Application of glycosaminoglycan-rich collagen peptide

Abstract

The present invention relates to the technical field of collagen peptides, and provides an application of a glycosaminoglycan-rich collagen peptide. The invention comprises the following steps: performing a high-pressure liquefaction reaction on fish skin or fish scales, bones, and connective tissues in combination with a microwave heating process, significantly improving the degree of hydrolysis of related raw materials on the one hand, promoting the complexation of polysaccharides and protein peptides in the raw materials on the other hand, and then preparing a collagen peptide-glycosaminoglycan complex with a low molecular weight and concentrated distribution through a step-by-step enzymatic hydrolysis technology, thereby increasing the added value of the production raw materials. The invention is suitable for large-scale production in enterprises, and can be used in preparing anti-skin allergy products, anti-skin aging products, skin repair products, and products for restoring skin moisture, improving skin elasticity, and reducing skin wrinkles. The glycosaminoglycan-rich collagen peptide comprises collagen peptides and glycosaminoglycans; and the collagen peptides account for 85-99% and 1-15% of the glycosaminoglycans in parts by weight. The multiple components act synergistically, are significantly active in improving skin problems, and have good application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen peptides, and in particular to an application of glycosaminoglycan-rich collagen peptides. Background Art

[0002] Proteoglycans are a vital component of the skin and, besides collagen, are the most abundant structural component of the skin's extracellular matrix. The basic units of proteoglycans include glycosaminoglycans and core proteins. Glycosaminoglycans are repeating disaccharide units composed of penturonic acid or hexuronic acid linked to N-acetylglucosamine or N-acetylgalactosamine, with some glycosaminoglycans substituted with sulfate groups at positions 1, 4, or 6. Glycosaminoglycans have a wide molecular weight range, ranging from a few thousand daltons to over one million daltons.

[0003] Because glycosaminoglycans contain many negatively charged carboxyl and sulfate groups, they play an important role in maintaining moisture in tissues. For example, it is reported that hyaluronic acid can accommodate water molecules up to 1,000 times its molecular weight. Proteoglycans composed of glycosaminoglycans and proteins are the main components of the skin. They are present in large quantities in the extracellular matrix of the dermis and are the core key to the skin's barrier function and physiological functions. Their regulatory function on cytokines and growth factors plays an important role in skin development, balance and regeneration.

[0004] Skin aging can be divided into intrinsic aging and photoaging based on its phenotype and mechanism. Intrinsic aging is characterized by epidermal thinning, which leads to fine lines, while photoaging is characterized by sagging skin, deep wrinkles, dilated capillaries, and rashes. Studies have reported that the proteoglycan content in the skin changes dramatically with age. Compared with young skin samples, the total sulfated glycosaminoglycan content in aged skin decreases, and the relative proportions of proteoglycans also change. In addition to changes in proteoglycan levels found in the skin, the pattern of proteoglycan deposition also changes. Studies have also found that the function of proteoglycans and collagen fiber organization in photoaged skin is weakened.

[0005] Collagen is primarily found in animal tissues such as bones, tendons, muscle sheaths, ligaments, fascia, skin, and cartilage. It is a crucial structural protein in connective tissue, supporting organs and protecting the body. It is the most abundant and widely distributed protein in mammals, accounting for 25% to 30% of the body's total protein. Collagen peptides are currently widely used in the food and pharmaceutical industries, with reference standards including QB 2732-2005 Hydrolyzed Collagen and GB 31645-2018 National Food Safety Standard Collagen Peptides. According to these standards, the current production process for collagen peptides primarily uses animal skins and bones as raw materials, soaking them in acid and alkali to produce gelatin, followed by enzymatic hydrolysis, or directly using acid and alkali treatment followed by enzymatic hydrolysis. Characteristic components of collagen peptides include protein, total nitrogen, and hydroxyproline. Because traditional gelatin processes or direct enzymatic hydrolysis processes require the use of acidic and alkaline reagents to treat the raw materials, the process causes the degradation and loss of animal mucopolysaccharides such as glycosaminoglycans in animal tissues. The final product contains almost no glycosaminoglycans, which greatly reduces the product's biological activity and use value. Therefore, it is of great significance to protect glycosaminoglycans during the production process, increase the glycosaminoglycan content in collagen peptides, and enhance and explore the efficacy value of the product. Summary of the Invention

[0006] Based on the above background, the purpose of the present invention is to provide an application of glycosaminoglycan-rich collagen peptide.

[0007] The present invention adopts the following technical solutions:

[0008] As one of the technical solutions, the use of glycosaminoglycan-rich collagen peptide in the preparation of anti-skin allergy products is provided. The glycosaminoglycan-rich collagen peptide is prepared by the following method:

[0009] S1: purification of raw materials, wherein the raw materials are rich in type I collagen material A and cartilage material B;

[0010] S2 High-pressure liquefaction fusion: Mix the processed type I collagen-rich material A and cartilage-like material B, place them in a magnetic high-pressure reactor, add deionized water, and stir them thoroughly with magnetic stirring; stir at a low speed of 10-30 MPa to form a collagen-polysaccharide complex;

[0011] S3 Microwave Polymerization: The high-pressure liquefied fusion reaction product is placed in a liquid microwave heater and subjected to microwave polymerization at a power of 100W-600W and a temperature range of 20-90°C to enhance the interaction between collagen and glycosaminoglycans.

