Method for preparing silk fibroin solution through microwave-assisted enzymolysis
Through microwave-assisted enzymatic solution, combined with microwave heating and ultrasonic oscillation, the problems of low efficiency and poor stability of traditional heating methods in preparing silk fibroprotein solutions are solved, efficient degumming and enzymatic solution are achieved, and low molecular weight and high purity silk fibroprotein solution is obtained, which extends its storage time and improves its application performance.
Patent Information
- Application Number
- CN202510187712.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-06-20
AI Technical Summary
When preparing silk fibroin solutions, traditional heating methods have problems such as slow heating speed, high energy consumption, radiation residue and uneven heating, and may have adverse effects on the molecular structure of silk fibroin and affect its subsequent application performance. At the same time, it is difficult to completely remove sericin by simply microwave heating, and the silk fibroin solution is prone to changes in molecular structure when environmental changes, resulting in aggregation phenomenon and short storage time.
The microwave-assisted enzymatic lysis method is used to disperse the silk in serine protease solution under microwave heating and degumming, and then dissolve and dialyze it in lithium bromide solution to obtain the regenerated silk fibroin solution. Then the hydrolyzed protease is used for enzymatic lysis treatment. Combined with microwave heating and ultrasonic oscillation, the dissolution time and enzymatic lysis time are shortened to ensure the stability and low molecular weight of silk fibroin.
The heating efficiency is improved and the efficient degumming and enzymatic decomposition of silk fibroin are achieved, and a low molecular weight and high purity silk fibroin solution is obtained, which enhances the stability of silk fibroin, extends its storage time, and improves its application performance in the field of medical surgery.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of silk fibroin preparation, and particularly relates to a method for preparing silk fibroin solution by microwave-assisted enzymatic hydrolysis. Background Art
[0002] Silk fibroin, also known as fibroin, is a natural polymeric fibrous protein extracted from silk. Silk protein fiber is mainly composed of silk fibroin and sericin. Among them, sericin is located outside and accounts for about 25% of the weight, while silk fibroin, as the main component of silk, accounts for up to 70%. Silk fibroin is composed of numerous peptide chains tightly bound by ionic bonds and hydrophobic bonds. Hydrolyzing it with specific enzymes can obtain sericin and silk fibroin, and further enzymatic hydrolysis can obtain silk peptides and serine with different molecular weights. In addition, silk fibroin is non-toxic, non-allergenic and non-irritating to the human body, has excellent biocompatibility, and its performance can be comparable to that of collagen.
[0003] Due to its unique properties such as good biocompatibility, low immunogenicity and biodegradability, silk protein has been widely used in the fields of biomedicine and sustainable development. The silk fibroin after removing sericin can be used as scaffold materials in medical surgeries, such as gels, surgical sutures and heart bypass materials. The environmental stability of silk fibroin and its ability to control the delivery and encapsulation of proteins and small molecule drugs make it have broad application prospects in the field of medical surgery. In the process of preparing silk fibroin solution, usually, the difference in solubility of silk fibroin and sericin in the aqueous solution system is utilized to treat silk with acids, alkalis, soaps, enzymes, etc., so that sericin dissolves in the solution system, and then silk fibroin is separated by methods such as centrifugation, filtration and dialysis. However, traditional heating methods, such as using other high-energy electromagnetic waves for heating, often have problems such as slow heating speed, high energy consumption, radiation residues and uneven heating, and may have an adverse impact on the molecular structure of silk fibroin, thus affecting its subsequent application performance.
[0004] Microwave-assisted heating, as a clean non-contact heating method, usually has a heating frequency of 2.45 GHz, and has the advantages of fast heating speed, uniform heating and high energy utilization rate. Microwave heating releases energy through the interaction of electric and magnetic fields. These energies are transmitted in the form of microwaves, causing the electromagnetic oscillation of dipoles inside the substance, thereby converting electromagnetic energy into heat energy to achieve rapid heating. Compared with traditional heating methods, microwave heating has the advantages of fast heating speed, uniformity and low energy consumption. Therefore, introducing microwave heating technology in the process of preparing silk fibroin solution is expected to improve the heating efficiency and dissolution effect.
