Skin repair dressing based on crocodile scale extract and preparation method thereof

Abstract

The invention discloses a skin repair dressing based on a crocodile scale extract and a preparation method thereof, and relates to the technical field of biological medicines and cosmetics, and the preparation method comprises the following steps: enzymatic hydrolysis cross-linking: decalcified crocodile scales are treated with lignin peroxidase under an acidic condition, hydroxypropyl-beta-cyclodextrin is synchronously added, and a collagen cross-linked structure is cracked; the pH is adjusted to be alkaline, and alkaline protease hydrolysis and trypsin hydrolysis are sequentially carried out after hydroxypropyl-beta-cyclodextrin is added; inclusion purification: adding hydroxypropyl-beta-cyclodextrin inclusion active peptide into the filtrate after ultrafiltration, and concentrating and drying to obtain collagen peptide dry powder; and dressing forming: mixing the peptide dry powder with a gel matrix, a humectant and a preservative, homogenizing and sterilizing to obtain a final product. The crocodile scale collagen is efficiently extracted and modified, so that the skin repair dressing with excellent biological activity and stability is prepared.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and cosmetics, and particularly relates to a skin repair dressing based on crocodile scale extract and a preparation method thereof. BACKGROUND

[0002] Skin, as the first barrier between the human body and the external environment, bears the key functions of protecting the organism and regulating body temperature, and is the largest organ in the human body. With the destruction of the earth's atmosphere, the skin is being threatened by various factors, such as burns, scalds, trauma, ultraviolet radiation, skin diseases, etc. Skin problems such as skin itching, blisters, allergies, ultraviolet damage, skin cancer, etc. are becoming more and more common. The repair of damaged skin is a complex physiological process that requires appropriate repair materials to assist in promoting healing. There are various skin repair materials on the market, such as gel dressings with hyaluronic acid as the main component, which have certain moisturizing effect, but the repair effect on deep damage skin is limited; traditional medical Vaseline gauze can only play a simple isolation and protection role, and lacks active ingredients to promote cell proliferation and tissue regeneration.

[0003] In the prior art, natural biological materials have limited repair efficiency due to their dense cross-linked structure and difficulty in fully releasing active ingredients. For example, collagen extracts generally have problems such as incomplete enzymatic hydrolysis and wide molecular weight distribution, resulting in low transdermal absorption rate and difficulty in meeting the repair needs of deep damage. Although biological resources such as crocodile scales are rich in repair active substances, their high-density cross-linked network hinders the dissolution and delivery of effective components, and conventional extraction processes cannot achieve efficient release and stable delivery of active ingredients, limiting their application value in repair dressings. SUMMARY

[0004] The present application provides a skin repair dressing based on crocodile scale extract and a preparation method thereof, which solves the limitations of skin repair materials in repairing damage and the deficiencies of crocodile scale extraction technology in the prior art. By innovatively introducing a synergistic system of lignin peroxidase (LiP) and hydroxypropyl-β-cyclodextrin (HP-β-CD), efficient extraction and modification of crocodile scale collagen are achieved, and a skin repair dressing with excellent biological activity and stability is prepared.

[0005] The present application provides a preparation method of a skin repair dressing based on crocodile scale extract, which includes the following steps: Enzymatic hydrolysis to break cross-linking: decalcified crocodile scales are treated with lignin peroxidase under acidic conditions, and hydroxypropyl-β-cyclodextrin is added simultaneously to crack the collagen cross-linking structure; Double enzymatic hydrolysis synergy: adjust the pH to alkaline, and then sequentially add alkaline protease and trypsin for hydrolysis after adding hydroxypropyl-β-cyclodextrin; Inclusion purification: After ultrafiltration, hydroxypropyl-β-cyclodextrin is added to the filtrate to include the active peptides, and the mixture is concentrated and dried to obtain collagen peptide powder; Dressing molding: The peptide powder is mixed with gel matrix, moisturizer, and preservative, and homogenized and sterilized to obtain the final product.

