Preparation method and application of anti-photoaging collagen polypeptide

Abstract

The invention discloses a preparation method and application of anti-photoaging collagen polypeptide. The preparation method comprises the following steps: unfreezing and cleaning silver carp skin, airing and cutting; naHCO3 is added, and soaking is carried out; adding Na2CO3 and carrying out ultrasonic impurity removal; performing ultrasonic-assisted treatment; adding protease for enzymolysis treatment; establishing an enzymolysis kinetic model under the alkaline protease condition; standing, cooling and centrifuging the enzymatic hydrolysate, collecting supernate, filtering, and performing vacuum freeze drying to obtain faint yellow SCSCP; the light aging resistant peptide SCSCP02 is separated, and the active peptide sequences of the light aging resistant peptide SCSCP02 are GPPGPPGTPGPQ, SGLPGPIGPPGPR and GLPGPIGPPGPR. The active substances which are extracted from the fish skin and have the anti-photoaging effect can reduce the risk of skin light damage caused by UVR by acting on various treatment targets, resource waste can be reduced, environmental pollution caused by biological resources can be relieved, and sustainable development of the industry can be promoted.

Description

Technical Field

[0001] The present invention relates to the field of collagen peptide preparation, and particularly to a preparation method and application of anti-photoaging collagen polypeptide. Background Art

[0002] Silver carp is one of the important economic freshwater fish species in Asian countries. Silver carp is not only an excellent edible fish, but also an important fishery resource, with extensive cultivation and utilization worldwide. Fish skin is the main by-product of fish processing, containing rich collagen, and is a good raw material for preparing bioactive collagen polypeptide. Utilizing the by-products generated by the aquatic product processing industry to isolate bioactive compounds beneficial to human health can effectively improve its added value and reduce the problem of resource waste.

[0003] Collagen polypeptide is a small molecular peptide substance obtained by cleaving proteins at specific amide bonds, with a molecular weight usually below 5000 Da, and has the ability to inhibit protein-protein interactions. Collagen polypeptide can enter the blood circulation through peptide transport systems such as active transport and non-energy-consuming exchange transport, with low or zero energy consumption; with the help of the peptide transport system, peptide segments containing 2-5 amino acid residues are more easily absorbed by the human body than free amino acids. Collagen polypeptide has been reported to have the functions of improving skin aging, preventing and treating osteoporosis and type II diabetes. In addition, it also has biological activities such as blood pressure lowering, immune regulation, antibacterial, antioxidant and anti-tumor, and is mostly applied in the fields of food additives, skin care formulations and medical treatments.

[0004] The commonly used methods for preparing collagen polypeptide at present are: hot water extraction method, alkali extraction method, acid extraction method, enzymatic hydrolysis method and composite extraction method. The hot water extraction process will cause irreversible denaturation of proteins, resulting in the breakage of the α-chain that maintains the collagen structure. Acid and alkali extraction methods belong to chemical hydrolysis methods, mainly by using chemical reagents to break the peptide chains of protein molecules to prepare collagen polypeptide; the alkali extraction method may cause certain damage to the biological activity of the polypeptide; the acid extraction method has a violent reaction, and the operation conditions during extraction should be strictly controlled, and at the same time, phenomena such as secondary pollution should be avoided. Peptides exist in the protein chain in an inactive form, and enzymes are used to cleave the covalent bonds between the terminal peptide chains of collagen, and the main body still maintains a triple helix structure. Due to the advantages of high product yield, safety, convenience and controllable reaction conditions, the enzymatic hydrolysis method has become the main method for preparing collagen polypeptide, and the differences in raw materials and protease types make the biological activities and effects of collagen polypeptide different.

[0005] It has been reported that the extracts and their collagen polypeptides isolated from silver carp skin have various biological activities and have great application potential in the health food and cosmeceutical industries. With the development and use of bioactive peptides, the impact on health care caused by bad living habits and population aging in the future may be improved.

[0006] In recent years, the research on anti-photoaging collagen polypeptides has become increasingly mature. Research shows that these active substances with anti-photoaging effects extracted from various animals and plants can reduce the risk of skin photo-damage caused by UVR by acting on various therapeutic targets. Therefore, researchers and the industry have begun to study natural active ingredients based on animals and plants.

[0007] This invention starts from the waste fish skin of silver carp, prepares collagen polypeptides with anti-photoaging activity by optimizing the enzymatic hydrolysis process, and explores their structural characterization and mechanism of action. The development and utilization of aquatic by-products can not only reduce resource waste, alleviate environmental pollution caused by biological resources, but also promote the sustainable development of the industry. Summary of the Invention

[0008] In view of this, this invention provides a preparation method and application of anti-photoaging collagen polypeptides.

[0009] To solve the above technical problems, this invention adopts the following technical solutions:

[0010] A preparation method of anti-photoaging collagen polypeptides, comprising the following steps:

[0011] Step S1: After thawing the silver carp skin, wash it repeatedly, remove the remaining fish scales and fish meat, and cut it after drying in the sun;

[0012] Step S2: Add NaHCO3 and soak; then add Na2CO3 and perform ultrasonic impurity removal, change the solution regularly, wash and dry for later use;

[0013] Step S3: Take the pretreated fish skin and add distilled water for ultrasonic-assisted treatment;

[0014] Step S4: Adjust the pH value, add protease for enzymatic hydrolysis treatment, and regularly adjust the pH of the enzymatic hydrolysis solution; after the enzymatic hydrolysis ends, boil in boiling water to terminate the enzyme activity;

[0015] Step S5: After the enzymatic hydrolysis solution is allowed to stand and cool, centrifuge at low temperature, collect the supernatant in the centrifuge tube, filter to remove impurities, take the filtered solution through a microporous filter membrane, and obtain light yellow SCSCP after vacuum freeze-drying. It has collagenase inhibitory ability, and the α-helix and β-sheet structures account for 17.70% and 22.65% respectively, and a stable α-helix structure is still retained during the enzymatic hydrolysis process;

