Animal-derived composite fermented skin-tendering protein peptide and preparation method thereof

Abstract

The invention belongs to the field of bioactive protein peptide materials, and provides an animal-derived composite fermented skin-tendering protein peptide and a preparation method thereof. According to the invention, bovine-derived collagen and fish-derived protein are subjected to synergistic fermentation to prepare a protein peptide core, and the outer surface of the protein peptide core is coated with an edible gelatin and chondroitin sulfate composite layer; the excellent performance that a small molecule peptide core with the molecular weight being 500-1500 Da, a stable coating layer with the coating rate being 80-99%, the glycine-proline-hydroxyproline sequence collagen peptide fragment accounting for 30-60% and the pH responsive swelling ratio being 1.5-3.0 are achieved, the technical problems that traditional protein peptide is poor in stability and low in bioavailability are solved, and the protein peptide has wide application value in the field of skin care products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biologically active protein peptide materials, in particular to an animal-derived composite fermented skin-smoothing protein peptide and a preparation method thereof. BACKGROUND

[0002] With the rapid development of modern biological beauty technology, protein peptide active ingredients are increasingly widely used in the field of skin care products, especially in high-end skin care products such as anti-aging, repair and skin-smoothing, and play a key role. In this application scenario, protein peptide materials need to have excellent stability to ensure the integrity of active ingredients in complex skin care product formula systems, and also need to have high bioavailability to achieve effective skin penetration and absorption. Stability requires protein peptides to maintain the integrity of the molecular structure under different pH values, temperature changes and light conditions, avoiding degradation and inactivation. High bioavailability requires protein peptides to effectively penetrate the skin barrier, reach the target site and exert the expected biological activity. Meeting these performance requirements is of great significance to improve the efficacy of skin care products, prolong the shelf life of products, and enhance the consumer experience. It is also a key technical basis for the development of protein peptide skin care products to higher quality and wider application range, providing strong support for the technological progress and industrial upgrading of the entire biological beauty industry.

[0003] However, there are currently technical bottlenecks of poor stability and low bioavailability of protein peptide materials in skin care applications, which seriously restricts their widespread use in high-end skin care products. In terms of stability, traditional protein peptides contain a large number of peptide bonds and active groups in their molecular structure, which are prone to hydrolysis, oxidation and aggregation in the complex environment of skin care products, leading to rapid degradation and failure of active ingredients. This is mainly due to the lack of effective protection mechanisms and stable structure design of protein peptide molecules. At the same time, the problem of low bioavailability is also prominent. Due to the large molecular weight and strong hydrophilicity of protein peptides, it is difficult to effectively penetrate the stratum corneum barrier of the skin, and most active ingredients remain on the skin surface and cannot reach the dermis to function. The root cause of this problem is the lack of suitable carrier systems and penetration promotion mechanisms. For example, Chinese Patent No. CN117547475A discloses a preparation method of a collagen peptide composition for beauty and skin care, but there is a problem of insufficient stability of protein peptides. For example, Chinese Patent No. CN120210313A discloses a preparation method and application of squid collagen peptide with beauty and skin care effects, but there is a problem of low bioavailability. These technical defects directly affect the actual application effect and market competitiveness of protein peptide skin care products. SUMMARY

[0004] (1) Technical problems solved The application aims to provide an animal source compound fermentation skin-smoothing protein peptide and a preparation method, and solve the problems of stability and bioavailability of the protein peptide.

[0005] (2) Technical scheme In order to achieve the above-mentioned purpose, the application provides the following technical scheme: An animal source compound fermentation skin-smoothing protein peptide, comprising a fermentation protein peptide core and an animal source compound coating layer covering the outer surface of the fermentation protein peptide core. The fermentation protein peptide core is prepared by synergistic fermentation of bovine collagen and fish protein, wherein the bovine collagen is extracted from bovine tissue, and the fish protein is extracted from fish tissue. The fish protein is one or both of fish collagen and fish elastin. The fermentation protein peptide core has a molecular weight of 500-1500 Da and an average particle size of 20-60 μm. The animal source compound coating layer is composed of edible gelatin and chondroitin sulfate, the edible gelatin is prepared by acid hydrolysis of bovine bone, and the chondroitin sulfate is prepared by enzymatic extraction of bovine cartilage. The animal source compound coating layer has an average thickness of 2.0-10.0 μm and a coating rate of 80-99%. The mass ratio of bovine protein solid to fish protein solid in the fermentation protein peptide core is 70-90:10-30.

[0006] The core-shell structure design is mainly used to enhance the stability and bioavailability of the protein peptide. The fermentation protein peptide core is prepared by synergistic fermentation of bovine collagen and fish protein, wherein the bovine collagen provides abundant glycine-proline-hydroxyproline sequences, and the fish collagen and fish elastin supplement specific amino acid composition and bioactive fragments. The synergistic fermentation of the two can produce more diverse active peptide segments than single protein source, significantly improving the overall bioactivity effect. The fermentation process degrades macromolecular proteins into small molecular peptides, effectively improving the bioavailability and making the active ingredients more easily absorbed by the skin. The outer animal source compound coating layer is composed of edible gelatin and chondroitin sulfate. The edible gelatin provides good film-forming property and protection as a natural polymer material, and the chondroitin sulfate has excellent moisturizing property and biocompatibility. The combination of the two forms a coating layer that not only effectively protects the internal protein peptide core from the adverse effects of the external environment and prevents oxidative degradation, but also regulates the release rate of the active ingredients to achieve a sustained-release effect. Through the cross-linking solidification treatment of glutamine transaminase, the coating layer structure is more stable, further enhancing the stability of the overall product and ensuring the activity of the protein peptide during storage and use.

[0007] Further, the preparation method of the fermented protein peptide core comprises the following steps: Step A1, take the bovine-derived collagen protein solution with a concentration of 15-35 mg / mL, add alkaline protease for pre-enzymolysis, the enzyme dosage is 0.1-0.15% of the weight of collagen protein, and the enzymolysis is carried out at a pH value of 8.5 and a temperature of 37°C for 2-4 h; Step A2, mix the pretreated collagen protein solution with the fish-derived protein hydrolysate according to the mass ratio of bovine-derived to fish-derived protein solids of 70-90:10-30, inoculate the fermentation microorganism, and synergistically ferment under the conditions of a pH value of 6.8-7.2 and a temperature of 30-37°C for 24-36 h; Step A3, form a coating layer by the method of complex co-precipitation of edible gelatin and chondroitin sulfate, and then cross-link and solidify by glutamine transaminase, and then perform fluidized bed drying; Among them, the degree of hydrolysis of the synergistic fermentation is controlled at 15-20%.

[0008] Further, the fermentation microorganism used in the synergistic fermentation process is one or more of Lactobacillus acidophilus, Saccharomyces cerevisiae and Bifidobacterium, the inoculation amount is 1-3% of the total weight of the fermentation system, and the stirring speed is 100-200 rpm.

[0009] Further, the concentration of the edible gelatin in the process of forming the animal-derived composite coating layer is 8-12 mg / mL, and the concentration of the chondroitin sulfate is 2-6 mg / mL; The edible gelatin and chondroitin sulfate are mixed according to a mass ratio of 2-5:1, the pH value is adjusted to 4.2-4.8 at a temperature of 25-35°C to form complex co-precipitation; The preparation method of the edible gelatin is as follows: the bovine bone is crushed to 1-3 mm, pretreated with a 0.3-1.5% citric acid solution for 12-24 h, then hydrolyzed and extracted at a temperature of 80-100°C for 4-8 h, and then filtered, concentrated and dried to obtain the edible gelatin; The preparation method of the chondroitin sulfate is as follows: the bovine cartilage is cut into pieces, and then enzymolyzed with protease at a pH value of 7.0-8.0 and a temperature of 50-60°C for 6-12 h, and then separated and purified to obtain the chondroitin sulfate; The preparation method of the fish-derived collagen protein is as follows: the fish skin or fish scales are pretreated with a dilute acid or dilute alkali solution for 8-16 h, and then hydrolyzed and extracted at a temperature of 70-90°C for 3-6 h, and then filtered, concentrated and dried to obtain the fish-derived collagen protein; The preparation method of the fish-derived elastin is as follows: the fish skin is enzymolyzed with trypsin at a pH value of 8.0-8.5 and a temperature of 40-50°C for 4-8 h, and then separated and purified to obtain the fish-derived elastin.

