Method for preparing functional self-assembled collagen fibers by combining coating and microfluidization

Abstract

The present invention belongs to the field of functional foods and discloses a method for preparing functional self-assembling collagen fibers by coating-microfluidization. Non-denatured type II collagen is extracted from chicken sternal cartilage by enzymatic hydrolysis, induced to self-assemble under appropriate conditions to form non-denatured type II collagen fibers, a sodium alginate coating is applied to the fiber surface by calcium ion induction, and finally homogenized by dynamic high-pressure microfluidization. The non-denatured type II collagen nanofibers coated with calcium ion sodium alginate have good thermal stability and dispersion stability, delaying the release of active fragments during simulated digestion. When used as a carrier for the hydrophobic active substance curcumin, the solubility and antioxidant capacity of curcumin can be significantly improved, achieving slow release of curcumin during simulated gastrointestinal digestion.

Description

Technical Field

[0001] The present application belongs to the field of functional foods. Specifically, the present application provides a method for preparing functional self-assembled collagen fibers by combining coating and microfluidization. Background Art

[0002] With the increasing global aging of the population and the prevalence of bone and joint diseases, the demand for preventive measures through functional foods is growing. Undenatured type II collagen (UC-II) is composed of three uniform α1 peptide chains, forming a complete triple helical structure. Previous studies have shown that UC-II can treat the autoimmune disease rheumatoid arthritis (RA) through "oral tolerance." Oral tolerance is an immune mechanism whereby foreign substances entering the body through the digestive system are recognized and reacted to by the immune system. However, the triple helical structure of UC-II is fragile and easily denatured by temperature, enzymes, and mechanical forces. Type II collagen with a disrupted triple helical structure loses its physiological activity. Zhao Mouming et al. (2021) proposed "a soluble undenatured type II collagen-polysaccharide complex with digestion-resistant properties and its preparation method." By forming a complex of undenatured type II collagen and polysaccharide, they aim to improve its digestive stability. However, they did not consider the thermal stability of UC-II and its potential applications. In thermodynamic equilibrium, collagen molecules can aggregate to form ordered fibers, a process known as collagen self-assembly. In recent years, collagen fibers have become an ideal material for constructing cell scaffolds and developing biomembranes due to their good biocompatibility, degradability and mechanical properties. In addition, food-grade protein fibers have a high aspect ratio and structural flexibility and have been used to encapsulate, protect and release bioactive compounds that are sensitive to environmental changes. It is reported that encapsulation within proteins or complexation with proteins is an effective way to improve the solubility of functional factors in aqueous solutions and increase their bioavailability. However, collagen fibers also have defects such as instability to external environmental stress, such as denaturation at high temperatures and rapid enzymatic hydrolysis under gastrointestinal digestion.

[0003] Coating structures are widely used to develop structured nano-delivery systems to encapsulate, protect and release bioactive substances. Zhang Hao et al. (2020) used electrospinning to prepare alcohol-soluble protein-based core-shell fibers in "Alcohol-soluble protein-based core-shell fiber membranes and food storage and preservation materials and preparation methods" and used them as carriers of curcumin. However, this method is complicated to operate and consumes a large amount of organic solvents, which is not environmentally friendly. In addition, dynamic high-pressure microfluidization technology can be used to improve the physical properties of proteins, such as stability and solubility. Wang Jinmei et al. (2018) proposed a method for "preparing stable soybean protein-sterol particles by combining heat treatment with high-pressure microfluidization treatment" to produce sterol delivery carriers with smaller particle size and higher stability. However, there have been no reports on the use of microfluidization technology for the stabilization of collagen fibers. Summary of the Invention

[0004] Sodium alginate is a natural polysaccharide that contains negatively charged carboxyl groups in its molecular structure. When calcium ions bind to negatively charged carboxyl groups, crosslinks are formed between the calcium ions and the sodium alginate. This crosslinked structure can form a three-dimensional network in the sodium alginate. Dynamic high-pressure microfluidization technology is an effective means to improve the structural and functional properties of macromolecular substances. In this application, calcium ion-induced sodium alginate coating is used in conjunction with dynamic high-pressure microfluidization technology to improve the stability of non-denatured type II collagen nanofibers and achieve the function of loading and slowly releasing the bioactive substance curcumin.