[0006] Furthermore, the lignin peroxidase comprises small and large particle sizes, with the small particle size being 0.5-1 μm and the large particle size being 10-20 μm, and the mass ratio of the small particle size to the large particle size being 6:4. Small-particle-size lignin peroxidase is used in the acidic enzymatic hydrolysis stage, while large-particle-size lignin peroxidase is used in the alkaline enzymatic hydrolysis stage.

[0007] Furthermore, the hydroxypropyl-β-cyclodextrin includes low-substituted hydroxypropyl-β-cyclodextrin and high-substituted hydroxypropyl-β-cyclodextrin; Low-substituted hydroxypropyl-β-cyclodextrin has a degree of substitution of 0.4-0.6 and is added during the acidic stage; Highly substituted hydroxypropyl-β-cyclodextrin has a degree of substitution of 1.0-1.2 and is added during the alkaline phase.

[0008] Furthermore, the sample was rinsed with pH 5.0 buffer before double enzymatic digestion.

[0009] Furthermore, the conditions for enzymatic hydrolysis to break cross-linking are pH 3.0-3.5, temperature 40-45℃, and reaction time 1-2 hours; the conditions for double enzymatic hydrolysis are pH 7.5-8.5, temperature 45-55℃, and hydrolysis time 2-4 hours.

[0010] Furthermore, in the inclusion purification step, the amount of hydroxypropyl-β-cyclodextrin added is 10%-20% of the peptide dry weight.

[0011] A skin repair dressing comprising the following components by weight percentage: Crocodile scale collagen peptides: 10%-40%; Moisturizer: 5%-15%; Gel matrix: 0.5%-3%; Preservatives: 0.1%-0.5%; The remainder is deionized water.

[0012] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages: Firstly, by introducing lignin peroxidase and hydroxypropyl-β-cyclodextrin, a synergistic system of oxidative cleavage, molecular inclusion, and transdermal delivery was constructed. LiP targets and cleaves the high-energy cross-links of crocodile scale collagen under acidic conditions, overcoming the structural limitations of traditional enzymatic hydrolysis processes. HP-β-CD includes hydrophobic fragments in the alkaline stage to relieve enzyme inhibition and stabilizes peptide conformation to eliminate aggregation during purification, ultimately achieving enhanced transdermal efficacy through the inclusion of Hyp fragments. The two work synergistically to form a combined mechanism of cross-link cleavage, enzyme activity protection, and enhanced transdermal efficacy. The active fragments generated by LiP are included by HP-β-CD, directionally enhancing the stratum corneum's permeability, thus achieving a comprehensive breakthrough at the molecular level from raw material deconstruction to functional delivery, overcoming the technical limitations of collagen extraction such as difficult cross-linking, low enzyme efficiency, and insufficient transdermal delivery. Secondly, a comprehensive regulatory system for molecular cleavage, dynamic inclusion, and conformation locking was constructed through further synergistic effects of lignin peroxidase (LiP) and differentially substituted hydroxypropyl-β-cyclodextrin (HP-β-CD). LiP cleaves collagen cross-links in an acidic environment, avoiding structural limitations of the enzymatic hydrolysis process; low-substituted HP-β-CD instantly includes hydrophobic fragments in an acidic environment to prevent recombination, and achieves safe isoelectric point transitions through pH 5.0 buffer rinsing; high-substituted HP-β-CD forms a hydrophilic barrier in the alkaline stage, protecting the exposed cleavage sites, and in the final stage, secondary inclusion stabilizes the peptide chain conformation, maintaining stability. These three components synergistically overcome the problems of residual cross-linked fragments, pH transition aggregation, and limited transdermal efficiency, simultaneously improving cleavage rate, enzymatic hydrolysis efficiency, and transdermal permeability. Thirdly, a regulatory system for targeted penetration, physical pore expansion, and dynamic inclusion was constructed through the dual synergy of lignin peroxidase particle size fractionation and differential substitution degree of hydroxypropyl-β-cyclodextrin. Small-particle-size LiP targets the deep pores of the cross-linking region to achieve molecular-level cutting, while low-substitution-degree HP-β-CD instantly includes and blocks fragment recombination; large-particle-size LiP physically disrupts the scaffold network, expanding the enzyme reaction interface, while high-substitution-degree HP-β-CD provides hydrophilic protection to ensure efficient enzymatic hydrolysis; and high-DS secondary inclusion in the final stage locks the peptide conformation and enhances transdermal absorption. These three factors synergistically overcome cross-linking residues, insufficient pore expansion, and transdermal limitations, improving the overall performance of the product. Detailed Implementation

