A multi-target microcollagen peptide-based composition, a preparation method therefor, and use thereof

Abstract

The present application relates to the technical fields of biological medicine and cosmetics, and discloses a composition based on multi-target microcollagen peptide, a preparation method and application thereof, wherein the composition and ingredient weight parts are as follows: microcollagen peptide 250-350 parts; chitosan 15-25 parts; sodium hyaluronate 4-6 parts; sodium chondroitin sulfate 2-4 parts; sodium tripolyphosphate 2-4 parts; and the rest is purified water, wherein the microcollagen peptide is composed of 90%-95% endogenous microcollagen peptide and 5%-10% exogenous microcollagen peptide; the composition is prepared through the steps of raw material glue extraction, step-by-step enzymolysis, membrane filtration and concentration, ion cross-linking self-assembly, sterilization filtration, spray drying and granulation; the composition realizes precise targeted delivery and on-demand release through a three-dimensional network structure, can greatly improve the bioavailability of microcollagen peptide, and thus promotes collagen gene expression; and the composition prepared by the present application is used for preparing anti-wrinkle and firming efficacy cosmetics, oral collagen peptide health food, repair type medical dressings or wound repair materials.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, specifically to a composition based on multi-target microcollagen peptides, its preparation method, and its application. Background Technology

[0002] Collagen is a major structural protein component of the extracellular matrix and connective tissue in animals. Secreted by fibroblasts, it is composed of procollagen molecules and often forms fibrous structures. Collagen fibers (also known as white fibers) are composed of type I collagen and are the main fibrous component in loose connective tissue; they are white when fresh. However, existing collagen products generally suffer from wide molecular weight distribution and unclear active sequences, making them difficult for the body to effectively absorb and utilize, resulting in low bioavailability. Furthermore, traditional collagen products lack precise delivery capabilities, failing to achieve targeted drug delivery and on-demand release, leading to unsatisfactory effects in applications such as tissue repair and anti-aging. Therefore, there is an urgent need to develop a composition and its preparation method that can improve collagen absorption efficiency, identify active components, and achieve precise delivery. Summary of the Invention

[0003] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a composition based on multi-target micro-collagen peptides, its preparation method, and its application. It has the advantages of precise targeted delivery, on-demand controllable release, high bioavailability, well-defined active sequences, and improved absorption efficiency, solving the problems of existing collagen proteins having a wide molecular weight distribution, unclear active sequences, lack of systematic delivery capabilities, and difficulty in achieving precise drug administration and effective absorption.

[0004] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a composition based on multi-target micro-collagen peptides, wherein the composition and its components are in the following weight parts: 250-350 parts micro-collagen peptides; 15-25 parts chitosan; 4-6 parts sodium hyaluronate; 2-4 parts sodium chondroitin sulfate; 2-4 parts sodium tripolyphosphate; and the remaining component is purified water.

[0005] Preferably, the microcollagen peptide is composed of 90% to 95% endogenous microcollagen peptide and 5% to 10% exogenous microcollagen peptide.

[0006] Preferably, the molecular weight distribution of the endogenous microcollagen peptide is 1500-2500 Da.

[0007] A method for preparing a composition based on multi-target micro-collagen peptides, comprising the following steps, according to the weight parts of the components of the above-mentioned composition based on multi-target micro-collagen peptides: Step 1: Select raw materials for collagen extraction: Select animal raw materials that are rich in type I collagen and easy to enzymatically hydrolyze, and perform collagen extraction on the animal raw materials to obtain crude collagen extract; Step 2, Filtration and initial filtration: The crude collagen extract is filtered and allowed to stand, and then filtered through cotton cake to remove large insoluble particles, resulting in a clear collagen solution. Step 3, Enzymatic hydrolysis: Add enzyme preparation to the clear gel solution for directional enzymatic hydrolysis, after enzyme inactivation, add activated carbon for decolorization, and obtain the enzymatic hydrolysis filtrate; Step 4, Filtration and Concentration: The enzymatic hydrolysis filtrate is purified by membrane filtration and then concentrated under vacuum to obtain collagen peptide concentrate, namely endogenous micro collagen peptides. Step 5, Loading and Assembly: Collagen peptide concentrate, GAGs glycosaminoglycans and natural polysaccharides are mixed and ionically cross-linked to self-assemble into a three-dimensional network system; Step 6, Sterilization and Drying: After sterilization and filtration, spray drying is performed to obtain a multi-target micro collagen peptide composition powder; Step 7: Granulation: Collect the dried multi-target micro collagen peptide composition powder, granulate and package it, and reject unqualified products by metal detection.

