Coprocessing method for improving stability of type III collagen

Abstract

The invention discloses a synergistic treatment method for improving the stability of three-type collagen, and belongs to the technical field of stability regulation and control of biomacromolecules. The method comprises the following steps: dissolving purified type III collagen in a buffer solution, and stirring until the type III collagen is completely dissolved; adjusting the pH value of the collagen solution to 5.5-6.5 by using a pH regulator, adding a composite protective agent into the collagen solution with the adjusted pH value, stirring until the collagen solution is completely dissolved, and then carrying out temperature gradient treatment to obtain a type III collagen solution subjected to temperature gradient treatment; and storing the type III collagen solution subjected to temperature gradient treatment under the condition that the temperature is 20-30 DEG C. Through the synergistic effect of pH regulation and control, addition of the composite protective agent and temperature gradient treatment, the structural stability of the type III collagen can be remarkably improved, and the effective preservation period of the type III collagen can be prolonged.

Description

Technical Field

[0001] This invention relates to a synergistic treatment method for improving the stability of type III collagen, belonging to the field of biomolecular stability regulation technology. Background Technology

[0002] Type III collagen, a fibrous protein with a unique triple helix structure, has been widely used in medical aesthetics, wound healing, and tissue engineering due to its excellent biocompatibility, low immunogenicity, and ability to promote cell proliferation and tissue repair. However, as a key protein for maintaining tissue elasticity and repair function, the stability of type III collagen directly determines the quality and effectiveness of related products. The molecular structure of type III collagen is extremely sensitive to external environmental factors. During post-production processing, storage, transportation, and actual application, it is prone to structural denaturation, molecular aggregation, or degradation due to pH fluctuations or temperature changes. This leads to reduced bioactivity, decreased solubility, and even loss of application value, significantly limiting its industrial application and extending product shelf life.

[0003] Currently, existing technologies for improving the stability of type III collagen mainly include genetic engineering, purification techniques, and freeze-drying processes. However, these methods are not only costly and complex, or prone to causing loss of type III collagen activity, but also offer limited regulation and cannot resist the influence of complex environments on the stability of type III collagen. For example, adding a single protective agent or storing at low temperatures can be problematic. A single protective agent has limited resistance to extreme pH or high temperature environments and cannot comprehensively inhibit collagen denaturation and degradation. While low-temperature freezing can extend shelf life, it increases storage and transportation costs, and during thawing, ice crystals can easily form, causing collagen molecules to aggregate, affecting its solubility and application effects.

[0004] Therefore, developing a multi-faceted synergistic method to improve stability is of great significance for solving the storage stability problem of type III collagen. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a synergistic treatment method for improving the stability of type III collagen. Through the synergistic effect of pH regulation, addition of compound protective agents, and temperature gradient treatment, the structural stability of type III collagen can be significantly improved, and its effective storage period can be extended.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: A synergistic treatment method for improving the stability of type III collagen includes the following steps: (1) Dissolve the purified type III collagen in buffer solution and stir until completely dissolved to obtain a collagen solution; (2) Adjust the pH of the collagen solution with a pH adjuster to obtain a pH-adjusted collagen solution; (3) Add a complex protective agent to the collagen solution that has been adjusted to pH, and stir until completely dissolved to obtain a collagen solution with added complex protective agent; (4) The collagen solution with added compound protective agent was subjected to temperature gradient treatment to obtain a temperature gradient treated type III collagen solution. (5) Store the type III collagen solution treated with temperature gradient at a temperature of 20-30℃.

[0007] Furthermore, in step (1), the type III collagen includes human type III collagen prepared by recombinant microbial fermentation and type III collagen extracted and purified from animal tissues. The type III collagen prepared by recombinant microbial fermentation is derived from recombinant Escherichia coli or yeast carrying the human type III collagen gene.

[0008] Furthermore, the purified type III collagen has a purity of ≥98% and a molecular weight of 45 kDa.

[0009] Furthermore, the purification involves loading the type III collagen stock solution onto a chromatography column and sequentially performing equilibration, washing, and elution operations.

[0010] Furthermore, in step (1), the stirring temperature is 4-10℃ and the stirring time is 30-60min.

[0011] Furthermore, in step (1), the concentration of the collagen solution is 0.5-2.0 mg / mL.

[0012] Further, in step (1), the buffer solution is a citrate-sodium citrate buffer solution with a concentration of 0.01-0.05 mol / L or a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a concentration of 0.01-0.05 mol / L.

[0013] Furthermore, the pH of both the citrate-sodium citrate buffer and the sodium dihydrogen phosphate-disodium hydrogen phosphate buffer is 6.

[0014] Furthermore, in step (2), the pH adjuster is an HCl solution or NaOH solution with a concentration of 0.1-1 mol / L; an automatic pH titrator is used for precise adjustment, with the adjustment accuracy controlled within ±0.05 pH units.

[0015] Furthermore, in step (2), the pH of the collagen solution is 5.5-6.5. The isoelectric point of type III collagen is pI≈5.8-6.2. This invention limits the pH to 5.5-6.5. At this pH, the net charge of collagen molecules is close to zero, the electrostatic repulsion between molecules is minimal, which is conducive to the collagen molecular chains approaching each other and the triple helix structure is stable.

[0016] Furthermore, in step (2), when adjusting the pH, the temperature is 4-10℃ and the stirring speed is 50-100rpm. Maintaining the temperature and stirring speed during pH adjustment can prevent sudden changes in local pH that could damage the structure of type III collagen.

[0017] Furthermore, in step (3), the mass ratio of the composite protective agent to type III collagen is 1:10.

[0018] Furthermore, in step (3), the composite protective agent is composed of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, hydroxytyrosol-gelatin conjugate, and hydroxypropyl-β-cyclodextrin.

[0019] Furthermore, the mass ratio of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, hydroxytyrosol-gelatin conjugate, and hydroxypropyl-β-cyclodextrin is (0.05-1.0):(0.01-0.5):(0.1-2.0):(0.04-1.5):(1.0-5.0).

[0020] Hydroxypropyl-β-cyclodextrin serves as the basic dispersed phase, encapsulating easily degradable components, adjusting solution viscosity, and reducing the risk of thermal motion and hydrolysis of collagen molecules. Hydroxytyrosol-gelatin conjugates form intermolecular covalent crosslinks with type III collagen, providing localized high-concentration antioxidant protection and enhancing the mechanical strength and anti-degradation ability of the collagen fiber network. Glutathione scavenge water-soluble free radicals, and fucoxanthin nanocomplexes scavenge lipid-soluble free radicals, together forming a comprehensive antioxidant system to protect collagen from oxidation. N-acetylneuraminic acid has extremely strong hydrophilicity, forming a dense hydration layer on the surface of collagen molecules, generating strong steric hindrance, increasing the electrostatic repulsion between collagen molecules, and further maintaining its dispersion stability.