[0016] Step S6: Use an ultrafiltration separation device and an ultrafiltration membrane with a molecular weight cut-off of 1-5 kDa to separate SCSCP;

[0017] Among them, the operating conditions of the ultrafiltration membrane separation equipment are: 0.06 Mpa, 25 °C, and the solution pH is maintained at 7.0 - 7.5; SCSCP02 is obtained; its molecular mass-to-charge ratio ranges from 636.14 m / z to 1541.43 m / z; 0.1 mg / mL SCSCP02 can reduce the ROS production induced by UVB from 364.92% to 232.41%, and the inhibition rate of MMP-1 reaches over 70%;

[0018] Step S7: Through mass spectrometry identification and database matching, three novel anti-photoaging peptides, GPPGPPGTPGPQ, SGLPGPIGPPGPR, and GLPGPIGPPGPR, are obtained. They all come from type I collagen α1 and have antioxidant, antithrombotic, α-glucosidase inhibitory, and DPP-IV inhibitory potentials.

[0019] Preferably, in step S2, 0.5% NaHCO3 is added at a solid-liquid ratio of 1:30, and 0.6 mol / L Na2CO3 is added at a solid-liquid ratio of 1:50.

[0020] Preferably, in step S2, the soaking time is 8 h; the ultrasonic impurity removal time is 12 h, and the solution is changed every 6 h.

[0021] Preferably, in step S3, ultrasonic-assisted heating is carried out at 80 °C for 30 min.

[0022] Preferably, in step S4, the protease is alkaline protease, and it is adjusted to the optimal pH of the protease using 1 mol / L HCL; the pH of the enzymatic hydrolysate is adjusted every 0.5 h.

[0023] Preferably, in step S4, the kinetic model of alkaline protease enzymatic hydrolysis is: DH = 3.839 ln[1 + (16.055E0 / S0 - 0.081)t], and the reaction rate R = (61.632E0 - 0.3109S0) exp[-0.2605(DH)];

[0024] where b = 0.2605, a = 61.632E0 / S0 - 0.3109, k d = 61.632 min -1 , f = 5.04×10 -3 ; at the same time, the enzyme inactivation kinetic constant k1 = 16.645 min during the alkaline protease hydrolysis of silver carp fish skin is obtained -1 ; the actual results fit well with the predicted values of this model.

[0025] Preferably, in step S5, low-temperature centrifugation is carried out at 4500 r / min for 30 min at 4 °C.

[0026] Preparation method of an anti-photoaging collagen polypeptide and application of the polypeptide in anti-skin photoaging

[0027] The present invention has achieved the following technical effects compared with the prior art:

[0028] (1) The preparation method of the anti-photoaging collagen polypeptide and the enzymatic hydrolysis kinetic model established by the present invention can specifically predict the reaction process between the enzyme and the substrate, improve the enzymatic hydrolysis rate, and reduce the industrial production cost;

[0029] (2) The active substance with anti-photoaging effect extracted from fish skin by the present invention can reduce the risk of skin photo-damage caused by UVR by acting on various therapeutic targets;

[0030] (3) The present invention can not only reduce the waste of resources, alleviate the environmental pollution caused by biological resources, but also promote the sustainable development of the industry. Brief Description of the Drawings

[0031] Figure 1 is the preparation flow chart of the present invention;

[0032] Figure 2 is the influence diagram of different proteases on the extraction of silver carp skin collagen polypeptide and the inhibition of collagenase activity in the present invention;

[0033] Note: Different lowercase letters indicate significant differences in peptide yield among components (p<0.05), and different uppercase letters indicate significant differences in collagenase activity inhibition rate among components (p<0.05);

[0034] Figure 3 (a) is the influence diagram of the solid-liquid ratio on the peptide yield and the collagenase activity inhibition rate in the present invention; Figure 3 (b) is the influence diagram of the enzymatic hydrolysis time on the peptide yield and the collagenase activity inhibition rate; Figure 3 (c) is the influence diagram of the enzyme addition amount on the peptide yield and the collagenase activity inhibition rate;

[0035] Note: Different lowercase letters indicate significant differences in peptide yield among components (p<0.05), and different uppercase letters indicate significant differences in collagenase activity inhibition rate among components (p<0.05);

[0036] Figure 4 is the response surface result diagram of the present invention; among them, (a) is the interaction between the enzyme addition amount and the solid-liquid ratio, (b) is the interaction between the enzyme addition amount and the enzymatic hydrolysis time, and (c) is the interaction between the enzymatic hydrolysis time and the solid-liquid ratio;

[0037] Figure 5 Variation trend diagram of the hydrolysis degree of silver carp skin under different initial substrate concentrations;

[0038] Figure 6Variation trend chart of the hydrolysis degree of silver carp skin at different initial enzyme concentrations;

[0039] Figure 7 Trend chart of the value changing with different E0 / S0 values;

[0040] Figure 8 Trend chart of R changing with time during enzymatic hydrolysis;

[0041] Figure 9 Trend chart of the ab value changing with different E0 / S0 values;

[0042] Figure 10 Kinetics model verification chart;

[0043] Among them, (a) is the comparison chart between the predicted DH and the actual DH of the model, and (b) is the correlation chart between the predicted DH value and the actual DH value of the model;

[0044] Figure 11 SCSCP diagram of different components of the present invention; among them, (a) is the SCSCP01 component (MW < 3 kDa), (b) is the SCSCP02 component (3 kDa < MW < 5 kDa), and (c) is the SCSCP03 component (MW > 5 kDa);