[0008] Preferably, the raw material selection and gum extraction process in step one includes: S1.1, Made from Alaskan cod skin or tilapia skin / scales; S1.2 Weigh 10-12 kg of Alaskan cod skin or tilapia skin / scales, remove impurities, cut into 1-2 cm pieces, and wash with purified water 3-5 times; S1.3 Add purified water at a material-to-liquid ratio of 1:4, adjust the pH to 4.9-5.0 with glacial acetic acid, heat to 50-55℃, and extract with constant temperature stirring for 2.8-3 hours; S1.4 After extraction, use a 200-300 mesh filter cloth for coarse filtration, collect the filtrate, and obtain crude collagen extract.

[0009] Preferably, in step two, after the crude collagen extract has been allowed to stand for 1-1.5 hours, the supernatant is coarsely filtered through a plate and frame filter press with a filter paper pore size of 10 μm, and then finely filtered through a cotton cake filter press with a cotton cake thickness of 3 cm and a pore size of 5 μm to obtain 30-31 L of clear collagen solution.

[0010] Preferably, the enzymatic hydrolysis in step three is as follows: S2.1. Heat the clarified gel solution to 48-50℃, adjust the pH to 7.5-7.55 using 0.5±0.002M sodium hydroxide, add 0.3±0.001% w / w alkaline protease, and enzymatically hydrolyze for 4-4.4 hours; S2.2, Continue adding 0.1±0.0005% w / w of protease, and then enzymatically hydrolyze for another 1-1.2 hours; S2.3, Heat to 88-90℃ to inactivate enzymes for 15-18 minutes, cool to 48-50℃, add 1.5±0.005%w / v activated carbon, stir and decolorize for 48-50 minutes; S2.4 Use a stainless steel plate and frame filter press to remove activated carbon and residue to obtain enzymatic hydrolysis filtrate.

[0011] Preferably, in step four, filtration and concentration are performed by passing the enzymatic hydrolysis filtrate through a microfiltration membrane and an ultrafiltration membrane in sequence, collecting the ultrafiltration permeate, and then concentrating it at 58-60°C and a vacuum degree of -0.09 to -0.08 MPa until the solid content is 24%-25% to obtain collagen peptide concentrate, i.e., endogenous micro collagen peptides.

[0012] Preferably, in step five, the load and assembly are performed as follows: S3.1 Preparation of mixed solution: Take 1±0.05L of collagen peptide concentrate obtained in step four, and add exogenous micro collagen peptide, sodium hyaluronate, sodium chondroitin sulfate and chitosan in the following proportions per liter of concentrate: 9-11g of exogenous micro collagen peptide, sodium hyaluronate, sodium chondroitin sulfate and chitosan in sequence. Stir thoroughly until completely dissolved to obtain a mixed solution system. S3.2 Adjusting pH and Ionic Crosslinking Self-Assembly: Adjust the pH of the above mixed solution system to 6.0-6.05 using glacial acetic acid and 0.5±0.002M sodium hydroxide. Under the condition of stirring speed of 300-400 rpm, slowly add sodium tripolyphosphate solution at a rate of 1-2 mL / min to a final concentration of 0.27%-0.33% w / v. After the addition is completed, continue stirring for 25-30 minutes. In step six, sterilization and drying are performed as follows: the composite solution is filtered through a 0.22μm sterilization filter membrane and then spray-dried in a clean area: the inlet air temperature is 175-180℃, the outlet air temperature is 80-85℃, the atomization pressure is 0.3-0.35MPa, the feed rate is 1.5-1.6L / h, and the dried multi-target micro collagen peptide composition powder is collected, with its moisture content controlled to ≤5%.

[0013] Application of a composition based on multi-target micro-collagen peptides: The composition prepared according to the above method is used to prepare anti-wrinkle and firming cosmetics, oral collagen peptide health foods, repair-type medical dressings, or wound repair materials.

[0014] Compared with the prior art, the present invention provides a composition based on multi-target micro-collagen peptides, its preparation method and application, which has the following beneficial effects: 1. This invention employs raw material extraction, stepwise enzymatic hydrolysis, and ultrafiltration membrane separation technologies to precisely control the molecular weight of micro-collagen peptides within the range of 1500–2500 Da. Simultaneously, it is rich in the characteristic Gly-Pro-Hyp tripeptide sequence, which improves the intestinal absorption rate and bioavailability of micro-collagen peptides (3–5 times higher than traditional collagen) and reduces immunogenicity (removal rate of macromolecular aggregates and antigenic epitopes ≥99%).

[0015] 2. This invention mixes endogenous and exogenous micro-collagen peptides with sodium hyaluronate, sodium chondroitin sulfate (Gags component), and chitosan, and cross-links them with sodium tripolyphosphate ions under pH 6.0–6.05 conditions to spontaneously assemble a three-dimensional network structure. This simulates the natural extracellular matrix environment, achieving precise encapsulation, targeted delivery, and on-demand release of micro-collagen peptides. It enhances the stability and retention time of the composition at the site of action, ultimately improving the expression of collagen genes (col1a1b and col1a2 promotion rate ≥68%).