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0014] Example 1: A skin repair dressing based on crocodile scale extract, comprising the following components: Crocodile scale collagen peptides: 10%-40%; Moisturizer: 5%-15%; Gel matrix: 0.5%-3%; Preservatives: 0.1%-0.5%; The remainder is deionized water; The molecular weight of the crocodile scale collagen peptide is <5000 Da, and the degree of hydrolysis is ≥25%.

[0015] The method for preparing the skin repair dressing includes the following steps: Step 1: Pre-treatment of crocodile scales; Take fresh crocodile scales, remove impurities and crush them to a particle size ≤ 5 mm. Add 0.3-0.6 mol / L citric acid solution at a material-to-liquid ratio of 1g:(5-10)mL. Decalcify at 20-40℃ for 1-3 hours, and wash with deionized water until neutral. Step 2: LiP enzymes break down cross-links; Add the decalcified flakes to deionized water at a material-to-liquid ratio of 1g:(5-10)mL; Adjust the pH to 3.0-3.5 (using 1M HCl or NaOH); Add 50-100 U / g of lignin peroxidase (LiP) (based on the dry weight of the scales); The reaction was carried out at a constant temperature of 40-45℃ with shaking for 1-2 hours, while 0.3-0.8 mM H2O2 was added dropwise simultaneously (flow rate 0.1-0.3 mL / min). Step 3: Dual-enzyme hydrolysis synergistically with HP-β-CD; Adjust the pH of the reaction solution to 7.5-8.5 (using 1M NaHCO3). Add 1%-3% HP-β-CD (based on dry weight of scales); Add the following in sequence: alkaline protease 0.3%-0.8% (enzymatic hydrolysis at 45-55℃ for 2-4 hours); trypsin 0.2%-0.5% (enzymatic hydrolysis at 45-55℃ for 2-4 hours); heat at 85-95℃ for 10-15 minutes to inactivate the enzymes. Step 4: Purification and inclusion; Centrifuge the enzyme inactivation solution at 8000-10000 r / min for 15-25 minutes; The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the filtrate was collected. Add 10%-20% HP-β-CD (based on peptide dry weight) to the filtrate and stir at 35-45℃ for 20-40 minutes; Step 5: Concentrate and dry; The filtrate was concentrated under reduced pressure at 40-50℃ to 15%-25% of its original volume; Spray drying (inlet air temperature 165-180℃, outlet air temperature 75-85℃) yields crocodile scale collagen peptide dry powder. Step 6: Dressing preparation; Add the gel matrix to deionized water and stir at 55-65℃ for 1-3 hours to dissolve it. Add crocodile scale collagen peptide powder, moisturizer, and preservative in sequence, and stir and mix at 45-55℃ for 0.5-1.5 hours; Homogenize (8000-12000 r / min, 10-20 minutes); After filling, sterilize at 121℃ for 15-20 minutes with moist heat, and then cool to obtain the finished product.

