Stable animal-derived collagen solution for pricking needle injection as well as preparation method and application of stable animal-derived collagen solution

Abstract

The invention discloses a stable animal-derived collagen solution for pricking needle injection as well as a preparation method and application of the stable animal-derived collagen solution. The preparation method comprises the following steps: enzymolysis, microfiltration and concentration, dilution and filtration, centrifugal separation, directional fixation and liquid preparation, homogenization and secondary fixation, and mixing and defoaming. By optimizing the preparation method of the collagen solution, the fiber state of the collagen is improved, so that the collagen fibers are uniformly dispersed in the system, the solution is uniform and fine in texture and good in flowability, and can be injected through a small needle head, so that the use requirements on the preparation performance in the beauty fields of hydro-optical needles and the like are met.

Description

Technical Field

[0001] This invention relates to the field of collagen preparation technology, and in particular to a stable animal-derived collagen solution for injection via a needle, its preparation method, and its application. Background Technology

[0002] Currently, injectable animal-derived collagen for medical use faces strict requirements regarding microbial limits, with almost all products needing to meet sterility standards. In the field of mesotherapy applications, the material structure and degradation properties must also meet the needs of injection procedures and repair cycles, thus placing higher demands on the formulation composition of collagen solutions and the stability of collagen fiber structures. To meet these requirements, the traditional approach is to add preservatives, antibiotics, or cross-linking agents to the collagen solution to extend shelf life and enhance collagen fibrosis. However, these additives may induce adverse reactions such as drug resistance and irritation. Especially for injectable collagen products, there are high risks of central nervous system toxicity and asphyxiation.

[0003] Patent CN 105063148A discloses a method for preparing collagen. This method produces collagen with a high concentration, making it unsuitable for mesotherapy. When formulated into lower concentration preparations, it easily generates numerous air bubbles, particularly with viscous animal-derived collagen, affecting injection accuracy. Patent CN108752465A discloses a method for preparing collagen solution, which involves reconstituted freeze-dried collagen sponges after high-temperature, high-pressure sterilization, ethylene oxide sterilization, or irradiation sterilization. This method significantly impacts collagen activity, reducing its ability to form collagen fibers.

[0004] In summary, current technologies for animal-derived collagen mostly employ terminal sterilization, which significantly impacts the activity of liquid collagen. However, in-process sterilization is limited by process conditions, resulting in low yields. Furthermore, current collagen injection products are primarily used for filler injections, exhibiting high concentrations and fiber content. For mesotherapy, dilution is often necessary, but direct dilution leads to inconsistent product homogeneity and stability, frequently causing aggregation, stratification, or thinning due to environmental changes. Large collagen particles are also unsuitable for injection via lancets. Therefore, a novel solution system is urgently needed that is sterile, harmless, and maintains collagen fiber stability. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a stable, needle-injectable animal-derived collagen solution, its preparation method, and its applications. The preparation method of this invention starts with readily available dried collagen, reducing the cumbersome collagen extraction process and enabling rapid production of sterile formulations. Furthermore, the collagen solution prepared by this method exhibits significantly improved fibrillation, with collagen fibers uniformly dispersed in the system. The solution has a homogeneous and fine texture, possesses good fluidity, and can be injected through fine needles, thus meeting the performance requirements of cosmetic procedures such as mesotherapy.

[0006] The technical solution of the present invention is as follows: The first aspect of this invention protects a method for preparing a stable, needle-injectable animal-derived collagen solution, the method comprising the following steps: (1) Enzymatic hydrolysis: Add the dried collagen product to the hydrochloric acid solution, stir to dissolve and obtain a collagen solution, add pepsin, continue stirring to react and obtain an enzymatic hydrolysis reaction solution; (2) Microfiltration concentration: The enzymatic reaction solution is microfiltered with a 30 μm microfilter to obtain microfiltrate. Then, hydrochloric acid solution is added to obtain hydrochloric acid solution of collagen. After ultrafiltration, elution and concentration, collagen concentrate is obtained. (3) Dilution and filtration: Add NaCl solution to the collagen concentrate, stir at low temperature, filter through a 0.2 μm filter, and collect the filtered solution; (4) Centrifugal separation: After adjusting the pH of the system to 6.5~7.0, let it stand at low temperature overnight, centrifuge, and collect the precipitate; (5) Directional fixation and solution preparation: Add phosphate buffer to the precipitate, let it stand, tilt the container and let it stand overnight to fix it and obtain collagen gel; then add water, stir evenly, and prepare a collagen solution with a collagen concentration of 5.0~15.0 g / mL with physiological saline. (6) Homogenization and secondary fixation: After homogenizing the prepared collagen solution, let it stand for fixation; (7) Mixing and degassing: After standing, mix thoroughly in a rotating and revolving mixing device, degas under vacuum, fill and seal to obtain a stable animal-derived collagen solution for injection.

[0007] Preferably, in step (1), The concentration of the hydrochloric acid solution is 8-12 mmol / L; The stirring and dissolving process is carried out at a temperature of 2~10 ℃, a speed of 150~200 rpm, and a time of 22~26 h. The concentration of the collagen solution is 4.0 mg / mL; The mass ratio of pepsin to dried collagen is (2~5):1; The stirring reaction is carried out at a temperature of 10~25 ℃, a rotation speed of 60~80 rpm, and a time of 24~48 h; Ultrasonic waves are applied during the stirring reaction. The power of the ultrasonic waves is 20-50W, and they operate intermittently in a cycle of 3 seconds on and 10 seconds off.

[0008] Preferably, in step (2), The concentration of the hydrochloric acid solution is 10 mmol / L; The volume ratio of the hydrochloric acid solution to the microfiltrate is (1~1.5):1; The ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 50~100kDa; The elution is performed using gradient elution, with the eluent being a 10 mmol / L hydrochloric acid solution, and the elution is performed 5 to 7 times with 1 to 2 times the volume of hydrochloric acid solution. The concentration is achieved using cross-flow concentration, with a transmembrane pressure of 0.5~1.0 MPa and a membrane surface velocity of 1~3 m / s; The concentration of the collagen concentrate is 3.5~4.0 mg / mL.

