Method for efficiently preparing water-soluble regenerated fibroin based on solvent replacement

Abstract

The invention discloses a method for efficiently preparing water-soluble regenerated fibroin based on solvent replacement, which comprises the following steps: dissolving degummed natural fibroin with a low-boiling-point and high-volatility organic solvent, adding water to stop reaction after complete dissolution, quickly separating the solvent from a regenerated fibroin aqueous solution through rotary evaporation or reduced pressure distillation, and recovering the solvent, thereby obtaining the water-soluble regenerated fibroin. After filtering, freeze-drying to obtain water-soluble regenerated fibroin which can be stored for a long time; the prepared regenerated fibroin is good in water solubility, the technical problems that in the preparation process of the water-soluble regenerated fibroin, the desalination difficulty is large, and the preparation efficiency is low are solved, 'salinization-free 'is achieved in the whole process, no wastewater is generated, the solvent can be recycled, the technological process is shorter, the production process is green and environmentally friendly, and the production efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to the field of natural polymer materials, and in particular to a method for efficiently preparing water-soluble regenerated silk fibroin. Background Art

[0002] Silk fibroin is the main component of silk. It has the advantages of excellent mechanical properties, good biocompatibility, biodegradability, and easy processing. It is an ideal source of biomedical materials and can be processed to prepare artificial nerves, artificial skin, artificial bones and other medical tissue materials. Natural silk fibroin is composed of a 391kDa H chain, a 28kDa L chain and a 25kDa P25 protein. The H chain and the L chain are connected by disulfide bonds at the carboxyl ends of the two subunits, and then combined with the P25 protein through non-covalent interactions such as hydrophobic bonds. The silk fibroin H chain protein is composed of a hydrophobic crystalline region and a hydrophilic non-crystalline region to form a long chain molecule. It forms an anti-parallel β-folding structure through hydrogen bonding. The molecular structure is stable, the mechanical properties are good, and it is insoluble in water. To process it into artificial nerves, artificial skin, artificial bones and other medical tissue materials, it must first be dissolved and regenerated. The solvents for dissolving natural silk fibroin mainly include strong acids, strong bases, high-concentration salts and their mixed solutions, organic solvents, ionic liquids, etc. Strong acids and strong bases have a strong hydrolysis effect on silk molecules, and the regenerated silk prepared has a low molecular weight and is not suitable for processing and preparing polymer materials. The acid and alkali as solvents need to be neutralized, and the generated salt needs to be desalted through a desalting column, which has a low desalination rate, and the desalting column needs to be cleaned regularly. The salt-containing wastewater needs to be treated and discharged, which is costly. High-concentration neutral salts such as lithium bromide and calcium chloride and their alcohol solutions can quickly dissolve silk to obtain high-molecular-weight regenerated silk protein, but how to efficiently separate a large amount of salt as a solvent from the regenerated silk has become a bottleneck problem in industrial preparation. The dialysis method takes a long time to desalinate and has a low flux. Dialysis and permeation membrane methods are prone to clogging and low efficiency, and are not suitable for industrial-scale production. The patent "Method for preparing regenerated silk protein with high efficiency and low cost (patent number ZL 2022 1 0182479.X)" provides a high-efficiency desalination technology for regenerated silk, but the regenerated silk protein prepared is water-insoluble, which will be limited in the processing of water-based silk materials. The patent "Method for dissolving natural silk fibers (CN 106589091 A)" provides a method for dissolving silk using a mixed solvent of an organic solvent and a salt, but does not mention a method for efficiently recovering and removing the organic solvent and the salt, which cannot meet the needs of industrial production.

[0003] Therefore, there is an urgent need for a high-efficiency, low-consumption method for preparing water-soluble regenerated silk fibroin to meet the needs of industrial production. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement. The method is simple, salt-free throughout the process, green and environmentally friendly, has a shorter process, high production efficiency, and is suitable for large-scale industrial production.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. A method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement, comprising the following steps:

[0007] (1) dissolving the degummed natural silk fibroin with an organic solvent, and then adding water to stop the reaction to obtain a dissolved product; the solvent is a low boiling point, high volatility organic solvent, and the low boiling point, high volatility organic solvent is trifluoroacetic acid, hexafluoroisopropanol, a mixture of one or two thereof, or an aqueous solution thereof;

[0008] (2) Separating the dissolved substance obtained in step (1) to recover the solvent to obtain a regenerated silk fibroin aqueous solution, filtering and drying to obtain a water-soluble regenerated silk fibroin.

