Silk fibroin aqueous solution and preparation method thereof

Abstract

The application provides a preparation method of a silk fibroin aqueous solution, comprising the following steps: S1. degumming silk to obtain degummed silk; S2. dissolving the degummed silk by using CaCl2 and formic acid to obtain a CaCl2-formic acid-silk fibroin dissolving solution; S3. placing the CaCl2-formic acid-silk fibroin dissolving solution prepared in the step S2 in an open container, placing the open container containing the CaCl2-formic acid-silk fibroin dissolving solution in a box with a steam hole, and introducing water vapor into the box for a certain time until a CaCl2-water-silk fibroin solution is obtained; and S4. placing the CaCl2-water-silk fibroin solution prepared in the step S3 in a dialysis bag for dialysis to remove CaCl2, and obtaining a silk fibroin aqueous solution. The application overcomes the problem that directly dialyzing the CaCl2-formic acid-silk fibroin dissolving solution can easily lead to gelation and the silk fibroin aqueous solution cannot be directly obtained.

Description

Technical Field

[0001] This invention relates to the field of natural polymer materials, and more specifically to a method for preparing an aqueous solution of silk fibroin. Background Technology

[0002] Silk has extremely wide applications, not only as textiles but also as non-textile materials. For example, fibroin can be obtained through certain methods and then processed into fibroin materials for use in the biomedical field. Mulberry silk is composed of fibroin (MSF) and sericin. Fibroin contains amino acids essential for the human body 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] Studies have shown that polar solvents (such as trifluoroacetic acid, acetone, or methanol) can alter the conformation of silk fibroin molecules, indicating a shift from silk I to silk II structures in silk fibroin crystals. Literature review reveals that the conformational shift of silk fibroin chains is primarily determined by the compatibility of the solvent and water; different solvents also affect the structural characteristics of silk fibroin. STM studies have found that molecular conformational changes occur during spinning, with random coils and α-helices eventually transforming into β-sheets as spinning stress increases. Changes in silk fibroin structure can also be related to external conditions such as temperature, ions, and pH.

[0004] Silk fibroin can dissolve in some water-soluble compounds (such as the ternary solvent of CaCl2 / H2O / C2H5OH), but it cannot dissolve in organic solvents or water. This is because the high concentration of salt in the compound disrupts the hydrogen bonds within the silk fibroin, causing the silk to swell and dissolve. There are many traditional dissolution methods: ① ternary method (CaCl2:CH3CH2OH:H2O, molar ratio 1:2:8), ② LiBr-CH3CH2OH-H2O solution, ③ LiBr-H2O solution: 9.0 mol / L LiBr, ④ CaCl2-FA dissolution. All these methods involve adding an appropriate amount of silk to the dissolution system and stirring for 3 hours. Afterwards, the dissolved membrane is cooled, and the MSF solution is placed in a dialysis bag for desalting, a process that lasts for 3 days. Finally, the solution is removed, coated onto a membrane, and dried to obtain four types of regenerated membrane samples.

[0005] After silk fibroin is dissolved into a solution, it is placed in a dialysis bag and the water is changed regularly to achieve a good desalting effect. The dissolved silk fibroin exhibits a random coil conformation in aqueous solution. The desalted silk fibroin solution forms a film with excellent film-forming properties and has applications in medicine, chemistry, and daily life.

[0006] In the first three dissolution systems, the desalting method involves placing the silk fibroin mixture directly into a dialysis bag and immersing it in deionized water for direct desalting, resulting in a silk fibroin aqueous solution. However, in the CaCl2-formic acid dissolution system, the desalting method requires first forming a membrane and then immersing the membrane in deionized water for desalting, resulting in a salt-free silk fibroin membrane. Currently, there is no further processing method to obtain a silk fibroin aqueous solution. Summary of the Invention

[0007] The technical problem to be solved: The purpose of this invention is to provide a method for preparing an aqueous solution of silk fibroin using a CaCl2-formic acid dissolution system, which can overcome the problem of gelation when directly dialyzing the CaCl2-formic acid-silk fibroin solution, and prevent organic solvents from inducing changes in the conformation of silk fibroin molecules during dialysis.

