CN116410302A - Application of glycosaminoglycan-rich collagen peptide - Google Patents
Application of glycosaminoglycan-rich collagen peptide Download PDFInfo
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- CN116410302A CN116410302A CN202310145385.XA CN202310145385A CN116410302A CN 116410302 A CN116410302 A CN 116410302A CN 202310145385 A CN202310145385 A CN 202310145385A CN 116410302 A CN116410302 A CN 116410302A
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- rich
- collagen
- glycosaminoglycan
- skin
- collagen peptide
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention relates to the technical field of collagen peptide, and provides an application of glycosaminoglycan-rich collagen peptide, which is characterized in that the hydrolysis degree of relevant raw materials is obviously improved on one hand by combining high-pressure liquefaction reaction of fish skin or fish scales, bones and connective tissues with microwave heating treatment, and on the other hand, the complexation of polysaccharide and protein peptide in the raw materials is promoted, and then collagen peptide-glycosaminoglycan compound with low molecular weight and concentrated distribution is prepared by a fractional enzymolysis technology, so that the added value of the production raw materials is improved, and the collagen peptide-glycosaminoglycan compound is suitable for large-scale production of enterprises, and can be applied to preparation of anti-skin allergy products, preparation of anti-skin aging products, preparation of skin repair products, preparation of skin moisture recovery products, improvement of skin elasticity and reduction of skin wrinkles; the glycosaminoglycan-rich collagen peptide comprises a collagen peptide and glycosaminoglycan; according to the parts by weight: the collagen peptide is 85-99%, the glycosaminoglycan is 1-15%, and the synergistic effect of multiple components is remarkable in activity in the aspects of improving skin problems and the like, and has good application potential.
Description
Technical Field
The invention relates to the technical field of collagen peptide, in particular to application of glycosaminoglycan-rich collagen peptide.
Background
Proteoglycans are an important component of the skin, being the most abundant structural tissue component in the extracellular matrix of the skin, except for collagen fibers. The basic units of proteoglycans include glycosaminoglycans and core proteins, wherein the glycosaminoglycans are repeating disaccharide units composed of pentose or hexose aldehyde acid linked to N-acetylglucosamine or N-acetylgalactosamine, and a part of the glycosaminoglycans are substituted by 1/4/6 sulfate groups; glycosaminoglycans range in molecular weight from a few kilodaltons to over a hundred thousand daltons.
Because glycosaminoglycans contain many negatively charged carboxyl and sulfate groups, they play an important role in maintaining moisture in tissues, e.g., hyaluronic acid is reported to be able to hold up to 1000 times its molecular weight of water molecules; glycosaminoglycans and proteoglycans, which are the major components of the skin and are present in large amounts in the extracellular matrix of the dermis, are central keys to the barrier and physiological functions of the skin, and have important roles in the regulation of cytokines and growth factors for skin development, balance and regeneration.
Skin aging can be classified into endogenous aging and photoaging according to the phenotype and mechanism of skin aging. Endogenous aging is characterized by thinning of the epidermis and thus by fine lines of the skin, while photoaging is characterized by slackening of the skin, deep wrinkles, telangiectasias, and rash. According to research reports, the proteoglycan content in skin changes strongly during aging of skin with age, the total sulfated glycosaminoglycan content level in aged skin is reduced compared with young skin samples, and furthermore, the relative proportion of proteoglycans also changes; in addition to the changes in proteoglycan levels found in skin, patterns of proteoglycan deposition were also changed, while studies have found reduced proteoglycan and collagen fibril tissue function in photoaged skin.
Collagen is mainly present in animal bone, tendon, myosheath, ligament, myomembrane, skin, cartilage and other tissues, is an extremely important structural protein in connective tissue, has the functions of supporting organs and protecting organisms, is the most abundant and widely distributed protein in mammals, and accounts for 25% -30% of the total protein in the bodies. At present, collagen peptide is widely used in the food and medicine industries and the like, and corresponding reference standards comprise QB 2732-2005 hydrolyzed collagen and GB 31645-2018 national food safety standard collagen peptide. According to the standard descriptions of QB 2732-2005 hydrolyzed collagen, GB 31645-2018 national food safety standard collagen peptide and the like, the current production process of the collagen peptide mainly adopts animal skin, bone grains and the like as raw materials, and adopts the raw materials to soak and decoct the raw materials into gelatin by acid and alkali and then carry out enzymolysis, or does not need to prepare gelatin but carries out enzymolysis after direct acid-base treatment, and the marked components are protein/total nitrogen, hydroxyproline and the like. Because the traditional gelatin process or the direct enzymolysis process needs to treat the raw materials by using acid and alkaline reagents, animal glycosaminoglycans such as glycosaminoglycans in animal tissues are degraded and lost in the process, and the final product basically does not contain glycosaminoglycan substances, so that the biological activity and the use value of the product are greatly reduced. Therefore, the method has important significance in protecting the glycosaminoglycan in the production process, increasing the content of the glycosaminoglycan in the collagen peptide and improving and exploring the efficacy value of the product.
Disclosure of Invention
Based on the background described above, an object of the present invention is to provide an application of glycosaminoglycan-rich collagen peptide.