[0021] Furthermore, in step (3), the preparation method of the composite protective agent is as follows: (3-1) Add hydroxypropyl-β-cyclodextrin to deionized water and stir for 20 min at 50-60℃ and 300 rpm. After clarification, cool to 35℃. (3-2) Add glutathione, stir for 15-20 min, then add N-acetylneuraminic acid, stir for 10-15 min, adjust the pH to 6.0 with 0.1 mol / L NaOH solution to obtain the solution; (3-3) Fucoxanthin nanocomposite and hydroxytyrosol-gelatin conjugate were dissolved in 50wt.% ethanol and sonicated at 300W for 5min to prepare a solution with a concentration of 110mg / mL. The solution was added dropwise to the solution in step (3-2) at a speed of 1mL / min and stirred at 500rpm for 20min to obtain a mixed solution. (3-4) The mixed solution was homogenized three times at 25 MPa and filtered through a 0.22 μm filter membrane to obtain the composite protective agent.

[0022] Furthermore, the preparation method of the hydroxytyrosol-gelatin conjugate is as follows: (a) Add PBS buffer to the gelatin and stir continuously in a water bath until completely dissolved to obtain a gelatin solution; (b) Dissolve hydroxytyrosol in PBS buffer to obtain a hydroxytyrosol solution; (c) Cool the gelatin solution to room temperature, add hydroxytyrosol solution dropwise while stirring continuously, mix well, then add tyrosinase solution, place in a constant temperature water bath and shake to react, to obtain a mixed solution; (d) The mixed solution was heated in a water bath and then cooled to room temperature in an ice-water bath to obtain a hydroxytyrosol-gelatin conjugate solution; (e) The hydroxytyrosol-gelatin conjugate solution was dialyzed, pre-frozen overnight, and then freeze-dried to obtain the hydroxytyrosol-gelatin conjugate.

[0023] Hydroxytyrosol is highly reactive and easily oxidized and degraded. Covalently binding it with the natural biopolymer gelatin to form a hydroxytyrosol-gelatin conjugate fundamentally solves the problem of hydroxytyrosol's chemical instability and prevents premature oxidation. Gelatin is structurally homologous to collagen and binds to type III collagen through hydrogen bonding and other interactions, allowing hydroxytyrosol to remain continuously around collagen molecules, exerting its antioxidant effect, filling in intermolecular gaps, and reducing conformational fluctuations. This conjugate can slowly release hydroxytyrosol under physiological conditions through hydrolysis or enzymatic hydrolysis, providing long-lasting antioxidant protection, retaining good free radical scavenging ability, and possessing the property of self-assembling into colloids or nanoparticles.

[0024] Furthermore, in step (a), the concentration of gelatin in the gelatin solution is 2.0 wt.%.

[0025] Furthermore, in step (a), the stirring temperature is 40°C, the stirring speed is 300 rpm, and the stirring time is 30-60 min.

[0026] Furthermore, in step (b), the concentration of the hydroxytyrosol solution is 20 mg / mL.

[0027] Furthermore, in step (c), the mass ratio of hydroxytyrosol to gelatin is 1:10.

[0028] Furthermore, in step (c), the tyrosinase solution is prepared by dissolving tyrosinase in ice-cold PBS buffer to prepare a solution with an activity of 5000 U / mL, which is the tyrosinase solution.

[0029] Furthermore, in step (c), the amount of tyrosinase added to the mixed solution is 100-200 U / mL.

[0030] Furthermore, in step (c), the temperature of the constant temperature water bath is 25°C; the rotation speed of the oscillation reaction is 150 rpm; and the oscillation reaction time is 24 h.

[0031] Furthermore, in step (d), the water bath temperature is 90°C; the heating time is 10 min.

[0032] Further, in step (e), the specific operation of dialysis is as follows: the hydroxytyrosol-gelatin conjugate solution is placed into a dialysis bag with a molecular weight cutoff of 8-10 kDa, and dialyzed with ultrapure water. The dialysis fluid is replaced every 6-8 hours at 4°C, and dialysis is performed continuously for 48-72 hours until no absorption peak is detected in the dialysis fluid at 270-280 nm using a UV spectrophotometer.

[0033] Furthermore, in step (e), the pre-freezing temperature is -80°C; the freeze-drying temperature is -50°C to -20°C.

[0034] Furthermore, the concentration of the PBS buffer is 0.1 mol / L and the pH is 7.0.

[0035] Furthermore, the preparation method of the fucoxanthin nanocomposite is as follows: (I) Dissolve fucoxanthin in anhydrous ethanol and vortex until completely dissolved to obtain a fucoxanthin solution; (II) Under high-speed stirring, inject the chitosan solution into the fucoxanthin solution, stir for 10 min, and continue stirring for 4-6 h to obtain a solution containing fucoxanthin-chitosan nanoparticles; (III) Under stirring, sodium alginate solution was slowly added dropwise to a solution containing fucoxanthin-chitosan nanoparticles, and the reaction was stirred to obtain a solution containing fucoxanthin nanocomposite. (IV) The solution containing fucoxanthin nanocomposite was centrifuged, washed, and freeze-dried to obtain fucoxanthin nanocomposite.

[0036] Fucoxanthin is easily degraded and has poor water solubility. Utilizing the electrostatic interaction between positively charged chitosan and negatively charged sodium alginate, a dense and stable polysaccharide composite protective layer is formed around the fucoxanthin nanonucleus. This significantly improves its stability and dispersibility in aqueous solutions, ensuring its bioavailability. It can more effectively interact with collagen fibers and slowly release fucoxanthin over a long period, providing long-lasting antioxidant protection. Simultaneously, chitosan and sodium alginate possess good biocompatibility, moisturizing properties, and film-forming properties, which synergistically work with the antioxidant and anti-inflammatory activities of fucoxanthin to jointly protect collagen.

[0037] Furthermore, in step (I), the ratio of fucoxanthin to anhydrous ethanol is 2 mg: 1 mL.

[0038] Furthermore, in step (II), the high-speed stirring speed is 800 rpm.

[0039] Furthermore, in step (II), the volume ratio of the chitosan solution to the fucoxanthin solution is 20:1.