[0045] Figure 12 Molecular weight distribution diagram of different components of SCSCP of the present invention; among them, (a) is the SCSCP01 component (MW < 3 kDa), (b) is the SCSCP02 component (3 kDa < MW < 5 kDa), and (c) is the SCSCP03 component (MW > 5 kDa);

[0046] Figure 13 Diagram of the influence of each component of SCSCP of the present invention on the activity of L929 cells; among them, (a) is the cell viability of the non-irradiated group, and (b) is the cell viability of the UVB-irradiated group;

[0047] Note: $ p < 0.05, $$$ p < 0.001, and $$$$ p < 0.0001 vs; normal cell group, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. UVB group, ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001 and ^^^^ p < 0.0001 vs. 0.1 mg / mL SCSCP treatment group, #p < 0.05 indicates a significant difference between the two connected groups of line segments;

[0048] Figure 14 This is a graph showing the effects of each component of the present invention's SCSCP on the ROS content induced by UVB; among them, (a) is a graph showing the relationship between the number of cells and the fluorescence intensity detected by DCFH-DA, and (b) is a histogram of the changes in ROS in cells;

[0049] Note: ^ p < 0.05, ^^^ p < 0.001 and ^^^^ p < 0.0001 vs; normal cell group, *** p < 0.001 and **** p < 0.0001 vs. UVB-treated group, # p < 0.05 indicates a significant difference between the two connected groups of line segments;

[0050] Figure 15 This is a graph showing the effects of each component of the present invention's SCSCP on the MMP-1 content induced by UVB;

[0051] Note: ^ p < 0.05, ^^^ p < 0.001 and ^^^^ p < 0.0001 vs; normal cell group, *** p < 0.001 and **** p < 0.0001 vs. UVB-treated group, # p < 0.05 indicates a significant difference between the two connected groups of line segments;

[0052] Figure 16 This is the peptide segment matching map and its structural diagram identified in the present invention; among them, (a) GPPGPPGTPGPQ, (b) SGLPGPIGPPGPR, (c) GLPGPIGPPGPR. Detailed implementation manners

[0053] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0054] As Figure 1 shown, the present invention discloses a preparation method and application of an anti-photoaging collagen polypeptide, including the following steps:

[0055] Step S1: After thawing the silver carp skin, wash it repeatedly to remove residual fish scales and fish meat. After drying in the sun, cut it into pieces of 1 cm×1 cm size.

[0056] Step S2: Add 0.5% NaHCO3 according to a solid-liquid ratio of 1:30 and soak for 8 h. Then add 0.6 mol / L Na2CO3 according to a solid-liquid ratio of 1:50, and remove impurities by ultrasonic treatment for 12 h. Replace the solution every 6 h. After cleaning, dry it for standby.

[0057] Step S3: Take the pretreated fish skin, add distilled water, and heat it with ultrasonic assistance at 80 °C for 30 min.

[0058] Step S4: Adjust to the optimal pH of the protease with 1 mol / L HCL, add protease for enzymatic hydrolysis treatment, and adjust the pH of the enzymatic hydrolysate every 0.5 h. After the enzymatic hydrolysis is completed, boil it in boiling water for 15 minutes to terminate the enzyme activity.

[0059] Step S5: After the enzymatic hydrolysate is allowed to stand and cool, centrifuge it at 4500 r / min at 4 °C for 30 min. Collect the supernatant in the centrifuge tube, filter to remove impurities, take the filtered solution through a 0.45 μm microporous filter membrane, and obtain light yellow SCSCP after vacuum freeze-drying.

[0060] Step S6: Use an ultrafiltration separation device and ultrafiltration membranes with a molecular weight cut-off of 1 kDa and 3 kDa to separate SCSCP. Among them, the operating conditions of the ultrafiltration membrane separation device are: 0.06 Mpa, 25 °C, and the solution pH is maintained at 7.0 - 7.5; obtain SCSCP02; the molecular mass-to-charge ratio range is 636.14 m / z - 1541.43 m / z; 0.1 mg / mL SCSCP02 can reduce the ROS production induced by UVB from 364.92% to 232.41%, and the inhibition rate of MMP-1 reaches more than 70%.

[0061] Step S7: Through mass spectrometry identification and database matching, obtain 3 novel anti-photoaging peptides GPPGPPGTPGPQ, SGLPGPIGPPGPR, and GLPGPIGPPGPR, which simultaneously have antioxidant, antithrombotic, α-glucosidase inhibitory, and DPP-IV inhibitory potentials.

[0062] Example:

[0063] (I) Protease screening

[0064] Weigh 5 g of silver carp skin, add distilled water according to a solid-liquid ratio of 1:25, with a unified enzyme addition amount of 4000 U / g and an enzymatic hydrolysis time of 6 h, and carry out enzymatic hydrolysis operations under the optimal enzymatic hydrolysis conditions of different proteases. The specific conditions are shown in Table 1.

[0065] Table 1:

[0066]

[0067] As Figure 2 shown, during the protease screening process, there were significant differences in the peptide yield between two different protease groups (p < 0.05), and the same was true for the inhibition rate of collagenase activity (p < 0.05). The highest inhibition rate of collagenase activity in the alkaline protease hydrolysate could reach 15.29%, while the inhibition rates of collagenase activity in the trypsin and neutral protease hydrolysates were 8.94% and 6.32% respectively. After enzymatic hydrolysis with a mixed enzyme, the peptide yield was significantly improved (p < 0.05), and its inhibition rate of collagenase activity was 12.85%, but the effect was still inferior to that of alkaline protease. Collagenase is a metalloprotease, and cysteine in the structural region at its N-terminus is connected to Zn 2+ to form the zinc site in the catalytic domain of the enzyme. During the active process, the active site may be exposed, and some collagenase inhibitors will add zinc chelating substances containing carbonyl, hydroxyl, carboxyl and other types, thereby preventing it from binding to the substrate. The hydrolysate obtained by alkaline protease has a higher inhibition rate of collagenase, probably because the enzyme has more cleavage sites, producing more peptide segments with inhibitory activity against collagenase. Therefore, alkaline protease was selected for enzymatic hydrolysis in subsequent experiments.

