Collagen freeze-drying preparation and preparation method thereof

Abstract

The invention discloses a collagen freeze-drying preparation and a preparation method thereof, and relates to the technical field of biological medicines. The preparation is composed of 0.01%-10% of collagen, 0.1%-10% of a freeze-drying protective additive 1, 0.1%-5% of a freeze-drying protective additive 2, a buffer salt, an osmotic pressure regulator, a diluent and a pH buffer solution, and solves the problems that in the prior art, a collagen triple helix structure is unstable and is easy to inactivate.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a collagen freeze-dried composition and its preparation method. Background Technology

[0002] In the freeze-drying protection of collagen, the traditional single freeze-drying protectants (such as mannitol and trehalose) have a significant insufficient protective effect on its structure. The retention rate of the triple helix structure of collagen after freeze-drying is less than 65%. The molecular structure of these protectants has a weak interaction with collagen and is difficult to effectively replace the hydrogen bond network formed between water molecules and the collagen triple helix. As a result, the original hydrogen bonds of collagen break during dehydration, thereby destroying the stable conformation of the triple helix.

[0003] Chinese patent CN116869849A, entitled "An Injectable Composite Liquid and Its Preparation Method and Use", discloses an injectable composite liquid comprising RGD peptide-modified sodium hyaluronate (HA-RGD), collagen and amino acids, as well as polydeoxyribonucleic acid (PDRN) and water-soluble vitamins. It has good moisturizing effect, but the preparation process of the composite liquid is complicated and costly, and the cosmetic effect still needs to be further improved.

[0004] Chinese patent CN118873432A, "A PDRN-Non-Crosslinked Collagen Composition and Its Preparation Method", discloses a freeze-dried composition comprising PDRN collagen peptides, aspartic acid, vitamin C and deionized water. However, the freeze-dried composition is treated with moist heat sterilization. High-temperature sterilization cannot guarantee the triple helix structure of collagen and the activity of PDRN, nor can it guarantee the stability of the collagen and PDRN complex solution after reconstitution.

[0005] To address the above problems, this invention provides a collagen freeze-dried formulation and its preparation method. Summary of the Invention

[0006] This application provides a collagen freeze-dried formulation and its preparation method, which solves the problems of unstable triple helix structure and easy inactivation of collagen in the prior art.

[0007] This application provides a collagen freeze-dried formulation, characterized in that it comprises the following components: 0.01% to 10% (w / w) collagen in the total freeze-dried formulation, 0.1% to 10% (w / w) freeze-drying protectant 1 in the total freeze-dried formulation, 0.1% to 5% (w / w) freeze-drying protectant 2 in the total freeze-dried formulation, buffer salts, osmotic pressure regulators, diluents, and pH buffer solutions.

[0008] As an optimization, the collagen is selected from one or more of natural collagen, the sources of which include one or more of bovine Achilles tendon (beef tendon), pig skin, chicken feet, fish skin, fish bones, chicken breast cartilage, and forest frog; natural collagen is classified according to function into one or more of type I, type II, and type III.

[0009] As an optimization, the freeze-drying protectant 1 is selected from one or more of mannitol, lactose, trehalose, sucrose, maltose, dextrin, stachyose, gelatin, pectin, sugar alcohol, sorbitol, proline, histidine, lysine, arginine, polyethylene glycol, or polyvinylpyrrolidine, preferably one or more of mannitol, lactose, trehalose, and sucrose.

[0010] As an optimization, the freeze-drying protectant 2 is selected from one or more of hydroxyproline, proline, histidine, lysine, and arginine.

[0011] As an optimization, the buffer salt is selected from one or more of disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution, dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer solution, disodium hydrogen phosphate / citric acid buffer solution, sodium bicarbonate buffer solution, or sodium bicarbonate / sodium carbonate buffer solution, preferably one or more of disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution or dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer solution.

[0012] As an optimization, the diluent includes, but is not limited to, one or more of water for injection, sodium chloride injection, glucose injection, lactated Ringer's solution, sucrose aqueous solution, xylitol aqueous solution, and pH buffer.

[0013] As an optimization, the sodium chloride injection solution includes, but is not limited to, 0.9% sodium chloride injection solution.

