A soybean protein amyloid fiber-chitosan 3D scaffold and its preparation method and application

Abstract

The present invention discloses a soy protein amyloid fiber-chitosan 3D cell meat scaffold and a preparation method and application thereof. The soy protein, chitosan and cross-linking agent in the soy protein amyloid fiber-chitosan 3D scaffold provided by the present invention are all safe and non-toxic ingredients, and can ensure edible safety as part of a food product. The present invention not only has a simple preparation process, but also the soy protein amyloid fiber-chitosan 3D scaffold has a certain thickness and high mechanical properties, which can support the adhesion, proliferation and differentiation of muscle cells in the scaffold, and can therefore be effectively applied to the field of cell cultured meat.

Description

Technical Field

[0001] The present invention relates to the preparation technology and application field of cell meat 3D scaffold, and in particular to a soybean protein amyloid fiber-chitosan 3D scaffold and a preparation method and application thereof. Background Art

[0002] Due to the rapid growth of the global population, the balance between traditional meat production systems and global meat demand is being broken. Traditional animal husbandry causes serious environmental burdens and also raises concerns about animal welfare and food safety. Cell-cultured meat is an emerging meat production technology that is expected to become a substitute for traditional meat. One of the key steps in the development of cell-cultured meat is to develop a scaffold that supports cell adhesion, proliferation, differentiation, and promotes oxygen and nutrient transport.

[0003] Among the raw materials for preparing scaffolds, protein amyloid fibers and chitosan are particularly attractive due to their biodegradability, wide range of sources, and safety and non-toxicity. Protein amyloid fibers are self-assembled aggregates produced by fibrillation of proteins under high temperature and low acidity conditions, and have advantages as building blocks in scaffold design. Scaffolds based on protein amyloid fibers have become a popular choice for constructing hybrid cell meat scaffolds due to their high nutritional value and excellent functional properties. However, the challenges faced by soy protein starch fiber scaffolds made from a single raw material are that they have low mechanical strength, fragility and structural instability. In addition, because they are water-soluble proteins, these scaffolds are not stable in culture media and there is a risk of dissolution and dispersion, so they are not suitable for large-scale production. Chitosan is an abundant natural polymer polysaccharide with a series of excellent properties, including flexibility, biocompatibility, biodegradability, antibacterial and osteogenic effects. Compared with scaffolds with protein amyloid fibers as the only raw material, the scaffolds prepared based on protein amyloid fibers-chitosan in the present invention have higher mechanical strength and are more suitable for cell adhesion, proliferation and differentiation on the scaffold.

[0004] Amyloid fibers are prepared using soy protein, and the soy protein amyloid fiber scaffold is combined with chitosan to develop an edible porous scaffold that can be used in the field of cell cultured meat. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and to propose a soy protein amyloid fiber-chitosan 3D scaffold and its preparation method and application. The soy protein, chitosan and cross-linking agent in the soy protein amyloid fiber-chitosan 3D scaffold provided by the present invention are all safe and non-toxic ingredients, which can ensure food safety as part of a food product. The present invention not only has a simple preparation process, but also the soy protein amyloid fiber-chitosan 3D scaffold has a certain thickness and high mechanical properties, which can support the adhesion, proliferation and differentiation of muscle cells in the scaffold, and can therefore be effectively applied to the field of cell cultured meat.

[0006] The specific technical solutions are as follows:

[0007] A method for preparing a soybean protein amyloid fiber-chitosan 3D scaffold comprises the following steps:

[0008] (a) dissolving soybean protein in ultrapure water, heating and stirring for a period of time under a pH value of 2, adjusting the pH value of the solution to obtain solution I; dissolving chitosan in an acetic acid solution, adjusting the pH value of the solution to obtain solution II; mixing solution I and solution II, and adding a crosslinking agent to the mixed solution to obtain solution III;

[0009] (b) placing solution III of step (a) in a well plate, incubating in a constant temperature and humidity incubator for a period of time to obtain a soybean protein amyloid fiber-chitosan 3D gel, freeze-drying the gel, and water annealing to obtain a soybean protein amyloid fiber-chitosan 3D scaffold.

[0010] In addition, on the basis of the above, the method may further include step (c) as follows:

[0011] (c) Soaking the 3D scaffold of step (b) in ethanol, irradiating it with an ultraviolet lamp, and finally washing it with a buffer solution to obtain a sterile soybean protein amyloid fiber-chitosan 3D scaffold.