[0009] Preferably, in step (3), The concentration of the NaCl solution is 0.2~0.5 mol / L; The volume of the NaCl solution used is 30% of the volume of the collagen concentrate. The low-temperature stirring is carried out at a temperature of 2~10 ℃, a speed of 80~100 rpm, and a time of 2~4 h.

[0010] Preferably, in step (4), Adjust the pH of the system to 6.5-7.0 using sodium hydroxide with a concentration of 0.2-0.5 mol / L; The temperature for overnight low-temperature standing is 2~10℃; The centrifugation speed is 7500~8500 rpm, and the time is 20~30 min.

[0011] Preferably, in step (5), The phosphate buffer solution is prepared from Na2HPO4, NaH2PO4, NaCl and water, wherein the concentration of Na2HPO4 is 0.26~0.43 mol / L, the concentration of NaH2PO4 is 0.14~0.19 mol / L, and the concentration of NaCl is 1.95~2.10 mol / L. The amount of the phosphate buffer solution used is 15% of the precipitate mass; The settling temperature is 28~37℃, and the time is 3~6h; The container is tilted at an angle of 35~45° and rotates at a speed of 5~10 rpm. The temperature for overnight storage is 28~37℃; The ratio of the mass of water to the mass of collagen gel is 1:1.

[0012] Preferably, in step (6), The homogenization process is carried out at a rotation speed of 2000~4000 rpm for a time of 45~60 min; The static fixation temperature is 28~37 ℃, and the time is 2~4 h.

[0013] Preferably, in step (7), In the aforementioned revolution-rotation mixing device, the rotation speed is 200~300 rpm, the revolution speed is 500~700 rpm, the revolution-rotation speed ratio is (2~2.5):1, the temperature is 10~25℃, the vacuum degree is increased from -50Kpa to -95Kpa, and the time is 5~10min.

[0014] The second aspect of this invention protects a stable animal-derived collagen solution for injection via a needle, said stable animal-derived collagen solution for injection via a needle is prepared by the preparation method described in the first aspect.

[0015] The third aspect of this invention protects the application of a stable, needle-injectable animal-derived collagen solution in the medical and aesthetic fields. The stable, needle-injectable animal-derived collagen solution is the stable, needle-injectable animal-derived collagen solution described in the second aspect, and / or a stable, needle-injectable animal-derived collagen solution prepared by the preparation method described in the first aspect.

[0016] The beneficial technical effects of this invention are as follows: This invention uses dried macromolecular collagen as raw material. Due to improper storage or transportation, the collagen in dried products often re-aggregates, affecting filtration. This invention reduces the adsorption of collagen on the filter membrane surface through enzymatic hydrolysis and sodium chloride treatment. Finally, a 0.2 μm filter can be used to filter the collagen, achieving a highly efficient sterilization effect.

[0017] This invention employs a graded fixation method and utilizes a rotational-revolutionary mixing device for degassing, effectively breaking down the cross-linking forces between molecules and fully agglomerating long collagen fibers into smaller collagen fiber segments, ensuring their uniform dispersion within the system. This process successfully solves the problems of unstable collagen fiber structure and easy stratification or aggregation in traditional collagen solutions. The obtained collagen solution maintains a certain level of turbidity even at low concentrations, and after being stored at 4–20°C for 3 months, no stratification or aggregation occurred, significantly improving the stability and applicability of the collagen solution.

[0018] The preparation method of this invention uses mild process conditions, which can effectively avoid sudden changes in local conditions, preserve the complete triple helix structure of collagen, and make the collagen solution more turbid under physiological conditions, exhibiting good self-assembly properties. Attached Figure Description

[0019] Figure 1 The images show the appearance of the animal-derived collagen solutions prepared in Examples 1-3 of this invention and filled into vials.

[0020] In the figure: A is the animal-derived collagen solution prepared in Example 1; B is the animal-derived collagen solution prepared in Example 2; C is the animal-derived collagen solution prepared in Example 3.

[0021] Figure 2 Electrophoretic images of collagen solutions before and after filtration by the 0.2 μm filter prepared in step (3) of Examples 1-3 of the present invention.

[0022] In the figure: 1 is a type I collagen standard; 2 is the collagen solution prepared in Example 1 with a 0.2 μm filter before filtration; 3 is the collagen solution prepared in Example 1 with a 0.2 μm filter after filtration; 4 is the collagen solution prepared in Example 2 with a 0.2 μm filter before filtration; 5 is the collagen solution prepared in Example 2 with a 0.2 μm filter after filtration; 6 is the collagen solution prepared in Example 3 with a 0.2 μm filter before filtration; 7 is the collagen solution prepared in Example 3 with a 0.2 μm filter after filtration.

[0023] Figure 3 Electrophoretic images of animal-derived collagen solutions prepared in Examples 1-3 of this invention and filled into vials.

[0024] In the figure: 1 is a type I collagen standard; 2 is an animal-derived collagen solution prepared in Example 1; 3 is an animal-derived collagen solution prepared in Example 2; and 4 is an animal-derived collagen solution prepared in Example 3.

[0025] Figure 4 The circular dichroism spectrum of the animal-derived collagen solution prepared in Example 1 of this invention and filled into vials.

[0026] Figure 5 The circular dichroism spectrum of the animal-derived collagen solution prepared in Example 2 of this invention and filled into vials.

[0027] Figure 6 The circular dichroism spectrum of the animal-derived collagen solution prepared in Example 3 of this invention and filled into vials.

[0028] Figure 7 The images show the appearance of the animal-derived collagen solutions prepared in Examples 2 and 2 of the present invention, filled into vials.

