A bovine protein peptide composition for anti-skin aging and its preparation method

Abstract

This invention belongs to the field of skincare technology and provides an anti-aging bovine protein peptide composition and its preparation method. Bovine protein peptides with a molecular weight of 300-800 Da are prepared using sequential enzymatic hydrolysis. Active peptides with arginine and lysine content ≥18% are directionally enriched by cation exchange chromatography. These peptides are then combined with sodium ascorbate phosphate, tocopheryl acetate, and phytic acid to construct an antioxidant synergistic network. A biomimetic liposome carrier is prepared using phosphatidylcholine, cholesterol, and ceramide 3. A multilayer responsive coating design is employed, sequentially coating the carrier surface with a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer, and grafting hyaluronic acid oligosaccharides to form a stable multilayer structure. The prepared composition has an average particle size of 120-280 nm, a drug loading efficiency ≥82%, and a 24-hour cumulative release rate of 58-82%, exhibiting excellent controlled-release performance and anti-aging activity, making it suitable for the development and application of high-end anti-aging skincare products.

Description

Technical Field

[0001] This invention relates to the field of skincare technology, specifically to an anti-skin aging bovine protein peptide composition and its preparation method. Background Technology

[0002] With the accelerating aging of the global population and the increasing awareness of skin health among consumers, the anti-aging skincare market is experiencing rapid growth. Among these, anti-aging products with bovine protein peptides as their core active ingredient have garnered significant attention due to their excellent biocompatibility and significant collagen-promoting effects. In modern skincare applications, bovine protein peptides, as an important class of bioactive materials, require highly efficient drug delivery capacity to ensure the full utilization of active ingredients, excellent skin penetration properties for effective delivery to deep tissues, sufficient bioavailability to maximize efficacy potential, good stability to guarantee product quality and shelf life, precise targeting to achieve selective action on specific skin cells, and long-lasting efficacy maintenance to meet the demand for long-lasting anti-aging. Meeting these performance requirements comprehensively is crucial for driving technological advancements in the anti-aging skincare industry. It not only significantly improves the overall performance and market competitiveness of products but also provides consumers with safer, more effective, and longer-lasting anti-aging solutions, while simultaneously opening up new technological pathways and application areas for the development of related biomaterials and pharmaceutical industries.

[0003] Despite the significant potential of bovine protein peptides in the field of anti-skin aging, current research and product development still face numerous technical challenges and performance limitations. For example, Chinese patent CN109735591B discloses a bovine bone collagen peptide and its production method, but it suffers from low drug loading efficiency. This is mainly because traditional physical mixing and simple encapsulation techniques cannot achieve efficient loading and stable maintenance of active peptides. Simultaneously, existing bovine protein peptide products generally suffer from poor skin permeability, primarily due to the relatively large molecular weight of the peptides and the lack of effective penetration-enhancing mechanisms, making it difficult for active ingredients to penetrate the skin's stratum corneum barrier and reach their target sites. Furthermore, insufficient bioavailability is a major bottleneck in current technological development, stemming from the lack of targeted carrier design and release control strategies, making it difficult to maintain effective concentrations of active ingredients locally on the skin. Poor stability also restricts the practical application of products; protein peptides are prone to oxidative degradation and structural damage during storage and use, mainly due to the lack of effective antioxidant protection systems. Weak targeting is reflected in the limited selective recognition and binding ability of existing products to specific skin cells, making it difficult to achieve precise efficacy delivery. Finally, the problem of short-lasting efficacy stems from the lack of an intelligent controlled-release mechanism, which causes the active ingredients to be released rapidly and thus fails to maintain a long-term effective anti-aging effect. Summary of the Invention

[0004] Technical problems to be solved

[0005] The purpose of this invention is to provide an anti-skin aging bovine protein peptide composition and its preparation method, thereby solving the technical problems of low drug loading efficiency, poor skin permeability, insufficient bioavailability, poor stability, weak targeting, and short duration of efficacy of bovine protein peptides in skin anti-aging applications.

[0006] Technical solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing an anti-skin aging bovine protein peptide composition includes the following steps:

[0009] Step S1: Sequential enzymatic hydrolysis to prepare bovine protein peptides: Using a mixture of bovine skin collagen and bovine whey protein as raw materials, the mixture is first hydrolyzed with alkaline protease, then hydrolyzed with papain. After enzyme inactivation treatment, the mixture is separated by a two-stage ultrafiltration membrane to obtain bovine protein peptides with a molecular weight of 300-800 Da.

[0010] Step S2, Targeted enrichment of active peptides: The protein peptides obtained in step S1 are processed by cation exchange chromatography, using pH gradient elution at pH 7.0~9.5, to enrich active peptides with arginine and lysine content ≥18%.

[0011] Step S3, Construction of Antioxidant Synergistic Network: The active peptide from step S2 is mixed with sodium ascorbate phosphate, tocopheryl acetate, and phytic acid, and an antioxidant synergistic system is prepared by high-shear emulsification technology;

[0012] Step S4, preparation of biomimetic carrier and mild drug loading: Phosphatidylcholine, cholesterol and ceramide 3 are mixed and formed into a thin film by rotary evaporation. The film is hydrated with phosphate buffer and S3 complex is added. The pH of the phosphate buffer is 5.0~6.0. The drug is passively loaded and ultrasonically dispersed. The particle size is controlled to be 65-90 nm by polycarbonate membrane extrusion.

[0013] S5. Multilayer response coating design: In step S4, a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer are sequentially coated on the surface of the carrier. A stable multilayer structure is formed through electrostatic interaction and hydrogen bonding. Hyaluronic acid oligosaccharides are grafted onto the surface of the polycaprolactone outer layer to obtain a bovine protein peptide composition for anti-skin aging.

[0014] This invention utilizes a multi-layered core-shell structure to prepare an anti-skin aging bovine protein peptide composition, primarily designed to enhance the drug loading efficiency, skin permeability, bioavailability, stability, targeting, and duration of efficacy of bovine protein peptides. The sequential enzymatic hydrolysis preparation technology, through the synergistic action of alkaline protease and papain, precisely cleaves specific sites in bovine collagen and bovine whey protein, obtaining active peptides with ideal molecular weight distribution. The dual-enzyme system fully leverages the specific advantages of different enzymes, resulting in enzymatic hydrolysates with higher bioactivity. The targeted enrichment technology for active peptides, through a cation exchange chromatography selective separation mechanism, specifically enriches alkaline peptides rich in arginine and lysine. These alkaline amino acid residues enhance the interaction between peptides and skin cell membranes and provide additional antioxidant activity. The antioxidant synergistic network construction technology combines active peptides with sodium ascorbate phosphate, tocopheryl acetate, and phytic acid to form a synergistic system with complementary antioxidant mechanisms. Sodium ascorbate phosphate provides water-soluble antioxidant protection, tocopheryl acetate is responsible for antioxidant protection in the lipid-soluble environment, and phytic acid blocks the oxidation reaction chain through its metal chelating ability. The organic combination of multiple antioxidant mechanisms significantly improves the overall antioxidant efficacy and prolongs the stability of active ingredients. The biomimetic carrier preparation technology uses a liposome system constructed from phosphatidylcholine, cholesterol, and ceramide 3 to simulate the composition of natural cell membranes. Phosphatidylcholine provides the basic framework of the membrane structure, cholesterol regulates the membrane's fluidity and stability, and ceramide 3 enhances compatibility with the stratum corneum of the skin. The biomimetic design improves the carrier's biocompatibility and optimizes drug encapsulation efficiency and release behavior. The multilayer responsive encapsulation design technology sequentially constructs a multilayer structure on the liposome surface, consisting of a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer. The poly-L-histidine layer utilizes pH-sensitive properties to provide intelligent responsive release. The hyaluronic acid layer enhances the carrier's moisturizing properties and skin affinity, improving skin penetration through natural transdermal absorption. The polycaprolactone outer layer acts as a protective barrier to prevent leakage of active ingredients and control the release rate. The outermost layer, grafted with hyaluronic acid oligosaccharides, strengthens the carrier's specific binding ability to the skin. This entire technical solution achieves organic unity of each functional module through precise structural design and material selection. The synergistic effect of multiple mechanisms significantly improves the final product's key performance indicators such as drug loading efficiency, stability, permeability, targeting, and sustained release, effectively overcoming the technical bottlenecks of traditional bovine protein peptides in skin anti-aging applications.

[0015] Furthermore, in step S1, bovine collagen and bovine whey protein are mixed at a weight ratio of 6.5~7.5:2.5~3.5, and the protein substrate concentration is 12~18wt%.

[0016] Furthermore, in step S1, the alkaline protease is enzymatically hydrolyzed at pH 8.3-8.7 and temperature 58-65°C for 1.5-2.5 hours, with an enzyme dosage of 2500-3500 U / g protein, and the papain is enzymatically hydrolyzed at pH 6.3-6.7 and temperature 48-52°C for 1.0-2.0 hours, with an enzyme dosage of 1500-2500 U / g protein.

[0017] Furthermore, in step S1, the molecular weight cutoff of the dual-stage ultrafiltration membrane is 200~250Da and 800~1000Da, the transmembrane pressure difference is 0.1~0.3MPa, and the degree of protein substrate hydrolysis is controlled at 15~28%.