[0012] The present invention introduces chitosan, a cationic polysaccharide, to form a strong three-dimensional network structure by using electrostatic adsorption and hydrogen bonding between molecules, and allows the soybean protein amyloid fiber-chitosan 3D scaffold to be added with Ca 2+ Further cross-linking with mTG provides a high-porosity 3D scaffold that can be used in cell meat culture.

[0013] In the present application, the concentration of solution I is 20-40 mg / mL, for example, 20 mg / mL, 30 mg / mL, 40 mg / mL; the concentration of solution II is 5-20 mg / mL, for example, 5 mg / mL, 7.5 mg / mL, 10 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL; the mixing volume ratio of solution I and II is (2-4):1, for example, 2:1, 3:1, 4:1;

[0014] Preferably, in step (a), the soy protein is SPI, 7S, or 11S, and the deacetylation degree of the chitosan is 80-90%, for example, 80%, 85%, 90%, etc.

[0015] Preferably, in step (a), the heating temperature of solution I is 80-90°C, for example, it can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, etc.

[0016] Preferably, in step (a), the stirring speed of the solution I is 100-200 rpm, for example, it can be 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, etc.

[0017] Preferably, in step (a), the heating time of solution I is 16 to 24 hours, for example, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, etc.

[0018] Preferably, in step (a), the pH of solutions I and II is adjusted to 5.0-7.0, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, etc.

[0019] Preferably, in step (a), the cross-linking agent is an aqueous solution of mTG enzyme and calcium chloride, the mTG enzyme content in the solution III is 12.5-200U / g, for example, it can be 12.5U / g, 25U / g, 50U / g, 100U / g, 200U / g; the concentration of the calcium chloride aqueous solution is 20-200mM, for example, it can be 20mM, 50mM, 100mM, 150mM, 200mM;

[0020] Preferably, in step (b), the temperature of the constant temperature and humidity chamber is 30-40°C, for example, it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C; the humidity is 40-50%, for example, it can be 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%; the incubation time is 20-24h, for example, it can be 20h, 20.5h, 21h, 21.5h, 22h, 22.5h, 23h, 23.5h, 24h;

[0021] Preferably, in step (b), the water annealing temperature is 120-125°C, for example, 120°C, 121, 122°C, 123°C, 124°C, 125°C; the time is 10-20min, for example, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min;

[0022] Preferably, in step (c), the ethanol is anhydrous ethanol or 75% ethanol, and the soaking time is 0.5-2h, for example, 0.5h, 1.0h, 1.5h, 2.0h;

[0023] Preferably, in step (c), the energy of the UV lamp is 20-40 mJ / cm 2 , for example, it can be 20mJ / cm 2 、25mJ / cm 2 、30mJ / cm 2 、35mJ / cm 2 、40mJ / cm 2 ; The irradiation time is 1-3h, for example, it can be 1h, 1.5h, 2.0h, 2.5h, 3h;

[0024] Preferably, in step (c), the buffer solution is sterile PBS.

[0025] Compared with the prior art, this application has the following beneficial effects:

[0026] (1) The raw materials of soybean protein and chitosan used in the soybean protein amyloid fiber-chitosan 3D scaffold provided in this application are good biocompatibility, safe and non-toxic, widely available, and low in preparation cost. Cross-linking can occur after simple modification with mTG enzyme and calcium ion, the preparation method is simple and portable, and the cross-linking agent is also a safe and non-toxic component, which can ensure the safety of food consumption.

[0027] (2) The soybean protein amyloid fiber-chitosan 3D scaffold provided in the present application has excellent mechanical properties and stability, and has a certain thickness and elasticity, which can well support the adhesion, proliferation, differentiation and maturation of cells on the scaffold, and can also provide a certain degree of taste for cell meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are pictures showing the effects of mouse myoblasts proliferating on the scaffold provided in the example on days 1, 4, and 7.

[0029] Figure 2 This is a diagram showing the differentiation effect of mouse myoblasts on the scaffold provided in the example on the 14th day. DETAILED DESCRIPTION

[0030] The present invention is further explained below in conjunction with specific examples. The present invention is implemented according to the technical solution of the invention, and detailed implementation methods and operating steps are given, but the protection scope of the present invention is not limited to the following examples. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions.

