Active collagen lyophilized formulations, methods of making and using the same

Abstract

The present application relates to the technical field of collagen, in particular to a kind of active collagen lyophilized preparation and its preparation method and application.The preparation method includes the following steps: S1, the aqueous solution of freeze-drying protective agent is prepared;S2, the initial emulsion of active collagen is diluted with physiological saline, to obtain the first solution, then mixed with the aqueous solution obtained in step S1, to obtain the second solution;S3, the second solution obtained in step S2 is added to the aqueous solution of step S1, and the third solution is prepared after mixing and emulsifying, to obtain mixed emulsion;S4, the mixed emulsion in step S3 is filled and freeze-dried, wherein the freeze-drying protective agent includes glycine, and the concentration of active collagen in the third solution is 5-15 mg / mL.The lyophilized preparation prepared by the preparation method has excellent reconstitution effect, and is convenient for transportation and storage.

Description

Technical Field

[0001] This invention relates to the field of collagen technology, and in particular to an active collagen freeze-dried formulation, its preparation method, and its application. Background Technology

[0002] Collagen is a natural structural protein that is widely found in animal connective tissues. Due to its good biocompatibility, biodegradability, and unique biological activities such as promoting cell adhesion and proliferation, it is widely used in biomedical materials, cosmetics, food and health products and other fields.

[0003] However, the inherent properties of collagen also present significant challenges to its long-term preservation and transportation. Firstly, collagen is a biological macromolecule whose function is highly dependent on its complex three-dimensional structure. At room temperature or high temperatures, it is extremely sensitive to factors such as temperature, humidity, oxygen, and microorganisms, readily undergoing denaturation, hydrolysis, and degradation, leading to the loss of its crucial biological activity and product failure. Therefore, traditional liquid or gel-based collagen products typically require strict low-temperature refrigeration conditions, significantly increasing storage and logistics costs.

[0004] In view of the above, CN115227597A discloses an active collagen lyophilized formulation, its preparation method, and its application. The method of this invention requires three lyophilization protectants to prepare lyophilized collagen: the first protectant is at least one of glycine and mannitol, the second protectant is ethyl acetate, and the third protectant is oxidized cellobiose. However, excessively high concentrations of the solubilizing agents (such as glycine and mannitol) can reduce the effective concentration of collagen, resulting in an overly dilute solution after reconstitution (e.g., <5 mg / mL), affecting injection filling or bioactivity. Furthermore, the amount of some solubilizing agents needs to be strictly controlled (e.g., 1 mg of collagen requires 2-6 mg of glycine as a solubilizing agent). Excessive solubilizing agents may damage the hydration layer of collagen molecules, thus delaying reconstitution.

[0005] In addition, CN112842931A discloses a freeze-dried animal collagen ball, a cosmetic kit, and a preparation process. This invention uses a single-stage heating process (5℃ / h to 25℃) during the freeze-drying stage, resulting in the freeze-dried collagen balls prepared by this method only achieving pore formation and failing to protect the freeze-dried activity.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an active collagen freeze-dried preparation, its preparation method, and its application, thereby solving at least one of the problems raised in the background art.

[0008] In a first aspect, the present invention provides a method for preparing an active collagen freeze-dried formulation, comprising the following steps:

[0009] S1. Prepare an aqueous solution of the lyophilization protectant;

[0010] S2. The initial emulsion of active collagen is diluted with physiological saline to obtain a first solution, which is then mixed with the aqueous solution of the lyophilization protectant obtained in step S1 to prepare a second solution.

[0011] S3. Add the second solution obtained in step S2 to the aqueous solution of the freeze-drying protectant obtained in step S1, mix to prepare the third solution, and then emulsify to obtain a mixed emulsion;

[0012] S4. The mixed emulsion from step S3 is filled and then freeze-dried.

[0013] The freeze-drying protectant includes glycine, and the concentration of the active collagen in the third solution is 5-15 mg / mL.

[0014] According to some embodiments, the mass concentration of glycine in the aqueous solution is 0.1% to 0.5%, preferably 0.2% to 0.3%; preferably, the aqueous solution is further subjected to filtration treatment.

[0015] According to some embodiments, the concentration of the active collagen in the third solution is 6-10 mg / mL.

