CN113735936B - A method for increasing the concentration of silk protein-based aqueous solution and the silk protein-based aqueous solution prepared by the method - Google Patents

Abstract

The present invention relates to a method for increasing the concentration of a silk protein-based aqueous solution and a silk protein-based aqueous solution prepared by the method. The method includes the steps of performing vacuum centrifugation after dialyzing and centrifuging the starting silk protein aqueous solution. The advantage of the method is that it can provide a pure silk protein-based aqueous solution and the method has the advantages of low cost, high efficiency and good controllability compared to the common preparation methods of the silk protein-based aqueous solution in the prior art.

Description

A method for increasing the concentration of silk protein-based aqueous solution and silk prepared by the method protein based aqueous solution

Technical field

The invention belongs to the technical field of silk protein-based material processing, and specifically relates to a method for increasing the concentration of a silk protein-based aqueous solution and a silk protein-based aqueous solution prepared by the method.

Background technique

Protein-based natural polymer materials (such as collagen, keratin, silk protein, etc.) have the characteristics of wide sources, good biocompatibility, and biodegradability. In recent years, they have received widespread attention and have been used in implantation of interventional materials and tissue repair. , tissue engineering, drug sustained release and many other biomedical and bioengineering fields.

Among many protein-based natural polymer materials, silk protein-based materials have become a hot research topic in recent years due to their excellent mechanical properties, biocompatibility and biodegradability.

In addition to directly utilizing natural silk fibers, one of the current main research directions is to process natural silk fibers to obtain regenerated silk proteins and then process them into various functional materials. Usually regenerated silk protein is extracted through an aqueous solution method, specifically dissolving natural silk fiber in a high-concentration inorganic salt (lithium bromide, sodium thiocyanate, etc.) solution or alcohol-salt mixed system (calcium chloride-ethanol-water, etc.) , and then undergo dialysis and purification to obtain a regenerated silk protein aqueous solution. Since the osmotic pressure is very high during the dialysis process, a large amount of water will enter the dialysis bag, so the concentration of the obtained silk protein aqueous solution is usually low (generally around 6wt.%). The low-concentration regenerated silk protein aqueous solution is prepared in the material preparation process. and production have been greatly restricted. In addition to silk protein aqueous solutions obtained through dissolution and regeneration, there are other silk protein-based aqueous solutions, such as aqueous solutions of regenerated silk protein derivatives obtained through chemical modification; composite silk protein-based solutions of regenerated silk protein/additives obtained through physical mixing ; Recombinant silk protein aqueous solutions obtained through genetic recombination technology will also face similar limitations. How to efficiently and controllably concentrate a low-concentration silk protein-based aqueous solution to obtain a higher-concentration solution has become an urgent problem that needs to be solved.

There are two main methods for concentrating silk protein aqueous solution in the prior art. One is the dialysis bag osmotic concentration method. This method mainly places the silk protein aqueous solution in a dialysis bag, and then injects it into a high molecular polymer (such as polyethylene). Dialysis is carried out in a concentrated solution of ethylene glycol, etc., during which the water in the dialysis bag can seep out to obtain a high-concentration silk protein solution (US8614293 B2). However, this method is time-consuming, requires the use of a large amount of polymer dialysate, and the concentration cost is extremely high. Moreover, polymers such as polyethylene glycol used in the dialysis process will have some small molecules or impurity ions remaining during the polymerization production process. These impurity ions may penetrate into the dialysis bag and affect the purity of the prepared silk protein solution. Another method of concentrating silk protein aqueous solution is to place the silk protein aqueous solution in a dialysis bag, then place it in a vacuum drying box and put a water-absorbing agent in the drying box, and vacuum dry it at a certain temperature. By regulating the vacuum The required different concentrations of silk protein solutions (CN 102167724) can be obtained by adjusting the time and vacuum degree. This method requires the use of dialysis bags and a large amount of water-absorbing agents, and the concentration cost is high.

Therefore, there is still a need for an efficient, pure, controllable, low-cost method for preparing a high-concentration silk protein-based aqueous solution with industrial prospects.