[0018] Furthermore, in step S2, the cation exchange chromatography resin has an exchange capacity of 4.2~5.0 mmol / g, elutes with a gradient of 0.1~0.5M NaCl, an elution flow rate of 0.8~1.5 BV / h, a column temperature of 15~25°C, and the enriched active peptides account for 60~85% of the total peptides.

[0019] Furthermore, in step S3, sodium ascorbate phosphate is 0.4~1.2wt%, tocopheryl acetate is 0.08~0.35wt%, phytic acid is 0.003~0.015wt%, and high-shear emulsification is performed at a temperature of 18~32°C and a rotation speed of 7500~13000rpm for 3~18 minutes, and the average droplet size is controlled at 0.1~0.8μm.

[0020] This invention employs a bovine protein peptide composition designed with precise process parameter control and multi-component synergistic formulation to enhance drug loading efficiency, release control, and synergistic antioxidant effects. By optimizing the weight ratio of bovine collagen and bovine whey protein, the two proteins achieve complementarity in amino acid composition and functional properties, resulting in a synergistic effect that significantly improves the overall bioactivity of the raw materials. In the sequential enzymatic hydrolysis process, alkaline protease cleaves hydrophobic amino acid residues under a slightly alkaline environment, while papain hydrolyzes aromatic amino acid sites under weakly acidic conditions. The synergistic effect of the two enzymes fully leverages the specificity and complementarity of the cleavage sites, ensuring the acquisition of peptides with ideal molecular weight distribution and bioactivity. Cation exchange chromatography, through precise parameter control, ensures highly selective enrichment of alkaline peptides rich in arginine and lysine. These alkaline amino acid residues enhance the cell membrane penetration ability of the peptides and provide additional antioxidant activity. In the construction of the antioxidant synergistic network, the precise ratio of sodium ascorbate phosphate, tocopheryl acetate, and phytic acid, along with the optimization of the high-shear emulsification process, achieves uniform dispersion of water-soluble and lipid-soluble antioxidants. Sodium ascorbate phosphate provides antioxidant protection for the extracellular matrix, tocopheryl acetate plays a membrane protection role, and phytic acid blocks free radical chain reactions by chelating metal ions. The synergistic effect of the three antioxidant mechanisms significantly enhances the overall antioxidant efficacy, and the synergistic combination of multiple antioxidants produces a comprehensive antioxidant effect far exceeding that of a single component.

[0021] Furthermore, in step S4, phosphatidylcholine, cholesterol, and ceramide 3 are mixed in a weight ratio of 68~72:23~27:4~6, with a lipid concentration of 45~85mM, and dissolved in chloroform-methanol at a volume ratio of 1.8~2.2:1.

[0022] Furthermore, in step S4, the rotary evaporation temperature is 35-45°C, the vacuum degree is -0.06 to -0.10 MPa, and the time is 20-40 minutes. The phosphate buffer solution is hydrated at 55-65°C for 25-45 minutes with a pH of 5.0-6.0. The active peptide complex is added for passive drug loading and binding, and the mixture is ultrasonically dispersed at 20 kHz for 10-15 minutes. The peptide to lipid mass ratio is 1:8-15, the phosphate buffer solution is pH 7.2-7.6, and the polycarbonate membrane is extruded using a stepwise reduction in pore size: the first stage pore size is 420-380 nm, the second stage pore size is 220-180 nm, and the third stage pore size is 120-80 nm. The drug loading efficiency is ≥82%.

[0023] Furthermore, in step S5, the poly-L-histidine layer has a pKa value of 5.8–6.2 and a thickness of 1.5–3.5 nm; the hyaluronic acid layer has a molecular weight of 45–120 kDa; the polycaprolactone outer layer has a molecular weight of 8000–18000 Da; the coating time for each layer is 25–70 minutes; the coating temperature is 20–32°C; the hyaluronic acid oligosaccharide has a molecular weight of 0.8–12 kDa; and the grafting density is 3–18 μg / cm³.2 The final average particle size is 120~280nm.

[0024] This invention also discloses a bovine protein peptide composition for anti-skin aging, wherein the composition is prepared by the above-described preparation method;

[0025] The composition has the following technical characteristics: average particle size distribution polydispersity index (PDI) less than 0.25, surface potential (Zeta potential) of -18mV to -22mV, cumulative release rate of 58% to 82% in vitro over 24 hours, and a promoting effect on skin collagen synthesis of 25% to 45%.

[0026] The composition is used in the preparation of anti-aging skin care products. The composition has a mass percentage of 0.1% to 5.0% in the skin care product composition, and the pH value of the skin care product composition is 5.5 to 6.8. The skin care product composition also contains the following active ingredients: sodium hyaluronate, with a mass percentage of 0.1% to 3.0%; niacinamide, with a mass percentage of 0.5% to 3.0%; vitamin C derivative, with a mass percentage of 0.1% to 1.5%; and ceramide complex, with a mass percentage of 0.5% to 2.5%.

[0027] This invention utilizes a biomimetic carrier construction and multilayer responsive encapsulation design to prepare an anti-skin aging bovine protein peptide composition, primarily aimed at enhancing drug loading efficiency, controlled release performance, and skin penetration. The biomimetic carrier preparation technology simulates the compositional characteristics of natural cell membranes through precise weight ratios of phosphatidylcholine, cholesterol, and ceramide 3. Phosphatidylcholine provides a stable bilayer framework, cholesterol regulates membrane fluidity and permeability, and ceramide 3 enhances compatibility with the stratum corneum. The synergistic effect of these three lipid components forms a carrier basis with excellent biocompatibility and stability. Precise control of rotary evaporation process parameters ensures complete solvent removal and the formation of a uniform lipid film. The hydration process of phosphate buffer under specific pH and temperature conditions promotes liposome self-assembly. Passive drug loading combined with ultrasonic dispersion technology achieves efficient encapsulation of active peptide complexes. The polycarbonate membrane extrusion process employs a stepwise reduction in pore size to precisely control the average particle size distribution of the carrier. The synergistic optimization of multiple process steps significantly improves drug loading efficiency. A multilayer responsive encapsulation design technique sequentially constructs a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer on the surface of liposomes. The poly-L-histidine layer provides pH-sensitive release function within a specific pKa range. The hyaluronic acid layer enhances the carrier's moisturizing properties and skin affinity, and improves skin permeability. The polycaprolactone outer layer acts as a protective barrier to prevent leakage of active ingredients and control the release rate. Grafting of hyaluronic acid oligosaccharides onto the surface strengthens the carrier's specific binding ability to skin cells. The prepared composition exhibits good uniformity in average particle size distribution and suitable surface potential, achieving excellent in vitro cumulative release behavior and a significant collagen synthesis-promoting effect. In skincare applications, it forms a synergistic system with active ingredients such as sodium hyaluronate, niacinamide, vitamin C derivatives, and ceramide complexes. The synergistic effects among the components significantly enhance the overall anti-skin aging effect.

[0028] Beneficial technical effects

[0029] 1. This invention achieves synergistic effects among various functional modules through the organic integration of sequential enzymatic hydrolysis, targeted enrichment, antioxidant synergistic network, and multi-layered core-shell structure, significantly improving drug loading efficiency, stability, permeability, and targeting, thus overcoming the bottlenecks of traditional protein peptide technology.

[0030] 2. This invention significantly improves drug loading efficiency and skin permeability through biomimetic carrier construction and multilayer responsive encapsulation design, solving the technical problems of poor stability and weak targeting of traditional protein peptides, achieving intelligent controlled release and precise delivery, and providing innovative technical support for high-end anti-aging skin care products. Attached Figure Description

[0031] Figure 1 This is a product morphology diagram from the process of preparing the anti-skin aging bovine protein peptide composition in Example 1 of the present invention.

[0032] Figure 1a is a morphology diagram of protein peptides after sequential enzymatic digestion in the S1 stage;

[0033] Figure 1 b is a morphology diagram of the active peptides after cation exchange chromatography enrichment in the S2 stage.

[0034] Figure 1 c is the morphology of the liposome carrier after preparation in the S4 stage;

[0035] Figure 1 d is the final product morphology diagram after the S5 stage multi-layer coating is completed;

[0036] Figure 2 This is a morphological image of the anti-skin aging bovine protein peptide composition prepared in Comparative Example 13 of the present invention.

[0037] Figure 3 This invention relates to the effect of protein substrate concentration on drug loading efficiency and collagen synthesis promotion effect.

[0038] Figure 4 This invention relates to the effect of the degree of hydrolysis of protein substrates on drug loading efficiency and the effect of promoting collagen synthesis.

[0039] Figure 5 This invention relates to the effect of alkaline protease hydrolysis temperature on drug loading efficiency and collagen synthesis promotion effect.

[0040] Figure 6 This invention relates to the effect of sodium ascorbate phosphate addition on drug loading efficiency and collagen synthesis promotion effect.

[0041] Figure 7 This invention relates to the effect of high shear emulsification speed on drug loading efficiency and collagen synthesis promotion effect.

[0042] Figure 8 This invention relates to the effect of lipid concentration on drug loading efficiency and collagen synthesis promotion effect.

[0043] Figure 9 This invention relates to the effect of phosphate buffer hydration pH on drug loading efficiency and collagen synthesis promotion effect.

[0044] Figure 10 The infrared Fourier transform infrared transmission mode spectrum of the lipid carrier in Example 1 of this invention;

[0045] Figure 11 The infrared Fourier transform infrared transmission mode spectrum of the multilayer coated empty system of Embodiment 1 of the present invention is shown.