[0016] Experiments were conducted on the technical solution of this embodiment; 1. The experimental groups are shown in Table 1 below; Table 1

[0017] 2. Experimental procedures and parameters: (1) Preparation of crocodile scale collagen peptides; Raw material: Fresh crocodile scales (particle size ≤ 5 mm); Fixed parameters: Decalcification: 0.45 mol / L citric acid, material-to-liquid ratio 1:8, 35℃×2h; Enzymatic hydrolysis: 0.5% alkaline protease, 0.3% trypsin, 50℃ × 2h, pH: 8.0 Purification: Ultrafiltration (5 kDa), concentrated to 20% solids content; LiP treatment: pH 3.0, 40℃, 1h, H2O2 0.5 mM; HP-β-CD addition: Enzymatic digestion stage: 1-3%; Purification stage: 15%; (2) Preparation of skin repair dressing; 25% peptide powder, 10% glycerol, 1.8% carbomer, 0.3% phenoxyethanol; Homogenize (10000 r / min × 15 min), sterilize (121℃ × 15 min); 3. The detection methods and indicators are shown in Table 2 below; Table 2

[0018] 4. The experimental results are shown in Table 3 below; Table 3

[0019] The technical solutions described in the embodiments of this application above have at least the following technical effects or advantages: In an acidic environment, lignin peroxidase activates its heme active center with hydrogen peroxide as a co-substrate. Through a long-range electron transfer mechanism, it targets and attacks the pyridinoline cross-links in crocodile collagen. These cross-links are composed of lysine and hydroxylysine residues connected by a C4-C4' covalent bond, which has high bond energy and is difficult to break effectively by conventional enzymatic hydrolysis. LiP precisely cleaves these high-energy bonds through a free radical catalytic reaction, decomposing the cross-linked collagen into soluble hydroxyproline fragments. This process directly breaks the rigid network structure of collagen fibers, allowing subsequent proteases to access the previously blocked cleavage sites. The cross-linking cleavage rate of collagen treated with LiP is significantly improved compared to traditional methods, removing the physical barrier of the cross-linking structure to the enzymatic hydrolysis reaction. Hydroxypropyl-β-cyclodextrin encapsulates the hydrophobic groups of collagen through its hydrophobic cavity, while its hydrophilic hydroxypropyl chains extend outward to form a hydration layer. During the enzymatic hydrolysis stage, HP-β-CD encapsulates the hydrophobic short peptides generated by enzymatic hydrolysis, preventing them from aggregating or competitively occupying the active site of the protease, thus ensuring the continuous and efficient operation of alkaline protease and trypsin. During the purification stage, the secondary addition of HP-β-CD stabilizes the α-helical conformation of small peptides through van der Waals forces and hydrogen bonds, inhibiting irreversible aggregation caused by β-sheet conversion. This dual effect improves peptide solubility, increases the degree of enzymatic hydrolysis, and avoids the risk of gel product stratification during storage. LiP and HP-β-CD form a molecular-level synergistic system of oxidative cleavage combined with inclusion delivery: hydrophobic Hyp fragments generated by the cleavage of cross-linking bonds during the acidic pretreatment stage of LiP are subsequently encapsulated and stabilized by HP-β-CD through a cavity. This inclusion effect prevents fragment recombination and cross-linking, while relieving its inhibition of the trypsin active site, further improving the enzymatic hydrolysis efficiency. When the HP-β-CD-encapsulated Hyp fragments penetrate the stratum corneum, their hydrophilic shell disrupts the orderly arrangement of the lipid bilayer, enhancing permeability. Hyp itself, as a collagen synthesis precursor, accelerates dermal repair. The synergy between the two increases the transdermal penetration rate, significantly improving the effect compared to a single component. The bilayer inclusion network, namely the low hydrophobic inner layer combined with the high hydrophilic outer layer, completely blocks the peptide chain aggregation pathway, resulting in a reduced precipitation rate and prolonged stability after 3 months of accelerated testing at 40℃. By introducing lignin peroxidase and hydroxypropyl-β-cyclodextrin, a synergistic system of oxidative cleavage, molecular inclusion, and transdermal delivery was constructed: LiP targets and cleaves the high-energy cross-links of crocodile scale collagen in an acidic environment, overcoming the structural limitations of traditional enzymatic hydrolysis processes; HP-β-CD includes hydrophobic fragments in an alkaline stage to relieve enzyme inhibition and stabilizes peptide conformation to eliminate aggregation in the purification stage, ultimately achieving transdermal enhancement by including Hyp fragments. The two work synergistically to form a combined mechanism of cross-link cleavage, enzyme activity protection, and transdermal enhancement. The active fragments generated by LiP are included by HP-β-CD to directionally enhance the stratum corneum's permeability, thereby achieving a comprehensive breakthrough from raw material deconstruction to functional delivery at the molecular level, overcoming the technical limitations of collagen extraction such as difficult cross-linking, low enzyme efficiency, and insufficient transdermal delivery.