[0008] Technical solution: A method for preparing an aqueous solution of silk fibroin, comprising the following steps: S1. Degumming the silk to obtain degummed silk; S2. Degummed silk was dissolved using CaCl2 and formic acid to obtain a CaCl2-formic acid-silk fibroin solution; S3. Place the CaCl2-formic acid-silk fibroin solution prepared in step S2 into an open container, place the open container containing the CaCl2-formic acid-silk fibroin solution into a box with a steam vent, introduce water vapor into the box, and after a certain period of time, obtain a CaCl2-water-silk fibroin solution. S4. The CaCl2-water-silk fibroin solution prepared in step S3 is placed in a dialysis bag for dialysis to remove CaCl2 and obtain an aqueous solution of silk fibroin.

[0009] Preferably, the silk is any one or a combination of two or more of mulberry silk, tussah silk, or castor silk.

[0010] Preferably, the formic acid concentration in step S2 is 80-100 wt.%.

[0011] Preferably, in step S2, the CaCl2 content in the CaCl2-formic acid-silk fibroin solution is 2wt.%-10wt.%, and the silk fibroin content is 0.1-50wt.%.

[0012] Preferably, the temperature of the water vapor is 0-60℃, and the relative humidity of the chamber is above 70%.

[0013] Preferably, the time for introducing water vapor is greater than 12 hours.

[0014] Preferably, the box with a steam vent includes a box body, the bottom of which is used to hold CaCl2-formic acid-silk fibroin solution. The box body is provided with a steam inlet and a steam outlet. The steam inlet supplies water vapor into the box body through a humidification device, and the steam outlet discharges formic acid volatilized from the box body and overflowing water vapor. The position of the steam outlet is higher than that of the steam inlet.

[0015] The silk fibroin aqueous solution prepared by any of the above preparation methods.

[0016] Beneficial effects: The method for preparing the silk fibroin aqueous solution of the present invention has the following advantages: This invention involves introducing water vapor into a box with a vent. The salt in the CaCl2-formic acid-silk fibroin solution absorbs the water vapor from the environment. Since formic acid is a volatile organic solvent, it also evaporates continuously during the introduction of water vapor. The evaporated formic acid leaves the CaCl2-formic acid-silk fibroin solution with the flowing water vapor. The salt absorbs the water vapor from the environment and gradually replaces the solvent in the CaCl2-formic acid-silk fibroin solution until the formic acid is completely evaporated, resulting in a CaCl2-water-silk fibroin solution. Dialysis of the CaCl2-water-silk fibroin solution directly yields an aqueous solution of silk fibroin, overcoming the problem that the silk fibroin solution formed by the CaCl2-formic acid dissolution system cannot form an aqueous solution, and also overcoming the problem that direct dialysis of the CaCl2-formic acid dissolution system will form a gel. Attached Figure Description

[0017] Figure 1 The diagram shows the reaction apparatus for preparing CaCl2-water-silk fibroin solution by dissolving CaCl2-formic acid-silk fibroin. The numbers in the diagram are: 1. Box body, 2. CaCl2-formic acid-silk fibroin solution, 3. Steam inlet, 4. Steam outlet, 5. Humidification device; Figure 2 The states of the solution after different humidification treatment times; Figure 3 The formula for calculating the rate of change of solution weight as a function of humidification treatment time is: ; Figure 4 The images are electron microscope images of the MSF films formed in Examples 1-5 and Comparative Example 1, with the appearance of the corresponding films shown in the upper right corner. Figure 5 The X-ray diffraction patterns of the MSF films formed in Examples 1-5 and Comparative Example 1 are shown below. Figure 6 The infrared absorption spectra of the MSF films formed in Examples 1-5 and Comparative Example 1 are shown below. Figure 7SDS-PAGE images of the MSF aqueous solutions formed in Examples 1-5 and Comparative Example 1. Detailed Implementation

[0018] 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.