[0010] Further, the amount of the transglutaminase used in step A3 is 0.03-0.08% of the total mass of the animal source composite coating layer, the crosslinking temperature is 30-38℃, the crosslinking time is 1.5-2.5 h, and the decrease rate of free amino groups is not less than 25% as the determination standard of the crosslinking end point.

[0011] Further, the fermentation protein peptide core contains collagen peptide fragments with glycine-proline-hydroxyproline sequences, and the collagen peptide fragments account for 30-60% of the total peptide content; the ratio of the swelling degree of the animal source composite coating layer at pH 4.5 to the swelling degree at pH 7.4 is 1.5-3.0.

[0012] Further, the animal source composite coating layer further contains at least one antioxidant and emulsifying auxiliary material selected from the group consisting of tocopherol, rosemary leaf extract and fish oil phospholipid, and the content is 0.005-0.5% based on the total weight of the animal source composite coating layer; the animal source composite coating layer further contains eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester, and the total amount is 0.02-0.20% based on the total weight of the animal source composite coating layer.

[0013] The preparation strategy of step-by-step enzymatic hydrolysis-synergistic fermentation-composite coating is mainly used for enhancing the biological activity and stability performance of protein peptides. The preparation process first performs pre-enzymatic hydrolysis treatment on bovine collagen protein by alkaline protease, effectively cuts the peptide bond of macromolecular protein, and creates favorable conditions for subsequent synergistic fermentation. Then, the pretreated collagen protein solution is mixed with fish protein hydrolysate at a specific ratio, and is inoculated with fermentation microorganisms such as Lactobacillus acidophilus, Saccharomyces cerevisiae and Bifidobacterium for synergistic fermentation. The multi-strain synergistic fermentation mechanism can fully exert the enzyme characteristics of different microorganisms, produce more abundant bioactive peptide segments than single-strain fermentation, and ensure ideal molecular weight distribution by controlling the degree of fermentation hydrolysis, thereby significantly improving the bioavailability of the final product. In the preparation process of the coating layer, edible gelatin and chondroitin sulfate form a composite co-precipitate under a specific pH condition, and the two form a stable composite structure through hydrogen bonding and electrostatic interaction. The edible gelatin provides good film-forming property, and the chondroitin sulfate contributes unique pH-responsive swelling properties, so that the coating layer can adjust the permeability according to the change of the environmental pH. The crosslinking solidification of transglutaminase further strengthens the mechanical strength and chemical stability of the coating layer, the decrease rate of free amino groups is monitored to ensure the full progress of the crosslinking reaction, and finally the effective protection and controlled release of the core protein peptide are realized.

[0014] The application further discloses a preparation method of the animal source composite fermentation skin-smoothing protein peptide. Step S1: Mix the fermented protein peptide core with edible gelatin and chondroitin sulfate, wherein the concentration of edible gelatin is 8-12 mg / mL and the concentration of chondroitin sulfate is 2-6 mg / mL. Step S2: Adjust the pH value to 4.2-4.8 at a temperature of 25-35℃ to form a composite co-precipitation coating layer of edible gelatin and chondroitin sulfate. Step S3: Add transglutaminase for cross-linking and curing. The amount of enzyme used is 0.03-0.08% of the total mass of the animal-derived composite coating layer. The cross-linking temperature is 30-38℃ and the cross-linking time is 1.5-2.5 h. Step S4: The cross-linked product is subjected to fluidized bed drying with an inlet air temperature of 55-65℃, an outlet air temperature of 45-55℃, a fluidizing air velocity of 2-5 m / s, and a spraying speed of 1-3 mL / min.

[0015] Furthermore, the process parameters for fluidized bed drying in step S4 are as follows: inlet air temperature is 55-65℃, outlet air temperature is 45-55℃, fluidization air velocity is 2-5 m / s, spraying speed is 1-3 mL / min, and coating efficiency is not less than 85%.

[0016] The application of an animal-derived compound fermented skin-rejuvenating protein peptide in skin care products, wherein the skin care products are one or more of face cream, serum, face mask and lotion, and the amount of the animal-derived compound fermented skin-rejuvenating protein peptide added to the skin care products is 0.5-3% of the total weight of the skin care products.

[0017] This invention employs an integrated preparation process of mixing-co-precipitation-crosslinking-drying, primarily aimed at enhancing the coating efficiency and product stability of protein peptides. This preparation method fully mixes the fermented protein peptide core with edible gelatin and chondroitin sulfate at specific concentrations, laying the foundation for subsequent coating layer formation. The synergistic effect of edible gelatin and chondroitin sulfate, under acidic pH conditions, enables the formation of a stable composite co-precipitation structure. The intermolecular interaction between the two materials produces a superior coating effect compared to using either material alone. The introduction of transglutaminase achieves crosslinking and solidification of the coating layer. Through enzymatic catalysis, a covalent crosslinking network is formed under mild conditions, significantly improving the mechanical strength and chemical stability of the coating layer and ensuring effective protection of the protein peptide core. The fluidized bed drying process utilizes precisely controlled temperature gradients and fluidization parameters, ensuring thorough drying of the product while avoiding the adverse effects of high temperatures on protein peptide activity. By optimizing the coordination of inlet and outlet air temperatures, fluidization velocity, and spray velocity, high coating efficiency is achieved while maintaining the product's bioactivity. The animal-derived complex fermented skin-nourishing protein peptides obtained by this preparation process can be widely used in various skin care products such as face creams, serums, masks, and lotions. By adding the appropriate amount, the expected skin care effects can be achieved, providing a feasible technical path for the industrial production of protein peptide skin care products.

[0018] (3) Beneficial technical effects 1. Significantly improves protein peptide stability: Through the core-shell structure design of the animal-derived composite coating layer and the fermented protein peptide core, the coating layer formed by edible gelatin and chondroitin sulfate effectively protects the internal protein peptide from the adverse effects of the external environment, preventing oxidative degradation and inactivation. At the same time, the cross-linking and solidification treatment of transglutaminase further enhances the structural stability of the coating layer, ensuring that the protein peptide maintains its activity for a long time during storage and use.

[0019] 2. Effectively improves bioavailability: Small molecule protein peptide cores are prepared through the synergistic fermentation of bovine collagen and fish protein, which degrades large molecule proteins into active peptides with a molecular weight of 500-1500 Da, significantly improving skin absorption efficiency. At the same time, the pH-responsive swelling properties of the coating layer can adjust the release rate of active ingredients according to changes in the skin environment, achieving better penetration and bioavailability.

[0020] 3. Significantly enhances bioactivity: Through a multi-strain synergistic fermentation mechanism, the enzyme system characteristics of Lactobacillus acidophilus, Saccharomyces cerevisiae, and Bifidobacterium are fully utilized to produce more diverse and abundant active peptides than a single protein source. Among them, the glycine-proline-hydroxyproline sequence collagen peptide fragment accounts for 30-60% of the total peptide content. Combined with the unique amino acid composition and bioactive fragments of fish-derived proteins, it synergistically enhances the overall skin-rejuvenating effect.

[0021] 4. Achieve efficient coating protection: The coating layer formed by the co-precipitation method of edible gelatin and chondroitin sulfate has an average thickness of 2.0-10.0μm and a coating rate of 80-99%. The coating efficiency is not less than 85% through a precisely controlled fluidized bed drying process, ensuring that the protein peptide core is fully and effectively protected, while maintaining the product's bioactivity and functionality.

[0022] 5. Expanding the application scope of skin care products: The animal-derived compound fermented skin-nourishing protein peptides prepared by this invention can be widely used in various skin care products such as face creams, serums, masks and lotions. The expected skin care effects can be achieved by adding an appropriate amount of 0.5-3%, providing a feasible technical solution for the industrial production and diversified application of protein peptide skin care products. Attached Figure Description

[0023] Figure 1 This is a cross-sectional view of the fermented protein peptide core prepared in Example 3 of the present invention.