[0016] 3. This invention provides a multi-purpose, highly active, and low-allergenic collagen supplementation platform by sterilizing and spray-drying the above composition into a dry powder, which can be applied as needed in the fields of cosmetics, oral health foods, medical dressings, and tissue engineering scaffolds. The composition can be used externally to reduce wrinkles and firm the skin and accelerate wound healing, and can also be taken internally to improve the level of collagen synthesis throughout the body, ultimately meeting the needs of different administration routes and efficacy. Attached Figure Description

[0017] Figure 1 These are diagrams showing the healing of cell scratches in different groups in Example 1 of the present invention; Figure 2 This is a graph showing the scratch healing rate of different groups in Example 1 of the present invention; Figure 3 This is a graph showing the HSF cell viability of Example 1 of the present invention. Figure 4 This is a flowchart of the zebrafish type I collagen gene expression promotion test in Example 2 of the present invention; Figure 5 This is a flowchart illustrating the preparation process of the composition of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Please see Figures 1-5A composition based on multi-target micro-collagen peptides, wherein the composition and its components are in the following weight parts: 250-350 parts micro-collagen peptides; 15-25 parts chitosan; 4-6 parts sodium hyaluronate; 2-4 parts sodium chondroitin sulfate; 2-4 parts sodium tripolyphosphate; and the remaining component is purified water.

[0020] Microcollagen peptides consist of 90%–95% endogenous microcollagen peptides and 5%–10% exogenous microcollagen peptides.

[0021] The molecular weight distribution of endogenous micro-collagen peptides is 1500-2500 Da, with a polydispersity index (PDI) ≤1.5. They are rich in the characteristic tripeptide repeat sequence of gly-proline-hydroxyproline (Gly-Pro-Hyp), which is highly homologous to the active fragment of human type I collagen. After deep enzymatic hydrolysis and ultrafiltration purification, the removal rate of macromolecular aggregates and potential antigenic epitopes is ≥99%. They have low sensitization and can directly pass through the small intestinal epithelial cell layer into the blood circulation. Their bioavailability is 3-5 times higher than that of traditional collagen.

[0022] Specifically, the function of each ingredient is shown in the table below: Table 1 A method for preparing a composition based on multi-target micro-collagen peptides, comprising the following steps, according to the weight proportions of the components of the aforementioned composition based on multi-target micro-collagen peptides: Step 1: Raw material selection and gum extraction treatment: S1.1. Alaskan cod skin or tilapia skin / scales were used as raw materials (Alaskan cod skin was provided by a marine biotechnology company in Shandong, and tilapia skin / scales were provided by a seafood processing plant in Hainan). Deep-sea cod skin or tilapia skin / scales were chosen as raw materials because: deep-sea cod skin has a high collagen content (≥85%), lives in a low-temperature environment, has low cross-linking of collagen molecules, and is easy to extract and enzymatically hydrolyze; tilapia skin / scales are abundant and low in cost, and the hydroxyapatite structure of fish scales can protect collagen from excessive damage. The collagen extracted from both is typical type I collagen with a relatively concentrated molecular weight distribution, which is suitable for subsequent preparation of micro-collagen peptides with controllable molecular weight. S1.2 Weigh 10-12 kg of Alaskan cod skin or tilapia skin / scales, remove impurities, cut into 1-2 cm pieces, and wash with purified water 3-5 times to remove surface impurities and soluble proteins. S1.3 Add purified water at a material-to-liquid ratio of 1:4, adjust the pH to 4.9-5.0 using glacial acetic acid (Tianjin Kemeio Chemical Reagent Co., Ltd.) (this pH condition can promote collagen swelling and avoid excessive acid hydrolysis), heat to 50-55℃, and extract with constant temperature stirring for 2.8-3 hours; S1.4 After extraction, use a 200-300 mesh filter cloth for coarse filtration, collect the filtrate, and obtain crude collagen extract.

[0023] The advantages are: by selecting deep-sea cod skin or tilapia skin / scales as raw materials, deep-sea cod skin has a high collagen content and the low temperature environment results in a low degree of cross-linking of collagen molecules, making it easy to extract and enzymatically hydrolyze; tilapia scales are abundant and low in cost, and their hydroxyapatite structure can protect collagen from excessive acid hydrolysis during the extraction process. Both are rich in typical type I collagen, and the molecular weight distribution is relatively concentrated, laying the foundation for obtaining high-purity, well-defined microcollagen peptides.

[0024] After the crude collagen extract in step two has been allowed to stand for 1-1.5 hours, the supernatant is coarsely filtered through a plate and frame filter press with a filter paper pore size of 10μm, and then finely filtered through a cotton cake filter press with a cotton cake thickness of 3cm and a pore size of 5μm to obtain 30-31L of clear collagen solution.