[0014] As an optimization, the glucose injection solution includes, but is not limited to, 5% glucose injection solution.

[0015] As an optimization, the pH buffer is a buffer with pH = 5.0 to 7.4, including but not limited to one or more of phosphate buffer, glycine buffer, histidine buffer, citrate buffer, and acetate buffer.

[0016] A method for preparing a lyophilized collagen formulation includes the following steps:

[0017] S1: Weigh each component according to its mass fraction;

[0018] S2. Add freeze-drying protectant 1 and freeze-drying protectant 2 to deionized water, stir until dissolved, at a stirring speed of 200-600 rpm for 20-30 min.

[0019] S3. Continue to add buffer salt and osmotic pressure regulator and stir until dissolved. The stirring speed is 200-600 rpm and the time is 20-30 min.

[0020] S4. Continue adding collagen and stirring until dissolved. Stir at 200-600 rpm for 20-30 minutes.

[0021] S5. The solution obtained in S4 is filled into vials, pre-frozen at -30℃ to -60℃ for 4 to 8 hours; then freeze-dried under vacuum at -10℃ to 30℃ for 8 to 24 hours; after being removed from the freezer and capped, it is sterilized by irradiation to obtain the collagen freeze-dried composition.

[0022] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0023] 1. In this invention, a certain amount of trehalose and hydroxyproline composition is added as a freeze-drying protectant, which can effectively stabilize the triple helix structure and activity of collagen. Trehalose can form a glassy protective film during freeze-drying, reducing ice crystal damage and maintaining the molecular spatial conformation during freeze-drying. After reconstitution, hydroxyproline strengthens the hydrogen bonds between collagen peptide chains through hydroxyl groups, directly stabilizing the triple helix domain (especially the Y site in the Gly-XY sequence) and activity, effectively solving the defects of unstable triple helix structure and easy inactivation of collagen in the prior art.

[0024] 2. The collagen freeze-dried composition of the present invention utilizes trehalose and hydroxyproline as freeze-drying protectants to synergistically stabilize the structure during the freeze-drying process and after reconstitution. After reconstitution, the collagen freeze-dried composition shows a 221nm characteristic peak retention rate of >90% in circular dichroism (CD) chromatography, indicating that the triple helix structure is intact, thus solving the problem of the stability of the entire collagen triple helix chain.

[0025] 3. The collagen freeze-dried composition of the present invention can significantly increase the collagen content in mice, has good safety after injection, no obvious inflammatory reaction, and is effective in improving skin condition, with a lasting cosmetic effect. Attached Figure Description

[0026] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0027] Figure 1 These are the two-color CD spectra of the lyophilized tapioca pearls of collagen compositions 1003 and 1012 of the present invention. Detailed Implementation

[0028] To better understand the above technical solution, the following detailed explanation will be provided through specific implementation methods.

[0029] Example 1: Investigation of triple helix optical rotation detection of collagen solution

[0030] Optical rotation determination: Different types of collagen stock solutions were diluted to 5 mg / mL, vortexed for 10 s, and allowed to stand at 4℃ for 2 h to fully hydrate. After centrifugation (10000 rpm, 10 min), the supernatant was collected for optical rotation detection. The heating rate was 1℃ / min, and the temperature range was 25~45℃.

[0031] Table 1 Results of Optical Rotation Detection

[0032]

[0033]

[0034] The results showed that porcine collagen and bovine collagen had similar optical rotation results, but the Tm was slightly lower, which may be related to differences in amino acid sequences between species (such as the content of hydroxyproline); the Tm of recombinant collagen was significantly lower than that of natural collagen, which may be related to insufficient post-translational modification.

[0035] Example 2: Study on freeze-drying of collagen

[0036] Weigh the prescribed amount of collagen according to Table 2, stir and dissolve until clear, dispense 2ml / bottle (vial), and freeze-dry according to Table 3.