[0024] Figure 2 This is a morphological image of the animal-derived compound fermented skin-rejuvenating protein peptide prepared in Example 3 of the present invention.

[0025] Figure 3 This is a morphological image of the animal-derived compound fermented skin-rejuvenating protein peptide prepared in Comparative Example 9 of this invention.

[0026] Figure 4 This invention relates to the effect of fermentation temperature on collagen peptide content and microbial activity.

[0027] Figure 5 This invention investigates the effect of enzyme dosage on the pass rate of collagen peptide content and molecular weight distribution.

[0028] Figure 6 This example compares the content of 500-1500 Da molecular weight peptides with pH response swelling ratios for both the example and the comparative examples.

[0029] Figure 7 The collagen peptide content and antioxidant activity IC50 are compared between the examples and the comparative examples.

[0030] Figure 8 This example compares the storage stability retention rate and 24-hour cumulative release rate between the embodiment and the comparative example. Detailed Implementation

[0031] 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.

[0032] Example 1: An animal-derived composite fermented skin-rejuvenating protein peptide comprises a fermented protein peptide core and an animal-derived composite coating layer covering the outer surface of the fermented protein peptide core in this embodiment. The fermented protein peptide core in this embodiment is prepared by co-fermentation of bovine collagen and fish protein, wherein the bovine collagen is extracted from bovine tissue and the fish protein is extracted from fish tissue; the fish protein in this embodiment is fish collagen. The molecular weight of the fermented protein peptide core in this embodiment is 500 Da, and the average particle size is 20 μm. The animal-derived composite coating layer in this embodiment is composed of edible gelatin and chondroitin sulfate. The edible gelatin in this embodiment is obtained by acid hydrolysis of bovine bone, and the chondroitin sulfate in this embodiment is obtained by enzymatic extraction from bovine cartilage. The average thickness of the animal-derived composite coating layer in this embodiment is 2.0 μm, and the coating rate is 80%. The mass ratio of bovine to fish protein solids in the fermented protein peptide core in this embodiment is 70:10.

[0033] The preparation method of the fermented protein peptide core in this embodiment includes the following steps: Step A1, take a bovine collagen solution with a concentration of 15 mg / mL, add alkaline protease for pre-enzymatic hydrolysis, the amount of enzyme used is 0.1% of the weight of collagen, and hydrolyze for 2 h at a pH of 8.5 and a temperature of 37°C; Step A2, mix the pretreated collagen solution with fish protein hydrolysate at a bovine to fish protein solid mass ratio of 70:10, inoculate with fermentation microorganisms, and co-ferment for 24 h at a pH of 6.8 and a temperature of 30°C; Step A3, form a coating layer by co-precipitation of edible gelatin and chondroitin sulfate, cross-link and solidify with transglutaminase, and then perform fluidized bed drying; wherein, the degree of hydrolysis of the co-fermentation in this embodiment is controlled at 15%.

[0034] In this embodiment, the fermentation microorganism used in the co-fermentation process is Lactobacillus acidophilus, the inoculum amount is 1% of the total weight of the fermentation system, and the stirring speed is 100 rpm.

[0035] In this embodiment, the concentration of edible gelatin in the formation of the animal-derived composite coating layer is 8 mg / mL, and the concentration of chondroitin sulfate is 2 mg / mL. The edible gelatin and chondroitin sulfate are mixed at a mass ratio of 2:1, and the pH is adjusted to 4.2 at 25°C to form a composite co-precipitate. The preparation method of the edible gelatin in this embodiment is as follows: bovine bone is crushed to 1 mm, pretreated with a 0.3% citric acid solution for 12 h, and then hydrolyzed and extracted at 80°C for 4 h. After filtration, concentration, and drying, edible gelatin is obtained. The preparation method of the chondroitin sulfate in this embodiment is as follows: bovine cartilage is chopped, enzymatically hydrolyzed with protease at pH 7.0 and 50°C for 6 h, and then purified to obtain chondroitin sulfate. The preparation method of the fish-derived collagen in this embodiment is as follows: fish skin is pretreated with a dilute acid solution for 8 h, and then hydrolyzed and extracted at 70°C for 3 h. After filtration, concentration, and drying, fish-derived collagen is obtained.

[0036] In step A3, the amount of transglutaminase used in this embodiment is 0.03% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 30°C, the cross-linking time is 1.5 h, and the determination criterion for the cross-linking endpoint is that the free amino group reduction rate is not less than 25%.

[0037] The fermented protein peptide core of this embodiment contains a collagen peptide fragment with a glycine-proline-hydroxyproline sequence, which accounts for 30% of the total peptide content; the ratio of the swelling degree of the animal-derived composite coating layer at pH 4.5 to that at pH 7.4 is 1.5.

[0038] The animal-derived composite coating layer of this embodiment also contains tocopherol as an antioxidant and emulsifying excipient, with a content of 0.005% based on the total weight of the animal-derived composite coating layer; the animal-derived composite coating layer of this embodiment also contains ethyl eicosapentaenoate and ethyl docosahexaenoate, with a total content of 0.02% based on the total weight of the animal-derived composite coating layer.

[0039] The preparation method of this embodiment includes the following steps: Step S1, mixing the fermented protein peptide core with edible gelatin and chondroitin sulfate, wherein the concentration of edible gelatin is 8 mg / mL and the concentration of chondroitin sulfate is 2 mg / mL; Step S2, adjusting the pH value to 4.2 at a temperature of 25°C, so that the edible gelatin and chondroitin sulfate form a composite co-precipitation coating layer; Step S3, adding transglutaminase for cross-linking and solidification, the enzyme dosage is 0.03% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 30°C, and the cross-linking time is 1.5 h; Step S4, subjecting the cross-linked product to fluidized bed drying, with an inlet air temperature of 55°C, an outlet air temperature of 45°C, a fluidizing air velocity of 2 m / s, a spraying speed of 1 mL / min, and a coating efficiency of not less than 85%.

[0040] In this embodiment, the skincare product is a face cream. The amount of animal-derived compound fermented skin-nourishing protein peptide added to the skincare product is 0.5% of the total weight of the skincare product.

[0041] Features of this embodiment: It uses the minimum parameter value combination, selects a single fish-derived protein (fish-derived collagen) and a single fermentation microorganism (Lactobacillus acidophilus), and the preparation process is relatively mild, making it suitable for basic skin care product application scenarios with high stability requirements, such as daily moisturizing creams and other gentle skin care products.

[0042] Example 2: An animal-derived composite fermented skin-rejuvenating protein peptide comprises a fermented protein peptide core and an animal-derived composite coating layer covering the outer surface of the fermented protein peptide core in this embodiment. The fermented protein peptide core in this embodiment is prepared by co-fermentation of bovine collagen and fish protein, wherein the bovine collagen is extracted from bovine tissue and the fish protein is extracted from fish tissue. The fish protein in this embodiment is a mixture of fish collagen and fish elastin (mass ratio 1:1). The molecular weight of the fermented protein peptide core in this embodiment is 800 Da, and the average particle size is 32 μm. The animal-derived composite coating layer in this embodiment is composed of edible gelatin and chondroitin sulfate. The edible gelatin in this embodiment is obtained by acid hydrolysis of bovine bone, and the chondroitin sulfate in this embodiment is obtained by enzymatic extraction from bovine cartilage. The average thickness of the animal-derived composite coating layer in this embodiment is 4.4 μm, and the coating rate is 86%. The mass ratio of bovine to fish protein solids in the fermented protein peptide core in this embodiment is 76:16.

[0043] The preparation method of the fermented protein peptide core in this embodiment includes the following steps: Step A1, take a bovine collagen solution with a concentration of 21 mg / mL, add alkaline protease for pre-enzymatic hydrolysis, the amount of enzyme used is 0.11% of the weight of collagen, and hydrolyze for 2.6 h at a pH of 8.5 and a temperature of 37°C; Step A2, mix the pretreated collagen solution with fish protein hydrolysate at a bovine to fish protein solid mass ratio of 76:16, inoculate with fermentation microorganisms, and co-ferment for 27 h at a pH of 6.9 and a temperature of 32°C; Step A3, form a coating layer by co-precipitation of edible gelatin and chondroitin sulfate, cross-link and solidify with transglutaminase, and then perform fluidized bed drying; wherein, the degree of hydrolysis of the co-fermentation in this embodiment is controlled at 16.5%.