[0005] In one aspect, the present application provides a method for preparing functional self-assembled collagen fibers by combining coating and microfluidization, the method comprising:

[0006] (1) dispersing non-denatured type II collagen in acetic acid at a mass ratio of 0.05-5%, then mixing evenly with PBS buffer, adjusting the pH to 4.0-7.0, and incubating to obtain non-denatured type II collagen nanofibers;

[0007] (2) dissolving sodium alginate in deionized water to obtain a sodium alginate solution with a mass fraction of 0.1-2%; injecting non-denatured type II collagen nanofibers into the sodium alginate solution under magnetic stirring to obtain a non-denatured type II collagen-sodium alginate mixed dispersion; and dripping a CaCl2 solution into the non-denatured type II collagen-sodium alginate mixed dispersion under magnetic stirring to obtain a non-denatured type II collagen dispersion with a calcium ion-induced sodium alginate coating;

[0008] (3) The dispersion obtained in step (2) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 8-24 hours to remove uncrosslinked calcium ions;

[0009] (4) Add the dispersion obtained in step (3) into a dynamic high-pressure microfluidizer, circulate and homogenize 1-3 times under 60-180 MPa conditions, and maintain the homogenization temperature at 20-30° C. to obtain non-denatured type II collagen nanofibers coated with sodium alginate.

[0010] Furthermore, the method further comprises: (1-2) preparing an 8-10 mg / mL curcumin stock solution with ethanol; and mixing the curcumin stock solution with the non-denatured type II collagen nanofibers under magnetic stirring.

[0011] On the other hand, the present application provides a high-stability collagen nanofiber, which is prepared according to the following method:

[0012] (1) dispersing non-denatured type II collagen in acetic acid at a mass ratio of 0.05-5%, then mixing evenly with PBS buffer, adjusting the pH to 4.0-7.0, and incubating to obtain non-denatured type II collagen nanofibers;

[0013] (2) dissolving sodium alginate in deionized water to obtain a sodium alginate solution with a mass fraction of 0.1-2%; injecting non-denatured type II collagen nanofibers into the sodium alginate solution under magnetic stirring to obtain a non-denatured type II collagen-sodium alginate mixed dispersion; and dripping a CaCl2 solution into the non-denatured type II collagen-sodium alginate mixed dispersion under magnetic stirring to obtain a non-denatured type II collagen dispersion with a calcium ion-induced sodium alginate coating;

[0014] (3) The dispersion obtained in step (2) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 8-24 hours to remove uncrosslinked calcium ions;

[0015] (4) Add the dispersion obtained in step (3) into a dynamic high-pressure microfluidizer, circulate and homogenize 1-3 times under 60-180 MPa conditions, and maintain the homogenization temperature at 20-30° C. to obtain non-denatured type II collagen nanofibers coated with sodium alginate.

[0016] On the other hand, the present application provides a highly stable collagen nanofiber with curcumin loading function, wherein the collagen nanofiber is prepared according to the following method:

[0017] (1) dispersing non-denatured type II collagen in acetic acid at a mass ratio of 0.05-5%, then mixing evenly with PBS buffer, adjusting the pH to 4.0-7.0, and incubating to obtain non-denatured type II collagen nanofibers;

[0018] (1-2) preparing a curcumin stock solution of 8-10 mg / mL in ethanol; mixing the curcumin stock solution with non-denatured type II collagen nanofibers under magnetic stirring;

[0019] (2) dissolving sodium alginate in deionized water to obtain a sodium alginate solution with a mass fraction of 0.1-2%; injecting non-denatured type II collagen nanofibers into the sodium alginate solution under magnetic stirring to obtain a non-denatured type II collagen-sodium alginate mixed dispersion; and dripping a CaCl2 solution into the non-denatured type II collagen-sodium alginate mixed dispersion under magnetic stirring to obtain a non-denatured type II collagen dispersion with a calcium ion-induced sodium alginate coating;

[0020] (3) The dispersion obtained in step (2) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 8-24 hours to remove uncrosslinked calcium ions and free curcumin;

[0021] (4) adding the dispersion obtained in step (3) into a dynamic high-pressure microfluidizer and performing circulation homogenization to obtain non-denatured type II collagen nanofibers with a sodium alginate coating loaded with curcumin.