[0046] Figure 12 The infrared Fourier transform infrared transmission mode spectrum of the final composition of Example 1 of the present invention is shown. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0048] Example 1

[0049] A method for preparing an anti-skin aging bovine protein peptide composition includes the following steps:

[0050] Step S1: Sequential enzymatic hydrolysis to prepare bovine protein peptides: Using a mixture of bovine skin collagen and bovine whey protein as raw materials, the peptides are first hydrolyzed with alkaline protease, then with papain. After enzyme inactivation treatment, the peptides are separated by a two-stage ultrafiltration membrane to obtain bovine protein peptides with a molecular weight of 300-800 Da. The bovine skin collagen and bovine whey protein are mixed at a weight ratio of 7.0:3.0, with a protein substrate concentration of 15 wt%. Alkaline protease hydrolysis is performed at pH 8.5 and 62°C for 2.0 hours, with an enzyme dosage of 3000 U / g protein. Papain hydrolysis is performed at pH 6.5 and 50°C for 1.5 hours, with an enzyme dosage of 2000 U / g protein. The molecular weight cutoffs of the two-stage ultrafiltration membranes are 225 Da and 900 Da, the transmembrane pressure difference is 0.2 MPa, and the degree of hydrolysis of the protein substrate is controlled at 22%.

[0051] Step S2, Targeted enrichment of active peptides: The protein peptides obtained in step S1 were processed by cation exchange chromatography with pH gradient elution at pH 8.2 to enrich active peptides with arginine and lysine content of 20%; wherein, the cation exchange chromatography resin had an exchange capacity of 4.6 mmol / g, elution was performed with a 0.3M NaCl gradient, the elution flow rate was 1.2 BV / h, the column temperature was 20°C, and the active peptides accounted for 72% of the total peptides after enrichment.

[0052] Step S3, Construction of Antioxidant Synergistic Network: The active peptide from step S2 was mixed with sodium ascorbate phosphate, tocopherol acetate, and phytic acid, and an antioxidant synergistic system was prepared by high-shear emulsification technology; wherein, sodium ascorbate phosphate was 0.8wt%, tocopherol acetate was 0.22wt%, and phytic acid was 0.009wt%, and high-shear emulsification was performed at 25°C and 10000rpm for 10 minutes, and the average droplet size was controlled at 0.4μm.

[0053] Step S4, Biomimetic Carrier Preparation and Mild Drug Loading: Phosphatidylcholine, cholesterol, and ceramide 3 are mixed and formed into a thin film by rotary evaporation. The film is then hydrated with phosphate buffer (pH 5.5) and the S3 complex is added. The drug is passively loaded and ultrasonically dispersed, and then extruded through a polycarbonate membrane to control the particle size range to 65–90 μm. nm; wherein, phosphatidylcholine, cholesterol, and ceramide 3 are mixed in a weight ratio of 70:25:5, with a lipid concentration of 65mM, dissolved in chloroform-methanol at a volume ratio of 2.0:1, and rotary evaporated at 40°C, vacuum degree of -0.08MPa, for 30 minutes. Phosphate buffer pH 5.5 is used for hydration at 60°C for 35 minutes, and active peptide complex is added for passive drug loading and binding. The mixture is then ultrasonically dispersed at 20kHz for 12 minutes. The peptide to lipid mass ratio is 1:12, and the phosphate buffer pH is 7.4. The polycarbonate membrane is extruded using a stepwise decreasing pore size method: the first pore size is 400nm, the second pore size is 200nm, and the third pore size is 100nm, with a drug loading efficiency of 85%.

[0054] Step S5, Multilayer Response Coating Design: Following Step S4, a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer are sequentially coated onto the carrier surface. A stable multilayer structure is formed through electrostatic interactions and hydrogen bonding. Hyaluronic acid oligosaccharides are then grafted onto the surface of the polycaprolactone outer layer to obtain an anti-skin aging bovine protein peptide composition. The poly-L-histidine layer has a pKa value of 6.0 and a thickness of 2.5 nm; the hyaluronic acid layer has a molecular weight of 80 kDa; the polycaprolactone outer layer has a molecular weight of 13000 Da; the coating time for each layer is 45 minutes; the coating temperature is 26°C; the hyaluronic acid oligosaccharide has a molecular weight of 6 kDa; and the grafting density is 10 μg / cm³. 2 The final average particle size is 200 nm.

[0055] The anti-skin aging bovine protein peptide composition of this embodiment has the following technical features: an average particle size distribution polydispersity index (PDI) of 0.22, a surface potential (Zeta potential) of -20 mV, a cumulative release rate of 70% over 24 hours in vitro, and a 35% promoting effect on skin collagen synthesis. The application of the anti-skin aging bovine protein peptide composition in the preparation of anti-skin aging skincare products is described, wherein the composition comprises 2.5% by mass in the skincare product composition, the pH value of the skincare product composition is 6.2, and the skincare product composition further contains the following active ingredients: sodium hyaluronate, 1.5% by mass; niacinamide, 2.0% by mass; vitamin C derivative, 0.8% by mass; and ceramide complex, 1.5% by mass.

[0056] Example 1 employs moderately conservative process parameters, reflecting a stability-first approach. This example selects a suitable protein substrate concentration (15wt%) and degree of hydrolysis (22%), coupled with moderate enzymatic hydrolysis conditions and emulsification parameters, ensuring process stability and reproducibility. Regarding carrier construction, a balanced lipid ratio and moderate drug loading efficiency (85%) are used, along with a rationally designed multilayer encapsulation structure. The final product exhibits a suitable average particle size (200nm) and good release characteristics (70%). This example is suitable for large-scale industrial production of anti-aging serums and creams, particularly suitable for the mid-to-high-end skincare market where product stability is paramount, providing a gentle yet effective anti-aging care solution for sensitive skin users.

[0057] Example 2

[0058] A method for preparing an anti-skin aging bovine protein peptide composition includes the following steps:

[0059] Step S1: Sequential enzymatic hydrolysis to prepare bovine protein peptides: Using a mixture of bovine skin collagen and bovine whey protein as raw materials, the peptides are first hydrolyzed with alkaline protease, then with papain. After enzyme inactivation, the peptides are separated by a two-stage ultrafiltration membrane to obtain bovine protein peptides with a molecular weight of 300-800 Da. The bovine skin collagen and bovine whey protein are mixed at a weight ratio of 6.8:3.2, with a protein substrate concentration of 12 wt%. Alkaline protease is used for hydrolysis at pH 8.3 and 58°C for 2.5 hours, with an enzyme dosage of 3500 U / g protein. Papain is used for hydrolysis at pH 6.7 and 52°C for 1.0 hour, with an enzyme dosage of 1500 U / g protein. The molecular weight cutoffs of the two-stage ultrafiltration membranes are 200 Da and 1000 Da, the transmembrane pressure difference is 0.3 MPa, and the degree of hydrolysis of the protein substrate is controlled at 15%.

[0060] Step S2, Targeted enrichment of active peptides: The protein peptides obtained in step S1 were processed by cation exchange chromatography with pH gradient elution at pH 9.5 to enrich active peptides with arginine and lysine content of 25%; wherein, the cation exchange chromatography resin had an exchange capacity of 5.0 mmol / g, elution was performed with a 0.5M NaCl gradient, the elution flow rate was 0.8 BV / h, the column temperature was 15°C, and the active peptides accounted for 85% of the total peptides after enrichment.

[0061] Step S3, Construction of Antioxidant Synergistic Network: The active peptide from step S2 was mixed with sodium ascorbate phosphate, tocopherol acetate, and phytic acid, and an antioxidant synergistic system was prepared by high-shear emulsification technology; wherein, sodium ascorbate phosphate was 1.2wt%, tocopherol acetate was 0.35wt%, and phytic acid was 0.015wt%, and high-shear emulsification was carried out at 18°C ​​and 13000rpm for 18 minutes, and the average droplet size was controlled at 0.1μm.

[0062] Step S4, Biomimetic Carrier Preparation and Mild Drug Loading: Phosphatidylcholine, cholesterol, and ceramide 3 are mixed and formed into a thin film by rotary evaporation. The film is then hydrated with phosphate buffer (pH 5.0) and the S3 complex is added. Drug loading is performed via passive loading combined with ultrasonic dispersion. The particle size is controlled to be 65–90 nm via polycarbonate membrane extrusion. nm; wherein, phosphatidylcholine, cholesterol, and ceramide 3 are mixed in a weight ratio of 68:27:5, with a lipid concentration of 85 mM, dissolved in chloroform-methanol at a volume ratio of 2.2:1, and rotary evaporated at 35°C, vacuum degree of -0.10 MPa, for 40 minutes. Phosphate buffer (pH 5.0) is hydrated at 65°C for 45 minutes, and the active peptide complex is added for passive drug loading and binding. The mixture is then ultrasonically dispersed at 20 kHz for 15 minutes. The peptide to lipid mass ratio is 1:8, and the phosphate buffer pH is 7.6. The polycarbonate membrane is extruded using a stepwise decreasing pore size method: the first pore size is 420 nm, the second pore size is 220 nm, and the third pore size is 80 nm, with a drug loading efficiency of 88%.