[0031] Example 1

[0032] A method for preparing a soybean protein 7S amyloid fiber-chitosan 3D scaffold, the specific steps are as follows:

[0033] (1) 600 mg of soybean protein 7S was dissolved in 20 mL of ultrapure water to prepare a soybean protein solution with a concentration of 30 mg / mL, which was adjusted to pH 2 and placed in a water bath at 85°C for 20 h and a stirring speed of 150 rpm to promote the formation of soybean protein 7S amyloid fibers. 200 mg of chitosan with a deacetylation degree of 85% was dissolved in 20 mL of 1% acetic acid solution to prepare a chitosan solution with a concentration of 10 mg / mL.

[0034] (2) The soy protein 7S amyloid fiber solution and chitosan solution of step (1) are adjusted to pH 6, and the soy protein 7S amyloid fiber solution and chitosan solution are mixed in a volume ratio of 3:1, and then mTG enzyme 50U / g and calcium chloride solution 50mM are added in sequence at a final concentration, and after magnetic stirring until they are completely dissolved, 600μl of slurry is added to each well of a 48-well plate, and the plate is placed in a constant temperature and humidity incubator at 37°C and 45% humidity for 24 hours to obtain 7S amyloid fiber-chitosan 3D gel.

[0035] (3) The 7S amyloid fiber-chitosan 3D gel in step (2) was freeze-dried and water annealed in an autoclave at 121°C for 15 min to obtain a 7S amyloid fiber-chitosan 3D scaffold. The 7S amyloid fiber-chitosan 3D scaffold was first immersed in anhydrous ethanol for 1 hour, then immersed in 75% ethanol for 1 hour, and then placed in a biosafety cabinet at 30 mJ / cm 2 The scaffold was irradiated with UV light for 2 h, and finally washed three times with sterile PBS to obtain a sterile 7S amyloid fiber-chitosan 3D scaffold.

[0036] Example 2

[0037] (1) 600 mg of soybean protein 7S was dissolved in 20 mL of ultrapure water to prepare a soybean protein solution with a concentration of 30 mg / mL, which was adjusted to pH 2 and placed in a water bath at a heating temperature of 85°C for 20 h and a stirring speed of 150 rpm to promote the formation of soybean protein 7S amyloid fibers. 200 mg of chitosan with a deacetylation degree of 80% was dissolved in 20 mL of 1% acetic acid solution to prepare a chitosan solution with a concentration of 10 mg / mL.

[0038] (2) The soy protein 7S amyloid fiber solution and chitosan solution of step (1) are adjusted to pH 6, and the soy protein 7S amyloid fiber solution and chitosan solution are mixed in a volume ratio of 3:1, and then mTG enzyme 50U / g and calcium chloride solution 50mM are added in sequence at a final concentration, and after magnetic stirring until they are completely dissolved, 600μl of slurry is added to each well of a 48-well plate, and the plate is placed in a constant temperature and humidity incubator at 37°C and 45% humidity for 24 hours to obtain 7S amyloid fiber-chitosan 3D gel.

[0039] (3) The 7S amyloid fiber-chitosan 3D gel in step (2) was freeze-dried and water annealed in an autoclave at 121°C for 15 min to obtain a 7S amyloid fiber-chitosan 3D scaffold. The 7S amyloid fiber-chitosan 3D scaffold was first immersed in anhydrous ethanol for 1 hour, then immersed in 75% ethanol for 1 hour, and then placed in a biosafety cabinet at 30 mJ / cm 2 The scaffold was irradiated with UV light for 2 h, and finally washed three times with sterile PBS to obtain a sterile 7S amyloid fiber-chitosan 3D scaffold.

[0040] Example 3

[0041] (1) 600 mg of soybean protein 7S was dissolved in 20 mL of ultrapure water to prepare a soybean protein solution with a concentration of 30 mg / mL, which was adjusted to pH 2 and placed in a water bath at a heating temperature of 85°C for 20 h and a stirring speed of 150 rpm to promote the formation of soybean protein 7S amyloid fibers. 200 mg of chitosan with a deacetylation degree of 90% was dissolved in 20 mL of 1% acetic acid solution to prepare a chitosan solution with a concentration of 10 mg / mL.