[0022] Furthermore, in step (1), non-denatured type II collagen is dispersed in acetic acid at a mass ratio of 0.1%.

[0023] Furthermore, in step (2), the mass ratio of non-denatured type II collagen nanofibers to sodium alginate in the non-denatured type II collagen-sodium alginate mixed dispersion obtained by injecting the non-denatured type II collagen nanofibers into the sodium alginate solution is 1:1.

[0024] Furthermore, the molar concentrations of acetic acid and PBS buffer in step (1) are both 0.1M.

[0025] Furthermore, in step (1), the non-denatured type II collagen is dispersed in acetic acid at a mass ratio of 0.05-5% in an ice bath, and then mixed evenly with a PBS buffer solution.

[0026] Furthermore, the incubation condition in step (1) is 37° C. for 60 min.

[0027] Furthermore, the magnetic stirring conditions in step (2) are all 600 rpm, 30 min.

[0028] Furthermore, the mass ratio of the non-denatured type II collagen nanofibers to the sodium alginate in the non-denatured type II collagen-sodium alginate mixed dispersion in step (2) is 0.1-10.

[0029] Furthermore, in step (2), the CaCl2 solution is dripped into the solution obtained from the non-denatured type II collagen-sodium alginate mixed dispersion. 2+ The concentration is 1-10 mM, preferably 5-10 mM.

[0030] Furthermore, the centrifugation condition in step (2) is 10000 rpm, 10 min.

[0031] Furthermore, the pH value of the PBS buffer in step (2) is 5.0, and the concentration of the non-denatured type II collagen in the non-denatured type II collagen-sodium alginate mixed dispersion is 0.1%.

[0032] Furthermore, in step (1-2), the mass ratio of curcumin to non-denatured type II collagen is 0.01-0.10.

[0033] Furthermore, the magnetic stirring condition in step (1-2) is 600 rpm for 30 min.

[0034] Furthermore, the dialysis time in step (3) is 20 hours.

[0035] Furthermore, in step (4), the homogenization pressure is 60-180 MPa, the homogenization times are 1-3 times, and the homogenization temperature is 20-30°C.

[0036] The non-denatured type II collagen material in this application can be prepared using any material known in the art, or purchased as a commercially available product. In a non-limiting manner, the following method is used for preparation:

[0037] Pretreatment: Remove meat and other tissues attached to the surface of the chicken breast cartilage, rinse with deionized water, and cut into small pieces. Degreasing: Soak the chicken breast cartilage pieces in 2-5 times the volume of 1-5% NaOH solution for 8-24 hours, centrifuge to obtain Precipitate I. Protein Removal: Treat with 2-5 times the volume of Precipitate I at 1-5 mol / L NaCl, stir for 8-24 hours, and centrifuge to obtain Precipitate II. Enzymatic Hydrolysis: Disperse Precipitate II in 2-5 times the volume of Precipitate II in 0.05 M acetic acid containing 0.01-0.1% pepsin, stir, centrifuge, and obtain the supernatant. Salting-out: Salt the supernatant with 1-5 M NaCl, centrifuge to obtain Precipitate III. Dialysis Desalting: Redissolve Precipitate III in 0.5 M acetic acid, dialyze against deionized water, and freeze-dry. This is preferably performed at 4°C and centrifuged at 12,000 rpm for 20 minutes. DETAILED DESCRIPTION

[0038] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to be limiting. These examples are for illustrative purposes only and in no way limit the scope of protection of the present invention.

[0039] Comparative Example 1

[0040] (1) Non-denatured type II collagen extraction:

[0041] Pretreatment: Remove meat and other tissues attached to the surface of the chicken sternal cartilage, wash with deionized water, and cut into small pieces; Degreasing: Soak the small pieces of chicken sternal cartilage in 5 times the volume of 1% NaOH solution for 15 hours, and centrifuge to obtain Precipitate I; Protein Removal: Treat with 1.5 mol / L NaCl 5 times the volume of Precipitate I, stir for 20 hours, and centrifuge to obtain Precipitate II; Enzymatic Hydrolysis: Disperse Precipitate II in 0.05 M acetic acid containing 0.05% pepsin 5 times the volume of Precipitate II, stir, centrifuge, and obtain the supernatant; Salting Out: Salt the supernatant with 3 M NaCl, and centrifuge to obtain Precipitate III; Dialysis Desalting: Redissolve Precipitate III in 0.5 M acetic acid, dialyze against deionized water, and freeze-dry.

