah silk has high breaking strength and tensile properties, but it is also difficult to be dissolved in conventional solvents. In view of the above problems, the present application provides a new dissolving system, which can efficiently and environmentally realize the dissolution of the tussah silk under mild conditions, and well maintain the structural integrity of the silk fibroin, thereby providing support for the application of the tussah silk in high-end textiles and biological materials.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology and relates to a dissolving solution, a dissolving method, and a tussah silk protein solution for dissolving tussah silk. Background Technology

[0002] As a natural protein fiber, tussah silk, with its excellent mechanical properties, outstanding biocompatibility, and biodegradability, shows broad application prospects in biomedical materials and high-end textiles. However, its further application is severely limited by its difficulty in dissolving. Compared with widely used mulberry silk, the two silk fibroin proteins have fundamental differences in molecular structure: mulberry silk fibroin is mainly composed of glycine (Gly, about 45%), alanine (Ala, about 30%), and serine (Ser, about 12%) with the repeating sequence Gly-Ala-Gly-Ala-Gly-Ser as the main chain. Its β-sheet crystalline region is relatively short and moderately regular, and the intermolecular forces are slightly weaker. In contrast, tussah silk fibroin has a significantly higher alanine (Ala) content (about 43%), and its characteristic (-Ala-)n repeating sequence is longer and more continuous. This allows tussah silk to form a denser, more regular, and highly stable β-sheet crystalline region. The molecules are tightly packed together through a stronger hydrogen bond network and the hydrophobic interaction of the nonpolar side chains, resulting in a more robust structure that is difficult for solvent molecules to penetrate and destroy. Therefore, its solubility is much higher than that of mulberry silk.

[0003] Currently, the core challenge in dissolving tussah silk lies in effectively dissociating its highly stable crystalline structure. Traditional solvent systems used for efficiently dissolving mulberry silk (such as the calcium chloride / ethanol / water ternary system) exhibit extremely low dissolution efficiency, or even near-insolubility, when faced with the more dense structure of tussah silk. To overcome this problem, existing technologies often turn to strong acids, strong bases, or high-concentration salt solutions. While these methods can achieve a certain degree of dissolution, they generally suffer from the following prominent issues: dissolution efficiency remains unsatisfactory; the reaction conditions are harsh (high temperature, high concentration), easily leading to hydrolysis of silk fibroin peptide chains or irreversible conformational damage, thus losing its inherent excellent properties; simultaneously, the solvents used are often highly toxic, corrosive, and difficult to recycle and regenerate, which is environmentally and equipment-insensitive, contradicting the principles of green chemistry and sustainable development.

[0004] Therefore, developing a novel dissolution method that can efficiently, gently, and environmentally friendly dissolve tussah silk while maximizing the preservation of the molecular structure and functional activity of silk fibroin is of vital practical significance for fully tapping the high added value application potential of tussah silk and promoting its practical application in the fields of high-end biomaterials and functional fibers. Summary of the Invention

[0005] In view of the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide a dissolving solution, a dissolving method and a tussah silk protein solution for dissolving tussah silk, wherein the tussah silk protein solution obtained by the dissolving method can retain the natural structure of silk protein.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] In a first aspect, the present invention provides a dissolving solution for dissolving tussah silk, said dissolving solution being prepared by mixing a eutectic solvent (DES) with formic acid, wherein the eutectic solvent is a mixed solution of choline chloride and oxalic acid. The DES composed of oxalic acid and choline chloride has a mild acidity, which can better preserve the properties of the silk fibroin while dissolving it.

[0008] In some embodiments of the present invention, the molar ratio of choline chloride to oxalic acid in the eutectic solvent is 1:(1-2).

[0009] In some embodiments of the present invention, the eutectic solvent and formic acid are mixed at a mass ratio of 1:(1-3) at room temperature.

[0010] In a second aspect, the present invention provides a method for dissolving tussah silk using the above-described dissolving solution, wherein the method comprises: adding shredded tussah silk to the dissolving solution and dissolving at 80-100°C for 3-8 hours to obtain a tussah silk protein solution.

[0011] In some embodiments of the present invention, the shredded tussah silk is deionized, dried, and then added to a dissolving solution.

[0012] In some embodiments of the present invention, the drying conditions are 50-60°C forced air drying for 2-3 hours.

