Method for obtaining silk fibroin powder through precipitation and application

Abstract

The invention relates to a method for obtaining silk fibroin powder through precipitation and application. Comprising the following steps: boiling silkworm cocoons in a first alkaline solution, and drying to obtain degummed silk; dissolving degummed silk in a phosphoric acid solution, and mixing to obtain a silk fibroin solution; diluting the silk fibroin solution with water, adding a second alkaline solution to adjust the pH value, separating and collecting silk fibroin precipitate; washing the silk fibroin precipitate with water to be neutral, and performing precooling and freeze drying to obtain silk fibroin powder. According to the preparation method, the silk fibroin is dissolved by phosphoric acid with the temperature of 10 DEG C below zero to 10 DEG C, the silk fibroin solution is subjected to deprotonation treatment and then freeze-dried, the silk fibroin powder of the random curled / spiral structure can be rapidly obtained, long-time dialysis is avoided, and the preparation time of the silk fibroin powder is shortened. The obtained regenerated silk fibroin powder can be used for preparing a high-strength silk fibroin material through hot press molding, and the bending strength reaches up to 179 MPa.

Description

Technical Field

[0001] This invention relates to the field of polymer materials technology, specifically to a method for obtaining silk fibroin powder by precipitation and its application. Background Technology

[0002] Silk fibroin powder, due to its excellent biocompatibility, biodegradability, and unique physicochemical properties, is widely used in cosmetic moisturizing and anti-aging, biomedical tissue repair, and material surface modification, with continuously growing market demand, especially in the high-end skincare products and medical dressings sectors where its penetration rate is constantly increasing. Currently, various methods for preparing silk fibroin powder have been developed, all revolving around the basic process of "degumming-dissolving-separation-drying," mainly including neutral salt dissolution-precipitation method, alkali treatment-pulverization method, and reverse emulsion curing method.

[0003] Chinese invention patent CN114621335B discloses a method for preparing regenerated silk fibroin at high efficiency and low cost. This method uses a high-concentration neutral salt solution or a salt-alcohol mixed solvent to dissolve natural silk fibroin. The resulting solution is diluted to a silk fibroin concentration below 2% to reduce the solvent effect. By adjusting the pH to 3.0-5.8, controlling the temperature at 15℃-65℃, and the rotation speed at 50-350 r / min, the silk fibroin is accelerated to precipitate. Solid-liquid separation can be completed in 6-8 hours, with a crude extraction yield of over 98%, and a purified yield of 81.4% with a purity ≥99.8%. This method requires no expensive specialized equipment and has potential for industrial application. While similar patents significantly shorten the time compared to traditional dialysis and column chromatography methods, the precipitation efficiency still has room for improvement, with a yield of only 25% in 2 hours, and the obtained regenerated silk fibroin powder cannot be further processed. Other methods, such as alkali treatment-pulverization, produce products with uneven particle size; reverse emulsion solidification methods have complex processes and poor batch stability; and desolvation methods suffer from particle agglomeration and narrow drug loading spectrum. Meanwhile, as downstream applications place higher demands on the purity, particle size uniformity, and production efficiency of silk fibroin powder, there is an urgent need to develop a more efficient, simple, and controllable precipitation preparation technology to meet the high-end needs of cosmetics, biomedical materials, and other fields, and to promote the large-scale application of silk fibroin powder. Summary of the Invention

[0004] The first aspect of this invention provides a method for obtaining silk fibroin powder by precipitation, comprising the following steps: boiling silkworm cocoons in a first alkaline solution and drying them to obtain degummed silk; dissolving the degummed silk in a phosphoric acid solution and mixing to obtain a silk fibroin solution; diluting the silk fibroin solution with water, adding a second alkaline solution to adjust the pH value, separating and collecting the silk fibroin precipitate; washing the silk fibroin precipitate with water until neutral, and freeze-drying it to obtain silk fibroin powder; wherein the mixing temperature is -10~10℃.