[0042] (2) Preparation of non-denatured type II collagen self-assembled fibers:

[0043] Non-denatured type II collagen was dispersed in 0.1 M acetic acid at a mass ratio of 0.1%, and then mixed evenly with an equal volume of 0.1 M PBS buffer to a final collagen concentration of 0.05%. The pH was adjusted to 5.0, and the mixture was incubated in a 37°C water bath for 60 minutes to obtain non-denatured type II collagen nanofibers.

[0044] Comparative Example 2

[0045] Steps (1) and (2) refer to Comparative Example 1.

[0046] (3) High-pressure microfluidization homogenization:

[0047] The dispersion obtained in step (2) was added to a dynamic high-pressure microfluidizer and circulated and homogenized twice at 120 MPa, with the homogenization temperature maintained at 25° C., to obtain non-denatured type II collagen nanofibers coated with sodium alginate without calcium ion induction.

[0048] Comparative Example 3

[0049] Steps (1) and (2) refer to Comparative Example 1.

[0050] (3) Calcium ion-induced sodium alginate coating:

[0051] Sodium alginate was dissolved in deionized water to obtain a sodium alginate stock solution with a mass fraction of 0.1%. Under the conditions of magnetic stirring at 600 rpm for 30 min, non-denatured type II collagen nanofibers were injected into the sodium alginate solution using a 2 mL syringe. The mass ratio of non-denatured type II collagen nanofibers to sodium alginate was 1:1, and calcium ion-induced sodium alginate-coated non-denatured type II collagen nanofibers were obtained. Under the conditions of magnetic stirring at 600 rpm for 30 min, 1% CaCl2 solution was added dropwise to the above non-denatured type II collagen-sodium alginate mixed dispersion using a 2 mL syringe. Finally, the mixed dispersion CaCl2 was added dropwise to the above non-denatured type II collagen-sodium alginate mixed dispersion. 2+ The concentration was 5 mM, and a non-denatured type II collagen dispersion was obtained for calcium ion-induced sodium alginate coating.

[0052] (4) Dialysis:

[0053] The dispersion obtained in step (3) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 20 hours to remove uncrosslinked calcium ions, thereby obtaining sodium alginate-coated non-denatured type II collagen nanofibers.

[0054] Example 1

[0055] Steps (1) and (2) refer to Comparative Example 1.

[0056] (3) Calcium ion-induced sodium alginate coating:

[0057] Sodium alginate was dissolved in deionized water to obtain a sodium alginate stock solution with a mass fraction of 0.1%. Under the conditions of magnetic stirring at 600 rpm for 30 min, non-denatured type II collagen nanofibers were injected into the sodium alginate solution using a 2 mL syringe. The mass ratio of non-denatured type II collagen nanofibers to sodium alginate was 1:1, and calcium ion-induced sodium alginate-coated non-denatured type II collagen nanofibers were obtained. Under the conditions of magnetic stirring at 600 rpm for 30 min, 1% CaCl2 solution was added dropwise to the above non-denatured type II collagen-sodium alginate mixed dispersion using a 2 mL syringe. Finally, the mixed dispersion CaCl2 was added dropwise to the above non-denatured type II collagen-sodium alginate mixed dispersion. 2+ The concentration was 5 mM, and a non-denatured type II collagen dispersion was obtained for calcium ion-induced sodium alginate coating.

[0058] (4) Dialysis:

[0059] The dispersion obtained in step (3) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 20 hours to remove uncrosslinked calcium ions, thereby obtaining sodium alginate-coated non-denatured type II collagen nanofibers.

[0060] (5) High-pressure microfluidization homogenization:

[0061] The dispersion obtained in step (4) was added into a dynamic high-pressure microfluidizer and circulated and homogenized twice at 120 MPa, with the homogenization temperature maintained at 25° C., to obtain sodium alginate-coated non-denatured type II collagen nanofibers.