[0040] Further, in step (II), the chitosan solution is prepared as follows: chitosan is dissolved in ultrapure water, 0.1% (v / v) glacial acetic acid is added to aid dissolution, and the mixture is magnetically stirred for at least 4 hours or overnight until completely dissolved. The pH is adjusted to 5.0 with a 1 mol / L NaOH solution, and then filtered through a 0.22 μm filter membrane for sterilization. The solution is stored at 4°C for later use to obtain a chitosan solution with a concentration of 1 mg / mL.

[0041] Furthermore, in step (III), the stirring speed is 400 rpm; the sodium alginate solution is slowly added at a rate of 1-2 mL / min; and the stirring reaction time is 30 min.

[0042] Furthermore, in step (III), the volume ratio of the solution containing fucoxanthin-chitosan nanoparticles to the sodium alginate solution is 1:1.

[0043] Furthermore, in step (IV), the centrifugation speed is 12000 rpm and the centrifugation time is 30 min.

[0044] Furthermore, in step (3), the stirring temperature is 4-10℃ and the stirring time is 20-40min.

[0045] Furthermore, in step (4), the temperature gradient treatment involves: treating the collagen solution with the added composite protectant at 15-20℃ for 1-2 hours, and then slowly cooling it to 4-8℃ at a cooling rate of 0.5-1℃ / min. The 15-20℃ constant temperature treatment allows collagen molecules sufficient energy for slow adjustment and rearrangement, providing ample time for the interaction between the composite protectant and collagen molecules. The slow cooling to 4-8℃ at 0.5-1℃ / min mimics the self-assembly process of collagen fibers in vivo; the extremely slow cooling rate allows sufficient time for collagen molecules and the composite protectant system to assemble into a regular fiber structure in a highly ordered and energy-efficient manner. Rapid cooling leads to high internal stress, resulting in a non-uniform and unstable structure.

[0046] Furthermore, in step (4), the temperature gradient treatment is as follows: the collagen solution with added composite protective agent is kept at 18°C ​​for 1.5h, and then slowly cooled to 6°C at a cooling rate of 0.8°C / min.

[0047] Furthermore, in step (5), when the type III collagen solution is used for subsequent product preparation or clinical application, if heat treatment is required, the heating temperature shall not exceed 45°C and the heating time shall not exceed 30 minutes.

[0048] This invention significantly enhances the structural stability of type III collagen and extends its effective shelf life through the synergistic effects of pH regulation, the addition of a composite protective agent, and temperature gradient treatment. First, by adjusting the pH to 5.5-6.5, a stable charged range for type III collagen, this invention maintains intermolecular electrostatic balance, protecting the triple helix structure of collagen and preventing hydrogen bond breakage and hydrophobic interaction imbalance. Second, this invention employs a self-made composite protective agent, with multiple components working synergistically to construct a multi-layered defense and enhancement system integrating covalent cross-linking (hydroxytyrosol-gelatin), physical encapsulation (HP-β-CD), comprehensive antioxidant effects (glutathione + fucoxanthin nanocomplex), and steric stabilization (N-acetylneuraminic acid), effectively improving the stability of type III collagen. Finally, this invention uses temperature gradient treatment to reduce molecular thermal motion, decrease hydrolysis and oxidation reaction rates, guide and solidify the optimized collagen-protective agent complex, forming a highly stable, near-natural supramolecular structure.

[0049] Therefore, pH regulation provides a suitable chemical environment to prevent the protective agent from failing or the collagen from denaturing prematurely; the composite protective agent protects the structure at multiple targets at the molecular level; and temperature gradient treatment combines promotion and low-temperature maintenance to consolidate the effect. These three elements form a chain of environmental adaptation, molecular protection, and state solidification, comprehensively and synergistically reducing the hydrolysis, oxidation, and conformational disorder of type III collagen, and significantly improving the stability of type III collagen.

[0050] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention can significantly improve the structural stability of type III collagen and extend its effective storage period through the synergistic effect of pH regulation, addition of composite protective agent and temperature gradient treatment, thus solving the problem that type III collagen is prone to denaturation, aggregation or degradation during storage, transportation and application.

[0051] (2) The present invention obtains type III collagen by self-assembling collagen and composite protectant under suitable pH and stability conditions. The resulting collagen has good biocompatibility, is closer to the natural state, and retains its biological activity and function to the greatest extent. It also avoids toxic substances such as chemical cross-linking agents, making it safe and environmentally friendly.

[0052] (3) The collagen solution processed by this invention has a stable structure and can be directly adapted to cosmetic formulations, biological dressings, and other subsequent processing without additional process adjustments; it has good stability, does not require a strict storage environment, reduces transportation and storage costs, and meets the needs of large-scale industrial applications. Attached Figure Description

[0053] Figure 1 This is an electrophoresis image of type III collagen during the purification process; Figure 2 These are high-performance liquid chromatograms of type III collagen before and after purification; Figure 3 This is a high-performance liquid chromatogram of type III collagen at pH=4.5; Figure 4 This is a high-performance liquid chromatogram of type III collagen at pH=6.0; Figure 5 This is a high-performance liquid chromatogram of type III collagen at pH=8.5; Figure 6 This is the effect of the components of the composite protective agent in Experiment Example 5 on the stability of type III collagen. Detailed Implementation

[0054] Example 1 The preparation method of hydroxytyrosol-gelatin conjugate is as follows: (1) Add PBS buffer with a concentration of 0.1 mol / L and a pH of 7.0 to the gelatin, and stir continuously at 300 rpm for 45 min in a 40℃ water bath until completely dissolved to obtain a gelatin solution with a concentration of 2.0 wt.%; (2) Dissolve hydroxytyrosol in PBS buffer to obtain a hydroxytyrosol solution with a concentration of 20 mg / mL; (3) Cool the gelatin solution to room temperature, and add hydroxytyrosol solution dropwise while stirring continuously. The mass ratio of hydroxytyrosol to gelatin is 1:10. Mix well, then add tyrosinase solution, and place in a 25°C constant temperature water bath and shake at 150 rpm for 24 hours to obtain a mixed solution. The amount of tyrosinase added in the mixed solution is 150 U / mL. (4) Heat the mixed solution in a 90°C water bath for 10 min, and then cool it to room temperature in an ice-water bath to obtain a hydroxytyrosol-gelatin conjugate solution; (5) The hydroxytyrosol-gelatin conjugate solution was placed in a dialysis bag with a molecular weight cutoff of 8-10 kDa and dialyzed with ultrapure water. The dialysate was replaced every 7 hours at 4°C and dialyzed continuously for 60 hours until the dialysate could not be detected by UV spectrophotometer at 270-280 nm. After pre-freezing at -80°C overnight, it was freeze-dried at -50°C to -20°C to obtain the hydroxytyrosol-gelatin conjugate.