[0005] Currently, the dissolution of silk fibroin relies on systems such as lithium bromide, formic acid-calcium chloride, and hexafluoroisopropanol. For example, patent CN103923200A discloses a method for preparing high molecular weight lyophilized silk fibroin powder. The silk fibroin powder is obtained by degumming silk, dissolving it with lithium bromide, dialysis, centrifugation, high-temperature and high-pressure sterilization, and freeze-drying. This system can obtain reprocessable amorphous silk fibroin powder, but due to its high salt concentration, it requires dialysis for 2-3 days to remove salt ions, which is time-consuming and water-intensive. The formic acid-calcium chloride system has good dissolution efficiency for degummed silk, but obtaining pure regenerated silk fibroin also requires a long dialysis process. Hexafluoroisopropanol has limited dissolving capacity and is usually used to dissolve regenerated silk fibroin. For example, patent CN101445971 discloses a method for preparing biomimetic extracellular matrix silk fibroin / chitosan composite nanofibers, in which the lyophilized silk fibroin powder is dissolved in hexafluoroisopropanol and then electrospun. This invention overcomes the aforementioned defects efficiently through the synergistic effect of low-temperature dissolution, isoelectric point precipitation, and freeze-fixation of the conformation: Firstly, a low-temperature phosphoric acid solution is used to dissolve degummed silk. The low-temperature environment prevents silk fibroin degradation, and phosphoric acid disrupts the hydrogen bonds and crystalline structure between protein molecules, achieving efficient dissolution without the need for high-concentration salts or toxic solvents. Secondly, after diluting the solution, the pH is adjusted to be close to the isoelectric point of silk fibroin, causing deprotonation of the protein and a sharp reduction in electrostatic repulsion. This allows for rapid formation of flocculent precipitates through hydrogen bonding and hydrophobic interactions, eliminating the need for prolonged dialysis and significantly shortening the process. Thirdly, the precipitate is freeze-dried after pre-freezing to fix its conformation, ultimately yielding a powder with a predominantly random coil / helical structure, exhibiting excellent processability and solving the problem of poor processability in traditional products.

[0006] The concentration of the phosphoric acid solution is 50-90 wt%.

[0007] Optionally, the mixing temperature is -5 to 0°C.

[0008] Optionally, the first alkaline solution includes a Na2CO3 solution with a concentration of 0.01-0.5 mol / L, a boiling time of 20-60 min, and a drying temperature of 40-80℃.

[0009] The concentration of silk fibroin in the silk fibroin solution is 5-25 wt%.

[0010] The dilution factor is 1-25 times.

[0011] Optionally, the dilution factor is 1-8 times.

[0012] The pH value is 3-6.

[0013] Optionally, the pH value is 4.5-5.

[0014] The second alkaline solution includes at least one of NaOH solution, KOH solution, and NH3·H2O solution.

[0015] Optionally, the alkaline solution includes a 0.1-3 mol / L NaOH solution.

[0016] The separation method is high-speed centrifugation or vacuum filtration, with the centrifuge speed being 7000-11000 rpm.

[0017] The pre-cooling temperature is below -80°C, and the freeze-drying time is 24-72 hours.

[0018] The second aspect of the present invention provides an application of precipitated silk fibroin powder for the preparation of silk fibroin-based materials; the method for preparing the silk fibroin-based materials includes the following steps: pre-treating the silk fibroin powder and then performing hot pressing treatment to obtain the silk fibroin-based materials.

[0019] The pretreatment includes the following steps: placing the silk fibroin powder in an environment with a temperature of 20-35℃ and a humidity of 50-90% for 15-50 minutes.

[0020] The hot pressing process is carried out at a temperature of 25-150℃, a pressure of 300-800MPa, and a time of 5-40min.

[0021] Optionally, the hot pressing treatment is performed at a temperature of 45-150℃ and a pressure of 300-632MPa.

[0022] Beneficial effects 1. This invention uses phosphoric acid at -10~10℃ to dissolve silk fibroin, deprotonates the silk fibroin solution, and then freeze-dries it to quickly obtain random coil / helical silk fibroin powder, avoiding prolonged dialysis and shortening the preparation time of silk fibroin powder.

[0023] 2. The present invention obtains regenerated silk fibroin powder with a conformation mainly of random coil / α-helix structure by limiting the dilution ratio to 1-8 times, the pH value to 4.5-5, and the alkaline solution including 0.1-3 mol / L NaOH solution, and has a high yield.

[0024] 3. The silk fibroin powder of the present invention can be used for the preparation of silk fibroin blocks through hot pressing, and can be processed into bent, twisted and wound states.