[0062] The Turbiscan was used to measure the intensity of the transmitted light and backscattered light of the sample within 24 hours to obtain the stability index (TSI), thereby quantifying the dispersion stability. The TSI reflects the combined changes in the sample volume concentration and particle size within a specified measurement time. The smaller the TSI, the better the dispersion stability. The thermal denaturation temperature (T d) The melting temperature (T m ). Circular dichroism was used to determine the retention rate of the triple helical structure of non-denatured type II collagen after gastrointestinal digestion. The results are shown in the following table:

[0063] Table 1 Stability index (TSI), thermal denaturation temperature (T d ), thermal melting temperature (T m ), triple helix structure retention rate after simulated gastrointestinal digestion in vitro.

[0064]

[0065] As shown in Table 1, the TSI values of Comparative Example 2 and Example 1 were lower than those of Comparative Examples 1 and 3, demonstrating that high-pressure microfluidization significantly improves the dispersion stability of UC-II. The minimal TSI of Example 1 further demonstrates that the presence of the calcium-induced sodium alginate coating has a positive effect on the dispersion stability of UC-II, likely due to the interaction of electrostatic repulsion. Comparative Example 3 and Example 1 exhibited improved thermal stability and higher post-digestion triple helical structure retention, demonstrating that the calcium-induced sodium alginate coating effectively improves the thermal stability of UC-II and protects the active structure of non-denatured type II collagen during gastrointestinal digestion. The combined use of the calcium-induced sodium alginate coating and dynamic high-pressure microfluidization technology yielded the best overall improvement in UC-II fiber stability, demonstrating a synergistic effect between the two.

[0066] Comparative Example 4

[0067] Steps (1) and (2) refer to Comparative Example 1.

[0068] (3) 8 mg / mL curcumin stock solution was prepared in ethanol; the curcumin stock solution was mixed with non-denatured type II collagen nanofibers under magnetic stirring, and the mass ratio of curcumin to UC-II in the dispersion was 0.06.

[0069] (4) Dialysis:

[0070] The dispersion obtained in step (3) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 20 hours to remove free curcumin.

[0071] Comparative Example 5

[0072] Steps (1) and (2) refer to Comparative Example 1.

[0073] (3) 8 mg / mL curcumin stock solution was prepared in ethanol; the curcumin stock solution was mixed with non-denatured type II collagen nanofibers under magnetic stirring, and the mass ratio of curcumin to UC-II in the dispersion was 0.06.

[0074] (4) Dialysis:

[0075] The dispersion obtained in step (3) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 20 hours to remove free curcumin.

[0076] (5) High-pressure microfluidization homogenization:

[0077] The dispersion obtained in step (4) was added to a dynamic high-pressure microfluidizer and circulated and homogenized twice at 120 MPa, with the homogenization temperature maintained at 25° C. to obtain non-denatured type II collagen nanofibers loaded with curcumin.

[0078] Comparative Example 6

[0079] Steps (1) and (2) refer to Comparative Example 1.

[0080] (3) 8 mg / mL curcumin stock solution was prepared in ethanol; the curcumin stock solution was mixed with non-denatured type II collagen nanofibers under magnetic stirring, and the mass ratio of curcumin to UC-II in the dispersion was 0.06.

[0081] (4) Calcium ion-induced sodium alginate coating:

[0082] Sodium alginate was dissolved in deionized water to obtain a sodium alginate stock solution with a mass fraction of 0.1%. Under the conditions of magnetic stirring at 600 rpm for 30 min, non-denatured type II collagen nanofibers were injected into the sodium alginate solution using a 2 mL syringe. The mass ratio of non-denatured type II collagen nanofibers to sodium alginate was 1:1, and calcium ion-induced sodium alginate-coated non-denatured type II collagen nanofibers were obtained. Under the conditions of magnetic stirring at 600 rpm for 30 min, 1% CaCl2 solution was added dropwise to the above non-denatured type II collagen-sodium alginate mixed dispersion using a 2 mL syringe. Finally, the mixed dispersion CaCl2 was added dropwise to the above non-denatured type II collagen-sodium alginate mixed dispersion. 2+ The concentration is 5mM.

[0083] (5) Dialysis:

[0084] The dispersion obtained in step (4) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 20 hours to remove free curcumin.