[0020] Example 2: The above Example 1 establishes a comprehensive regulatory system from molecular structure to functional delivery by introducing lignin peroxidase (LiP) and hydroxypropyl-β-cyclodextrin (HP-β-CD): LiP cleaves the high-strength cross-linking bonds of crocodile scale collagen in an acidic environment, overcoming the structural limitations of the enzymatic hydrolysis process; HP-β-CD encapsulates hydrophobic fragments in the alkaline stage to maintain enzyme activity, and stabilizes peptide conformation, eliminates aggregation and layering in the final product, and enhances transdermal efficiency. The two work together through a molecular relay mechanism of electron transfer catalytic cleavage and cavity inclusion directional delivery, which significantly improves the cross-linking cleavage rate, enzymatic hydrolysis efficiency and transdermal absorption rate, and achieves long-term stable storage of the product. Further improvements are made based on Example 1 to further improve the overall performance of the product.

[0021] The HP-β-CD includes both low and high substitution degrees; Low-substitution degree HP-β-CD: DS 0.4-0.6, added during the acidic pretreatment stage, at a rate of 1%-2% of the dry weight of the scales; High-substitution HP-β-CD: DS 1.0-1.2, added during the alkaline enzymatic hydrolysis stage, at a rate of 1%-2% of the dry weight of the scales; The total addition of low-substitution and high-substitution products is 1%-3%; The specific steps for adding are as follows: Step 2: LiP enzymes break down cross-links; Add 50-100 U / g of lignin peroxidase (LiP) (based on the dry weight of the scales); Simultaneously add 1%-2% (based on dry weight of flakes) of low-substitution degree HP-β-CD (DS 0.4-0.6); Step 3: Dual-enzyme hydrolysis synergistically with HP-β-CD; The reaction solution was filtered, and the filter residue was rinsed three times with pH 5.0 phosphate buffer (each rinse ≤3 minutes). Adjust the pH of the reaction solution to 7.5-8.5 (using 1M NaHCO3). Add 1%-2% (dry weight of flakes) of high DS HP-β-CD (DS 1.0-1.2); Step 4: Purification and inclusion; Centrifuge the enzyme inactivation solution at 8000-10000 r / min for 15-25 minutes; The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the filtrate was collected. Add 10-20% (based on peptide dry weight) of high DS HP-β-CD (DS 1.0-1.2) to the filtrate and stir at 35-45℃ for 20-40 minutes.

[0022] This embodiment is based on the experiment of group E in Example 1, and is referred to as group E-1. The difference between the experiments in group E-1 and group E is that the HP-β-CD includes low-substitution degree and high-substitution degree; low-substitution degree HP-β-CD: DS 0.4-0.6, added in the acidic pretreatment stage, with an addition amount of 1%; high-substitution degree HP-β-CD: DS 1.0-1.2, added in the alkaline enzymatic hydrolysis stage, with an addition amount of 1%; the experimental results are shown in Table 4 below; Table 4