[0016] According to some embodiments, the concentration of active collagen in the first solution in step S2 is 30~50 mg / mL, preferably 35~45 mg / mL; preferably, the volume ratio of the aqueous solution of the lyophilization protectant used in step S2 to the aqueous solution of the lyophilization protectant used in step S3 is 1:0.5~2.

[0017] According to some embodiments, the emulsification in step S3 is performed using a high-pressure homogenizer; preferably, the pressure during emulsification is set to 200~500 bar.

[0018] According to some embodiments, the amount of filling in step S4 is 1 / 3 to 1 / 2 of the volume of the filling container used.

[0019] According to some embodiments, the freeze-drying in step S4 includes pre-freezing and sublimation drying;

[0020] Preferably, the pre-freezing includes two temperature gradients, and the sublimation drying includes five temperature gradients; the pre-freezing includes: first maintaining at 0~2℃ for 0.3~1 h, and then maintaining at -50~-40℃ for 3~7 h.

[0021] According to some embodiments, the sublimation drying includes: first drying at a first temperature for 10-15 min, then drying at a second temperature for 2-5 min, then drying at a third temperature for 10-20 min, then drying at a fourth temperature for 1-4 min, and finally drying at a fifth temperature for 5-8 min, wherein the second temperature is 3-7°C higher than the first temperature, the third temperature is 3-7°C higher than the second temperature, the fourth temperature is 10-20°C higher than the third temperature, and the fifth temperature is 20-40°C higher than the fourth temperature; preferably, the sublimation drying includes: first heating at 15-25°C / min to -30°C to -20°C, and drying at that temperature for 10-15 min; then heating at 5-15°C / min to -30°C to -10°C, and drying at that temperature for 2-5 min; then heating at 5-15°C / min to -20°C to -10°C, and drying at that temperature for 10-20 min; then heating at 25-35°C / min to -5°C to 5°C, and drying at that temperature for 1-4 min. Then, increase the temperature to 20℃~35℃ at a rate of 25~35℃ / min and dry at this temperature for 5~8 min.

[0022] In a second aspect, the present invention provides an active collagen freeze-dried formulation prepared by the above-described preparation method.

[0023] A third aspect of the present invention provides an application of the above-described active collagen freeze-dried formulation in medical aesthetics.

[0024] The preparation method of the present invention uses a suitable protective agent and a suitable concentration, combined with a specific concentration of active collagen before filling, and follows a specific preparation process and freeze-drying process parameters, so that the freeze-dried preparation has excellent reconstitution effect, and the obtained freeze-dried preparation is easy to transport and store.

[0025] Specifically, this invention uses high-pressure homogenization to break collagen particles down to the submicron level (e.g., <1 μm), enabling the homogenized emulsion to form a thermodynamically stable system. This eliminates the agglomeration phenomenon caused by traditional stirring, ensuring no flocculation during reconstitution of the freeze-dried formulation. By using a glycine protectant to uniformly encapsulate collagen molecules, ice crystal damage is inhibited during freeze-drying, resulting in a triple helix structure retention rate of over 95%. By setting an appropriate concentration of active collagen before filling, the freeze-dried formulation exhibits excellent reconstitution performance after reconstitution. This invention represents a leapfrog innovation in molecular activity protection and freeze-drying technology, providing a new approach for the production of freeze-dried active collagen formulations. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a flowchart illustrating the preparation of an active collagen freeze-dried formulation in one embodiment of the present invention.

[0028] Figure 2 This is a flowchart illustrating the preparation of an active collagen freeze-dried formulation in another embodiment of the present invention;

[0029] Figure 3 This is a diagram of the third solution in the preparation process of an active collagen freeze-dried formulation according to one embodiment of the present invention, wherein the bottom of the bottle is gelatin;

[0030] Figure 4 This is a diagram of the mixed emulsion after emulsification during the preparation of an active collagen freeze-dried formulation in one embodiment of the present invention, wherein the bottom of the bottle is gelatin;

[0031] Figure 5 This is a circular dichroism chromatogram of an active collagen lyophilized preparation after reconstitution according to an embodiment of the present invention;

[0032] Figure 6 This is a comparison chart of triple helix activity test standards for freeze-dried active collagen formulations.