[0005] However, although microwave heating technology has many advantages in preparing silk fibroin solution, there are still some problems in practical application. For example, simple microwave heating technology cannot completely remove sericin in the preparation of silk fibroin solution. And the silk fibroin solution currently on the market faces a series of challenges in the preparation and storage process. For example, the molecular structure of silk fibroin solution is prone to change when the environment changes (such as high temperature or shock), resulting in aggregation, which is specifically manifested as gel formation or precipitation. Even under relatively mild conditions, such as room temperature storage, the storage time of purified silk fibroin solution is usually not more than two weeks. For large molecular weight regenerated silk fibroin solution, even if it is stored in a 4°C refrigerator, its structure is slowly changing and the molecules are constantly aggregating. This aggregation phenomenon may cause the viscosity of the silk fibroin solution to increase, the fluidity to decrease, and even the formation of insoluble precipitates, which directly affects the performance of the subsequent prepared materials.
[0006] Therefore, how to improve the stability and storage time of silk fibroin solution and avoid the occurrence of molecular aggregation is a technical problem that needs to be solved urgently in the current field of silk fibroin preparation and application. Summary of the invention
[0007] In order to solve the above technical problems, the purpose of the present invention is to provide a method for preparing silk fibroin solution by microwave-assisted enzymatic hydrolysis.
[0008] The above object of the present invention is achieved through the following technical solutions:
[0009] The present invention provides a method for preparing a silk fibroin solution by microwave-assisted enzymatic hydrolysis, comprising the following steps:
[0010] (1) dispersing silk in a serine protease solution under the condition of heating to 60-80° C. at a microwave of 5-10 kW, performing ultrasonic degumming treatment, and after degumming, heating to 100-110° C. and ultrasonicating for 5-60 minutes to obtain degummed silk;
[0011] (2) dispersing the degummed silk obtained in step (1) in a lithium bromide solution, heating and dissolving at 55-65° C., and performing a dialysis treatment to obtain a regenerated silk fibroin solution;
[0012] (3) adding protease to the regenerated silk fibroin solution obtained in step (2), adjusting the pH value of the obtained mixed solution to 7.5-8.5, heating the mixed solution to 50-60° C. at a microwave temperature of 5-10 kW, heating the mixed solution to 100-110° C. after the reaction, and performing ultrasonic treatment for 5-10 min to inactivate the protease, performing solid-liquid separation, and collecting the supernatant to obtain a silk fibroin solution, wherein the molecular weight of the silk fibroin in the silk fibroin solution is less than 20 kDa.
[0013] As a non-contact heating method, microwave heating has the advantages of fast heating speed, uniform heating, and high energy utilization rate. During the preparation of regenerated silk fibroin solution, microwave heating can directly penetrate the material itself without changing the bonding energy inside the molecules, and its energy is much lower than the bond energy between ions. Therefore, it can dissolve silk efficiently and cleanly. Compared with traditional heating methods, microwave heating shows better degumming effect under the same degumming conditions, making the silk surface smoother and easier to dissolve. Acting synergistically with serine protease, it shortens the dissolution time and enzymatic hydrolysis time, reducing costs. Under suitable temperature and pH conditions, during microwave heating, the hydrolytic protease reacts fully with the regenerated silk fibroin solution, and the intermolecular frictional heat transfer causes the liquid temperature to rise, increasing the enzymatic hydrolysis rate. During the ultrasonic oscillation process, the enzymatic hydrolysis precipitate precipitates faster, obtaining a low molecular weight silk fibroin solution.
[0014] In the process of preparing silk fibroin solution in the present invention, two enzymes are used and microwave-assisted enzymatic hydrolysis is adopted, which not only speeds up the degumming rate, but also removes sericin completely, enhances the stability of silk fibroin, and obtains a low molecular weight silk fibroin solution. Microwave heating has a repairing effect on the number of amino acids in the silk fibroin solution. After being blended with other antibacterial functional substances, it can enhance the stability and extend the storage time of the silk fibroin solution.
[0015] Further, in step (1), the solid-liquid ratio of the silk and the serine protease solution is (30 - 50) g : (1 - 3) L.