The invention adopts the following technical scheme:
as one of the technical schemes, the application of the glycosaminoglycan-rich collagen peptide in preparing the anti-skin allergy product is that the glycosaminoglycan-rich collagen peptide is prepared by the following steps:
s1, purifying a raw material, wherein the raw material is rich in a type I collagen substance A and a cartilage substance B;
s2, high-pressure liquefaction and fusion: mixing the treated collagen-rich material A rich in type I with the cartilage material B, placing the mixture in a magnetic high-pressure reaction kettle, adding deionized water, and magnetically stirring the mixture to be fully and uniformly mixed; stirring at low speed under 10-30MPa to form collagen-polysaccharide complex;
s3, microwave polymerization: placing the high-pressure liquefied and fused reaction product in a liquid microwave heater, and carrying out microwave polymerization reaction at the power of 100W-600W and the temperature of 20-90 ℃ to strengthen the interaction between collagen and glycosaminoglycan;
s4, composite enzymolysis: cooling the microwave polymerized product to 40-60 deg.c, further controlling the temperature at 50-60 deg.c, adding one or several of alkaline proteinase, trypsin and pepsin in the amount of 0.02-0.2% and enzymolysis for 0.5-1.0 hr; then adding one or more of neutral protease, papain and bromelain, wherein the addition amount of the enzyme is 0.02% -0.2%, and the total enzymolysis time is 3-5h, so as to prepare the glycosaminoglycan-rich collagen hydrolysate containing specific peptide fragments;
the polysaccharide-rich collagen peptide has GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF characteristic peptide fragments.
As one of the technical schemes, the application of the glycosaminoglycan-rich collagen peptide in preparing the skin aging resistant product is that the glycosaminoglycan-rich collagen peptide is prepared by the following steps:
s1, purifying raw materials, wherein the raw materials are substances A rich in type I collagen and substances B;
s2, high-pressure liquefaction and fusion: mixing the treated collagen-rich material A rich in type I with the cartilage material B, placing the mixture in a magnetic high-pressure reaction kettle, adding deionized water, and magnetically stirring the mixture to be fully and uniformly mixed; stirring at low speed under 10-30MPa to form collagen-polysaccharide complex;
s3, microwave polymerization: placing the high-pressure liquefied and fused reaction product in a liquid microwave heater, and carrying out microwave polymerization reaction at the power of 100W-600W and the temperature of 20-90 ℃ to strengthen the interaction between collagen and glycosaminoglycan;
s4, composite enzymolysis: cooling the microwave polymerized product to 40-60 deg.c, further controlling the temperature at 50-60 deg.c, adding one or several of alkaline proteinase, trypsin and pepsin in the amount of 0.02-0.2% and enzymolysis for 0.5-1.0 hr; then adding one or more of neutral protease, papain and bromelain, wherein the addition amount of the enzyme is 0.02% -0.2%, and the total enzymolysis time is 3-5h, so as to prepare the glycosaminoglycan-rich collagen hydrolysate containing specific peptide fragments;
the polysaccharide-rich collagen peptide has GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF characteristic peptide fragments.
As one of the technical schemes, the application of the glycosaminoglycan-rich collagen peptide in preparing skin repair products is that the glycosaminoglycan-rich collagen peptide is prepared by the following steps:
s1, purifying raw materials, wherein the raw materials are substances A rich in type I collagen and substances B;
s2, high-pressure liquefaction and fusion: mixing the treated collagen-rich material A rich in type I with the cartilage material B, placing the mixture in a magnetic high-pressure reaction kettle, adding deionized water, and magnetically stirring the mixture to be fully and uniformly mixed; stirring at low speed under 10-30MPa to form collagen-polysaccharide complex;
s3, microwave polymerization: placing the high-pressure liquefied and fused reaction product in a liquid microwave heater, and carrying out microwave polymerization reaction at the power of 100W-600W and the temperature of 20-90 ℃ to strengthen the interaction between collagen and glycosaminoglycan;
s4, composite enzymolysis: cooling the microwave polymerized product to 40-60 deg.c, further controlling the temperature at 50-60 deg.c, adding one or several of alkaline proteinase, trypsin and pepsin in the amount of 0.02-0.2% and enzymolysis for 0.5-1.0 hr; then adding one or more of neutral protease, papain and bromelain, wherein the addition amount of the enzyme is 0.02% -0.2%, and the total enzymolysis time is 3-5h, so as to prepare the glycosaminoglycan-rich collagen hydrolysate containing specific peptide fragments;
the polysaccharide-rich collagen peptide has GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF characteristic peptide fragments.
As one of the technical schemes, the application of the glycosaminoglycan-rich collagen peptide in preparing products for restoring skin moisture, improving skin elasticity and reducing skin wrinkles is provided, and the glycosaminoglycan-rich collagen peptide is prepared by the following steps:
s1, purifying raw materials, wherein the raw materials are substances A rich in type I collagen and substances B;
s2, high-pressure liquefaction and fusion: mixing the treated collagen-rich material A rich in type I with the cartilage material B, placing the mixture in a magnetic high-pressure reaction kettle, adding deionized water, and magnetically stirring the mixture to be fully and uniformly mixed; stirring at low speed under 10-30MPa to form collagen-polysaccharide complex;
s3, microwave polymerization: placing the high-pressure liquefied and fused reaction product in a liquid microwave heater, and carrying out microwave polymerization reaction at the power of 100W-600W and the temperature of 20-90 ℃ to strengthen the interaction between collagen and glycosaminoglycan;
s4, composite enzymolysis: cooling the microwave polymerized product to 40-60 deg.c, further controlling the temperature at 50-60 deg.c, adding one or several of alkaline proteinase, trypsin and pepsin in the amount of 0.02-0.2% and enzymolysis for 0.5-1.0 hr; then adding one or more of neutral protease, papain and bromelain, wherein the addition amount of the enzyme is 0.02% -0.2%, and the total enzymolysis time is 3-5h, so as to prepare the glycosaminoglycan-rich collagen hydrolysate containing specific peptide fragments;
the polysaccharide-rich collagen peptide has GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF characteristic peptide fragments.
