A method for preparing controllable molecular weight silk fibroin freeze-dried balls

Abstract

This invention relates to the field of lyophilized formulation technology and discloses a method for preparing controllable molecular weight silk fibroin lyophilized spheres, comprising the following steps: preparing a mixture of a lyophilization protectant, a protein conformation stabilizer, and silk fibroin solutions of different molecular weights. The lyophilization protectant includes sugars and sugar alcohols, and the protein conformation stabilizer is a polyol. After pre-freezing the mixture into spheres, a surface coating modification is performed by adding an ethanol solution containing cellulose, followed by a second pre-freezing in liquid nitrogen, and finally, the lyophilized product is controlled by a specific program in a vacuum freeze dryer. This invention employs specific lyophilization protectants, protein conformation stabilizers, surface coating modification techniques, and freeze-drying techniques, enabling the prepared silk fibroin lyophilized spheres to have a better appearance and improving their resistance to moisture absorption and shrinkage, as well as their rapid reconstitution.

Description

Technical Field

[0001] This invention relates to the field of lyophilized formulation technology, and in particular to a method for preparing lyophilized silk fibroin balls with controllable molecular weight. Background Technology

[0002] Silk is a natural protein polymer approved by the U.S. Food and Drug Administration (FDA) for medical use. Silk fibroin is processed from mulberry silk after removing the outer sericin layer; when combined with other substances, it may trigger an immune response. Silk fibroin possesses excellent biocompatibility, controllable biodegradability, significant mechanical strength, and low immunogenicity. Therefore, it has been widely used in tissue engineering and regenerative medicine applications such as cartilage and corneal repair. Due to its hemostatic properties, low inflammatory potential, and permeability to oxygen and water vapor, silk fibroin is also used as a biomaterial for skin repair. Silk fibroin nanofibers have been reported to promote the adhesion of human keratinocytes and fibroblasts in vitro and enhance type I collagen deposition. Further studies have shown that in small animal models, silk fibroin membrane dressings or sponge dressings promote wound healing and skin regeneration more rapidly than hydrogel dressings or porcine dermal / dermal matrix dressings. Furthermore, silk fibroin does not cause acute skin toxicity, acute skin irritation, or skin sensitization.

[0003] Currently, there are two main types of vacuum freeze-drying technology for silk fibroin: in-situ freeze-drying and non-in-situ freeze-drying. In-situ freeze-drying involves directly loading the mixed freeze-drying solution into the required packaging material for freeze-drying. However, due to the nature of the packaging materials themselves, the freeze-drying technology varies greatly between different packaging materials, generally allowing only small-scale production. Furthermore, vacuum sealing is required after exiting the freeze chamber, resulting in products that are typically irregular in shape and difficult to repackage and transport during use. Non-in-situ freeze-drying, on the other hand, involves pre-freezing the mixed freeze-drying solution into a specified shape in liquid nitrogen, then placing the pre-cooled and shaped product onto a carrier such as a vial, and finally transferring it to a freeze dryer for freeze-drying. This is followed by partial capping or final sealing, thus overcoming the drawbacks of traditional in-situ freeze-drying and enabling continuous, large-scale production. However, the freeze-drying protectants currently used are made of trehalose, sucrose, PEG, BSA, etc., which easily form freeze-dried products with irregular shapes. If more protectants are added, they will have a certain inhibitory effect on silk fibroin and will also reduce the mechanical properties of freeze-dried products, making them prone to flaking during subsequent packaging and transportation, thus affecting the consistency and stability of freeze-dried products.

[0004] CN105030561A discloses a silk fibroin microsphere encapsulated with arbutin and vitamin C, composed of the following raw material components: 20%–60% silk fibroin, 2%–8% arbutin, 10%–20% vitamin C, 20%–60% lyophilization protectant, and 1%–4% distilled water. This method encapsulates arbutin and vitamin C in silk fibroin and prepares nanoscale microspheres. However, the preparation method is relatively complex, involves an aqueous phase system, and the obtained microspheres have too small a particle size, making them unsuitable for current requirements.

[0005] CN114159555A discloses a freeze-dried antigen protein microsphere and its preparation method. This patent involves mixing a freeze-drying protective solution with an antigen protein solution and then freeze-drying the resulting freeze-dried antigen protein microsphere, with a volume ratio of freeze-drying protective solution to antigen protein solution of 5:1 to 9:1. These freeze-dried microspheres can be directly mixed with the sample to be tested, avoiding measurement errors caused by liquid sampling errors; they also do not adhere to the tube wall and can be transferred, especially when multiple antigen proteins are mixed, allowing for rapid combination as needed, and their particulate form facilitates carrying and storage. However, this patent requires a large amount of freeze-drying protective agent, and accelerated aging preservation is only possible at 37°C, resulting in poor high-temperature resistance; the diameter deviation of the freeze-dried microspheres is within ±2.5 mm, indicating instability.

[0006] CN103923200A and CN103910789B disclose a method for preparing high molecular weight silk fibroin freeze-dried powder. The freeze-dried powder is obtained by degumming silk, dissolving it in lithium bromide, dialysis, centrifugation, high-temperature and high-pressure sterilization, and freeze-drying. The average molecular weight of the silk fibroin in the freeze-dried powder is greater than 100,000 Daltons, and after purification, it exists in solution as particles of 50-300 nanometers. The silk fibroin powder prepared by this method has no porosity, is easily hygroscopic, and has a long reconstitution time after absorbing moisture.

[0007] In existing technologies, silk fibroin is generally in the form of a solution. Silk fibroin solutions have problems such as easy β-sheet formation, difficulty in storage and transportation, and short shelf life. Currently, silk fibroin is often directly prepared into powder for transportation and storage. However, silk fibroin powder also has problems such as excessively long dissolution time (>60s), easy moisture absorption and clumping. After absorbing moisture, silk fibroin is prone to β-sheet formation and cannot be completely redissolved. Summary of the Invention

[0008] To address the technical problems of existing silk fibroin solutions being difficult to transport and preserve, and prone to moisture absorption and shrinkage during storage, making reconstitution impossible, this invention provides a method for preparing controllable molecular weight silk fibroin freeze-dried spheres. This method employs specific freeze-drying protectants, silk fibroin conformation stabilizers, surface coating moisture-proof modification techniques, and freeze-drying technology. This results in freeze-dried silk fibroin spheres with improved appearance and enhanced resistance to moisture absorption and shrinkage. This enables long-term storage and transportation of silk fibroin, rapid dissolution, and protection of its properties, thereby extending its shelf life.