[0055] Example 2 The preparation method of fucoxanthin nanocomposites is as follows: (1) Dissolve fucoxanthin in anhydrous ethanol at a ratio of 2 mg: 1 mL, and vortex until completely dissolved to obtain a fucoxanthin solution; (2) Dissolve chitosan in ultrapure water, add 0.1% (v / v) glacial acetic acid to aid dissolution, stir magnetically for at least 4 hours or overnight until completely dissolved, adjust the pH to 5.0 with 1 mol / L NaOH solution, filter with a 0.22 μm filter membrane for sterilization, store at 4℃ for later use, and obtain a chitosan solution with a concentration of 1 mg / mL. (3) Under high-speed stirring at 800 rpm, inject the chitosan solution into the fucoxanthin solution at a volume ratio of 20:1. After stirring for 10 min, continue stirring for 4-6 h to obtain a solution containing fucoxanthin-chitosan nanoparticles. (4) Under the stirring state of 400 rpm, sodium alginate solution was slowly added dropwise to the solution containing fucoxanthin-chitosan nanoparticles at a dropping rate of 1.5 mL / min, and the reaction was stirred for 30 min to obtain a solution containing fucoxanthin nanocomposite. (5) The solution containing fucoxanthin nanocomposite was centrifuged at 12000 rpm for 30 min, washed, and freeze-dried to obtain fucoxanthin nanocomposite.

[0056] Example 3 The preparation method of the composite protective agent is as follows: (1) Add hydroxypropyl-β-cyclodextrin to deionized water and stir for 20 min at 60℃ and 300 rpm. After clarification, cool to 35℃. (2) Add glutathione, stir for 15 min, then add N-acetylneuraminic acid, stir for 10 min, adjust pH to 6.0, and obtain the solution; (3) The fucoxanthin nanocomposite and the hydroxytyrosol-gelatin conjugate were dissolved in 50wt.% ethanol and sonicated at 300W for 5min to prepare a solution with a concentration of 110mg / mL. The solution was added dropwise to the solution in step (2) at a speed of 1mL / min and stirred at 500rpm for 20min to obtain a mixed solution. (4) The mixed solution was homogenized three times at 25 MPa and filtered through a 0.22 μm filter membrane to obtain a composite protective agent; (5) Mix the composite protective agent with a type III collagen solution with a concentration of 1 mg / mL at a mass ratio of 1:10.

[0057] Example 4 A synergistic treatment method for improving the stability of type III collagen includes the following steps: (1) The purified type III collagen was dissolved in a buffer solution and stirred at 4°C for 60 min until completely dissolved to obtain a collagen solution with a concentration of 0.5 mg / mL; the buffer solution was a 0.01 mol / L citrate-sodium citrate buffer solution. (2) Under the conditions of a temperature of 4℃ and a stirring speed of 100rpm, the pH of the collagen solution was adjusted to 6.5 to obtain a collagen solution with adjusted pH. (3) Add a composite protective agent to the collagen solution with pH adjusted, and stir at 4°C for 40 min until completely dissolved to obtain a collagen solution with added composite protective agent; the composite protective agent is composed of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, hydroxytyrosol-gelatin conjugate, and hydroxypropyl-β-cyclodextrin in a mass ratio of 0.05:0.01:0.1:0.04:1.0; (4) The collagen solution with added compound protective agent was kept at 15℃ for 2 hours, and then slowly cooled to 4℃ at a cooling rate of 0.5℃ / min to obtain a type III collagen solution with temperature gradient treatment. (5) The type III collagen solution treated with temperature gradient was stored at a temperature of 20°C.

[0058] Example 5 A synergistic treatment method for improving the stability of type III collagen includes the following steps: (1) The purified type III collagen was dissolved in a buffer solution and stirred at 10°C for 30 min until completely dissolved to obtain a collagen solution with a concentration of 2.0 mg / mL; the buffer solution was a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a concentration of 0.05 mol / L. (2) Under the conditions of a temperature of 4℃ and a stirring speed of 50rpm, the pH of the collagen solution was adjusted to 5.5 to obtain a collagen solution with adjusted pH. (3) Add a composite protective agent to the collagen solution with pH adjusted, and stir at 10°C for 20 min until completely dissolved to obtain a collagen solution with added composite protective agent; the composite protective agent is composed of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, hydroxytyrosol-gelatin conjugate, and hydroxypropyl-β-cyclodextrin in a mass ratio of 1.0:0.5:2.0:1.5:5.0; (4) The collagen solution with added compound protective agent was kept at 20°C for 1 hour, and then slowly cooled to 8°C at a cooling rate of 1°C / min to obtain a type III collagen solution with temperature gradient treatment. (5) The type III collagen solution treated with temperature gradient was stored at a temperature of 30°C.

[0059] Example 6 A synergistic treatment method for improving the stability of type III collagen includes the following steps: (1) The purified type III collagen was dissolved in a buffer solution and stirred at 7°C for 45 min until completely dissolved to obtain a collagen solution with a concentration of 1.2 mg / mL; the buffer solution was a 0.03 mol / L citrate-sodium citrate buffer solution. (2) Under the conditions of a temperature of 7℃ and a stirring speed of 75rpm, the pH of the collagen solution was adjusted to 6.0 to obtain a collagen solution with adjusted pH. (3) Add a composite protective agent to the collagen solution with pH adjusted, and stir at 7°C for 30 min until completely dissolved to obtain a collagen solution with added composite protective agent; the composite protective agent is composed of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, hydroxytyrosol-gelatin conjugate, and hydroxypropyl-β-cyclodextrin in a mass ratio of 0.07:0.2:1.0:1.0:2.5; (4) The collagen solution with added compound protective agent was kept at 17°C for 1.5 h, and then slowly cooled to 6°C at a cooling rate of 0.7°C / min to obtain a type III collagen solution with temperature gradient treatment. (5) The type III collagen solution treated with temperature gradient was stored at a temperature of 25°C.