[0016] Further, in step (1), the concentration of serine protease in the serine protease solution is 0.5 - 5 wt%.
[0017] Further, in step (1), the serine protease solution is an aqueous solution of serine protease.
[0018] Further, in step (1), the time of ultrasonic treatment is 10 - 20 min.
[0019] Specifically, in step (1), under the condition of microwave heating, the silk is dispersed in the serine protease solution, and ultrasonic treatment is carried out for multiple degumming treatments. After washing and drying, degummed silk is obtained.
[0020] Further, in step (2), the solid-liquid ratio of the degummed silk and the lithium bromide solution is (20 - 30) g : (60 - 90) mL.
[0021] Further, in step (2), the concentration of the lithium bromide solution is 9 - 10 mol / L.
[0022] Further, in step (2), the lithium bromide solution is an aqueous solution of lithium bromide.
[0023] Further, in step (2), dialysis is performed until the conductivity of the final solution is less than 20 μS / cm and lithium bromide is completely removed to obtain a regenerated silk fibroin solution.
[0024] Further, in step (3), the mass ratio of the regenerated silk fibroin solution to the hydrolytic protease is 100:(0.2 - 0.5).
[0025] Further, in step (3), the reaction is carried out for 20 - 40 min under the condition of microwave heating to 50 - 60 °C.
[0026] Further, in step (3), when the pH value of the obtained mixed solution < 8.5, sodium hydroxide is used to adjust the pH value; when the pH value of the obtained mixed solution > 8, acetic acid is used to adjust the pH value.
[0027] Advantages of the present invention:
[0028] 1. In the traditional enzymatic hydrolysis process, heating is carried out first and then oscillation. The preparation time of silk fibroin is too long. Generally, the supernatant of silk fibroin needs to wait at least 12 h before it can be taken out. However, the present invention optimizes the degumming process, uses the combined action of microwave heating and serine protease, followed by dialysis, then treatment with hydrolytic protease, and utilizes microwave heating and ultrasonic oscillation, which shortens the dissolution time and enzymatic hydrolysis time, and can quickly obtain a clarified high-purity low-molecular-weight silk fibroin solution.
[0029] 2. The present invention uses the microwave heating method to improve the solubility of silk fibroin, ensure the stability of silk fibroin, and the microwave heating has a repairing effect on the number of amino acids in the dissolved silk fibroin solution. After being blended with other antibacterial functional substances, it can enhance the stability and extend the storage time of the silk fibroin solution.
[0030] 3. The present invention enhances the functionality of the silk fibroin solution. By adding different functional components, it can meet the diverse needs of different fields for silk fibroin materials. Description of the drawings
[0031] Figure 1 It is a physical diagram of the degummed silk obtained in step (1) in Examples 1 - 2 and Comparative Examples 1 - 2; among them, (a) is Comparative Example 1, (b) is Comparative Example 2, (c) is Example 1, and (d) is Example 2.
[0032] Figure 2 It is a comparison diagram of the storage conditions of the silk fibroin solutions prepared in Examples 1 - 2 and Comparative Examples 1 - 2, from left to right are Comparative Example 1, Comparative Example 2, Example 1, and Example 2.
[0033] Figure 3 It is a test data diagram of the molecular weight of silk fibroin in the silk fibroin solutions prepared in Example 1 and Comparative Example 2.
[0034] Figure 4 FTIR spectra of the silk fibroin solutions prepared in Examples 1-2 and Comparative Examples 1-2. Detailed implementation manners
[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of this invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.
[0036] The present invention will be 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 be able to implement it, but the exemplified embodiments are not intended to limit the present invention.
[0037] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels. In the following examples and comparative examples, the hydrolytic protease used is Alcalase 2.4 L (Novozymes hydrolytic protease 2.4 L).
[0038] Example 1
[0039] A method for preparing a silk fibroin solution by microwave-assisted enzymatic hydrolysis, comprising the following steps:
[0040] (1) Heat 40 g of silk cocoons to 60 °C with 10 kW of microwave, dissolve them in 2 L of an aqueous solution of serine protease with a concentration of 2%, ultrasonicate for 20 min, raise the temperature to 100 °C, ultrasonicate for 5 min to inactivate the serine protease, fish out the silk cocoons, wash them twice, and then fish them out and dry them to obtain degummed silk.
