A method for preparing a peptide composition and use thereof

Abstract

This invention relates to the field of bio-health technology, specifically a method for preparing and applying a peptide composition. The preparation method includes the following steps: S1, preparation of hydrolyzed fish collagen as a raw material for the peptide composition; S11, enzymatic hydrolysis of fish skin collagen; S12, purification using ion exchange resin; S2, mixing and preparing the peptide composition. The peptide composition prepared by this method is used in skin brightening and spot-fading products, with a peptide content of 0.5-10%. The hydrolyzed fish collagen produced by this invention is highly compatible with palmitoyl oligopeptides without precipitation. Furthermore, this invention uses a 0.22-micron polypropylene membrane for filtration, which uses less water and is more efficient than ultrafiltration or nanofiltration, thus preventing bacterial contamination from prolonged filtration of protein peptides. This invention combines hydrolyzed fish collagen with palmitoyl oligopeptides such as palmitoyl tripeptide-5, resulting in excellent anti-wrinkle activity when applied to skincare products.

Description

Technical Field

[0001] This invention relates to the field of bio-health technology, specifically a method for preparing and applying a peptide composition. Background Technology

[0002] Peptides possess a variety of biological activities, such as scavenging free radicals, anti-oxidation, and anti-aging. They can not only promote the proliferation of skin cells, but also provide nutrients to the skin, delay skin aging, and repair the skin barrier. Therefore, they are widely used in anti-wrinkle cosmetics.

[0003] Regarding the application of peptide compositions, the invention patent with publication number CN113749967A discloses a peptide composition of hydrolyzed collagen peptide and palmitoyl oligopeptide. This technical solution, by compounding hydrolyzed collagen with palmitoyl polypeptide, produces a peptide composition that not only has a good wrinkle-reducing effect but also has the effect of fading age spots and brightening the skin. By using a compound of palmitoyl tripeptide-5 and palmitoyl dipeptide-7, combined with hydrolyzed pigskin collagen, the synergistic effect of the three can further improve the wrinkle-reducing effect and significantly enhance the effect of fading age spots and brightening the skin tone.

[0004] However, hydrolyzed collagen peptides, especially hydrolyzed fish collagen, currently have poor compatibility with palmitoyl oligopeptides such as palmitoyl tripeptide-5 and palmitoyl tetrapeptide-7, and the two are prone to forming flocculent precipitates in aqueous solutions.

[0005] Furthermore, current methods for preparing hydrolyzed collagen mostly employ ultrafiltration and nanofiltration. These methods are expensive, time-consuming, and require large amounts of water, resulting in water waste. Additionally, prolonged filtration of protein peptides increases the risk of bacterial contamination, reducing the shelf life of the final product. Summary of the Invention

[0006] The present invention adopts the following technical solution to solve the above-mentioned technical problems: a method for preparing a peptide composition, comprising the following steps: S1, preparing hydrolyzed fish collagen as a raw material for the peptide composition.

[0007] S11. Enzymatic hydrolysis of fish skin collagen: Fish skin collagen powder is reconstituted with water, and then enzymatic hydrolysis is carried out by adding protease. After the enzymatic hydrolysate is inactivated, it is filtered through a 0.22-micron polypropylene membrane to obtain the enzymatic hydrolysate.

[0008] S12. Ion exchange resin purification: Adjust the pH of the enzymatic hydrolysis filtrate to 3-6, load the sample onto a weak anion exchange resin column, and elute sequentially with water, 30% ethanol, and 60% ethanol; collect the 30% ethanol eluent and recover the solvent to obtain hydrolyzed fish collagen.

[0009] S2. Preparation of the peptide composition: The peptide composition consists of the following components in parts by weight: 0.3-0.7 parts hydrolyzed fish collagen, 0.01-0.2 parts palmitoyl tripeptide-5, 98.9-99.6 parts solvent, and 0.08-0.12 parts moisturizer.