[0037] Table 2 Freeze-dried Collagen

[0038]

[0039] Table 3 Freeze-drying methods

[0040] Step number type Temperature / °C vacuum / μbar Time / h 1 Pre-freezing -45 / 0.5 2 Pre-freezing -45 / 3 3 Empty / 150 / 4 dry -5 150 8 5 dry -5 150 8 6 dry 25 150 2 7 dry 25 150 2 8 dry 25 5 0.017 9 dry 25 5 1

[0041] Optical polarization determination: The lyophilized powder was reconstituted with 4 mL of physiological saline at room temperature, vortexed for 10 s, and allowed to stand at 4 °C for 2 h to fully hydrate. After centrifugation (10000 rpm, 10 min), the supernatant was collected at 2 h, 24 h, and 72 h after reconstitution for optical polarization determination.

[0042] Table 4 Results of Optical Rotation Detection

[0043]

[0044] The results showed that natural collagen (bovine / porcine) decreased in temperature (Tm) by 2–3°C within 2 hours after reconstitution due to hydroxyproline cross-linking, while reconstituted collagen decreased by more than 3°C, indicating partial triple helix unwinding. Reconstituted collagen is more prone to time-dependent denaturation at physiological pH. Natural collagen should be used as soon as possible after reconstitution, while reconstituted collagen requires the addition of stabilizers or should be prepared and used immediately.

[0045] Example 3: Investigation of conventional freeze-dried compositions

[0046] Weigh freeze-drying protectant 1, freeze-drying protectant 2, and purified water according to Table 5, mix and stir until dissolved at 200 rpm for 20 min; add bovine type I collagen weighed according to Table 5, stir at 200 rpm for 30 min until dissolved, pour into vials, and freeze-dry according to Table 3. Sterilize by irradiation to obtain the freeze-dried collagen composition.

[0047] Freeze-drying protectant 1 is one of trehalose, sucrose, and mannitol, and freeze-drying protectant 2 is hydroxyproline.

[0048] Table 5 Freeze-dried compositions

[0049]

[0050] Optical rotation determination: The lyophilized powder of the composition was reconstituted with 4 mL of physiological saline at room temperature, vortexed for 10 s, and allowed to stand at 4 °C for 2 h to fully hydrate. After centrifugation (10000 rpm, 10 min), the supernatant was collected for optical rotation detection. The heating rate was 1 °C / min, and the temperature range was 25–45 °C.

[0051] Table 6 Results of Optical Rotation Detection

[0052]

[0053]

[0054] The results showed that the temperature range (Tm) of bovine type I collagen decreased after the addition of trehalose, sucrose, mannitol, and hydroxyproline as freeze-drying protectants. The Tm decreased by more than 10°C after the addition of mannitol as a freeze-drying protectant, indicating that some triple helixes unwind. Mannitol is prone to crystallization during freeze-drying, and the crystallization process may compress collagen molecules, leading to aggregation or denaturation.

[0055] Example 4: Investigation of the lyophilized composition

[0056] Weigh freeze-drying protectant 1, freeze-drying protectant 2, and purified water according to Table 7, mix and stir until dissolved at 300 rpm for 20 min; add bovine type I collagen weighed according to Table 7, stir at 300 rpm for 30 min until dissolved, pour into vials, and freeze-dry according to Table 3. Perform irradiation sterilization to obtain the freeze-dried collagen composition.

[0057] Freeze-drying protectant 1 is one of trehalose, sucrose, and mannitol, and freeze-drying protectant 2 is hydroxyproline.

[0058] Table 7 Freeze-dried compositions

[0059]

[0060] Optical polarization determination: The lyophilized powder of the composition was reconstituted with 4 mL of physiological saline at room temperature, vortexed for 10 s, and allowed to stand at 4℃ for 2 h and 24 h to fully hydrate. After centrifugation (10000 rpm, 10 min), the supernatant was collected for optical polarization detection. The heating rate was 1℃ / min, and the temperature range was 25~45℃.

[0061] Table 8 Results of Optical Rotation Detection

[0062]

[0063]

[0064] The results showed that, unexpectedly, the addition of a combination of trehalose and hydroxyproline as a freeze-drying protectant to bovine type I collagen significantly stabilized the Tm value (°C) of the reconstituted collagen and maintained the triple helix structure of the collagen.