Further, the substance A rich in the type I collagen is fish skin and/or fish scales; the cartilage substance B is edible animal bone and/or connective tissue.
Further, the cartilage substance B is livestock cartilage and/or fish cartilage.
In one aspect of the invention, the product is a food, a health product, a dietary supplement, a pharmaceutical product, a cosmetic product.
In one aspect of the invention, the glycosaminoglycan-rich collagen peptide comprises collagen peptide and glycosaminoglycan.
In one aspect of the invention, the glycosaminoglycan-rich collagen peptide comprises 85-99% of collagen peptide and 1-15% of glycosaminoglycan.
In one aspect of the invention, the collagen peptide in the polysaccharide-rich collagen peptide of the invention has an average molecular weight of less than 1500Da.
The invention has the beneficial effects that:
(1) According to the invention, the high-pressure liquefaction reaction of the fish skin or the fish scales, the bones and the connective tissues is combined with the microwave heating treatment, so that on one hand, the hydrolysis degree of relevant raw materials is remarkably improved, on the other hand, the complexation of polysaccharide and protein peptide in the raw materials is promoted, and then the collagen peptide-glycosaminoglycan compound with low molecular weight and concentrated distribution is prepared through the fractional enzymolysis technology, so that the added value of the raw materials is improved, and the method is suitable for large-scale production of enterprises, and can be applied to the preparation of anti-skin allergy products, the preparation of anti-skin aging products, the preparation of skin repair products, the preparation of skin moisture restoration products, the improvement of skin elasticity and the reduction of skin wrinkles;
(2) The glycosaminoglycan-rich collagen peptide comprises collagen peptide and glycosaminoglycan; according to the parts by weight: the collagen peptide is 85-99%, the glycosaminoglycan is 1-15%, and the synergistic effect of multiple components is remarkable in activity in the aspects of improving skin problems and the like, and has good application potential.
Drawings
FIG. 1 shows nuclear magnetic resonance spectroscopy of glycosaminoglycan-rich collagen peptide of the present invention;
FIG. 2 is a chromatographic analysis of glycosaminoglycan-rich collagen peptide according to the present invention;
FIG. 3 is a graph showing the results of HE staining of glycosaminoglycan-rich collagen peptide of the present invention for skin epidermis repair;
FIG. 4 shows the IF staining of the glycosaminoglycan-rich collagen peptide of the present invention for proteins of interest in the epidermis of the skin;
FIG. 5 is an immunofluorescence of various groups of AQP3 expression;
FIG. 6 is an immunofluorescence of different sets of CD44 expression;
FIG. 7 is an immunofluorescence of ZO-1 expression for different groups;
FIG. 8 is an immunofluorescence of different groups of clDN1 expression.
Detailed Description
The polysaccharide-rich collagen peptide according to the present invention was developed by the company of the present unit and the company of the science and technology of Meta (Qingdao). The present invention will be specifically described with reference to examples below:
example 1:
preparation of glycosaminoglycan-rich collagen peptide
1. Pretreatment of raw materials:
taking 4.5kg of frozen decalcified fish scales, adding deionized water according to a water-to-water ratio of 1:5, thawing, soaking for 10 hours, and draining water for later use. 1kg of connective tissue (livestock cartilage) is taken, washed and thawed by deionized water, and the cartilage is soaked by the deionized water, wherein the solid-to-liquid ratio is 1:5, treating for 6 hours at room temperature, draining, and crushing into uniform particles with the diameter ranging from 0.1 cm to 0.3cm for standby.
2. High-pressure liquefaction fusion and microwave treatment stage:
putting the treated fish scales and cartilage raw materials into a magnetic high-pressure reaction kettle according to a water-to-care ratio of 2:1 adding deionized water, stirring at a low speed under 10MPa, and reacting for 8 hours; and adding the treated feed liquid into a liquid microwave heater, and heating for 20min at 90 ℃ and 600W.
3. And (3) a composite enzymolysis stage:
and cooling the product of the last step to 55 ℃, and stirring at a speed of 100rpm to mix thoroughly. Adding trypsin with the addition amount of 0.05%, performing enzymolysis for 1h, and then adding neutral protease and papain for continuous enzymolysis for 2.5h, wherein the addition amounts of the enzymes are respectively 0.025%. And after the enzymolysis is finished, heating the enzymolysis liquid to 90 ℃ for 20min to terminate the enzymolysis reaction.
4. Raw material refining:
and (3) primarily separating enzymolysis residues from the enzymolysis liquid in a 6000rpm centrifuge, and taking supernatant. The disc filter further clarifies and filters to remove fine particles, thereby achieving the effect of further purification. Adding 0.5% decolorized active carbon and 0.5% deodorized active carbon particles into the supernatant, and performing adsorption reaction at 52-54 ℃ for 1h. And (3) centrifuging the activated carbon adsorption liquid at a high speed at 70000 rpm for 30min, and taking the centrifugate and further performing decarbonization treatment through a disc filter and a cardboard filter. Separating the clarified filtrate with nanofiltration membrane to remove low-valence cations, removing ash and improving taste, and further filtering with 0.22 μm pore size filter membrane to remove impurities.