[0085] Example 2

[0086] Steps (1) and (2) refer to Comparative Example 1.

[0087] (3) 8 mg / mL curcumin stock solution was prepared in ethanol; the curcumin stock solution was mixed with non-denatured type II collagen nanofibers under magnetic stirring, and the mass ratio of curcumin to UC-II in the dispersion was 0.06.

[0088] (4) Calcium ion-induced sodium alginate coating:

[0089] Sodium alginate was dissolved in deionized water to obtain a sodium alginate stock solution with a mass fraction of 0.1%. Under the conditions of magnetic stirring at 600 rpm for 30 min, non-denatured type II collagen nanofibers were injected into the sodium alginate solution using a 2 mL syringe. The mass ratio of non-denatured type II collagen nanofibers to sodium alginate was 1:1, and calcium ion-induced sodium alginate-coated non-denatured type II collagen nanofibers were obtained. Under the conditions of magnetic stirring at 600 rpm for 30 min, 1% CaCl2 solution was added dropwise to the above non-denatured type II collagen-sodium alginate mixed dispersion using a 2 mL syringe. Finally, the mixed dispersion CaCl2 was added dropwise to the above non-denatured type II collagen-sodium alginate mixed dispersion. 2+ The concentration was 5 mM, and a non-denatured type II collagen dispersion was obtained for calcium ion-induced sodium alginate coating.

[0090] (5) Dialysis:

[0091] The dispersion obtained in step (4) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 20 hours to remove uncrosslinked calcium ions and free curcumin.

[0092] (6) High-pressure microfluidization homogenization:

[0093] The dispersion obtained in step (5) was added to a dynamic high-pressure microfluidizer and circulated and homogenized twice at 120 MPa, with the homogenization temperature maintained at 25° C. to obtain non-denatured type II collagen nanofibers coated with sodium alginate loaded with curcumin.

[0094] UV-visible spectrophotometry at 420 nm was used to evaluate the curcumin loading capacity of the UC-II nanofibers and the amount of curcumin released during in vitro gastrointestinal digestion. DPPH and ABTS free radical scavenging assays were used to test the antioxidant activity of curcumin. The results are shown in the table below:

[0095] Table 2 shows the solubility, antioxidant capacity and release rate of curcumin during in vitro gastrointestinal digestion of comparative examples 4-6 and example 2.

[0096]

[0097] As shown in Table 2, the solubility of curcumin in Comparative Examples 4, 5, and 6 was 37.36, 38.61, and 48.37 μg / mL, respectively, while the solubility of curcumin in Example 2 reached 50.22 μg / mL. This demonstrates that non-denatured type II collagen nanofibers are an excellent carrier for curcumin, and that sodium alginate coating and high-pressure microfluidization significantly enhance the ability of non-denatured type II collagen nanofibers to load curcumin. Consequently, curcumin's antioxidant capacity, including its DPPH and ABTS free radical scavenging abilities, was also enhanced, primarily due to the increased availability of water-dissolved curcumin. Due to the calcium-induced physical interception and hindrance of digestive enzymes by the sodium alginate coating, Comparative Example 6 and Example 2 exhibited slower curcumin release during in vitro simulated gastrointestinal digestion, indicating a high degree of curcumin-to-non-denatured type II collagen nanofiber complexation. Dynamic high-pressure microfluidization did not negatively impact curcumin's antioxidant capacity or digestive sustained-release properties. In general, the coating-microfluidization combination is an effective technical means to prepare highly stable self-assembled non-denatured type II collagen fibers with the function of loading the functional factor curcumin.

Claims

1. A method for preparing functional self-assembled collagen fibers by combining coating and microfluidization, characterized in that: The method comprises: (1) Dispersing non-denatured type II collagen at a mass ratio of 0.1% in 0.1 M acetic acid in an ice bath, then mixing evenly with an equal volume of 0.1 M PBS buffer, adjusting the pH to 5.0, and incubating to obtain non-denatured type II collagen nanofibers; (2) Sodium alginate is dissolved in deionized water to obtain a sodium alginate solution with a mass fraction of 0.1%; under magnetic stirring, non-denatured type II collagen nanofibers are injected into the sodium alginate solution to obtain a non-denatured type II collagen-sodium alginate mixed dispersion, wherein the mass ratio of non-denatured type II collagen nanofibers to sodium alginate is 1:1; under magnetic stirring, CaCl2 solution is dropped into the non-denatured type II collagen-sodium alginate mixed dispersion until CaCl2 solution is dissolved in the sodium alginate solution. 2+ The concentration is 5 mM; (3) The dispersion obtained in step (2) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 8-24 hours; (4) adding the dispersion obtained in step (3) into a dynamic high-pressure microfluidizer and homogenizing the dispersion in a cycle to obtain non-denatured type II collagen nanofibers coated with sodium alginate.