[0060] Example 7 A synergistic treatment method for improving the stability of type III collagen includes the following steps: (1) The purified type III collagen was dissolved in a buffer solution and stirred at 8°C for 45 min until completely dissolved to obtain a collagen solution with a concentration of 1.0 mg / mL; the buffer solution was a 0.02 mol / L citrate-sodium citrate buffer solution. (2) Under the conditions of 8℃ and 60rpm, the pH of the collagen solution was adjusted to 6.0 with 0.5mol / L HCl solution and 0.5mol / L NaOH solution to obtain the pH adjusted collagen solution. (3) Add a composite protective agent to the collagen solution with pH adjusted, and stir at 8°C for 30 min until completely dissolved to obtain a collagen solution with added composite protective agent; the composite protective agent is composed of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, hydroxytyrosol-gelatin conjugate, and hydroxypropyl-β-cyclodextrin in a mass ratio of 0.2:0.05:0.6:0.5:2.0; (4) The collagen solution with added compound protective agent was kept at 18°C ​​for 1.5 h, and then slowly cooled to 6°C at a cooling rate of 0.8°C / min to obtain a type III collagen solution with temperature gradient treatment. (5) The type III collagen solution treated with temperature gradient was stored at a temperature of 25°C.

[0061] Comparative Example 1 The steps are the same as in Example 7, except that in step (2), the pH is adjusted to 8.0.

[0062] Comparative Example 2 The steps are the same as in Example 7, except that no composite protective agent is added in step (3).

[0063] Comparative Example 3 The steps are the same as in Example 7, except that the temperature treatment in step (4) is to directly cool down to 6°C.

[0064] Comparative Example 4 The steps are the same as in Example 7, except that in step (3), only glutathione is added as a protectant.

[0065] Comparative Example 5 The steps are the same as in Example 7, except that no protective agent is added in step (3) and the temperature treatment in step (4) is to directly cool down to 6°C.

[0066] Experimental Example 1 After storing the type III collagen from Examples 7 and Comparative Examples 1-5 at 6°C for 6 months, their activity retention rate at 37°C for 24 hours, the intensity of the negative absorption peak at 222 nm, and their solubility were measured. The results are listed in Table 1.

[0067] Among them, the 24-hour activity retention rate at 37℃ refers to the percentage of native conformation activity retained by type III collagen after 6 months of storage at 6℃ and incubation for 24 hours in a simulated physiological environment (37℃, pH 7.4) relative to the initial activity before storage; the negative absorption peak intensity at 222nm (relative value) is the ratio of the negative absorption peak intensity at 222nm of type III collagen after 6 months of storage at 6℃ to the initial negative absorption peak intensity at 222nm; the solubility refers to the percentage of collagen mass that remains dissolved (no visible aggregates, no precipitation) in the buffer solution (0.02mol / L citrate-sodium citrate buffer, pH consistent with the storage setting) of type III collagen after 6 months of storage at 6℃, relative to the initial collagen mass before storage.

[0068] Table 1. Comparison of the stability of type III collagen As shown in Table 1, compared with Example 7, the activity retention and solubility of type III collagen were significantly reduced in Comparative Example 1 (pH adjusted to 8.0), Comparative Example 2 (no composite protective agent added), Comparative Example 3 (directly cooled to 6℃), and Comparative Example 4 (added with the single protective agent glutathione). The intensity of the negative absorption peak at 222nm was also significantly reduced. Comparative Example 5 (no protective agent added, directly cooled to 6℃) showed the worst stability of type III collagen. This indicates that the process of the present invention, through the synergistic effect of pH control, addition of composite protective agent, and temperature gradient treatment, can significantly improve the structural stability of type III collagen, achieve long-term stable storage of type III collagen for 6 months, and simultaneously ensure its conformational stability, functional activity retention rate, and physical solubility, thus solving the problems of denaturation and difficult storage of type III collagen.

[0069] Experiment Example 2: Effect of pH on the stability of type III collagen The steps are the same as in Example 7, except that the pH value in step (2) is changed to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 respectively. After storing the type III collagen at 6°C for 6 months, its activity retention rate at 37°C for 24 hours, the intensity of the negative absorption peak at 222 nm, and its solubility are measured. The results are listed in Table 2. Simultaneously, the high-performance liquid chromatograms of the type III collagen at pH=4.5, pH=6, and pH=8.5 are shown in the attached table. Figure 3-5 As shown.

[0070] Table 2. Effect of pH on the stability of type III collagen Table 2 shows that as the pH increased from 4.0 to 8.5, the activity retention rate, solubility, and negative absorption peak intensity at 222 nm of type III collagen all first increased and then decreased. Type III collagen exhibited the best stability between pH 5.5 and 6.5, with the best stability at pH 6.0, showing an activity retention rate of 92.5%, a negative absorption peak intensity at 222 nm that was 93% of its initial value, and a solubility of 97.0%. Furthermore, combined with the attached... Figure 3-5 It can be seen that, compared with type III collagen at pH=6, the molecular weight distribution of type III collagen at pH=4.5 and pH=8.5 is wider and degradation fragments appear, indicating that pH significantly affects the triple helix structure of collagen molecules. When the pH is too low or too high, its triple helix structure undergoes significant unwinding.

[0071] Experiment Example 3: Effect of Temperature Gradient Treatment on the Stability of Type III Collagen The steps are the same as in Example 7, except that the temperature gradient treatment in step (4) is changed. The experimental design is as follows: Group A: Same as Example 7; Group B: The collagen solution with added compound protective agent was kept at 18℃ for 1.5h and then directly cooled to 6℃; Group C: The collagen solution with added compound protective agent was directly cooled to 6℃; Then, after storing type III collagen at 6°C for 6 months, its activity retention rate at 37°C for 24 hours, the intensity of the negative absorption peak at 222 nm, and its solubility were measured. The results are listed in Table 3.

[0072] Table 3. Effect of temperature gradient treatment on the stability of type III collagen As shown in Table 3, Group B, which did not undergo slow cooling at the rate specified in this invention, experienced a significant decrease in the retention rate of type III collagen activity, the intensity of the negative absorption peak at 222 nm, and solubility, resulting in poor stability. Group C, which directly cooled to 6℃, also experienced a significant decrease in the retention rate of type III collagen activity, the intensity of the negative absorption peak at 222 nm, and solubility. This indicates that the temperature gradient treatment in this invention, which involves isothermal treatment of the collagen solution with added composite protective agent at 15-20℃ for 1-2 hours, followed by slow cooling to 4-8℃ at a rate of 0.5-1℃ / min, can improve the stability of type III collagen. This is because isothermal treatment at 18℃ can prevent type III collagen from denaturing due to high temperature and ensure that type III collagen molecules fully contact the composite protective agent to form a stable "collagen-protective agent complex." Slow cooling allows collagen molecules to fully combine with the protective agent, forming a denser and more ordered complex.

