Keratin for filling and preparation method thereof

Abstract

The preparation method comprises the following steps that broken hair is sequentially subjected to alkali washing, sterilization, shearing treatment, ultrasonic treatment, enzymolysis, dialysis and finally drying, and the keratin for filling is obtained. The invention provides a novel method for extracting keratin, shearing treatment and ultrasonic treatment are introduced, previous reduction treatment is omitted, and finally enzymolysis dialysis is performed to obtain the keratin, the filling material prepared from the keratin has good degradation capacity after a certain time while maintaining supporting stability, and particularly has the effects of promoting proliferation of fibroblasts and improving the keratin extraction rate. And the technical progress of self-filling is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials and relates to a method for protein extraction, and particularly to a keratin for filling and a preparation method thereof. Background Art

[0002] Keratin is a fibrous protein rich in cysteine, and its structure contains a large number of binding groups beneficial to cell attachment and proliferation, such as leucine - aspartic acid - valine (LDV), glutamic acid - aspartic acid - serine (EDS), and arginine - glycine - aspartic acid (RGD). Keratin can be extracted from wool, feathers, and cattle horns, but the cysteine content of keratin extracted from human hair is relatively high. Keratin has a stable chemical structure, with the molecular chains presenting a tight packing of α - helix and β - sheet, and there are extensive disulfide cross - links between and within macromolecules. Therefore, it is difficult for keratin to dissolve in common polar solvents or non - polar solvents, and the dissolution and extraction of keratin is a difficult process. CN105924654A describes a method for extracting human hair keratin using L - cysteine, including adding an L - cysteine solution to washed and pulverized hair fibers, followed by precipitation, centrifugation, dialysis, and freeze - drying, with the reaction temperature being 65 - 95°C. Due to the high reaction temperature, the energy consumption is also high. In addition, the above document also includes a precipitation method using an acetic acid solution. Due to the use of excessive chemicals, high reaction temperature, and centrifugation, the above method has a high cost. CN118217443A uses sodium hydroxide and mercaptoethanol to break the disulfide bonds in human hair keratin, reduce it to mercapto groups, and extract soluble keratin therefrom. By utilizing the inherent mercapto groups in keratin and cross - linking with oxygen to form disulfide bonds, the cross - linking degree is controlled by adjusting the solid content of keratin to achieve sponge synthesis, but this method is not suitable for preparing keratin for filling. The inventor previously combined high - temperature and high - pressure treatment, alkali treatment, reduction treatment, and enzyme dissolution to obtain keratin with a molecular weight of up to 70,000. Although the prepared filling material has good stability, it is not easily degraded. As a soft tissue filling material, in addition to being stable and having good support performance during initial application, it also needs to have the ability to gradually degrade in order to promote the proliferation of fibroblasts and achieve autologous filling. Summary of the Invention

[0003] There are many methods for extracting keratin in the prior art, and different methods exist for different purposes. As a filling material, keratin needs to have good gel properties, good stability, and strong support, and also needs to have the ability to promote the proliferation of fibroblasts to achieve autologous filling. Currently, the keratin obtained from the disclosed keratin or extraction methods still needs to improve the technical effect of promoting the proliferation of fibroblasts to achieve autologous filling when used for tissue filling, especially for eye wrinkle filling. The present invention proposes a new method for extracting keratin, introducing shear treatment and ultrasonic treatment, omitting the previous reduction treatment, and finally enzymatically hydrolyzing and dialyzing to obtain keratin. The filling material prepared therefrom has good degradation ability after a certain period of time while maintaining support stability, and especially achieves the technical progress of promoting the proliferation of fibroblasts to achieve autologous filling.

[0004] The present invention adopts the following technical solutions.

[0005] A method for preparing keratin for filling, comprising the following steps: broken hair is successively subjected to alkali washing, sterilization, shear treatment, ultrasonic treatment, enzymatic hydrolysis, dialysis, and finally drying to obtain keratin for filling.

[0006] In the present invention, the broken hair is human hair debris, and its acquisition is prior art.