5. Sterilization, concentration spray drying stage:
the feed liquid after the multistage filtration and purification treatment is sterilized, concentrated and spray-dried to obtain white-like granular powder, and the glycosaminoglycan-rich collagen peptide is obtained.
6. Identification of glycosaminoglycan-rich collagen peptides
6.1 determination of glycosaminoglycan content of glycosaminoglycan-rich collagen peptide
The glycosaminoglycan content was measured to be 4.0g/100g with reference to the light industry Standard QB/T4576-2013 of the people's republic of China.
6.2 structural analysis of glycosaminoglycans contained in glycosaminoglycan-rich collagen peptides
The results of the nuclear magnetic resonance spectroscopy analysis of the glycosaminoglycan-rich collagen peptide are shown in figure 1. Wherein the diagrams A and B are respectively 1 HNMR and 13 c NMR and corresponding spectral peak assignments. The result shows that the glycosaminoglycan in the glycosaminoglycan-rich collagen peptide prepared by the invention is mainly sulfated glycosaminoglycan formed by connecting glucuronic acid with N-acetylglucosamine or N-acetylgalactosamine.
6.3 molecular weight distribution of Polypeptides contained in glycosaminoglycan-rich collagen peptides
The high-efficiency gel exclusion chromatography analysis is utilized, and substances with molecular weight less than 3.5kDa in the molecular weight distribution of the polypeptide contained in the glycosaminoglycan-rich collagen peptide of the present inventionThe ratio exceeds 90%. By high performance gel exclusion chromatography (shown in FIG. 2), the standard curve is obtained by taking the logarithm of the molecular weight of the standard substance and the retention time as standard curve, and the regression equation of the standard curve is y= -0.18997x+7.1197 (R 2 = 0.9555), the average molecular weight of the analyzed sample is 827.29Da, below 1500Da, consistent with the characteristics of small molecule peptides.
6.4 identification of characteristic Polypeptides in glycosaminoglycan-rich collagen peptides
The mass spectrum detection is carried out on the glycosaminoglycan-rich collagen peptide obtained by the invention, then the Data Dependency Acquisition (DDA) is carried out on a mass spectrum method, and the data acquired by the mass spectrum is searched in a software Protein Pilot, wherein the searched library is a collagen library downloaded from NCBI websites. And (3) carrying out data processing by using MaxQuant software, and detecting the generated result against a database in UNIPOT. The results show that the reliability of peptide fragments of the glycosaminoglycan-rich collagen peptide is higher than 95%, wherein 11 peptide fragments modified by N-glycosylation modification are identified, and the characteristic glycosylated collagen peptide contained in the glycosaminoglycan-rich collagen peptide is shown in table 1.
TABLE 1 characteristic glycosylated collagen peptide contained in glycosaminoglycan-rich collagen peptide obtained in example 1
The results based on signal intensity show that the characteristic peptide segment of the glycosaminoglycan-rich collagen peptide is GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF.
Test example: the activity detection of the glycosaminoglycan-rich collagen peptide comprises the following steps:
test example 1. Skin soothing, improvement of sensitive skin (antiallergic) efficacy test
The test uses 300mJ/cm 2 Dosage of UVB radiationThe soothing efficacy of the product of the invention was evaluated by measuring the change in the levels of pro-inflammatory factors (IL-6, IL-8, TNF-a) after the action of the sample against keratinocytes (lot number Ep220707, supplied by Guangdong Boxi Biotechnology Co., ltd.).
The main reagent comprises:
KC2500 culture (Guangdong Boxi organism), PBS (Soy pal), dexamethasone (Sigma), IL-6ELISA kit (Abeam), IL-8ELISA kit (Abeam), TNF-a ELISA kit (Abeam)
The testing method comprises the following steps:
1) Cell inoculation: according to 2.2X10 5 Seeding density of individual/well keratinocytes were seeded into 6-well plates and incubated overnight in an incubator;
2) Preparing test object working solution according to the test group (see table 2);
table 2 skin antiallergic test group
3) According to the test group in Table 1, when the cell plating rate in the 6-hole plate reaches 40% -60%, group drug administration is carried out, each hole is added with 2mL of sample, and each group is provided with 3 compound holes. After the completion of the administration, the 6-well plate was placed in an incubator (37 ℃, 5% CO) 2 ) Is cultured for 24 hours.
4) UVB irradiation: according to the test group, the groups with UVB irradiation were subjected to 300mJ/cm 2 Is irradiated with UVB.
5) ELISA detection: after the irradiation, cell culture supernatant was collected and ELISA was performed according to ELISA kit instructions.
6) Results statistical analysis: graphPad Prism was used to map and the results were expressed as mean±sd. The comparisons between groups were analyzed using F-test statistics. Statistical analysis was double tailed. PV <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.
Test results
The skin antiallergic test results are shown in Table 3. Compared to BC group, significance is denoted by #, P-value <0.05 is denoted by #, and P-value <0.01 is denoted by #; compared to NC groups, significance is expressed as x, P-value <0.05 is expressed as x, and P-value <0.01 is expressed as x.