[0044] In this embodiment, the fermentation microorganism used in the co-fermentation process is a mixed strain of Lactobacillus acidophilus and Saccharomyces cerevisiae (mass ratio 1:1), with an inoculum amount of 1.6% of the total weight of the fermentation system and a stirring speed of 130 rpm.

[0045] In this embodiment, the concentration of edible gelatin in the formation of the animal-derived composite coating layer is 9.2 mg / mL, and the concentration of chondroitin sulfate is 3.2 mg / mL. The edible gelatin and chondroitin sulfate are mixed at a mass ratio of 2.9:1, and the pH is adjusted to 4.4 at 28°C to form a composite co-precipitate. The preparation method of the edible gelatin in this embodiment is as follows: bovine bone is crushed to 1.6 mm, pretreated with a 0.66% citric acid solution for 15.6 h, and then hydrolyzed and extracted at 86°C for 5.2 h. After filtration, concentration, and drying, edible gelatin is obtained. The preparation method of the chondroitin sulfate in this embodiment is as follows: bovine cartilage is chopped, enzymatically hydrolyzed with protease at pH 7.3 and 53°C for 7.8 h, and then purified to obtain chondroitin sulfate. The preparation method of the fish-derived collagen in this embodiment is as follows: fish scales are pretreated with a dilute alkaline solution for 10.4 h, and then hydrolyzed and extracted at 76°C for 3.9 h. h, after filtration, concentration and drying, fish-derived collagen is obtained; the preparation method of fish-derived elastin in this embodiment is as follows: fish skin is enzymatically hydrolyzed with trypsin at pH 8.1 and temperature 42℃ for 5.2 h, and then separated and purified to obtain fish-derived elastin.

[0046] In step A3, the amount of transglutaminase used in this embodiment is 0.045% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 32°C, the cross-linking time is 1.8 h, and the determination criterion for the cross-linking endpoint is that the free amino group reduction rate is not less than 25%.

[0047] The fermented protein peptide core of this embodiment contains a collagen peptide fragment with a glycine-proline-hydroxyproline sequence, which accounts for 39% of the total peptide content; the ratio of the swelling degree of the animal-derived composite coating layer at pH 4.5 to that at pH 7.4 is 2.0.

[0048] The animal-derived composite coating layer of this embodiment also contains rosemary leaf extract as an antioxidant and emulsifying excipient, with a content of 0.153% based on the total weight of the animal-derived composite coating layer; the animal-derived composite coating layer of this embodiment also contains ethyl eicosapentaenoate and ethyl docosahexaenoate, with a total content of 0.074% based on the total weight of the animal-derived composite coating layer.

[0049] The preparation method of this embodiment includes the following steps: Step S1, mixing the fermented protein peptide core with edible gelatin and chondroitin sulfate, wherein the concentration of edible gelatin is 9.2 mg / mL and the concentration of chondroitin sulfate is 3.2 mg / mL; Step S2, adjusting the pH value to 4.4 at a temperature of 28℃, so that the edible gelatin and chondroitin sulfate form a composite co-precipitation coating layer; Step S3, adding transglutaminase for cross-linking and solidification, the amount of enzyme used is 0.045% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 32℃, and the cross-linking time is 1.8 h; Step S4, subjecting the cross-linked product to fluidized bed drying, with an inlet air temperature of 58℃, an outlet air temperature of 48℃, a fluidizing air velocity of 2.9 m / s, a spraying speed of 1.6 mL / min, and a coating efficiency of not less than 85%.

[0050] In this embodiment, the skincare product is a serum. The amount of animal-derived compound fermented skin-nourishing protein peptide added to the skincare product is 1.25% of the total weight of the skincare product.

[0051] Features of this embodiment: It adopts a combination of moderate to low parameter values, uses mixed fish-derived proteins and a dual-strain fermentation system, which balances bioactivity and stability, making it suitable for functional skincare applications, such as anti-aging serums and other products that require the synergistic effect of multiple active ingredients.

[0052] Example 3: An animal-derived composite fermented skin-rejuvenating protein peptide comprises a fermented protein peptide core and an animal-derived composite coating layer covering the outer surface of the fermented protein peptide core in this embodiment. The fermented protein peptide core in this embodiment is prepared by co-fermentation of bovine collagen and fish protein, wherein the bovine collagen is extracted from bovine tissue and the fish protein is extracted from fish tissue. The fish protein in this embodiment is a mixture of fish collagen and fish elastin (mass ratio 3:2). The molecular weight of the fermented protein peptide core in this embodiment is 1100 Da, and the average particle size is 44 μm. The animal-derived composite coating layer in this embodiment is composed of edible gelatin and chondroitin sulfate. The edible gelatin in this embodiment is obtained by acid hydrolysis of bovine bone, and the chondroitin sulfate in this embodiment is obtained by enzymatic extraction from bovine cartilage. The average thickness of the animal-derived composite coating layer in this embodiment is 6.8 μm, and the coating rate is 91%. The mass ratio of bovine to fish protein solids in the fermented protein peptide core in this embodiment is 82:22.

[0053] The preparation method of the fermented protein peptide core in this embodiment includes the following steps: Step A1, take a bovine collagen solution with a concentration of 27 mg / mL, add alkaline protease for pre-enzymatic hydrolysis, the amount of enzyme used is 0.13% of the weight of collagen, and hydrolyze for 3.2 h at a pH of 8.5 and a temperature of 37°C; Step A2, mix the pretreated collagen solution with fish protein hydrolysate at a bovine to fish protein solid mass ratio of 82:22, inoculate with fermentation microorganisms, and co-ferment for 30 h at a pH of 7.0 and a temperature of 34°C; Step A3, form a coating layer by co-precipitation of edible gelatin and chondroitin sulfate, cross-link and solidify with transglutaminase, and then dry in a fluidized bed; wherein, the degree of hydrolysis of the co-fermentation in this embodiment is controlled at 18%.

[0054] In this embodiment, the fermentation microorganisms used in the co-fermentation process are a mixed strain of Lactobacillus acidophilus, Saccharomyces cerevisiae and Bifidobacterium (mass ratio 1:1:1), the inoculum amount is 2.2% of the total weight of the fermentation system, and the stirring speed is 160 rpm.

[0055] In this embodiment, the concentration of edible gelatin in the formation of the animal-derived composite coating layer is 10.4 mg / mL, and the concentration of chondroitin sulfate is 4.4 mg / mL. The edible gelatin and chondroitin sulfate are mixed at a mass ratio of 3.8:1, and the pH is adjusted to 4.6 at 31°C to form a composite co-precipitate. The preparation method of the edible gelatin in this embodiment is as follows: bovine bone is crushed to 2.2 mm, pretreated with a 1.02% citric acid solution for 19.2 h, and then hydrolyzed and extracted at 92°C for 6.4 h. After filtration, concentration, and drying, edible gelatin is obtained. The preparation method of the chondroitin sulfate in this embodiment is as follows: bovine cartilage is chopped, enzymatically hydrolyzed with protease at pH 7.6 and 56°C for 9.6 h, and then purified to obtain chondroitin sulfate. The preparation method of the fish-derived collagen in this embodiment is as follows: fish skin is pretreated with a dilute acid solution for 12.8 h, and then hydrolyzed and extracted at 82°C for 4.8 h. h, after filtration, concentration and drying, fish-derived collagen is obtained; the preparation method of fish-derived elastin in this embodiment is as follows: fish skin is enzymatically hydrolyzed with trypsin at pH 8.3 and temperature 46℃ for 6.4 h, and then separated and purified to obtain fish-derived elastin.

[0056] In step A3, the amount of transglutaminase used in this embodiment is 0.06% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 34°C, the cross-linking time is 2.1 h, and the determination criterion for the cross-linking endpoint is that the free amino group reduction rate is not less than 25%.

[0057] The fermented protein peptide core of this embodiment contains a collagen peptide fragment with a glycine-proline-hydroxyproline sequence, which accounts for 48% of the total peptide content; the ratio of the swelling degree of the animal-derived composite coating layer at pH 4.5 to that at pH 7.4 is 2.5.