[0025] 4. By limiting the hot pressing parameters, the bending strength of the silk fibroin block can be controlled, and it can be used as a bone material in the field of tissue engineering. Attached Figure Description

[0026] Figure 1The image shows the preparation process of silk fibroin powder in Example 1. The left image shows the silk fibroin solution; the middle image shows the silk fibroin aggregate; and the right image shows the obtained silk fibroin powder.

[0027] Figure 2 a) Fourier transform infrared spectroscopy (FTIR) characterization of silk fibroin powder in Example 1 and Comparative Example 2; b) Comparison of the content of different conformations of silk fibroin powder.

[0028] Figure 3 The images shown are of silk fibroin powder after hot pressing in Example 1. The left image shows the recycled silk fibroin block material; the middle image shows the recycled silk fibroin circular film; and the right image shows the recycled twisted silk fibroin plastic.

[0029] Figure 4 The mechanical properties of the regenerated silk fibroin-based materials in Examples 7-9 are characterized at different hot-pressing temperatures. Detailed Implementation

[0030] Example 1 A method for obtaining silk fibroin powder by precipitation, such as Figure 1 The following steps are shown: S1. Mix the degummed silk with a pre-cooled phosphoric acid solution until homogeneous, and stir with a kneader for 40 minutes at 0°C to obtain a silk fibroin solution with a concentration of 25 wt%; the phosphoric acid concentration is 85 wt%. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:5. Add 1 mol / L NaOH solution to adjust the pH to 4.5, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH is 7. Pre-freeze the precipitate and then freeze-dry it to obtain silk fibroin powder, denoted as SF-freeze-dried.

[0031] An application of precipitated silk fibroin powder for the preparation of silk fibroin-based materials is disclosed. The preparation method includes the following steps: hot pressing the silk fibroin powder (SF-freeze-dried) obtained in step S3 for 15 min at a pressure of 632 MPa and a temperature of 25 °C to obtain silk fibroin-based materials.

[0032] Example 2 The specific implementation method is the same as in Example 1, except that the hot pressing temperature is 60°C.

[0033] Example 3 The specific implementation method is the same as in Example 1, except that the hot pressing temperature is 80°C.

[0034] Example 4 The specific implementation method is the same as in Example 1, except that the hot pressing temperature is 100°C.

[0035] Example 5 The specific implementation method is the same as in Example 1, except that the hot pressing temperature is 120°C.

[0036] Example 6 The specific implementation method is the same as in Example 1, except that the hot pressing temperature is 145°C.

[0037] Example 7 The specific implementation method is the same as in Example 4, except that the hot pressing time is 30 minutes.

[0038] Example 8 The specific implementation method is the same as in Example 5, except that the hot pressing time is 30 minutes.

[0039] Example 9 The specific implementation method is the same as in Example 6, except that the hot pressing time is 30 minutes.

[0040] Comparative Example 1 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Mix the degummed silk with a pre-cooled phosphoric acid solution until homogeneous, and stir with a kneader for 40 minutes at 25°C to obtain a silk fibroin solution with a concentration of 25 wt%; the phosphoric acid concentration is 85 wt%. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:5. Add 1 mol / L NaOH solution to adjust the pH to 4.5, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH reaches 7, and then freeze-dry it to obtain silk fibroin powder.

[0041] Comparative Example 2 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Mix the degummed silk with a pre-cooled phosphoric acid solution until homogeneous, and stir with a kneader for 40 minutes at 0°C to obtain a silk fibroin solution with a concentration of 25 wt%; the phosphoric acid concentration is 85 wt%. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:5. Ethanol solution is added to adjust the pH to 4.5, so that the silk fibroin precipitates out in the form of flocculent precipitate. The precipitate is separated and collected by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH is 7. Pre-freeze the precipitate and then freeze-dry it to obtain silk fibroin powder with β-sheet as the main component, denoted as SF-ethanol induced.

[0042] Comparative Example 3 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Mix the degummed silk with a pre-cooled phosphoric acid solution until homogeneous, and stir with a kneader for 40 minutes at 0°C to obtain a silk fibroin solution with a concentration of 25 wt%; the phosphoric acid concentration is 85 wt%. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:5. Add 1 mol / L NaOH solution to adjust the pH to 4.5, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. The silk fibroin precipitate collected in step S2 is washed with deionized water until the pH reaches 7, and then dried at room temperature to obtain silk fibroin powder with a β-sheet structure as the main component, denoted as SF-room temperature dried.