[0033] Figure 7 This is a graph showing the triple helix activity test results of an active collagen freeze-dried formulation prepared according to an embodiment of the present invention. Detailed Implementation

[0034] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form includes the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0038] One aspect of the present invention provides a method for preparing a freeze-dried formulation of active collagen, such as... Figure 1 As shown, the preparation method includes the following steps:

[0039] S1. Prepare an aqueous solution of the lyophilization protectant;

[0040] S2. The initial emulsion of active collagen is diluted with physiological saline to obtain a first solution, which is then mixed with the aqueous solution of the lyophilization protectant obtained in step S1 to prepare a second solution.

[0041] S3. Add the second solution from step S2 to the aqueous solution of the freeze-drying protectant obtained in step S1, mix to prepare a third solution, and then emulsify the third solution to obtain a mixed emulsion.

[0042] S4. The mixed emulsion from step S3 is filled and then freeze-dried.

[0043] The lyophilization protectant includes glycine, and the concentration of active collagen in the third solution is 5–15 mg / mL, for example, 7 mg / mL, 8.5 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, etc. Glycine, as a lyophilization protectant, has good biocompatibility. The above-mentioned concentrations of active collagen show good results upon reconstitution, maintaining the collagen's activity and allowing it to retain its triple helix structure. Figure 2 and Figure 3 As shown, the gelatin content in the solution before and after emulsification (homogenization) is basically the same, indicating that the sample did not denature after homogenization. This makes the concentration of active collagen in the mixed emulsion approximately the same as the concentration of active collagen in the third solution. Therefore, the concentration of active collagen in the mixed emulsion before filling can be controlled by controlling the concentration of active collagen in the third solution.

[0044] As can be seen from the above method, mixing the first solution containing active collagen with the glycine aqueous solution in two stages can prevent over-addition and ensure more uniform coating of collagen by glycine. By using glycine as a protectant to uniformly coat collagen molecules, ice crystal damage is inhibited during freeze-drying, and the triple helix structure retention rate is >95%. This represents a significant innovation in molecular activity protection and freeze-drying technology, providing a new approach for the production of freeze-dried active collagen formulations.

[0045] In some embodiments, the concentration of active collagen in the third solution is preferably 6-10 mg / mL. This range allows for even better reconstitution.

[0046] To ensure collagen dissolution, the optimal concentration of glycine is used for dissolution. Too high a glycine concentration affects osmotic pressure, while too low a concentration renders collagen insoluble and lacks a triple helix structure. Therefore, in some embodiments, the mass concentration of glycine in the aqueous solution can be 0.1% to 0.5%, for example, 0.15%, 0.22%, 0.28%, 0.35%, 0.4%, 0.45%, etc., preferably 0.2% to 0.3%.

[0047] In some embodiments, the aqueous solution of glycine is further subjected to filtration to improve the stability of the prepared formulation.

[0048] The concentration of active collagen relative to the concentration of glycine should not be too low; otherwise, as mentioned above, the glycine concentration will be too high, affecting the osmotic pressure. The concentration of active collagen in the initial emulsion will also not reach a very high concentration. Moreover, in order to facilitate the calculation of the fill volume, in some embodiments, the concentration of active collagen in the first solution in step S2 can be 30~50 mg / mL, for example, 33 mg / mL, 37 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, etc., preferably 35~45 mg / mL.

[0049] In some embodiments, the volume ratio of the aqueous solution of the lyophilizing protectant used in step S2 to the aqueous solution of the lyophilizing protectant used in step S3 is 1:0.5~2, for example, 1:0.7, 1:0.9, 1:1, 1:1.4, 1:1.8, etc. This specific ratio of the two-step mixing allows the glycine protectant to more uniformly encapsulate collagen molecules, thereby inhibiting ice crystal damage during lyophilization and ensuring a triple helix structure retention rate >95%.

[0050] In some embodiments, emulsification in step S3 is performed using a high-pressure homogenizer. Using a high-pressure homogenizer for emulsification allows for better dispersion of collagen, resulting in improved subsequent freeze-drying. Furthermore, high-pressure homogenization breaks down collagen particles to submicron levels (<1 μm), enabling the homogenized emulsion to form a thermodynamically stable system. This eliminates agglomeration caused by traditional stirring, ensuring no flocculation during reconstitution of the freeze-dried formulation.

[0051] In some embodiments, the emulsification pressure is set to 200-500 bar, such as 220 bar, 290 bar, 350 bar, 400 bar, 450 bar, etc. This emulsification pressure range ensures effective emulsification while preventing machine overheating from affecting collagen activity. The emulsification (homogenization) time can be 15 minutes or more, for example, 20-40 minutes, and an appropriate value can be selected based on the emulsification pressure.