[0058] The animal-derived composite coating layer of this embodiment also contains fish oil phospholipids as antioxidant and emulsifying excipients, with a content of 0.302% based on the total weight of the animal-derived composite coating layer; the animal-derived composite coating layer of this embodiment also contains ethyl eicosapentaenoate and ethyl docosahexaenoate, with a total content of 0.128% based on the total weight of the animal-derived composite coating layer.

[0059] The preparation method of this embodiment includes the following steps: Step S1, mixing the fermented protein peptide core with edible gelatin and chondroitin sulfate, wherein the concentration of edible gelatin is 10.4 mg / mL and the concentration of chondroitin sulfate is 4.4 mg / mL; Step S2, adjusting the pH value to 4.6 at a temperature of 31°C, so that the edible gelatin and chondroitin sulfate form a composite co-precipitation coating layer; Step S3, adding transglutaminase for cross-linking and solidification, the enzyme dosage is 0.06% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 34°C, and the cross-linking time is 2.1 h; Step S4, subjecting the cross-linked product to fluidized bed drying, with an inlet air temperature of 61°C, an outlet air temperature of 51°C, a fluidizing air velocity of 3.8 m / s, a spraying speed of 2.2 mL / min, and a coating efficiency of not less than 85%.

[0060] In this embodiment, the skincare product is a face mask. The amount of animal-derived compound fermented skin-nourishing protein peptide added to the skincare product is 2% of the total weight of the skincare product.

[0061] Features of this embodiment: It adopts a combination of medium to high parameter values, uses a three-strain synergistic fermentation system and a high content of active ingredients, which enhances the product's bioactivity and efficacy. It is suitable for high-end functional skincare product application scenarios, such as intensive repair masks and other products that require strong functions.

[0062] Example 4: An animal-derived composite fermented skin-rejuvenating protein peptide comprises a fermented protein peptide core and an animal-derived composite coating layer covering the outer surface of the fermented protein peptide core in this embodiment. The fermented protein peptide core in this embodiment is prepared by co-fermentation of bovine collagen and fish protein, wherein the bovine collagen is extracted from bovine tissue and the fish protein is extracted from fish tissue. The fish protein in this embodiment is a mixture of fish collagen and fish elastin (mass ratio 2:3). The molecular weight of the fermented protein peptide core in this embodiment is 1500 Da, and the average particle size is 60 μm. The animal-derived composite coating layer in this embodiment is composed of edible gelatin and chondroitin sulfate. The edible gelatin in this embodiment is obtained by acid hydrolysis of bovine bone, and the chondroitin sulfate in this embodiment is obtained by enzymatic extraction from bovine cartilage. The average thickness of the animal-derived composite coating layer in this embodiment is 10.0 μm, and the coating rate is 99%. The mass ratio of bovine to fish protein solids in the fermented protein peptide core in this embodiment is 90:30.

[0063] The preparation method of the fermented protein peptide core in this embodiment includes the following steps: Step A1, take a bovine collagen solution with a concentration of 35 mg / mL, add alkaline protease for pre-enzymatic hydrolysis, the amount of enzyme used is 0.15% of the weight of collagen, and hydrolyze for 4 h at a pH of 8.5 and a temperature of 37°C; Step A2, mix the pretreated collagen solution with fish protein hydrolysate at a bovine to fish protein solid mass ratio of 90:30, inoculate with fermentation microorganisms, and co-ferment for 36 h at a pH of 7.2 and a temperature of 37°C; Step A3, form a coating layer by co-precipitation of edible gelatin and chondroitin sulfate, cross-link and solidify with transglutaminase, and then perform fluidized bed drying; wherein, the degree of hydrolysis of the co-fermentation in this embodiment is controlled at 20%.

[0064] In this embodiment, the fermentation microorganisms used in the co-fermentation process are a mixed strain of Lactobacillus acidophilus, Saccharomyces cerevisiae and Bifidobacterium (mass ratio 2:1:2), the inoculation amount is 3% of the total weight of the fermentation system, and the stirring speed is 200 rpm.

[0065] In this embodiment, the concentration of edible gelatin in the formation of the animal-derived composite coating layer is 12 mg / mL, and the concentration of chondroitin sulfate is 6 mg / mL. The edible gelatin and chondroitin sulfate are mixed at a mass ratio of 5:1, and the pH is adjusted to 4.8 at 35°C to form a composite co-precipitate. The preparation method of the edible gelatin in this embodiment is as follows: bovine bone is crushed to 3 mm, pretreated with a 1.5% citric acid solution for 24 h, and then hydrolyzed and extracted at 100°C for 8 h. After filtration, concentration, and drying, edible gelatin is obtained. The preparation method of the chondroitin sulfate in this embodiment is as follows: bovine cartilage is chopped, enzymatically hydrolyzed with protease at pH 8.0 and 60°C for 12 h, and then purified to obtain chondroitin sulfate. The preparation method of the fish-derived collagen in this embodiment is as follows: fish scales are pretreated with a dilute alkaline solution for 16 h, and then hydrolyzed and extracted at 90°C for 6 h. h, after filtration, concentration and drying, fish-derived collagen is obtained; the preparation method of fish-derived elastin in this embodiment is as follows: fish skin is enzymatically hydrolyzed with trypsin at pH 8.5 and temperature 50℃ for 8 h, and then separated and purified to obtain fish-derived elastin.

[0066] In step A3, the amount of transglutaminase used in this embodiment is 0.08% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 38°C, the cross-linking time is 2.5 h, and the determination criterion for the cross-linking endpoint is that the free amino group reduction rate is not less than 25%.

[0067] The fermented protein peptide core of this embodiment contains a collagen peptide fragment with a glycine-proline-hydroxyproline sequence, which accounts for 60% of the total peptide content; the ratio of the swelling degree of the animal-derived composite coating layer at pH 4.5 to that at pH 7.4 is 3.0.

[0068] The animal-derived composite coating layer of this embodiment also contains a mixture of tocopherol, rosemary leaf extract and fish oil phospholipids (mass ratio 1:1:1) as an antioxidant and emulsifying excipient, with a content of 0.5% based on the total weight of the animal-derived composite coating layer; the animal-derived composite coating layer of this embodiment also contains ethyl eicosapentaenoate and ethyl docosahexaenoate, with a total content of 0.20% based on the total weight of the animal-derived composite coating layer.

[0069] The preparation method of this embodiment includes the following steps: Step S1, mixing the fermented protein peptide core with edible gelatin and chondroitin sulfate, wherein the concentration of edible gelatin is 12 mg / mL and the concentration of chondroitin sulfate is 6 mg / mL; Step S2, adjusting the pH value to 4.8 at a temperature of 35℃, so that the edible gelatin and chondroitin sulfate form a composite co-precipitation coating layer; Step S3, adding transglutaminase for cross-linking and solidification, the enzyme dosage is 0.08% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 38℃, and the cross-linking time is 2.5 h; Step S4, subjecting the cross-linked product to fluidized bed drying, with an inlet air temperature of 65℃, an outlet air temperature of 55℃, a fluidizing air velocity of 5 m / s, a spraying speed of 3 mL / min, and a coating efficiency of not less than 85%.

[0070] In this embodiment, the skincare product is a combination of face cream, serum, mask and lotion. The amount of animal-derived complex fermented skin-nourishing protein peptide added to the skincare product is 3% of the total weight of the skincare product.

[0071] Features of this embodiment: It adopts the maximum parameter value combination, uses the highest proportion of fish-derived elastin and a three-strain optimized fermentation system, has the highest active ingredient content and the strongest encapsulation protection effect, and is suitable for high-end products that require ultimate efficacy, such as high-concentration anti-aging sets.

[0072] Comparative Example 1: It is basically the same as Example 1, except that the pre-enzymatic hydrolysis temperature in step A1 is set to 25°C, while other process parameters remain unchanged.

[0073] Comparative Example 2: It is basically the same as Example 1, except that the pre-enzymatic hydrolysis time in step A1 is set to 0.5 h, while other process parameters remain unchanged.