[0063] Step S5, Multilayer Response Coating Design: Following Step S4, a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer are sequentially coated onto the carrier surface. A stable multilayer structure is formed through electrostatic interactions and hydrogen bonding. Hyaluronic acid oligosaccharides are then grafted onto the surface of the polycaprolactone outer layer to obtain an anti-skin aging bovine protein peptide composition. The poly-L-histidine layer has a pKa value of 5.8 and a thickness of 1.5 nm; the hyaluronic acid layer has a molecular weight of 120 kDa; the polycaprolactone outer layer has a molecular weight of 18000 Da; the coating time for each layer is 70 minutes; the coating temperature is 20°C; the hyaluronic acid oligosaccharide has a molecular weight of 12 kDa; and the grafting density is 18 μg / cm³. 2 The final average particle size is 280 nm.

[0064] The anti-skin aging bovine protein peptide composition of this embodiment has the following technical features: an average particle size distribution polydispersity index (PDI) of 0.18, a surface potential (Zeta potential) of -18 mV, a cumulative release rate of 58% over 24 hours in vitro, and a 45% promoting effect on skin collagen synthesis. The application of the anti-skin aging bovine protein peptide composition in the preparation of anti-skin aging skincare products is described, wherein the composition comprises 5.0% by mass in the skincare product composition, the pH value of the skincare product composition is 6.8, and the skincare product composition further contains the following active ingredients: sodium hyaluronate (3.0% by mass); niacinamide (3.0% by mass); vitamin C derivative (1.5% by mass); and ceramide complex (2.5% by mass).

[0065] Example 2 highlights the technical features of efficient drug loading and controlled release, employing a relatively aggressive combination of process parameters. This example achieved the highest proportion of active peptides (85%) and drug loading efficiency (88%) through high enzyme dosage (3500 U / g) and enhanced enrichment conditions (pH 9.5). Simultaneously, the use of the highest molecular weight hyaluronic acid layer (120 kDa) and polycaprolactone outer layer (18000 Da) resulted in the lowest release rate (58%) and the highest collagen-promoting effect (45%). This example is suitable for high-end anti-aging ampoule serums and professional medical-grade skincare products, particularly suitable for intensive repair treatment of mature skin and severe aging problems, demonstrating excellent long-lasting anti-aging efficacy in a high-concentration formula (5.0%).

[0066] Example 3

[0067] A method for preparing an anti-skin aging bovine protein peptide composition includes the following steps:

[0068] Step S1: Sequential enzymatic hydrolysis to prepare bovine protein peptides: Using a mixture of bovine skin collagen and bovine whey protein as raw materials, the peptides are first hydrolyzed with alkaline protease, then with papain. After enzyme inactivation treatment, the peptides are separated by a two-stage ultrafiltration membrane to obtain bovine protein peptides with a molecular weight of 300-800 Da. The bovine skin collagen and bovine whey protein are mixed at a weight ratio of 7.5:2.5, with a protein substrate concentration of 18 wt%. Alkaline protease is used for hydrolysis at pH 8.7 and 65°C for 1.5 hours, with an enzyme dosage of 2500 U / g protein. Papain is used for hydrolysis at pH 6.3 and 48°C for 2.0 hours, with an enzyme dosage of 2500 U / g protein. The molecular weight cutoffs of the two-stage ultrafiltration membranes are 250 Da and 800 Da, the transmembrane pressure difference is 0.1 MPa, and the degree of hydrolysis of the protein substrate is controlled at 28%.

[0069] Step S2, Targeted enrichment of active peptides: The protein peptides obtained in step S1 were processed by cation exchange chromatography with pH gradient elution at pH 7.0 to enrich active peptides with arginine and lysine content of 18%; wherein, the cation exchange chromatography resin had an exchange capacity of 4.2 mmol / g, elution was performed with a 0.1M NaCl gradient at a flow rate of 1.5 BV / h and a column temperature of 25°C, and the active peptides accounted for 60% of the total peptides after enrichment.

[0070] Step S3, Construction of Antioxidant Synergistic Network: The active peptide from step S2 was mixed with sodium ascorbate phosphate, tocopherol acetate, and phytic acid, and an antioxidant synergistic system was prepared by high-shear emulsification technology; wherein, sodium ascorbate phosphate was 0.4wt%, tocopherol acetate was 0.08wt%, and phytic acid was 0.003wt%, and high-shear emulsification was performed at 32°C and 7500rpm for 3 minutes, and the average droplet size was controlled at 0.8μm.

[0071] Step S4, preparation of biomimetic carrier and gentle drug loading: Phosphatidylcholine, cholesterol, and ceramide 3 are mixed and formed into a thin film by rotary evaporation. The film is then hydrated with phosphate buffer and the S3 complex is added. The phosphate buffer has a pH of 6.0. The drug is passively loaded and ultrasonically dispersed, and the average particle size is controlled to be 65–90 nm by polycarbonate membrane extrusion. nm; wherein, phosphatidylcholine, cholesterol, and ceramide 3 are mixed in a weight ratio of 72:23:5, with a lipid concentration of 45 mM, and dissolved in chloroform-methanol at a volume ratio of 1.8:1. The mixture is rotary evaporated at 45°C, under a vacuum of -0.06 MPa for 20 minutes. The phosphate buffer (pH 6.0) is hydrated at 55°C for 25 minutes. The active peptide complex is then added for passive drug loading and dispersion by ultrasonication at 20 kHz for 10 minutes. The peptide to lipid mass ratio is 1:15, and the phosphate buffer (pH 7.2) is used. The polycarbonate membrane is extruded using a stepwise reduction in pore size: the first pore size is 380 nm, the second pore size is 180 nm, and the third pore size is 120 nm, with a drug loading efficiency of 82%.

[0072] Step S5, Multilayer Response Coating Design: Following Step S4, a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer are sequentially coated onto the carrier surface. A stable multilayer structure is formed through electrostatic interactions and hydrogen bonding. Hyaluronic acid oligosaccharides are then grafted onto the surface of the polycaprolactone outer layer to obtain an anti-skin aging bovine protein peptide composition. The poly-L-histidine layer has a pKa value of 6.2 and a thickness of 3.5 nm; the hyaluronic acid layer has a molecular weight of 45 kDa; the polycaprolactone outer layer has a molecular weight of 8000 Da; the coating time for each layer is 25 minutes; the coating temperature is 32°C; the hyaluronic acid oligosaccharide has a molecular weight of 0.8 kDa; and the grafting density is 3 μg / cm³. 2 The final average particle size is 120 nm.

[0073] The anti-skin aging bovine protein peptide composition of this embodiment has the following technical features: an average particle size distribution polydispersity index (PDI) of 0.24, a surface potential (Zeta potential) of -22 mV, a cumulative release rate of 82% over 24 hours in vitro, and a 25% promoting effect on skin collagen synthesis. The application of the anti-skin aging bovine protein peptide composition in the preparation of anti-skin aging skincare products is described, wherein the composition comprises 0.1% by mass in the skincare product composition, the pH value of the skincare product composition is 5.5, and the skincare product composition further contains the following active ingredients: sodium hyaluronate (0.1% by mass); niacinamide (0.5% by mass); vitamin C derivative (0.1% by mass); and ceramide complex (0.5% by mass).

[0074] Example 3 emphasizes a technology-driven approach focused on rapid penetration and immediate results, employing optimized parameters to promote skin absorption. This example achieved the highest degree of hydrolysis (28%) through high-temperature enzymatic hydrolysis (65°C) and prolonged papain treatment (2.0 hours). Combined with the smallest average droplet size (0.8 μm) and the thinnest coating layer structure, it achieved the smallest average carrier particle size (120 nm) and the fastest release rate (82%). A low molecular weight hyaluronic acid layer (45 kDa) and outer layer design further enhance skin penetration. This example is suitable for fast-acting anti-aging masks and emergency repair products, particularly ideal for occasions requiring immediate skin improvement, such as pre-event skincare and daily rapid hydration and anti-aging needs.

[0075] Example 4

[0076] A method for preparing an anti-skin aging bovine protein peptide composition includes the following steps:

[0077] Step S1: Sequential enzymatic hydrolysis to prepare bovine protein peptides: Using a mixture of bovine skin collagen and bovine whey protein as raw materials, the peptides were first hydrolyzed with alkaline protease, then with papain. After enzyme inactivation treatment, the peptides were separated by a two-stage ultrafiltration membrane to obtain bovine protein peptides with a molecular weight of 300-800 Da. The bovine skin collagen and bovine whey protein were mixed at a weight ratio of 6.5:3.5, with a protein substrate concentration of 14 wt%. Alkaline protease was used for hydrolysis at pH 8.6 and 61°C for 1.8 hours, with an enzyme dosage of 2800 U / g protein. Papain was used for hydrolysis at pH 6.4 and 49°C for 1.3 hours, with an enzyme dosage of 1800 U / g protein. The molecular weight cutoff of the two-stage ultrafiltration membranes was 230 Da and 850 Da, the transmembrane pressure difference was 0.15 MPa, and the degree of hydrolysis of the protein substrate was controlled at 20%.

[0078] Step S2, Targeted enrichment of active peptides: The protein peptides obtained in step S1 were processed by cation exchange chromatography with pH gradient elution at pH 8.8 to enrich active peptides with arginine and lysine content of 22%; wherein, the cation exchange chromatography resin had an exchange capacity of 4.4 mmol / g, elution was performed with a 0.35M NaCl gradient at a flow rate of 1.0 BV / h and a column temperature of 22°C, and the active peptides accounted for 75% of the total peptides after enrichment.