[0052] In some embodiments, the amount filled in step S4 is 1 / 3 to 1 / 2 of the volume of the filling container used, for example, 2 / 5, 7 / 15, etc. Filling amounts within the above range result in better morphology of the prepared lyophilized formulation and ensure thorough lyophilization, making it easier to obtain lyophilized products with good performance.

[0053] In some embodiments, freeze drying in step S4 includes pre-freezing and sublimation drying.

[0054] In some embodiments, pre-freezing includes two temperature gradients, and sublimation drying includes five temperature gradients. The two-step freeze-drying process allows sufficient time for ice crystals to grow, forming a large, interconnected ice crystal network; and leaves behind a porous, low-resistance framework after drying, which facilitates rapid water vapor escape and shortens sublimation time.

[0055] In some embodiments, pre-freezing includes: holding at 0~2°C for 0.3~1 h, for example, holding at 0°C for 0.4 h, holding at 1°C for 0.5 h, holding at 1.5°C for 0.9 h, etc., and then holding at -50~-40°C for 3~7 h, for example, holding at -48°C for 3.5 h, holding at -45°C for 5 h, holding at -42°C for 6.5 h, etc.

[0056] In some embodiments, drying is first performed at a first temperature for 10-15 minutes, followed by drying at a second temperature for 2-5 minutes, then drying at a third temperature for 10-20 minutes, then drying at a fourth temperature for 1-4 minutes, and finally drying at a fifth temperature for 5-8 minutes. The second temperature is 3-7°C higher than the first temperature, the third temperature is 3-7°C higher than the second temperature, the fourth temperature is 10-20°C higher than the third temperature, and the fifth temperature is 20-40°C higher than the fourth temperature. For example, the first temperature can be -30°C to -20°C, the second temperature can be -25°C to -15°C, the third temperature can be -18°C to -12°C, the fourth temperature can be -5°C to 5°C, and the fifth temperature can be 25°C to 35°C, etc.

[0057] Sublimation temperature is divided into primary drying and secondary drying. Excessively high temperatures may degrade and denature heat-sensitive active ingredients (such as proteins), hindering a smooth transition to the secondary drying (desorption drying) stage, which requires higher temperatures but may have different vacuum levels. Rapid heating can also cause the product to collapse. This invention, by setting appropriate temperature gradients and heating rates, achieves optimal shape retention in the freeze-dried formulation, resulting in a clear and transparent solution after reconstitution, and the highest collagen activity retention rate (all >95%).

[0058] In some preferred embodiments, sublimation drying includes: first, heating to -30°C to -20°C at a rate of 15-25°C / min and drying at that temperature for 10-15 min; then heating to -30°C to -10°C at a rate of 5-15°C / min and drying at that temperature for 2-5 min; then heating to -20°C to -10°C at a rate of 5-15°C / min and drying at that temperature for 10-20 min; then heating to -5°C to 5°C at a rate of 25-35°C / min and drying at that temperature for 1-4 min; and finally heating to 20°C to 35°C at a rate of 25-35°C / min and drying at that temperature for 5-8 min.

[0059] In the above embodiment, the starting temperature of the temperature gradient overlaps with the ending temperature of the previous gradient.

[0060] like Figure 2 As shown, the preparations obtained after freeze-drying can then undergo processes such as capping, light inspection, packaging, finished product testing, and warehousing.

[0061] A second aspect of the present invention provides an active collagen freeze-dried formulation, prepared according to the above-described preparation method.

[0062] A third aspect of the present invention provides the application of the above-mentioned lyophilized active collagen preparation in medical aesthetics.

[0063] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0064] Example 1: Preparation of an active collagen freeze-dried formulation

[0065] 1. Preparation of an aqueous solution of lyophilization protectant

[0066] Weigh 12.5 g of glycine, dissolve it in an appropriate amount of water for injection, stir until completely dissolved, and bring the volume to 5 L to obtain a 0.25% glycine aqueous solution. Then, filter the solution using a capsule filter and transfer it to a first emulsification bag.

[0067] 2. Prepare the initial emulsion and mixed solution of active collagen.