[0013] In some embodiments of the present invention, the length of the tussah silk after being cut into pieces is ≤5mm, so as to facilitate full soaking and dissolution.

[0014] In some embodiments of the present invention, the mass ratio of tussah silk to the dissolving solution is 1g: (50-100)g.

[0015] A third aspect of the present invention provides a tussah silk protein solution obtained by the above-described method. The solution is clear and has good fluidity.

[0016] Furthermore, the tussah silk protein solution retains the natural structure of silk protein, and the infrared spectrum shows a sharp β-sheet characteristic peak near 1625 cm⁻¹, indicating that the unique stable structure of tussah silk has not been damaged.

[0017] Furthermore, the crystallinity of the tussah silk protein solution is 19-26%, and the tensile strength is 17 MPa-23 MPa;

[0018] Furthermore, the tussah silk protein solution did not deteriorate after being left for a long time, indicating that the solution has good stability.

[0019] The tussah silk dissolving method and dissolving system of the present invention have the following advantages and beneficial effects:

[0020] (1) This invention is the first to propose a composite system of eutectic solvent and formic acid for dissolving tussah silk. Through hydrogen bond breaking, protonation and synergistic penetration, this system can efficiently break down the dense inter-chain stacking in the crystalline region of tussah silk, allowing the silk fibroin molecular chains to be uniformly dispersed while preserving the integrity of the β-sheet conformation. This solves the technical problem of low solubility or even insolubility of tussah silk in traditional solvent systems.

[0021] (2) The solution system of the present invention is environmentally friendly. The eutectic solvent used has the characteristics of low toxicity, biodegradability and easy recycling. The concentration and total amount of formic acid used are significantly reduced, which reduces the pollution of the environment and the corrosion of equipment by strong acid, and meets the requirements of green chemical industry and sustainable development.

[0022] (3) The dissolution method of the present invention is mild and can achieve efficient dissolution at medium and low temperatures. It can maintain the molecular integrity of silk fibroin to the greatest extent, which is beneficial to subsequent spinning, film formation and other processing applications. The solution has good stability and is suitable for large-scale production. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] (1) Weigh 0.6g of tussah silk, cut it into pieces to a length of ≤5 mm, wash it and dry it in a forced-air dryer at 55℃ for 2.5 h.

[0026] (2) 30 g of DES solvent was obtained by reacting choline chloride and oxalic acid in a 1:1 molar ratio at 90 °C.

[0027] (3) Mix the DES solvent obtained in step (2) with formic acid at a mass ratio of 1:1 to obtain 60 g of tussah silk dissolving solution.

[0028] (4) Add the tussah silk obtained in step (1) to the solution obtained in step (3) and dissolve at 100°C for 5 h to obtain tussah silk protein solution.

[0029] (5) The tussah silk protein solution obtained in step (4) was allowed to stand. Observation showed that the solution was slightly thin, clear, and had good fluidity. The tussah silk protein solution was used to prepare a transparent protein film with a thickness of 0.5 mm. Observation by scanning electron microscopy (SEM) showed that the film surface was smooth, with excellent transparency and no obvious particle aggregation. Fourier transform infrared spectroscopy (FTIR) test showed a sharp β-sheet characteristic peak near 1625 cm⁻¹, indicating that the unique stable structure of tussah silk was not destroyed. The tensile strength of the protein film was 21 MPa according to the Instron universal tensile testing machine. The crystallinity of the protein film was 23% according to X-ray diffraction (XRD). Particle size analysis results showed that the particle size in the tussah silk protein solution was moderate. The solution did not deteriorate after long-term storage and had good stability.

[0030] Example 2

[0031] (1) Weigh 0.8 g of tussah silk, cut it into pieces to a length of ≤5 mm, wash it and dry it in a forced-air dryer at 55℃ for 2.5 h.

[0032] (2) 20 g of DES solvent was obtained by reacting choline chloride and oxalic acid in a 1:1 molar ratio at 90 °C.

[0033] (3) The DES solvent obtained in step (2) is mixed with formic acid at a mass ratio of 1:3 to obtain 80 g of tussah silk dissolving solution.

[0034] (4) Add the tussah silk obtained in step (1) to the solution obtained in step (3) and dissolve at 100°C for 5 h to obtain tussah silk protein solution.