[0029] In the figure: A is the animal-derived collagen solution prepared in Example 2; B is the animal-derived collagen solution prepared in Comparative Example 2.

[0030] Figure 8 The changes in optical density values ​​of the animal-derived collagen solutions prepared in Examples 2 and 2 of this invention and filled into vials are shown.

[0031] Figure 9The images show the appearance of the animal-derived collagen solutions prepared in Examples 2 and 3 of this invention and filled into syringes.

[0032] In the figure: A is the animal-derived collagen solution prepared in Example 2; B is the animal-derived collagen solution prepared in Comparative Example 3. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0034] A method for preparing a stable, needle-injectable animal-derived collagen solution, the method comprising the following steps: (1) Enzymatic hydrolysis: Add the dried collagen product to the hydrochloric acid solution, stir to dissolve and obtain a collagen solution, add pepsin, continue stirring to react and obtain an enzymatic hydrolysis reaction solution; (2) Microfiltration concentration: The enzymatic reaction solution is microfiltered with a 30 μm microfilter to obtain microfiltrate. Then, hydrochloric acid solution is added to obtain hydrochloric acid solution of collagen. After ultrafiltration, elution and concentration, collagen concentrate is obtained. (3) Dilution and filtration: Add NaCl solution to the collagen concentrate, stir at low temperature, filter through a 0.2 μm filter, and collect the filtered solution; (4) Centrifugal separation: After adjusting the pH of the system to 6.5~7.0, let it stand at low temperature overnight, centrifuge, and collect the precipitate; (5) Directional fixation and solution preparation: Add phosphate buffer to the precipitate, let it stand, tilt the container and let it stand overnight to fix it and obtain collagen gel; then add water, stir evenly, and prepare a collagen solution with a collagen concentration of 5.0~15.0 g / mL with physiological saline. (6) Homogenization and secondary fixation: After homogenizing the prepared collagen solution, let it stand for fixation; (7) Mixing and degassing: After standing, mix thoroughly in a rotating and revolving mixing device, degas under vacuum, fill and seal to obtain a stable animal-derived collagen solution for injection.

[0035] Dried collagen products often re-aggregate due to improper storage or transportation, affecting filtration. This invention uses low-frequency ultrasound and pepsin in synergy to make enzymatic hydrolysis more thorough, fundamentally reducing collagen solution aggregation. After multiple ultrafiltrations, cross-flow concentrations, and low-concentration sodium chloride treatments, the adsorption of collagen on the filter membrane surface is reduced. Finally, a 0.2 μm filter can be used for filtration, achieving a highly efficient sterilization effect.

[0036] Furthermore, this invention employs a method of "slowly rotating an inclined container" during the initial fixation phase to guide collagen molecules towards oriented alignment and form a fiber structure. Subsequently, homogenization, secondary fixation, and the shear forces generated by revolution and rotation effectively break down the cross-linking forces between molecules, fully aggregating and breaking down long collagen fibers into smaller collagen fiber segments, which are stably distributed within the system. This method significantly improves the dispersion stability of collagen fibers and avoids the layering and aggregation phenomena that easily occur in traditional processes.

[0037] The entire process of this invention is controlled at a temperature of 2~37℃. Low-frequency stirring and ultrasonication, pH adjustment, dilution and vacuum mixing are all carried out in a "gradient manner", which can avoid sudden changes in local conditions and preserve the complete triple helix structure of collagen.

[0038] In some embodiments, in step (4), the pH of the system is adjusted to 6.5-7.0 using sodium hydroxide with a concentration of 0.2-0.5 mol / L. Preferably, the pH of the system is gradually adjusted to 6.5-7.0 using a sodium hydroxide solution with a concentration of 0.2-0.5 mol / L.

[0039] The gradual adjustment is as follows: sodium hydroxide is slowly added dropwise, adjusting the pH by 0.2-0.3 units every 10 minutes. When the pH reaches 4.0-4.5 and 5.5-6.0, the mixture is stirred for 3-5 minutes, and then sodium hydroxide is added dropwise until the pH reaches 6.5-7.0. The sodium hydroxide used is sterilized by moist heat and cooled before use.

[0040] In some embodiments, in step (5), the phosphate buffer and physiological saline are sterilized by moist heat and then cooled before use.

[0041] In some embodiments, in step (5), preferably, a collagen solution with a collagen concentration of 5.0~15.0 g / mL is prepared by adding physiological saline through gradient dilution. The gradient dilution is performed by adding 10% of the system volume each time, stirring for 10 minutes before adding the next batch.

[0042] The stable animal-derived collagen solution prepared by this invention for injection can be used in the medical and aesthetic fields, such as for mesotherapy.

[0043] The dried collagen used in the following embodiments of the present invention was purchased from Wuxi Bedy Biotechnology Co., Ltd. The type I collagen standard used was purchased from the China National Institutes for Food and Drug Control.

[0044] Example 1 A method for preparing a stable, needle-injectable animal-derived collagen solution includes the following steps: (1) Enzymatic hydrolysis: 80 g of dried collagen was added to 20 L of hydrochloric acid solution with a concentration of 12 mmol / L and stirred at 2℃ and 200 rpm for 22 h to obtain a collagen solution with a concentration of 4.0 mg / mL. 400 g of pepsin was added and reacted at 25℃, stirring at 80 rpm, with an ultrasonic power of 50W and a working cycle of "3s on / 10s off" for 24 h to obtain the enzymatic hydrolysis reaction solution.