[0009] The method for preparing controllable molecular weight silk fibroin freeze-dried balls according to the present invention includes:

[0010] S1: A mixture of lyophilization protectant, protein conformation stabilizer, and silk fibroin solution is prepared; the lyophilization protectant includes sugars and sugar alcohols, the protein conformation stabilizer is a polyol, and the silk fibroin has a narrow distribution and controllable molecular weight, with a molecular weight range of 10kDa to 80kDa, 80kDa to 100kDa, 100kDa to 120kDa, 120kDa to 160kDa, 160kDa to 200kDa, and >200kDa;

[0011] S2: Pre-cool the prepared mixture (15-45 μL) and shape it into a 5.00 mm thick sheet. 3 -40.00mm 3 The small ball;

[0012] S3: Place the pre-cooled and shaped small balls from step S2 into a mixture of a treatment agent and anhydrous ethanol for surface coating; the treatment agent is one or more of hydroxypropyl methylcellulose, sodium hyaluronate, and sodium carboxymethylcellulose.

[0013] S4: Place the small balls after surface coating in step S3 into liquid nitrogen for secondary molding;

[0014] S5: Place the pre-cooled and shaped pellets in a freeze dryer for freeze drying. The freeze drying process is as follows:

[0015] S5.1: The freeze dryer should be kept at -49 to -51℃ for 1.5 to 2 hours; raised to -28 to -31℃ and kept at 1 to 1.5 hours; lowered to -49 to -51℃ and kept at 1 to 1.5 hours.

[0016] S5.2: Turn on the vacuum; raise the temperature to -43 to -45℃ and hold for 14 to 16 hours; raise the temperature to -38 to -42℃ and hold for 9 to 11 hours; raise the temperature stepwise to -24 to -26℃, raising it by 4 to 5℃ and holding for 1 to 1.5 hours before continuing to raise the temperature; raise the temperature to -1 to 1℃ and hold for 1.0 to 1.5 hours.

[0017] S5.3: Raise to 19-21℃ and maintain for 5-5.5 hours; raise to 29-31℃ and maintain for 4-4.5 hours;

[0018] S6: The freeze-dried product is repackaged in a glove box;

[0019] S7: The repackaged product is capped and labeled.

[0020] Furthermore, in step S1, the mass ratio of silk fibroin: carbohydrates: sugar alcohols: polyols is 1:1:1:1 to 3:2:1:1, where carbohydrates include compounds represented by formula I and / or formula II, and sugar alcohols have the structural formula shown in formula III.

[0021] Formula I: C 12 H 22 O 11 Examples include trehalose, lactose, sucrose, maltose, and caramel.

[0022] Formula II: C 12 H 22 O 11 • 2H2O, such as trehalose dihydrate;

[0023] Formula III: HOCH2(CHOH) n CH2OH n≥1, for example mannitol, sorbitol, xylitol, erythritol;

[0024] Formula IV: C n H 2n+2-x (OH) x (n≥1, x≥2), for example, glycerol, ethylene glycol, butylene glycol;

[0025] Furthermore, in step S1, the molecular weight of the silk fibroin solution is 10kDa~80kDa, 80kDa~100kDa, 100kDa~120kDa, 120kDa~160kDa, 160kDa~200kDa, or >200kDa.

[0026] Furthermore, in step S2, the method for pre-freezing the mixture (15–45 μL) into spheres involves dropping the mixture into liquid nitrogen, wherein each lyophilized sphere has a volume of 5.00 mm. 3 -40.00mm 3 Its freeze-dried particle size is 1-5 mm.

[0027] Furthermore, in step S3, the pre-cooled and shaped spheres are transferred to a mixture of treatment agent and anhydrous ethanol for surface coating, and the surface coating time is ≤60s.

[0028] Furthermore, in step S3, a certain volume of liquid nitrogen is added to the pre-cooled freeze-dried spheres during the transfer process, wherein the volume ratio of the pre-cooled freeze-dried spheres to the liquid nitrogen is 1:1 to 2:1.

[0029] Furthermore, in step S3, the hydroxypropyl methylcellulose content of the treatment agent-anhydrous ethanol mixture is 1%-5%.

[0030] Furthermore, the time for the product undergoing secondary molding in step S4 is ≤3 minutes;

[0031] Furthermore, in step S5.1, the heating or cooling rate for raising the temperature to -28 to -31°C and lowering it to -49 to -51°C is higher than 0.5°C / min.

[0032] In the further step S5.2, the heating or cooling rates for raising the temperature to -43 to -45°C, raising the temperature to -38 to -42°C, raising the temperature stepwise to -24 to -26°C, and raising the temperature to -1 to 1°C are all less than 0.5°C / min.

[0033] Furthermore, in step S5.3, the heating or cooling rates to 19–21°C and 29–31°C are both less than 0.5°C / min.

[0034] Furthermore, the humidity of the glove box in step S6 is ≤15%.

[0035] Furthermore, the finished product in step S7 should undergo a sealing test using a color water method.

[0036] A type of lyophilized silk fibroin ball was prepared using the above-described preparation method.

[0037] The aforementioned freeze-dried spheres specifically include those with a volume of 5.00 mm². 3 -40.00mm 3 The particle size is 1-5 mm, preferably the surface roundness of the freeze-dried balls is ≥95%, and more preferably the internal structure is loose and porous with an internal pore size of less than 10 mm and a porosity of ≥70%.

[0038] The application of the above-mentioned silk fibroin freeze-dried balls is characterized in that the applications include: water-light products, dressing products, and filler products.