[0068] The initial emulsion containing active collagen was diluted with physiological saline to obtain a first solution. The first solution was then mixed with the aqueous solution obtained in step S1 to prepare a second solution, which was transferred to a second emulsification bag. The concentration of active collagen in the first solution was 40 mg / mL.

[0069] Proteins can be obtained according to methods in the prior art, such as the method described in CN115466322A. Specifically, they can be obtained according to... Figure 2 The method described involves, for example, soaking fresh cowhide after removing hair and grease, then homogenizing it with digestive enzymes, followed by adsorption filtration using an adsorption filter aid, and collecting the filtrate. The collected filtrate is then subjected to a series of processes including concentration and liquid replacement, ion exchange chromatography, concentration and mixing, filtration for sterilization, and precipitation centrifugation to obtain the protein product. The sterilized solution can then be tested for its properties using solution analysis.

[0070] 3. Mixing and emulsification

[0071] The glycine solution in the first emulsification bag was connected to the second emulsification bag (containing 48,000 mg of active collagen, a value that remained constant throughout the preparation process) via the outlet. A third solution was obtained by transferring the second solution from the second emulsification bag to the glycine solution in the first emulsification bag using a peristaltic pump. The first emulsification bag was then connected to a high-pressure homogenizer, and the cooling water system was turned on. After the material flowed out steadily, the pressure was gradually adjusted to the process set value (300-400 bar) for emulsification (homogenization) for 30 minutes to obtain a mixed emulsion. Measurements showed that the concentration of active collagen in both the third solution and the mixed emulsion was approximately 8 mg / mL, indicating that the homogenization treatment did not denature the collagen.

[0072] 4. Filling and freeze-drying

[0073] Install the aseptic components of the filling machine; connect the dispensing pipe to the emulsion bag, check the parameters, start the equipment, and set the filling volume to 4 mL.

[0074] Place the sample tray into the freeze dryer, set the freeze-drying process parameters according to Table 1 below, and start the vacuum device and freeze dryer. Monitor the temperature and vacuum level (10-20 Pa) of the drying chamber to ensure parameter stability. After the sample is dried, turn off the vacuum system and remove the sample.

[0075] Table 1

[0076]

[0077] Example 2: Comparison of the effects of glycine concentration

[0078] This example aims to illustrate the importance of glycine concentration.

[0079] Following the same method as in Example 1, aqueous solutions of the freeze-drying protectant with glycine concentrations of 0.15%, 0.25% (Example 1), and 0.40% were prepared, and lyophilized formulations of active collagen were prepared. The results showed that when the glycine concentration was in the range of 0.2% to 0.3%, the lyophilized cake exhibited the best shape retention, the solution was clear and transparent after reconstitution, and the collagen activity retention rate was the highest (all >95%). When the concentration decreased to 0.15%, the lyophilized cake showed slight shrinkage; when the concentration increased to 0.40%, the lyophilized cake structure became too rigid, and the reconstitution time was slightly prolonged. This indicates that the preferred range of the present invention has unexpected technical effects.

[0080] Example 3: Preparation of different concentrations of active collagen

[0081] This example illustrates the adjustability of the final concentration of active collagen.

[0082] Referring to the method of Example 1, by adjusting the amount of active collagen added in step 2, third solutions with final concentrations of approximately 6 mg / mL, 8 mg / mL (Example 1), and 10 mg / mL were prepared respectively (the concentration of the mixed emulsion was consistent), and then filled and lyophilized.

[0083] The results showed that the freeze-drying process proceeded smoothly within this concentration range, and the resulting products all met quality requirements. At a concentration of 6 mg / mL, the freeze-dried cake was more porous; at a concentration of 10 mg / mL, the freeze-dried cake had higher mechanical strength. Users can choose the appropriate concentration based on their specific application needs. Concentrations that are too high or too low will affect the reconstitution effect.

[0084] Determination of triple helix structure

[0085] The reconstituted sample of the lyophilized formulation from Example 1 (diluted to 8 mg / mL with physiological saline) was tested using circular dichroism spectroscopy, and the results are as follows: Figure 5As shown in the figure, the sample has a negative peak at around 195 nm and a positive peak at around 221 nm, indicating that the freeze-dried formulation prepared by the method of the present invention retains the triple helix structure of collagen.