[0012] S4 composite enzymatic hydrolysis: the microwave-polymerized product is cooled to 40-60°C, and then the temperature is further controlled at 50-60°C. One or more of alkaline protease, trypsin, and pepsin are first added at an enzyme addition amount of 0.02%-0.2%, and the enzymatic hydrolysis is carried out for 0.5-1.0 h. Subsequently, one or more of neutral protease, papain, and bromelain are added at an enzyme addition amount of 0.02%-0.2%, and the total enzymatic hydrolysis time is 3-5 h to obtain a glycosaminoglycan-rich collagen hydrolyzate containing specific peptide fragments;

[0013] The polysaccharide-rich collagen peptide has characteristic peptide segments of GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

[0014] As one of the technical solutions, the use of glycosaminoglycan-rich collagen peptide in the preparation of anti-skin aging products is provided. The glycosaminoglycan-rich collagen peptide is prepared by the following method:

[0015] S1: purification of raw materials, wherein the raw materials are material A and material B rich in type I collagen;

[0016] S2 High-pressure liquefaction fusion: Mix the processed type I collagen-rich material A and cartilage-like material B, place them in a magnetic high-pressure reactor, add deionized water, and stir them thoroughly with magnetic stirring; stir at a low speed of 10-30 MPa to form a collagen-polysaccharide complex;

[0017] S3 Microwave Polymerization: The high-pressure liquefied fusion reaction product is placed in a liquid microwave heater and subjected to microwave polymerization at a power of 100W-600W and a temperature range of 20-90°C to enhance the interaction between collagen and glycosaminoglycans.

[0018] S4 composite enzymatic hydrolysis: the microwave-polymerized product is cooled to 40-60°C, and then the temperature is further controlled at 50-60°C. One or more of alkaline protease, trypsin, and pepsin are first added at an enzyme addition amount of 0.02%-0.2%, and the enzymatic hydrolysis is carried out for 0.5-1.0 h. Subsequently, one or more of neutral protease, papain, and bromelain are added at an enzyme addition amount of 0.02%-0.2%, and the total enzymatic hydrolysis time is 3-5 h to obtain a glycosaminoglycan-rich collagen hydrolyzate containing specific peptide fragments;

[0019] The polysaccharide-rich collagen peptide has characteristic peptide segments of GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

[0020] As one of the technical solutions, the use of glycosaminoglycan-rich collagen peptide in the preparation of skin repair products is provided. The glycosaminoglycan-rich collagen peptide is prepared by the following method:

[0021] S1: purification of raw materials, wherein the raw materials are material A and material B rich in type I collagen;

[0022] S2 High-pressure liquefaction fusion: Mix the processed type I collagen-rich material A and cartilage-like material B, place them in a magnetic high-pressure reactor, add deionized water, and stir them thoroughly with magnetic stirring; stir at a low speed of 10-30 MPa to form a collagen-polysaccharide complex;

[0023] S3 Microwave Polymerization: The high-pressure liquefied fusion reaction product is placed in a liquid microwave heater and subjected to microwave polymerization at a power of 100W-600W and a temperature range of 20-90°C to enhance the interaction between collagen and glycosaminoglycans.

[0024] S4 composite enzymatic hydrolysis: the microwave-polymerized product is cooled to 40-60°C, and then the temperature is further controlled at 50-60°C. One or more of alkaline protease, trypsin, and pepsin are first added at an enzyme addition amount of 0.02%-0.2%, and the enzymatic hydrolysis is carried out for 0.5-1.0 h. Subsequently, one or more of neutral protease, papain, and bromelain are added at an enzyme addition amount of 0.02%-0.2%, and the total enzymatic hydrolysis time is 3-5 h to obtain a glycosaminoglycan-rich collagen hydrolyzate containing specific peptide fragments;

[0025] The polysaccharide-rich collagen peptide has characteristic peptide segments of GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

[0026] As one of the technical solutions, the use of glycosaminoglycan-rich collagen peptide in the preparation of a product for restoring skin moisture, improving skin elasticity, and reducing skin wrinkles, the glycosaminoglycan-rich collagen peptide is prepared by the following method:

[0027] S1: purification of raw materials, wherein the raw materials are material A and material B rich in type I collagen;

[0028] S2 High-pressure liquefaction fusion: Mix the processed type I collagen-rich material A and cartilage-like material B, place them in a magnetic high-pressure reactor, add deionized water, and stir them thoroughly with magnetic stirring; stir at a low speed of 10-30 MPa to form a collagen-polysaccharide complex;

[0029] S3 Microwave Polymerization: The high-pressure liquefied fusion reaction product is placed in a liquid microwave heater and subjected to microwave polymerization at a power of 100W-600W and a temperature range of 20-90°C to enhance the interaction between collagen and glycosaminoglycans.

[0030] S4 composite enzymatic hydrolysis: the microwave-polymerized product is cooled to 40-60°C, and then the temperature is further controlled at 50-60°C. One or more of alkaline protease, trypsin, and pepsin are first added at an enzyme addition amount of 0.02%-0.2%, and the enzymatic hydrolysis is carried out for 0.5-1.0 h. Subsequently, one or more of neutral protease, papain, and bromelain are added at an enzyme addition amount of 0.02%-0.2%, and the total enzymatic hydrolysis time is 3-5 h to obtain a glycosaminoglycan-rich collagen hydrolyzate containing specific peptide fragments;

[0031] The polysaccharide-rich collagen peptide has characteristic peptide segments of GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

[0032] Furthermore, the type I collagen-rich substance A is fish skin and / or fish scales; and the cartilage-like substance B is edible animal bones and / or connective tissue.