[0042] (2) The soy protein 7S amyloid fiber solution and chitosan solution of step (1) are adjusted to pH 6, and the soy protein 7S amyloid fiber solution and chitosan solution are mixed in a volume ratio of 3:1, and then mTG enzyme 50U / g and calcium chloride solution 50mM are added in sequence at a final concentration, and after magnetic stirring until they are completely dissolved, 600μl of slurry is added to each well of a 48-well plate, and the plate is placed in a constant temperature and humidity incubator at 37°C and 45% humidity for 24 hours to obtain 7S amyloid fiber-chitosan 3D gel.

[0043] (3) The 7S amyloid fiber-chitosan 3D gel in step (2) was freeze-dried and water annealed in an autoclave at 121°C for 15 min to obtain a 7S amyloid fiber-chitosan 3D scaffold. The 7S amyloid fiber-chitosan 3D scaffold was first immersed in anhydrous ethanol for 1 hour, then immersed in 75% ethanol for 1 hour, and then placed in a biosafety cabinet at 30 mJ / cm 2 The scaffold was irradiated with UV light for 2 h, and finally washed three times with sterile PBS to obtain a sterile 7S amyloid fiber-chitosan 3D scaffold.

[0044] Example 4

[0045] (1) 600 mg of soy protein SPI was dissolved in 20 mL of ultrapure water to prepare a soy protein solution with a concentration of 30 mg / mL, which was adjusted to a pH of 2 and placed in a water bath at a heating temperature of 85°C for 20 h and a stirring speed of 150 rpm to promote the formation of soy protein SPI amyloid fibers. 200 mg of chitosan with a deacetylation degree of 80% was dissolved in 20 mL of 1% acetic acid solution to prepare a chitosan solution with a concentration of 10 mg / mL.

[0046] (2) The soy protein SPI amyloid fiber solution and chitosan solution of step (1) are adjusted to pH 6, and the soy protein SPI amyloid fiber solution and chitosan solution are mixed in a volume ratio of 2:1, and then mTG enzyme 50U / g and calcium chloride solution 50mM are added in sequence at a final concentration, and after magnetic stirring until all dissolved, 600μl of slurry is added to a 48-well plate in each well, and the plate is placed in a constant temperature and humidity incubator at 37°C and 45% humidity for 24 hours to obtain SPI amyloid fiber-chitosan 3D gel.

[0047] (3) The SPI amyloid fiber-chitosan 3D gel in step (2) was freeze-dried and water annealed in an autoclave at 121°C for 15 min to obtain a SPI amyloid fiber-chitosan 3D scaffold. The SPI amyloid fiber-chitosan 3D scaffold was first immersed in anhydrous ethanol for 1 hour, then immersed in 75% ethanol for 1 hour, and then placed in a biosafety cabinet at 30 mJ / cm 2 The scaffold was irradiated with UV light for 2 h, and finally washed three times with sterile PBS to obtain a sterile SPI amyloid fiber-chitosan 3D scaffold.

[0048] Example 5

[0049] (1) 600 mg of soybean protein 11S was dissolved in 20 mL of ultrapure water to prepare a soybean protein solution with a concentration of 30 mg / mL, which was adjusted to pH 2 and placed in a water bath at a heating temperature of 85°C for 20 h and a stirring speed of 150 rpm to promote the formation of soybean protein 11S amyloid fibers. 200 mg of chitosan with a deacetylation degree of 80% was dissolved in 20 mL of 1% acetic acid solution to prepare a chitosan solution with a concentration of 10 mg / mL.

[0050] (2) The soy protein 11S amyloid fiber solution and chitosan solution of step (1) were adjusted to pH 6, and the soy protein 11S amyloid fiber solution and chitosan solution were mixed in a volume ratio of 4:1, and then mTG enzyme 50U / g and calcium chloride solution 50mM were added in sequence at a final concentration, and after magnetic stirring until they were completely dissolved, 600μl of slurry was added to each well of a 48-well plate, and the plate was placed in a constant temperature and humidity incubator at 37°C and 45% humidity for 24h to obtain 7S amyloid fiber-chitosan 3D gel.

[0051] (3) The 11S amyloid fiber-chitosan 3D gel in step (2) was freeze-dried and water annealed in an autoclave at 121°C for 15 min to obtain a 11S amyloid fiber-chitosan 3D scaffold. The 11S amyloid fiber-chitosan 3D scaffold was first immersed in anhydrous ethanol for 1 hour, then immersed in 75% ethanol for 1 hour, and then placed in a biosafety cabinet at 30 mJ / cm 2 The scaffold was irradiated with UV light for 2 h, and finally washed three times with sterile PBS to obtain a sterile 11S amyloid fiber-chitosan 3D scaffold.