[0039] Specifically, the mechanisms and effects of the freeze-drying protectant, silk fibroin conformation stabilizer, surface moisture-proof coating modification technology, and freeze-drying technology of the present invention are as follows:

[0040] (1) The synergistic effect among carbohydrates, sugar alcohols, and polyols:

[0041] During freeze-drying, sugars can fill the pores of protein molecules, thereby inhibiting changes in the internal structure of the protein molecules and preventing protein inactivation during freeze-drying. Based on this, by adding sugar alcohols and sugars, skeletal support is achieved for the material; sugars act as protein protectants for the main framework, while sugar alcohols act as supporting materials, increasing the glass transition temperature of the material; the synergistic effect of sugars enhances the material's resistance to high-temperature damage, enabling low-temperature storage of silk fibroin. Since existing freeze-dried spheres are all low-molecular-weight products, low molecular weight substances are less prone to precipitation and folding. However, they have narrow distribution controllable molecular weights of 10kDa–80kDa, 80kDa–100kDa, 100kDa–120kDa, 120kDa–160kDa, 160kDa–200kDa, and >200kDa. Silk fibroin is a high-molecular-weight substance, prone to irreversible β-sheet formation. Adding polyols can prevent high-molecular-weight silk fibroin from undergoing β-sheet formation. Furthermore, because the mixed freeze-drying solution has a high viscosity, it can form instantly upon being dropped into the liquid. However, due to the impact force, the shape of the freeze-dried spheres is prone to change. Adding polyols can lower the freezing point and reduce shape changes.

[0042] (2) Reasons for applying a surface coating

[0043] 1. Lubricity and Film-Forming Properties of HPMC: As a high-molecular polymer, HPMC possesses excellent lubricity and film-forming properties. When used as a coating material, it can form a smooth and uniform film on the surface of silk fibroin freeze-dried pellets. This film not only makes the freeze-dried pellets smoother but also reduces their direct contact with the external environment, thereby lowering the risk of surface contamination or oxidation. 2. Hydrophobicity of HPMC: HPMC exhibits a certain degree of hydrophobicity, meaning it can effectively prevent moisture from penetrating into the interior of the freeze-dried pellets. By adding an HPMC coating, the stability of the freeze-dried pellets in humid environments is significantly improved, and their surface is less prone to changes or dissolution due to water absorption. 3. Interaction between HPMC and Silk Fibrous Protein: Certain interaction forces, such as hydrogen bonds and van der Waals forces, may exist between HPMC and silk fibroin. These interactions help enhance the adhesion between the HPMC coating and the surface of the silk fibroin freeze-dried pellets, making the coating more firmly attached. A robust coating prevents peeling or damage during use, thus maintaining the smoothness and stability of the freeze-dried pellet surface.

[0044] (3) Add freeze-drying protectants and silk fibroin conformation stabilizers, and combine with specific freeze-drying technology:

[0045] This invention reveals that by combining the aforementioned freeze-drying protectant and silk fibroin conformation stabilizer with the freeze-drying step of this invention, the freeze-drying protectant provides a robust supporting framework for the freeze-dried pellets, resulting in a smooth, dense, and loosely structured pellet. The protein conformation stabilizer causes the silk fibroin to tend towards a random structure, preventing β-sheet formation, while simultaneously reducing the likelihood of ice crystal formation and further decreasing silk fibroin precipitation. The silk fibroin freeze-dried pellets prepared using this process overcome the drawbacks of traditional silk fibroin solutions, such as difficulty in preservation and transportation, while retaining many of the advantages of traditional silk fibroin solutions. Moreover, compared to traditional silk fibroin solutions, the silk fibroin freeze-dried pellets prepared using the method of this invention can reduce protein damage.

[0046] Preferably, in step S1, the molecular weight of the silk fibroin solution used is (10kDa~80kDa, 80kDa~100kDa, 100kDa~120kDa, 120kDa~160kDa, 160kDa~200kDa, >200kDa). The test method refers to Appendix B of YY / T1950-2024 "Tissue Engineering Medical Devices Silk Fiber". The rheological data and viscosity data are imported into Favorsun SmartMolFit "Multi-directional Intelligent Calculation" commercial software for calculating the molecular weight of biopolymers to calculate the number-average molecular weight and weight-average molecular weight.

[0047] Preferably, in step S1, the sugars in the mixture include, but are not limited to, trehalose, lactose, sucrose, maltose, caramel, and other disaccharides.

[0048] Preferably, in step S1, the sugar alcohols in the mixture include, but are not limited to, mannitol, sorbitol, xylitol, erythritol and other sugar alcohols.

[0049] Preferably, in step S1, the polyols in the mixture include, but are not limited to, glycerol, ethylene glycol, butanediol and other polyols.

[0050] Sugars can provide structural support, maintain protein activity, and preserve the product's appearance. However, excessive addition can affect the product's appearance and storage stability, causing it to wrinkle and collapse during storage.

[0051] Adding an appropriate amount of sugar alcohols can improve the reconstitution of freeze-dried pellets. However, if the content of sugar alcohols is too high, it will affect the appearance of the product, making it less smooth and rougher. It will also affect the storage stability of the product, making it prone to problems such as yellowing, shrinkage, and reduced protein activity during storage.

[0052] Further preferred, in step S1, the ratio of sugars:sugar alcohols:sugar alcohols in the mixture is 2:1:1 to 1:1:1.

[0053] When the total amount of sugars, sugar alcohols, and polyols is fixed, if the proportion of sugar alcohols is too high, freeze-dried pellets are prone to problems during storage, such as moisture absorption and collapse, and reduced protein activity.

[0054] Preferably, in step S2, the method for pre-freezing the mixture into spheres is to drip the mixture into liquid nitrogen using a continuous dispenser.

[0055] Preferably, the freeze-dried pellets have a volume of 15–45 μL and a particle size of 1–5 mm. More preferably, the volume is 20–40 μL and the particle size is 3.2–3.4 mm.

[0056] Preferably, in step S3, the pre-cooled and shaped microspheres are transferred to a mixture of hydroxypropyl methylcellulose and anhydrous ethanol for surface coating, and the surface coating time is ≤60s. More preferably, it is 30s-60s.

[0057] Preferably, a certain volume of liquid nitrogen is added to the pre-cooled freeze-dried spheres during the transfer process, wherein the volume ratio of the pre-cooled freeze-dried spheres to the liquid nitrogen is 1:1 to 2:1. More preferably, the ratio is 1:1.

[0058] Preferably, the hydroxypropyl methylcellulose-anhydrous ethanol mixture used in step S3 has a hydroxypropyl methylcellulose content of 1%-5%. More preferably, it is 2%-3%.

[0059] Preferably, the time for secondary molding of the product in step S4 is ≤3 min. More preferably, it is 1-1.5 min.

[0060] Preferably, in step S5.1, the rate of raising the temperature to -28 to -31°C and lowering it to -49 to -51°C is higher than 0.5°C / min. More preferably, it is 0.7 to 1°C / min.