[0033] Furthermore, the cartilage substance B is poultry and livestock cartilage and / or fish cartilage.

[0034] In one aspect of the present invention, the product of the present invention is a medicine or a cosmetic.

[0035] In one aspect of the present invention, the glycosaminoglycan-rich collagen peptide of the present invention comprises collagen peptide and glycosaminoglycan.

[0036] In one aspect of the present invention, the glycosaminoglycan-rich collagen peptide of the present invention comprises 85-99% collagen peptide and 1-15% glycosaminoglycan.

[0037] In one aspect of the present invention, the average molecular weight of the collagen peptide in the polysaccharide-rich collagen peptide of the present invention is less than 1500 Da.

[0038] The present invention has the following beneficial effects:

[0039] (1) The present invention combines high-pressure liquefaction reaction with microwave heating treatment on fish skin or fish scales, bones and connective tissues, thereby significantly improving the hydrolysis degree of the relevant raw materials on the one hand, and promoting the complexation of polysaccharides and protein peptides in the raw materials on the other hand, and then preparing collagen peptide-glycosaminoglycan complexes with low molecular weight and concentrated distribution through step-by-step enzymatic hydrolysis technology, thereby increasing the added value of the production raw materials, being suitable for large-scale production in enterprises, and being applicable to the preparation of anti-skin allergy products, anti-skin aging products, skin repair products, and products for restoring skin moisture, improving skin elasticity, and reducing skin wrinkles;

[0040] (2) The glycosaminoglycan-rich collagen peptide of the present invention comprises collagen peptide and glycosaminoglycan; in terms of weight: collagen peptide is 85-99%, glycosaminoglycan is 1-15%. The multiple ingredients act synergistically, and are significantly active in improving skin problems and other aspects, and have good application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the nuclear magnetic resonance spectroscopy analysis of the glycosaminoglycan-rich collagen peptide of the present invention;

[0042] Figure 2 Chromatographic analysis of the glycosaminoglycan-rich collagen peptides of the present invention;

[0043] Figure 3 This is the HE staining result of the glycosaminoglycan-rich collagen peptide of the present invention on the skin epidermal repair ability;

[0044] Figure 4 This is the IF staining of related proteins in the skin epidermis by the glycosaminoglycan-rich collagen peptide of the present invention;

[0045] Figure 5 Immunofluorescence images of AQP3 expression in different groups;

[0046] Figure 6 Immunofluorescence images of CD44 expression in different groups;

[0047] Figure 7 Immunofluorescence images of ZO-1 expression in different groups;

[0048] Figure 8 Immunofluorescence images of CLDN1 expression in different groups. DETAILED DESCRIPTION

[0049] It should be noted that the polysaccharide-rich collagen peptide involved in the present invention is the result of joint research and development by our company and Mattel Technology (Qingdao) Co., Ltd. The present invention is described in detail below with reference to the following examples:

[0050] Example 1:

[0051] Preparation of Glycosaminoglycan-Rich Collagen Peptides

[0052] 1. Raw material pretreatment:

[0053] Thaw 4.5 kg of frozen decalcified fish scales in deionized water at a material-to-water ratio of 1:5, soak for 10 hours, and drain thoroughly before use. Thaw 1 kg of connective tissue (poultry and livestock cartilage) in deionized water, soak the cartilage in deionized water at a solid-to-liquid ratio of 1:5, and treat at room temperature for 6 hours. Drain thoroughly and crush into uniform particles with a diameter of 0.1-0.3 cm. Set aside.

[0054] 2. High-pressure liquefaction fusion and microwave treatment stage:

[0055] The treated fish scale and cartilage raw materials were placed in a magnetic high-pressure reactor, deionized water was added at a material-water ratio of 2:1, and the reaction was carried out under low-speed stirring at 10 MPa for 8 hours; the treated liquid was added to a liquid microwave heater and microwave-heated at 90°C and 600W for 20 minutes.

[0056] 3. Compound enzymatic hydrolysis stage:

[0057] Cool the product from the previous step to 55°C while stirring at 100 rpm to thoroughly mix. Add trypsin at a concentration of 0.05% and continue enzymatic hydrolysis for 1 hour. Then, add neutral protease and papain at a concentration of 0.025% each for another 2.5 hours. After the enzymatic hydrolysis is complete, heat the solution to 90°C and terminate the reaction for 20 minutes.

[0058] 4. Raw material refining stage:

[0059] The enzymatic hydrolysis liquid is initially separated from the enzymatic hydrolysis residue in a 6000rpm centrifuge, and the supernatant is taken. The disc filter further clarifies and filters to remove fine particles, thereby further purifying the liquid. 0.5% decolorizing activated carbon and 0.5% deodorizing activated carbon particles are added to the supernatant, and the adsorption reaction is carried out at 52-54°C for 1 hour. The activated carbon adsorption liquid is centrifuged at 7000rpm for 30 minutes, and the centrifuge is further decarbonized by a disc filter and a cardboard filter. The above-mentioned clarified filtrate is separated by a nanofiltration membrane to remove low-valent cations, remove ash and improve the taste, and further filtered with a 0.22μm pore size filter membrane to purify and remove impurities.