[0041] (2) Place 75 mL of a 9.3 mol / L lithium bromide aqueous solution in a beaker, preheat it at 60 °C for 30 min, loosen 25 g of degummed silk and add it to the lithium bromide aqueous solution, stir with a glass rod to make them fully contact, cover with a plastic wrap, dissolve at 60 °C for 30 min to obtain a degummed silk solution, and cool it to room temperature; fold one end of the dialysis bag several times and clamp it, then slowly pour the degummed silk solution from the other end, and then clamp it with a dialysis clip. Place the dialysis bag in a large beaker filled with deionized water for dialysis. Change the water every 2 h at the beginning of dialysis. Dialyze for a total of 3 days until the conductivity of the final solution is less than 20 μS / cm and the lithium bromide is completely removed to obtain a regenerated silk fibroin solution.
[0042] (3) Add 0.2 g of hydrolytic protease to 100 g of the regenerated silk fibroin solution. Use a pH tester to measure the pH value and stabilize the pH of the regenerated silk fibroin solution at 8. Heat it to 55 °C with 10 kW of microwave for 30 min. After the reaction, raise the temperature to 100 °C and ultrasonicate for 5 min to inactivate the hydrolytic protease. Let it stand for 20 min, filter, and take the supernatant to obtain the silk fibroin solution. The molecular weight of the silk fibroin in the silk fibroin solution is measured to be 10 - 20 kDa by gel electrophoresis.
[0043] Example 2
[0044] A method for preparing a silk fibroin solution by microwave-assisted enzymatic hydrolysis, comprising the following steps:
[0045] (1) Heat 40 g of silk cocoons to 80 °C with 5 kW of microwave and dissolve them in 2 L of an aqueous solution of serine protease with a concentration of 5%. Ultrasonicate for 10 min, raise the temperature to 100 °C, and ultrasonicate for 5 min to inactivate the serine protease. Fish out the silk cocoons, wash them twice, and then take them out to dry to obtain degummed silk.
[0046] (2) Place 75 mL of 9.3 mol / L lithium bromide aqueous solution in a beaker and preheat it at 60 °C for 30 min. Loosen 25 g of the degummed silk and add it to the lithium bromide aqueous solution. Stir with a glass rod to make them fully contact. Cover with a plastic wrap and dissolve at 60 °C for 30 min to obtain a degummed silk solution. Cool it to room temperature. Fold one end of the dialysis bag several times and clamp it, then slowly pour the degummed silk solution from the other end and clamp it with a dialysis clip. Place the dialysis bag in a large beaker filled with deionized water for dialysis. Change the water every 2 h at the beginning of dialysis. Dialyze for 3 days in total to make the conductivity of the final solution less than 20 μS / cm and completely remove the lithium bromide to obtain the regenerated silk fibroin solution.
[0047] (3) Add 0.2 g of hydrolytic protease to 100 g of the regenerated silk fibroin solution. Use a pH tester to measure the pH value and stabilize the pH of the regenerated silk fibroin solution at 8. Heat it to 50 °C with 10 kW of microwave for 30 min. After the reaction, raise the temperature to 100 °C and ultrasonicate for 5 min to inactivate the hydrolytic protease. Let it stand for 20 min, filter, and take the supernatant to obtain the silk fibroin solution. The molecular weight of the silk fibroin in the silk fibroin solution is measured to be 10 - 15 kDa by gel electrophoresis.
[0048] Example 3
[0049] A method for preparing a silk fibroin solution by microwave-assisted enzymatic hydrolysis, comprising the following steps:
[0050] (1) Dissolve 40 g of silk in 2 L of an aqueous solution of serine protease with a concentration of 7% by heating with microwave at 10 kW to 60 °C and ultrasonicate for 20 min. Then raise the temperature to 110 °C and ultrasonicate for 5 min to inactivate the serine protease. Fish out the silk, wash it twice, and then take it out to dry, obtaining degummed silk.