[0061] Preferably, in step S5.2, the rates of heating to -43 to -45°C, heating to -38 to -42°C, step heating to -24 to -26°C, and heating to -1 to 1°C are all less than 0.5°C / min. More preferably, the rates are 0.2 to 0.4°C / min.

[0062] Preferably, in step S5.3, the rates of raising the temperature to 19–21°C and to 29–31°C are both less than 0.5°C / min. More preferably, the rates are 0.1–0.2°C / min.

[0063] Preferably, in step S5.2, the vacuum level is controlled to be ≤15 Pa. More preferably, during the process of raising the temperature to -1 to 1°C and maintaining it for 1 to 1.5 hours, the vacuum level is controlled to be ≤10 Pa.

[0064] Preferably, in step S5.3, the vacuum level is controlled to be ≤1 Pa.

[0065] In this invention, "vacuum degree" refers to the gauge pressure of the freeze dryer, that is, the pressure inside the freeze dryer chamber.

[0066] Preferably, the moisture content of the freeze-dried pellets should be ≤3% when they leave the warehouse.

[0067] Preferably, in step S6, the humidity of the glove box should be ≤15%. More preferably, it should be 2%-10%.

[0068] Preferably, in step S7, the product with the rolled cap should pass a sealing test.

[0069] Another objective of this invention is achieved through the following technical solution:

[0070] An application of a type of silk fibroin freeze-dried ball, the application including: water-light products, dressing products and filler products.

[0071] Compared with the prior art, the present invention has the following advantages:

[0072] (1) In the process of preparing silk fibroin freeze-dried balls, the present invention uses specific freeze-drying protectants, silk fibroin conformation stabilizers, surface moisture-proof modification technology and freeze-drying technology to obtain round shape, moisture absorption and shrinkage resistance, smoothness, and easy shedding and deformation during the conveying process.

[0073] (2) The present invention prepares a protein microsphere with a narrow molecular weight distribution of silk fibroin and a molecular weight range of 80kDa to 200kDa. Narrowly distributed and controllable proteins have good therapeutic effects, but they are prone to β-sheet formation and are difficult to re-dissolve. The present invention solves the problem of rapid re-dissolving.

[0074] (3) HPMC coating is added to the surface of silk fibroin freeze-dried balls. Due to the lubricity, film-forming properties, hydrophobicity, interaction with silk fibroin, and optimization of the microstructure of the freeze-dried balls, the coating results in better appearance and performance of the freeze-dried balls.

[0075] (4) In the process of preparing silk fibroin freeze-dried balls, the present invention can improve the appearance and moisture-absorbing shrinkage resistance of freeze-dried balls and enhance their resolubility by adjusting the proportion of sugars, sugar alcohols and polyols in the freeze-drying mixed solution, while also effectively protecting the protein.

[0076] (5) After the silk fibroin is made into freeze-dried balls by the method of the present invention, the stability of the silk fibroin can be improved, and it can be applied to water-light products, dressing products and filler products in the medical beauty field.

[0077] (6) Using the freeze-drying protectant, surface coating method and specific freeze-drying procedure of the present invention, the obtained silk fibroin freeze-dried balls can have the following characteristics: 1) beautiful appearance, round, without wrinkles or collapse; 2) easy to pack and transport, without slag or deformation; 3) the product has good stability, will not absorb moisture and collapse, can be taken out of the chamber under normal low humidity conditions, and can be stored at room temperature, while also maintaining the protein activity in the silk fibroin well. Attached image description:

[0078] Figure 1 Example 1: Appearance of freeze-dried silk fibroin balls

[0079] Figure 2 Example 1: Image of the internal pores of a silk fibroin freeze-dried sphere.

[0080] Figure 3 Example 1: Measured Fourier Transform Infrared Absorption Spectrum (FT-IR) of Lyophilized Silk Fibroin Spheres

[0081] Figure 4 Example 1: X-ray diffraction (XRD) pattern of freeze-dried silk fibroin bulbs

[0082] Figure 5 Example 1: Schematic diagram of the structure of freeze-dried silk fibroin spheres Detailed implementation method:

[0083] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0084] Preparation of silk fibroin freeze-dried spheres:

[0085] Example 1:

[0086] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, and the molecular weight of silk fibroin is 80kDa~100kDa), 2g of trehalose, 1g of mannitol, 1g of glycerol, and the rest are balanced with purified water to 100g. After balancing, mix evenly and set aside.

[0087] (2) The mixture is dispersed into 30 μL of solution per drop by a continuous separator and dropped into liquid nitrogen for pre-cooling and forming into spheres; the pre-cooled spheres are transferred to a hydroxypropyl methylcellulose-anhydrous ethanol mixture with a volume ratio of 1:1 for surface coating (hydroxypropyl methylcellulose accounts for 2.5%), the surface coating time is 40s, and the coated product is placed in liquid nitrogen for 1min for secondary pre-cooling and forming.

[0088] (3) Transfer the pre-cooled and shaped pellets along with liquid nitrogen to a pre-cooled stainless steel tray. Then, place the stainless steel tray into a vacuum freeze dryer that has been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 1) to freeze-dry, obtaining silk fibroin freeze-dried pellets with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 1. In Table 1, the temperature change rate for each step refers to the rate of temperature change from the previous process to the current process. The processing time for each step is based on the time the current process temperature is maintained.

[0089] Table 1 Freeze-drying procedure in Example 1

[0090]

[0091]

[0092] (4) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0093] (5) The repackaged products are capped by a capping machine and then labeled and packaged.

[0094] The appearance of the silk fibroin freeze-dried balls in Example 1 after freeze-drying is as follows: Figure 1 As shown, the surface is smooth and glossy, and its internal porosity is as follows: Figure 2 As shown, the internal structure is loose and porous, with pores less than 10 mm and a porosity ≥70%. The lyophilized silk fibroin pellets were analyzed using FT-IR and XRD to verify whether lyophilization affected the chemical and physical structures of the silk fibroin. The chemical structure was characterized and analyzed using infrared spectroscopy (e.g., FT-IR and XRD). Figure 3 As shown in the figure, the FT-IR measured band of silk fibroin is at 1651.73 cm⁻¹. -1 1543.74cm -1 and 1236.25cm -1Characteristic peaks exist at these locations, corresponding to the amide I, amide II, and amide III bands of silk fibroin, respectively; the physical structure of the lyophilized silk fibroin spheres was characterized and analyzed by XRD (e.g., Figure 4 As shown, silk fibroin has two aggregated structures: crystalline and amorphous. The crystalline state has two spatial configurations, referred to as α-type and β-type, also known as Silk I and Silk II. X-ray diffraction (XRD) patterns at 2θ = 20° show the Silk II crystalline structure of silk fibroin. This indicates that the freeze-drying process did not damage the physicochemical properties of silk fibroin and can effectively protect it.