[0060] 5. Sterilization, concentrated spray drying stage:

[0061] The liquid material after multi-stage filtration and purification is sterilized, concentrated, and spray-dried to obtain an off-white granular powder, namely, the glycosaminoglycan-rich collagen peptide.

[0062] 6. Identification of Glycosaminoglycan-Rich Collagen Peptides

[0063] 6.1 Determination of glycosaminoglycan content in glycosaminoglycan-rich collagen peptides

[0064] With reference to the Light Industry Standard of the People's Republic of China QB / T 4576-2013, the glycosaminoglycan content was determined to be 4.0 g / 100 g.

[0065] 6.2 Structural Analysis of Glycosaminoglycans in Glycosaminoglycan-Rich Collagen Peptides

[0066] The results of nuclear magnetic resonance spectroscopy analysis of glycosaminoglycan-rich collagen peptides are shown in Figure 1 As shown. Figure A and Figure B are 1 HNMR and 13 C NMR and corresponding spectral peak identification. The results indicate that the glycosaminoglycans in the glycosaminoglycan-rich collagen peptide prepared by the present invention are mainly sulfated mucopolysaccharides composed of glucuronic acid and N-acetylglucosamine or N-acetylgalactosamine.

[0067] 6.3 Molecular Weight Distribution of Polypeptides Contained in Glycosaminoglycan-Rich Collagen Peptides

[0068] Using high performance gel exclusion chromatography analysis, the molecular weight distribution of the polypeptides contained in the glycosaminoglycan collagen peptide of the present invention is less than 3.5KDa. The proportion of substances with a molecular weight of more than 90%. Using high performance gel exclusion chromatography analysis (see Figure 2 As shown), the logarithm of the molecular weight of the standard and the retention time were used to draw a standard curve, and the regression equation of the standard curve was y = -0.18997x + 7.1197 (R 2 =0.9555), and the average molecular weight of the analyzed sample was 827.29 Da, which was lower than 1500 Da, consistent with the characteristics of small molecule peptides.

[0069] 6.4 Identification of Characteristic Peptides in Glycosaminoglycan-Rich Collagen Peptides

[0070] The glycosaminoglycan-rich collagen peptides obtained by the present invention were subjected to mass spectrometry detection, and then the mass spectrometry method was subjected to data-dependent acquisition (DDA). The data collected by mass spectrometry were searched in the software Protein Pilot, and the search library was a collagen library downloaded from the NCBI website. The data was processed using MaxQuant software, and the generated results were tested against the database in UNIPROT. The results showed that the peptide segments of the glycosaminoglycan-rich collagen peptides had a credibility higher than 95%, of which 11 peptide segments modified by N-glycosylation were identified. The characteristic glycosylated collagen peptides contained in the obtained glycosaminoglycan-rich collagen peptides are shown in Table 1.

[0071] Table 1 Characteristic glycosylated collagen peptides contained in the glycosaminoglycan-rich collagen peptides obtained in Example 1

[0072]

[0073]

[0074] The results based on signal intensity showed that the representative characteristic peptide segments of the glycosaminoglycan-rich collagen peptide of the present invention include GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

[0075] Test Example: Activity detection of the glycosaminoglycan-rich collagen peptide of the present invention:

[0076] Test Example 1. Skin soothing and improving sensitive skin (anti-allergy) efficacy testing

[0077] This test uses 300mJ / cm 2 The soothing effect of the product of the present invention was evaluated by detecting the changes in the levels of pro-inflammatory factors (IL-6, IL-8, TNF-a) after irradiation of keratinocytes with a dose of UVB (Batch No. Ep220707, provided by Guangdong Boxi Biotechnology Co., Ltd.).

[0078] Main reagents:

[0079] KC2500 culture medium (Guangdong Boxi Biotechnology), PBS (Solaibao), dexamethasone (Sigma), IL-6 ELISA kit (Abeam), IL-8 ELISA kit (Abeam), TNF-a ELISA kit (Abeam)

[0080] Test method:

[0081] 1) Cell seeding: 2.2×10 5 Keratinocytes were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator overnight;

[0082] 2) Prepare the test substance working solution according to the test group (see Table 2);

[0083] Table 2 Skin allergy test groups

[0084]

[0085] 3) Assemble the test groups according to Table 1. When the cell plating rate in the 6-well plate reaches 40% to 60%, administer the drug to each group. Add 2 mL of sample to each well, with three replicates per group. After administration, place the 6-well plate in an incubator (37°C, 5% CO2) and incubate for 24 hours.

[0086] 4) UVB irradiation: According to the experimental group, the group with UVB irradiation was irradiated with 300mJ / cm 2 of UVB radiation.

[0087] 5) ELISA assay: After irradiation, the cell culture supernatant was collected and ELISA assay was performed according to the ELISA kit instructions.

[0088] 6) Statistical Analysis: Graphs were generated using GraphPad Prism, and results are expressed as mean ± SD. Intergroup comparisons were analyzed using the F-test. All statistical analyses were two-tailed. A P<0.05 was considered a significant difference, and a P<0.01 was considered a highly significant difference.