Contents of the invention

The purpose of the present invention is to provide a method for preparing a high-concentration silk protein-based aqueous solution that is efficient, pure, controllable, low-cost, and has industrial prospects.

The invention provides a method for increasing the concentration of silk protein-based aqueous solution, which method includes the following steps:

(1) Put the starting silk protein aqueous solution into a dialysis bag for dialysis, and the dialyzed solution is centrifuged at the first centrifugal speed under ambient pressure, and the precipitate is discarded to obtain a concentration of 4.0-8.0w/v%, preferably 4.5-6.5w/v% silk protein based aqueous solution;

(2) Place the silk protein-based aqueous solution obtained in step (1) under a vacuum environment and perform centrifugal concentration at a second centrifugal speed, thereby obtaining a silk protein-based aqueous solution with a concentration greater than 10 w/v%.

In specific embodiments, the starting silk protein aqueous solution in step (1) can be a natural silk protein aqueous solution; an aqueous solution of regenerated silk protein derivatives obtained through chemical modification; regenerated silk protein/additives (such as drugs) obtained through physical mixing , growth factors, antibiotics, etc.) composite silk protein-based solution; recombinant silk protein aqueous solution, etc.

In a specific embodiment, the starting natural silk protein aqueous solution is prepared by the following method: put chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and cook it for 30-180 min at a temperature of 60-100°C, thereby The silkworm cocoons are degummed, and then the dried degummed silk is dissolved in 9-10M, preferably 9.3M LiBr aqueous solution, and placed in a 60-150°C oven for 2-8 hours to completely dissolve.

In a specific embodiment, in step (1), the starting silk protein aqueous solution is dialyzed under the following dialysis conditions: the dialysis time is 2-5 days, the dialysate is deionized water, and the molecular weight cutoff of the dialysis bag is 3500 Da Dayton (Da).

In a specific embodiment, in step (1), the first centrifugal speed is 5000-11000 rpm, preferably 9000 rpm.

In a specific embodiment, before step (2), additives for improving the properties of silk protein-based material products can be added to the silk protein aqueous solution, and the additives are selected from the group consisting of glycerol, inorganic salts, bioactive molecules (such as enzymes, antibiotics, drugs, etc.), inorganic materials, organic molecules. The concentration of additives may range from 0.1 to 50 wt%. The properties of silk protein-based material products, such as physical, chemical and biological properties, can be improved by adding additives, but will not affect the concentration effect.

In a specific embodiment, in step (2), vacuum centrifugation is performed under the following conditions: the second centrifugal speed is 100-3000rpm, preferably 300-2000rpm, the concentration temperature is 5-60°C, and the vacuum degree is 0.1-1000mbar, Concentration time is 3-48h.

In a specific embodiment, the concentration of the silk protein-based aqueous solution obtained in step (2) is 10-50w/v%, preferably 25-40w/v%. Preferably, the silk protein-based aqueous solution obtained in step (2) The concentration of the aqueous solution is 6 times the concentration of the silk protein-based aqueous solution obtained in step (1).

In a specific embodiment, the method further includes: after step (2), performing a third centrifugal operation at a third centrifugal speed to separate the precipitated silk protein.

In a specific embodiment, the third centrifugation operation is performed under the following conditions: centrifugation time is 5-30 min, centrifugation temperature is 4-30°C, and the third centrifugation speed is 5000-14000 rpm, preferably 9000 rpm.

In another aspect, the present invention provides a silk protein-based aqueous solution prepared by the above method.

In yet another aspect, the present invention provides a silk protein-based material made from the above-mentioned silk protein-based aqueous solution.

In specific embodiments, the silk protein-based material can be in the form of any one of film, fiber, sponge, hydrogel, and powder.