[0095] The overall score for freeze-dried pellets is 5. The scores for each item are shown in Table 2. The scoring criteria are detailed in Table 14.

[0096] Table 2: Evaluation Table for Freeze-Dried Balls in Example 1

[0097]

[0098] Example 2:

[0099] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, with a molecular weight of 100kDa to 120kDa), 2g of trehalose, 1g of mannitol, 1g of glycerol, and the rest are balanced with purified water to 100g. After balancing, mix evenly and set aside.

[0100] (2) The mixture is dispersed into 30 μL of solution per drop by a continuous separator and dropped into liquid nitrogen for pre-cooling and forming into spheres; the pre-cooled spheres are transferred to a mixture of sodium carboxymethyl cellulose and anhydrous ethanol at a volume ratio of 1:1 for surface coating (hydroxypropyl methyl cellulose accounts for 2.5%), the surface coating time is 40s, and the coated product is placed in liquid nitrogen for 1min for secondary pre-cooling and forming.

[0101] (3) Transfer the pre-cooled and shaped pellets along with liquid nitrogen to a pre-cooled stainless steel tray. Then, place the stainless steel tray into a vacuum freeze dryer that has been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 2) to freeze-dry, obtaining silk fibroin freeze-dried pellets with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 3. In Table 3, the temperature change rate for each step refers to the rate of temperature change from the previous process to the current process. The processing time for each step is based on the time the current process temperature is maintained.

[0102] Table 3 Freeze-drying procedure in Example 2

[0103]

[0104] (4) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0105] (5) The repackaged products are capped by a capping machine and then labeled and packaged.

[0106] The overall score for freeze-dried pellets is 4.75. The scores for each item are shown in Table 4. The scoring criteria are detailed in Table 14.

[0107] Table 4: Evaluation Table for Freeze-Dried Balls in Example 2

[0108]

[0109]

[0110] Example 3:

[0111] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, with a molecular weight of 120kDa to 140kDa), 2g of sucrose, 1g of sorbitol, 1g of ethylene glycol, and the remainder is balanced with purified water to 100g. After balancing, mix evenly and set aside.

[0112] (2) The mixture was dispersed into a solution of 30 μL per drop by a continuous separator and dropped into liquid nitrogen for pre-cooling and forming into spheres. The pre-cooled spheres were then transferred to a sodium hyaluronate-anhydrous ethanol mixture at a volume ratio of 1:1 for surface coating (hydroxypropyl methylcellulose accounted for 3.0%). The surface coating time was 45 s. The coated product was then placed in liquid nitrogen for 1.5 min for secondary pre-cooling and forming.

[0113] (3) Transfer the pre-cooled and shaped pellets along with liquid nitrogen to a pre-cooled stainless steel tray. Then, place the stainless steel tray into a vacuum freeze dryer that has been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 1) to freeze-dry, obtaining silk fibroin freeze-dried pellets with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 1. In Table 1, the temperature change rate for each step refers to the rate of temperature change from the previous process to the current process. The processing time for each step is based on the time the current process temperature is maintained.

[0114] (4) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0115] (5) The repackaged products are capped by a capping machine and then labeled and packaged.

[0116] The overall score for freeze-dried pellets is 4.25. The scores for each item are shown in Table 5. The scoring criteria are detailed in Table 14.

[0117] Table 5: Evaluation Table for Freeze-Dried Balls in Example 3

[0118]

[0119] Example 4:

[0120] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, with a molecular weight of 140kDa to 160kDa), 1.5g of sucrose, 1g of sorbitol, 1g of butanediol, and the remainder is balanced with purified water to 100g. After balancing, mix evenly and set aside.

[0121] (2) The mixture is dispersed into 30 μL of solution per drop by a continuous separator and dropped into liquid nitrogen for pre-cooling and forming into spheres; the pre-cooled spheres are transferred to a hydroxypropyl methylcellulose-anhydrous ethanol mixture with a volume ratio of 1:1 for surface coating (hydroxypropyl methylcellulose accounts for 2.5%), the surface coating time is 60s, and the coated product is placed in liquid nitrogen for 1min for secondary pre-cooling and forming.

[0122] (3) Transfer the pre-cooled and shaped pellets along with liquid nitrogen to a pre-cooled stainless steel tray. Then, place the stainless steel tray into a vacuum freeze dryer that has been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 2) to freeze-dry, obtaining silk fibroin freeze-dried pellets with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 2. In Table 2, the temperature change rate for each step refers to the rate of temperature change from the previous process to the current process. The processing time for each step is based on the time the current process temperature is maintained.

[0123] (4) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0124] (5) The repackaged products are capped by a capping machine and then labeled and packaged.

[0125] The overall score for freeze-dried pellets is 4.75. The scores for each item are shown in Table 6. The scoring criteria are detailed in Table 14.

[0126] Table 6: Evaluation Table for Freeze-Dried Balls in Example 4

[0127]

[0128] Example 5:

[0129] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, with a molecular weight of 160kDa to 200kDa), 1g of lactose, 1g of erythritol, 1g of pentylene glycol, and the rest are balanced with purified water to 100g. After balancing, mix evenly and set aside.

[0130] (2) The mixture was dispersed into a solution of 30 μL per drop by a continuous separator and dropped into liquid nitrogen for pre-cooling and forming into spheres. The pre-cooled spheres were then transferred to a hydroxypropyl methylcellulose-anhydrous ethanol mixture at a volume ratio of 1:1 for surface coating (hydroxypropyl methylcellulose accounted for 2.0%). The surface coating time was 50 s. The coated product was then placed in liquid nitrogen for 1.4 min for secondary pre-cooling and forming.