TABLE 3 skin antiallergic test results
The results show that: the glycosaminoglycan-rich collagen peptide product of the invention can obviously inhibit the expression of inflammatory factors IL-6, IL-8 and TNF-a in skin cells, and has the effects of relieving skin, improving skin sensibility and resisting allergy.
Test example 2. Detection of skin aging and skin repair Capacity
2.1 anti-skin aging detection
The test uses 30J/cm 2 The anti-aging efficacy of the sample to be tested is evaluated by measuring changes in the viability of type I Collagen (Collagen I), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) by irradiating fibroblasts with a dose of UVA.
The cells used in this test were fibroblasts, lot number: fbl9052002 by Guangdong Boxi Biotechnology Co.
DMEM broth (Gibco), PBS (solebao), vitamin E (VE, sigma), collagen I ELISA kit (Cusabio), SOD kit (bi yun), CAT kit (south kyo build), GSH-Px kit (south kyo build).
The testing method comprises the following steps:
1) Cell inoculation: according to 2.2X10 5 Seeding density of individual/well keratinocytes were seeded into 6-well plates and incubated overnight in an incubator;
2) Preparing test object working solution according to the test group (see table 4);
table 4 anti-skin aging test group
3) And (3) carrying out drug administration, namely carrying out group drug administration according to test groups when the cell plating rate in the 6-hole plate reaches 40% -60%, and adding 2mL of sample into each hole, wherein 3 compound holes are formed in each group. After the completion of the administration, the 6-well plate was placed in an incubator (37 ℃, 5% CO) 2 ) Is cultured for 24 hours.
4) UVA irradiation: according to the test group, UVA irradiation is carried out on the group needing irradiation, and the irradiation dose is 30J/cm 2 . After the irradiation, the mixture was placed in an incubator (37 ℃ C., 5% CO) 2 ) The culture was continued for 24 hours.
5) ELISA detection: after the irradiation, cell culture supernatant was collected and ELISA was performed according to ELISA kit instructions.
6) HPLC detection: after digestion of the cells treated under different conditions, they were transferred to 1.5mL centrifuge tubes, each tube was charged with 500uL of 0.2mg/mL protein kinase K, and placed in a 50℃water bath at lh. Adding 250uL of methanol into each tube, and performing ultrasonic treatment for 30min; then, the mixture was centrifuged at 14000rpm for lO min. Evaporating methanol at 60deg.C, and storing at 4deg.C for preparation.
7) And (3) enzyme activity detection: after the UVA irradiation is finished, collecting cell culture supernatant, and detecting according to the corresponding kit operation instructions of SOD, CAT and GSH-Px.
8) Results statistical analysis: graphPad Prism was used to map and the results were expressed as mean±sd. The comparisons between groups were analyzed using F-test statistics. Statistical analysis was double tailed. PV <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.
The results of the anti-skin aging test are shown in Table 5. Compared to BC group, significance is denoted by #, P-value <0.05 is denoted by #, and P-value <0.01 is denoted by #; compared to NC groups, significance is expressed as x, P-value <0.05 is expressed as x, and P-value <0.01 is expressed as x.
TABLE 5 anti-skin aging test results
In addition, the invention also takes the commercial collagen peptide as a sample, tests the influence of the commercial collagen peptide on MDA in UVA-induced aging cells, the inhibition rate of the commercial collagen peptide on MDA is 30.96%, and the inhibition effect of the product of the invention is obviously different from that of the commercial collagen peptide.
Furthermore, the effect of the product of the invention on the proliferation capacity of cells is also measured, and the proliferation capacity of aged cells is also influenced.
2.2 detection of skin repair Capacity
SLS (sodium dodecyl sulfate) stimulated epidermis model (EpiKutis 3D epidermis model) was constructed, and then HE staining and IF (immunofluorescence) were used to detect skin cell conditions and expression of skin barrier associated proteins (FLG, LOR) to assess the reparative ability of the product of the invention to skin. The skin repair capability test group is shown in table 6.
Table 6 skin repair capability test packet
The skin repair capability test results are shown in fig. 3 and 4, wherein the HE staining results for the skin epidermis repair capability are shown in fig. 3; the IF staining for the relevant proteins in the epidermis of the skin is shown in FIG. 4.
As can be seen from HE staining and IF immunofluorescence staining, compared with BC group, NC group was stimulated by SLS, the stratum corneum was loose and thickened, the living cell layer was damaged, and cavitation appeared, which indicates that the stimulation condition of this test was effective. Compared with NC group, PC group has clear structure boundary, compact arrangement of living cell layer, loose and thickened horny layer, and obviously improved damage condition of living cell layer, which shows that the test detection system is effective. Compared with NC group, the product (sample 1) can also obviously improve the loose thickening of the stratum corneum and the damage of living cells, so that the product can play a role in repairing skin and restoring cell activity and rebuild skin barrier.
Thus, the product of the invention can increase the amount of type I collagen and Catalase (CAT) in cells, superoxide dismutase (SOD), and greatly inhibit the generation of Malondialdehyde (MDA) in cells without affecting glutathione peroxidase (GSH-Px). The product of the invention plays an anti-aging role through an anti-oxidation pathway, and the inhibition of Malondialdehyde (MDA) is obviously superior to that of the collagen peptide sold in the market. Meanwhile, the product provided by the invention can quickly stimulate cell proliferation to play roles in proliferation, repair and anti-aging. The product of the invention can also obviously improve the loose thickening of the horny layer and the damage condition of living cells, and has the effects of repairing and restoring the cell activity of the skin and reconstructing the skin barrier.