[0045] (2) Microfiltration concentration: 20 L of the enzymatic hydrolysis reaction solution was microfiltered using a 30 μm microfilter. After microfiltration, 20 L of 10 mmol / L hydrochloric acid solution was added to obtain approximately 40 L of collagen hydrochloric acid solution. This solution was then passed through an ultrafiltration membrane module with a molecular weight cutoff of 50 kDa, using 10 mmol / L hydrochloric acid solution as the eluent. When the volume of collagen hydrochloric acid solution reached 20 L, 20 L of 10 mmol / L hydrochloric acid solution was added to complete one ultrafiltration cycle. This process was repeated 4 times. Then, 15 L of 10 mmol / L hydrochloric acid solution was added to eluent until the volume of collagen hydrochloric acid solution reached 15 L. This process was repeated 3 times. After ultrafiltration, the solution was concentrated at a transmembrane pressure of 1.0 MPa and a membrane flow rate of 3 m / s until the collagen concentration reached 4.0 mg / mL.

[0046] (3) Dilution and filtration: Take 16 L of collagen concentrate with a concentration of 4.0 mg / mL, add 4.8 L of 0.5 mol / L NaCl solution, stir at 2 ℃ and 100 rpm for 4 h, then filter once with a 0.2 μm filter and collect the filtered solution.

[0047] (4) Centrifugation: Take 18.5 L of the filtered solution and slowly add 0.5 mol / L sodium hydroxide solution. Adjust the pH by 0.2~0.3 units every 10 min. When the pH of the system is adjusted to 4.5, stir for 5 min. Then continue to add sodium hydroxide solution to adjust the pH of the system to 6.0. Stir for 5 min and then continue to add sodium hydroxide solution to adjust the pH of the system to 7.0. After stirring evenly, let it stand overnight at 4℃. Then centrifuge at 8500 rpm for 20 min, discard the supernatant, and retain the precipitate (the mass of the precipitate is 6% of the mass of the solution before centrifugation) and detect the protein content.

[0048] (5) Directional fixation and solution preparation: Take 1110 g of the collected centrifuged precipitate into a container, add 166.5 g of phosphate buffer (phosphate buffer formula: 0.43 mol / L Na2HPO4, 0.19 mol / L NaH2PO4, 2.10 mol / L NaCl), mix well, let stand at 37 ℃ for 3 h, then tilt the container at 45° and slowly rotate it at 10 rpm, and leave it at 37 ℃ overnight to obtain collagen gel; then add 1276.5 g of water for injection, stir well, and gradually add physiological saline, each time adding 10% of the system mass, stir for 10 min, and then add the next batch of physiological saline to finally prepare a collagen solution with a collagen concentration of 15.0 mg / mL.

[0049] (6) Secondary fixation: Take the prepared collagen solution with a concentration of 15.0 mg / mL, homogenize it at 4000 rpm for 60 min, and then let it stand at 37 ℃ for 2 h to complete the secondary fixation.

[0050] (7) Mixing and degassing: After settling, the collagen solution is transferred to a rotating and revolving mixing device for mixing and degassing under vacuum. The mixing and degassing parameters are: rotation speed 300 rpm, revolution speed 600 rpm, temperature 10℃, vacuum degree increased from -50Kpa to -95Kpa, and time 10 min. Then, it is filled and sealed to obtain a stable animal-derived collagen solution suitable for injection.

[0051] Example 2 A method for preparing a stable, needle-injectable animal-derived collagen solution includes the following steps: (1) Enzymatic hydrolysis: 60 g of dried collagen was added to 15 L of hydrochloric acid solution with a concentration of 10 mmol / L and stirred at 6℃ and 180 rpm for 24 h to obtain a collagen solution with a concentration of 4.0 mg / mL. 210 g of pepsin was added and reacted at 18℃, stirring at 70 rpm, with an ultrasonic power of 30W and a working cycle of "3s on / 10s off" for 36 h to obtain the enzymatic hydrolysis reaction solution.

[0052] (2) Microfiltration concentration: 15 L of the enzymatic hydrolysis reaction solution was microfiltered using a 30 μm microfilter. After microfiltration, 18 L of 10 mmol / L hydrochloric acid solution was added to obtain approximately 33 L of collagen hydrochloric acid solution. This solution was then passed through an ultrafiltration membrane module with a molecular weight cutoff of 100 kDa, using 10 mmol / L hydrochloric acid solution as the eluent. When the volume of collagen hydrochloric acid solution reached 15 L, 18 L of 10 mmol / L hydrochloric acid solution was added to complete one ultrafiltration cycle. This process was repeated three times. After ultrafiltration, the solution was concentrated at a transmembrane pressure of 0.8 MPa and a membrane flow rate of 2 m / s until the collagen concentration reached 3.7 mg / mL.

[0053] (3) Dilution and filtration: Take 13.5 L of collagen concentrate with a concentration of 3.7 mg / mL, add 4.05 L of 0.3 mol / L NaCl solution, stir at 6 ℃ and 90 rpm for 3 h, then filter once with a 0.2 μm filter and collect the filtered solution.

[0054] (4) Centrifugation: Take 15L of filtered solution and slowly add 0.35 mol / L sodium hydroxide solution. Adjust the pH by 0.2~0.3 units every 10 min. When the pH of the system is adjusted to 4.2, stir for 5 min. Then continue to add sodium hydroxide solution to adjust the pH of the system to 5.8. Stir for 5 min and then continue to add sodium hydroxide solution to adjust the pH of the system to 6.8. After stirring evenly, let it stand overnight at 6 ℃. Then centrifuge at 8000 rpm for 25 min, discard the supernatant, and retain the precipitate (the mass of the precipitate is 8% of the mass of the solution before centrifugation) and detect the protein content.

[0055] (5) Directional fixation and solution preparation: Take 1200 g of the collected centrifuged precipitate into a container, add 180 g of phosphate buffer (phosphate buffer formula: 0.36 mol / L Na2HPO4, 0.16 mol / L NaH2PO4, 2.0 mol / L NaCl), mix well, let stand at 33 ℃ for 5 h, then tilt the container at 40° and slowly rotate it at 8 rpm, and place it at 33 ℃ overnight to obtain collagen gel; then add 1380 g of water for injection, stir well, and gradually add physiological saline, each time adding 10% of the system mass, stir for 10 min, and then add the next batch of physiological saline to finally prepare a collagen solution with a collagen concentration of 8.0 mg / mL.