[0065] Example 5: Investigation of the lyophilized composition of trehalose and hydroxyproline

[0066] Weigh freeze-drying protectant 1, freeze-drying protectant 2, and purified water according to Table 9, mix and stir until dissolved at 600 rpm for 20 min; add bovine type I collagen weighed according to Table 9, stir at 400 rpm for 30 min until dissolved, pour into vials, and freeze-dry according to Table 3. Perform irradiation sterilization to obtain the collagen freeze-dried composition;

[0067] Lyophilization protectant 1 is trehalose, and lyophilization protectant 2 is hydroxyproline.

[0068] Table 9 Freeze-dried compositions

[0069]

[0070] Optical polarization determination: The lyophilized powder of the composition was reconstituted with 4 mL of physiological saline at room temperature, vortexed for 10 s, and allowed to stand at 4℃ for 2 h and 24 h to fully hydrate. After centrifugation (10000 rpm, 10 min), the supernatant was collected for optical polarization detection. The heating rate was 1℃ / min, and the temperature range was 25~45℃.

[0071] Table 10 Results of Optical Rotation Detection

[0072]

[0073]

[0074] The results showed that adding trehalose and hydroxyproline as freeze-drying protectants to bovine type I collagen significantly improved the stability of the reconstituted solution within 24 hours in the mass ratio range of 1:1 to 10:1, with the Tm value decreasing by less than 2℃, effectively maintaining the triple helix structure of collagen.

[0075] Example 6: Investigation of different collagens

[0076] Weigh freeze-drying protectant 1, freeze-drying protectant 2, and purified water according to Table 11, mix and stir until dissolved at 600 rpm for 30 min; add collagen according to Table 11, stir at 600 rpm for 30 min until dissolved, pour into vials, and freeze-dry according to Table 3. Sterilize by irradiation to obtain the freeze-dried collagen composition.

[0077] The collagen is one of bovine type I collagen, porcine type I collagen, or recombinant type III humanized collagen. The freeze-drying protectant 1 is trehalose, and the freeze-drying protectant 2 is hydroxyproline.

[0078] Table 11 Freeze-dried compositions

[0079]

[0080]

[0081] Optical polarization determination: The lyophilized powder of the composition was reconstituted with 4 mL of physiological saline at room temperature, vortexed for 10 s, and allowed to stand at 4℃ for 2 h and 24 h to fully hydrate. After centrifugation (10000 rpm, 10 min), the supernatant was collected for optical polarization detection. The heating rate was 1℃ / min, and the temperature range was 25~45℃.

[0082] Table 12 Results of Optical Rotation Detection

[0083]

[0084] The results showed that bovine type I collagen, porcine type I collagen, and recombinant type III humanized collagen could maintain their triple helix structure after 24 hours of reconstitution using trehalose and hydroxyproline as freeze-drying protectants.

[0085] Example 7: Investigation of Buffer Salts and Osmotic Pressure Regulators

[0086] Weigh freeze-drying protectant 1, freeze-drying protectant 2, and purified water according to Table 13, mix and stir until dissolved at 500 rpm for 20 min; add buffer salts, osmotic pressure regulators, citric acid, and sodium chloride from Table 13, stir at 500 rpm for 30 min until dissolved, add collagen from Table 13, stir at 200 rpm for 30 min until dissolved, pour into vials, and freeze-dry according to Table 3. Perform irradiation sterilization to obtain the freeze-dried collagen composition.

[0087] The collagen is bovine type I collagen, the freeze-drying protectant 1 is trehalose, the freeze-drying protectant 2 is hydroxyproline, and the buffer salt is one of disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and disodium hydrogen phosphate.

[0088] Table 13 Lyophilized Compositions

[0089]

[0090]

[0091] Optical polarization determination: The lyophilized powder of the composition was reconstituted with 4 mL of water for injection at room temperature, vortexed for 10 s, and allowed to stand at 4℃ for 2 h and 24 h to fully hydrate. After centrifugation (10000 rpm, 10 min), the supernatant was collected for optical polarization detection. The heating rate was 1℃ / min, and the temperature range was 25~45℃.

[0092] Table 14 Results of Optical Rotation Detection

[0093]

[0094] The results showed that the combination of collagen, trehalose, and hydroxyproline could maintain the triple helix structure of collagen after being reconstituted with different buffer solutions.