[0035] (5) Let the tussah silk protein solution obtained in step (4) stand. Observation shows that the solution is thin, clear, and has good fluidity. [The text abruptly ends here, likely due to an incomplete sentence or missing information.]

[0036] A transparent protein film with a thickness of 0.5 mm was prepared from the solution. Scanning electron microscopy (SEM) revealed a smooth surface, good transparency, and no particle aggregation. Fourier transform infrared spectroscopy (FTIR) showed a sharp β-sheet characteristic peak near 1625 cm⁻¹, indicating that the unique stable structure of tussah silk remained intact. Tensile testing using an Instron universal tensile testing machine yielded a tensile strength of 19 MPa. X-ray diffraction (XRD) analysis showed a crystallinity of 25%. Particle size analysis revealed that the tussah silk protein solution contained particles of moderate size. The solution showed no deterioration after prolonged storage, exhibiting good stability.

[0037] Example 3

[0038] (1) Weigh 0.6 g of tussah silk, cut it into pieces to a length of ≤5 mm, wash it and dry it in a forced-air dryer at 55℃ for 2.5 h.

[0039] (2) 30 g of DES solvent was obtained by reacting choline chloride and oxalic acid in a molar ratio of 1:2 at 90 °C.

[0040] (3) Mix the DES solvent obtained in step (2) with formic acid at a mass ratio of 1:1 to obtain 60 g of tussah silk dissolving solution.

[0041] (4) Add the tussah silk obtained in step (1) to the solution obtained in step (3) and dissolve at 100°C for 7 h to obtain tussah silk protein solution.

[0042] (5) The tussah silk protein solution obtained in step (4) was allowed to stand. Observation showed that the solution was of moderate viscosity, clear and had good fluidity. The tussah silk protein solution was prepared into a transparent protein film with a thickness of 0.5 mm. Observation by scanning electron microscopy (SEM) showed that the surface of the film was smooth, with good transparency and no particle aggregation. Fourier transform infrared spectroscopy (FTIR) test showed a sharp β-sheet characteristic peak near 1625 cm⁻¹, indicating that the unique stable structure of tussah silk was not damaged. The tensile strength of the protein film was 22 MPa according to the Instron universal tensile tester. The crystallinity of the protein film was 19% according to X-ray diffraction (XRD). Particle size analysis results showed that the particle size in the tussah silk protein solution was moderate, and no deterioration occurred after long-term storage, indicating that the solution had good stability.

[0043] Example 4

[0044] (1) Weigh 0.8g of tussah silk, cut it into pieces to a length of ≤5 mm, wash it and dry it in a forced-air dryer at 55℃ for 2.5 h.

[0045] (2) 20 g of DES solvent was obtained by reacting choline chloride and oxalic acid in a molar ratio of 1:2 at 90 °C.

[0046] (3) The DES solvent obtained in step (2) is mixed with formic acid at a mass ratio of 1:3 to obtain 80 g of tussah silk dissolving solution.

[0047] (4) Add the tussah silk obtained in step (1) to the solution obtained in step (3) and dissolve at 100°C for 7 h to obtain tussah silk protein solution.

[0048] (5) The tussah silk protein solution obtained in step (4) was allowed to stand. Observation showed that the solution was of moderate viscosity, clear and had good fluidity. The tussah silk protein solution was prepared into a transparent protein film with a thickness of 0.5 mm. Observation by scanning electron microscopy (SEM) showed that the surface of the film was smooth, with good transparency and no particle aggregation. Fourier transform infrared spectroscopy (FTIR) test showed a sharp β-sheet characteristic peak near 1625 cm⁻¹, indicating that the unique stable structure of tussah silk was not damaged. The tensile strength of the protein film was 17 MPa according to the Instron universal tensile test. The crystallinity of the protein film was 21% according to X-ray diffraction (XRD). Particle size analysis results showed that the particle size in the tussah silk protein solution was moderate, and no deterioration occurred after long-term storage, indicating that the solution had good stability.

[0049] Example 5

[0050] (1) Weigh 1.2 g of tussah silk, cut it into pieces to a length of ≤5 mm, wash it and dry it in a forced-air dryer at 55℃ for 2.5 h.

[0051] (2) 30 g of DES solvent was obtained by reacting choline chloride and oxalic acid in a molar ratio of 1:2 at 90 °C.