[0089] Test results

[0090] The results of the skin anti-allergy test are shown in Table 3. Compared with the BC group, significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0091] Table 3 Skin anti-allergy test results

[0092]

[0093]

[0094] The results showed that the glycosaminoglycan-rich collagen peptide product of the present invention can significantly inhibit the expression of inflammatory factors IL-6, IL-8, and TNF-a in skin cells, and has the effects of soothing the skin, improving skin sensitivity, and anti-allergic.

[0095] Test Example 2. Anti-skin aging and skin repair ability testing

[0096] 2.1 Anti-skin aging test

[0097] This test uses 30J / cm 2 The anti-aging efficacy of the test samples was evaluated by irradiating fibroblasts with UVA of different doses and detecting the changes in the activities of type I collagen (Collagen I), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px).

[0098] The cells used in this test were fibroblasts, batch number: Fbl9052002, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0099] DMEM culture medium (Gibco), PBS (Solaibao), vitamin E (VE, Sigma), Collagen I ELISA kit (Cusabio), SOD kit (Biyuntian), CAT kit (Nanjing Jiancheng), GSH-Px kit (Nanjing Jiancheng).

[0100] Test method:

[0101] 1) Cell seeding: 2.2×10 5 Keratinocytes were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator overnight;

[0102] 2) Prepare the test substance working solution according to the test group (see Table 4);

[0103] Table 4 Anti-skin aging test groups

[0104]

[0105] 3) Dosing: When the cell plating rate in the 6-well plate reaches 40% to 60%, administer the drug to each group according to the test group. Add 2 mL of sample to each well, with three replicates per group. After dosing, place the 6-well plate in an incubator (37°C, 5% CO2) and incubate for 24 hours.

[0106] 4) UVA irradiation: According to the test group, the group that needs irradiation is irradiated with UVA, with an irradiation dose of 30J / cm 2 After irradiation, the cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 hours.

[0107] 5) ELISA assay: After irradiation, the cell culture supernatant was collected and ELISA assay was performed according to the ELISA kit instructions.

[0108] 6) HPLC Assay: After digestion, cells treated with different conditions were transferred to 1.5 mL centrifuge tubes. 500 μL of 0.2 mg / mL protein kinase K was added to each tube and incubated in a 50°C water bath for 1 hour. 250 μL of methanol was added to each tube and sonicated for 30 minutes. The tube was then centrifuged at 14,000 rpm for 10 minutes. The methanol was evaporated at 60°C and stored at 4°C until ready for analysis.

[0109] 7) Enzyme activity detection: After UVA irradiation, the cell culture supernatant was collected and assayed according to the instructions of the corresponding kits for SOD, CAT, and GSH-Px.

[0110] 8) Statistical Analysis: Graphs were generated using GraphPad Prism, and results are expressed as mean ± SD. Intergroup comparisons were analyzed using the F-test. All statistical analyses were two-tailed. A P<0.05 was considered a significant difference, and a P<0.01 was considered a highly significant difference.

[0111] The results of the anti-aging test are shown in Table 5. Compared with the BC group, significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0112] Table 5 Anti-skin aging test results

[0113]

[0114]

[0115] In addition, the present invention also uses commercially available collagen peptides as samples to test the effects of commercially available collagen peptides on MDA in UVA-induced aging cells. The inhibition rate of commercially available collagen peptides on MDA is 30.96%. There is a significant difference in the inhibitory effect between the product of the present invention and the commercially available collagen peptides.

[0116] Furthermore, the present invention also determines the effect of the product of the present invention on the proliferation ability of cells. The proliferation ability of aging cells will also be affected. The present invention uses the MTT method to detect the effect of the product of the present invention on the proliferation of keratinocytes. Compared with the blank control without the addition of the test substance, the proliferation of keratinocytes increased by 9.23% at 24 hours, 36.61% at 48 hours, and 26.22% at 72 hours. This shows that the product of the present invention can quickly stimulate cell proliferation and play a role in proliferation, repair and anti-aging.

[0117] 2.2 Skin repair ability test

[0118] An SLS (sodium lauryl sulfate)-stimulated epidermal model (EpiKutis 3D epidermal model) was constructed. HE staining and immunofluorescence (IF) were then used to examine skin cell status and the expression of skin barrier-related proteins (FLG, LOR) to evaluate the skin repair ability of the product of the present invention. The skin repair ability test groups are shown in Table 6.

[0119] Table 6 Skin repair ability test groups

[0120]

[0121] Skin repair ability test results are shown in Figure 3 and Figure 4 As shown, the HE staining results of skin epidermal repair ability are shown in Figure 3 As shown; for the IF staining of related proteins in the skin epidermis, see Figure 4 shown.

[0122] As can be seen from HE staining and IF immunofluorescence staining, compared with the BC group, after SLS stimulation, the stratum corneum of the NC group was loose and thickened, the living cell layer was damaged, and vacuoles appeared, indicating that the stimulation conditions of this experiment were effective. Compared with the NC group, the structural boundaries of the PC group were clear, the living cell layer was tightly arranged, the stratum corneum was loose and thickened, and the damage to the living cell layer was significantly improved, indicating that this experimental detection system is effective. Compared with the NC group, the product of the present invention (sample 1) can also significantly improve the loosening and thickening of the stratum corneum and the damage to living cells. Therefore, the product of the present invention can play a role in skin repair, restoring cell vitality, and rebuilding the skin barrier.