[0043] Example 10 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Mix the degummed silk with a pre-cooled phosphoric acid solution until homogeneous, and stir with a kneader for 40 minutes at 0°C to obtain a silk fibroin solution with a concentration of 25 wt%; the phosphoric acid concentration is 85 wt%. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:10. Add 1 mol / L NaOH solution to adjust the pH to 4.5, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH reaches 7, and then freeze-dry it to obtain silk fibroin powder.

[0044] Example 11 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Mix the degummed silk with a pre-cooled phosphoric acid solution until homogeneous, and stir with a kneader for 40 minutes at 0°C to obtain a silk fibroin solution with a concentration of 25 wt%; the phosphoric acid concentration is 85 wt%. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:15. Add 1 mol / L NaOH solution to adjust the pH to 4.5, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH reaches 7. Pre-freeze the precipitate and then freeze-dry it to obtain silk fibroin powder.

[0045] Example 12 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Dissolve the degummed silk in a phosphoric acid solution with a concentration of 85 wt%. After the silk is dissolved, a silk fibroin solution with a concentration of 25 wt% is obtained. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:5. Add 1 mol / L NaOH solution to adjust the pH to 3, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH is 7. Pre-freeze the precipitate and then freeze-dry it to obtain silk fibroin powder with random coil / helical structure as the main component, denoted as SF-3.

[0046] Example 13 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Dissolve the degummed silk in a phosphoric acid solution with a concentration of 85 wt%. After the silk is dissolved, a silk fibroin solution with a concentration of 25 wt% is obtained. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:5. Add 1 mol / L NaOH solution to adjust the pH to 4, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH is 7. Pre-freeze the precipitate and then freeze-dry it to obtain silk fibroin powder with random coil / helical structure as the main component, denoted as SF-4.

[0047] Example 14 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Dissolve the degummed silk in a phosphoric acid solution with a concentration of 85 wt%. After the silk is dissolved, a silk fibroin solution with a concentration of 25 wt% is obtained. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:5. Add 1 mol / L NaOH solution to adjust the pH to 6, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH is 7. Pre-freeze the precipitate and then freeze-dry it to obtain silk fibroin powder with random coil / helical structure as the main component, which is denoted as SF-6.

[0048] Example 15 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Mix the degummed silk with a pre-cooled phosphoric acid solution until homogeneous, and stir with a kneader for 40 minutes at 0°C to obtain a silk fibroin solution with a concentration of 25 wt%; the phosphoric acid concentration is 85 wt%. S2. After diluting the silk fibroin solution, the volume ratio of the silk fibroin solution to deionized water is 1:5. Add 1 mol / L KOH solution to adjust the pH to 4.5, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH reaches 7, and then freeze-dry it to obtain silk fibroin powder.

[0049] Example 16 A method for obtaining silk fibroin powder by precipitation comprises the following steps: S1. Mix the degummed silk with a pre-cooled phosphoric acid solution until homogeneous, and stir with a kneader for 40 minutes at 0°C to obtain a silk fibroin solution with a concentration of 25 wt%; the phosphoric acid concentration is 85 wt%. S2. After diluting the silk fibroin solution, the volume ratio of silk fibroin solution to deionized water is 1:5. Add 2% NH3·H2O solution to adjust the pH to 4.5, so that the silk fibroin precipitates out in the form of flocculent precipitate. Separate and collect the precipitate by centrifugation. S3. Wash the silk fibroin precipitate collected in step S2 with deionized water until the pH reaches 7, and then freeze-dry it to obtain silk fibroin powder.

[0050] Performance testing methods 1. Structural analysis and Fourier transform infrared spectroscopy (FTIR) testing were performed on the silk fibroin and silk-based materials obtained in the examples and comparative examples: An appropriate amount of silk fibroin powder was taken and subjected to attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) at a resolution of 2 cm⁻¹. -1 Sample spectra were acquired under conditions of 32 scans, ranging from 400 to 4000 cm⁻¹. -1 The obtained infrared spectra were analyzed using Peak Fit software in the 1580-1720 cm⁻¹ range. -1 Peak fitting was performed on the absorption peaks within the range to obtain the content of β-sheet, α-helix / random coil, and β-turn structures. The characteristic peak of the secondary structure of silk fibroin was assigned to 1620 cm⁻¹. -1With 1700cm -1 Corresponding to β-sheet, 1650cm -1 Corresponding to random coils or helixes, and 1680cm -1 The test data corresponding to β-turn are listed in Table 1.