In specific embodiments, the preparation method of silk protein-based materials in the form of films, fibers, sponges, hydrogels or powders is not limited, and conventional methods in the art for preparing these forms from silk protein-based aqueous solutions can be used.

beneficial effects

Compared with the methods in the prior art, the method for improving the solubility of silk protein-based aqueous solutions provided by this application has the following advantages: (1) No polymers, such as polyethylene glycol, are used, so the resulting high-concentration silk protein-based solution is There will be no residual small molecules or impurity ions in the polymer production process, making it purer; (2) No water absorbing agents and dialysis bags are used during the vacuum concentration process, which effectively reduces the cost of concentration.

In addition, since no additional impurity small molecules or impurity ions are incorporated in the method for increasing the concentration of silk protein-based aqueous solution provided in this application, the high-concentration silk protein-based aqueous solution obtained thereby is purer, and therefore can be used more advantageously It can be used in silk protein-based materials (such as films, fibers, sponges, hydrogels, powders, etc.) in the fields of biomedicine, tissue engineering, textiles, flexible electronic devices, etc., and thus provides silk protein-based materials compared with existing technologies. Additional advantages of silk aqueous solution.

Description of drawings

Figure 1: Relationship between concentration time and concentration of silk protein aqueous solution.

Figure 2: Relationship between shear rate and shear viscosity in pure silk protein aqueous solutions with different concentrations.

Figure 3: Plot of modulus versus angular velocity in 28 wt.% silk protein aqueous solution.

Figure 4: Comparison of the initial concentration solution of silk protein and the concentrated high-concentration solution in Example 4.

Detailed ways

The process of the present invention is described in detail below through specific examples. However, these examples are only representative examples to illustrate the method of the present invention and are not intended to limit the scope of the present invention.

the term:

As used herein, the term "high-concentration silk protein-based aqueous solution" means a silk protein-based aqueous solution with a concentration greater than 10 w/v%.

In this article, the term "concentration time" refers to the time the instrument runs when the dilute concentration solution reaches the specified high concentration solution during the centrifugal concentration process.

In this article, the term "silk protein" may be understood as "silk fibroin".

The manufacturers of the main experimental reagents and instruments used in the following examples are listed in Table 1 below.

Table 1 Main experimental reagents and experimental instruments

Example 1

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk.

Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water. The dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm frequently to discard the bottom sediment. An aqueous silk protein solution with an initial concentration of 5.0% (w/v) was obtained.

(2) Place the low-concentration silk protein aqueous solution prepared above in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. The concentration temperature is 45°C, the centrifugal speed is 2000rpm, the vacuum degree is 20mbar, the initial solution volume is 120mL, and the concentration time is 11h.

(3) The concentrated silk protein aqueous solution is placed in a centrifuge for centrifugation, and the bottom sediment is discarded for purification for subsequent processing. The centrifugal speed is 5000 rpm, the centrifugation time is 10 min, and the centrifugation temperature is 4°C. The final concentration of the silk protein aqueous solution is 17wt.%, and can be stably stored at low temperature.

Example 2

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk.

Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water. The dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm frequently to discard the bottom sediment. An aqueous silk protein solution with an initial concentration of 5.4% (w/v) was obtained.

(2) Place the low-concentration silk protein aqueous solution prepared above in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. The concentration temperature is 45°C, the centrifugal speed is 2000rpm, the vacuum degree is 20mbar, the initial solution volume is 150mL, and the concentration time is 14h.

(3) The concentrated silk protein aqueous solution is placed in a centrifuge for centrifugation, and the bottom sediment is discarded for purification for subsequent processing. The centrifugal speed is 7000rpm, the centrifugation time is 15min, and the centrifugation temperature is 4°C. The concentration of the finally prepared silk protein aqueous solution is 28wt.%, and can be stably stored at low temperature.

Example 3

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk.

Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water. The dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm frequently to discard the bottom sediment. An aqueous silk protein solution with an initial concentration of 6.1% (w/v) was obtained.

(2) Place the low-concentration silk protein aqueous solution prepared above in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. The concentration temperature is 45°C, the centrifugal speed is 2000rpm, the vacuum degree is 20mbar, the initial solution volume is 120mL, and the concentration time is 13h.