[0009] Preferably, in step (1), the silk fibroin is one or more of cocoon shells, cocoon silk, spun silk, raw silk, waste silk derived from silkworms, wild silkworms or transgenic silkworms, or processed insoluble regenerated silk fibroin, micro-nano fiber, silk fibroin powder; the wild silkworms are tussah silkworms, ricin silkworms, silkworms of Celastrus, willow silkworms and camphor silkworms.

[0010] Preferably, the amount of the dissolving solvent added is 1:2.5 to 1:10 in terms of the mass volume ratio of silk fibroin to solvent, in units of g:mL.

[0011] Preferably, the dissolving is carried out at -10°C to 25°C for 1 to 60 minutes.

[0012] Preferably, the step of adding water to stop the reaction is to add pre-cooled pure water in a volume equivalent to 5 to 10 times the volume of the dissolving solvent to stop the reaction.

[0013] Preferably, the recovered solvent is subjected to rotary evaporation or reduced pressure distillation.

[0014] Preferably, the rotary evaporation or reduced pressure distillation is carried out at a temperature of 10°C to 65°C and a vacuum degree of 0.1 to 70 mbar for 1 to 2 hours.

[0015] Preferably, the drying is freeze-drying after freezing at -80°C for 24 hours.

[0016] The beneficial effects of the present invention are:

[0017] 1. Use organic solvents to dissolve silk fibroin, no salt in the whole process, no wastewater generated, the solvent can be reused, the production process is green and environmentally friendly, no wastewater treatment, and low cost;

[0018] 2. The dissolution process does not require high temperature, and has lower energy consumption than dissolution using lithium bromide or ternary solvent (60°C ~ 80°C); the silk dissolution and solvent separation time is short (<2h), which is shorter than desalination methods such as dialysis (72h) and permeation dialysis (about 9h), greatly improving production efficiency, and the process is short, making it suitable for industrial production.

[0019] 3. The regenerated silk fibroin extracted by this method has good water solubility and has a wider range of applications compared to insoluble regenerated silk fibroin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0021] Figure 1 Flow chart for the preparation of regenerated silk fibroin.

[0022] Figure 2 It is the SDS-PAGE electrophoresis diagram of regenerated silk fibroin protein (M is marker protein, lanes 1-4 are solid-liquid ratios (g:mL) of 1:2.5, 1:3, 1:5 and 1:7.5, respectively, and lane 5 is the water-soluble regenerated silk fibroin prepared by dialysis method).

[0023] Figure 3 It is the SDS-PAGE electrophoresis diagram of regenerated silk fibroin (M is marker protein, lanes 1-3 are silk fibroin prepared by dissolving in 100% trifluoroacetic acid at a solid-liquid ratio (g:mL) of 1:10, 1:7.5, and 1:5, respectively; lanes 4-6 are silk fibroin prepared by dissolving in 90% trifluoroacetic acid at a solid-liquid ratio (g:mL) of 1:10, 1:7.5, and 1:5, respectively).

[0024] Figure 4 is the yield of regenerated silk under different conditions.

[0025] Figure 5 is the solubility of regenerated silk fibroin (A: high molecular weight silk fibroin; B: water-soluble regenerated silk fibroin prepared by dialysis; C: insoluble regenerated silk fibroin prepared by patent method (publication number: CN114621335A)).

[0026] Figure 6 This is the infrared spectrum of the regenerated silk sample prepared in Example 1.

[0027] Figure 7 This is a photograph of the aqueous solution of the regenerated silk fibroin sample prepared in Example 3 (solvent: trifluoroacetic acid / hexafluoroisopropanol volume ratio is 9:1).

[0028] Figure 8 This is the infrared spectrum of the regenerated silk sample prepared in Example 4. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] The present invention obtains natural silk fibroin by degumming silk cocoons, then dissolves the silk fibroin in a low-boiling-point and high-volatile organic solvent under controlled conditions, adds water to stop the reaction after the dissolution is complete, removes the solvent (recovery) by negative pressure or the like, obtains a regenerated silk fibroin aqueous solution, and then filters and dries to obtain the regenerated silk fibroin. The specific process is as follows Figure 1 shown.