[0131] (3) Transfer the pre-cooled and shaped pellets along with liquid nitrogen to a pre-cooled stainless steel tray. Then, place the stainless steel tray into a vacuum freeze dryer that has been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 1) to freeze-dry, obtaining silk fibroin freeze-dried pellets with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 1. In Table 1, the temperature change rate for each step refers to the rate of temperature change from the previous process to the current process. The processing time for each step is based on the time the current process temperature is maintained.

[0132] (4) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0133] (5) The repackaged products are capped by a capping machine and then labeled and packaged.

[0134] The overall score for freeze-dried pellets is 4.25. The scores for each item are shown in Table 7. The scoring criteria are detailed in Table 14.

[0135] Table 7: Evaluation Table for Freeze-Dried Balls in Example 5

[0136]

[0137] Example 6:

[0138] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, with a molecular weight of 120kDa to 140kDa), 1.5g of caramel, 1g of xylitol, 1g of glycerol, and the rest are balanced with purified water to 100g. After balancing, mix evenly and set aside.

[0139] (2) The mixture was dispersed into a solution of 30 μL per drop by a continuous separator and dropped into liquid nitrogen for pre-cooling and forming into spheres. The pre-cooled spheres were then transferred to a hydroxypropyl methylcellulose-anhydrous ethanol mixture at a volume ratio of 1:1 for surface coating (hydroxypropyl methylcellulose accounted for 2.8%). The surface coating time was 48 s. The coated product was then placed in liquid nitrogen for 1 min for secondary pre-cooling and forming.

[0140] (3) Transfer the pre-cooled and shaped pellets along with liquid nitrogen to a pre-cooled stainless steel tray. Then, place the stainless steel tray into a vacuum freeze dryer that has been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 1) to freeze-dry, obtaining silk fibroin freeze-dried pellets with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 1. In Table 1, the temperature change rate for each step refers to the rate of temperature change from the previous process to the current process. The processing time for each step is based on the time the current process temperature is maintained.

[0141] (4) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0142] (5) The repackaged products are capped by a capping machine and then labeled and packaged.

[0143] The overall score for freeze-dried pellets is 4.25. The scores for each item are shown in Table 8. The scoring criteria are detailed in Table 14.

[0144] Table 8: Evaluation Table for Freeze-Dried Balls in Example 6

[0145]

[0146] Comparative Example 1:

[0147] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, and the molecular weight of silk fibroin is 80kDa~100kDa), and balance the rest with purified water to 100g. Mix well after balancing and set aside.

[0148] (2) The mixture is dispersed into 30 μL of solution per drop by a continuous separator and dropped into liquid nitrogen for pre-cooling and forming into spheres; the pre-cooled spheres are transferred to a hydroxypropyl methylcellulose-anhydrous ethanol mixture with a volume ratio of 1:1 for surface coating (hydroxypropyl methylcellulose accounts for 2.5%), the surface coating time is 40s, and the coated product is placed in liquid nitrogen for 1min for secondary pre-cooling and forming.

[0149] (3) Transfer the pre-cooled and shaped pellets along with liquid nitrogen to a pre-cooled stainless steel tray. Then, place the stainless steel tray into a vacuum freeze dryer that has been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 1) to freeze-dry, obtaining silk fibroin freeze-dried pellets with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 1. In Table 1, the temperature change rate for each step refers to the rate of temperature change from the previous process to the current process. The processing time for each step is based on the time the current process temperature is maintained.

[0150] (4) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0151] (5) The repackaged products are capped by a capping machine and then labeled and packaged.

[0152] There is no comprehensive score for freeze-dried balls, as shown in Table 9. For detailed scoring criteria, please refer to Table 14.

[0153] Table 9: Scoring Table for Freeze-Dried Balls of Comparative Example 1

[0154]

[0155] Comparative Example 2:

[0156] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, and the molecular weight of silk fibroin is 80kDa~100kDa), 2g of trehalose, 1g of mannitol, 1g of glycerol, and the rest are balanced with purified water to 100g. After balancing, mix evenly and set aside.

[0157] (2) The mixture is dispersed into 30 μL drops using a continuous separator and pre-cooled into spheres in liquid nitrogen. The pre-cooled spheres, along with the liquid nitrogen, are transferred to a pre-cooled stainless steel tray. The stainless steel tray is then placed in a vacuum freeze dryer that has been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 1) to freeze-dry, obtaining silk fibroin freeze-dried spheres with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 1. In Table 1, the temperature change rate for each step refers to the rate of temperature change from the previous step to the current step. The processing time for each step is based on the time the current step temperature is maintained.

[0158] (3) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0159] (4) The repackaged products are capped by a capping machine and then labeled and packaged.

[0160] The reasons why freeze-dried balls did not receive a comprehensive score are shown in Table 10, and the scoring criteria are detailed in Table 14.

[0161] Table 10: Scoring Table for Freeze-Dried Balls in Comparative Example 2

[0162]

[0163] Comparative Example 3:

[0164] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, and the molecular weight of silk fibroin is 80kDa~100kDa), 2g of trehalose, 1g of mannitol, and the rest are balanced with purified water to 100g. After balancing, mix evenly and set aside.

[0165] (2) The mixture was dispersed into 30 μL drops using a continuous separator and pre-cooled into spheres in liquid nitrogen. The pre-cooled spheres were then transferred to a hydroxypropyl methylcellulose-anhydrous ethanol mixture (2.5% hydroxypropyl methylcellulose) for surface coating (40 s). The coated spheres were then placed in liquid nitrogen for a second pre-cooling and shaping process for 1 min. The pre-cooled spheres, along with the liquid nitrogen, were transferred to a pre-cooled stainless steel tray. The tray was then placed in a vacuum freeze dryer that had been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 1) for freeze-drying to obtain lyophilized silk fibroin spheres with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 1. In Table 1, the temperature change rate for each step refers to the rate of temperature change from the previous step to the current step. The time for each step is based on the time the current step temperature is maintained.

[0166] (3) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0167] (4) The repackaged products are capped by a capping machine and then labeled and packaged.

[0168] There is no comprehensive score for freeze-dried balls, as shown in Table 11. For detailed scoring criteria, please refer to Table 14.

[0169] Table 11: Scoring Table for Freeze-Dried Balls in Comparative Example 3

[0170]

[0171]

[0172] Comparative Example 4:

[0173] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution contains 3.5g of silk fibroin per 100g of solution, and the molecular weight of silk fibroin is 80kDa~100kDa), 1g of glycerol, and balance the rest with purified water to 100g. Mix well after balancing and set aside.