[0068] (2) Single-factor experiments

[0069] After determining the optimal protease, the inhibition rate of collagenase activity and the peptide yield were used as screening indicators to investigate the effects of the material-liquid ratio, enzyme dosage, and hydrolysis time on protease hydrolysis. Weighed 2 g of pretreated fish skin material, and under the enzymatic hydrolysis conditions of the optimal protease, any two of the investigated parameters were fixed respectively to study the effects of different material-liquid ratios (1:18, 1:20, 1:22, 1:24, 1:26, g / mL), enzyme dosages (3000, 3500, 4000, 4500, 5000, U / g), and hydrolysis times (4, 5, 6, 7, 8, h) on the inhibitory activity of collagenase and the peptide yield.

[0070] As Figure 3 (a) shows, when the solid-liquid ratio was 1:24, the inhibition rate of collagenase activity reached the highest level of 27.14%, which was significantly different from other groups (p < 0.05). In the range of solid-liquid ratios from 1:22 to 1:26, the inhibition rate of collagenase activity showed a trend of first increasing and then decreasing with the increase of the solid-liquid ratio. When the solid-liquid ratio was too low, the viscosity of the enzymatic hydrolysate was too high, which was not conducive to the full reaction of the enzyme with the substrate.

[0071] As Figure 3As shown in

[0072] (b), the peptide yield of SCSCP increased significantly with the increase of enzymatic hydrolysis time (p<0.05). The inhibition rate of SCSCP on collagenase activity reached the highest of 30.69% at 6 h, and then decreased significantly after 6 h. This may be because within too short enzymatic hydrolysis time, bioactive small peptides have not been completely decomposed, while too long enzymatic hydrolysis time leads to excessive hydrolysis of peptide segments with collagenase activity inhibition, resulting in a decrease in the inhibition rate.

[0072] As Figure 3 As shown in Figure 3 (c), with the increase of enzyme dosage, the peptide yield of SCSCP increased in a dependent manner, while the inhibition rate of collagenase activity showed a trend of increasing first and then decreasing, reaching the maximum value of 39.01% when the enzyme dosage was 4000 U / g. When the enzyme dosage was low, all raw materials could not be converted into enzymatic hydrolysis products, so the inhibition rate of collagenase activity was low; with the increase of enzyme dosage, there was more contact space between raw materials and enzymes, and the enzymatic hydrolysis was more sufficient, producing more substances with collagenase activity inhibition; continuing to increase the enzyme dosage, phenomena such as saturation of the binding sites between enzymes and substrates and competitive inhibition may occur, resulting in a decrease in the inhibition rate of enzymatic hydrolysis products on collagenase activity. Based on the results of the single-factor experiment histograms, the optimal enzymatic hydrolysis conditions were determined as a solid-liquid ratio of 1:24 g / mL, an enzyme dosage of 4000 U / g, and an enzymatic hydrolysis time of 6 h. At this time, the highest inhibition rate of collagenase activity was 39.01%, showing a significant increase compared with the highest inhibition rate of collagenase activity under the enzyme screening conditions.

[0073] (III) Response surface optimization experiment

[0074] Using Design-Expert 10.0.4 software, based on the results of the single-factor experiment, the effects of three controlled independent variables, namely enzyme dosage, material-liquid ratio, and enzymatic hydrolysis time, and their interactions on the inhibitory activity of collagenase were designed and determined to obtain the factor and level table of the response surface experiment design, as shown in Table 2. The effects of each parameter and its interaction on the enzymatic hydrolysis effect were evaluated, and the optimal parameter levels affecting the inhibition rate of collagenase activity were predicted by this method.

[0075] Table 2:

[0076]

[0077] A response surface model design was carried out for the enzymatic hydrolysis process of silver carp skin. The experimental grouping design and the corresponding inhibition rate of collagenase activity are shown in Table 3, and the variance analysis of the regression model is shown in Table 4.

[0078] Table 3:

[0079]

[0080] Table 4:

[0081]

[0082] Note: * indicates p < 0.05, ** indicates p < 0.01, ns indicates not significant

[0083] According to the magnitude of the F values in Table 4, it can be judged that the order of the influence intensity on the inhibition rate of collagenase activity is: enzyme addition amount (A) > solid-liquid ratio (B) > enzymolysis time (C). Perform a quadratic multiple regression model analysis on the data in Table 4, and fit to determine the quadratic polynomial regression equation of the collagenase activity inhibition rate with the enzyme addition amount (A), solid-liquid ratio (B), and enzymolysis time (C):

[0084] Y = 52.89 + 6.30A + 4.74B + 1.50C - 9.38AB - 1.90AC + 1.38BC - 2.63A2 - 8.76B2 - 14.49C2. The P value of the prediction model is significant and the lack-of-fit term is not significant. The R2 of this model = 0.9381 indicates that the model has a good fit and subsequent optimization design can be carried out.