[0086] Determination of triple helix activity

[0087] Triple helix activity was determined using a commercially available rapid identification kit for active collagen (colorimetric method). Figure 6 The diagram shows the results for negative and positive tests. A negative result is indicated by the absence of red precipitate (-) or a non-red flocculent precipitate (-), which means the sample does not contain active collagen or the concentration of active collagen is too low (below 0.1 mg / mL). A positive result is indicated by the presence of red precipitate (+) as shown in the diagram, which means the sample contains active collagen (with a complete triple helix structure).

[0088] The lyophilized formulation prepared in the examples was reconstituted (diluted to 8 mg / mL with physiological saline) and subjected to a triple helix activity test. The results are as follows: Figure 7 As shown in the figure, the presence of a red precipitate indicates that the lyophilized formulation prepared in this example is active.

[0089] Comparative Example 1: Without glycine

[0090] Except for using an equal amount of water for injection to completely replace the glycine aqueous solution in step 1, the remaining steps are exactly the same as in Example 1.

[0091] The results showed that the sample without glycine collapsed severely after freeze-drying, the freeze-dried cake shrank and stuck to the walls, the solution was turbid after reconstitution, and a small amount of insoluble precipitate was present. Analysis revealed that the triple helix structure of collagen was severely damaged, and the activity retention rate was less than 70%. This comparative example fully demonstrates the indispensable role of glycine as a freeze-drying protectant in this invention.

[0092] Experimental Example: Verification of Product Application Effect

[0093] The lyophilized formulation prepared in Example 1 was reconstituted with 2 mL of physiological saline, and then subjected to cell compatibility testing (CCK-8 assay) and animal skin irritation testing. The results showed that the formulation significantly promoted the proliferation of human fibroblasts and was non-irritating and non-sensitizing to rabbit skin. In a clinical observation of its application in facial hyaluronic acid injection, the subjects showed significantly better improvement in skin moisture, elasticity, and wrinkles compared to the control group, demonstrating the promising application prospects of this product in the medical aesthetics field.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an active collagen freeze-dried formulation, characterized in that, Includes the following steps: S1. Prepare an aqueous solution of a lyophilization protectant, wherein the lyophilization protectant is composed of glycine, and the mass concentration of glycine in the aqueous solution is 0.1~0.5%; S2. Dilute the initial emulsion of active collagen with physiological saline to obtain a first solution with an active collagen concentration of 30~50mg / mL, and then mix it with the aqueous solution of the lyophilization protectant obtained in step S1 to prepare a second solution. S3. The second solution obtained in step S2 is added to the aqueous solution of the lyophilization protectant obtained in step S1, and the mixture is mixed to obtain a third solution. The mixture is then emulsified using a high-pressure homogenizer at a pressure of 200-500 bar to obtain a mixed emulsion. The concentration of the active collagen in the third solution is 5-15 mg / mL. The volume ratio of the aqueous solution of the lyophilization protectant used in step S2 to the aqueous solution of the lyophilization protectant used in step S3 is 1:0.5-2. S4. The mixed emulsion from step S3 is filled and then freeze-dried.

2. The preparation method according to claim 1, characterized in that, In the third solution, the concentration of the active collagen is 6-10 mg / mL.

3. The preparation method according to claim 1 or 2, characterized in that, In step S4, the amount of filling is 1 / 3 to 1 / 2 of the volume of the filling container used.

4. The preparation method according to any one of claims 1-3, characterized in that, In step S4, the freeze-drying includes pre-freezing and sublimation drying; The pre-freezing includes two temperature gradients, and the sublimation drying includes five temperature gradients; The pre-freezing process includes: first maintaining the temperature at 0~2℃ for 0.3~1 h, and then maintaining the temperature at -50~-40℃ for 3~7 h.

5. The preparation method according to claim 4, characterized in that, The sublimation drying process includes: first drying at a first temperature for 10-15 min, then drying at a second temperature for 2-5 min, then drying at a third temperature for 10-20 min, then drying at a fourth temperature for 1-4 min, and then drying at a fifth temperature for 5-8 min. The second temperature is 3-7°C higher than the first temperature, the third temperature is 3-7°C higher than the second temperature, the fourth temperature is 10-20°C higher than the third temperature, the fifth temperature is 20-40°C higher than the fourth temperature, and the first temperature is -30°C to -20°C.

6. A freeze-dried formulation of active collagen, characterized in that, Prepared according to the preparation method of any one of claims 1-5.

Images