[0079] Step S3, Construction of Antioxidant Synergistic Network: The active peptide from step S2 was mixed with sodium ascorbate phosphate, tocopherol acetate, and phytic acid, and an antioxidant synergistic system was prepared by high-shear emulsification technology; wherein, sodium ascorbate phosphate was 0.6wt%, tocopherol acetate was 0.15wt%, and phytic acid was 0.008wt%, and high-shear emulsification was performed at 28°C and 9500rpm for 8 minutes, and the average droplet size was controlled at 0.3μm.

[0080] Step S4, preparation of biomimetic carrier and gentle drug loading: Phosphatidylcholine, cholesterol, and ceramide 3 are mixed and formed into a thin film by rotary evaporation. The film is then hydrated with phosphate buffer (pH 5.2) and the S3 complex is added. Drug loading is performed via passive loading combined with ultrasonic dispersion. The average particle size is controlled to be 65–90 nm via polycarbonate membrane extrusion. nm; wherein, phosphatidylcholine, cholesterol, and ceramide 3 were mixed in a weight ratio of 69:26:5, with a lipid concentration of 58 mM, and dissolved in chloroform-methanol at a volume ratio of 1.9:1. The mixture was rotary evaporated at 38°C, under a vacuum of -0.075 MPa, for 35 minutes. The phosphate buffer (pH 5.2) was hydrated at 58°C for 32 minutes. The active peptide complex was then added for passive drug loading and dispersion by ultrasonication at 20 kHz for 11 minutes. The peptide to lipid mass ratio was 1:10, and the phosphate buffer (pH 7.3) was used. The polycarbonate membrane was extruded using a stepwise reduction in pore size: the first pore size was 390 nm, the second pore size was 190 nm, and the third pore size was 95 nm, with a drug loading efficiency of 86%.

[0081] Step S5, Multilayer Response Coating Design: Following Step S4, a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer are sequentially coated onto the carrier surface. A stable multilayer structure is formed through electrostatic interactions and hydrogen bonding. Hyaluronic acid oligosaccharides are then grafted onto the surface of the polycaprolactone outer layer to obtain an anti-skin aging bovine protein peptide composition. The poly-L-histidine layer has a pKa value of 5.9 and a thickness of 2.8 nm; the hyaluronic acid layer has a molecular weight of 95 kDa; the polycaprolactone outer layer has a molecular weight of 12000 Da; the coating time for each layer is 55 minutes; the coating temperature is 24°C; the hyaluronic acid oligosaccharide has a molecular weight of 4.5 kDa; and the grafting density is 12 μg / cm³. 2 The final average particle size is 165 nm.

[0082] The anti-skin aging bovine protein peptide composition of this embodiment has the following technical features: an average particle size distribution polydispersity index (PDI) of 0.20, a surface potential (Zeta potential) of -19 mV, a cumulative release rate of 65% over 24 hours in vitro, and a 32% promoting effect on skin collagen synthesis. The application of the anti-skin aging bovine protein peptide composition in the preparation of anti-skin aging skincare products is described, wherein the composition comprises 1.8% by mass in the skincare product composition, the pH value of the skincare product composition is 6.0, and the skincare product composition further contains the following active ingredients: sodium hyaluronate (0.8% by mass); niacinamide (1.2% by mass); vitamin C derivative (0.4% by mass); and ceramide complex (1.2% by mass).

[0083] Example 4 embodies the technical concept of comprehensive balance and personalized customization, achieving an optimized combination of performance through refined parameter control. This example employs moderate yet precise process conditions, ensuring good drug loading efficiency (86%) while achieving balanced release behavior (65%) and stable promoting effects (32%) through reasonable molecular weight selection and encapsulation time control. The medium average particle size (165nm) design balances stability and permeability requirements. This example is suitable for personalized anti-aging skincare products and functional day and night creams, particularly suitable for daily care routines requiring long-term use. It provides gentle and lasting anti-aging effects for different skin types, making it an excellent choice for basic anti-aging care.

[0084] Comparative Example 1: It is basically the same as Example 1, except that only a single alkaline protease is used for enzymatic hydrolysis in step S1, and the papain hydrolysis step is omitted. The alkaline protease is used for hydrolysis at pH 8.5 and temperature 62°C for 3.5 hours, and the enzyme dosage is 3000 U / g protein.

[0085] Comparative Example 2: It is basically the same as Example 1, except that the alkaline protease hydrolysis temperature in step S1 is reduced to 45°C, while other hydrolysis conditions remain unchanged.

[0086] Comparative Example 3: Basically the same as Example 1, except that the weight ratio of bovine collagen and bovine whey protein in step S1 was adjusted to 5.0:5.0, while other preparation conditions remained unchanged.

[0087] Comparative Example 4: It is basically the same as Example 1, except that the degree of hydrolysis of the protein substrate in step S1 is controlled at 5%, which is achieved by shortening the enzymatic hydrolysis time to 0.5 hours.

[0088] Comparative Example 5: It is basically the same as Example 1, except that the cation exchange chromatography directional enrichment step is omitted in step S2, and the protein peptides obtained in step S1 are directly used for subsequent antioxidant synergistic network construction.

[0089] Comparative Example 6: Basically the same as Example 1, except that the pH value of cation exchange chromatography elution in step S2 is reduced to 5.5, while other chromatographic conditions remain unchanged.

[0090] Comparative Example 7: Basically the same as Example 1, except that in step S3 only 0.8 wt% sodium ascorbate phosphate was added as an antioxidant, and the addition of tocopherol acetate and phytic acid was omitted.

[0091] Comparative Example 8: Basically the same as Example 1, except that the high-shear emulsification speed in step S3 is reduced to 3000 rpm and the processing time is extended to 30 minutes.

[0092] Comparative Example 9: Basically the same as Example 1, except that the weight ratio of phosphatidylcholine, cholesterol and ceramide 3 in step S4 is adjusted to 80:15:5, while the other carrier preparation conditions remain unchanged.

[0093] Comparative Example 10: Basically the same as Example 1, except that the ceramide 3 component was omitted in step S4, and only phosphatidylcholine and cholesterol were used to construct the carrier system in a weight ratio of 75:25.

[0094] Comparative Example 11: Basically the same as Example 1, except that the pH of the phosphate buffer was adjusted to 7.5 in step S4, while other drug loading conditions remained unchanged.

[0095] Comparative Example 12: It is basically the same as Example 1, except that in step S4, an active drug delivery method is used instead of a passive drug delivery method. The drug delivery is achieved in 30 minutes under pH 4.0 conditions by pH gradient method.

[0096] Comparative Example 13: It is basically the same as Example 1, except that the coating of the poly-L-histidine layer is omitted in step S5, and the hyaluronic acid layer and the polycaprolactone outer layer are directly coated on the surface of the carrier.

[0097] Comparative Example 14: It is basically the same as Example 1, except that the hyaluronic acid layer is omitted in step S5, and only the poly-L-histidine layer and the polycaprolactone outer layer are sequentially coated on the surface of the carrier.

[0098] Comparative Example 15: Basically the same as Example 1, except that the grafting step of hyaluronic acid oligosaccharide is omitted in step S5, and the outer surface of polycaprolactone is not modified in any way.

[0099] Performance testing:

[0100] Average Particle Size Distribution and Polydispersity Test: The test subject was a suspension of an anti-skin aging bovine protein peptide composition dispersed in deionized water at a concentration of 1 mg / mL. The purpose of the test was to evaluate the uniformity of the average particle size distribution and the dispersion stability of the composition. The test principle was based on dynamic light scattering technology, which determined the average particle size distribution by analyzing the intensity fluctuations of scattered light caused by Brownian motion. The experimental method used a laser particle size analyzer, under a constant temperature of 25°C, with a 532 nm laser as the light source and a scattering angle of 90°, and 10 consecutive measurements were taken and the average value was recorded. The standard was ISO 22412:2017 "Particle size analysis—Dynamic light scattering method". Key parameters included a test temperature of 25 ± 0.1°C, a sample pretreatment time of 5 minutes, and a measurement duration of 120 seconds per measurement. Data processing involved calculating the Z-mean average particle size, the polydispersity index (PDI), and the average particle size distribution curve. A PDI value less than 0.3 indicated good dispersibility.

[0101] Drug loading efficiency determination: The test subject was a newly prepared anti-skin aging bovine protein peptide composition drug loading system. The purpose of the test was to quantitatively evaluate the composition's encapsulation ability of active peptides. The test principle involved separating the free drug and drug-loaded particles, and then determining the encapsulated protein peptide content using the BCA protein quantification method. Experimental method: First, the drug-loaded particles and free protein peptides were separated using ultracentrifugation (100,000 g, 4°C, 1 hour). Then, the protein content in the supernatant and precipitate was determined using a BCA kit. Key parameters included centrifugation temperature 4 ± 1°C, centrifugation time 60 minutes, BCA colorimetric reaction temperature 37°C, and reaction time 30 minutes. Data processing: Drug loading efficiency was calculated using the formula: Drug loading efficiency (%) = (Total protein content - Free protein content) / Total protein content × 100%. The measurement was repeated three times, and the average value was taken.