[0025] The advantages are: by employing a two-stage fine filtration combination, plate and frame filtration quickly removes large suspended particles, while cotton cake filtration utilizes its deep-layer retention mechanism to further remove fine insoluble matter and colloidal impurities, resulting in a highly clear solution. This process effectively reduces the interference of impurities on enzyme activity in the subsequent enzymatic hydrolysis system, avoids the risk of membrane filtration system clogging, significantly improves enzymatic hydrolysis efficiency and product purity, and ensures the stability of solution quality between batches.

[0026] Enzymatic hydrolysis in step three: S2.1. Heat the clarified gel solution to 48-50℃, adjust the pH to 7.5-7.55 using 0.5±0.002M sodium hydroxide (Xilong Scientific), add 0.3±0.001% w / w alkaline protease (Alcalase 2.4L, based on protein), and enzymatically hydrolyze for 4-4.4 hours. S2.2, Continue adding 0.1±0.0005% w / w of flavorzyme, and then enzymatically hydrolyze for another 1-1.2 hours; S2.3, Heat to 88-90℃ to inactivate enzymes for 15-18 minutes, cool to 48-50℃, add activated carbon (YP-2 type) 1.5±0.005% w / v, stir and decolorize for 48-50 minutes; S2.4 Use a stainless steel plate and frame filter press (filtration accuracy 1-5μm, filter cloth material is polypropylene) to remove activated carbon and residue, and obtain enzymatic hydrolysis filtrate.

[0027] The advantages are: by adopting a two-step enzymatic hydrolysis strategy of alkaline protease and flavor protease, alkaline protease first efficiently cleaves collagen peptide bonds to produce medium molecular weight peptides; flavor protease further hydrolyzes terminal hydrophobic amino acid residues, reducing the formation of bitter peptides, improving the taste of the product, and thus enhancing the sensory quality of the final product.

[0028] Step 4: Filtration and Concentration: The enzymatic hydrolysis filtrate is filtered sequentially through a microfiltration membrane and an ultrafiltration membrane. The microfiltration membrane has a pore size of 0.22 μm and is made of polyethersulfone, while the ultrafiltration membrane has a molecular weight cutoff of 3000 Da and is made of regenerated cellulose. The ultrafiltration permeate (containing micro-collagen peptides with a molecular weight range of 1500–2500 Da) is collected and then concentrated to a solid content of 24%–25% under conditions of 58–60 °C and a vacuum degree of -0.09 to -0.08 MPa to obtain a collagen peptide concentrate, i.e., endogenous micro-collagen peptides.

[0029] The advantages are: by using combined membrane separation technology: microfiltration removes bacteria and tiny particles, ensuring the flux and lifespan of the subsequent ultrafiltration membrane; the ultrafiltration membrane accurately retains large molecular weight peptides with a molecular weight greater than 3000 Da and undigested proteins, and collects permeate with a molecular weight of 1500-2500 Da, achieving narrow distribution fractionation of microcollagen peptides.

[0030] Step 5: Loading and Assembly S3.1 Preparation of mixed solution: Take 1±0.05L of the collagen peptide concentrate obtained in step four, and add exogenous micro-collagen peptides, sodium hyaluronate, sodium chondroitin sulfate, and chitosan in the following proportions per liter of concentrate (i.e., final concentrations of 0.9%-1.1% w / v, 0.45%-0.55% w / v, 0.27%-0.33% w / v, and 1.8%-2.2% w / v, respectively). Add exogenous micro-collagen peptides (collagen peptides or laboratory-made low molecular weight collagen peptides), sodium hyaluronate (Bloomage Biotechnology, molecular weight 50kDa), sodium chondroitin sulfate (Shandong Weikang, purity 95%), and chitosan (Zhejiang Jinke, degree of deacetylation 92%) in sequence, and stir thoroughly until completely dissolved to obtain a mixed solution system. S3.2 Adjusting pH and Ionic Crosslinking Self-Assembly: Adjust the pH of the above mixed solution system to 6.0-6.05 using glacial acetic acid and 0.5±0.002M sodium hydroxide. Under stirring speed of 300-400 rpm, slowly add sodium tripolyphosphate solution (Hubei Xingfa, 10% w / v) at a rate of 1-2 mL / min to a final concentration of 0.27%-0.33% w / v (i.e. 0.3±0.03% w / v). After the addition is complete, continue stirring for 25-30 minutes. The system spontaneously forms a three-dimensional network structure. The advantages are: by using collagen peptide concentrate as a matrix, exogenous micro-collagen peptides, sodium hyaluronate, sodium chondroitin sulfate, and chitosan are precisely added in proportion to construct a three-dimensional network system that simulates the natural extracellular matrix. Sodium hyaluronate and sodium chondroitin sulfate together simulate the glycosaminoglycan environment in the extracellular matrix, enhancing the biocompatibility and bioavailability of micro-collagen peptides; chitosan, as a natural polysaccharide carrier, pre-binds with micro-collagen peptides and Gags components through hydrogen bonding and electrostatic interactions under pH 6.0-6.05 conditions. Slowly adding sodium tripolyphosphate forms ionic crosslinks, and the amino groups of chitosan undergo electrostatic crosslinking with the phosphate groups of sodium tripolyphosphate, spontaneously assembling into a stable three-dimensional network structure. This achieves precise encapsulation and on-demand release of micro-collagen peptides. This process is mild (room temperature, neutral pH), requires no organic solvents, and has high self-assembly efficiency, significantly improving the targeted delivery capability and in vivo stability of the composition.