[0023] The technical solutions described in the embodiments of this application above have at least the following technical effects or advantages: In an acidic environment, lignin peroxidase catalyzes the decomposition of hydrogen peroxide through its heme active site, generating a high-valent ferro-oxygen intermediate (Compound I). This active substance precisely breaks the strong pyridinline cross-links in crocodile scale collagen through a long-range electron transfer mechanism, decomposing the cross-linked structure into soluble hydroxyproline fragments. This overcomes the dense network of collagen fibers, creating fully exposed cleavage sites for subsequent enzymatic hydrolysis. Simultaneously, low-substituted hydroxypropyl-β-cyclodextrin is added during the acidic pretreatment stage. Its low-substitution characteristic exposes the strongly hydrophobic cavity of native β-cyclodextrin, which efficiently encapsulates the hydrophobic cross-linked fragments generated by LiP cleavage through van der Waals forces, forming stable inclusion complexes that block fragment recombination and cross-linking, thereby increasing the cross-linking cleavage rate. Before the system is transferred to an alkaline environment, a safe transition is achieved by rinsing with pH 5.0 phosphate buffer: the rinsing solution maintains the potential, avoids the collagen isoelectric point aggregation window, and protects the low-substitution degree HP-β-CD inclusion complex from dissociation, significantly reducing the aggregate particle size and eliminating a large amount of aggregation loss in traditional processes. In the alkaline enzymatic hydrolysis stage, high-substitution degree HP-β-CD is added. Its abundant hydroxypropyl groups form a hydrophilic hydration layer, which blocks the hydrophobic aggregation of nascent peptides through steric hindrance, reduces the local viscosity of the solution, increases the protease diffusion coefficient, reduces the masking rate of the enzyme cleavage site, and increases the degree of hydrolysis. During the purification stage, a second addition of highly substituted HP-β-CD locks the α-helix conformation of the peptide chain through hydrogen bonds, inhibiting β-sheet conversion, thereby improving the solubility of the final product and achieving further stable storage. Simultaneously, the Hyp fragment generated by LiP is encapsulated by the highly substituted HP-β-CD to form an amphiphilic complex: its hydrophilic shell disrupts the orderly arrangement of lipids in the stratum corneum, lowering the diffusion barrier; its hydrophobic core acts as a collagen synthesis precursor, reaching directly into the dermis, further enhancing transdermal permeability. The single degree of substitution (DS) HP-β-CD has limitations in cross-pH enzymatic hydrolysis processes. First, there is a conflict between the requirements for hydrophobicity and hydrophilicity; a single DS value cannot simultaneously meet the high hydrophobic inclusion requirement in acidic environments and the high hydrophilic protection requirement in alkaline environments. Second, when the pH jumps abruptly from acidic (3.0) to alkaline (8.0), a single DS HP-β-CD cannot inhibit the violent aggregation of collagen at its isoelectric point. In addition, in terms of conformational stability, a single DS HP-β-CD has insufficient ability to lock the conformation of peptide chains, resulting in limitations in the solubility and transdermal efficiency of the final product. By further synergistically combining lignin peroxidase (LiP) and differentially substituted hydroxypropyl-β-cyclodextrin (HP-β-CD), a comprehensive regulatory system for molecular cleavage, dynamic inclusion, and conformational locking was constructed: LiP cleaves collagen cross-links in an acidic environment, avoiding structural limitations of the enzymatic hydrolysis process; low-substituted HP-β-CD is used to instantly include hydrophobic fragments in an acidic environment to prevent recombination, and achieves safe isoelectric point transitions through pH 5.0 buffer rinsing; high-substituted HP-β-CD forms a hydrophilic barrier in the alkaline stage to protect the exposed cleavage sites, and in the final stage, secondary inclusion stabilizes the peptide chain conformation, maintaining stability. The three components synergistically overcome the problems of residual cross-linked fragments, pH transition aggregation, and limited transdermal efficiency, simultaneously improving cleavage rate, enzymatic hydrolysis efficiency, and transdermal permeability.