[0056] (6) Secondary fixation: Take the prepared collagen solution with a concentration of 8.0 mg / mL, homogenize it at 3000 rpm for 45 min, and then let it stand at 33 ℃ for 3 h to complete the secondary fixation.

[0057] (7) Mixing and degassing: After settling, the collagen solution is transferred to a rotating and revolving mixing device for mixing and degassing under vacuum. The mixing and degassing parameters are: rotation speed 250 rpm, revolution speed 550 rpm, temperature 20℃, vacuum degree increased from -50Kpa to -95Kpa, and time 7min. Then, it is filled and sealed to obtain a stable animal-derived collagen solution suitable for injection.

[0058] Example 3 A method for preparing a stable, needle-injectable animal-derived collagen solution includes the following steps: (1) Enzymatic hydrolysis: 48 g of dried collagen was added to 12 L of hydrochloric acid solution with a concentration of 8 mmol / L and stirred at 10℃ and 150 rpm for 26 h to obtain a collagen solution with a concentration of 4.0 mg / mL. 96 g of pepsin was added and reacted at 10℃, stirring at 60 rpm, with an ultrasonic power of 20W and a working cycle of "3s on / 10s off" for 48 h to obtain the enzymatic hydrolysis reaction solution.

[0059] (2) Microfiltration concentration: 12 L of enzymatic hydrolysis reaction solution was microfiltered using a 30 μm microfilter. After microfiltration, 18 L of 10 mmol / L hydrochloric acid solution was added to obtain approximately 30 L of collagen hydrochloric acid solution. This solution was then passed through an ultrafiltration membrane module with a molecular weight cutoff of 100 kDa, using 10 mmol / L hydrochloric acid solution as the eluent. When the volume of collagen hydrochloric acid solution reached 12 L, 18 L of 10 mmol / L hydrochloric acid solution was added to complete one ultrafiltration. This process was repeated 3 times. Then, 15 L of 10 mmol / L hydrochloric acid solution was added to eluent. When the volume of collagen hydrochloric acid solution reached 12 L, 15 L of 10 mmol / L hydrochloric acid solution was added to complete one ultrafiltration. This process was repeated 2 times. After ultrafiltration, the solution was concentrated at a transmembrane pressure of 0.5 MPa and a membrane flow rate of 1 m / s until the collagen concentration reached 3.5 mg / mL.

[0060] (3) Dilution and filtration: Take 10 L of collagen concentrate with a concentration of 3.5 mg / mL, add 3 L of 0.2 mol / L NaCl solution, stir at 10 ℃ and 80 rpm for 2 h, then filter once with a 0.2 μm filter and collect the filtered solution.

[0061] (4) Centrifugation: Take 10.5L of filtered solution and slowly add 0.2 mol / L sodium hydroxide solution. Adjust the pH by 0.2~0.3 units every 10 min. When the pH of the system is adjusted to 4.0, stir for 5 min. Then continue to add sodium hydroxide solution to adjust the pH of the system to 5.5. Stir for 5 min and then continue to add sodium hydroxide solution to adjust the pH of the system to 6.5. After stirring evenly, let it stand overnight at 10℃. Then centrifuge at 7500 rpm for 30 min, discard the supernatant, and retain the precipitate (the mass of the precipitate is 10% of the mass of the solution before centrifugation) and detect the protein content.

[0062] (5) Directional fixation and solution preparation: Take 1050 g of the collected centrifuged precipitate into a container, add 157.5 g of phosphate buffer (phosphate buffer formula: 0.26 mol / L Na2HPO4, 0.14 mol / L NaH2PO4, 1.95 mol / L NaCl), mix well, and let stand at 28 ℃ for 6 h. Then tilt the container at 35° and slowly rotate it at 5 rpm, and leave it at 28 ℃ overnight to obtain collagen gel. Then add 1207.5 g of water for injection, stir well, and gradually add physiological saline, each time adding 10% of the system mass. Stir for 10 min, and then add the next batch of physiological saline to finally prepare a collagen solution with a collagen concentration of 5.0 mg / mL.

[0063] (6) Secondary fixation: Take the prepared collagen solution with a concentration of 5.0 mg / mL, homogenize it at 2000 rpm for 60 min, and then let it stand at 28 ℃ for 4 h to complete the secondary fixation.

[0064] (7) Mixing and degassing: After settling, the collagen solution is transferred to a rotating and revolving mixing device for mixing and degassing under vacuum. The mixing and degassing parameters are: rotation speed 200 rpm, revolution speed 500 rpm, temperature 25℃, vacuum degree increased from -50Kpa to -95Kpa, and time 5min. Then, it is filled and sealed to obtain a stable animal-derived collagen solution suitable for injection.

[0065] Comparative Example 1 This comparative example provides a method for preparing an animal-derived collagen solution. The preparation method is basically the same as that in Example 2, except that: in step (1) of this comparative example, pepsin is not added for enzymatic hydrolysis, and there is no dilution with sodium chloride in steps (2) and (3). The dried collagen product is directly prepared into a solution with a collagen concentration of 3.0 mg / mL using 10 mmol / L hydrochloric acid solution. The specific steps include the following: (1) Dissolution: 30 g of dried collagen was added to 10 L of hydrochloric acid solution with a concentration of 10 mmol / L and stirred at 6℃ and 180 rpm for 24 h to obtain a collagen solution with a concentration of 3.0 mg / mL.

[0066] (2) Filtration: Take the dissolved collagen solution of 3.0 mg / mL, stir at 6℃ and 90 rpm for 3h, then filter it once with a 0.2 μm filter and collect the filtered solution.

[0067] Result: The collagen solution clogged the filter and could not pass through the 0.2 μm filter.