[0007] In the present invention, the alkali washing is carried out using an aqueous sodium hydroxide solution; preferably, the concentration of the aqueous sodium hydroxide solution is 0.05 - 0.2 M, and further preferably, the concentration of the aqueous sodium hydroxide solution is 0.08 - 0.15 M.

[0008] In the present invention, during alkali washing, the mass ratio of broken hair to the aqueous sodium hydroxide solution is 1∶(10 - 30), and preferably, the mass ratio of broken hair to the aqueous sodium hydroxide solution is 1∶(15 - 25).

[0009] In the present invention, the temperature of alkali washing is room temperature to 50 °C, and the time is 5 minutes to 10 hours. Preferably, the time of alkali washing is 1 - 9 hours, further preferably, the time of alkali washing is 2 - 8 hours, and still more preferably, the time of alkali washing is 3 - 8 hours.

[0010] In the present invention, the sterilization is high-temperature sterilization; preferably, the sterilization temperature is 100 - 130 °C, and the time is 5 - 20 minutes. Further preferably, the sterilization temperature is 120 - 125 °C, and the time is 5 - 15 minutes. Preferably, after sterilization, keep warm at 60 - 80 °C for 0.5 - 2 hours.

[0011] In the present invention, the rotation speed of the shear treatment is 25000 - 30000 rpm, and the time is 10 - 15 minutes; the power of the ultrasonic treatment is 3000 - 3500 W, the frequency is 20 - 25 KHz, and the time is 60 - 90 minutes.

[0012] In the present invention, enzymatic hydrolysis is carried out in keratin. Preferably, the temperature of enzymatic hydrolysis is 30 - 40 °C and the time is 5 - 15 hours. More preferably, the temperature of enzymatic hydrolysis is 35 - 40 °C and the time is 5 - 8 hours.

[0013] In the present invention, the dialysis time is 40 - 60 hours.

[0014] In the present invention, drying is a conventional technique, and the keratin for filling can be obtained by conventional drying.

[0015] The present invention discloses the application of the above keratin for filling in the preparation of filling materials.

[0016] The present invention discloses the application of the above keratin for filling in the preparation of medical filling materials.

[0017] The present invention discloses the application of the above keratin for filling in the preparation of medical soft tissue filling materials.

[0018] The present invention discloses the application of the above keratin for filling in the preparation of medical wrinkle filling materials.

[0019] The present invention discloses the application of the above keratin for filling in the preparation of medical eye wrinkle filling materials.

[0020] Keratin is widely sourced in nature and exists in animal hair, horns, nails, claws, beaks, etc. Keratin biomaterials have been widely applied in various biomedical fields such as hemostasis, wound repair, and drug delivery due to their excellent biological activity and biocompatibility. Therefore, developing an efficient and rapid keratin extraction method that is conducive to application can not only improve the utilization efficiency of waste proteins but also effectively promote the development of keratin in the biomedical field. There are many existing methods for extracting keratin, and different methods exist for different uses. As a filling material, keratin needs to have good gel properties, good stability, strong support, and the ability to promote the proliferation of fibroblasts to achieve autologous filling. Currently, the keratin obtained from the disclosed keratin or extraction methods still needs to improve the technical effect of promoting the proliferation of fibroblasts to achieve autologous filling when used for tissue filling, especially for eye wrinkle filling. The present invention proposes a new method for extracting keratin, introducing shear treatment and ultrasonic treatment, omitting the previous reduction treatment, and finally performing enzymatic hydrolysis and dialysis to obtain keratin. The filling material prepared therefrom has good degradation ability after a certain period while maintaining support stability, and in particular, achieves the technical progress of promoting the proliferation of fibroblasts to achieve autologous filling. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the injection site of the gel for mice.

[0022] Figure 2It is a 20×10 magnification photo under light microscope of Group C at 8 weeks.

[0023] Figure 3 It is a 20×10 magnification photo under light microscope of Group A at 8 weeks.