[0024] Example 3: Example 2 utilizes a differentiated substitution degree design, specifically using low-substitution-degree HP-β-CD in an acidic environment to enhance hydrophobic inclusion capacity, and high-substitution-degree HP-β-CD in an alkaline environment to enhance hydrophilic protection. This solves the problem that single-substitution-degree HP-β-CD cannot simultaneously meet the dual requirements of hydrophobic capture and hydrophilic shielding in cross-pH enzymatic hydrolysis processes. Simultaneously, it avoids the uncontrollable aggregation of collagen near its isoelectric point caused by charge abrupt changes, resulting in a simultaneous improvement in lysis rate, enzymatic hydrolysis efficiency, and transdermal penetration rate. To further improve the overall performance of the product, further improvements were made based on Example 2.

[0025] The LiP includes small-sized and large-sized particles, with small-sized particles being 0.5-1 μm and large-sized LiP particles being 10-20 μm. The mass ratio of small to large particle size is 6:4; small particles are added during the acidic pretreatment stage; large particles are added during the alkaline enzymatic hydrolysis stage.

[0026] The preparation of small-particle-size LiP (0.5-1 μm) involves the following specific steps: The LiP solution (50 U / mL) was mixed with sodium alginate (1.5%) and then broken up using a high-pressure microjet system (pressure 150 MPa). Spray drying (inlet air 160℃, outlet air 60℃) yields powder with a particle size of 0.5-1μm and an encapsulation rate of ≥85%. The specific steps for preparing large-particle-size LiP (10-20 μm) are as follows: The LiP solution (50 U / mL) was mixed with PLGA (10%), emulsified, and then solidified. Freeze-drying yields porous microparticles with a particle size of 10-20 μm and an encapsulation efficiency of ≥90%. This embodiment is based on the experiment of group E-1 in Example 1, and is designated as group E-2. The difference between group E-2 and group E-1 is that the LiP includes both small and large particle sizes. The small particle size is 0.5-1 μm, and the large particle size LiP is 10-20 μm; the mass ratio of small to large particle sizes is 6:4; the small particle size is added during the acidic pretreatment stage, and the large particle size is added during the alkaline enzymatic hydrolysis stage. The experimental results are shown in Table 5 below. Table 5

[0027] The technical solutions described in the embodiments of this application above have at least the following technical effects or advantages: Lignin peroxidase achieves synergistic functional combination through particle size classification design: small-particle-size LiP, under acidic conditions, leverages its nanoscale size advantage to penetrate the submicron pores of the cross-linked region of crocodile scales, targeting deep pyridine-line bonds that traditional enzyme molecules cannot reach, and precisely cleaving the cross-linked structure through catalytic oxidation reaction, further reducing the fragmentation rate; large-particle-size LiP, in the alkaline stage, releases kinetic energy through mechanical collision, physically breaking the rigid network of the scale skeleton, increasing porosity, and creating highly permeable reaction channels for protease; Low-substituted HP-β-CD encapsulates hydrophobic fragments generated by the cleavage of small-particle LiP in the acidic stage, and its strong hydrophobic cavity blocks the fragment recombination pathway. High-substituted HP-β-CD forms a hydrophilic barrier in the alkaline stage, which, together with the high diffusion environment after the pore expansion of large-particle LiP, further improves the enzymatic hydrolysis efficiency of the protease. The two work together through the matching of different particle sizes and different degrees of substitution: after small-particle LiP completes molecular-level cleavage, low-DS HP-β-CD immediately encapsulates and stabilizes the product; after large-particle LiP physically expands the pores, high-DS HP-β-CD blocks the aggregation of nascent peptides, forming a combination of enzymatic hydrolysis and protection. In the final product stage, the high-DS HP-β-CD secondary inclusion locks the α-helical conformation, inhibits β-sheet transformation, and stabilizes and improves solubility; its included Hyp fragment forms an amphiphilic complex, the hydrophilic chain disrupts the lipid arrangement of the stratum corneum, and the hydrophobic core reaches the dermis directly, increasing the transdermal rate. The synergistic combination of LiP particle size fractionation and HP-β-CD substitution degree enables the enzymatic hydrolysis process to form a coordinated process of targeted penetration, physical pore expansion, and dynamic inclusion; By employing a dual synergistic approach of lignin peroxidase particle size fractionation and hydroxypropyl-β-cyclodextrin substitution degree differentiation, a regulatory system for targeted penetration, physical pore expansion, and dynamic inclusion was constructed: small-particle-size LiP targets the deep pores of the cross-linking region to achieve molecular-level cutting, while low-substitution-degree HP-β-CD instantly includes and blocks fragment recombination; large-particle-size LiP physically breaks down the scaffold network to expand the enzyme reaction interface, while high-substitution-degree HP-β-CD provides hydrophilic protection to ensure efficient enzymatic hydrolysis; and high-DS secondary inclusion in the final stage locks the peptide conformation and enhances transdermal absorption. These three aspects work synergistically to overcome cross-linking residues, insufficient pore expansion, and transdermal limitations, further improving the overall performance of the product.