[0095] Example 8: Detection of Triple Helix Structure in CD Spectroscopy

[0096] After reconstitution of the lyophilized collagen compositions 1003 and 1012, the triple helix structure was detected by CD spectroscopy using a circular dichroism spectroscopy system. Figure 1 Scanning range: 190–250 nm, scanning speed: 50–100 nm / min.

[0097] from Figure 1 It can be seen that the circular dichroism (CD) spectrum of the collagen sample of the composition shows a negative peak near 195 nm and a positive peak near 220 nm, which is the same as the CD spectrum of bovine type I collagen reference standard from the National Institutes for Food and Drug Control. This indicates that the composition retains the triple helix structure of bovine type I collagen, and the collagen sample of the composition does not have an obvious triple helix structure.

[0098] Example 9

[0099] (1) Collagen production test

[0100] Samples of ordinary collagen compositions 1006 and 1007 (untreated and 28 days treated) and collagen compositions 1012, 1019, and 1020 (containing trehalose and hydroxyproline lyophilization protectants) were added to RPMⅠ1640 culture medium containing 20% ​​newborn calf serum, respectively. Mouse fibroblasts were then added and cultured. A blank control group was also set up, in which the collagen composition samples were replaced with an equal volume of physiological saline. After 24 and 48 hours of culture, the supernatant culture medium was collected, and the collagen content was recorded and measured using a modified HPR assay. The measured results for each example are shown in Table 15 below.

[0101] Table 15 Collagen Content

[0102]

[0103] As shown in Table 15, compositions 1006 and 1007 of the present invention, compared with compositions 1012, 1019, and 1020, all exhibited varying degrees of collagen-promoting effects compared to the blank control group. Compositions 1012, 1019, and 1020 showed collagen content increases of over 170% after 24 hours and 28 days of storage, respectively, and approximately 400% after 48 hours, demonstrating excellent stability and the ability to promote the body's own collagen synthesis. Compositions 1006 and 1007, being ordinary collagen compositions, did not show an increase in collagen content, but their degradation rate was slower, with little difference in performance between the two groups after 28 days of storage.

[0104] (2) Animal experiments

[0105] Compositions 1012, 1019, and 1020 of the present invention were reconstituted with 2 ml of physiological saline. 200 μL of the collagen composition prepared in this invention was subcutaneously injected into five-week-old female nude mice. Epidermal tissue samples were collected from the backs of the mice on days 7, 14, and 28 for pathological analysis, reaching the subcutaneous layer (including the epidermis, dermis, and part of the subcutaneous tissue). The mice were sutured post-surgery. 1 g of epidermal tissue was weighed and mixed with 9 g of PBS (pH 7.2-7.4). The sample was homogenized thoroughly using a homogenizer and centrifuged for approximately 20 minutes (2000-3000 rpm). The supernatant was carefully collected, and the absorbance at 450 nm was measured using an ELISA kit. The concentration of the inflammatory factor IL-1α was recorded and determined.

[0106] Table 16 Results of Inflammatory Response Detection of Collagen Compositions

[0107]

[0108] No abnormalities were observed in female nude mice 28 days after injection of samples 1012, 1019, and 1020. The IL-1α factor content was low, and there were no abnormal changes in the surrounding tissues. No intradermal reaction was induced in the female nude mice, indicating that the intradermal reaction test of the PDRN-non-crosslinked collagen composition was safe and qualified.

[0109] Example 10: Beauty Effect Test

[0110] The performance of compositions 1012, 1019, and 1020 from the embodiments of this application was tested. The samples prepared in different groups were vortexed and dispersed evenly with 2 ml of physiological saline and then allowed to stand at 4°C for 2 hours for hydration. They were then given to 10 volunteers for full-face application, twice a day, 2 mL each time, for 30 consecutive days. The volunteers' skin moisture content, skin elasticity, wrinkle depth, pigmentation, and skin luster were measured.