[0051] (2) Place 75 mL of an aqueous solution of lithium bromide at 9.3 mol / L in a beaker and preheat it at 60 °C for 30 min. Loosen 25 g of degummed silk and add it to the aqueous solution of lithium bromide. Stir with a glass rod to make them fully contact. Cover with a plastic wrap and dissolve at 60 °C for 30 min to obtain a degummed silk solution, and then cool it to room temperature. Fold one end of the dialysis bag several times and clamp it, then slowly pour the degummed silk solution from the other end and clamp it with a dialysis clip. Place the dialysis bag in a large beaker filled with deionized water for dialysis. Change the water every 2 h at the beginning of dialysis, and the dialysis lasts for 3 days until the conductivity of the final solution is less than 20 μS / cm and the lithium bromide is completely removed, obtaining a regenerated silk fibroin solution.
[0052] (3) Add 0.2 g of hydrolytic protease to 100 g of the regenerated silk fibroin solution. Use a pH tester to measure the pH value and make the pH of the regenerated silk fibroin solution stable at 8. Heat with microwave at 5 kW to 60 °C and react for 30 min. After that, raise the temperature to 110 °C and ultrasonicate for 10 min to inactivate the hydrolytic protease. Let it stand for 20 min, filter, and take the supernatant to obtain a silk fibroin solution. The molecular weight of the silk fibroin in the silk fibroin solution is measured to be 10 - 20 kDa by gel electrophoresis.
[0053] Comparative Example 1
[0054] A method for preparing a silk fibroin solution by an alkaline method, comprising the following steps:
[0055] (1) Heat the aqueous solution to 100 °C, add anhydrous sodium carbonate and boil for 10 min to obtain an aqueous solution of anhydrous sodium carbonate with a concentration of 0.5%. Add 40 g of silk to 2000 g of the aqueous solution of anhydrous sodium carbonate and boil for 30 min. Fish out the silk, wash it twice, and then take it out to dry, obtaining degummed silk.
[0056] (2) Place 75 mL of an aqueous solution of lithium bromide at 9.3 mol / L in a beaker and preheat it at 60 °C for 30 min. Loosen 25 g of degummed silk and add it to the aqueous solution of lithium bromide. Stir with a glass rod to make them fully contact. Cover with a plastic wrap and dissolve at 60 °C for 30 min to obtain a degummed silk solution, and then cool it to room temperature. Fold one end of the dialysis bag several times and clamp it, then slowly pour the degummed silk solution from the other end and clamp it with a dialysis clip. Place the dialysis bag in a large beaker filled with deionized water for dialysis. Change the water every 2 h at the beginning of dialysis, and the dialysis lasts for 3 days until the conductivity of the final solution is less than 20 μS / cm and the lithium bromide is completely removed, obtaining a regenerated silk fibroin solution.
[0057] (3) Add 0.2 g of hydrolytic protease to 100 g of regenerated silk fibroin solution. Use a pH tester to measure the pH value and stabilize the pH of the regenerated silk fibroin solution at 8. Heat it to 55 °C with 10 kW of microwave for 30 min. After the reaction, raise the temperature to 100 °C and sonicate for 5 min to inactivate the hydrolytic protease. Let it stand for 20 min, filter, and take the supernatant to obtain a silk fibroin solution. The molecular weight of the silk fibroin in the silk fibroin solution is measured to be 10 - 250 kDa by gel electrophoresis.
[0058] Comparative Example 2
[0059] A method for preparing silk fibroin solution by induction cooker-assisted enzymatic hydrolysis, comprising the following steps:
[0060] (1) Heat 40 g of silk in 2 L of an aqueous solution of serine protease with a concentration of 2% to 60 °C using an induction cooker, boil for 30 min on the induction cooker, raise the temperature to 100 °C, and sonicate for 5 min to inactivate the serine protease. Take out the silk, wash it twice, and then take it out and dry it to obtain degummed silk.