(3) The concentrated silk protein aqueous solution is placed in a centrifuge for centrifugation, and the bottom sediment is discarded for purification for subsequent processing. The centrifugal speed is 9000rpm, the centrifugation time is 15min, and the centrifugation temperature is 4°C. The final concentration of the silk protein aqueous solution is 24% and can be stored stably at low temperatures.

Example 4

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk.

Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water. The dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm frequently to discard the bottom sediment. An aqueous silk protein solution with an initial concentration of 6.3% (w/v) was obtained.

(2) Place the low-concentration silk protein aqueous solution prepared above in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. The concentration temperature is 45°C, the centrifugal speed is 2000rpm, the vacuum degree is 20mbar, the initial solution volume is 120mL, and the concentration time is 16h.

(3) The concentrated silk protein aqueous solution is placed in a centrifuge for centrifugation, and the bottom sediment is discarded for purification for subsequent processing. The centrifugal speed is 9000rpm, the centrifugation time is 20min, and the centrifugation temperature is 4°C. The final concentration of the silk protein aqueous solution is 36wt.%, and can be stably stored at low temperature.

Comparative photos of the silk protein-based aqueous solution obtained after the above concentration process and the silk protein-based aqueous solution before concentration are shown in Figure 4.

Example 5

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk.

Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water. The dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm frequently to discard the bottom sediment. An aqueous silk protein solution with an initial concentration of 5.8% (w/v) was obtained.

(2) Add 20wt% glycerol to the low-concentration silk protein aqueous solution prepared above. After the two solutions are evenly mixed, place them in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. The concentration temperature is 45°C, the centrifugal speed is 1500rpm, the vacuum degree is 20mbar, the initial solution volume is 150mL, and the concentration time is 17h.

(3) The concentrated silk protein aqueous solution is placed in a centrifuge and centrifuged to obtain a supernatant to remove the precipitate at the bottom. The centrifugal speed is 9000rpm, the centrifugation time is 20min, and the centrifugation temperature is 4°C. The final composite solution has a solid content of 31 wt.% and can be stably stored at low temperature.

Example 6

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk.

Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water. The dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm frequently to discard the bottom sediment. An aqueous silk protein solution with an initial concentration of 5.4% (w/v) was obtained.

(2) Add 10wt% glycerol to the low-concentration silk protein aqueous solution prepared above. After the two solutions are evenly mixed, place them in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. The concentration temperature is 30°C, the centrifugal speed is 1500rpm, the vacuum degree is 15mbar, the initial solution volume is 90mL, and the concentration time is 23h.

(3) The concentrated silk protein aqueous solution is placed in a centrifuge for centrifugation, and the bottom sediment is discarded for purification for subsequent processing. The centrifugal speed is 9000rpm, the centrifugation time is 20min, and the centrifugation temperature is 4°C. The final composite solution has a solid content of 37 wt.% and can be stably stored at low temperature.

Example 7

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk.

Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water. The dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm frequently to discard the bottom sediment. An aqueous silk protein solution with an initial concentration of 6.2% (w/v) was obtained.

(2) Add 5wt% calcium chloride solution to the low-concentration silk protein aqueous solution prepared above. After the two solutions are evenly mixed, place them in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. The concentration temperature is 30°C, the centrifugal speed is 2000rpm, the vacuum degree is 10mbar, the initial solution volume is 150mL, and the concentration time is 22h.

(3) The concentrated silk protein aqueous solution is placed in a centrifuge for centrifugation, and the bottom sediment is discarded for purification for subsequent processing. The centrifugal speed is 9000rpm, the centrifugation time is 20min, and the centrifugation temperature is 4°C. The final composite solution has a solid content of 43 wt.% and can be stored stably at low temperatures.

Example 8

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk.

Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water. The dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm frequently to discard the bottom sediment. An aqueous silk protein solution with an initial concentration of 4.3% (w/v) was obtained.