[0102] In vitro release behavior test: The test subject was a drug-loaded bovine protein peptide composition for anti-skin aging, dispersed in phosphate buffer solutions at pH 5.5 and pH 7.4. The purpose of the test was to evaluate the drug release characteristics and release mechanism of the composition under different pH conditions. The test principle used a dialysis bag method to simulate the in vitro release environment, and the concentration of protein peptides in the release medium was detected by sampling at regular intervals. Experimental method: The drug-loaded composition was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, immersed in a release medium at a constant temperature of 37°C, and shaken at 100 rpm. Samples were taken at 0.5, 1, 2, 4, 8, 12, and 24 hours. Key parameters were: release temperature 37 ± 0.5°C, shaking frequency 100 ± 5 rpm, and a sample volume of 3 mL with timely replenishment of an equal volume of fresh medium. Data processing: Protein concentration was determined by ultraviolet spectrophotometry at a wavelength of 280 nm. A cumulative release rate-time curve was plotted, and the release mechanism was fitted using zero-order, first-order, and Higuchi equations.

[0103] Thermal stability analysis: The test subject was a freeze-dried anti-skin aging bovine protein peptide composition powder, with a sample amount of 5-10 mg. The purpose of the test was to evaluate the thermal decomposition temperature, thermal stability, and phase transition behavior of the composition. The test principle employed thermogravimetric analysis (TGA) to analyze thermal stability by measuring the change in sample mass during a programmed temperature rise. The experimental method used a TGA analyzer under a nitrogen protective atmosphere, heating from room temperature to 600°C at a rate of 10°C / min, and recording the sample mass change curve. Key parameters were: heating rate 10 ± 0.5°C / min, nitrogen flow rate 50 mL / min, temperature range 25-600°C, and weighing accuracy ± 0.01 mg. Data processing and analysis included the thermal decomposition onset temperature (T5%, temperature corresponding to 5% weight loss), the maximum decomposition rate temperature, and the char residue, and plotting TG and DTG curves.

[0104] Cytotoxicity Assay: Different concentrations of the anti-skin aging bovine protein peptide composition were used as test subjects, with concentration gradients of 1, 10, 50, 100, and 500 μg / mL. The purpose of the assay was to evaluate the cytotoxic effects and biosafety of the composition. The assay principle employed the MTT assay, which reflects cell viability by detecting mitochondrial succinate dehydrogenase activity. Human keratinocytes (HaCaT) were seeded in 96-well plates. After 24 hours, different concentrations of the test sample were added, and after further culture for 24, 48, and 72 hours, MTT solution was added for colorimetric reaction. Key parameters included culture temperature 37±1°C, CO2 concentration 5±0.5%, relative humidity 95%, MTT concentration 5 mg / mL, and colorimetric reaction time 4 hours. Data Processing: Cell viability was calculated at each concentration as (experimental group OD value / control group OD value) × 100%. A viability rate greater than 70% was considered to indicate no significant toxicity.

[0105] Skin permeability evaluation: The test subject was a bovine protein peptide composition for anti-skin aging loaded with fluorescently labeled protein peptides, while the control group was a solution of free fluorescent protein peptides. The purpose of the test was to evaluate the promoting effect of the composition carrier on the skin penetration of the active ingredients. The test principle used a Franz diffusion cell and a porcine skin model, and evaluated the penetration effect by detecting changes in fluorescence intensity in the receiving solution. Experimental method: Fresh porcine skin was fixed in a Franz diffusion cell, the test sample was added to the supply cell, and the receiving solution was phosphate buffer (pH 7.4). Under constant temperature of 32°C, samples were taken at regular intervals to detect fluorescence intensity. Key parameters were: skin temperature 32±1°C, stirring speed 300 rpm, sampling time points of 1, 2, 4, 6, 8, 12, and 24 hours, and skin thickness controlled at 400-600 μm. Data processing: The cumulative penetration amount, penetration rate, and penetration enhancement ratio were calculated, and a cumulative penetration amount-time curve was plotted.

[0106] Antioxidant Activity Assay: The test subject was a water-soluble extract of the anti-skin aging bovine protein peptide composition, with concentration gradients of 0.1, 0.5, 1.0, 2.0, and 5.0 mg / mL. The purpose of the test was to evaluate the synergistic antioxidant capacity of the antioxidant components in the composition. The test principle employed the DPPH and ABTS free radical scavenging methods, evaluating antioxidant activity by measuring the free radical scavenging rate. Experimental Methods: DPPH and ABTS working solutions were prepared separately and mixed with test samples of different concentrations. After reacting for 30 minutes under light-protected conditions, the absorbance change was measured. Key parameters were: DPPH concentration 0.1 mM, detection wavelength 517 nm; ABTS concentration 7 mM, detection wavelength 734 nm; reaction temperature 25 ± 2°C. Data Processing: The free radical scavenging rate was calculated as: Scavenging rate (%) = (Control group absorbance - Sample group absorbance) / Control group absorbance × 100%, and the IC50 was calculated. 50 Values ​​are used to evaluate antioxidant strength.

[0107] The performance of the anti-skin aging bovine protein peptide compositions of Examples 1-4 and Comparative Examples 1-15 is summarized in Table 1. As can be seen from the table, Comparative Example 1, which used a single alkaline protease hydrolysis method omitting the papain step, resulted in uneven molecular weight distribution of the protein peptides and insufficient exposure of active sites, leading to deteriorated average particle size dispersion, reduced drug loading efficiency, and weakened bioactivity. The low-temperature hydrolysis conditions in Comparative Example 2 severely inhibited enzyme activity, resulting in incomplete hydrolysis, carrier structural defects, and decreased thermal stability. The protein ratio imbalance in Comparative Example 3 disrupted the synergistic effect of collagen and whey protein, affecting the carrier formation mechanism and stability. The extremely low degree of hydrolysis in Comparative Example 4 limited the release of active peptides, leading to insufficient drug loading capacity and significantly reduced bioactivity. The omission of the directional enrichment step in Comparative Example 5 reduced the content of active peptides, affecting the selective encapsulation and functional expression of the carrier. The low pH elution conditions in Comparative Example 6 failed to effectively enrich positively charged amino acid residues, reducing the surface charge density and stability of the carrier. Comparative Example 7, using only a single antioxidant, lacked the synergistic effect of multiple components, significantly weakening the construction and protective effect of the antioxidant network. Comparative Example 8, with its low shear conditions, resulted in incomplete emulsification, leading to larger average particle size and uneven distribution, affecting the stability and release performance of the carrier. In Comparative Example 9, the altered lipid ratio disrupted the optimal composition of the membrane structure, affecting the encapsulation efficiency and membrane stability of the carrier. Comparative Example 10, omitting the ceramide component, weakened the integrity and biocompatibility of the membrane structure, reducing the stability and skin affinity of the carrier. Comparative Example 11, with its high pH drug loading environment, may have caused protein peptide denaturation and carrier structure damage, affecting drug loading efficiency and the maintenance of biological activity. Comparative Example 12, employing an active drug loading method, may have damaged pH-sensitive protein peptides, affecting their structural integrity and biological function. Comparative Example 13, lacking a poly-L-histidine layer, weakened pH responsiveness and carrier stability, affecting controlled release and skin permeability. Comparative Example 14, omitting the hyaluronic acid layer, reduced the moisturizing effect and skin compatibility of the carrier, lowering the overall skincare efficacy. Comparative Example 15 showed that the absence of hyaluronic acid oligosaccharide grafting reduced surface functionalization modifications, affecting the interaction between the carrier and the skin and the targeting performance. These structural and process defects together led to a comprehensive decline in key performance indicators such as drug loading efficiency, stability, release performance, skin permeability and bioactivity.

[0108] Table 1. Performance summary of the anti-skin aging bovine protein peptide compositions of Examples 1-4 and Comparative Examples 1-15

[0109]

[0110] The results in the accompanying drawings fully verify the effectiveness and superiority of the technical solution of the present invention.

[0111] Figure 1The images show the morphology of the products at different stages of the preparation process of the anti-skin aging bovine protein peptide composition prepared in Example 1. SEM image analysis shows that this preparation method achieves controllable evolution of morphology from raw materials to finished products. After sequential enzymatic hydrolysis in stage S1, the protein peptides exhibit an irregular granular structure, indicating that enzymatic hydrolysis effectively breaks down the protein molecular chains. Figure 1 a) The conversion of active peptides into finer particles after cation exchange chromatography enrichment in the S2 stage confirms the purification and homogenization effects of chromatographic separation. Figure 1 b) The formation of microparticle structures after the preparation of liposome carriers in the S4 stage verifies the particle size control effect of polycarbonate membrane extrusion technology. Figure 1 c) After the S5 stage multi-layer coating is completed, the final product exhibits a complete spherical core-shell structure and a smooth surface, indicating that the multi-layer coating process has successfully constructed a stable carrier system. Figure 1 d). The entire preparation process achieved a gradual improvement in morphological uniformity, evolving from an irregular structure to a regular spherical core-shell structure. This demonstrates that the process route can effectively control the morphology of the carrier, achieve controllable structural transformation, and obtain nanocarrier products with smooth surfaces and uniform particle sizes, thus verifying the technical feasibility and process stability of the preparation method. In contrast, Figure 2 Comparative Example 13 exhibited significant structural defects due to the omission of the poly-L-histidine coating layer. The carrier surface was rough and uneven, the coating layer was not firmly bonded, peeling occurred in some areas, and the average particle size distribution was uneven, which confirmed the importance of complete coating design.