[0061] (2) Place 75 mL of 9.3 mol / L lithium bromide aqueous solution in a beaker, preheat it at 60 °C for 30 min. Loosen 25 g of degummed silk and add it to the lithium bromide aqueous solution. Stir with a glass rod to make them fully contact, cover it with a plastic wrap, and dissolve it at 60 °C for 30 min to obtain a degummed silk solution. Cool it to room temperature; fold one end of the dialysis bag several times and clamp it, then slowly pour the degummed silk solution from the other end, and then clamp it with a dialysis clip. Place the dialysis bag in a large beaker filled with deionized water for dialysis. Change the water every 2 h at the beginning of dialysis. Dialyze for a total of 3 days until the conductivity of the final solution is less than 20 μS / cm and the lithium bromide is completely removed to obtain a regenerated silk fibroin solution.
[0062] (3) Add 0.2 g of hydrolytic protease to 100 g of regenerated silk fibroin solution. Use a pH tester to measure the pH value and stabilize the pH of the regenerated silk fibroin solution at 8. Heat it to 55 °C with 10 kW of microwave for 30 min. After the reaction, raise the temperature to 100 °C and sonicate for 5 min to inactivate the hydrolytic protease. Let it stand for 20 min, filter, and take the supernatant to obtain a silk fibroin solution. The molecular weight of the silk fibroin in the silk fibroin solution is measured to be 15 - 70 kDa by gel electrophoresis.
[0063] Comparative Example 3
[0064] A method for preparing silk fibroin solution by induction cooker-assisted enzymatic hydrolysis, comprising the following steps:
[0065] (1) Heat 40 g of silk in an induction cooker to 80 °C and dissolve it in 2 L of an aqueous solution of serine protease with a concentration of 5%. Boil it in the induction cooker for 30 min, then raise the temperature to 110 °C and ultrasonicate for 5 min to inactivate the serine protease. Take out the silk, wash it twice, and then take it out to dry, obtaining degummed silk.
[0066] (2) Place 75 mL of 9.3 mol / L lithium bromide aqueous solution in a beaker and preheat it at 60 °C for 30 min. Loosen 25 g of degummed silk and add it to the lithium bromide aqueous solution. Stir with a glass rod to make them fully contact. Cover it with a plastic wrap and dissolve it at 60 °C for 30 min to obtain a degummed silk solution, and then cool it to room temperature. Fold one end of the dialysis bag several times and clamp it, then slowly pour the degummed silk solution from the other end, and then clamp it with a dialysis clip. Place the dialysis bag in a large beaker filled with deionized water for dialysis. Change the water every 2 h at the beginning of dialysis. Dialyze for a total of 3 days until the conductivity of the final solution is less than 20 μS / cm and the lithium bromide is completely removed, obtaining a regenerated silk fibroin solution.
[0067] (3) Add 0.2 g of hydrolytic protease to 100 g of the regenerated silk fibroin solution. Use a pH tester to measure the pH value and make the pH of the regenerated silk fibroin solution stable at 8. Heat it to 50 °C with a microwave of 10 kW and react for 30 min. After that, raise the temperature to 100 °C and ultrasonicate for 5 min to inactivate the hydrolytic protease. Let it stand for 20 min, filter it, and take the supernatant to obtain a silk fibroin solution. The molecular weight of the silk fibroin in the silk fibroin solution is measured to be 15 - 70 kDa by gel electrophoresis.
[0068] Figure 1 It is the physical diagram of the degummed silk obtained in step (1) of Examples 1 - 2 and Comparative Examples 1 - 2; among them, (a) is Comparative Example 1, (b) is Comparative Example 2, (c) is Example 1, and (d) is Example 2. The degummed silk obtained by alkaline degumming in Comparative Example 1 is dry and wrinkled, and it is easy to have residues when dissolved; the degummed silk obtained by heating with an induction cooker and adding serine protease with a concentration of 2% for enzymatic hydrolysis in Comparative Example 2 is fluffier and easier to dissolve than Comparative Example 1; the degummed silk obtained by combining enzymatic hydrolysis with 2% and 5% serine protease and microwave heating in Examples 1 - 2 is even fluffier and easier to dissolve, and it is not slippery to the touch, indicating that the degumming is relatively clean and the damage to silk is not significant.