(2) Add 15wt% calcium chloride solution to the low-concentration silk protein aqueous solution prepared above. After the two solutions are evenly mixed, place them in a vacuum environment for centrifugal concentration to prepare a high-concentration silk protein aqueous solution. The concentration temperature is 30°C, the centrifugal speed is 1500rpm, the vacuum degree is 20mbar, the initial solution volume is 120mL, and the concentration time is 25h.

(3) The concentrated silk protein aqueous solution is placed in a centrifuge for centrifugation, and the bottom sediment is discarded for purification for subsequent processing. The centrifugal speed is 9000rpm, the centrifugation time is 20min, and the centrifugation temperature is 4°C. The final composite solution has a solid content of 48 wt.% and can be stably stored at low temperature.

Test example 1

In order to explore the relationship between the change of solution concentration and vacuum centrifugation time in vacuum centrifugation, the following test examples were carried out.

(1) Put the chopped silkworm cocoons into a 0.02M Na ₂ CO ₃ aqueous solution and boil them at a temperature of 100°C for 30 minutes to degumm the silkworm cocoons. Then the dried degummed silk was dissolved in 9.3M LiBr aqueous solution and placed in a 60°C oven for 4 hours to completely dissolve the degummed silk. Then put the completely dissolved solution into a dialysis bag with a molecular weight cutoff of 3500 daltons (Da), in which the dialysate is deionized water, and the dialysis time is 3 days. The dialyzed solution is centrifuged at 9000 rpm to obtain an initial concentration of 5.0 % (w/v) silk protein aqueous solution.

(2) Place the low-concentration silk protein aqueous solution prepared above in a vacuum environment for centrifugal concentration, where the concentration temperature is 45°C, the centrifugal speed is 1500 rpm, the vacuum degree is 20 mbar, and the initial solution volume is 120 mL. In this process, an appropriate amount of solution needs to be taken at a certain time interval, and its mass is weighed with a balance, which is the wet weight. Then it is placed in a 60°C oven to dry, and the mass of the sample at this time is weighed with a balance, which is the dry weight. . The mass fraction of the solution under a certain concentration time is calculated through formula (1). The test results are shown in Figure 1.

Among them, ω is the mass fraction of the solution, in wt.%; m is the dry weight of the test sample, and M is the wet weight of the test sample, both in g.

(3) It can be seen from Figure 1 that as the concentration time increases, the increase rate of the solution concentration gradually increases. And when the solution concentration reaches 31wt.%, the required concentration time is only 14.5h. Therefore, compared with other methods in the prior art, the concentration time is greatly shortened and the concentration efficiency is significantly improved. In addition, the above results also show that the method of the present invention can be used to obtain a silk protein-based aqueous solution with a certain concentration by adjusting the concentration time, so that the method of the present invention shows good controllability and good repeatability in the concentrated silk protein-based aqueous solution. advantage.

Test example 2

Test method: In the process of testing the rheological properties of the solution, an ARES-G2 rotational rheometer (TA-Waters, USA) was used. During the test, a parallel splint with a diameter of 25mm was used, the distance between the upper and lower plate clamps was set to 0.5mm, and the test temperature was 25°C.

In the shear rheology experiment, the shear rate was scanned from low to high, and the shear rate range was 10 ^-2 -10 ² s ^-1 . The solutions tested during the experiment were solutions with concentrations of 17wt.%, 24wt.%, and 36wt.% prepared in the above example, respectively. The test results are shown in Figure 2.

During the small-amplitude frequency sweep test, the strain amplitude in the linear region is 1%, the frequency sweep is performed from high to low, and the sweep range is 10 ² -10 ^-1 rad/s. The 28 wt.% silk protein solution prepared in the above embodiment was selected for a small amplitude frequency scanning test. The test results are shown in Figure 3.

Result analysis:

It can be seen from Figure 2 that under different concentration conditions, as the shear rate increases, the solution exhibits shear thinning behavior. And high-concentration solutions have higher shear viscosity than low-concentration solutions under different shear rates.

It can be seen from Figure 3 that both the storage modulus G’ and the loss modulus G” increase with the increase of frequency. When the frequency is about 3rad/s, the storage modulus G’ and the loss modulus G” intersect. On the left side of the intersection point (that is, in the lower frequency range), G'>G" is shown; on the right side of the intersection point (that is, in the higher frequency range), G'<G" is shown.