[0123] Thus, the product of the present invention can increase the amount of type I collagen, catalase (CAT), and superoxide dismutase (SOD) in cells, and significantly inhibit the production of malondialdehyde (MDA) in cells, while having no effect on glutathione peroxidase (GSH-Px). The product of the present invention exerts an anti-aging effect through an antioxidant pathway, and its inhibition of malondialdehyde (MDA) is significantly better than commercially available collagen peptides. At the same time, the product of the present invention can rapidly stimulate cell proliferation to achieve the effect of proliferation, repair, and anti-aging. The product of the present invention can also significantly improve the loosening and thickening of the stratum corneum and the damage of living cells, play a role in skin repair, restoring cell vitality, and rebuilding the skin barrier.

[0124] Test Example 3: Efficacy testing for restoring skin moisture, improving skin elasticity, and reducing skin wrinkles

[0125] The cells used in this test were keratinocytes, batch number: Ep220707, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0126] Main reagents: KC2500 (Guangdong Boxi Biotechnology), PBS (Solaibao), paraformaldehyde (Biosharp), AQP3 antibody (Abcam), CD44 antibody (Abcam), ZO-1 antibody (Proteintech), CLDN1 antibody (Proteintech), WY14643 (Sigma).

[0127] Test method:

[0128] 1) Cell seeding: 5×10 4 Keratinocytes were seeded into 24-well plates at a seeding density of 100 wells and incubated in an incubator (37° C., 5% CO 2 ) overnight.

[0129] 2) Solution preparation: Prepare the working solutions of the test substances according to the test groups (see Table 7).

[0130] Table 7 Test groups for restoring skin moisture, improving skin elasticity, and reducing skin wrinkles

[0131]

[0132] 3) Dosing: When the cell plating rate in the 24-well plate reaches 40% to 60%, the cells are dosed according to the test group, with three replicate wells per group. After dosing, the 24-well plate is placed in an incubator (37°C, 5% CO2) and cultured for 24 hours.

[0133] 4) UVB irradiation: After washing the cells with PBS, the UVB irradiated groups were irradiated with 300 mJ / cm 2 of UVB stimulation.

[0134] 5) Post-incubation: After washing the cells with PBS, the 24-well plate was placed in an incubator (37° C., 5% CO 2 ) and incubated for 24 h.

[0135] 6) Immunofluorescence detection: The cells were fixed with 4% paraformaldehyde for 30 minutes, and then immunofluorescence detection was performed. The cells were photographed using a fluorescence microscope and analyzed using Image-ProSPlus image processing software.

[0136] 7) Statistical Analysis: Graphs were generated using GraphPad Prism, and results are expressed as mean ± SD. Comparisons between groups were analyzed using the t-test. P < 0.05 was considered a significant difference, and P < 0.01 was considered a highly significant difference.

[0137] The immunofluorescence images of AQP3 expression in different groups are shown in Figure 5 As shown; immunofluorescence images of CD44 expression in different groups are shown in Figure 6 As shown; immunofluorescence images of ZO-1 expression in different groups are shown in Figure 7 As shown; immunofluorescence images of CLDN1 expression in different groups are shown in Figure 8 shown.

[0138] The results of skin moisture restoration, skin elasticity improvement, and skin wrinkle reduction tests are shown in Table 8. Compared with the BC group, significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0139] Table 8 Test results of restoring skin moisture, improving skin elasticity and reducing skin wrinkles

[0140]

[0141] CLDN1 is a gene associated with tight junctions, which are crucial for maintaining the selective permeability barrier function of epidermal cells. Furthermore, the present invention also tested the effects of the present invention's product and commercially available collagen on CLDN1 in glial cells without UVB stimulation using the same immunofluorescence method as described above. The results showed that the present invention increased CLDN1 protein expression in keratinocytes by 31%, while the commercial collagen increased it by 22%, a significant difference between the two (P-value < 0.05).

[0142] The primary function of MMPs (matrix metalloproteinases) is to degrade collagen. When MMP levels are reduced, collagen degradation is inhibited, thereby achieving an anti-wrinkle effect. COL-1 (type I collagen) is the primary component of collagen in the dermis, accounting for approximately 80% and contributing to the skin's mechanical support. With aging or stress-induced aging, type I collagen levels decrease significantly. Elastin is the primary protein that makes up elastic fibers. Decreased levels of elastin lead to thinning of elastic fibers and decreased skin elasticity. Elastic fibers, composed of microfibrils and elastin, are highly elastic but lack toughness. They cross-link with collagen fibers to maintain the skin's elasticity and toughness. At the molecular level, immediate effects can be attributed to elastic fibers in the dermis, while delayed effects can be primarily attributed to collagen fibers and the extracellular matrix, which is primarily composed of proteoglycans and glycosaminoglycans. Hyaluronic acid is a major component of the skin's extracellular matrix. Among its many biological functions, hydration is one of hyaluronic acid's functions, which determines the skin's viscoelastic properties. Hyaluronic acid synthase is the key to HA synthesis. There are three main synthases in the skin, namely HAS1, HAS2 and HAS3. Therefore, the present invention also uses fibroblasts to test the effect of UVA 30J / cm 2 Under stimulation conditions, the effects of adding the product of the present invention on the COL-1, Elastin, HAS1, HAS2, HAS3, MMP-1, and MMP-3 proteins in the cells are shown in Table 9.