[0031] Example 1

[0032] The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement comprises the following steps:

[0033] (1) Cut the silk cocoons into pieces, degumming them twice with a 0.5% (w / v) sodium carbonate aqueous solution at a ratio of 1 g:100 mL, each time for 30 min, fully washing away the sericin, and then air-drying or low-temperature drying to obtain natural silk fibroin;

[0034] (2) Weigh 0.40 g of dried natural silk fibroin and dissolve it in 2 to 4 mL of a 90% to 100% trifluoroacetic acid aqueous solution at -10°C to room temperature (25°C). After dissolving for 5 to 25 min, add 5 to 10 times the volume of pre-cooled pure water to terminate the reaction;

[0035] (3) The mixed solution is subjected to rotary evaporation or vacuum negative pressure for 1 to 2 hours at a temperature of 10°C to 65°C and a vacuum degree of 0.1 to 70 mbar, and the solvent is recovered to obtain a regenerated silk fibroin solution, which is then centrifuged and filtered, and then frozen at -80°C for 24 hours and freeze-dried. The prepared regenerated silk fibroin solution is subjected to SDS-PAGE electrophoresis.

[0036] According to the same method, solid-liquid ratios (g:mL) of 1:1.25, 1:3, 1:5 and 1:7.5 were used, and the ternary solvent dissolution dialysis method was used for comparison. The results are as follows Figure 2 The results show that the regenerated silk prepared by this method has a high molecular weight. The yield of regenerated silk under different conditions was calculated by weight method, and the results are shown in Figure 4 The results show that the yields under different solid-liquid ratios are not much different and the lowest yield is 97.5%, so the method of the present invention has a high yield of regenerated silk fibroin.

[0037] The silk fibroin can be one or more of cocoon shells, cocoon silk, spun silk, raw silk, and waste silk of wild silkworms such as mulberry silkworms or tussah silkworms, or insoluble regenerated silk fibroin prepared by other methods.

[0038] Drying can also be replaced by spray drying, preferably freeze drying.

[0039] Example 2

[0040] The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement comprises the following steps:

[0041] (1) Cut the silk cocoons into pieces, and degummed them twice with a 0.5% (w / v) sodium carbonate aqueous solution at a ratio of 1 g:100 mL, each time for 30 min, and then fully wash away the sericin and air dry or low-temperature dry to obtain natural silk fibroin.

[0042] (2) Weigh 0.40 g of dried natural silk fibroin and dissolve it in 2 to 4 mL of a 100% to 80% trifluoroacetic acid aqueous solution at 0°C to room temperature (25°C). After dissolving for 1 to 60 min, add 5 to 10 times the volume of pre-cooled pure water to terminate the reaction;

[0043] (3) The mixed solution was subjected to rotary evaporation or vacuum negative pressure for 1 to 2 hours at a temperature of 10°C to 65°C and a vacuum degree of 0.1 to 70 mbar, and the solvent was recovered to obtain a regenerated silk fibroin solution, which was centrifuged and filtered, and then frozen at -80°C for 24 hours and then freeze-dried. The prepared regenerated silk fibroin solution was subjected to SDS-PAGE electrophoresis detection. The results are as follows: Figure 3 The results show that the regenerated silk prepared by this method has a low molecular weight.

[0044] The freeze-dried regenerated silk fibroin prepared in Example 1 and Example 2 was dissolved in an aqueous solution, and the insoluble regenerated silk fibroin protein prepared by the patent method with publication number CN114621335A was used as a control to detect the water solubility. The results are as follows: Figure 5 The results show that the regenerated silk prepared by the present invention has good water solubility and can be quickly dissolved in water to prepare regenerated silk solutions of various concentrations. Under the condition of a solid-liquid ratio of 1g / 10mL, its solubility in water can reach 94%. The obtained regenerated silk was tested by infrared spectroscopy, and the results are as follows Figure 6 The results show that silk fibroin has a -1 and 1530cm -1 There is strong absorption at the peak, indicating that its secondary structure is mainly random coil and helical structure, so it has good water solubility and no solvent residual peak (CF3 stretching vibration peak 1278cm -1 、1148cm -1 )exist.

[0045] Example 3

[0046] The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement comprises the following steps:

[0047] (1) Insoluble regenerated silk fibroin is prepared by a patented method with publication number CN114621335A; the main process includes degumming, ternary solvent dissolution, high-efficiency desalting, water washing, freeze drying, etc.