[0052] Comparative Example 1

[0053] (1) 600 mg of soy protein SPI was dissolved in 20 mL of ultrapure water to prepare a soy protein solution with a concentration of 30 mg / mL, which was adjusted to pH 2 and placed in a water bath at 85 °C for 20 h and a stirring speed of 150 rpm to promote the formation of soy protein SPI amyloid fibers.

[0054] (2) The soy protein SPI amyloid fiber solution of step (1) was adjusted to a pH of 6, and mTG enzyme 100U / g and calcium chloride solution 100mM were added in sequence, and magnetic stirring was performed until all dissolved. Then, 600μl of slurry was added to each well of a 48-well plate, and the plate was placed in a constant temperature and humidity incubator at 37°C and 45% humidity for 24h to obtain SPI amyloid fiber 3D gel.

[0055] (3) The SPI amyloid fiber 3D gel in step (2) was freeze-dried and water annealed in an autoclave at 121°C for 15 min to obtain a SPI amyloid fiber 3D scaffold. The SPI amyloid fiber 3D scaffold was first immersed in anhydrous ethanol for 1 hour, then immersed in 75% ethanol for 1 hour, and then placed in a biosafety cabinet at 30 mJ / cm 2 The scaffold was irradiated with UV light for 2 h, and finally washed three times with sterile PBS to obtain a sterile SPI amyloid fiber 3D scaffold.

[0056] Comparative Example 2

[0057] (1) 600 mg of soy protein SPI was dissolved in 20 mL of ultrapure water to prepare a soy protein solution with a concentration of 30 mg / mL, which was adjusted to a pH of 2 and placed in a water bath at a heating temperature of 85°C for 20 h and a stirring speed of 150 rpm to promote the formation of soy protein SPI amyloid fibers. 200 mg of chitosan with a deacetylation degree of 80% was dissolved in 20 mL of 1% acetic acid solution to prepare a chitosan solution with a concentration of 10 mg / mL.

[0058] (2) The soy protein SPI amyloid fiber solution of step (1) was adjusted to a pH of 6, and mTG enzyme and calcium chloride solution were added in sequence at a final concentration of 50 U / g, and magnetic stirring was performed until all dissolved. Then, 600 μl of slurry was added to each well of a 48-well plate, and the plate was placed in a constant temperature and humidity incubator at 37°C and 45% humidity for 24 hours to obtain SPI amyloid fiber 3D gel.

[0059] (3) Immerse the SPI amyloid fiber 3D gel in step (2) in the chitosan solution in step (1), and perform water annealing in an autoclave at 121°C for 15 min to obtain a SPI amyloid fiber-chitosan coated scaffold. The SPI amyloid fiber-chitosan coated scaffold was first immersed in anhydrous ethanol for 1 hour, then immersed in 75% ethanol for 1 hour, and then placed in a biosafety cabinet at 30 mJ / cm 2 The scaffold was irradiated with UV light for 2 h, and finally washed three times with sterile PBS to obtain a sterile SPI amyloid fiber-chitosan coated scaffold.

[0060] The Young's modulus of the scaffolds obtained in the above-mentioned embodiments and comparative examples, and the quantitative data of the proliferation, differentiation and growth of mouse myoblasts thereon are shown in Table 1, Table 2 and Table 3, respectively.

[0061]

[0062]

[0063] Table 1

[0064]

[0065] Table 2

[0066] sample Fusion index (%) Maturity index (%) Example 1 68.58 80.93 Example 2 55.85 67.60 Example 3 60.19 77.54 Example 4 15.35 18.94 Example 5 16.81 25.16

[0067] Table 3

[0068] The soy protein amyloid fiber-chitosan 3D scaffold provided by the present invention is a mixture of soy protein, chitosan and a cross-linking agent as a material, which not only provides the nutritional value of the scaffold, but also ensures the edible safety as a part of the food product. The preparation process of the present invention is simple, and the soy protein amyloid fiber-chitosan 3D scaffold has a certain thickness and high mechanical properties, and can support the adhesion, proliferation and differentiation of muscle cells in the scaffold.