[0143] Table 9 Effects on COL-1, Elastin, HAS1, HAS2, HAS3, MMP-1, and MMP-3 proteins in cells

[0144]

[0145] The results showed that compared with NC, the sample group to which the product of the present invention was added could increase the amount of COL-1, elastin, and hyaluronic acid synthase, and increase the inhibition of MMP-1 and MMP-3 degradation, thereby retaining collagen and hyaluronic acid in the skin and playing an anti-wrinkle role.

[0146] Test Example 4: Comparative evaluation of human trials on restoring skin moisture, improving skin elasticity, and reducing skin wrinkles:

[0147] 120 volunteers aged 35 to 45 years who met the test requirements were selected as subjects and randomly divided into 4 groups, with 30 subjects in each group. The evaluation methods of "T / ZHCA003-2018 Test Method for the Effect of Cosmetics on Transepidermal Water Loss", "T / ZHCA005-2019 Test Method for the Effect of Cosmetics on Skin Elasticity" and "T / ZHCA006-2019 Test Method for the Anti-wrinkle Efficacy of Cosmetics" were referred to. Before product intervention, the moisture, elasticity and wrinkle data of the subjects' skin were measured as baseline values. Then, 30 days after the corresponding product intervention, the skin condition values ​​of the subjects were measured again using the same method. The changes in skin moisture, elasticity and wrinkles of the same subjects before and after intervention were analyzed using statistical software, and scores were given according to the significance of the statistical results of each group. The main differences in the test formulas are shown in Table 10, and the corresponding scoring criteria are shown in Table 11.

[0148] Table 10 Main differences in test substance formulations

[0149]

[0150] Table 11: Significant difference score table of statistical results

[0151]

[0152] After 30 days of intervention with the corresponding products, the scores of skin moisture, elasticity and wrinkle changes in each group of subjects before and after intervention are shown in Table 12.

[0153] Table 12 Results of skin moisture, elasticity and wrinkle changes before and after intervention

[0154]

[0155] As can be seen from the scoring results in Table 12, after 30 days of intervention with 3g / d of collagen peptide, there was no significant difference in the changes in skin moisture, skin elasticity and skin wrinkles in the group taking traditional collagen peptides, with a comprehensive score of 1 point; there was a significant difference in the changes in skin moisture in the group taking the new collagen peptides, with a comprehensive score of 4 points, indicating that the new collagen peptides can significantly improve skin moisture, while also showing an improvement trend in increasing skin elasticity and reducing skin wrinkles. After 30 days of intervention with 5g / d of collagen peptides, there was a significant difference in the skin moisture and skin elasticity values ​​of the group taking traditional collagen peptides, with a comprehensive score of 5 points, but there was no significant difference in reducing skin wrinkles; there was a significant difference in the skin moisture, skin elasticity and skin wrinkles of the group taking the new collagen peptides, with a comprehensive score of 9 points, indicating that the new collagen peptides of the present invention can significantly reduce skin moisture loss, increase skin elasticity and reduce skin wrinkles.

[0156] In summary, the product of the present invention can increase the expression of aquaporin AQP3 and tight junction proteins (ZO-1, CLDN1) in cells, which helps to absorb and retain water, while having a limited effect on the hyaluronic acid receptor CD44, that is, it does not achieve the purpose of improving skin by increasing the absorption of hyaluronic acid. The product of the present invention is significantly superior to commercially available collagen peptides in terms of the increase in tight junction protein CLDN1. The sample group of the product of the present invention can increase the amount of COL-1, elastin, and hyaluronic acid synthase, and increase the inhibition of MMP-1 and MMP-3 degradation, thereby retaining collagen and hyaluronic acid in the skin to play an anti-wrinkle role. The glycosaminoglycan-rich collagen peptide can be used to prepare anti-skin allergy products, anti-skin aging products, skin repair products, and products for restoring skin moisture, improving skin elasticity, and reducing skin wrinkles. The glycosaminoglycan-rich collagen peptide contains collagen peptide and glycosaminoglycan. In terms of weight, the collagen peptide accounts for 85-99% and the glycosaminoglycan accounts for 1-15%. The multiple ingredients work synergistically, have significant activity in improving skin problems, and have good application potential.

[0157] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the scope of protection of the present invention.

Claims

1. The use of glycosaminoglycan-rich collagen peptide in the preparation of anti-skin allergy products, characterized by: The glycosaminoglycan-rich collagen peptide is prepared by the following method: S1: purification of raw materials, wherein the raw materials are rich in type I collagen material A and cartilage material B; S2 High-pressure liquefaction fusion: The processed type I collagen-rich material A and cartilage-like material B are mixed and placed in a magnetic high-pressure reactor. Deionized water is added and magnetic stirring is used to fully mix the mixture. The mixture is stirred at a low speed of 10 MPa for 8 hours to form a collagen-polysaccharide complex. S3 microwave polymerization: The high-pressure liquefied fusion reaction product was placed in a liquid microwave heater and microwave-heated at a power of 600 W and a temperature of 90°C for 20 minutes to perform microwave polymerization reaction and enhance the interaction between collagen and glycosaminoglycans; S4 composite enzymatic hydrolysis: The microwave-polymerized product was cooled to 40-60°C and then further controlled at 50-60°C. Trypsin was first added at an enzyme dosage of 0.05% and enzymatic hydrolysis was performed for 1.0 h. Neutral protease and papain were then added at an enzyme dosage of 0.025% for 2.5 h to obtain a glycosaminoglycan-rich collagen hydrolyzate containing specific peptide fragments. The polysaccharide-rich collagen peptide has characteristic peptide segments of GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