2. The method according to claim 1, wherein the incubation conditions in step (1) are 37°C for 60 min.

3. The method according to claim 1, wherein in step (4), the homogenization pressure is 60-180 MPa, the homogenization times are 1-3 times, and the homogenization temperature is 20-30°C.

4. A high-stability collagen nanofiber, characterized in that: The collagen nanofibers are prepared according to the following method: (1) Dispersing non-denatured type II collagen at a mass ratio of 0.1% in 0.1 M acetic acid in an ice bath, then mixing evenly with an equal volume of 0.1 M PBS buffer, adjusting the pH to 5.0, and incubating to obtain non-denatured type II collagen nanofibers; (2) dissolving sodium alginate in deionized water to obtain a sodium alginate solution with a mass fraction of 0.1%; Under magnetic stirring conditions, non-denatured type II collagen nanofibers were injected into sodium alginate solution to obtain a non-denatured type II collagen-sodium alginate mixed dispersion, wherein the mass ratio of non-denatured type II collagen nanofibers to sodium alginate was 1:1; under magnetic stirring conditions, CaCl2 solution was dropped into the non-denatured type II collagen-sodium alginate mixed dispersion until CaCl2 solution was added to the dispersion. 2+ The concentration is 5 mM; (3) The dispersion obtained in step (2) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 8-24 hours; (4) adding the dispersion obtained in step (3) into a dynamic high-pressure microfluidizer and homogenizing the dispersion in a cycle to obtain non-denatured type II collagen nanofibers coated with sodium alginate.

5. The collagen nanofibers according to claim 4, wherein the incubation conditions in step (1) are 37°C for 60 min.

6. The collagen nanofibers according to claim 4, wherein in step (4), the homogenization pressure is 60-180 MPa, the number of homogenizations is 1-3 times, and the homogenization temperature is 20-30°C.

7. A highly stable, curcumin-loaded collagen nanofiber, characterized in that: The collagen nanofibers are prepared according to the following method: (1) Dispersing non-denatured type II collagen in 0.1 M acetic acid at a mass ratio of 0.1% in an ice bath, then mixing evenly with an equal volume of 0.1 M PBS buffer, adjusting the pH to 5.0, and incubating to obtain non-denatured type II collagen nanofibers; preparing an 8-10 mg / mL curcumin stock solution with ethanol; mixing the curcumin stock solution with the non-denatured type II collagen nanofibers under magnetic stirring; in the dispersion of the mixed curcumin stock solution and the non-denatured type II collagen nanofibers, the mass ratio of curcumin to non-denatured type II collagen is 0.01-0.10; (2) dissolving sodium alginate in deionized water to obtain a sodium alginate solution with a mass fraction of 0.1%; Under magnetic stirring conditions, the curcumin stock solution obtained in step (1) and the dispersion mixed with the non-denatured type II collagen nanofibers were injected into the sodium alginate solution to obtain a non-denatured type II collagen-sodium alginate mixed dispersion, wherein the mass ratio of the non-denatured type II collagen nanofibers to the sodium alginate was 1:1; under magnetic stirring conditions, the CaCl2 solution was dropped into the non-denatured type II collagen-sodium alginate mixed dispersion until the CaCl2 solution was dissolved in the solution. 2+ The concentration is 5 mM; (3) The dispersion obtained in step (2) was sealed in a 10 kDa dialysis bag and dialyzed in deionized water for 8-24 hours; (4) adding the dispersion obtained in step (3) into a dynamic high-pressure microfluidizer and homogenizing the dispersion in a cycle to obtain non-denatured type II collagen nanofibers coated with sodium alginate.

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