[0010] Furthermore, in step S11, the mass ratio of fish skin collagen powder to water is 1:5 to 30, and the protease is one or more of papain, trypsin, neutral protease, alkaline protease, and acidic protease; the amount of protease used is 0.1 to 0.5% of the weight of the fish skin collagen powder aqueous solution.

[0011] Furthermore, the enzymatic hydrolysis temperature is 25–50°C, the pH value for enzymatic hydrolysis is 4–9, and the enzymatic hydrolysis time is 4–8 hours.

[0012] Furthermore, the weak anion exchange resins used in step S12 include, but are not limited to: D941, D301, D380, D392, LX-94, LX-T5, and LXD-762.

[0013] Furthermore, the moisturizer is ethylhexylglycerin; the solvent is a mixture of water and polyol, wherein the mixing ratio of water and polyol is 70-90:10-30; and the polyol is selected from at least one of 1,2-hexanediol, 1,3-butanediol, and glycerol.

[0014] Furthermore, in step S12, the ion exchange resin purification is intelligently monitored using an ion exchange resin purification monitoring and analysis system to analyze the quality of the hydrolyzed fish collagen.

[0015] Furthermore, the ion exchange resin purification monitoring and analysis system includes an eluent monitoring module, an eluent analysis module, an elution process monitoring module, an elution process analysis module, an ion exchange column monitoring module, an ion exchange column analysis module, a comprehensive evaluation module, and a storage database.

[0016] The eluent monitoring module is used to acquire images of the initial condition of the eluent, thus obtaining an initial image of the eluent.

[0017] The eluent analysis module reads the initial image of the eluent and uses image processing to obtain the amount of floating matter and precipitate in the eluent, thereby determining whether the eluent is qualified. Unqualified eluent should be replaced or reprocessed immediately.

[0018] The ion exchange column monitoring module is used to collect defect data of the ion exchange column before elution.

[0019] The ion exchange column analysis module is used to analyze the defect data of the ion exchange column to obtain the pass / fail coefficient of the ion exchange column.

[0020] The elution process monitoring module is used to collect elution parameters during the elution process of the eluent.

[0021] The elution process analysis module is used to read elution parameters and analyze them to obtain the elution process pass coefficient.

[0022] The comprehensive evaluation module is used to analyze the quality of hydrolyzed fish collagen based on the pass coefficient of the elution process.

[0023] The storage database is used to store various standard parameters during the elution process.

[0024] Furthermore, the elution parameters include elution temperature, elution flow rate, and pH value during elution.

[0025] Furthermore, the defect data of the ion exchange column is obtained by high-definition image acquisition of the ion exchange column, and the contamination area and wear area of ​​the ion exchange column are analyzed by image processing.

[0026] The peptide composition prepared by the above method is used in anti-wrinkle products, and the content of the peptide composition is 0.5-10%.

[0027] The beneficial effects of this invention are as follows: First, the hydrolyzed fish collagen produced by this invention is well compatible with palmitoyl oligopeptide and does not produce precipitation. Furthermore, this invention uses a 0.22-micron polypropylene membrane for filtration. Compared with ultrafiltration and nanofiltration, this filtration method uses less water, is more efficient, and has a simpler process, thereby preventing the protein peptide components from being contaminated by bacteria due to excessively long filtration time.

[0028] II. The present invention also provides an application of hydrolyzed fish collagen, wherein hydrolyzed fish collagen combined with palmitoyl oligopeptides such as palmitoyl tripeptide-5 has excellent anti-wrinkle activity.

[0029] Third, the present invention employs an ion exchange resin purification monitoring and analysis system to intelligently monitor the ion exchange resin purification step. This system analyzes the process from two aspects: before elution and during the ion exchange resin purification process. It analyzes the quality of hydrolyzed fish collagen, thereby increasing the monitoring accuracy of ion exchange resin purification and reducing the defect rate of the finished product.

[0030] Fourth, the ion exchange resin purification monitoring and analysis system of the present invention can comprehensively monitor the elution process of the eluent, including elution temperature, elution flow rate, pH value and ion concentration, thereby increasing the purity of hydrolyzed fish collagen and the effectiveness of peptide compositions. Attached Figure Description

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] Figure 1 This is a flowchart of the preparation method of the peptide composition of the present invention.