[0068] Comparative Example 2 This comparative example provides a method for preparing an animal-derived collagen solution. The preparation method is basically the same as that in Example 2, except that: after adding phosphate buffer and stirring evenly in step (5), no directional fixation is performed to obtain collagen gel. The specific steps include the following: (1) Enzymatic hydrolysis: 60 g of dried collagen was added to 15 L of hydrochloric acid solution with a concentration of 10 mmol / L and stirred at 6℃ and 180 rpm for 24 h to obtain a collagen solution with a concentration of 4.0 mg / mL. 210 g of pepsin was added and reacted at 18℃, stirring at 70 rpm, with an ultrasonic power of 30W and a working cycle of "3s on / 2s off" for 36 h to obtain the enzymatic hydrolysis reaction solution.

[0069] (2) Microfiltration concentration: 15 L of the enzymatic hydrolysis reaction solution was microfiltered using a 30 μm microfilter. After microfiltration, 18 L of 10 mmol / L hydrochloric acid solution was added to obtain approximately 33 L of collagen hydrochloric acid solution. This solution was then passed through an ultrafiltration membrane module with a molecular weight cutoff of 100 kDa, using 10 mmol / L hydrochloric acid solution as the eluent. When the volume of collagen hydrochloric acid solution reached 15 L, 18 L of 10 mmol / L hydrochloric acid solution was added to complete one ultrafiltration cycle. This process was repeated three times. After ultrafiltration, the solution was concentrated at a transmembrane pressure of 0.8 MPa and a membrane flow rate of 2 m / s until the collagen concentration reached 3.7 mg / mL.

[0070] (3) Dilution and filtration: Take 13.5 L of collagen concentrate with a concentration of 3.7 mg / mL, add 4.05 L of 0.3 mol / L NaCl solution, stir at 6 ℃ and 90 rpm for 3 h, then filter once with a 0.2 μm filter and collect the filtered solution.

[0071] (4) Centrifugation: Take 15L of filtered solution and slowly add 0.35 mol / L sodium hydroxide solution. Adjust the pH by 0.2~0.3 units every 10 min. When the pH of the system is adjusted to 4.2, stir for 5 min. Then continue to add sodium hydroxide solution to adjust the pH of the system to 5.8. Stir for 5 min and then continue to add sodium hydroxide solution to adjust the pH of the system to 6.8. After stirring evenly, let it stand overnight at 6 ℃. Then centrifuge at 8000 rpm for 25 min, discard the supernatant, and retain the precipitate (the mass of the precipitate is 8% of the mass of the solution before centrifugation) and detect the protein content.

[0072] (5) Solution preparation: Take 1200 g of the collected centrifuged precipitate into a container, add 180 g of phosphate buffer (phosphate buffer formula: 0.36 mol / L Na2HPO4, 0.16 mol / L NaH2PO4, 2.0 mol / L NaCl), mix well, and let stand overnight at 20℃ to obtain collagen gel; then add 1380 g of water, stir well, and gradually add physiological saline, each time adding 10% of the system mass. After stirring for 10 min, add the next batch of physiological saline to finally prepare a collagen solution with a collagen concentration of 8.0 mg / mL.

[0073] (6) Secondary fixation: Take the prepared collagen solution with a concentration of 8.0 mg / mL, homogenize it at 3000 rpm for 45 min, and then let it stand at 33 ℃ for 3 h to complete the secondary fixation.

[0074] (7) Mixing and degassing: After settling, the collagen solution is transferred to a rotating and revolving mixing device for mixing and degassing under vacuum. The mixing and degassing parameters are: rotation speed 250 rpm, revolution speed 550 rpm, temperature 20℃, vacuum degree increased from -50Kpa to -95Kpa, and time 7min. Then, it is filled and sealed to obtain a stable animal-derived collagen solution suitable for injection.

[0075] Comparative Example 3 This comparative example provides a method for preparing an animal-derived collagen solution. The preparation method is basically the same as that in Example 2, except that: this comparative example does not undergo the secondary fixation and mixing / degassing in steps (6) and (7). Instead, it directly takes the prepared collagen solution with a concentration of 8.0 mg / mL, fills it into a vial or syringe, and seals it to obtain the animal-derived collagen solution. The steps include: A method for preparing a stable, needle-injectable animal-derived collagen solution includes the following steps: (1) Enzymatic hydrolysis: 60 g of dried collagen was added to 15 L of hydrochloric acid solution with a concentration of 10 mmol / L and stirred at 6℃ and 180 rpm for 24 h to obtain a collagen solution with a concentration of 4.0 mg / mL. 210 g of pepsin was added and reacted at 18℃, stirring at 70 rpm, with an ultrasonic power of 100W and a working cycle of "3s on / 2s off" for 36 h to obtain the enzymatic hydrolysis reaction solution.

[0076] (2) Microfiltration concentration: 15 L of the enzymatic hydrolysis reaction solution was microfiltered using a 30 μm microfilter. After microfiltration, 18 L of 10 mmol / L hydrochloric acid solution was added to obtain approximately 33 L of collagen hydrochloric acid solution. This solution was then passed through an ultrafiltration membrane module with a molecular weight cutoff of 100 kDa, using 10 mmol / L hydrochloric acid solution as the eluent. When the volume of collagen hydrochloric acid solution reached 15 L, 18 L of 10 mmol / L hydrochloric acid solution was added to complete one ultrafiltration cycle. This process was repeated three times. After ultrafiltration, the solution was concentrated at a transmembrane pressure of 0.8 MPa and a membrane flow rate of 2 m / s until the collagen concentration reached 3.7 mg / mL.

[0077] (3) Dilution and filtration: Take 13.5 L of collagen concentrate with a concentration of 3.7 mg / mL, add 4.05 L of 0.3 mol / L NaCl solution, stir at 6 ℃ and 90 rpm for 3 h, then filter once with a 0.2 μm filter and collect the filtered solution.