[0074] Comparative Example 3: It is basically the same as Example 1, except that the pre-enzymatic hydrolysis pH value in step A1 is set to 6.0, while other process parameters remain unchanged.

[0075] Comparative Example 4: It is basically the same as Example 1, except that the co-fermentation temperature in step A2 is set to 20°C, while other process parameters remain unchanged.

[0076] Comparative Example 5: It is basically the same as Example 1, except that the co-fermentation time in step A2 is set to 12 h, while other process parameters remain unchanged.

[0077] Comparative Example 6: It is basically the same as Example 1, except that the pH value of co-fermentation in step A2 is set to 5.5, while other process parameters remain unchanged.

[0078] Comparative Example 7: It is basically the same as Example 1, except that the degree of hydrolysis of co-fermentation in step A2 is controlled at 8%, while other process parameters remain unchanged.

[0079] Comparative Example 8: Basically the same as Example 1, except that the stirring speed in step A2 is set to 50 rpm, while other process parameters remain unchanged.

[0080] Comparative Example 9: Basically the same as Example 1, except that the crosslinking temperature of transglutaminase in step A3 is set to 15°C, while other process parameters remain unchanged.

[0081] Comparative Example 10: Basically the same as Example 1, except that the glutamine transaminase crosslinking time in step A3 was set to 0.5 h, while other process parameters remained unchanged.

[0082] Comparative Example 11: Basically the same as Example 1, except that in step S4 the inlet air temperature of the fluidized bed dryer is set to 100°C and the outlet air temperature is set to 90°C, while other process parameters remain unchanged.

[0083] Comparative Example 12: Basically the same as Example 1, except that the mass ratio of bovine to fish protein solids in the fermented protein peptide core was set to 95:5, while other process parameters remained unchanged.

[0084] Comparative Example 13: Basically the same as Example 1, except that edible gelatin and chondroitin sulfate are mixed at a mass ratio of 10:1, while other process parameters remain unchanged.

[0085] Comparative Example 14: Basically the same as Example 1, except that only Escherichia coli was used as the fermentation microorganism in the co-fermentation process, and the inoculum amount was 1% of the total weight of the fermentation system, while other process parameters remained unchanged.

[0086] Comparative Example 15: Basically the same as Example 1, except that no antioxidants or emulsifying agents are added to the animal-derived composite coating layer, while other process parameters remain unchanged.

[0087] Performance testing: Molecular Weight Distribution Test: The test subject was the core component of fermented protein peptides in animal-derived compound fermented skin-rejuvenating protein peptides. Test Objective: To accurately determine the molecular weight distribution range of the core fermented protein peptides and verify whether its molecular weight meets the design requirement of 500-1500 Da. Test Principle: Based on the principle of gel permeation chromatography, protein peptides of different molecular weights have different elution times in the chromatographic column. The molecular weight distribution of the sample is determined by comparison with standard molecular weight substances. Experimental Method: 0.1 g of sample was dissolved in 10 mL of deionized water, filtered through a 0.22 μm filter membrane, and analyzed using a high-performance liquid chromatograph equipped with a TSK-GEL G2000SW column. The mobile phase was 0.1 M phosphate buffer (pH 7.0), the flow rate was 1.0 mL / min, the detection wavelength was 280 nm, and the column temperature was 30℃. Key Parameters: Injection volume was 20 μL, analysis time was 60 minutes, and a molecular weight standard curve was established using standard proteins such as insulin (5.8 kDa) and cytochrome C (12.4 kDa).

[0088] pH Response Swelling Performance Test: The test subject was an animal-derived composite coating layer of animal-derived fermented skin-rejuvenating peptides. The purpose of the test was to evaluate the swelling behavior of the coating layer under different pH environments and verify whether the swelling degree at pH 4.5 was 1.5-3.0 times that at pH 7.4. The test principle was based on the pH response mechanism of polymer gels and the principle of volume change measurement. Experimental Method: The material sample was immersed in acetate buffer at pH 4.5 and phosphate buffer at pH 7.4, respectively, and equilibrated at a constant temperature of 37℃ for 24 hours. The mass change before and after swelling was measured by gravimetric method, and the swelling degree was calculated. Simultaneously, the particle volume change was observed under a microscope. Key parameters included buffer ionic strength of 0.15M, temperature of 37±0.5℃, test time of 24 hours, and sample mass of approximately 100mg. Data processing: The swelling degree was calculated using the formula Q=(Ws-Wd) / Wd×100%, where Ws is the mass after swelling and Wd is the dry mass. The pH response ratio was required to be within the design range and have good reproducibility.

[0089] Collagen peptide content testing experiment: The test object is the glycine-proline-hydroxyproline sequence collagen peptide fragment in the core of animal-derived compound fermented skin-rejuvenating protein peptide. Test objective: To quantitatively detect the content of characteristic collagen peptide sequences, verify that they account for 30-60% of the total peptide content, and evaluate the content of the product's active ingredients. Test principle: High-performance liquid chromatography (HPLC) is used to separate different peptide fragments, and the content of the target sequence is quantitatively analyzed by comparison with a standard collagen peptide reference. Experimental method: After double hydrolysis with pepsin and trypsin, the sample is separated using a C18 reversed-phase column. Mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution, with gradient elution, a flow rate of 1.0 mL / min, a detection wavelength of 214 nm, and a column temperature of 35℃. Key parameters: Injection volume 10 μL, analysis time 45 min, and a standard curve is established using Gly-Pro-Hyp standard tripeptide with a concentration range of 10-1000 μg / mL. Data processing: The collagen peptide content is calculated based on the peak area. The requirement is that the glycine-proline-hydroxyproline sequence collagen peptides account for 30-60% of the total peptide content, and the detection limit is not higher than 5 μg / mL.

[0090] Antioxidant Activity Test Experiment: The test subject was the antioxidant and emulsifying excipients added to the animal-derived compound fermented skin-rejuvenating protein peptide. Test Objective: To evaluate the product's antioxidant capacity, verify the efficacy of the antioxidant excipients, and provide a scientific basis for the product's anti-aging function. Test Principle: The DPPH free radical scavenging method was used. The antioxidants bind to DPPH free radicals, causing their purple color to fade. The free radical scavenging rate was calculated by measuring the change in absorbance. Experimental Method: 1×10⁻⁶ ppm was prepared. -4 Different concentrations of test solutions were prepared by dissolving the sample in mol / L DPPH ethanol solution. These solutions were then mixed with an equal volume of DPPH solution and reacted in the dark for 30 minutes. The absorbance was measured at 517 nm, with vitamin C as a positive control. Key parameters: reaction temperature 25±1℃, reaction time 30±1 min, operation in the dark, sample concentration range 0.1-10 mg / mL. Data processing: Free radical scavenging rate = (control group absorbance - sample group absorbance) / control group absorbance × 100%. The IC50 value was calculated; an IC50 value less than 1 mg / mL indicated good antioxidant activity.

[0091] Storage Stability Test: The test subject is the stability of animal-derived compound fermented skin-rejuvenating protein peptides under different storage conditions. Test Objective: To determine the effective shelf life and optimal storage conditions of the product, providing a technical basis for product packaging and distribution. Test Principle: Through accelerated aging tests and real-time stability tests, the physicochemical properties of the product under different temperature and humidity conditions are monitored. Experimental Method: Three storage conditions are set: 25℃ / 60%RH, 40℃ / 75%RH, and 60℃ / 75%RH. Samples are taken at 1, 3, 6, and 12 months to measure changes in key indicators such as moisture content, pH value, microbial indicators, and collagen peptide content. Key Parameters: Temperature control accuracy ±2℃, relative humidity control accuracy ±5%RH, sealed container packaging, and a minimum sample size of 10g per test. Data Processing: Curves of change of each indicator over time are established. The Arrhenius equation is used to predict the shelf life at room temperature. Under recommended storage conditions, the shelf life is required to be at least 24 months, and the change rate of key indicators ≤10%.