[0174] (2) The mixture was dispersed into 30 μL drops using a continuous separator and pre-cooled into spheres in liquid nitrogen. The pre-cooled spheres were then transferred to a hydroxypropyl methylcellulose-anhydrous ethanol mixture (2.5% hydroxypropyl methylcellulose) for surface coating (40 s). The coated spheres were then placed in liquid nitrogen for a second pre-cooling and shaping process for 1 min. The pre-cooled spheres, along with the liquid nitrogen, were transferred to a pre-cooled stainless steel tray. The tray was then placed in a vacuum freeze dryer that had been pre-cooled (i.e., the "shelf pre-cooling" procedure in Table 1) for freeze-drying to obtain lyophilized silk fibroin spheres with a particle size of 3.2–3.4 mm. The detailed freeze-drying steps are listed in Table 1. In Table 1, the temperature change rate for each step refers to the rate of temperature change from the previous step to the current step. The time for each step is based on the time the current step temperature is maintained.

[0175] (3) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0176] (4) The repackaged products are capped by a capping machine and then labeled and packaged.

[0177] There is no comprehensive score for freeze-dried balls, as shown in Table 12. The scoring criteria are detailed in Table 14.

[0178] Table 12: Comparative Example 4 Freeze-dried Ball Scoring Table

[0179]

[0180] Comparative Example 5:

[0181] The antigen protein freeze-dried microspheres and their preparation method are described in CN114159555A. Since this patent involves certain freeze-drying protectants, they are not applicable to this scope of application; therefore, the application is limited to areas where protectants are available. Freeze-drying is performed according to the mannitol:trehalose (mass ratio) ratio of 1:1 specified in Example 1 of the patent.

[0182] (1) Preparation of freeze-dried mixture: Take 87.72g of 3.5% silk fibroin solution (3.5% silk fibroin solution means that each 100g solution contains 3.5g of silk fibroin), 1g of trehalose, 1g of mannitol, and the rest are balanced with purified water to 100g. After balancing, mix evenly for later use.

[0183] (2) The mixture is dispersed into 30 μL of solution per drop by a continuous separator and dropped into liquid nitrogen for pre-cooling and forming into spheres; the pre-cooled spheres are transferred to a hydroxypropyl methylcellulose-anhydrous ethanol mixture with a volume ratio of 1:1 for surface coating (hydroxypropyl methylcellulose accounts for 2.5%), the surface coating time is 40s, and the coated product is placed in liquid nitrogen for 1min for secondary pre-cooling and forming.

[0184] (3) The pre-cooled and shaped microspheres, along with liquid nitrogen, are transferred to a pre-cooled stainless steel tray. The stainless steel tray is then placed in a pre-cooled vacuum freeze dryer for freeze drying to obtain silk fibroin freeze-dried microspheres with a particle size of 3.2–3.4 mm. The freeze drying procedure is the freeze drying procedure in CN114159555A antigen protein freeze-dried microspheres and its preparation method, which is as follows: 1. Set the temperature of the shelf to -45°C and maintain this temperature for 4 hours; 2. Set the temperature of the shelf to -35°C, and maintain this temperature for 10 hours after evacuating the freeze dryer. 3. Set the shelf temperature to -30℃ and maintain this temperature for 10 hours; 4. Set the shelf temperature to -25℃ and maintain this temperature for 10 hours; 5. Set the shelf temperature to -20℃ and maintain this temperature for 1.5 hours; 6. Set the shelf temperature to -10℃ and maintain this temperature for 1.5 hours; 7. Set the shelf temperature to 0℃ and maintain this temperature for 4 hours; 8. Set the shelf temperature to 10℃ and maintain this temperature for 1.5 hours; 9. Set the shelf temperature to 25℃ and maintain this temperature for 6 hours; 10. The vacuum degree formed by evacuating the freeze dryer in step S412 is less than 20 Pa.

[0185] (4) The freeze-dried products are packaged in a glove box with a humidity of 10%;

[0186] (5) The repackaged products are capped by a capping machine and then labeled and packaged.

[0187] There is no comprehensive score for freeze-dried balls, as shown in Table 13. The scoring criteria are detailed in Table 14.

[0188] Table 13: Comparative Example 5 Freeze-dried Ball Scoring Table

[0189]

[0190] Compared to CN114159555A, the differences and advantages of this patent are as follows: 1. This patent is in the field of lyophilized formulations and is applicable to the medical aesthetics industry, allowing for safer use of protective agents; 2. This product contains less lyophilized protective agent, making it safer; 3. The accelerated aging conditions of this patent can be set to a temperature of 60℃ and a humidity of 60% RH, while the accelerated aging conditions of CN114159555A are 37℃, and higher temperatures will lead to inactivation. This patent has better high-temperature resistance and is more stable; 4. The diameter deviation range of the lyophilized pellets in this patent is ±1, while that of CN114159555A is ±2.5, indicating that the technology of this patent is more stable.

[0191] Table 14 Evaluation Table for Silk Fibroin Freeze-Dried Balls

[0192]

[0193] Note:

[0194] 1. The reconstitution time is the time required for 33 lyophilized silk fibroin beads to completely dissolve in 1 ml of purified water or 1 ml of physiological saline.

[0195] 2. Accelerated stability testing is conducted because silk fibroin may degrade over time, leading to pH changes. The greater the degree of degradation, the greater the pH change. The temperature and humidity used for accelerated stability testing were conducted according to the relevant requirements in YY / T0681.1-2018, where 10 days of accelerated aging is equivalent to 105 days of storage at room temperature.

[0196] Table 13 Summary of Humidity of Freeze-Dried Silk Fiber Balls After Storage

[0197]

[0198]

[0199] Note: The humidity of the freeze-dried bulbs upon exiting the chamber is the water content inside a freeze-dried bulb at 0 moments after exiting the chamber, measured by the Karl Fischer method.