[0031] Step six involves sterilization and drying: The composite solution is filtered through a 0.22μm sterile filter membrane (polyethersulfone), and then spray-dried in a clean area: inlet air temperature 175-180℃, outlet air temperature 80-85℃, atomization pressure 0.3-0.35MPa, feed rate 1.5-1.6L / h, and the dried multi-target micro collagen peptide composition powder is collected, with its moisture content controlled to ≤5%.

[0032] In step seven, granulation: Collect the spray-dried multi-target micro-collagen peptide composition powder in a Class D clean area, granulate it through a 30-mesh sieve using a dry granulator, and package the granules into food-grade aluminum foil bags (Huangshan Yongxin, 500g per bag), heat-seal the bags; then detect them with a metal detector (sensitivity Fe 0.5mm, SUS 1.0mm), reject unqualified products, and store qualified products in a cool and dry place.

[0033] Application of a composition based on multi-target micro-collagen peptides: The composition prepared according to the above method is used to prepare anti-wrinkle and firming cosmetics, oral collagen peptide health foods, repair-type medical dressings, or wound repair materials.

[0034] The following examples demonstrate the efficacy of cell scratch assays in promoting zebrafish collagen gene expression: Example 1 (Cell Scratch Assay) Cell migration refers to the movement of cells after receiving migration signals or sensing stimulation from certain substances. Increased cell migration ability indicates a repair-promoting effect. This experiment evaluates the potential repair effect of the test substance by comparing the rate of cell scratch healing and calculating the scratch healing rate.

[0035] Experimental reagents (1) Cell line: HSF (human skin fibroblasts); (2) Culture medium: DMEM / F12 medium containing 10% FBS; (3) Culture conditions: Cultured at 37℃, 5% CO2, and saturated humidity; (4) Solutions and controls: Blank control: Cell culture medium; Positive control (PC): TGF-β1 (10 ng / mL): Test sample (TS): TGF-β1 was purchased from Sigma-Aldrich and diluted sequentially with culture medium to the required test concentration. Main instruments: CO2 incubator (Thermo, 150I), ultra-clean workbench (Sujing Antai, SW-CJ-1F), Olympus CK53 microscope, multi-functional microplate reader (TECAN, SPARK). The experimental method is as follows: T1. Sample preparation: The test substance is dissolved in ultrapure water, filtered through a 0.22 μm filter membrane, and diluted to the required concentration using cell culture medium; T2. Cell preparation: Cells were cultured at 37℃ and 5% CO2, with the cell density controlled at 1.0 × 10⁶ cells / mL. 4 ~5.0×10 5 Individual samples were used for biological activity assay 24–36 hours after passage. T3. Screening of cell tolerance concentrations of test substances: Cells were seeded at 6000 cells / well in 96-well cell culture plates and cultured for 24 hours. The old culture medium was removed, and complete culture medium containing different concentrations of test substances was added and incubated for 24 hours. MTT was added and incubated for 4 hours. The liquid in the culture plate was removed, DMSO was added and mixed, and the absorbance at a wavelength of 490 nm was measured using a microplate reader. The results were recorded. T4, scratch test Cells were seeded in 6-well plates, with the cell count adjusted to achieve confluence every other day. After 24 hours of adherent culture, straight scratches were made on the bottom of the culture plate using a 20 μL plastic pipette tip, ensuring consistent pressure, angle, and thickness. Floating cells were washed away with PBS, and fresh culture medium (containing 1% fetal bovine serum) was added. The blank control group received no intervention, the positive control group received medium containing 10 ng / mL TGF-β1, and the sample groups received culture medium containing different concentrations of the test substance. Locations with consistent scratch thickness and cell density were selected, their coordinates were recorded, and photographs were taken at different time points to compare the scratch healing speed of each group. The scratch area of ​​each group was measured using ImageJ software, and the scratch healing rate was calculated using the following formula: Scratch healing rate / % = (0h scratch area - scratch area at each time point) / 0h scratch area × 100%.