[0024] Figure 4 It is a 20×10 magnification photo under light microscope of Group B at 8 weeks. Detailed implementation manners

[0025] Compared with other natural proteins, keratin has some unique advantages. For example, keratin has a high cysteine content, which makes it have convenient modifiability. It is a natural protein derived from the animal cutin layer, so it has good biocompatibility. Keratin can be recognized and decomposed by the human immune system, without causing obvious immune responses and rejection reactions. It also has certain biological activities, such as promoting cell growth, adhesion and proliferation, etc. It can be used as a good tissue engineering scaffold material, and the source of keratin is wide. In the application of keratin, the extraction method has different effects on the application effect. During the extraction process, the disulfide bonds are broken and the amino acids are also damaged, which have different effects on the activity, structure and molecular weight of the obtained keratin, and thus bring different application effects. The present invention proposes a new method for extracting keratin, introducing shear treatment and ultrasonic treatment, omitting the previous reduction treatment, and finally enzymatic hydrolysis and dialysis to obtain keratin. The filling material prepared therefrom has good degradation ability after a certain period of time while maintaining the support stability, especially achieving the technological progress of promoting the proliferation of fibroblasts and realizing autologous filling.

[0026] The present invention is realized through the following technical solutions.

[0027] A preparation method of keratin for filling, comprising the following steps: (1) Add human hair debris to an NaOH aqueous solution with a concentration of 0.08 - 0.12 mol / L, and stir conventionally to make the solution and the human hair fiber debris mix evenly; (2) Put the mixed system of the above solution and human hair fiber debris into an autoclave for sterilization, then keep warm, and after naturally cooling to room temperature, filter it. The filter cake is repeatedly washed with distilled water until the pH value is about 7 to obtain a hair fragment dispersion; (3) Add the hair fragment dispersion to a blender, and then perform ultrasonic treatment; the rotation speed for breaking the wall is 25000 - 30000 rpm, and the time is 10 - 15 minutes; the power of ultrasonic treatment is 3000 - 3500 W, the frequency is 20 - 25 KHz, and the time is 60 - 90 minutes; (4) Filter the system after ultrasonic treatment to obtain a filter cake; (5) Add the filter cake to a keratinase solution, and stir and react conventionally at 37°C for 5 - 8 hours. After the treatment is completed, filter it with a 300 - mesh nylon filter cloth; (6) Place the filtrate in a dialysis bag for dialysis for 40 - 60 h, changing the distilled water every 12 h during this period. Pour the solution obtained from dialysis into a beaker and dry it in a vacuum drying oven to obtain light yellow keratin powder.

[0028] The following illustrates the technical progress of the present invention through specific experiments. The raw materials used are all existing products, and the specific preparation operations and performance tests are all conventional technologies; the animal experiments comply with the requirements of conventional ethics.

[0029] Preparation of protease solution: Dissolve keratinase (Fengtai Biology) in PBS solution with a pH of 7.0, adjust the concentration to 0.5% according to the mass - volume ratio, fully dissolve at 4°C for 4 hours, and centrifuge at 3000 rpm for 30 minutes to collect the supernatant for standby.

[0030] Wash human hair, dry it, and cut it into pieces of 2 cm to obtain human hair debris (broken hair), which is a conventional method.

[0031] Preferably, the temperature of alkali washing is 30 - 40°C and the time is 4 - 8 h; in the following experiments, unless otherwise specified, the temperature of alkali washing is 35°C and the time is 6 h.

[0032] The autoclave, cell disruptor, and ultrasonic device are conventional products and can be operated according to the instructions.

[0033] Filtration is carried out using a nylon filter screen with 300 meshes, which is an existing product.