[0019] The preparation of the CaCl2-water-silk fibroin solution in the following examples all require specific containers, such as... Figure 1 As shown, the container is a box with a steam vent. The box with the steam vent includes a box body 1. The bottom of the box body is used to place the CaCl2-formic acid-silk fibroin solution 2. The box body is provided with a steam inlet 3 and a steam outlet 4. The steam inlet 3 supplies water vapor into the box body through a humidification device 5. The steam outlet 4 outputs the formic acid volatilized from the box body and the overflowing water vapor. The position of the steam outlet is higher than that of the steam inlet. Example 1

[0020] A method for preparing an aqueous solution of silk fibroin, characterized by comprising the following steps: S1. Degumming is performed on mulberry silk to obtain degummed mulberry silk; S2. Degummed silk was dissolved using CaCl2 and formic acid, with a formic acid concentration of 98 wt.%, to obtain a CaCl2-formic acid-silk fibroin solution, wherein the CaCl2 content was 2 wt.% and the silk fibroin content was 8 wt.%. S3. Place the CaCl2-formic acid-silk fibroin solution prepared in step S2 into an open container, place the open container containing the CaCl2-formic acid-silk fibroin solution into a box with a steam vent, introduce water vapor at a temperature of 0℃ into the sealed box, and after the water vapor is introduced, the relative humidity of the box is 80%, and the water vapor is introduced for 12 hours to obtain CaCl2-water-silk fibroin solution. S4. The CaCl2-water-silk fibroin solution prepared in step S3 is placed in a dialysis bag for dialysis to remove CaCl2 and obtain an aqueous solution of silk fibroin. Example 2

[0021] A method for preparing an aqueous solution of silk fibroin, characterized by comprising the following steps: S1. Degumming is performed on mulberry silk to obtain degummed mulberry silk; S2. Degummed mulberry silk was dissolved using CaCl2 and formic acid, with a formic acid concentration of 98 wt.%, to obtain a CaCl2-formic acid-silk fibroin solution, wherein the CaCl2 content was 2.5 wt.% and the silk fibroin content was 10 wt.%. S3. Place the CaCl2-formic acid-silk fibroin solution prepared in step S2 into an open container, place the open container containing the CaCl2-formic acid-silk fibroin solution into a box with a steam vent, introduce water vapor at a temperature of 20°C into the sealed box, and after the water vapor is introduced, the relative humidity of the box is 80%. The water vapor is introduced for 12 hours to obtain CaCl2-water-silk fibroin solution. S4. The CaCl2-water-silk fibroin solution prepared in step S3 is placed in a dialysis bag for dialysis to remove CaCl2 and obtain an aqueous solution of silk fibroin. Example 3

[0022] A method for preparing an aqueous solution of silk fibroin, characterized by comprising the following steps: S1. Degumming is performed on mulberry silk to obtain degummed mulberry silk; S2. Degummed mulberry silk was dissolved using CaCl2 and formic acid, with a formic acid concentration of 98 wt.%, to obtain a CaCl2-formic acid-silk fibroin solution, wherein the CaCl2 content was 2.8 wt.% and the silk fibroin content was 12 wt.%. S3. Place the CaCl2-formic acid-silk fibroin solution prepared in step S2 into an open container, place the open container containing the CaCl2-formic acid-silk fibroin solution into a box with a steam vent, introduce water vapor at a temperature of 40°C into the sealed box, and after the water vapor is introduced, the relative humidity of the box is 80%. The water vapor is introduced for 12 hours to obtain CaCl2-water-silk fibroin solution. S4. The CaCl2-water-silk fibroin solution prepared in step S3 is placed in a dialysis bag for dialysis to remove CaCl2 and obtain an aqueous solution of silk fibroin. Example 4

[0023] A method for preparing an aqueous solution of silk fibroin, characterized by comprising the following steps: S1. Degumming is performed on mulberry silk to obtain degummed mulberry silk; S2. Degummed mulberry silk was dissolved using CaCl2 and formic acid, with a formic acid concentration of 98 wt.%, to obtain a CaCl2-formic acid-silk fibroin solution, wherein the CaCl2 content was 3 wt.% and the silk fibroin content was 15 wt.%. S3. Place the CaCl2-formic acid-silk fibroin solution prepared in step S2 into an open container, place the open container containing the CaCl2-formic acid-silk fibroin solution into a box with a steam vent, introduce water vapor at a temperature of 50°C into the sealed box, and after the water vapor is introduced, the relative humidity of the box is 80%. The water vapor is introduced for 12 hours to obtain CaCl2-water-silk fibroin solution. S4. The CaCl2-water-silk fibroin solution prepared in step S3 is placed in a dialysis bag for dialysis to remove CaCl2 and obtain an aqueous solution of silk fibroin. Example 5