[0112] Figure 3 To investigate the effect of protein substrate concentration on drug loading efficiency and collagen synthesis promotion, the following parameters were fixed: bovine collagen to bovine whey protein weight ratio of 7.0:3.0, alkaline protease pH 8.5, enzymatic hydrolysis at 62°C for 2.0 hours, and enzyme dosage of 3000 U / g. -1 Papain was hydrolyzed at pH 6.5 and temperature 50°C for 1.5 hours, with an enzyme dosage of 2000 U / g. -1The optimal parameters for drug loading were: cation exchange elution pH 8.2, sodium ascorbate phosphate 0.8 wt%, high shear rotation speed 10000 rpm, lipid concentration 65 mM, hydration pH 5.5, poly-L-histidine layer thickness 2.5 nm, hyaluronic acid layer molecular weight 80 kDa, and polycaprolactone outer layer molecular weight 13000 Da. The protein substrate concentration varied from 10 wt% to 20 wt%. At substrate concentrations of 14.5 to 16.5 wt%, the drug loading efficiency reached 83% to 85%, and the collagen-promoting effect remained at 33% to 35%, exhibiting a synergistic optimal range. At concentrations below 12 wt%, insufficient substrate limited the total amount of enzymatic hydrolysates, resulting in a drug loading efficiency below 79% and a promotion effect of only 29%. At concentrations above 18 wt%, excessive system viscosity inhibited enzyme diffusion, causing uneven hydrolysis and reducing the drug loading efficiency to 81%. This demonstrates that a moderate substrate concentration can simultaneously ensure the yield of active peptides and the encapsulation capacity of the carrier.

[0113] Figure 4 To investigate the effect of the degree of hydrolysis of the protein substrate on drug loading efficiency and the collagen synthesis-promoting effect, the following parameters were fixed: bovine collagen to bovine whey protein weight ratio of 7.0:3.0, substrate concentration of 15 wt%, alkaline protease pH of 8.5, temperature of 62°C, and enzyme dosage of 3000 U / g. -1 Papain, pH 6.5, temperature 50°C, enzyme dosage 2000 U / g -1 The parameters included cation exchange elution at pH 8.2, sodium ascorbate phosphate at 0.8 wt%, high shear rotation speed at 10,000 rpm, lipid concentration at 65 mM, hydration pH at 5.5, poly-L-histidine layer thickness at 2.5 nm, hyaluronic acid layer molecular weight at 80 kDa, and polycaprolactone outer layer molecular weight at 13,000 Da. The degree of protein substrate hydrolysis ranged from 12% to 32%. At a degree of hydrolysis of 20-24%, the drug loading efficiency remained at 84-85%, and the collagen-promoting effect reached 34-35%, achieving a peak performance plateau. Below a degree of hydrolysis of 15%, the proportion of large peptides was too high, resulting in insufficient exposure of active sites and a drug loading efficiency of only 76%, with the promoting effect decreasing to 25%. Above a degree of hydrolysis of 28%, excessive degradation produced too many small molecular fragments, leading to the loss of active domains and a drop in the promoting effect to 32%. This validates that moderate hydrolysis ensures optimal yield and bioactivity retention of target peptides ranging from 300 to 800 Da.

[0114] Figure 5 To investigate the effect of alkaline protease hydrolysis temperature on drug loading efficiency and collagen synthesis promotion effect, the following parameters were fixed: bovine skin collagen to bovine whey protein weight ratio of 7.0:3.0, substrate concentration of 15 wt%, substrate degree of hydrolysis of 22%, alkaline protease pH of 8.5, hydrolysis time of 2.0 hours, and enzyme dosage of 3000 U·g. -1 Papain was hydrolyzed at pH 6.5 and temperature 50°C for 1.5 hours, with an enzyme dosage of 2000 U / g.-1 The optimal parameters for the alkaline protease hydrolysis were: cation exchange elution pH 8.2, sodium ascorbate phosphate 0.8 wt%, high shear rotation speed 10000 rpm, lipid concentration 65 mM, hydration pH 5.5, poly-L-histidine layer thickness 2.5 nm, hyaluronic acid layer molecular weight 80 kDa, and polycaprolactone outer layer molecular weight 13000 Da. The hydrolysis temperature ranged from 54°C to 68°C. At hydrolysis temperatures of 60-64°C, the drug loading efficiency remained at 82-85%, and the collagen-promoting effect reached 32-35%, representing the optimal window for enzyme activity. Below 58°C, enzyme activity significantly decreased, leading to insufficient reaction rate and a drug loading efficiency below 77%, with a promotion effect of only 27%. Above 65°C, partial enzyme inactivation and substrate denaturation triggered non-specific hydrolysis, causing the promotion effect to drop back to 33%. This indicates that precise temperature control is fundamental to achieving efficient and specific enzymatic hydrolysis and selective enrichment of active peptides.

[0115] Figure 6 To investigate the effect of sodium ascorbate phosphate addition on drug loading efficiency and collagen synthesis promotion, the following parameters were fixed: bovine collagen to bovine whey protein weight ratio 7.0:3.0, substrate concentration 15 wt%, substrate hydrolysis degree 22%, alkaline protease pH 8.5, hydrolysis at 62°C for 2.0 h, papain pH 6.5, hydrolysis at 50°C for 1.5 h, cation exchange elution pH 8.2, tocopheryl acetate 0.22 wt%, phytic acid 0.009 wt%, high shear speed 10000 rpm, lipid concentration 65 mM, hydration pH 5.5, poly-L-histidine layer thickness 2.5 nm, hyaluronic acid layer molecular weight 80 kDa, and polycaprolactone outer layer molecular weight 13000 Da. The ascorbate phosphate addition was varied from 0.2 wt% to 1.5 wt%. When the dosage of sodium ascorbate phosphate is 0.7 to 0.9 wt%, the drug loading efficiency is stable at 84 to 85%, and the collagen promotion effect reaches 34 to 35%, showing the optimal ratio of antioxidant synergy. When the dosage is less than 0.4 wt%, insufficient antioxidant protection leads to oxidative damage to active peptides, resulting in a reduction of the promotion effect to below 28%. When the dosage is greater than 1.2 wt%, excessive antioxidants interfere with lipid membrane assembly, causing the drug loading efficiency to drop back to 83%. This demonstrates that an appropriate antioxidant concentration can balance the protection of active ingredients and the stability of the carrier structure.

[0116] Figure 7To investigate the effect of high-shear emulsification speed on drug loading efficiency and collagen synthesis promotion, the following parameters were fixed: bovine collagen to bovine whey protein weight ratio 7.0:3.0, substrate concentration 15 wt%, substrate hydrolysis degree 22%, alkaline protease pH 8.5, hydrolysis at 62°C for 2.0 h, papain pH 6.5, hydrolysis at 50°C for 1.5 h, cation exchange elution pH 8.2, sodium ascorbate phosphate 0.8 wt%, emulsification temperature 25°C for 10 min, lipid concentration 65 mM, hydration pH 5.5, poly-L-histidine layer thickness 2.5 nm, hyaluronic acid layer molecular weight 80 kDa, and polycaprolactone outer layer molecular weight 13000 Da. The high-shear emulsification speed was varied from 6000 rpm to 15000 rpm. When the emulsification speed is 9500 to 10500 rpm, the drug loading efficiency reaches 84 to 85% and the collagen promoting effect is maintained at 34 to 35%, achieving the optimal window for uniform emulsification. When the speed is below 7500 rpm, the insufficient shear force leads to incomplete emulsification and the formation of larger particle size, resulting in a drug loading efficiency of less than 80%. When the speed is above 13000 rpm, the excessive shear energy causes local overheating and shear degradation, causing the promoting effect to drop back to 32%. This verifies that moderate shear strength can simultaneously ensure the uniformity of nanoemulsions and the structural integrity of active peptides.

[0117] Figure 8 To investigate the effect of lipid concentration on drug loading efficiency and collagen synthesis promotion, the following parameters were fixed: bovine collagen to bovine whey protein weight ratio 7.0:3.0, substrate concentration 15 wt%, substrate hydrolysis degree 22%, alkaline protease pH 8.5, temperature 62°C, enzymatic hydrolysis for 2.0 h, papain pH 6.5, temperature 50°C, enzymatic hydrolysis for 1.5 h, cation exchange elution pH 8.2, sodium ascorbate phosphate 0.8 wt%, high shear speed 10000 rpm, phosphatidylcholine to cholesterol to ceramide 3 weight ratio 70:25:5, rotary evaporation temperature 40°C, vacuum degree -0.08 MPa, time 30 min, hydration pH 5.5, poly-L-histidine layer thickness 2.5 nm, hyaluronic acid layer molecular weight 80 kDa, and polycaprolactone outer layer molecular weight 13000 Da. The lipid concentration was varied from 35 mM to 95 mM. When the lipid concentration is 60 to 70 mM, the drug loading efficiency remains at 84 to 85% and the collagen-promoting effect reaches 34 to 35%, showing the optimal concentration range for liposome formation. When the concentration is below 45 mM, insufficient lipids limit the carrier encapsulation volume, resulting in a drug loading efficiency of less than 79%. When the concentration is above 85 mM, the film is too thick, making hydration difficult and forming multilayer vesicles and aggregates, causing the drug loading efficiency to drop back to 82%. This indicates that a moderate lipid concentration is a structural prerequisite for the formation of uniformly sized monolayer liposomes and efficient drug loading.