[0036] Statistical analysis: GraphPad Prism 8.0 was used for statistical analysis. Quantitative variables were described as mean ± standard deviation, and one-way ANOVA was performed for comparisons among multiple groups.

[0037] The experimental results are shown in Table 2 below: Table 2 Comparison of scratch healing rates among different groups (±s, %) Note: vs Control: p < 0.05; vs Control: p < 0.01.

[0038] Experimental results are as follows Figure 1 Table 2 Figure 2 As shown, compared with the blank control group, the healing rate of the positive control group (10 ng / mL LTGF-β1) was significantly increased at 24 h and 48 h, with a significant difference (p < 0.01), proving the effectiveness of the experimental system. Furthermore, the 1.00 mg / mL multi-target micro-collagen peptide composition showed a significant upward trend in cell scratch healing rate at 24 h, with a significant difference compared with the blank control group (p < 0.01); the 0.50 mg / mL Marinepep® collagen peptide showed an upward trend in cell scratch healing rate at 48 h, with a statistically significant difference compared with the blank control group (p < 0.05); and the 1.00 mg / mL Marinepep® collagen peptide showed a significant upward trend in cell scratch healing rate at 48 h, with a significant difference compared with the blank control group (p < 0.01). These combined results indicate that 0.50 mg / mL and 1.00 mg / mL Marinepep® collagen peptides can enhance the migration ability of human skin fibroblasts and have potential repair effects.

[0039] Table 3 Screening results were based on cell tolerance concentration (Table 3). Figure 3 The study concluded that Marinepep® collagen peptides, based on HSF cells, did not negatively impact cell growth at concentrations <3.75 mg / mL.

[0040] Example 2 (Efficacy verification of multi-target micro-collagen peptide composition in promoting collagen gene expression) Experimental materials Test substance: A multi-target micro-collagen peptide-based composition (spray-dried powder) prepared according to the method of Example 1, dissolved and diluted with fish embryo culture medium to the test concentration (100 μg / mL, based on collagen peptide content) before use.

[0041] Experimental animals: Healthy zebrafish (wild-type AB strain, provided by the Institute of Hydrobiology, Chinese Academy of Sciences) 6 days after fertilization were incubated in a constant temperature incubator at 28±1℃ until they reached 6 days of age.

[0042] Main reagents: Fish embryo culture medium (self-prepared: 5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM MgSO4, 0.1% methylene blue, pH 7.2); RNAlater solution (Thermo Fisher, AM7021); TRIzol reagent (Invitrogen, 15596026); chloroform, isopropanol, ethanol (Sinopharm Group, analytical grade); DNase / RNase-free ultrapure water (self-made); PrimeScript RT kit (containing gDNA Eraser, Takara, RR047A); iTaq UniversalSYBR Green Supermix (Bio-Rad, 1725125); primers (Shanghai Sangon Biotech).

[0043] Consumables: 24-well plate (Corning, 3526); 1.5mL centrifuge tube (Axygen, MCT-150-C); 96-well PCR plate (Applied Biosystems, 4306737); optical film (Bio-Rad, MSB1001); pellets (Beijing Lanbolide).

[0044] Experimental methods Test grouping and exposure management: A blank control group and a test group were set up, with 36 zebrafish in each group, and the experiment was repeated 3 times independently.

[0045] Blank control group: 36 zebrafish were randomly selected and transferred to 24-well plates, 12 fish per well, and 2.5 mL of fish embryo culture medium was added to each well.

[0046] Test group: 36 zebrafish were randomly selected and transferred to 24-well plates, 12 fish per well. 2.5 mL of test solution (100 μg / mL, prepared with fish embryo culture medium) was added to each well.

[0047] Incubate the 24-well plate in a constant temperature incubator at 28±1℃ for 24±1 hours. After the culture is completed, collect 12 zebrafish from each well into a 1.5mL centrifuge tube, remove the culture medium, add 0.5mL of RNAlater solution, and store frozen (-80℃).

[0048] RNA extraction: Remove the stored sample, aspirate the RNAlater solution, and wash three times with PBS solution; place on ice, aspirate the PBS, and add 500 μL of TRIzol reagent; homogenize the embryo using a pellet mill, and incubate on ice for 10 minutes; add 100 μL of chloroform, vortex for 1 minute, and incubate on ice for 5 minutes; centrifuge at 12000 rpm for 20 minutes at 4°C, transfer the upper aqueous phase to a new 1.5 mL centrifuge tube, add 250 μL of isopropanol, vortex for 1 minute, and incubate on ice for 10 minutes; centrifuge again (4°C, 12000 rpm, 20 minutes), discard the supernatant; add 500 μL of TRIzol reagent. 75% ethanol (prepared with DNase / RNase-free water), vortex for 1 minute, centrifuge for 5 minutes (4℃, 12000rpm), discard the ethanol; repeat washing 3 times; after the last centrifugation, remove the ethanol, and air dry in a fume hood for 10 minutes with the lid open; add 10μL of DNase / RNase-free ultrapure water, heat at 55℃ for 15 minutes to dissolve the RNA, and determine the RNA concentration and purity.