[0085] The interaction of each factor on the collagenase activity inhibition rate is as Figure 4 shown, and the highest point appears on each response surface. The response surface contour map can show the interaction between factors, and the steepness of the curved surface of the three-dimensional response surface map reflects whether the influence is significant. The "P value" of the AB index is less than 0.05, and the AB contour map tends to be elliptical, indicating that there is an interaction between the enzyme addition amount and the solid-liquid ratio. In the three-dimensional response surface map between BC, the enzymolysis time surface is more inclined, and the contour map is more circular. The interaction between BC has no significant influence on the collagenase activity inhibition rate, and the influence of enzymolysis time is more significant than that of the solid-liquid ratio. The interaction between AC has a small influence on the inhibition rate of collagenase activity. Use Design Expert 8.0.6 software to optimize the combination of process parameters, as Figure 4 (a)(b)(c) shown. The optimal process conditions are: enzyme addition amount 4500 U / g, solid-liquid ratio 1:23.47 g / mL, enzymolysis time 5.97 h, and the predicted collagenase activity inhibition rate is 57.19%. In order to verify the effectiveness of the model, combined with the actual situation, the optimal process conditions are modified to: enzyme addition amount 4500 U / g, material-liquid ratio 1:24 g / mL, enzymolysis time 6 h. Under these process conditions, the collagenase activity inhibition rate is 54.47%, and there is no significant difference from the predicted value (p > 0.05), indicating that the optimization effect of this model has a high credibility. It is reported that the inhibition rate of the enzymolysis product from donkey-hide gelatin on collagenase can reach 54.09%, and the inhibition rates of plant extracts such as green tea, lavender, rose water, and pomegranate on collagenase are between 11% - 47%, while the inhibition rates of the polypeptides prepared in this experiment on collagenase are higher than the above substances.

[0086] (4) Establishment and verification of enzymatic hydrolysis kinetic model

[0087] Weigh 2.5 g of silver carp fish skin and perform enzymatic hydrolysis using alkaline protease. Fix the substrate mass concentration at 30 g / L and investigate the variation law of DH during the enzymatic hydrolysis process under different enzyme concentration conditions (0.6, 0.8, 1.0, 1.2, 1.4, mol / L). Fix the enzyme concentration at 1 mL / L and investigate the variation law of DH during the enzymatic hydrolysis process under different substrate mass concentration conditions (10, 20, 30, 40, 50, g / L).

[0088] Effect of initial substrate concentration and initial enzyme concentration on the degree of hydrolysis of SCSCP

[0089] To determine the parameters in the kinetic model, it is necessary to study the effect of initial substrate concentration and initial enzyme concentration on the degree of hydrolysis. Under the conditions of temperature 55 °C and pH 8.5, investigate the situation of alkaline protease catalyzing the hydrolysis to obtain SCSCP under different substrate concentrations and initial enzyme concentrations, as Figure 5 shown. As can be seen from Figure 5 , during the hydrolysis process under different initial substrate concentrations, the DH value increases significantly with the extension of enzymatic hydrolysis time within 0 - 20 min, while the increasing trend of the DH value gradually flattens after 20 min; the DH value shows a dose-dependent inhibition trend with different substrate concentrations, that is, the greater the initial substrate concentration, the lower the DH value of the reaction process and at the saturation point. The DH value of the substrate concentration of 10 g / L reaches the highest 17.74% at 120 min. This may be because the viscosity of the reaction system increases with the increase of concentration, slowing down the diffusion of the substrate to the enzyme active center and resulting in the loss of enzyme activity; as the enzymatic hydrolysis time increases, the enzymatic hydrolysis sites in the substrate gradually reach saturation. When the substrate concentration is fixed at 30 g / L, the DH value of silver carp fish skin under different initial enzyme concentrations increases rapidly within the first 20 min. As the enzymatic hydrolysis time increases, the growth of the DH value begins to slow down and gradually tends to the saturation value, and the DH value increases with the increase of the initial enzyme concentration. As shown in Figure 6 , the DH of silver carp fish skin can reach 17.66% under the condition of an initial enzyme concentration of 1.4 mol / L. The reason for this phenomenon may be that the deconstruction and unfolding of proteins are greater, and the contact area between the enzyme and the substrate is larger, thereby increasing the degree of subsequent protein hydrolysis.

[0090] Determination of kinetic model parameters

[0091] During the process of alkaline protease hydrolyzing to obtain SCSCP, use the nonlinear regression model of SPSS software to calculate the DH data of different initial substrate concentrations and protease concentrations, and thus obtain the corresponding kinetic parameters a and b during the process. The results are shown in Table 5.

[0092] Table 5

[0093]

[0094] As can be seen from Table 5, the kinetic parameter b changes little with the change of E0 or S0, and the value is close to a constant, fluctuating slightly around its average value of 0.2605. Therefore, on the premise of a constant temperature system, b can be approximately regarded as a constant, that is, b = 0.2605. This is consistent with the kinetic model derivation in the above text. The kinetic parameter a shows a concentration-dependent inhibitory relationship with the initial substrate concentration, while showing a concentration-dependent increasing trend with the initial enzyme concentration. According to the equation derivation, during the enzymatic hydrolysis process at a constant temperature, the value of a is linearly related to the ratio of the initial protease concentration to the initial substrate concentration (E0 / S0). Therefore, with E0 / S0 as the abscissa and the value of a as the ordinate, linear fitting is performed on the relevant data to obtain the a-E0 / S0 change trend diagram as shown in Figure 7 Figure 6, and the slope is the enzymatic hydrolysis reaction rate constant k d .

[0095] The equation corresponding to the a-E0 / S0 linear relationship can be obtained from the figure:

[0096] a = 61.632E0 / S0 - 0.3109, and solving for k d = 61.632 min -1 , f = 5.04×10 -3

[0097] Substituting the kinetic parameters a and b into the equation respectively, the kinetic model of SCSCP is obtained as:

[0098] DH = 3.839ln[1 + (16.055E0 / S0 - 0.081)t]

[0099] R = (61.632E0 - 0.3109S0)exp[-0.2605(DH)]

[0100] Enzymatic hydrolysis reaction rate:

[0101] The reaction rate R of protease hydrolyzing silver carp skin is related to both the initial protease concentration and the substrate concentration. It increases with the increase of protease concentration and decreases with the increase of substrate concentration; as the reaction proceeds, R will decrease with the increase of the DH value, which is also consistent with the results of Figure 5 . Under the enzymatic hydrolysis conditions of fixing E0 = 1.0 mL / L and S0 = 30 g / L, substituting the DH values measured at different enzymatic hydrolysis times t into the equation, the fitting curve of t and the enzymatic hydrolysis rate R is obtained, as shown in Figure 7 .