[0118] Figure 9To investigate the effect of phosphate buffer hydration pH on drug loading efficiency and collagen synthesis promotion, the following parameters were fixed: bovine collagen to bovine whey protein weight ratio 7.0:3.0, substrate concentration 15 wt%, substrate degree of hydrolysis 22%, alkaline protease pH 8.5, hydrolysis at 62°C for 2.0 h, papain pH 6.5, hydrolysis at 50°C for 1.5 h, cation exchange elution pH 8.2, sodium ascorbate phosphate 0.8 wt%, high shear speed 10000 rpm, lipid concentration 65 mM, phosphatidylcholine to cholesterol to ceramide 3 weight ratio 70:25:5, hydration temperature 60°C for 35 min, poly-L-histidine layer thickness 2.5 nm, hyaluronic acid layer molecular weight 80 kDa, and polycaprolactone outer layer molecular weight 13000 Da. The phosphate buffer hydration pH was varied from 4.5 to 6.5. When the hydration pH is 5.3 to 5.7, the drug loading efficiency remains at 84 to 85% and the collagen-promoting effect reaches 33 to 35%, representing the optimal balance point of charge state. When the pH is below 5.0, the excessive protonation of protein peptides enhances electrostatic repulsion, causing the drug loading efficiency to drop below 78%. When the pH is above 6.0, the ionization state of the phospholipid head groups changes, affecting membrane stability and causing the drug loading efficiency to drop back to 81%. This demonstrates that a weakly acidic hydration environment can ensure a mild and efficient drug loading process for the interaction between protein peptides and lipid membranes.

[0119] Combination Figure 10 , Figure 11 and Figure 12 It can be seen that the lipid carrier is located at approximately 2920–2925 cm⁻¹. -1 With 2850–2855 cm -1 —CH2 asymmetric / symmetric stretching vibrations occur at approximately 1740–1735 cm. -1 The ester group C=O stretches, approximately 1465–1470 cm. -1 With 1375–1380 cm -1 The curve is formed by the -CH2 / -CH3 group, approximately 1235–1245 cm. -1 With 1160–1175 cm -1 For C–O–C and C–O stretching, approximately 1060–1090 cm -1 Absorbed by aliphatic C–O / C–C; in multilayer coated empty systems, the —OH / —NH broadband band extends from approximately 3400–3300 cm⁻¹. -1 Enhanced and slightly redshifted to ≈3350–3280 cm -1 The C=O group of the ester group has a length of approximately 1740–1735 cm⁻¹. -1 Slight redshift to ≈1732–1728 cm -1 Furthermore, with the increase in bandwidth, the C–O–C region changes from approximately 1238 cm to approximately 1232 cm. -1≈1168→≈1162cm -1 A slight movement was observed, and the distance was 2920 / 2850 cm. -1 The changes in peak intensity ratio and full width at half maximum (FWHM) suggest the formation of hydrogen bonds and electrostatic interactions; in the final composition, the —OH / —NH broadband band further broadens and shifts center to approximately 3320–3260 cm⁻¹. -1 A weak shoulder peak appears; the C=O main peak is located at approximately 1730–1725 cm. -1 And the amide / carboxyl-related absorption is at ≈1715–1685 cm⁻¹ -1 Signs of coupling are observed; the amide I band is approximately 1655–1635 cm². -1 Amide II band approximately 1550–1530 cm -1 It appears and intensifies; —CH2 stretching is maintained at ≈2922 / 2852 cm. -1 However, the half-width at half-maximum increases; the C–O–C and C–O regions are approximately 1240–1228 cm⁻¹. -1 With ≈1170–1158 cm -1 Cooperative migration occurs, ≈1090–1040 cm -1 The bandwidth has increased. The systematic redshift of the above peaks, the increase in bandwidth, and the emergence of new amide-related bands, along with the absence of new strong covalent bond characteristic peaks (such as new C≡N or C=C features), collectively indicate that the physical complexing and layer-by-layer self-assembly process, mainly based on hydrogen bonds, ion pairs, and van der Waals interactions, actually occurs in the system. This proves the rationality and stability of the multilayer coating strategy and the final composition construction of this invention.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an anti-skin aging bovine protein peptide composition, characterized in that, Includes the following steps: Step S1: Sequential enzymatic hydrolysis to prepare bovine protein peptides: Using a mixture of bovine skin collagen and bovine whey protein as raw materials, the mixture is first hydrolyzed with alkaline protease, then hydrolyzed with papain. After enzyme inactivation treatment, the mixture is separated by a two-stage ultrafiltration membrane to obtain bovine protein peptides with a molecular weight of 300-800 Da. Step S2, Targeted enrichment of active peptides: The protein peptides obtained in step S1 are processed by cation exchange chromatography, using pH gradient elution at pH 7.0~9.5, to enrich active peptides with arginine and lysine content ≥18%. Step S3, Construction of Antioxidant Synergistic Network: The active peptide from step S2 is mixed with sodium ascorbate phosphate, tocopheryl acetate, and phytic acid, and an antioxidant synergistic system is prepared by high-shear emulsification technology; Step S4, preparation of biomimetic carrier and mild drug loading: Phosphatidylcholine, cholesterol and ceramide 3 are mixed and formed into a thin film by rotary evaporation. The film is hydrated with phosphate buffer and S3 complex is added. The pH of the phosphate buffer is 5.0~6.

0. The drug is passively loaded and ultrasonically dispersed. The particle size is controlled to be 65-90 nm by polycarbonate membrane extrusion. Step S5, Multilayer Response Coating Design: In step S4, a poly-L-histidine layer, a hyaluronic acid layer, and a polycaprolactone outer layer are sequentially coated on the surface of the carrier. A stable multilayer structure is formed through electrostatic interaction and hydrogen bonding. Hyaluronic acid oligosaccharides are grafted onto the surface of the polycaprolactone outer layer to obtain an anti-skin aging bovine protein peptide composition. In step S1, the molecular weight cutoff of the dual-stage ultrafiltration membrane is 200-250 Da and 800-1000 Da, the transmembrane pressure difference is 0.1-0.3 MPa, and the degree of protein substrate hydrolysis is controlled at 15-28%. In step S2, the cation exchange chromatography resin has an exchange capacity of 4.2–5.0 mmol / g, and elution is performed using a 0.1–0.5 M NaCl gradient at a flow rate of 0.8–1.5 BV / h and a column temperature of 15–25°C. After enrichment, the active peptides account for 60–85% of the total peptides. In step S3, sodium ascorbate phosphate is 0.4-1.2 wt%, tocopheryl acetate is 0.08-0.35 wt%, and phytic acid is 0.003-0.015 wt%. High-shear emulsification is carried out at a temperature of 18-32°C and a rotation speed of 7500-13000 rpm for 3-18 minutes, and the average droplet size is controlled at 0.1-0.8 μm. In step S4, phosphatidylcholine, cholesterol, and ceramide 3 are mixed in a weight ratio of 68~72:23~27:4~6, with a lipid concentration of 45~85mM, and dissolved in chloroform-methanol at a volume ratio of 1.8~2.2:

1. In step S5, the poly-L-histidine layer has a pKa value of 5.8-6.2 and a thickness of 1.5-3.5 nm; the hyaluronic acid layer has a molecular weight of 45-120 kDa; the polycaprolactone outer layer has a molecular weight of 8000-18000 Da; the coating time for each layer is 25-70 minutes; the coating temperature is 20-32°C; the hyaluronic acid oligosaccharide has a molecular weight of 0.8-12 kDa; the grafting density is 3-18 μg / cm²; and the final average particle size is 120-280 nm. In step S1, bovine collagen and bovine whey protein are mixed at a weight ratio of 6.5-7.5:2.5-3.5, with a protein substrate concentration of 12-18 wt%. In step S1, alkaline protease is enzymatically hydrolyzed at pH 8.3-8.7 and temperature 58-65°C for 1.5-2.5 hours, with an enzyme dosage of 2500-3500 U / g protein. Papain is enzymatically hydrolyzed at pH 6.3-6.7 and temperature 48-52°C for 1.0-2.0 hours, with an enzyme dosage of 1500-2500 U / g protein.

2. The method for preparing an anti-skin aging bovine protein peptide composition as described in claim 1, characterized in that, In step S4, the rotary evaporation temperature is 35-45°C, the vacuum degree is -0.06 to -0.10 MPa, and the time is 20-40 minutes. The phosphate buffer solution is hydrated at 55-65°C for 25-45 minutes with a pH of 5.0-6.

0. The active peptide complex is added for passive drug loading and binding, and the mixture is ultrasonically dispersed at 20 kHz for 10-15 minutes. The peptide to lipid mass ratio is 1:8-15, the phosphate buffer solution is pH 7.2-7.6, and the polycarbonate membrane is extruded using a stepwise reduction in pore size: the first stage pore size is 420-380 nm, the second stage pore size is 220-180 nm, and the third stage pore size is 120-80 nm. The drug loading efficiency is ≥82%.

3. The composition prepared by the method for preparing an anti-skin aging bovine protein peptide composition according to any one of claims 1-2, characterized in that, The composition was prepared using the method described above. The composition has the following technical characteristics: average The particle size distribution polydispersity index (PDI) is less than 0.25, the surface potential (Zeta potential) is -18mV to -22mV, the cumulative release rate in vitro over 24 hours is 58% to 82%, and the promoting effect on skin collagen synthesis is 25% to 45%.

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