[0073] Experiment Example 4: Temperature Gradient Parameter Optimization The steps are the same as in Experiment Example 6, but with 4 sets of variable formulations, only the temperature gradient parameter is changed. The scheme design is as follows: Option 1: Cool down to 6℃ at a rate of 0.8℃ / min from 18℃ / 1.5h; Option 2: Cool down to 6℃ at a rate of 0.5℃ / min, from 15℃ / 1h. Option 3: Cool down to 6℃ at a rate of 1.0℃ / min from 20℃ / 2h; Option 4: Directly lower the temperature to 6℃.

[0074] Then, after storing type III collagen at 6°C for 6 months, its activity retention rate at 37°C for 24 hours, its thermal stability at 45°C for 30 minutes, and its triple helix structure integrity were measured. The results are listed in Table 4.

[0075] The thermal stability at 45℃ for 30 min refers to the percentage of native conformational activity retained by type III collagen after storage at 6℃ for 6 months, following a specific heat stress treatment (45℃ for 30 min), compared to its pre-heat stress activity. This directly reflects collagen's ability to resist heat denaturation and is positively correlated with the stability of its triple helix structure. The reagents and instruments used to quantify thermal stability are the same as those used for the activity retention rate at 37℃ for 24 h.

[0076] Triple helix integrity refers to the degree to which type III collagen retains its natural triple helix structure (a right-handed triple helix formed by Gly-XY repeating sequences) after storage at 6°C for 6 months. This is assessed using circular dichroism spectroscopy (CD) to detect the intensity, shape, and integrity of characteristic absorption peaks, categorized into three levels: "excellent," "good," and "poor." It is a core direct indicator of structural stability. Higher peak intensity, more symmetrical peak shape, and the absence of extraneous peaks (degradation peaks) indicate a more complete triple helix structure.

[0077] Table 4. Effects of temperature gradient parameters on the stability of type III collagen As shown in Table 4, after 6 months of storage at 6℃, compared with Scheme 4 which directly cooled to 6℃, Schemes 1-3 all effectively improved the activity retention rate of type III collagen at 37℃ for 24 hours, the thermal stability at 45℃ for 30 minutes, and protected its triple helix structure. This indicates that the temperature gradient treatment of the present invention can effectively improve the stability of type III collagen. Direct cooling causes a sharp drop in the mobility of collagen molecules, preventing the orderly recombination of the hydrogen bond network of the triple helix structure, exposing and aggregating hydrophobic groups, ultimately leading to deformation of the triple helix structure, broadening of peaks, loss of functional activity, and inability to resist thermal stress due to structural incompleteness. Among them, Scheme 1 showed a 92.5% activity retention rate of type III collagen at 37℃ for 24 hours and a thermal stability of 72.3% after 45℃ for 30 minutes, with clear triple helix characteristic peaks, significantly higher than the other three groups. It is evident that the temperature gradient treatment of type III collagen in this invention can improve the stability of collagen. Under the optimal gradient treatment scheme defined in this invention: constant temperature at 18℃ for 1.5h, followed by cooling to 6℃ at a rate of 0.8℃ / min, the constant temperature parameter and cooling rate achieve optimal synergy, promoting the binding of collagen with the complex protective agent and locking the natural triple helix conformation, resulting in the best stability of type III collagen.

[0078] Experimental Example 5: Effect of the components of the composite protective agent on the stability of type III collagen To investigate the effects of the components of the composite protective agent of this invention on type III collagen, the following experiment was designed: 1. Grouping: Group 1: Glutathione 2.0g, Fucoxanthin nanocomplex 0.5g, N-acetylneuraminic acid 5.0g, Hydroxytyrosol-gelatin conjugate 5.0g, Hydroxypropyl-β-cyclodextrin 20.0g; Group 2: Fucoxanthin nanocomplex 0.5g, N-acetylneuraminic acid 5.0g, hydroxytyrosol-gelatin conjugate 5.0g, hydroxypropyl-β-cyclodextrin 20.0g; Group 3: Glutathione 2.0g, N-acetylneuraminic acid 5.0g, hydroxytyrosol-gelatin conjugate 5.0g, hydroxypropyl-β-cyclodextrin 20.0g; Group 4: Glutathione 2.0g, Fucoxanthin nanocomplex 0.5g, N-acetylneuraminic acid 5.0g, Hydroxypropyl-β-cyclodextrin 20.0g; Group 5: Glutathione 2.0g, Fucoxanthin nanocomplex 0.5g, Hydroxytyrosol-gelatin conjugate 5.0g, Hydroxypropyl-β-cyclodextrin 20.0g; Group 6: Glutathione 2.0g, Fucoxanthin 0.5g, N-acetylneuraminic acid 5.0g, Hydroxytyrosol-gelatin conjugate 5.0g, Hydroxypropyl-β-cyclodextrin 20.0g; Group 7: Glutathione 2.0g, Fucoxanthin nanocomplex 0.5g, N-acetylneuraminic acid 5.0g, Hydroxytyrosol 5.0g, Hydroxypropyl-β-cyclodextrin 20.0g; 2. Test methods: Weigh out the components of the composite protective agent in groups 1-7 respectively, and prepare the composite protective agent according to the preparation method in Example 3. Mix the composite protective agent with type III collagen at a mass ratio of 1:10. After thorough mixing, store at 6°C for 6 months.

[0079] (3) Performance testing: The heat denaturation temperature (Td), activity retention rate after 6 months of storage at 6℃, retention rate after 24 hours of MMP-1 enzymatic hydrolysis, and stability after 5 freeze-thaw cycles at -20℃ were determined for the three types of collagen mentioned above. The test results are shown in Table 5 and Appendix. Figure 6 .

[0080] Table 5. Effects of the components of the compound protective agent on the stability of type III collagen. As shown in Table 5, compared with Group 1, the stability of all three types of collagen in Groups 2-5 decreased. This indicates that the absence of a single component in the composite protective agent of the present invention leads to a significant decrease in the stability of the three types of collagen. The quaternary combination of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, and hydroxytyrosol-gelatin conjugate in the composite protective agent of the present invention forms a protective network with complementary functions and synergistic mechanisms. A single component cannot replace the overall synergistic effect.

[0081] Group 2 lacked glutathione, resulting in reduced heat denaturation temperature, activity retention rate, and MMP-1 enzymatic hydrolysis retention rate over 24 hours. Glutathione is a major antioxidant and structural stabilizer that can effectively scavenge free radicals and prevent oxidative stress damage to collagen. This is crucial for maintaining long-term storage stability (activity retention rate) and resisting heat denaturation.