[0092] Release Kinetics Test: The test subject was the release behavior of active ingredients of animal-derived compound fermented skin-rejuvenating peptides in a simulated skin environment. Test Objective: To study the sustained-release characteristics of the coating layer, evaluate the product's efficacy persistence and bioavailability, and optimize product design. Test Principle: A Franz diffusion cell was used to simulate the skin penetration process. The cumulative release of active ingredients was analyzed by timed sampling to establish a release kinetics model. Experimental Method: A Franz diffusion cell was used. The receiving cell was filled with PBS buffer (pH 7.4), and the dosing cell was filled with the sample dispersion. A cellulose acetate membrane was used as a separator. The cell was kept at a constant temperature of 37°C, and collagen peptide concentration was analyzed by timed sampling over 24 hours. Key Parameters: Membrane pore size 0.45 μm, effective diffusion area 1.77 cm². 2 The dosage was 100 μL, and sampling time points were 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, with 1 mL sample taken each time. Data processing: The cumulative release-time curve was plotted, and zero-order, first-order, and Higuchi models were fitted. The release rate constant and 24-hour cumulative release rate were calculated, requiring obvious sustained-release characteristics, with a 24-hour release rate in the range of 60-85%.

[0093] The performance of the examples and comparative examples is summarized in Table 1. In Comparative Example 1, lowering the pre-enzymatic hydrolysis temperature to 25°C significantly inhibited alkaline protease activity, resulting in incomplete protein degradation, decreased efficiency in generating target molecular weight peptides, and reduced availability of the substrate for subsequent fermentation. Ultimately, this led to a decrease in molecular weight distribution, collagen peptide content, and overall efficacy. In Comparative Example 2, shortening the pre-enzymatic hydrolysis time to 0.5 hours resulted in insufficient enzymatic hydrolysis, failing to effectively break down protein macromolecules, directly affecting the fermentation effect and the uniformity of the final product's molecular weight distribution. In Comparative Example 3, the pre-enzymatic hydrolysis pH deviated from the optimal condition to 6.0, disrupting the catalytic microenvironment of alkaline protease, significantly reducing enzyme activity and protein hydrolysis efficiency. This resulted in insufficient available nitrogen source during subsequent fermentation, affecting the generation of target peptides. In Comparative Example 4, the excessively low fermentation temperature of 20°C severely inhibited microbial metabolic activity and enzyme activity, leading to slow and incomplete fermentation, low protein conversion rate, and reduced generation of the target product. Furthermore, the low temperature was unfavorable for intermolecular interactions and peptide chain rearrangement. In Comparative Example 5, the fermentation time was less than 12 hours, resulting in insufficient microbial metabolism, incomplete protein fermentation and conversion, and a significant deficiency in the production of characteristic peptides. Consequently, the product's bioactivity and the content of its active ingredients decreased significantly. In Comparative Example 6, the fermentation pH was too low (5.5), deviating from the optimal growth environment for the fermenting microorganisms. This inhibited microbial activity and enzyme expression, and the acidic environment may have led to the degradation of some sensitive peptides, affecting product quality. In Comparative Example 7, the degree of hydrolysis was too low (8%), resulting in insufficient protein degradation and an excessively high proportion of macromolecular peptides, which is detrimental to subsequent encapsulation and bioavailability, while also limiting the release of characteristic functional peptides. In Comparative Example 8, the stirring speed was too low (50 rpm), causing uneven mixing of the reaction system, poor mass transfer, and affecting the sufficient contact between the enzyme and substrate, leading to reduced reaction efficiency and uneven product quality. In Comparative Example 9, the cross-linking temperature was too low (15℃), severely affecting the activity of transglutaminase. The cross-linking reaction proceeded slowly and incompletely, resulting in a loose coating layer structure with insufficient mechanical strength and chemical stability, affecting the product's storage stability and sustained-release properties. In Comparative Example 10, shortening the cross-linking time to 0.5 hours resulted in incomplete cross-linking, insufficient cross-linking network density within the coating layer, poor structural stability, and susceptibility to damage during storage and use, affecting the product's performance durability. In Comparative Example 11, an excessively high drying temperature of 100°C may have caused protein denaturation and damage to the coating layer structure. High temperatures may also have caused degradation of some active ingredients, affecting the product's bioactivity and stability. In Comparative Example 12, the extreme protein ratio of 95:5 disrupted the synergistic effect between bovine and fish proteins. A single protein source could not provide a diverse amino acid composition and functional peptides, reducing the product's overall efficacy. In Comparative Example 13, the imbalanced gelatin-chondroitin sulfate ratio of 10:1 disrupted their optimal synergistic effect. Excessive gelatin may have resulted in an overly dense coating layer, hindering the release of active ingredients, while insufficient chondroitin sulfate would reduce the coating layer's pH responsiveness and biocompatibility.Comparative Example 14, by replacing beneficial fermentation bacteria with *E. coli*, completely altered the fermentation metabolic pathway. *E. coli* lacks a specific protease system, making it unable to effectively produce the target functional peptides and potentially generating harmful metabolites, severely impacting product safety and efficacy. Comparative Example 15, by completely removing antioxidant excipients, caused the product to lose its antioxidant protection. The protein peptides are prone to oxidative degradation during storage and use, affecting not only the stability of the active ingredients but also potentially generating harmful oxidation products. Furthermore, the lack of emulsifying excipients also affects the product's dispersibility and stability in skincare products.

[0094] Table 1 Summary of the performance of protein peptide materials in Examples 1-4 and Comparative Examples 1-12

[0095] Figure 1 The results clearly demonstrate that the fermented protein peptide core prepared in Example 3 has a dense and uniform internal structure, and the core particles are regularly spherical, indicating that the synergistic fermentation process parameters are set reasonably, the protein is fully converted, and a stable peptide network structure is formed. Figure 2 The animal-derived composite fermented skin-rejuvenating protein prepared in Example 3 completely covered the core surface without damage or detachment. The coating layer was tightly bonded to the core interface, demonstrating that under the conditions of a cross-linking temperature of 30-38℃ and a cross-linking time of 1.5-2.5 hours, transglutaminase can effectively catalyze the formation of a stable cross-linked network between gelatin and chondroitin sulfate, achieving a coating efficiency of over 85%. In comparison, Figure 3 In Comparative Example 9, the crosslinking temperature was too low (15℃), significantly inhibiting glutamine transaminase activity. This resulted in a loose and uneven coating layer structure, with obvious cracks, holes, and incomplete areas on the surface. The coating layer thickness varied considerably, and localized detachment occurred, exposing part of the core particles. The coating integrity was severely compromised, and this structural defect directly affects the product's storage stability and efficacy release control. The comparison of the three images visually verifies the scientific nature of the process parameters set in this invention, particularly the decisive role of key parameters such as crosslinking temperature and time on coating quality. It also demonstrates the feasibility of the co-fermentation preparation of the core and the composite co-precipitation coating technology route, ensuring that the final product has good morphological characteristics and structural integrity, providing reliable technical support for the industrial production and quality control of the product.

[0096] based on Figure 4 , Figure 5 The single-factor optimization experimental results fully demonstrate the rationality, reliability, and effectiveness of the technical solution of this invention. Figure 4The results show that within the fermentation temperature range of 30-37℃, collagen peptide content and microbial activity exhibit a significant temperature dependence, with the optimal temperature being 35.4-36.2℃. When the fermentation temperature is below 30℃, microbial metabolic activity is severely inhibited, enzyme activity decreases, collagen peptide content drops to 28.7%, and microbial activity is only 72.4%. The experimental data are completely consistent with the low-temperature fermentation results of Comparative Example 4. When the temperature exceeds 37℃, although microbial activity may remain at a high level in the short term, the sustained high-temperature environment will lead to a gradual decrease in microbial activity, while increasing the risk of protein denaturation and product quality deterioration. Figure 5 This study revealed the precise control effect of enzyme dosage within the range of 0.1-0.15% on the content and molecular weight distribution qualification rate of collagen peptides. The optimal enzyme dosage was 0.131-0.137%. When the enzyme dosage was below 0.1%, protein degradation was insufficient, the generation efficiency of target molecular weight peptides was severely inadequate, the collagen peptide content dropped below 32.1%, and the molecular weight distribution qualification rate was only 78.3%, completely consistent with the performance of Comparative Example 3. However, when the enzyme dosage exceeded 0.15%, excessive enzymatic hydrolysis may damage the functional structure of the protein, leading to increased non-specific degradation and affecting product quality. Single-factor optimization experiments of the two key parameters showed a significant performance decrease when the parameters exceeded the set range, fully demonstrating the scientific rationality of the parameter settings and the accuracy of the boundary selection, providing a reliable theoretical basis and experimental support for the industrial application of the technical solution of this invention.