Test example 3 efficacy test for skin moisture recovery, skin elasticity improvement, skin wrinkle reduction
The cells used in this test were keratinocytes, lot number: ep220707, available from guangdong bosch biotechnology limited.
The main reagent comprises: KC2500 (guangdong bosch organism), PBS (solebao), paraformaldehyde (Biosharp), AQP3 antibody (Abcam), CD44 antibody (Abcam), ZO-1 antibody (protentitech), CLDN1 antibody (protentitech), WY14643 (Sigma).
The testing method comprises the following steps:
1) Cell inoculation: according to 5X 10 4 Inoculation Density of individual wells keratinocytes were inoculated into 24 well plates, incubator (37 ℃, 5% CO) 2 ) Incubate overnight.
2) Preparing liquid: test working solutions were prepared separately from the test groups (see table 7).
TABLE 7 test packets for restoring skin moisture, improving skin elasticity, reducing skin wrinkles
3) Administration: according to the test grouping, when the cell plating rate in the 24 pore plates reaches 40% -60%, grouping drug administration is carried out, and 3 compound pores are arranged in each group. After completion of the administration, the 24-well plate was placed in an incubator (37 ℃, 5% co 2) and cultured for 24 hours.
4) UVB irradiation: after PBS washing the cells, the groups with UVB irradiation were subjected to 300mJ/cm according to the test group 2 Is not shown.
5) Post-incubation: after washing the cells with PBS, the 24-well plate was placed in an incubator (37 ℃ C., 5% CO) 2 ) Incubation was performed for 24h after the medium.
6) Immunofluorescence detection: fixation with 4% paraformaldehyde was performed for 30min, after which immunofluorescence detection was performed, photographed by a fluorescence microscope and analyzed using Image-proslus Image processing software.
7) Results statistical analysis: graphPad Prism was used to map and the results were expressed as Mean SD. Comparisons between groups were performed using t-test statistical analysis. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.
Immunofluorescence of AQP3 expression for different groups is shown in fig. 5; immunofluorescence for different sets of CD44 expression is shown in fig. 6; immunofluorescence maps of ZO-1 expression for the different groups are shown in FIG. 7; immunofluorescence of CLDN1 expression for different groups is shown in fig. 8.
The results of the skin moisture recovery, skin elasticity improvement, and skin wrinkle reduction test are shown in Table 8. Compared to BC group, significance is denoted by #, P-value <0.05 is denoted by #, and P-value <0.01 is denoted by #; compared to NC groups, significance is expressed as x, P-value <0.05 is expressed as x, and P-value <0.01 is expressed as x.
TABLE 8 test results for restoring skin moisture, improving skin elasticity, reducing skin wrinkles
CLDN1 is a tight junction related gene, tight junctions between cells are critical to maintaining permselective barrier function of epidermal cells. Furthermore, the invention also tests the effect of the product of the invention and commercial collagen on CLDN1 in glial forming cells without UVB stimulation, using the same immunofluorescence method as described above, and the results show that the expression enhancement rate of the invention on CLDN1 protein in keratinocytes is 31%, whereas the enhancement rate of commercial collagen is 22%, with a significant difference (P-value < 0.05).
The main function of MMP (matrix metalloproteinase) is to degrade collagen, and after MMP content is reduced, the degradation degree of collagen can be inhibited, thereby achieving anti-wrinkle effect. COL-1 (type I collagen) is a major constituent of collagen in the dermis layer of the skin, accounting for about 80% of the total composition, and participates in mechanical support of the skin, and the content of type I collagen is significantly reduced when the skin ages or is subjected to stress aging. Elastin (Elastin) is the major protein that makes up Elastin, and is reduced in content, resulting in less elastic fiber and less skin elasticity. The elastic fiber is composed of microfibril and elastin, has elasticity but poor toughness, and is crosslinked with collagen fiber to keep the elasticity and toughness of skin. At the molecular level, the immediate response can be attributed to elastic fibers in the dermis, the delayed response can be attributed primarily to collagen fibers, and the extracellular matrix consisting primarily of proteoglycans and glycosaminoglycans. Hyaluronic acid is the main component of the extracellular matrix of skin. Among the many biological functions, hydration is one of the functions of hyaluronic acid, which determines the viscoelasticity of the skin. The hyaluronate synthase is key to HA synthesis, and there are 3 major synthetases in the skin, HAs1, HAs2 and HAs3, respectively. Thus, the present invention also tested at UVA 30J/cm using fibroblasts 2 Under the condition of stimulation, the effect of adding the product of the invention on COL-1, elastin, HAS1, HAS2, HAS3, MMP-1 and MMP-3 proteins in cells is shown in Table 9.
TABLE 9 influence of COL-1, elastin, HAS1, HAS2, HAS3, MMP-1, MMP-3 proteins in cells
As a result, it was found that the sample group to which the product of the present invention was added was able to increase the amount of COL-1, elastin, and hyaluronic acid synthase and to increase the inhibition of MMP-1, MMP-3 degradation, compared with NC, thereby retaining collagen and hyaluronic acid in the skin for anti-wrinkle effect.