[0082] Group 3 lacked fucoxanthin nanocomplex, while Group 6 added fucoxanthin. The stability of Group 6's type III collagen was significantly better than Group 3, but worse than Group 1. This is because fucoxanthin is highly lipid-soluble and disperses unevenly in aqueous systems, failing to form a highly efficient "water-soluble-lipid-soluble" antioxidant network with glutathione. However, nano-processing improved the dispersibility of fucoxanthin, allowing it to fully exert its synergistic antioxidant effect, proving that nano-modification is key to optimizing its efficacy. This invention nano-processing of fucoxanthin can improve its dispersibility and antioxidant synergy.

[0083] The hydroxytyrosol-gelatin conjugate is the core of the anti-enzymatic hydrolysis activity and affects the overall structural stability. Group 4 did not contain the hydroxytyrosol-gelatin conjugate, while Group 7 contained hydroxytyrosol. The stability of type III collagen in Group 7 was significantly lower than in Group 1, indicating that the gelatin conjugate enhances the binding ability of hydroxytyrosol to type III collagen, making it more competitively bound to the MMP-1 active site and improving the anti-enzymatic hydrolysis effect. In contrast, ordinary hydroxytyrosol has weak binding affinity to collagen, resulting in decreased anti-enzymatic hydrolysis efficacy, suggesting that the gelatin conjugate can play a targeted optimization role. In Group 4, the absence of the hydroxytyrosol-gelatin conjugate resulted in the lowest MMP-1 retention rate after 24 hours of enzymatic hydrolysis, with a Td decreasing to 52.4℃, an activity retention rate of 58.3%, and a large amount of precipitate after freeze-thaw cycles (turbidity 3.5 NTU). This indicates that the gelatin conjugate structure not only enhances anti-enzymatic hydrolysis activity but also helps maintain the triple helix structure through interaction with collagen.

[0084] Group 5 lacked N-acetylneuraminic acid, resulting in a decrease in all indicators of type III collagen. This is because N-acetylneuraminic acid can increase the thickness of the hydration layer on the surface of collagen molecules, inhibit aggregation caused by hydrophobic interactions, improve freeze-thaw stability, and form hydrogen bonds with collagen molecules to help stabilize the triple helix structure and improve activity retention. It also partially covers the enzymatic sites of collagen and slightly enhances the ability to resist MMP-1 degradation.

[0085] Therefore, the components of the composite protective agent of this invention have complementary functions and synergistic effects, which can achieve high stability of type III collagen in multiple scenarios and solve the technical pain points of type III collagen being prone to thermal denaturation, enzymatic hydrolysis, aggregation, and inactivation during long-term storage.

[0086] Experimental Example 6 The type III collagen stock solution was loaded onto a chromatography column, and equilibration, washing, and elution were performed sequentially. The purified type III collagen was then subjected to SDS-PAGE protein chromatography. The results are shown in the attached figure. Figure 1 As shown.

[0087] In the figure, the marker is the protein molecular weight standard, the stock solution is the type III collagen solution before column loading, the flow-through is the unbound protein after column loading, the washing is the collagen after washing away non-specifically bound impurities, the elution is the target protein that is washed off the chromatography column, and the CIP is the impurity washed off by the alkaline solution at the end.

[0088] from Figure 1 As can be seen, the target protein specifically eluted from the chromatography column has a clear protein band with a molecular weight of about 45KD. The band is free of impurities, indicating that the purity of the type III collagen is good after purification.

[0089] Experimental Example 7 The three types of collagen before and after purification were analyzed by high-performance liquid chromatography (HPLC). The test results are attached. Figure 2 As shown.

[0090] The test conditions were as follows: mobile phase (phase B): phosphate buffer-acetonitrile (10:1) (Mobile phase preparation method: Dissolve 17.32g of anhydrous disodium hydrogen phosphate and 12.15g of sodium dihydrogen phosphate dihydrate in water and bring the volume to 2L. Filter through a 0.22-micron filter membrane, add 200mL of acetonitrile, mix well, and degas by sonication.) Detection conditions: phase B isocratic elution, 0.5 mL / min, time 35 min, detection wavelength 220 nm, column temperature 25-30 ℃.

[0091] from Figure 2 It can be seen that before purification, the three types of collagen contained a large number of impurities and had low purity; after purification, the impurity peaks disappeared in the liquid chromatogram of collagen, and the peaks were single and narrow and long, indicating that purification effectively improved the purity of the three types of collagen.

[0092] The specific testing method in the implementation is as follows: (1) The method for testing the activity retention rate at 37℃ for 24 hours is as follows: Enzyme-linked immunosorbent assay (ELISA) is used for detection: ① Take 1 mL of collagen solution (initial concentration 1.0 mg / mL) stored at 6℃ for 6 months, dilute it to 0.1 mg / mL with pH=7.4 PBS buffer, and incubate it in a constant temperature incubator at 37℃ for 24 hours (simulating the stability under physiological conditions); centrifuge at 4℃ and 10000 rpm for 10 min, and take the supernatant; cover the supernatant on a 96-well plate, block it with bovine serum albumin, and bind it to the first and second antibodies in sequence. Measure the OD value according to the displayed color; take the fresh type III collagen solution before storage as a positive control, and the activity retention rate (%) = (OD value of type III collagen after storage / OD value of fresh type III collagen before storage) × 100%.

[0093] (2) The method for determining the heat denaturation temperature (Td) is as follows: ① Sample preparation: Take a compound protective agent + type III collagen solution (mass ratio 1:10, final collagen concentration 0.1 mg / mL), dilute to 0.05 mg / mL with 0.02 mol / L citrate-sodium citrate buffer (pH=6.0), and shake well; ② Sample loading: Inject 20 μL of sample into an aluminum sample dish and seal; the blank control is the same volume of buffer solution; ③ DSC detection: Set parameters: nitrogen atmosphere (flow rate 50 mL / min), heating rate 10℃ / min, scanning range 25-80℃; ④ Data reading: Analyze the endothermic curve using DSC software and read the temperature corresponding to the peak, which is Td. Three parallel samples are set for each group, and the average value is taken (RSD≤1%).