[0102] Figure 8During the enzymatic hydrolysis process, the changing trend of R gradually decreases with the increase of t. The reaction rate R reaches its maximum value at the initial stage. When hydrolyzed for 80 min, R is 0.98 mol / (L·min), and at this time the reaction rate is as low as below 1 mol / (L·min); at 120 min, R is only 0.66 mol / (L·min), and the enzymatic hydrolysis reaction tends to the termination state. The decrease in the enzymatic hydrolysis rate may be caused by enzyme inactivation and substrate differentiation.

[0103] Derivation of the protease inactivation constant:

[0104] By plotting the value of E0 / S0 on the abscissa and the ab value on the ordinate, we get Figure 9 and the linear relationship equation of ab - E0 / S0 can be obtained. Substituting the kinetic parameters The kinetic parameter b = k i k M / k d Multiplying them gives:

[0105]

[0106] The slope is the inactivation constant of alkaline protease during the hydrolysis process, that is, k1 = k i ×k M .

[0107] Performing linear fitting on the ab value and gives: The equation corresponding to the relationship curve between the ab value and the E0 / S0 value is:

[0108] ab = 16.645E0 / S0 + 0.0391

[0109] From this, the kinetic constant k1 of alkaline protease inactivation is solved as k1 = 16.645 min -1 . According to the trend of the graph, the higher the initial enzyme concentration, the greater the inactivation rate. This is because in an environment with a high enzyme concentration, more enzymes attached to the silver carp skin and the products generated are also more, and the activity of the protease is correspondingly lower.

[0110] Verification of the enzyme kinetic model:

[0111] To confirm the rationality of the model, we carried out three repeated enzymatic hydrolyses at an initial substrate concentration of 30 g / L and a protease concentration of 1.0 mL / L, and recorded the DH value. Comparing the model prediction results with the actual results, we obtained the differences as shown in Figure 10 . Verify the practical application significance of the kinetic model.

[0112] As Figure 10As shown in (a), both the actual and predicted DH values showed a rapid growth trend at the initial stage of enzymatic hydrolysis, then gradually slowed down, and the actual and predicted values were in good agreement. This indicates that at the initial stage of enzymatic hydrolysis, alkaline protease was in full contact with silver carp skin collagen, and the hydrolysis rate increased sharply; thereafter, due to the massive consumption of protein, the hydrolysis rate decreased. As Figure 10 shown in (b), it shows a linear relationship between the actual DH value and the model prediction result, and the correlation coefficient R 2 = 0.9847. It can be seen that the model fitting degree is good and the relative error is low, which indicates that the established kinetic model of enzymatic hydrolysis can accurately describe the enzymatic hydrolysis process and has good practical application value.

[0113] All in all, based on the experimental results of alkaline protease enzymatic hydrolysis, the kinetic model of its enzymatic hydrolysis reaction was obtained: DH = 3.839ln[1 + (16.055E0 / S0 - 0.081)t], and the reaction rate R = (61.632E0 - 0.3109S0)exp[-0.2605(DH)]. Among them, b = 0.2605, a = 61.632E0 / S0 - 0.3109, k d = 61.632 min -1 , f = 5.04×10 -3 . At the same time, the kinetic constant of enzyme inactivation k1 = 16.645 min -1 during the alkaline protease hydrolysis of silver carp skin was obtained. Through the above series of model verification experiments, it was found that the actual results were in good agreement with the model prediction values, proving that the enzymatic hydrolysis kinetic model was reliable and had practical value.

[0114] (V) Ultrafiltration separation

[0115] SCSCP was obtained by alkaline protease hydrolysis of silver carp skin. The ultrafiltration membrane separation equipment and ultrafiltration membranes with a molecular weight cut-off of 5 kDa and 3 kDa were used to filter and separate SCSCP. Equipment operating conditions: 0.06 Mpa, 25 °C, and the solution pH was maintained at 7.0 - 7.5. Finally, SCSCP01 fraction (MW < 3 kDa), SCSCP02 fraction (3 kDa < MW < 5 kDa), and SCSCP03 fraction (MW > 5 kDa) were obtained. Each fraction was freeze-dried and stored in a -20 °C refrigerator for later use.

[0116] (VI) Contents of each fraction after ultrafiltration of SCSCP

[0117] After ultrafiltration separation, the enzymolysis solutions of SCSCP01 (MW < 3 kDa), SCSCP02 (3 kDa < MW < 5 kDa) and SCSCP03 (MW > 5 kDa) were collected. The different components were freeze-dried to obtain peptide powders. The peptide powders of SCSCP01 component (MW < 3 kDa), SCSCP02 component (3 kDa < MW < 5 kDa) and SCSCP03 component (MW > 5 kDa) are respectively as Figure 11 (a), (b) and (c) shown; their colors gradually become lighter as the cut-off molecular weight decreases. After enzymolysis and ultrafiltration of 50 g of raw fish skin, SCSCP01, 02 and 03 components with masses of 6.51 g, 16.08 g and 12.37 g can be obtained respectively. Among them, the proportion of SCSCP02 (3 kDa < MW < 5 kDa) component is the highest.