[0034] Example 1 A preparation method of keratin for filling, which is the following steps: (1) Add human hair debris to a 0.1 mol / L NaOH aqueous solution according to a bath ratio of 1:20, and stir conventionally to make the solution and human hair fiber debris mix evenly. (2) Put the mixed system of the above - mentioned solution and human hair fiber debris into an autoclave, sterilize at 121°C for 10 minutes, then keep it warm at 70°C for 1 h, naturally cool to room temperature and then filter. Wash the filter cake repeatedly with distilled water until the pH value is 7.1 to obtain a broken - hair dispersion. (3) Add the broken - hair dispersion to a cell disruptor and process it at 30000 rpm for 10 minutes; then perform ultrasonic treatment. The power of ultrasonic treatment is 3500 W, the frequency is 20 KHz, and the time is 60 minutes. (4) Filter the system after ultrasonic treatment to obtain a filter cake. (5) Add the filter cake to a 0.5% keratinase solution (PBS pH 7.0), stir and react conventionally at 37°C for 6 hours. After the treatment, filter it with a nylon filter cloth with 300 meshes. (6) Place the filtrate in a dialysis bag for dialysis (molecular weight cut-off 20KD) to retain keratinase. The dialysis time is 48h, and the distilled water is changed every 12h during this period. Pour the solution obtained by dialysis into a beaker and dry it in a vacuum drying oven to obtain keratin powder.

[0035] Comparative Example 1 A preparation method of keratin for filling is as follows: (1) Add human hair debris to a 0.1mol / L NaOH aqueous solution at a bath ratio of 1:20, and stir conventionally to make the solution and human hair fiber debris mix evenly; (2) Put the mixed system of the above solution and human hair fiber debris into an autoclave, sterilize at 121°C for 10 minutes, then keep warm at 70°C for 1h, naturally cool to room temperature and then filter. Wash the filter cake repeatedly with distilled water until the pH value is 7.0 to obtain a hair fragment dispersion; (3) Ultrasonically treat the hair fragment dispersion. The power of ultrasonic treatment is 3500W, the frequency is 20KHz, and the time is 60 minutes; (4) Filter the ultrasonically treated system to obtain a filter cake; (5) Add the filter cake to a 0.5% keratinase solution (PBS pH7.0), stir and react conventionally at 37°C for 6 hours. After the treatment, filter with a 300-mesh nylon filter cloth; (6) Place the filtrate in a dialysis bag for dialysis (molecular weight cut-off 20KD), the dialysis time is 48h, and the distilled water is changed every 12h during this period. Pour the solution obtained by dialysis into a beaker and dry it in a vacuum drying oven to obtain keratin powder.

[0036] Comparative Example 2 Refer to Example CN2024115616344 to prepare keratin powder (1) Add human hair debris to a 0.1mol / L NaOH aqueous solution at a bath ratio of 1:20, and stir conventionally to make the solution and human hair fiber debris mix evenly; (2) Put the mixed system of the above solution and human hair fiber debris into an autoclave, sterilize at 121°C for 10 minutes, then keep warm at 70°C for 1h, naturally cool to room temperature and then filter. Wash the filter cake repeatedly with distilled water until the pH value is 7.1 to obtain a hair fragment dispersion; (3) Add the hair fragment dispersion to a reduction system containing 0.5mol / L Na 2 SO 3 and 0.5mol / L urea at a bath ratio of 1:15, and perform treatment at 56°C for 2h. Filter the treated mixture and repeatedly wash the insoluble matter with distilled water until the pH value is 7.0; (4) Add the insoluble matter to a 0.5% keratinase solution (PBS pH 7.0), stir and react conventionally at 37 °C for 8 hours. After the treatment, filter with a 300-mesh nylon filter cloth; (5) Put the filtrate into a dialysis bag for dialysis (cut-off molecular weight 50KD), the dialysis time is 48h, and replace the distilled water every 12h. Pour the solution obtained by dialysis into a beaker and dry it in a vacuum drying oven to obtain keratin powder.

[0037] Comparative Example 3 Research on the extraction process of human hair keratin Refer to Comparative Example 2, omit the sterilization step, and obtain keratin powder.

[0038] Comparative Example 4 Acid hydrolysis method (refer to the existing method) (1) Acid hydrolysis reaction: First add deionized water to the stirring kettle, turn on the stirring, and then pump in concentrated sulfuric acid (final concentration 6mol / L). Add human hair debris according to the bath ratio of 1:20, and carry out acid hydrolysis at 90 °C for 12h to obtain an acid hydrolysis solution; (2) Neutralization reaction: After the acid hydrolysis is completed, add barium hydroxide, pour it into the acid hydrolysis solution to adjust the pH value to 5.0, and continuously stir until the pH value reaches stability; (3) Activated carbon decolorization: After cooling to 65 °C, add activated carbon and stir, then filter; Pump the filtrate into a sterilization pot, sterilize at 121 °C for 10 minutes, and dry naturally after the sterilization is completed to obtain a powder with too low molecular weight to be used as an injection product.