Test example 4: skin moisture recovery, skin elasticity improvement, skin wrinkle reduction human body test comparative evaluation:
120 volunteers 35-45 years old meeting the test requirements are selected as test subjects, the volunteers are randomly divided into 4 groups, 30 of each group are respectively referred to the evaluation methods of T/ZHCA 003-2018 cosmetics influence skin moisture loss test method, T/ZHCA 005-2019 cosmetics influence skin elasticity test method and T/ZHCA 006-2019 cosmetics anti-wrinkle efficacy test method, the data of moisture, elasticity, wrinkles and the like of the skin of a subject are respectively measured as reference values before the intervention of the products are given, then the skin state values of the subject are measured by the same method after the intervention of the corresponding products for 30 days, the skin moisture, elasticity and wrinkle change values of the same subject are subjected to differential analysis by adopting statistical software, the scores are carried out according to the significance of the statistical results of each group, the main grading standard of the test subject formulas is shown in a table 10, and the corresponding grading standard is shown in a table 11.
TABLE 10 major differences in subject formulation
Table 11 table of significance difference scores for statistical results
The skin moisture, elasticity and wrinkle change scores before and after each group of subjects had been intervened for 30 days after the intervention with the corresponding products are shown in table 12.
TABLE 12 skin moisture, elasticity and wrinkle Change scoring results before and after intervention
As can be seen from the scoring results in table 12, the skin moisture, skin elasticity and skin wrinkle changes of the group tested with the conventional collagen peptide were not significantly different after the intervention of 3g/d for 30 days, and the overall score was 1 score; the skin moisture changes of the group tested by taking the novel collagen peptide are obviously different, the comprehensive score is 4 points, which shows that the novel collagen peptide can obviously improve the skin moisture, and meanwhile, the novel collagen peptide has an improvement trend in the effects of increasing the skin elasticity and reducing the skin wrinkles. The skin moisture and skin elasticity values of the population tested by taking the traditional collagen peptide are remarkably different after the intervention of 5g/d of the collagen peptide for 30 days, the comprehensive score is 5 points, but the skin wrinkles are reduced without remarkable difference; the skin moisture, skin elasticity and skin wrinkles of the tested group of people taking the novel collagen peptide have obvious differences, and the comprehensive score is 9 minutes, so that the novel collagen peptide can obviously reduce the loss of skin moisture, increase the skin elasticity and reduce the skin wrinkles.
In summary, the product of the invention can promote the expression of aquaporin AQP3 and claudin (ZO-1 and CLDN 1) in cells, is helpful for the absorption and maintenance of water, and has limited influence on the hyaluronic acid receptor CD44, namely, the purpose of improving the skin is achieved by not increasing the absorption of hyaluronic acid. The product of the invention is significantly superior to the commercially available collagen peptide in terms of the promotion of the claudin 1. The sample group of the product can increase the amount of COL-1, elastin and hyaluronic acid synthetase, and increase the inhibition of MMP-1 and MMP-3 degradation, thereby retaining collagen and hyaluronic acid in skin and playing an anti-wrinkle role. The glycosaminoglycan-rich collagen peptide can be applied to preparing an anti-skin allergy product, an anti-skin aging product, a skin repair product and a product for recovering skin moisture, improving skin elasticity and reducing skin wrinkles; the glycosaminoglycan-rich collagen peptide comprises a collagen peptide and a glycosaminoglycan; according to the parts by weight: the collagen peptide is 85-99%, the glycosaminoglycan is 1-15%, and the synergistic effect of multiple components is remarkable in activity in the aspects of improving skin problems and the like, and has good application potential.
It should be understood that the above description is not intended to limit the invention to the particular embodiments disclosed, but to limit the invention to the particular embodiments disclosed, and that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications, adaptations, additions and alternatives falling within the spirit and scope of the invention.
Claims (10)
1. The application of the glycosaminoglycan-rich collagen peptide in preparing the anti-skin allergy product is characterized in that: the glycosaminoglycan-rich collagen peptide is prepared by the following method:
s1, purifying a raw material, wherein the raw material is rich in a type I collagen substance A and a cartilage substance B;
s2, high-pressure liquefaction and fusion: mixing the treated collagen-rich material A rich in type I with the cartilage material B, placing the mixture in a magnetic high-pressure reaction kettle, adding deionized water, and magnetically stirring the mixture to be fully and uniformly mixed; stirring at low speed under 10-30MPa to form collagen-polysaccharide complex;
s3, microwave polymerization: placing the high-pressure liquefied and fused reaction product in a liquid microwave heater, and carrying out microwave polymerization reaction at the power of 100W-600W and the temperature of 20-90 ℃ to strengthen the interaction between collagen and glycosaminoglycan;
s4, composite enzymolysis: cooling the microwave polymerized product to 40-60 deg.c, further controlling the temperature at 50-60 deg.c, adding one or several of alkaline proteinase, trypsin and pepsin in the amount of 0.02-0.2% and enzymolysis for 0.5-1.0 hr; then adding one or more of neutral protease, papain and bromelain, wherein the addition amount of the enzyme is 0.02% -0.2%, and the total enzymolysis time is 3-5h, so as to prepare the glycosaminoglycan-rich collagen hydrolysate containing specific peptide fragments;
the polysaccharide-rich collagen peptide has GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF characteristic peptide fragments.