[0069] Figure 2Comparison chart of the preservation of the silk fibroin solutions prepared in Examples 1-2 and Comparative Examples 1-2. From left to right are Comparative Example 1, Comparative Example 2, Example 1, and Example 2. The pure silk fibroin solution without enzymatic hydrolysis in Comparative Example 1 solidified within one month, while the silk fibroin solution with enzymatic hydrolysis had a longer storage time. The silk fibroin solution obtained by enzymatic hydrolysis with only 2% serine protease in Comparative Example 2 was a bit turbid and had suspensions in the liquid, while the silk fibroin solutions obtained by combining enzymatic hydrolysis with 2% and 5% serine protease and microwave heating in Examples 1-2 have not become turbid yet since they were produced on July 17, 2024.
[0070] The molecular weights of the silk fibroin in the silk fibroin solutions prepared in Example 1 and Comparative Example 2 were tested using gel electrophoresis. The test results are as Figure 3 shown. It was found that the molecular weight range of the silk fibroin in the silk fibroin solution obtained by microwave heating enzymatic hydrolysis was smaller than that in the silk fibroin solution obtained by induction cooker heating enzymatic hydrolysis.
[0071] Figure 4 Fourier Transform Infrared Spectroscopy (FTIR) diagrams of the silk fibroin solutions prepared in Examples 1-2 and Comparative Examples 1-2. It can be seen from Figure 4 them that the structures of the silk fibroin in the silk fibroin solutions have not changed.
[0072] Obviously, the above examples of the present invention are only illustrations for clearly explaining the present invention, and are not limitations on the implementation modes of the present invention. Those skilled in the art should understand that other different forms of changes or variations can be made on the basis of the above description. It is not necessary and impossible to enumerate all implementation modes here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.
Claims
1. A method for preparing a silk fibroin solution by microwave-assisted enzymatic hydrolysis, characterized in that: The following steps are involved: (1) dispersing silk in a serine protease solution under the condition of heating to 60-80° C. at a microwave of 5-10 kW, performing ultrasonic degumming treatment, and after degumming, heating to 100-110° C. and ultrasonicating for 5-60 minutes to obtain degummed silk; (2) dispersing the degummed silk obtained in step (1) in a lithium bromide solution, heating and dissolving at 55-65° C., and performing a dialysis treatment to obtain a regenerated silk fibroin solution; (3) adding protease to the regenerated silk fibroin solution obtained in step (2), adjusting the pH value of the obtained mixed solution to 7.5-8.5, heating the mixed solution to 50-60° C. at a microwave temperature of 5-10 kW, heating the mixed solution to 100-110° C. after the reaction, and performing ultrasonic treatment for 5-10 min to inactivate the protease, performing solid-liquid separation, and collecting the supernatant to obtain a silk fibroin solution, wherein the molecular weight of the silk fibroin in the silk fibroin solution is less than 20 kDa.
2. The method according to claim 1, characterized in that In step (1), the solid-liquid ratio of the silk and serine protease solution is (30-50) g: (1-3) L.
3. The method according to claim 1, characterized in that: In step (1), the concentration of serine protease in the serine protease solution is 0.5-5wt%.
4. The method according to claim 1, characterized in that: In step (1), the ultrasonic time is 10-20 minutes.
5. The method according to claim 1, characterized in that In step (1), the silk is dispersed in a serine protease solution under microwave heating, subjected to ultrasonic degumming treatment for multiple times, and then washed and dried to obtain degummed silk.
6. The method according to claim 1, characterized in that In step (2), the solid-to-liquid ratio of the degummed silk to the lithium bromide solution is (20-30) g:(60-90) mL.
7. The method according to claim 1, characterized in that In step (2), the concentration of the lithium bromide solution is 9-10 mol / L.
8. The method according to claim 1, characterized in that In step (3), the mass ratio of the regenerated silk fibroin solution to the hydrolyzing protease is 100:(0.2-0.5).
9. The method according to claim 1, characterized in that: In step (3), the reaction is carried out under microwave heating at 50-60° C. for 20-40 min.
10. The method according to claim 1, characterized in that In step (3), when the pH value of the obtained mixed solution is less than 8.5, sodium hydroxide is used to adjust the pH value; when the pH value of the obtained mixed solution is greater than 8, acetic acid is used to adjust the pH value.