[0118] (VII) Molecular weights of each component of SCSCP

[0119] As Figure 12 shown, the molecular weight distributions of each component of SCSCP are all in the range of 500 - 3000 Da, mainly small molecular polypeptides. As Figure 12 (a) shown, the detection signals of SCSCP01 component are stronger at 612.99 m / z, 663.06 m / z and 887.41 m / z; as Figure 12 (b) shown, SCSCP02 mainly has stronger relative abundances at 636.14 m / z, 662.65 m / z, 980.01 m / z, 1165.59 m / z and 1541.41 m / z; as Figure 12 (c) shown, SCSCP03 has higher intensities at 1164.96 m / z and 1487.69 m / z. Among them, the relative intensity of low molecular weight peptide segments in SCSCP02 component is the highest. The relative molecular mass of polypeptides determines their antioxidant activities. It is generally believed that components with lower relative molecular masses have stronger antioxidant activities.

[0120] (VIII) Establishment of UVB irradiation model and detection of cell viability

[0121] The proliferation ability of skin fibroblasts plays a decisive role in the normal repair of the skin. L929 cells, which are mouse upper epidermal fibroblast-like cells, are also one of the commonly used cells for evaluating cytotoxicity. Take L929 cells in good growth state and inoculate them in 96-well plates at a density of 5×10 3 / well. At the same time, set the solvent group as the blank control and culture overnight at 37 °C. Add 100 μl of sterile PBS into the wells around the cell wells to avoid the influence of drug concentration caused by water evaporation on the results. Use a Philips TL20W / 01-RS UVB lamp tube for irradiation treatment (100 mJ / cm 2)Establish an irradiation model. The experiment was set up with two major groups: the non-irradiated group and the UVB group. Culture media containing SCSCP01, 02, and 03 with concentration gradients of 0, 0.1, 0.5, 1.0, and 2.0 mg / mL were added respectively, and incubated in an incubator at 37°C and 5% CO2 for 24 h for subsequent experiments. EGCG was used as a positive control. After the culture, 10 μL of CCK8 was added to each well and reacted at 37°C for 1 h. The OD values at 450 nm of each well were measured using an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the effects of each component of SCSCP on the viability of L929 cells. At the same time, the morphological changes of L929 cells were observed under an inverted microscope.

[0122] 1. Effects of SCSCP on the viability of L929 cells

[0123] Under non-irradiated conditions, with the increase in the concentration of SCSCP, the cell viability showed a gradually decreasing trend as Figure 13 (a) shown. SCSCP01 and 02 at 0.1 mg / mL increased the viability of L929 cells by 8.31% and 14.96% respectively, which were significantly higher than the EGCG group (p < 0.0001). The UVB irradiation group showed obvious cytotoxicity. As Figure 13 (b) shown, treatment with SCSCP01, 02, and 03 at 0.1 - 1.0 mg / mL could significantly restore the cell viability after UVB irradiation (p < 0.001), and showed a dose-dependent decreasing trend. When the concentration was 0.1 mg / mL, each component of SCSCP could restore the viability of L929 cells by 23.86%, 27.04%, and 16.88% respectively, all of which were higher than the recovery rate of the EGCG group (p < 0.0001). Therefore, 0.1 mg / mL of SCSCP was selected for subsequent experiments.

[0124] 2. Inhibitory effect of SCSCP on UVB-induced ROS

[0125] As Figure 14As shown in Fig. (a) and Fig. 14(b), the ROS level of the control group of L929 cells was set at 100%, and after UVB irradiation, the ROS level of the cells increased to 364.92%. When the concentration was 0.1 mg / mL, the treatments of SCSCP01, 02, 03 and EGCG all significantly (p < 0.0001) inhibited the ROS activity induced by UVB. Among them, the inhibition rate of SCSCP02 on ROS (232.41%) was significantly higher than that of other groups (p < 0.001). UVB irradiation can stimulate the production of ROS in epidermal fibroblasts. Excessive ROS can cause oxidative damage to proteins, DNA, etc. by subtracting electrons, thus leading to damage of the main cell components. This may be because SCSCP has good antioxidant activity and can relieve the oxidative damage of cells caused by UVB. The study found that 0.1 mg / mL of SCSCP02 had the best effect on ROS scavenging and could restore the oxidative damage caused by UVB by scavenging the excess ROS in damaged L929 cells.

[0126] 3. Inhibitory effect of SCSCP on MMP-1 induced by UVB

[0127] As Figure 15 shown, after UVB irradiation, the content of MMP-1 in L929 cells increased significantly (p < 0.0001) to 2438.39 pg / mL. Each component of 0.1 mg / mL SCSCP could significantly (p < 0.0001) inhibit the secretion of MMP-1 induced by UVB; among them, SCSCP02 had the most significant inhibitory effect, and the highest inhibition rate could reach 70.17%. This may be because SCSCP02 contains more hydrophobic amino acids, and hydrophobic amino acids can act as hydrogen donors to react with metal ions to reduce the oxidation rate, showing better antioxidant ability. Excessive ROS stimulates dermal fibroblasts to synthesize and express MMP-1, which plays a key role in the process of photoaging. Peng et al. reported that oyster protein polypeptide could significantly reduce the degradation of collagen fibers by inhibiting the expression of MMP-1. This study shows that SCSCP02 has a significant inhibitory effect on the secretion of MMP-1 in L929 cells induced by UVB, so the SCSCP02 component was selected for the next step of research.

[0128] 4. Identification of characteristic peptide segments in SCSCP02

[0129] Peptide segments of SCSCP02 were identified using LC / MS. The PEAKS software was used to match peptides and proteins in the Uniprot Protein Database, and finally 8 major proteins were searched, and the species source was mainly Hypophthalmichthys molitrix, as shown in Table 6.

[0130] Table 6:

[0131]

[0132]

[0133] Note: -10lgP, the confidence score of the protein; OS, species name; GN, gene name; PE, reliability of protein presence, the smaller the number, the more reliable.