[0048] (2) Weigh 0.1-0.4 g of dry insoluble regenerated silk fibroin and dissolve it in 1-4 mL of 90%-100% trifluoroacetic acid aqueous solution or trifluoroacetic acid / hexafluoroisopropanol mixed solution (volume ratio 9:1, 4:1) at room temperature (25°C). After dissolving for 10-60 min, add 5-10 times the volume of pre-cooled pure water to stop the reaction;

[0049] (3) The mixed solution was subjected to rotary evaporation or vacuum negative pressure for 1 to 2 hours at a temperature of 10°C to 65°C and a vacuum degree of 0.1 to 70 mbar. The solvent was recovered to obtain a regenerated silk solution, which was centrifuged and filtered. The solution was frozen at -80°C for 24 hours and then freeze-dried to obtain a water-soluble regenerated silk protein. 0.01 to 0.1 g of freeze-dried regenerated silk was added to 1 mL of water. The results were as follows: Figure 7 As shown, the sample dissolved rapidly, the aqueous solution was clear and transparent, and there was no obvious insoluble matter after centrifugation.

[0050] Example 4

[0051] The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement comprises the following steps:

[0052] (1) Insoluble regenerated silk fibroin is prepared by a patented method with publication number CN114621335A; the main process includes degumming, ternary solvent dissolution, high-efficiency desalting, water washing, freeze drying, etc.

[0053] (2) Weigh 0.1-0.4 g of dry insoluble regenerated silk fibroin into 1-10 mL of hexafluoroisopropanol solution and dissolve it at 50° C. for 12-24 h. After it is completely dissolved, add 2-5 times the volume of precooled pure water to stop the reaction;

[0054] (3) The mixed solution was subjected to rotary evaporation or vacuum negative pressure for 1 to 2 hours at a temperature of 10°C to 65°C and a vacuum degree of 0.1 to 70 mbar, and the solvent was recovered to obtain a regenerated silk solution, which was centrifuged and filtered, and then frozen at -80°C for 24 hours and freeze-dried to obtain a water-soluble regenerated silk protein. Infrared spectrum test results ( Figure 8 ) showed that its structure was mainly random coil and helical structure, with good water solubility and no obvious solvent residue.

[0055] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement, characterized in that: The steps include: (1) The degummed natural silk fibroin is fully dissolved in an organic solvent, and then water is added to terminate the reaction to obtain a dissolved product; the organic solvent is a low boiling point, high volatility organic solvent, and the low boiling point, high volatility organic solvent is trifluoroacetic acid, hexafluoroisopropanol, a mixture of one or two thereof, or an aqueous solution thereof. (2) Separating the dissolved substance obtained in step (1) to recover the solvent to obtain a regenerated silk fibroin aqueous solution, filtering and drying to obtain a water-soluble regenerated silk fibroin.

2. The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement according to claim 1, characterized in that: In step (1), the silk fibroin is one or more of cocoon shells, cocoon silk, spun silk, raw silk, waste silk from domestic silkworms, wild silkworms or genetically modified silkworms, or processed insoluble regenerated silk fibroin, micro-nano fiber or silk fibroin powder; the wild silkworm is tussah silkworm, ricin silkworm, silkworm of Ceropegia serrata, willow silkworm or camphor silkworm.

3. The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement according to claim 1, characterized in that: The amount of the dissolving solvent added is based on a mass volume ratio of silk fibroin to solvent of 1:2.5 to 1:10, in units of g:mL.

4. The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement according to claim 1, characterized in that: The dissolving is carried out at -10°C to 25°C for 1 to 60 minutes.

5. The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement according to claim 1, characterized in that: The step of adding water to stop the reaction is to add pre-cooled pure water in a volume equivalent to 5 to 10 times the volume of the dissolving solvent to stop the reaction.

6. The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement according to claim 1, characterized in that: The recovered solvent is rotary evaporated or distilled under reduced pressure.

7. The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement according to claim 6, characterized in that: The rotary evaporation or reduced pressure distillation is carried out at a temperature of 10° C. to 65° C. and a vacuum degree of 0.1 to 70 mbar for 1 to 2 hours.

8. The method for efficiently preparing water-soluble regenerated silk fibroin based on solvent replacement according to claim 1, characterized in that: The drying is freeze-drying after freezing at -80°C for 24 hours.

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