[0078] (4) Centrifugation: Take 15L of filtered solution and slowly add 0.35 mol / L sodium hydroxide solution. Adjust the pH by 0.2~0.3 units every 10 min. When the pH of the system is adjusted to 4.2, stir for 5 min. Then continue to add sodium hydroxide solution to adjust the pH of the system to 5.8. Stir for 5 min and then continue to add sodium hydroxide solution to adjust the pH of the system to 6.8. After stirring evenly, let it stand overnight at 6 ℃. Then centrifuge at 8000 rpm for 25 min, discard the supernatant, and retain the precipitate (the mass of the precipitate is 8% of the mass of the solution before centrifugation) and detect the protein content.

[0079] (5) Directional fixation and solution preparation: Take 1200 g of the collected centrifuged precipitate into a container, add 180 g of phosphate buffer (phosphate buffer formula: 0.36 mol / L Na2HPO4, 0.16 mol / L NaH2PO4, 2.0 mol / L NaCl), mix well, and let stand at 33 ℃ for 5 h. Then tilt the container at 40° and slowly rotate it at 8 rpm, and leave it at 33 ℃ overnight to obtain collagen gel. Then add 1380 g of water for injection, stir well, and gradually add physiological saline, each time adding 10% of the system mass. Stir for 10 min, and then add the next batch of physiological saline to finally prepare a collagen solution with a collagen concentration of 8.0 mg / mL. Then homogenize at 3000 rpm for 45 min, fill into syringes and seal to obtain animal-derived collagen solution.

[0080] Test case (1) Detection of protein structure and properties of animal-derived collagen solution The animal-derived collagen solutions prepared in Examples 1-3 and filled into vials were photographed and their appearance observed. The results are as follows: Figure 1 As shown. From Figure 1 The results show that in the animal-derived collagen solution prepared by the method of the present invention, the collagen fibers are uniformly distributed in the system, and the collagen solutions of different concentrations all exhibit a liquid state ranging from light white to white, and the texture is uniform.

[0081] Take an appropriate amount of collagen solution prepared in step (3) of Examples 1-3 of this invention before and after filtration through a 0.2 μm filter. Dilute the sample with 0.01 mol / L hydrochloric acid to a collagen concentration of 1-2 mg / mL. Perform the determination according to the fifth method of electrophoresis in Part IV, 0541 of the Pharmacopoeia of the People's Republic of China (2020 edition), using SDS-polyacrylamide gel electrophoresis. The separating gel concentration is 7.5%, and the sample volume is 20 μL. Compare the bands of the type I collagen standard and the collagen solution prepared in step (3) of Examples 1-3 before and after filtration through SDS-polyacrylamide gel electrophoresis. The results are as follows:Figure 2 As shown. From Figure 2 The results show that the bands before and after filtration are basically the same, with four to six bands for molecules with a molecular weight of 95 kDa or higher, showing no significant difference. This indicates that the collagen has a complete triple helix structure, has not undergone denaturation or degradation, and possesses biological activity.

[0082] The animal-derived collagen solutions prepared in Examples 1-3 of this invention and filled into vials were subjected to SDS-polyacrylamide gel electrophoresis using the same method described above. The results are as follows: Figure 3 As shown. From Figure 3 The results show that the SDS-polyacrylamide gel electrophoresis bands of the collagen solutions prepared in Examples 1-3 are consistent with the bands of the type I collagen standard control.

[0083] The animal-derived collagen solutions prepared in Examples 1-3 of this invention and filled into vials were diluted with 0.05% acetic acid to a collagen concentration of 0.03 mg / mL. The samples were then scanned using a circular dichroism spectroscopy spectrometer in the range of 190–260 nm. The results are as follows: Figures 4-6 As shown. From Figures 4-6 The results show that the animal-derived collagen solutions prepared in Examples 1-3 have a significant negative absorption peak at around 197 nm and a relatively weak positive absorption peak at around 221 nm. Furthermore, the ratio of the absolute values ​​of the CD values ​​of the positive peak at 221 nm and the negative peak at 197 nm in the animal-derived collagen solutions prepared in Examples 1-3 is 0.13, indicating that the collagen solutions retain the complete triple helix structure.

[0084] Take the animal-derived collagen solution prepared in Examples 1-3 of this invention and fill it into a 1 mL syringe. Remove the hard screw cap of the syringe, install a 32G injection needle, and then fix the sample on the medical syringe sliding tester. The pushing force is measured at a pushing speed of 30 mm / min. The results are shown in Table 1.

[0085] Table 1: Extrusive force of animal-derived collagen solutions in Examples 1-3

[0086] As can be seen from the results in Table 1, the animal-derived collagen solutions prepared in Examples 1-3 can be passed through a 32G fine needle with a pushing force of less than 15N, indicating that the solutions have good fluidity and can improve injection efficiency and accuracy, making them particularly suitable for mesotherapy.

[0087] (2) Turbidity detection of animal-derived collagen solution Animal-derived collagen solutions prepared in Example 2 and Comparative Example 2 were used. The optical density (OD) value at 313 nm wavelength was measured using a microplate reader to monitor the turbidity change of the collagen solution due to fiber formation. During microplate readings, 100 µL of sample was added to a 96-well plate. Under controlled temperature of 37 °C, the OD value was recorded as a function of time on the instrument at least every 30 seconds, and the OD value was measured over 60 minutes. The samples were photographed before testing to observe their appearance. The results are shown in Table 2. Figure 7 and Figure 8 As shown.

[0088] Table 2: Optical density values ​​of animal-derived collagen solutions in Example 2 and Comparative Example 2

[0089] From Table 2 and Figure 7 and 8 The results show that the turbidity of Example 2 is significantly higher than that of Comparative Example 2, indicating that the stability of the fiber structure is significantly improved. Moreover, the turbidity gradually increases at 37 °C and reaches stability after 60 min, indicating that the solution can self-assemble to form collagen fibers under conditions close to human body temperature.