[0111] Skin moisture content was measured using a skin moisture meter; skin elasticity was measured using a skin elasticity meter; wrinkle depth was measured using a 3D skin surface scanner; skin pigmentation was measured using a skin pigment concentration meter; and skin gloss was measured using a skin gloss meter. The ambient temperature was controlled at 20–25℃, and the ambient humidity at 40–60%. Test sites were the forehead and cheeks; measurements were taken in the morning after cleansing, before product use, and 4 weeks after product use. Average values ​​were recorded. Specific performance test results are shown in Table 17 below.

[0112] Table 17 Skin Improvement Effects

[0113]

[0114] As shown in Table 17 above, compositions 1012, 1019, and 1020 of this application have the best effect on improving skin quality. Their overall performance is significantly better than that of compositions 1006 and 1007. That is, within the technical solution defined in this application, the collagen composition containing trehalose and hydroxyproline lyophilized protective agent has excellent overall performance.

[0115] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A lyophilized collagen preparation, characterized in that, It contains the following components: 0.01% to 10% (w / w) collagen in the total lyophilized formulation, 0.1% to 10% (w / w) lyophilization protectant 1 in the total lyophilized formulation, 0.1% to 5% (w / w) lyophilization protectant 2 in the total lyophilized formulation, buffer salts, osmotic pressure regulators, diluents, and pH buffers.

2. The collagen freeze-dried formulation as described in claim 1, characterized in that, The collagen is selected from one or more of natural collagen, the sources of which include one or more of beef Achilles tendon, pig skin, chicken feet, fish skin, fish bones, chicken breast cartilage, and forest frog. Natural collagen is classified into one or more types I, II, and III according to its function.

3. The collagen freeze-dried formulation as described in claim 1, characterized in that, The freeze-drying protectant 1 is selected from one or more of mannitol, lactose, trehalose, sucrose, maltose, dextrin, stachyose, gelatin, pectin, sugar alcohol, sorbitol, proline, histidine, lysine, arginine, polyethylene glycol, or polyvinylpyrrolidine, preferably one or more of mannitol, lactose, trehalose, and sucrose.

4. The collagen freeze-dried formulation as described in claim 1, characterized in that, The freeze-drying protectant 2 is selected from one or more of hydroxyproline, proline, histidine, lysine, and arginine.

5. The collagen freeze-dried formulation as described in claim 1, characterized in that, The buffer salt is selected from one or more of the following: disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution, dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer solution, disodium hydrogen phosphate / citric acid buffer solution, sodium bicarbonate buffer solution, or sodium bicarbonate / sodium carbonate buffer solution, preferably one or more of the following: disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution, dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer solution.

6. The collagen freeze-dried formulation as described in claim 1, characterized in that, The diluents include, but are not limited to, one or more of the following: water for injection, sodium chloride injection, glucose injection, lactated Ringer's solution, sucrose aqueous solution, xylitol aqueous solution, and pH buffer.

7. The collagen freeze-dried formulation as described in claim 6, characterized in that, The sodium chloride injection solution includes, but is not limited to, 0.9% sodium chloride injection solution.

8. The collagen freeze-dried formulation as described in claim 6, characterized in that, The glucose injection solution includes, but is not limited to, 5% glucose injection solution.

9. The collagen freeze-dried formulation as described in claim 1, characterized in that, The pH buffer solution is a buffer solution with pH = 5.0 to 7.4, including but not limited to one or more of phosphate buffer, glycine buffer, histidine buffer, citrate buffer, and acetate buffer.

10. A method for preparing a lyophilized collagen formulation, characterized in that, Includes the following steps: S1: Weigh each component according to its mass fraction; S2. Add freeze-drying protectant 1 and freeze-drying protectant 2 to deionized water, stir until dissolved, at a stirring speed of 200-600 rpm for 20-30 min; S3. Continue to add buffer salt and osmotic pressure regulator and stir until dissolved. The stirring speed is 200-600 rpm and the time is 20-30 min. S4. Continue adding collagen and stirring until dissolved. Stir at 200-600 rpm for 20-30 minutes. S5. The solution obtained in S4 is filled into vials, pre-frozen at -30℃ to -60℃ for 4 to 8 hours; then freeze-dried under vacuum at -10℃ to 30℃ for 8 to 24 hours; after being removed from the freezer and capped, it is sterilized by irradiation to obtain the collagen freeze-dried composition.

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