[0049] cDNA synthesis: Dilute the RNA of each sample to 1000 ng / 7 μL using DNase / RNase-free ultrapure water; follow the instructions of the PrimeScript RT kit (including gDNA Eraser): take 7 μL of RNA (1000 ng), add 2 μL of 5×gDNA Eraser Buffer and 1 μL of gDNA Eraser, and bring the volume to 10 μL. Incubate at 42℃ for 2 minutes; then add 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of RT Primer Mix, 4 μL of 5×PrimeScript Buffer 2 and 4 μL of DNase / RNase-free water, incubate at 37℃ for 15 minutes, and then inactivate at 85℃ for 5 seconds to obtain cDNA, which is stored at -20℃.

[0050] Real-time RT-PCR detection: cDNA was diluted 10-fold with DNase / RNase-free water. Primer sequences are shown in Table 4 (5'→3'). Table 4 Prepare the reaction mixture in a 96-well PCR plate as follows: 9 μL SYBR Green premix (containing Taq enzyme, dNTPs, and SYBR Green), 1 μL diluted cDNA template, and primer mixture (each primer to a final concentration of 0.2 μM). Perform triple replicates per sample. Seal with optical gel and centrifuge for 5 minutes. Amplify using a real-time PCR instrument: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 10 seconds, annealing / extension at 60°C for 30 seconds, for 40 cycles. Melting curve analysis: 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

[0051] Data calculation: Using β-actin as an internal reference, the relative expression levels of col1a1b and col1a2 genes in each sample were calculated using the 2-ΔΔCt method. The collagen gene expression promotion rate was calculated using the following formula: Promotion rate = ×100% In the formula, T represents the relative expression level of collagen gene in the test group; C represents the relative expression level of collagen gene in the blank control group. The two-tailed t-test was used to compare the differences between the two groups, and p < 0.05 was considered statistically significant.

[0052] The experimental results are shown in Table 5: Table 5. The promoting effect of the test substances on the expression of collagen genes in zebrafish. Note: Data are expressed as mean ± standard deviation, n=3; relative expression levels were normalized with the blank control group as 1.

[0053] After 24 hours of exposure, the survival rate of all fish embryos was greater than 95%, meeting the test validity requirements (≥90%). The analysis results showed that, compared with the blank control group, the composition of the present invention could significantly upregulate the expression of col1a1b and col1a2 genes in zebrafish (p<0.05), with promotion rates of 72% and 68%, respectively.

[0054] Example 2 demonstrates that the multi-target micro-collagen peptide-based composition prepared according to the present invention can effectively promote the expression of collagen genes (col1a1b and col1a2) in a zebrafish model with statistically significant differences. This result provides direct supporting evidence for the composition to promote collagen regeneration and achieve anti-wrinkle and firming effects.

[0055] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A composition based on multi-target microcollagen peptides, characterized in that, The composition and its components are as follows by weight: 250-350 parts of microcollagen peptides; 15-25 parts of chitosan; 4-6 parts of sodium hyaluronate; 2-4 parts of sodium chondroitin sulfate; 2-4 parts of sodium tripolyphosphate; and the remaining component is purified water.

2. The composition based on multi-target microcollagen peptides according to claim 1, characterized in that, The microcollagen peptides consist of 90%–95% endogenous microcollagen peptides and 5%–10% exogenous microcollagen peptides.

3. The composition based on multi-target microcollagen peptides according to claim 1, characterized in that, The molecular weight distribution of the endogenous microcollagen peptides is 1500–2500 Da.

4. A method for preparing a composition based on multi-target microcollagen peptides, characterized in that, The composition based on multi-target micro-collagen peptides according to claim 1 is prepared by the following steps: Step 1: Select raw materials for collagen extraction: Select animal raw materials that are rich in type I collagen and easy to enzymatically hydrolyze, and perform collagen extraction on the animal raw materials to obtain crude collagen extract; Step 2, Filtration and initial filtration: The crude collagen extract is filtered and allowed to stand, and then filtered through cotton cake to remove large insoluble particles, resulting in a clear collagen solution. Step 3, Enzymatic hydrolysis: Add enzyme preparation to the clear gel solution for directional enzymatic hydrolysis, after enzyme inactivation, add activated carbon for decolorization, and obtain the enzymatic hydrolysis filtrate; Step 4, Filtration and Concentration: The enzymatic hydrolysis filtrate is purified by membrane filtration and then concentrated under vacuum to obtain collagen peptide concentrate, namely endogenous micro collagen peptides. Step 5, Loading and Assembly: Collagen peptide concentrate, GAGs glycosaminoglycans and natural polysaccharides are mixed and ionically cross-linked to self-assemble into a three-dimensional network system; Step 6, Sterilization and Drying: After sterilization and filtration, spray drying is performed to obtain a multi-target micro collagen peptide composition powder; Step 7: Granulation: Collect the dried multi-target micro collagen peptide composition powder, granulate and package it, and reject unqualified products by metal detection.