2. The application of the glycosaminoglycan-rich collagen peptide in preparing the skin aging resistant product is characterized in that: the glycosaminoglycan-rich collagen peptide is prepared by the following method:
s1, purifying raw materials, wherein the raw materials are substances A rich in type I collagen and substances B;
s2, high-pressure liquefaction and fusion: mixing the treated collagen-rich material A rich in type I with the cartilage material B, placing the mixture in a magnetic high-pressure reaction kettle, adding deionized water, and magnetically stirring the mixture to be fully and uniformly mixed; stirring at low speed under 10-30MPa to form collagen-polysaccharide complex;
s3, microwave polymerization: placing the high-pressure liquefied and fused reaction product in a liquid microwave heater, and carrying out microwave polymerization reaction at the power of 100W-600W and the temperature of 20-90 ℃ to strengthen the interaction between collagen and glycosaminoglycan;
s4, composite enzymolysis: cooling the microwave polymerized product to 40-60 deg.c, further controlling the temperature at 50-60 deg.c, adding one or several of alkaline proteinase, trypsin and pepsin in the amount of 0.02-0.2% and enzymolysis for 0.5-1.0 hr; then adding one or more of neutral protease, papain and bromelain, wherein the addition amount of the enzyme is 0.02% -0.2%, and the total enzymolysis time is 3-5h, so as to prepare the glycosaminoglycan-rich collagen hydrolysate containing specific peptide fragments;
the polysaccharide-rich collagen peptide has GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF characteristic peptide fragments.
3. The application of the glycosaminoglycan-rich collagen peptide in preparing skin repair products is characterized in that: the glycosaminoglycan-rich collagen peptide is prepared by the following method:
s1, purifying raw materials, wherein the raw materials are substances A rich in type I collagen and substances B;
s2, high-pressure liquefaction and fusion: mixing the treated collagen-rich material A rich in type I with the cartilage material B, placing the mixture in a magnetic high-pressure reaction kettle, adding deionized water, and magnetically stirring the mixture to be fully and uniformly mixed; stirring at low speed under 10-30MPa to form collagen-polysaccharide complex;
s3, microwave polymerization: placing the high-pressure liquefied and fused reaction product in a liquid microwave heater, and carrying out microwave polymerization reaction at the power of 100W-600W and the temperature of 20-90 ℃ to strengthen the interaction between collagen and glycosaminoglycan;
s4, composite enzymolysis: cooling the microwave polymerized product to 40-60 deg.c, further controlling the temperature at 50-60 deg.c, adding one or several of alkaline proteinase, trypsin and pepsin in the amount of 0.02-0.2% and enzymolysis for 0.5-1.0 hr; then adding one or more of neutral protease, papain and bromelain, wherein the addition amount of the enzyme is 0.02% -0.2%, and the total enzymolysis time is 3-5h, so as to prepare the glycosaminoglycan-rich collagen hydrolysate containing specific peptide fragments;
the polysaccharide-rich collagen peptide has GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF characteristic peptide fragments.
4. The application of the glycosaminoglycan-rich collagen peptide in preparing a product for recovering skin moisture, improving skin elasticity and reducing skin wrinkles is characterized in that: the glycosaminoglycan-rich collagen peptide is prepared by the following method:
s1, purifying raw materials, wherein the raw materials are substances A rich in type I collagen and substances B;
s2, high-pressure liquefaction and fusion: mixing the treated collagen-rich material A rich in type I with the cartilage material B, placing the mixture in a magnetic high-pressure reaction kettle, adding deionized water, and magnetically stirring the mixture to be fully and uniformly mixed; stirring at low speed under 10-30MPa to form collagen-polysaccharide complex;
s3, microwave polymerization: placing the high-pressure liquefied and fused reaction product in a liquid microwave heater, and carrying out microwave polymerization reaction at the power of 100W-600W and the temperature of 20-90 ℃ to strengthen the interaction between collagen and glycosaminoglycan;
s4, composite enzymolysis: cooling the microwave polymerized product to 40-60 deg.c, further controlling the temperature at 50-60 deg.c, adding one or several of alkaline proteinase, trypsin and pepsin in the amount of 0.02-0.2% and enzymolysis for 0.5-1.0 hr; then adding one or more of neutral protease, papain and bromelain, wherein the addition amount of the enzyme is 0.02% -0.2%, and the total enzymolysis time is 3-5h, so as to prepare the glycosaminoglycan-rich collagen hydrolysate containing specific peptide fragments;
the polysaccharide-rich collagen peptide has GSRGEPGPNGAVGPVGPS, GANGDKGEGGSF, ARGPNGYSGPVGPPGPPGLPGPPGPA, SGSPGENGSPGPMGPR, IQVPDEESNGIFAA, RNEEPVLF characteristic peptide fragments.
5. Use according to any one of claims 1 to 4, wherein the collagen-rich material a is fish skin and/or fish scales; the cartilage substance B is edible animal bone and/or connective tissue.
6. The use according to claim 5, wherein the cartilage substance B is cartilage of livestock and/or fish.
7. The use according to any one of claims 1 to 4, wherein the product is a food product, a health product, a dietary supplement, a pharmaceutical product, a cosmetic product.
8. The use according to any one of claims 1 to 4, wherein the glycosaminoglycan-rich collagen peptide comprises a collagen peptide, a glycosaminoglycan.
9. The use according to claim 8, wherein the glycosaminoglycan-rich collagen peptide comprises 85-99% collagen peptide and 1-15% glycosaminoglycan.
10. The use according to claim 8, wherein the collagen peptide of the polysaccharide-rich collagen peptide has an average molecular weight of less than 1500Da.
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