[0090] (3) Stability test of animal-derived collagen solution The animal-derived collagen solutions prepared in Examples 2 and 3 of this invention were filled into syringes, and their appearance was observed by photographing them after three months. The results are as follows. Figure 9 As shown. From Figure 9 The results show that the animal-derived collagen solution prepared in Example 2 is uniformly distributed, does not separate into layers, and has good stability; while the animal-derived collagen solution prepared in Comparative Example 3 shows collagen fiber precipitation and poor stability.

[0091] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for preparing a stable, needle-injectable animal-derived collagen solution, characterized in that, The preparation method includes the following steps: (1) Enzymatic hydrolysis: Add the dried collagen product to the hydrochloric acid solution, stir to dissolve and obtain a collagen solution, add pepsin, continue stirring to react and obtain an enzymatic hydrolysis reaction solution; (2) Microfiltration concentration: The enzymatic reaction solution is microfiltered with a 30 μm microfilter to obtain microfiltrate. Then, hydrochloric acid solution is added to obtain hydrochloric acid solution of collagen. After ultrafiltration, elution and concentration, collagen concentrate is obtained. (3) Dilution and filtration: Add NaCl solution to the collagen concentrate, stir at low temperature, filter through a 0.2 μm filter, and collect the filtered solution; (4) Centrifugal separation: After adjusting the pH of the system to 6.5~7.0, let it stand at low temperature overnight, centrifuge, and collect the precipitate; (5) Directional fixation and solution preparation: Add phosphate buffer to the precipitate, let it stand, tilt the container and let it stand overnight to fix it and obtain collagen gel; then add water, stir evenly, and prepare a collagen solution with a collagen concentration of 5.0~15.0 g / mL with physiological saline. (6) Homogenization and secondary fixation: After homogenizing the prepared collagen solution, let it stand for fixation; (7) Mixing and degassing: After standing, mix thoroughly in a rotating and revolving mixing device, degas under vacuum, fill and seal to obtain a stable animal-derived collagen solution for injection.

2. The preparation method according to claim 1, characterized in that, In step (1), The concentration of the hydrochloric acid solution is 8-12 mmol / L; The stirring and dissolving process is carried out at a temperature of 2~10 ℃, a speed of 150~200 rpm, and a time of 22~26 h. The concentration of the collagen solution is 4.0 mg / mL; The mass ratio of pepsin to dried collagen is (2~5):1; The stirring reaction is carried out at a temperature of 10~25 ℃, a rotation speed of 60~80 rpm, and a time of 24~48 h; Ultrasonic waves are applied during the stirring reaction. The power of the ultrasonic waves is 20-50W, and they operate intermittently in a cycle of 3 seconds on and 10 seconds off.

3. The preparation method according to claim 1, characterized in that, In step (2), The concentration of the hydrochloric acid solution is 10 mmol / L; The volume ratio of the hydrochloric acid solution to the microfiltrate is (1~1.5):1; The ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 50~100kDa; The elution is performed using gradient elution, with the eluent being a 10 mmol / L hydrochloric acid solution, and the elution is performed 5 to 7 times with 1 to 2 times the volume of hydrochloric acid solution. The concentration is achieved using cross-flow concentration, with a transmembrane pressure of 0.5~1.0 MPa and a membrane surface velocity of 1~3 m / s; The concentration of the collagen concentrate is 3.5~4.0 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (3), The concentration of the NaCl solution is 0.2~0.5 mol / L; The volume of the NaCl solution used is 30% of the volume of the collagen concentrate. The low-temperature stirring is carried out at a temperature of 2~10 ℃, a speed of 80~100 rpm, and a time of 2~4 h.

5. The preparation method according to claim 1, characterized in that, In step (4), Adjust the pH of the system to 6.5-7.0 using sodium hydroxide with a concentration of 0.2-0.5 mol / L; The temperature for overnight low-temperature standing is 2~10℃; The centrifugation speed is 7500~8500 rpm, and the time is 20~30 min.

6. The preparation method according to claim 1, characterized in that, In step (5), The phosphate buffer solution is prepared from Na2HPO4, NaH2PO4, NaCl and water, wherein the concentration of Na2HPO4 is 0.26~0.43 mol / L, the concentration of NaH2PO4 is 0.14~0.19 mol / L, and the concentration of NaCl is 1.95~2.10 mol / L. The amount of the phosphate buffer solution used is 15% of the precipitate mass; The settling temperature is 28~37℃, and the time is 3~6h; The container is tilted at an angle of 35~45° and rotates at a speed of 5~10 rpm. The temperature for overnight storage is 28~37℃; The ratio of the mass of water to the mass of collagen gel is 1:

1.

7. The preparation method according to claim 1, characterized in that, In step (6), The homogenization process is carried out at a rotation speed of 2000~4000 rpm for a time of 45~60 min; The static fixation temperature is 28~37 ℃, and the time is 2~4 h.

8. The preparation method according to claim 1, characterized in that, In step (7), In the aforementioned revolution-rotation mixing device, the rotation speed is 200~300 rpm, the revolution speed is 500~700 rpm, the revolution-rotation speed ratio is (2~2.5):1, the temperature is 10~25℃, the vacuum degree is increased from -50Kpa to -95Kpa, and the time is 5~10min.

9. A stable, needle-injectable animal-derived collagen solution, characterized in that, The stable, needle-injectable animal-derived collagen solution is prepared by the preparation method according to any one of claims 1 to 8.

10. The application of a stable, needle-injectable animal-derived collagen solution in the medical and aesthetic fields, characterized in that... The stable, needle-injectable animal-derived collagen solution is the stable, needle-injectable animal-derived collagen solution as described in claim 9, and / or the stable, needle-injectable animal-derived collagen solution prepared by any one of the preparation methods described in claims 1 to 8.

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