[0039] Application Example At room temperature, accurately weigh 0.3 g of keratin powder and 0.3 g of hydroxypropyl methylcellulose HPMC50, dissolve them in 10 ml of PBS (pH = 7.0), stir well, and sterilize by irradiation to prepare a keratin gel for standby.

[0040] 12 nude mice at 6-7 weeks of age, half male and half female, select 4 injection points on the back of each nude mouse, 2 injection points on each side: Group A: composed of 400 ul of PBS solution containing 3% hair keratin (Example 1); Group B: composed of 400 ul of PBS gel containing 3% hair keratin (Comparative Example 2) and 3% hydroxypropyl methylcellulose HPMC50; Group C: composed of 400 ul of PBS gel containing 3% hair keratin (Example 1) and 3% hydroxypropyl methylcellulose HPMC50.

[0041] After 12 nude mice were injected with the keratin gel filler, their growth conditions were good, and there was no obvious redness, swelling or exudation on the skin after the operation; At 8 weeks, the injection area was marked with picric acid (3%), see Figure 1 And Figure 2 、Figure 3 , Figure 4 : Group C: The dermis is the thickest, with an average thickness of about 0.58 mm at 8 weeks. A large number of keratin granules are aggregated under the dermis. There is growth of cells and fibrous tissue between the keratin, and a small number of multinucleated giant cells, which phagocytize keratin granules.

[0042] Group A: The dermis is relatively thick, with an average thickness of about 0.45 mm at 8 weeks. A large number of keratin granules are aggregated under the dermis. There is less growth of cells and fibrous tissue between the granules. The granules are aggregated into clumps, and a small number of multinucleated giant cells, which phagocytize keratin granules.

[0043] Group B: The dermis is thicker than that of normal nude mice, with an average thickness of about 0.50 mm at 8 weeks. A small amount of cell and fibrous tissue growth can be seen under the dermis, significantly less than that in Group C, and no multinucleated giant cells are seen.

[0044] Comparative animal experiment Refer to the above method: Group D: Composed of a 400 μl PBS solution containing 3% hair keratin (Comparative Example 1) and 3% hydroxypropyl methylcellulose HPMC50; Group E: Composed of a 400 μl PBS gel containing 3% hair keratin (Comparative Example 3) and 3% hydroxypropyl methylcellulose HPMC50; Group F: Composed of a gel of 400 μl PBS containing 3% hair keratin (Example 1) and 3% hydroxypropyl methylcellulose HPMC50.

[0045] At 8 weeks, the injection area was marked with picric acid (3%). The average thickness of the dermis in Group D and Group E and the growth of cells and fibrous tissue between the keratin were inferior to those in Group F, and even Group E was inferior to Group B.

[0046] Example 2 A preparation method of keratin for filling, which is the following steps: (1) Add human hair debris to a 0.1 mol / L NaOH aqueous solution according to a bath ratio of 1:20, and stir conventionally to make the solution and human hair fiber debris mix evenly; (2) Put the mixed system of the above solution and human hair fiber debris into an autoclave, sterilize at 121 °C for 10 minutes, then keep warm at 70 °C for 1 h, and filter after naturally cooling to room temperature. The filter cake is washed repeatedly with distilled water until the pH value is 7.1 to obtain a hair fragment dispersion; (3) Add the hair fragment dispersion to a blender and process it at 25000 rpm for 10 minutes; then perform ultrasonic treatment. The power of the ultrasonic treatment is 3500 W, the frequency is 20 KHz, and the time is 70 minutes; (4) Filter the ultrasonicated system to obtain a filter cake; (5) Add the filter cake to a 0.5% keratinase solution (PBS pH 7.0), stir the reaction conventionally at 37 °C for 6 hours, and filter with a 300-mesh nylon filter cloth after the treatment is completed; (6) Put the filtrate into a dialysis bag for dialysis (cut-off molecular weight 20KD), the dialysis time is 48h, replace the distilled water every 12h during this period, pour the solution obtained by dialysis into a beaker, and dry it in a vacuum drying oven to obtain keratin powder.