2. The use of glycosaminoglycan-rich collagen peptides in the preparation of anti-skin aging products, characterized by: The glycosaminoglycan-rich collagen peptide is prepared by the following method: S1: purification of raw materials, wherein the raw materials are rich in type I collagen material A and cartilage material B; S2 High-pressure liquefaction fusion: The processed type I collagen-rich material A and cartilage-like material B are mixed and placed in a magnetic high-pressure reactor. Deionized water is added and magnetic stirring is used to fully mix the mixture. The mixture is stirred at a low speed of 10 MPa for 8 hours to form a collagen-polysaccharide complex. S3 microwave polymerization: The high-pressure liquefied fusion reaction product was placed in a liquid microwave heater and microwave-heated at a power of 600 W and a temperature of 90°C for 20 minutes to perform microwave polymerization reaction and enhance the interaction between collagen and glycosaminoglycans; S4 composite enzymatic hydrolysis: The microwave-polymerized product was cooled to 40-60°C and then further controlled at 50-60°C. Trypsin was first added at an enzyme dosage of 0.05% and enzymatic hydrolysis was performed for 1.0 h. Neutral protease and papain were then added at an enzyme dosage of 0.025% for 2.5 h to obtain a glycosaminoglycan-rich collagen hydrolyzate containing specific peptide fragments. The polysaccharide-rich collagen peptide has characteristic peptide segments of GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

3. The use of glycosaminoglycan-rich collagen peptides in the preparation of skin repair products, characterized by: The glycosaminoglycan-rich collagen peptide is prepared by the following method: S1: purification of raw materials, wherein the raw materials are rich in type I collagen material A and cartilage material B; S2 High-pressure liquefaction fusion: The processed type I collagen-rich material A and cartilage-like material B are mixed and placed in a magnetic high-pressure reactor. Deionized water is added and magnetic stirring is used to fully mix the mixture. The mixture is stirred at a low speed of 10 MPa for 8 hours to form a collagen-polysaccharide complex. S3 microwave polymerization: The high-pressure liquefied fusion reaction product was placed in a liquid microwave heater and microwave-heated at a power of 600 W and a temperature of 90°C for 20 minutes to perform microwave polymerization reaction and enhance the interaction between collagen and glycosaminoglycans; S4 composite enzymatic hydrolysis: The microwave-polymerized product was cooled to 40-60°C and then further controlled at 50-60°C. Trypsin was first added at an enzyme dosage of 0.05% and enzymatic hydrolysis was performed for 1.0 h. Neutral protease and papain were then added at an enzyme dosage of 0.025% for 2.5 h to obtain a glycosaminoglycan-rich collagen hydrolyzate containing specific peptide fragments. The polysaccharide-rich collagen peptide has characteristic peptide segments of GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

4. The use of glycosaminoglycan-rich collagen peptides in the preparation of products for restoring skin moisture, improving skin elasticity, and reducing skin wrinkles, characterized by: The glycosaminoglycan-rich collagen peptide is prepared by the following method: S1: purification of raw materials, wherein the raw materials are rich in type I collagen material A and cartilage material B; S2 High-pressure liquefaction fusion: The processed type I collagen-rich material A and cartilage-like material B are mixed and placed in a magnetic high-pressure reactor. Deionized water is added and magnetic stirring is used to fully mix the mixture. The mixture is stirred at a low speed of 10 MPa for 8 hours to form a collagen-polysaccharide complex. S3 microwave polymerization: The high-pressure liquefied fusion reaction product was placed in a liquid microwave heater and microwave-heated at a power of 600 W and a temperature of 90°C for 20 minutes to perform microwave polymerization reaction and enhance the interaction between collagen and glycosaminoglycans; S4 composite enzymatic hydrolysis: The microwave-polymerized product was cooled to 40-60°C and then further controlled at 50-60°C. Trypsin was first added at an enzyme dosage of 0.05% and enzymatic hydrolysis was performed for 1.0 h. Neutral protease and papain were then added at an enzyme dosage of 0.025% for 2.5 h to obtain a glycosaminoglycan-rich collagen hydrolyzate containing specific peptide fragments. The polysaccharide-rich collagen peptide has characteristic peptide segments of GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, and RNEEPVLF.

5. The use according to any one of claims 1 to 4, characterized in that The type I collagen-rich substance A is fish skin and / or fish scales; the cartilage-like substance B is edible animal bones and / or connective tissue.

6. The use according to claim 5, characterized in that The cartilage substance B is poultry and livestock cartilage and / or fish cartilage.

7. The use according to any one of claims 1 to 4, characterized in that The products described are medicines and cosmetics.

8. The use according to any one of claims 1 to 4, characterized in that The glycosaminoglycan-rich collagen peptide comprises collagen peptide and glycosaminoglycan.

9. The use according to claim 8, characterized in that The glycosaminoglycan-rich collagen peptide contains 85-99% collagen peptide and 1-15% glycosaminoglycan.

10. The use according to claim 8, characterized in that The average molecular weight of the collagen peptides in the polysaccharide-rich collagen peptides is lower than 1500 Da.

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