[0069] from Figure 1 and Figure 2 It can be seen that the three soybean protein amyloid fiber-chitosan 3D scaffolds prepared from chitosan with three different deacetylation degrees provided in Examples 1-5 can support the adhesion, proliferation and differentiation of mouse myoblasts on the scaffold into slender myotubes. Among them, Example 1, that is, the scaffold prepared from chitosan with a deacetylation degree of 85% and 7S amyloid fiber, showed the best cell support ability. However, the cells could not adhere, proliferate, and differentiate on Comparative Examples 1-2. Tables 2 and 3 specifically quantify the various data of cell growth on Examples 1-5, further proving that Example 1 is most beneficial to cell growth, followed by Example 1, Example 3, Example 5 and Example 4.

[0070] From the results in Table 1, it can be seen that the soybean protein amyloid fiber-chitosan 3D scaffolds provided by Examples 1-5 have a certain elasticity, and the specific Young's modulus is 15.322, 4.8286, 12.275, 2.6425, and 3.3893 kPa. Example 1 shows the best Young's modulus, so the scaffold prepared in Example 1 can provide the best growth support for cell meat. At the same time, each scaffold can provide a certain taste support for the cultured cell meat to a certain extent. The specific Young's modulus of Comparative Examples 1-2 is 0.3425 and 0.6778 kPa, which are much lower than that of Examples 1-5, so Comparative Examples 1-2 cannot provide support for cell growth.

Claims

1. A method for preparing a soybean protein amyloid fiber-chitosan 3D cell meat scaffold, characterized in that: These include: (a) dissolving soybean protein in ultrapure water, heating and stirring for a period of time under a pH value of 2, adjusting the pH value of the solution to obtain solution I; dissolving chitosan in an acetic acid solution, adjusting the pH value of the solution to obtain solution II; mixing solution I and solution II, and adding a crosslinking agent to the mixed solution to obtain solution III; (b) placing solution III of step (a) in a well plate, incubating in a constant temperature and humidity incubator for a period of time to obtain a soybean protein amyloid fiber-chitosan 3D gel, freeze-drying the gel, and water annealing to obtain a soybean protein amyloid fiber-chitosan 3D scaffold; In step (a), the heating temperature of the solution I is 80-90° C.; the stirring speed is 100-200 rpm; the heating time is 16-24 h, the pH of the solutions I and II is adjusted to 5.0-7.0, the cross-linking agent is an aqueous solution of mTG enzyme and calcium chloride, the mTG enzyme content in the solution III is 12.5-200 U / g, and the concentration of the calcium chloride aqueous solution is 20-200 mM.

2. The method for preparing the soybean protein amyloid fiber-chitosan 3D scaffold according to claim 1, characterized in that: The method further comprises: (c) Soaking the 3D scaffold of step (b) in ethanol, irradiating it with an ultraviolet lamp, and finally washing it with a buffer solution to obtain a sterile soybean protein amyloid fiber-chitosan 3D scaffold.

3. The method for preparing the soybean protein amyloid fiber-chitosan 3D scaffold according to claim 1, characterized in that: In step (a), the concentration of solution I is 20-40 mg / mL, the concentration of solution II is 5-20 mg / mL, and the mixed volume ratio of solution I and II is (2-4):

1.

4. The method for preparing the soybean protein amyloid fiber-chitosan 3D scaffold according to claim 1, characterized in that: In step (a), the soybean protein is SPI, 7S, or 11S, and the deacetylation degree of the chitosan is 80-90%.

5. The method for preparing the soybean protein amyloid fiber-chitosan 3D scaffold according to claim 1, characterized in that: In step (b), the temperature of the constant temperature and humidity chamber is 30-40° C., the humidity is 40-50%, and the incubation time is 20-24 hours; the temperature of the water annealing is 120-125° C., and the time is 10-20 minutes.

6. The method for preparing the soybean protein amyloid fiber-chitosan 3D scaffold according to claim 1, characterized in that: In step (c), the ethanol is anhydrous ethanol or 75% ethanol, and the immersion time is 0.5-2h respectively; the energy of the ultraviolet lamp is 20-40mJ / cm 2 , the irradiation time is 1-3h; the buffer solution is sterile PBS.

7. A soybean protein amyloid fiber-chitosan 3D scaffold, characterized in that: The method is prepared by the method according to any one of claims 1 to 6.

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