[0024] A method for preparing an aqueous solution of silk fibroin, characterized by comprising the following steps: S1. Degumming is performed on mulberry silk to obtain degummed mulberry silk; S2. Degummed mulberry silk was dissolved using CaCl2 and formic acid, with a formic acid concentration of 98 wt.%, to obtain a CaCl2-formic acid-silk fibroin solution, wherein the CaCl2 content was 3 wt.% and the silk fibroin content was 18 wt.%. S3. Place the CaCl2-formic acid-silk fibroin solution prepared in step S2 into an open container, place the open container containing the CaCl2-formic acid-silk fibroin solution into a box with a steam vent, introduce water vapor at a temperature of 60°C into the sealed box, and after the water vapor is introduced, the relative humidity of the box is 80%. The water vapor is introduced for 12 hours to obtain CaCl2-water-silk fibroin solution. S4. The CaCl2-water-silk fibroin solution prepared in step S3 is placed in a dialysis bag for dialysis to remove CaCl2 and obtain an aqueous solution of silk fibroin.

[0025] The silk fibroin aqueous solution prepared in Examples 1-5 was poured into a petri dish with a diameter of 15 cm and evaporated at room temperature to prepare a silk fibroin membrane. The silk fibroin membrane was then immersed in deionized water to remove CaCl2, resulting in a pure silk fibroin membrane.

[0026] Comparative Example 1 A method for preparing a silk fibroin membrane, characterized by comprising the following steps: S1. Degumming is performed on mulberry silk to obtain degummed mulberry silk; S2. Degummed mulberry silk was dissolved using CaCl2 and formic acid, with a formic acid concentration of 98 wt.%, to obtain a CaCl2-formic acid-silk fibroin solution, wherein the CaCl2 content was 3 wt.% and the silk fibroin content was 15 wt.%. S3. Pour the CaCl2-formic acid-silk fibroin solution prepared in step S2 into a petri dish with a diameter of 15 cm and evaporate it at room temperature to prepare a silk fibroin membrane; S4. Immerse the silk fibroin membrane prepared in step S3 in deionized water to remove CaCl2 and obtain a pure silk fibroin membrane.

[0027] Figure 2 The graph shows the states of a CaCl2-formic acid-silk fibroin solution after different humidification times. As can be seen from the graph, the solution gradually becomes clearer with increasing humidification time. Figure 3 The value in the middle represents the rate of change of the weight of the CaCl2-formic acid-silk fibroin solution with increasing humidification time. The rate of change of the solution is relatively gradual as time increases.

[0028] Figure 4 The images shown are of pure silk fibroin membranes from Examples 1-5 and Comparative Example 1, respectively. There is not much difference in appearance between the membranes formed in the examples and the comparative example. The pure silk fibroin membranes obtained by both preparation methods have good film-forming properties.

[0029] To verify that the pure silk fibroin membrane prepared in this example has similar or close performance to the silk fibroin membrane prepared by the conventional method (i.e., desalting after membrane formation) in the comparative example, XRD, infrared, and electrophoresis tests were performed on the silk fibroin membranes of the example and the comparative example. The specific test results are analyzed as follows: Figure 5 As can be seen, there is a strong diffraction peak at 2θ = 20.64° and a weaker diffraction peak at 2θ = 23.27°. The diffraction peaks of the Silk II structure are mainly around 2θ = 9.1°, 18.9°, 20.7°, and 24.3°. Therefore, these two diffraction peaks are characteristic peaks of Silk II (β-sheet), indicating that the main conformation of silk fibroin fibers is β-sheet, and the main conformation of the silk fibroin membranes prepared by the two methods is still the same.