[0097] comprehensive Figure 6 – Figure 8 The comparison results show that the embodiment has a significantly higher content of small molecule peptides (500–1500 Da) than the comparative example, and exhibits a more favorable pH-responsive swelling ratio (controlled swelling under acidic conditions and structural stability under neutral conditions), indicating that its carrier structure is more compatible with the peptide distribution. In terms of functional performance, the embodiment has a higher collagen peptide content and a lower IC50 for antioxidant activity, demonstrating stronger free radical scavenging ability. In terms of application stability, the embodiment has a higher storage stability retention rate and a more controllable 24-hour cumulative release rate (small initial burst release and stable subsequent drug release), comprehensively demonstrating that this process is superior to the comparative example in terms of active ingredient enrichment, functional activity enhancement, and stable and controllable release of the formulation.

[0098] 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. An animal-derived compound fermented skin-rejuvenating protein peptide, characterized in that, It includes a fermented protein peptide core and an animal-derived composite coating layer covering the outer surface of the fermented protein peptide core; The fermented protein peptide core is obtained by co-fermentation of bovine collagen and fish protein, wherein the bovine collagen is extracted from bovine tissue and the fish protein is extracted from fish tissue. The fish-derived protein is one or both of fish-derived collagen and fish-derived elastin. The molecular weight of the fermented protein peptide core is 500-1500 Da, and the average particle size is 20-60 μm; The animal-derived composite coating layer is composed of edible gelatin and chondroitin sulfate. The edible gelatin is obtained by acid hydrolysis of bovine bone, and the chondroitin sulfate is obtained by enzymatic extraction from bovine cartilage. The average thickness of the animal-derived composite coating layer is 2.0-10.0 μm, and the coating rate is 80-99%. The mass ratio of bovine to fish protein solids in the fermented protein peptide core is 70-90:10-30.

2. The animal-derived compound fermented skin-rejuvenating protein peptide as described in claim 1, characterized in that, The preparation method of the fermented protein peptide core includes the following steps: Step A1: Take a bovine collagen solution with a concentration of 15-35 mg / mL, add alkaline protease for pre-enzymatic hydrolysis, the amount of enzyme used is 0.1-0.15% of the weight of collagen, and hydrolyze for 2-4 h at a pH of 8.5 and a temperature of 37℃. Step A2: Mix the pretreated collagen solution with fish protein hydrolysate at a bovine to fish protein solids mass ratio of 70-90:10-30, inoculate with fermentation microorganisms, and co-ferment at pH 6.8-7.2 and temperature 30-37℃ for 24-36 h. Step A3: A coating layer is formed by co-precipitation of edible gelatin and chondroitin sulfate, and then cross-linked and cured by transglutaminase before being dried in a fluidized bed. The degree of hydrolysis in the co-fermentation is controlled at 15-20%.

3. The animal-derived compound fermented skin-rejuvenating protein peptide as described in claim 2, characterized in that, The fermentation microorganisms used in the co-fermentation process are one or more of Lactobacillus acidophilus, Saccharomyces cerevisiae, and Bifidobacterium, with an inoculation amount of 1-3% of the total weight of the fermentation system and a stirring speed of 100-200 rpm.

4. The animal-derived compound fermented skin-rejuvenating protein peptide as described in claim 1, characterized in that, During the formation of the animal-derived composite coating layer, the concentration of the edible gelatin is 8-12 mg / mL, and the concentration of the chondroitin sulfate is 2-6 mg / mL. The edible gelatin and chondroitin sulfate are mixed at a mass ratio of 2-5:1, and the pH value is adjusted to 4.2-4.8 at a temperature of 25-35℃ to form a composite coprecipitate; The method for preparing the edible gelatin is as follows: crush cattle bones to 1-3 mm, pretreat them with a 0.3-1.5% citric acid solution for 12-24 h, then hydrolyze and extract them at a temperature of 80-100℃ for 4-8 h, and obtain edible gelatin by filtration, concentration and drying. The preparation method of the chondroitin sulfate is as follows: bovine cartilage is chopped and enzymatically hydrolyzed with protease at a pH of 7.0-8.0 and a temperature of 50-60℃ for 6-12 h, and then separated and purified to obtain chondroitin sulfate. The preparation method of the fish-derived collagen is as follows: fish skin or fish scales are pretreated with dilute acid or dilute alkali solution for 8-16 h, and then hydrolyzed and extracted at a temperature of 70-90℃ for 3-6 h. After filtration, concentration and drying, fish-derived collagen is obtained. The method for preparing the fish-derived elastin is as follows: fish skin is enzymatically hydrolyzed with trypsin at a pH of 8.0-8.5 and a temperature of 40-50℃ for 4-8 hours, and then separated and purified to obtain the fish-derived elastin.

5. The animal-derived compound fermented skin-rejuvenating protein peptide as described in claim 2, characterized in that, Includes the following steps: In step A3, the amount of transglutaminase used is 0.03-0.08% of the total mass of the animal-derived composite coating layer, the cross-linking temperature is 30-38℃, the cross-linking time is 1.5-2.5 h, and the determination criterion for the cross-linking endpoint is that the free amino group reduction rate is not less than 25%.

6. The animal-derived compound fermented skin-rejuvenating protein peptide as described in claim 1, characterized in that, The fermented protein peptide core contains a collagen peptide fragment with a glycine-proline-hydroxyproline sequence, which accounts for 30-60% of the total peptide content; the ratio of the swelling degree of the animal-derived composite coating at pH 4.5 to that at pH 7.4 is 1.5-3.

0.

7. The animal-derived compound fermented skin-rejuvenating protein peptide as described in claim 5, characterized in that, The animal-derived composite coating layer also contains at least one antioxidant and emulsifying excipient selected from tocopherol, rosemary leaf extract and fish oil phospholipids, with a content of 0.005-0.5% based on the total weight of the animal-derived composite coating layer; the animal-derived composite coating layer also contains ethyl eicosapentaenoate and ethyl docosahexaenoate, with a total content of 0.02-0.20% based on the total weight of the animal-derived composite coating layer.

8. A method for preparing an animal-derived compound fermented skin-rejuvenating protein peptide as described in any one of claims 1 to 7, characterized in that, Includes the following steps: Step S1: Mix the fermented protein peptide core with edible gelatin and chondroitin sulfate, wherein the concentration of edible gelatin is 8-12 mg / mL and the concentration of chondroitin sulfate is 2-6 mg / mL. Step S2: Adjust the pH value to 4.2-4.8 at a temperature of 25-35℃ to form a composite co-precipitation coating layer of edible gelatin and chondroitin sulfate. Step S3: Add transglutaminase for cross-linking and curing. The amount of enzyme used is 0.03-0.08% of the total mass of the animal-derived composite coating layer. The cross-linking temperature is 30-38℃ and the cross-linking time is 1.5-2.5 h. Step S4: The cross-linked product is subjected to fluidized bed drying with an inlet air temperature of 55-65℃, an outlet air temperature of 45-55℃, a fluidizing air velocity of 2-5 m / s, and a spraying speed of 1-3 mL / min.

9. The method for preparing an animal-derived compound fermented skin-rejuvenating protein peptide as described in claim 8, characterized in that, The process parameters for fluidized bed drying in step S4 are as follows: inlet air temperature is 55-65℃, outlet air temperature is 45-55℃, fluidization air velocity is 2-5 m / s, spraying speed is 1-3 mL / min, and coating efficiency is not less than 85%.

10. The application of an animal-derived compound fermented skin-nourishing protein peptide as described in any one of claims 1 to 7 in skincare products, characterized in that, The skincare product is one or more of face cream, serum, face mask and lotion, and the amount of animal-derived compound fermented skin-nourishing protein peptide added to the skincare product is 0.5-3% of the total weight of the skincare product.

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