[0047] Example 3 A preparation method of keratin for filling, the steps are as follows: (1) Add human hair debris to a 0.1mol / L NaOH aqueous solution according to a bath ratio of 1:20, stir conventionally to make the solution and human hair fiber debris mix evenly; (2) Put the mixed system of the above solution and human hair fiber debris into an autoclave, sterilize at 121 °C for 10 minutes, then keep warm at 70 °C for 1h, filter after naturally cooling to room temperature, and wash the filter cake repeatedly with distilled water until the pH value is 7.1 to obtain a hair fragment dispersion; (3) Add the hair fragment dispersion to a blender and process it at 30000rpm for 10 minutes; then perform ultrasonic treatment, the power of ultrasonic treatment is 3000W, the frequency is 20KHz, and the time is 90 minutes; (4) Filter the system after ultrasonic treatment to obtain a filter cake; (5) Add the filter cake to a 0.5% keratinase solution (PBS pH 7.0), stir the reaction conventionally at 37 °C for 6 hours, and filter with a 300-mesh nylon filter cloth after the treatment is completed; (6) Put the filtrate into a dialysis bag for dialysis (cut-off molecular weight 20KD), the dialysis time is 48h, replace the distilled water every 12h during this period, pour the solution obtained by dialysis into a beaker, and dry it in a vacuum drying oven to obtain keratin powder.

[0048] Example 4 A preparation method of keratin for filling, the steps are as follows: (1) Add human hair debris to a 0.1mol / L NaOH aqueous solution according to a bath ratio of 1:20, stir conventionally to make the solution and human hair fiber debris mix evenly; (2) Put the mixed system of the above solution and human hair fiber debris into an autoclave, sterilize at 121 °C for 10 minutes, then keep warm at 70 °C for 1h, filter after naturally cooling to room temperature, and wash the filter cake repeatedly with distilled water until the pH value is 7.1 to obtain a hair fragment dispersion; (3) Add the hair fragment dispersion to a blender and process it at 30000rpm for 10 minutes; then perform ultrasonic treatment, the power of ultrasonic treatment is 3300W, the frequency is 20KHz, and the time is 80 minutes; (4) Filter the system after ultrasonic treatment to obtain a filter cake. (5) Add the filter cake to a 0.5% keratinase solution (PBS pH 7.0), and stir the reaction at 37 °C for 7 hours. After the treatment, filter it with a 300-mesh nylon filter cloth. (6) Put the filtrate into a dialysis bag for dialysis (cut-off molecular weight 20KD), the dialysis time is 48h, and replace the distilled water every 12h during this period. Pour the solution obtained by dialysis into a beaker and dry it in a vacuum drying oven to obtain keratin powder.

[0049] Example 5 A preparation method of keratin for filling, the steps are as follows: (1) Add human hair debris to a 0.1 mol / L NaOH aqueous solution according to a bath ratio of 1:20, and stir it conventionally to make the solution and human hair fiber debris mix evenly. (2) Put the mixed system of the above solution and human hair fiber debris into an autoclave, sterilize at 121 °C for 10 minutes, then keep it warm at 70 °C for 1h, and filter it after natural cooling to room temperature. Wash the filter cake repeatedly with distilled water until the pH value is 7.1 to obtain a fragmented hair dispersion. (3) Add the fragmented hair dispersion to a blender and process it at 30000 rpm for 10 minutes; then perform ultrasonic treatment, the power of ultrasonic treatment is 3200W, the frequency is 20KHz, and the time is 75 minutes. (4) Filter the system after ultrasonic treatment to obtain a filter cake. (5) Add the filter cake to a 0.5% keratinase solution (PBS pH 7.0), and stir the reaction at 37 °C for 6 hours. After the treatment, filter it with a 300-mesh nylon filter cloth. (6) Put the filtrate into a dialysis bag for dialysis (cut-off molecular weight 20KD), the dialysis time is 48h, and replace the distilled water every 12h during this period. Pour the solution obtained by dialysis into a beaker and dry it in a vacuum drying oven to obtain keratin powder.