[0030] Depend on Figure 6 It can be concluded that all pure silk fibroin membranes are at 692.54 cm. -1 1288.37 cm -1 1540.01 cm -1 1645.69 c m-1 A distinct infrared absorption peak appears at 692.54 cm⁻¹. -1 It has an amide V β-sheet structure and a length of 1288.37 cm. -1 It has an amide III β-sheet structure and a diameter of 1645.69 cm. -1 It has an amide I β-sheet structure, 1540.01 cm⁻¹ -1 The absorption peaks are irregularly coiled. The infrared spectra of the MSF films obtained by the two methods are similar, and the differences between the Silk I and Silk II structures are not visible in the infrared spectrum.

[0031] The molecular weight of the membrane from Comparative Example 1 was measured after dissolving it using a ternary method. Figure 4 As shown. By Figure 7 It can be seen that for bands a, b, and c, the molecular weight of the band in the 180-25 kDa range decreases and the band color becomes lighter with increasing calcium chloride concentration. This can be attributed to the fact that under the influence of CaCl2 salt concentration, the higher the concentration, the more severe the heavy chain breakage, and thus the more severe the damage to the silk fibroin macromolecules. Among these three bands, the band in the 25-11 kDa range shows little change, indicating that the addition of CaCl2 has little effect on the light chains of silk fibroin. In band f, the band color is slightly lighter after secondary dissolution using the ternary method because the secondary dissolution during the preparation of the silk fibroin aqueous solution causes secondary damage to the silk fibroin.

[0032] To verify the mechanical properties of the film-forming products (pure silk fibroin films) prepared by the two methods using aqueous solutions of silk fibroin, the tensile strength, modulus, and elongation at break were tested. The specific results are shown in the table below. Table 1 Tensile Mechanical Properties of MSF Membrane Example 1 8.157±2.714 118.234±64.584 72.127±40.849 Example 2 6.761±2.273 82.101±45.707 97.573±16.674 Example 3 6.640±2.685 61.129±27.180 85.894±14.970 Example 4 6.586±4.032 55.498±46.364 110.881±37.579 Example 5 6.410±1.197 57.294±11.714 80.787±28.532 Comparative Example 1 6.336±0.965 46.059± 7.518 100.580±35.306 As can be seen from Table 1 above, there is no significant difference in the mechanical properties of pure silk fibroin membranes prepared by the two different desalting and deformic acid methods.

[0033] 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 preparing an aqueous solution of silk fibroin, characterized in that, Includes the following steps: S1. Degumming the silk to obtain degummed silk; S2. Degummed silk was dissolved using CaCl2 and formic acid to obtain a CaCl2-formic acid-silk fibroin solution; S3. Place the CaCl2-formic acid-silk fibroin solution prepared in step S2 into an open container. Place the open container containing the CaCl2-formic acid-silk fibroin solution into a box with a steam vent. Introduce water vapor into the box. After a certain period of time, a CaCl2-water-silk fibroin solution is obtained. The box with the steam vent includes a box body. The bottom of the box body is used to place the CaCl2-formic acid-silk fibroin solution. The box body is provided with a steam inlet and a steam outlet. The steam inlet delivers water vapor into the box body through a humidification device. The steam outlet outputs the formic acid that has evaporated from the box body and the overflowing water vapor. The position of the steam outlet is higher than the position of the steam inlet. S4. The CaCl2-water-silk fibroin solution prepared in step S3 is placed in a dialysis bag for dialysis to remove CaCl2 and obtain an aqueous solution of silk fibroin.

2. The method for preparing the silk fibroin aqueous solution 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 preparing the silk fibroin aqueous solution according to claim 1, characterized in that: In step S2, the concentration of formic acid is 80-100 wt.

4. The method for preparing the silk fibroin aqueous solution according to claim 1, characterized in that: In step S2, the CaCl2 content in the CaCl2-formic acid-silk fibroin solution is 2wt.%-10wt.%, and the silk fibroin content is 0.1-50wt.%.

5. The method for preparing the silk fibroin aqueous solution according to claim 1, characterized in that: The temperature of the water vapor is 20-60℃, and the relative humidity of the chamber is above 70%.

6. The method for preparing the silk fibroin aqueous solution according to claim 1, characterized in that: The time for introducing water vapor is greater than 12 hours.

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