[0051] 2. Mechanical Property Testing: The silk fibroin-based materials prepared in the examples and comparative examples were placed on the three-point bending fixture of a universal testing machine for testing. The sample size was set to 14×6×1mm (±0.1mm). Before testing, the length, width, and thickness were measured three times at different positions, and the average value was taken. The testing speed was set to 1mm / min, and the environmental conditions were 25℃ and 50% relative humidity (RH). Each group of samples was tested three times, and the final average value was taken as the mechanical property test result. The test data are listed in Table 1, with blank spaces indicating no test was performed.

[0052] Performance test data Table 1

[0053] As shown in Table 1, the secondary structure of the silk fibroin powder prepared in the embodiments of the present invention is mainly random coil / helical structure. The content of α-helical / random coil structure and the yield of silk fibroin are both good. The silk fibroin-based material prepared from the silk fibroin powder has excellent mechanical properties. In Comparative Example 1, the viscosity of the silk fibroin solution at 25°C is low, and the silk fibroin solution is easily degraded in an environment of 25°C; for example... Figure 2 As shown, in Comparative Example 2, silk fibroin induced by ethanol yielded silk fibroin with a predominantly β-sheet structure, accounting for 59 wt%, while the content of α-helix / random coil structure was 18 wt%, which is unfavorable for subsequent processing and application. In Comparative Example 3, the silk fibroin powder obtained by room temperature drying showed that its structure was predominantly β-sheet, accounting for 50 wt%. This structure of silk fibroin is stable, but unfavorable for subsequent processing and application. The results of Examples 10-11 indicate that the initial concentration of the silk fibroin solution had no significant effect on the structure of the silk fibroin powder, but had a significant negative impact on the yield. The results of Examples 12-14 indicate that the content of the β-sheet structure in silk fibroin obtained at pH outside the preferred range was lower, and the yield was also lower. The results of Examples 15-16 indicate that the use of other alkaline solutions had a significant negative impact on the yield.

Claims

1. A method for obtaining silk fibroin powder by precipitation, characterized in that, Includes the following steps: Silkworm cocoons are boiled in a first alkaline solution and then dried to obtain degummed silk. Degummed silk is dissolved in a phosphoric acid solution and mixed to obtain a silk fibroin solution; After diluting the silk fibroin solution with water, a second alkaline solution was added to adjust the pH value, and the silk fibroin precipitate was separated and collected. The silk fibroin precipitate was washed with water until neutral, pre-cooled, and freeze-dried to obtain silk fibroin powder; the mixing temperature was -10~10℃.

2. The method for obtaining silk fibroin powder by precipitation according to claim 1, characterized in that, The alkaline solution is a Na2CO3 solution with a concentration of 0.01-0.5 mol / L, a boiling time of 20-60 min, and a drying temperature of 40-80℃.

3. The method for obtaining silk fibroin powder by precipitation according to claim 1, characterized in that, The concentration of silk fibroin in the silk fibroin solution is 5-25 wt%.

4. The method for obtaining silk fibroin powder by precipitation according to claim 1, characterized in that, The dilution factor is 1-25 times.

5. The method for obtaining silk fibroin powder by precipitation according to claim 4, characterized in that, The dilution factor is 1-8 times.

6. The method for obtaining silk fibroin powder by precipitation according to claim 1, characterized in that, The pH value is 3-6.

7. The method for obtaining silk fibroin powder by precipitation according to claim 6, characterized in that, The pH value is 4.5-5.

8. The method for obtaining silk fibroin powder by precipitation according to claim 1 or 7, characterized in that, The second alkaline solution comprises a 0.1-3 mol / L sodium hydroxide solution.

9. An application of the precipitation method for obtaining silk fibroin powder according to any one of claims 1-8, characterized in that, The method for preparing silk fibroin-based materials includes the following steps: pretreating the silk fibroin powder and then hot-pressing it to obtain the silk fibroin-based material.

10. The application of the silk fibroin powder obtained by precipitation according to claim 9, characterized in that, The hot pressing process is carried out at a temperature of 25-150℃, a pressure of 300-800MPa, and a time of 5-40min.

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