5. The method for preparing a composition based on multi-target micro-collagen peptides according to claim 4, characterized in that, The raw material selection and gum extraction process in step one: S1.1, Made from Alaskan cod skin or tilapia skin / scales; S1.2 Weigh 10-12 kg of Alaskan cod skin or tilapia skin / scales, remove impurities, cut into 1-2 cm pieces, and wash with purified water 3-5 times; S1.3 Add purified water at a material-to-liquid ratio of 1:4, adjust the pH to 4.9-5.0 with glacial acetic acid, heat to 50-55℃, and extract with constant temperature stirring for 2.8-3 hours; S1.4 After extraction, use a 200-300 mesh filter cloth for coarse filtration, collect the filtrate, and obtain crude collagen extract.

6. The method for preparing a composition based on multi-target micro-collagen peptides according to claim 4, characterized in that, In step two, after the crude collagen extract has been allowed to stand for 1-1.5 hours, the supernatant is coarsely filtered through a plate and frame filter press with a filter paper pore size of 10 μm, and then finely filtered through a cotton cake filter press with a cotton cake thickness of 3 cm and a pore size of 5 μm to obtain 30-31 L of clear collagen solution.

7. The method for preparing a composition based on multi-target micro-collagen peptides according to claim 4, characterized in that, Enzymatic hydrolysis in step three: S2.

1. Heat the clarified gel solution to 48-50℃, adjust the pH to 7.5-7.55 using 0.5±0.002M sodium hydroxide, add 0.3±0.001% w / w alkaline protease, and enzymatically hydrolyze for 4-4.4 hours; S2.2, Continue adding 0.1±0.0005% w / w of protease, and then enzymatically hydrolyze for another 1-1.2 hours; S2.3, Heat to 88-90℃ to inactivate enzymes for 15-18 minutes, cool to 48-50℃, add 1.5±0.005% w / v activated carbon, stir and decolorize for 48-50 minutes; S2.4 Use a stainless steel plate and frame filter press to remove activated carbon and residue to obtain enzymatic hydrolysis filtrate.

8. The method for preparing a composition based on multi-target micro-collagen peptides according to claim 4, characterized in that, In step four, filtration and concentration are performed as follows: the enzymatic hydrolysis filtrate is filtered sequentially through a microfiltration membrane and an ultrafiltration membrane, the ultrafiltration permeate is collected, and then concentrated to a solid content of 24%-25% under conditions of 58-60℃ and a vacuum degree of -0.09 to -0.08MPa to obtain a collagen peptide concentrate, namely endogenous micro collagen peptides.

9. A method for preparing a composition based on multi-target micro-collagen peptides according to claim 4, characterized in that, The loading and assembly in step five: S3.1 Preparation of mixed solution: Take 1±0.05L of collagen peptide concentrate obtained in step four, and add exogenous micro collagen peptide, sodium hyaluronate, sodium chondroitin sulfate and chitosan in the following proportions per liter of concentrate: 9-11g of exogenous micro collagen peptide, sodium hyaluronate, sodium chondroitin sulfate and chitosan in sequence. Stir thoroughly until completely dissolved to obtain a mixed solution system. S3.2 Adjusting pH and Ionic Crosslinking Self-Assembly: Adjust the pH of the above mixed solution system to 6.0-6.05 using glacial acetic acid and 0.5±0.002M sodium hydroxide. Under the condition of stirring speed of 300-400 rpm, slowly add sodium tripolyphosphate solution at a rate of 1-2 mL / min to a final concentration of 0.27%-0.33% w / v. After the addition is completed, continue stirring for 25-30 minutes. In step six, sterilization and drying are performed as follows: the composite solution is filtered through a 0.22μm sterilization filter membrane and then spray-dried in a clean area: the inlet air temperature is 175-180℃, the outlet air temperature is 80-85℃, the atomization pressure is 0.3-0.35MPa, the feed rate is 1.5-1.6L / h, and the dried multi-target micro collagen peptide composition powder is collected, with its moisture content controlled to ≤5%.

10. An application of a composition based on multi-target microcollagen peptides, characterized in that, The composition prepared according to claim 4 is used to prepare anti-wrinkle and firming cosmetics, oral collagen peptide health foods, repair-type medical dressings, or wound repair materials.

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