[0028] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a skin repair dressing based on crocodile scale extract, characterized in that, Includes the following steps: Enzymatic hydrolysis to break cross-links: Decalcified crocodile scales were treated with lignin peroxidase under acidic conditions, and hydroxypropyl-β-cyclodextrin was added simultaneously to lyse the collagen cross-linked structure; Synergistic dual-enzyme hydrolysis: After adjusting the pH to alkaline, hydroxypropyl-β-cyclodextrin was added, followed by sequential hydrolysis with alkaline protease and trypsin; Inclusion purification: After ultrafiltration, hydroxypropyl-β-cyclodextrin is added to the filtrate to include the active peptides, and the solution is concentrated and dried to obtain collagen peptide powder. Dressing formation: Mix the peptide powder with the gel matrix, moisturizer, and preservative, then homogenize and sterilize to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The lignin peroxidase includes small and large particle sizes, with small particle sizes of 0.5-1 μm and large particle sizes of 10-20 μm, and the mass ratio of small to large particle sizes is 6:

4. Small-particle-size lignin peroxidase is used in the acidic enzymatic hydrolysis stage, while large-particle-size lignin peroxidase is used in the alkaline enzymatic hydrolysis stage.

3. The preparation method according to claim 1, characterized in that, The hydroxypropyl-β-cyclodextrin includes low-substituted hydroxypropyl-β-cyclodextrin and high-substituted hydroxypropyl-β-cyclodextrin; Low-substituted hydroxypropyl-β-cyclodextrin has a degree of substitution of 0.4-0.6 and is added during the acidic stage; Highly substituted hydroxypropyl-β-cyclodextrin has a degree of substitution of 1.0-1.2 and is added during the alkaline phase.

4. The preparation method according to claim 1, characterized in that, Wash with pH 5.0 buffer before double enzymatic digestion.

5. The preparation method according to claim 1, characterized in that, The conditions for enzymatic hydrolysis to break cross-links are pH 3.0-3.5, temperature 40-45℃, and reaction time 1-2 hours; the conditions for double enzymatic hydrolysis are pH 7.5-8.5, temperature 45-55℃, and hydrolysis time 2-4 hours.

6. The preparation method according to claim 1, characterized in that, In the inclusion purification step, the amount of hydroxypropyl-β-cyclodextrin added is 10%-20% of the peptide dry weight.

7. A skin repair dressing prepared by the preparation method according to any one of claims 1-6, characterized in that, It contains the following components by mass percentage: Crocodile scale collagen peptides: 10%-40%; Moisturizer: 5%-15%; Gel matrix: 0.5%-3%; Preservatives: 0.1%-0.5%; The remainder is deionized water.

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