The above results prove that compared with conventionally prepared silk protein solutions, the silk protein solution obtained by the present application has excellent and adjustable rheological properties.

Claims (13)

1. A method of increasing the concentration of an aqueous silk fibroin-based solution, the method comprising the steps of:

(1) Putting the initial silk fibroin aqueous solution into a dialysis bag for dialysis, centrifuging the dialyzed solution at a first centrifugal speed under the ambient pressure, and discarding the precipitate to obtain a silk fibroin-based aqueous solution with the concentration of 4.0-8.0w/v%, wherein the first centrifugal speed is 5000-11000rpm;

(2) Placing the silk fibroin-based aqueous solution obtained in the step (1) in a vacuum environment for centrifugal concentration at a second centrifugal rotating speed to obtain the silk fibroin-based aqueous solution with the concentration of more than 10w/v%, wherein the second centrifugal rotating speed is 1500-2000rpm; and

(3) A third centrifugation of the aqueous silk fibroin-based solution obtained in step (2) at a third centrifugation speed to separate precipitated silk proteins, wherein the third centrifugation speed is 5000-14000rpm,

wherein, in step (2), vacuum centrifugation is performed under the following conditions: the concentration temperature is 30-60 ℃, the vacuum degree is 0.1-20mbar, and the concentration time is 3-48h.

2. The method according to claim 1, wherein an aqueous silk fibroin-based solution having a concentration of 4.5-6.5w/v% is obtained in step (1).

3. The method of claim 1, wherein the initial aqueous silk protein solution in step (1) is selected from the group consisting of: an aqueous solution of natural silk fibroin; an aqueous solution of the regenerated silk protein derivative obtained through chemical modification; the regenerated silk protein/additive composite silk protein base solution is obtained through physical mixing; recombinant silk protein aqueous solution.

4. The method of claim 2, wherein in step (1), the starting aqueous solution of natural silk proteins is naturalAn aqueous silk protein solution, and which is prepared by the following method: placing the cut cocoons into 0.02M Na ₂ CO ₃ Boiling in water solution at 60-100deg.C for 30-180min to degumm silk cocoon, dissolving the degummed silk in 9-10M LiBr water solution, and placing in 60-150deg.C oven for 2-8 hr to dissolve completely.

5. The method according to claim 4, wherein in the step (1), the dried degummed silk is dissolved in a 9.3M aqueous solution of LiBr and is put into an oven at 60-150 ℃ for 2-8 hours to be completely dissolved.

6. The method of claim 1, wherein in step (1), the aqueous starting silk protein solution is dialyzed under the following dialysis conditions: the dialysis time is 2-5 days, the dialysis solution is deionized water, and the cut-off molecular weight of the dialysis bag is 3500Da.

7. The method of claim 1, wherein in step (1), the first centrifugal rotational speed is 9000rpm.

8. The method according to claim 1, wherein an additive for improving the properties of the silk protein-based material product is added to the silk protein aqueous solution before step (2), the additive being selected from the group consisting of glycerin and calcium chloride, wherein the glycerin is added at a concentration of 10-20wt% and the calcium chloride is added at a concentration of 5-15wt%.

9. The method according to claim 1, wherein the concentration of the aqueous silk fibroin-based solution obtained in step (2) is 10-50w/v%.

10. The process according to claim 1, wherein the concentration of the aqueous silk fibroin-based solution obtained in step (2) is 25-40w/v%.

11. The method according to claim 1, wherein the concentration of the aqueous silk fibroin-based solution obtained in step (2) is 6 times the concentration of the aqueous silk fibroin-based solution obtained in step (1).

12. The method of claim 1, wherein in step (3), the third centrifugation operation is performed under the following conditions: the centrifugation time is 5-30min, and the centrifugation temperature is 4-30 ℃.

13. The method of claim 1, wherein the third centrifugal speed is 9000rpm.