[0094] (3) The method for determining the retention rate of MMP-1 after 24 hours of enzymatic hydrolysis is as follows: ① Preparation of enzymatic hydrolysis system: Take 200 μL of type III collagen solution-protectant mixture (final type III collagen concentration 0.5 mg / mL), add 20 μL of MMP-1 enzyme solution (concentration 10 ng / mL), and supplement with 50 mmol / L MMP-1 buffer (Tris-HCl, pH 7.5, containing 10 mmol / L CaCl2) to 500 μL, and vortex to mix; ② Enzymatic hydrolysis reaction: Incubate at 37℃ for 24 h, add 100 μL of 5×SDS loading buffer, and stop the reaction by boiling in a water bath for 5 min; ③ SDS-PAGE Detection: Prepare a 12% separating gel, load 20 μL of sample, perform electrophoresis (stacking gel 80V, separating gel 120V, 90 min), stain → destain, and then take pictures using a gel imaging system; ④ Gray value analysis: Analyze the gray value of the 45 kDa target band using ImageJ software, with "unenzyme-free collagen solution" as a control. Enzyme retention rate (%) = (gray value of the band after enzyme digestion / gray value of the control band) × 100%.

[0095] (4) The method for determining the stability after 5 freeze-thaw cycles at -20℃ is as follows: ① Freeze-thaw cycle: Take 1 mL of collagen-protectant mixture, freeze it in a -20℃ freezer for 2 hours, then thaw it in a 25℃ water bath for 1 hour. Repeat 5 times. ② Turbidity detection: After freeze-thaw, measure the turbidity of the solution using a turbidimeter (the blank control is buffer solution with turbidity ≤ 0.1 NTU). ③ Activity detection: Measure the activity retention rate of collagen after freeze-thaw according to the ELISA method.

Claims

1. A synergistic treatment method for improving stability of type III collagen, characterized by: It comprises the following steps: (1) Dissolve the purified type III collagen in a buffer solution, and stir until completely dissolved to obtain a collagen solution; (2) Adjust the pH of the collagen solution with a pH adjuster to obtain a collagen solution with adjusted pH; (3) Add a complex protective agent to the collagen solution with adjusted pH, and stir until completely dissolved to obtain a collagen solution with added complex protective agent; (4) Perform temperature gradient treatment on the collagen solution with added complex protective agent to obtain a type III collagen solution treated by temperature gradient; (5) Store the type III collagen solution treated by temperature gradient at a temperature of 20-30℃.

2. The synergistic treatment method for improving stability of type 3 collagen according to claim 1, characterized by: In step (2), the pH of the collagen solution is 5.5-6.

5.

3. The synergistic treatment method for improving stability of type 3 collagen according to claim 1, characterized by: In step (3), the complex protective agent consists of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, hydroxytyrosol-gelatin conjugate, and hydroxypropyl-beta-cyclodextrin; the mass ratio of glutathione, fucoxanthin nanocomplex, N-acetylneuraminic acid, hydroxytyrosol-gelatin conjugate, and hydroxypropyl-beta-cyclodextrin is (0.05-1.0):(0.01-0.5):(0.1-2.0):(0.04-1.5):(1.0-5.0).

4. The synergistic treatment method for improving stability of type 3 collagen according to claim 3, characterized by: The preparation method of the hydroxytyrosol-gelatin conjugate is as follows: (a) Add PBS buffer solution to gelatin, and continuously stir in a water bath until completely dissolved to obtain a gelatin solution; (b) Dissolve hydroxytyrosol in PBS buffer solution to obtain a hydroxytyrosol solution; (c) Cool the gelatin solution to room temperature, and add the hydroxytyrosol solution drop by drop under continuous stirring, mix uniformly, then add tyrosinase solution, and place in a constant-temperature water bath for shaking reaction to obtain a mixed solution; (d) Heat the mixed solution in a water bath, then cool to room temperature in an ice water bath to obtain a hydroxytyrosol-gelatin conjugate solution; (e) Dialyze the hydroxytyrosol-gelatin conjugate solution, freeze overnight, and then freeze-dry to obtain the hydroxytyrosol-gelatin conjugate.

5. The synergistic treatment method for improving stability of type 3 collagen according to claim 4, characterized by: In step (c), the mass ratio of hydroxytyrosol to gelatin is 1:

10.

6. The synergistic treatment method for improving stability of type 3 collagen according to claim 3, characterized by: The preparation method of the fucoxanthin nanocomplex is as follows: (I) Dissolve fucoxanthin in anhydrous ethanol, and vortex until completely dissolved to obtain a fucoxanthin solution; (II) Under high-speed stirring, inject the fucoxanthin solution into a chitosan solution, stir for 10 min, then continue stirring for 4-6 h to obtain a solution containing fucoxanthin-chitosan nanoparticles; (III) Under stirring, slowly add sodium alginate solution to the solution containing fucoxanthin-chitosan nanoparticles, and stir to react to obtain a solution containing fucoxanthin nanocomplex; (IV) Centrifuge the solution containing fucoxanthin nanocomplex, wash, and freeze-dry to obtain the fucoxanthin nanocomplex.

7. The synergistic treatment method for improving stability of type 3 collagen according to claim 6, characterized by: In step (III), the volume ratio of the solution containing fucoxanthin-chitosan nanoparticles to the sodium alginate solution is 1:

1.

8. The synergistic treatment method for improving stability of type 3 collagen according to claim 1, characterized by: In step (3), the preparation method of the complex protective agent is as follows: (3-1) Add hydroxypropyl-beta-cyclodextrin to deionized water, and stir at 60℃ and 300 rpm for 20 min, then clarify and cool to 35℃; (3-2) Glutathione was added and stirred for 15 min, then N-acetylneuraminic acid was added and stirred for 10 min, and the pH was adjusted to 6.0 with 0.1 mol / L NaOH solution to obtain a solution; (3-3) The fucoxanthin nanocomposite and hydroxytyrosol-gelatin conjugate were dissolved in 50 wt.% ethanol, and a solution with a concentration of 110 mg / mL was prepared by ultrasonicating at a power of 300 W for 5 min, and then the solution was added dropwise to the solution of step (3-2) at a speed of 1 mL / min, and stirred at 500 rpm for 20 min to obtain a mixed solution; (3-4) The mixed solution was homogenized at 25 MPa for 3 times, and then filtered with a 0.22 μm filter membrane to obtain a composite protective agent.

9. The synergistic treatment method for improving stability of type 3 collagen according to claim 1, characterized by: In step (4), the temperature gradient treatment is that the collagen solution after adding the composite protective agent is treated at a constant temperature of 15-20 °C for 1-2 h, and then slowly cooled to 4-8 °C at a cooling rate of 0.5-1 °C / min.

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