[0134] A0A077B3P8 and A0A2H4ZEX8 are identified as highly abundant proteins, annotated as collagen type I alpha 1 and collagen type I alpha 2 respectively, and the gene names are COL1A1 and COL1A2 respectively. PE = 2, indicating that the protein has good credibility. 65 matching characteristic peptide segments in these two proteins were identified, and the results are shown in Table 7. The molecular weight of the peptide segments is mainly distributed between 358.68 Da and 863.89 Da, and the biological activity of fish skin peptides has been proven to be related to low molecular weight peptides.

[0135] Table 7:

[0136]

[0137]

[0138]

[0139] Note: -10lgP: confidence of the peptide segment; ppm, mass error; RT, retention time.

[0140] The characteristic peptide segments in Table 7 were matched for biological activity through the BIOPEP-UWM online database. The results show that the above characteristic peptide segments mainly contain peptides such as antioxidant, alpha-glucosidase inhibitor, ACE inhibitor, DPP-IV inhibitor, antithrombotic, anti-amnesia, and regulation. Sequences with a peptide ranker response value greater than 0.8 were screened through the peptide ranker system. As shown in Table 8, combined with the mass spectrometry identification results, the peptide sequences with high matching degree and potential anti-photoaging activity were finally screened out as: GPPGPPGTPGPQ, SGLPGPIGPPGPR, and GLPGPIGPPGPR, corresponding to m / z of 529.76, 601.33, and 557.82, all from collagen type I alpha 1.

[0141] The matching maps and structures of each peptide segment are as Figure 16 shown.

[0142] Table 8:

[0143]

[0144]

[0145] As described above, it is only the preferred embodiment of the present invention, and does not impose any limitation on the technical scope of the present invention. Therefore, any minor modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A preparation method of anti-photoaging collagen polypeptide, characterized in that It includes the following steps: Step S1: After thawing, the silver carp skin is repeatedly washed to remove residual fish scales and fish meat, and then cut after drying in the sun. Step S2: Add NaHCO3 and soak; then add Na2CO3 for ultrasonic impurity removal, change the solution regularly, wash and dry for standby. Step S3: Take the pretreated fish skin, add distilled water, and perform ultrasonic-assisted treatment. Step S4: Adjust the pH value, add protease for enzymatic hydrolysis, and adjust the pH of the enzymatic hydrolysate regularly; after the enzymatic hydrolysis ends, boil in boiling water to terminate the enzyme activity. Step S5: After the enzymatic hydrolysate is allowed to stand and cool, centrifuge at low temperature, collect the supernatant in the centrifuge tube, filter to remove impurities, pass the filtered solution through a microporous filter membrane, and obtain light yellow SCSCP after vacuum freeze-drying. It has collagenase inhibitory ability, and the α-helix and β-sheet structures account for 17.70% and 22.65% respectively, and a stable α-helix structure is still retained during the enzymatic hydrolysis process. Step S6: Use an ultrafiltration separation device and an ultrafiltration membrane with a molecular weight cut-off of 1 - 5 kDa to separate SCSCP. Among them, the operating conditions of the ultrafiltration membrane separation device are: 0.06 Mpa, 25 °C, and the solution pH is maintained at 7.0 - 7.5; SCSCP02 is obtained; its molecular mass-to-charge ratio range is 636.14 m / z - 1541.43 m / z; 0.1 mg / mL SCSCP02 can reduce the ROS production induced by UVB from 364.92% to 232.41%, and the inhibition rate of MMP-1 reaches more than 70%. Step S7: Through mass spectrometry identification and database matching, three novel anti-photoaging peptides, GPPGPPGTPGPQ, SGLPGPIGPPGPR, and GLPGPIGPPGPR, are obtained. They all come from type I collagen α1 and have antioxidant, antithrombotic, α-glucosidase inhibitory, and DPP-IV inhibitory potentials.

2. The preparation method of an anti-photoaging collagen polypeptide according to claim 1, wherein In the said step S2, 0.5% NaHCO3 is added according to a solid-liquid ratio of 1:30, and 0.6 mol / L Na2CO3 is added according to a solid-liquid ratio of 1:

50.

3. The preparation method of an anti-photoaging collagen polypeptide according to claim 1, wherein, In the said step S2, the soaking time is 8 h; the ultrasonic impurity removal time is 12 h, and the solution is changed every 6 h.

4. The preparation method of an anti-photoaging collagen polypeptide according to claim 1, characterized in that, In the said step S3, ultrasonic-assisted heating is carried out at 80 °C for 30 min.

5. The preparation method of an anti-photoaging collagen polypeptide according to claim 1, wherein, In the said step S4, the protease is alkaline protease, and it is adjusted to the optimal pH of the protease with 1 mol / L HCL; the pH of the enzymatic hydrolysate is adjusted once every 0.5 h.

6. The preparation method of an anti-photoaging collagen polypeptide according to claim 1, wherein, In the said step S4, the enzymatic hydrolysis reaction kinetic model of the alkaline protease is: DH = 3.839ln[1+(16.055E0 / S0 - 0.081)t], and the reaction rate R = (61.632E0 - 0.3109S0)exp[-0.2605(DH)]; where b = 0.2605, a = 61.632E0 / S0 - 0.3109, k d = 61.632 min -1 , f = 5.04×10 -3 ; meanwhile, the enzyme inactivation kinetic constant k1 = 16.645 min in the process of alkaline protease hydrolyzing silver carp skin was obtained -1 ; the actual results are in good agreement with the predicted values of this model.

7. The preparation method of an anti-photoaging collagen polypeptide according to claim 1, characterized in that In the said step S5, centrifuge at low temperature at 4500 r / min for 30 min under the condition of 4 °C.

8. A preparation method of an anti-photoaging collagen polypeptide as claimed in claims 1 - 7 and the application of this peptide in anti-skin photoaging.

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