[0052] (3) Mix the DES solvent obtained in step (2) with formic acid at a mass ratio of 1:1 to obtain 60 g of tussah silk dissolving solution.

[0053] (4) Add the tussah silk obtained in step (1) to the solution obtained in step (3) and dissolve at 100°C for 8 hours to obtain tussah silk protein solution.

[0054] (5) The tussah silk protein solution obtained in step (4) was allowed to stand. It was observed that the solution was relatively viscous, but clear and still had good fluidity. The tussah silk protein solution was prepared into a transparent protein film with a thickness of 0.5 mm. The film was observed by scanning electron microscopy (SEM). The surface of the film was smooth, with good transparency and no particle aggregation. Fourier transform infrared spectroscopy (FTIR) test showed a sharp β-sheet characteristic peak near 1625 cm⁻¹. The stable structure of tussah silk was not damaged. The tensile strength of the protein film was 23 MPa according to the Instron universal tensile test. The crystallinity of the protein film was 26% according to X-ray diffraction (XRD). The particle size analysis test showed that the particle size of the tussah silk protein solution was moderate. It did not deteriorate after a long period of time and the solution had good stability.

[0055] Comparative Example 1

[0056] Weigh 0.4 g of cleaned and dried tussah silk (length ≤ 5 mm) and prepare 40 g of a ternary solvent of calcium chloride / ethanol / water with a molar ratio of 8:2:1. Add the tussah silk to the ternary solvent and dissolve at 100℃ for 8 h to obtain a tussah silk protein solution. After the obtained tussah silk protein solution is allowed to stand, it is observed that the tussah silk is undissolved and clumps together, making it impossible to form a film.

[0057] Comparative Example 2

[0058] 0.6 g of washed and dried tussah silk (length ≤ 5 mm) was weighed, and 60 g of eutectic solvent (DES) was prepared by mixing choline chloride and oxalic acid in a 1:1 molar ratio. The tussah silk was added to the DES and dissolved at 100 °C for 8 h to obtain a tussah silk protein solution. After standing, the obtained tussah silk protein solution was observed to be incompletely dissolved, resulting in a turbid solution. A protein film with a thickness of 0.5 mm was prepared from this solution. Scanning electron microscopy (SEM) revealed a rough surface, poor transparency, and a large aggregation of tussah silk fibers. Particle size analysis showed uneven particle size distribution in the solution. The solution exhibited precipitation after prolonged standing, indicating poor stability.

[0059] Comparative Example 3

[0060] 0.6 g of washed and dried tussah silk (length ≤ 5 mm) was weighed, and 60 g of formic acid was weighed as the dissolving solution. The tussah silk was added to the formic acid and dissolved at 100℃ for 8 h to obtain a tussah silk protein solution. After the obtained tussah silk protein solution was allowed to stand, it was observed that the tussah silk was not completely dissolved, and the solution was turbid. A protein film with a thickness of 0.5 mm was prepared from this solution. Scanning electron microscopy (SEM) revealed a rough surface, poor transparency, and a large amount of aggregated tussah silk fibers. Particle size analysis showed that the particles in the solution were uneven in size. The solution showed precipitation after prolonged standing, indicating poor stability.

Claims

1. A dissolving solution for dissolving tussah silk, characterized in that, The solution is prepared by mixing a eutectic solvent with formic acid, wherein the eutectic solvent is a mixed solution of choline chloride and oxalic acid.

2. The solution according to claim 1, characterized in that, In the eutectic solvent, the molar ratio of choline chloride to oxalic acid is 1:(1-2).

3. The solution according to claim 2, characterized in that, The eutectic solvent and formic acid are mixed at a mass ratio of 1:(1-3) at room temperature.

4. A method for dissolving tussah silk using the dissolving solution as described in any one of claims 1-3, characterized in that, The method is as follows: add the shredded tussah silk to the dissolving solution and dissolve at 80~100℃ for 3~8 hours to obtain a tussah silk protein solution.

5. The method according to claim 4, characterized in that, The length of the tussah silk after being cut into pieces is ≤5mm.

6. The method according to claim 4, characterized in that, The mass ratio of tussah silk to the dissolving solution is 1g: (50-100)g.

7. A solution of tussah silk protein obtained by dissolving according to any one of claims 4-6.