[0050] Example 6 A preparation method of keratin for filling, the steps are as follows: (1) Add human hair debris to a 0.1 mol / L NaOH aqueous solution according to a bath ratio of 1:20, and stir it conventionally to make the solution and human hair fiber debris mix evenly. (2) Put the mixed system of the above solution and human hair fiber debris into an autoclave, sterilize at 121 °C for 10 minutes, then keep it warm at 70 °C for 1h, and filter it after natural cooling to room temperature. Wash the filter cake repeatedly with distilled water until the pH value is 7.1 to obtain a fragmented hair dispersion. (3) Add the broken hair dispersion liquid to a blender and process it at 30,000 rpm for 12 minutes; then perform ultrasonic treatment. The power of the ultrasonic treatment is 3500 W, the frequency is 25 KHz, and the time is 60 minutes; (4) Filter the system after ultrasonic treatment to obtain a filter cake; (5) Add the filter cake to a 0.5% keratinase solution (PBS pH 7.0), and carry out a conventional stirring reaction at 37 °C for 6 hours. After the treatment, filter it with a 300-mesh nylon filter cloth; (6) Put the filtrate into a dialysis bag for dialysis (the molecular weight cut-off is 20 KD), the dialysis time is 48 h, and the distilled water is changed every 12 h during this period. Pour the solution obtained by dialysis into a beaker and dry it in a vacuum drying oven to obtain keratin powder.

[0051] The keratin of hair contains sulfur, and there are extensive and tight cross-links between its peptide chains, showing a high degree of spatial stability. It has stable properties and is not easily absorbed and degraded by tissues quickly. This is a factor affecting keratin as a wrinkle filling material. The present invention proposes a new method for extracting keratin, introducing shear treatment and ultrasonic treatment, omitting the previous reduction treatment, and finally enzymatically hydrolyzing and dialyzing to obtain keratin. The filling material prepared therefrom has good degradation ability after a certain period of time while maintaining the support stability, especially achieving the technical progress of promoting the proliferation of fibroblasts and realizing autologous filling.

Claims

1. A method for preparing keratin for filling, comprising the following steps: broken hair is sequentially subjected to alkali washing, sterilization, shearing treatment, ultrasonic treatment, enzymolysis, dialysis, and finally drying to obtain keratin for filling.

2. The method for preparing keratin for filling according to claim 1, characterized in that: The alkali washing adopts a sodium hydroxide aqueous solution; during the alkali washing, the mass ratio of the broken hair to the sodium hydroxide aqueous solution is 1: (10-30); the temperature of the alkali washing is room temperature-50°C, and the time is 5 minutes-10 hours.

3. The method for preparing keratin for filling according to claim 1, characterized in that: Sterilization is high temperature sterilization.

4. The method for preparing keratin for filling according to claim 1, characterized in that: After sterilization, keep warm at 60-80℃ for 0.5-2 hours.

5. The method for preparing keratin for filling according to claim 1, characterized in that: The rotation speed of the shearing treatment is 25000-30000 rpm, and the time is 10-15 minutes; the power of the ultrasonic treatment is 3000-3500 W, the frequency is 20-25 KHz, and the time is 60-90 minutes.

6. The method for preparing keratin for filling according to claim 1, characterized in that: The enzymatic hydrolysis is carried out in keratinase.

7. The method for preparing keratin for filling according to claim 1, characterized in that: The dialysis time is 40 to 60 hours.

8. Keratin for filling prepared by the method for preparing keratin for filling according to claim 1.

9. Use of the keratin for filling according to claim 8 in preparing filling materials.

10. Use of the keratin for filling according to claim 8 in the preparation of medical filling materials.

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