[0200] Table 14: Summary of Reconstitution Time for Lyophilized Silk Fibroin Balls

[0201] Group Reconstitution time Example 1 11s Example 2 21s Example 3 22s Example 4 24s Example 5 25s Example 6 23s Comparative Example 1 Cannot be completely reconstituted Comparative Example 2 Cannot be completely reconstituted Comparative Example 3 Cannot be completely reconstituted Comparative Example 4 Cannot be completely reconstituted Comparative Example 5 Cannot be completely reconstituted

[0202] Table 15: pH difference before and after aging of silk fibroin freeze-dried pellets

[0203]

[0204]

[0205] In summary, the product obtained in Example 1 is the optimal product. Compared with Example 2, Example 1 demonstrates that a lower freeze-drying temperature and longer time result in better solubility of the protein microspheres. Compared with Examples 3, 4, 5, and 6, Example 1 shows that the protein microspheres prepared using trehalose, mannitol, and glycerol in combination have better appearance, solubility, and stability. Increasing the freeze-drying protection dose and reducing the protein molecular weight are beneficial for improving solubility.

Claims

1. A method for preparing controllable molecular weight silk fibroin freeze-dried spheres, characterized in that, Includes the following steps: S1: A mixture of lyophilization protectant, protein conformation stabilizer, and silk fibroin solution is prepared; the lyophilization protectant includes sugars and sugar alcohols, the protein conformation stabilizer is a polyol, and the silk fibroin has a narrow distribution and controllable molecular weight, with a molecular weight range of 10kDa to 80kDa, 80kDa to 100kDa, 100kDa to 120kDa, 120kDa to 160kDa, 160kDa to 200kDa, and >200kDa; S2: Pre-cool and shape the prepared mixture into a volume of 5.00 mm. 3 -40.00 mm 3 The small ball; S3: Place the pre-cooled and shaped small balls from step S2 into a mixture of a treatment agent and anhydrous ethanol for surface coating; the treatment agent is one or more of hydroxypropyl methylcellulose, sodium hyaluronate, and sodium carboxymethylcellulose. S4: Place the small balls after surface coating in step S3 into liquid nitrogen for secondary molding; S5: Place the pre-cooled and shaped pellets in a freeze dryer for freeze drying. The freeze drying process is as follows: S5.1: Freeze dryer in 49~ Maintain at 51℃ for 1.5~2 hours; then increase to... 28~ Maintain at 31℃ for 1~1.5 h; then reduce to 31℃. 49~ Maintain at 51℃ for 1~1.5 h; S5.2: Open the vacuum; rise to 43~ Maintain at 45℃ for 14-16 hours; then increase to... 38~ Maintain at 42℃ for 9-11 hours; gradually increase the temperature to... 24~ 26℃, increasing the temperature by 4-5℃ each time, maintaining this temperature for 1-1.5 hours before continuing to increase it; until... Maintain at 1~1℃ for 1.0~1.5 h; S5.3: Raise to 19~21℃ and hold for 5~5.5 h; raise to 29~31℃ and hold for 4~4.5 h; S6: The freeze-dried product is repackaged in a glove box; S7: Packaging of repackaged products with crimped caps and labels; The freeze-dried pellets have a particle size of 1~5 mm; In step S1, the mass ratio of silk fibroin: carbohydrates: sugar alcohols: polyols is 1:1:1:1~3:2:1:

1. The carbohydrates include compounds shown in Formula I and / or Formula II, the sugar alcohols have the structural formula shown in Formula III, and the polyols have the structural formula shown in Formula IV. Formula I: C 12 H 22 O 11 , Formula II: C 12 H 22 O 11 ·2H2O, Formula III: HOCH2(CHOH) n CH2OH, where n≥1 Formula IV: C n H 2n+2-x (OH) x , where n≥1, x≥2.

2. The preparation method according to claim 1, characterized in that, Formula 1 is trehalose, lactose, sucrose, maltose, and caramel; Formula II is trehalose dihydrate; Formula III is mannitol, sorbitol, xylitol, and erythritol; Formula IV is glycerol, ethylene glycol, and butylene glycol. The volume of the mixed liquid prepared in step S2 is 15~45 μL.

3. The preparation method according to claim 1, characterized in that, In step S2, the method for pre-freezing the mixture into spheres involves dropping the mixture into liquid nitrogen, with each freeze-dried sphere having a volume of 5.00 mm². 3 -40.00 mm 3 Its freeze-dried particle size is 1~5mm.

4. The preparation method according to claim 1, characterized in that, In step S3, the pre-cooled and shaped spheres are transferred to a mixture of treatment agent and anhydrous ethanol for surface coating. The surface coating time is ≤60s.

5. The preparation method according to claim 1, characterized in that, In step S3, a certain volume of liquid nitrogen is added to the pre-cooled freeze-dried spheres during the transfer process, wherein the volume ratio of the pre-cooled freeze-dried spheres to the liquid nitrogen is 1:1 to 2:

1.

6. The preparation method according to claim 1, characterized in that, The treatment agent used in step S3, anhydrous ethanol mixture, has a treatment agent ratio of 1%-5%.

7. The preparation method according to claim 1, characterized in that, The time for the product undergoing secondary molding in step S4 is ≤3 min.

8. The preparation method according to claim 1, characterized in that, In step S5.1, the elevation to 28~ 31℃, dropped to 49~ The heating or cooling rate at 51℃ is higher than 0.5℃ / min.

9. The preparation method according to claim 1, characterized in that, In step S5.2, the elevation to 43~ 45℃, rise to 38~ 42℃, stepped temperature increase to 24~ 26℃ and rise to The heating or cooling rate of 1~1℃ is less than 0.5℃ / min.

10. The preparation method according to claim 1, characterized in that, In step S5.3, the heating or cooling rates to 19~21℃ and 29~31℃ are both less than 0.5℃ / min.

11. The preparation method according to claim 1, characterized in that, The humidity of the glove box in step S6 should be ≤15%; the finished product after crimping in step S5 should be checked for sealing using the color water method.

12. A type of lyophilized silk fibroin sphere, characterized in that: It is prepared by the preparation method described in any one of claims 1-7.

13. The silk fibroin freeze-dried pellets according to claim 12, characterized in that: The freeze-dried pellets have a volume of 15~45μL and a particle size of 1~5mm.

14. The freeze-dried sphere according to claim 13, wherein the surface roundness of the freeze-dried sphere is ≥95%.

15. The freeze-dried sphere according to claim 13, wherein the freeze-dried sphere is loosely porous inside, with an internal pore size of less than 10 mm and a porosity of ≥70%.

16. The application of the silk fibroin freeze-dried balls according to any one of claims 12-15, characterized in that, The applications include: water-light products, dressing products, and filler products.

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