[0033] Figure 2 This is a module connection diagram of the ion exchange resin purification monitoring and analysis system.

[0034] Figure 3 This is a comparison diagram of the peptide compositions prepared in the examples and comparative examples.

[0035] Figure 4 This is a verification image of the anti-wrinkle properties of the peptide composition prepared in this invention.

[0036] Figure 5 These are images showing the anti-wrinkle effects of two typical subjects in the human anti-wrinkle efficacy evaluation.

[0037] Figure 6 This is a graph showing the cell viability results from an anti-aging experiment using fibroblasts.

[0038] Figure 7 These are microscope images of cells stained with MTT at 72 hours during an anti-aging assay for fibroblasts.

[0039] Figure 8 This is a flowchart of the ELISA results in the fibroblast anti-aging assay.

[0040] Figure 9 This is a graph showing the expression levels of COLI synthesized by cells treated with 0.25% and 0.5% PTC in an anti-aging assay for fibroblasts.

[0041] Figure 10 This is a graph showing the expression levels of COL III synthesized by cells treated with 0.25% and 0.5% PTC in an anti-aging assay for fibroblasts.

[0042] Figure 11 This is a bar chart showing the intracellular and extracellular COL VII content detected by ELISA in the evaluation of type VII collagen results. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] like Figure 1 As shown, a method for preparing a peptide composition includes the following steps: S1, preparing hydrolyzed fish collagen as a raw material for the peptide composition.

[0045] S11. Enzymatic hydrolysis of fish skin collagen: Fish skin collagen powder is reconstituted with water, and then enzymatic hydrolysis is carried out by adding protease. After the enzymatic hydrolysate is inactivated, it is filtered through a 0.22-micron polypropylene membrane to obtain the enzymatic hydrolysate.

[0046] The mass ratio of the fish skin collagen powder to water is 1:5 to 30. The protease is one or more of papain, trypsin, neutral protease, alkaline protease, and acidic protease. The amount of protease used is 0.1 to 0.5% of the weight of the fish skin collagen powder aqueous solution. The enzymatic hydrolysis temperature is 25 to 50°C, the pH value for enzymatic hydrolysis is 4 to 9, and the enzymatic hydrolysis time is 4 to 8 hours.

[0047] S12. Ion exchange resin purification: Adjust the pH of the enzymatic hydrolysis filtrate to 3-6, load the sample onto a weak anion exchange resin column, and elute sequentially with water, 30% ethanol, and 60% ethanol; collect the 30% ethanol eluent and recover the solvent to obtain hydrolyzed fish collagen.

[0048] The aforementioned weak anion exchange resins include, but are not limited to: D941, D301, D380, D392, LX-94, LX-T5, and LXD-762.

[0049] S2. Preparation of the peptide composition: The peptide composition consists of the following components in parts by weight: 0.3-0.7 parts hydrolyzed fish collagen, 0.01-0.2 parts palmitoyl tripeptide-5, 98.9-99.6 parts solvent, and 0.08-0.12 parts moisturizer.

[0050] The humectant is ethylhexylglycerin; the solvent is a mixture of water and polyol, with a mixing ratio of 70-90:10-30; the polyol is selected from at least one of 1,2-hexanediol, 1,3-butanediol, and glycerol.

[0051] The preparation method of the peptide composition of the present invention yields a peptide composition that can be used in anti-wrinkle products, wherein the content of the peptide composition is 0.5-10%.

[0052] In step S12, the ion exchange resin purification is intelligently monitored using an ion exchange resin purification monitoring and analysis system to analyze the quality of hydrolyzed fish collagen. The ion exchange resin purification monitoring and analysis system analyzes the process from two aspects: before elution and during the ion exchange resin purification process, thereby increasing the monitoring accuracy of ion exchange resin purification.

[0053] like Figure 2As shown, the ion exchange resin purification monitoring and analysis system includes an eluent monitoring module, an eluent analysis module, an elution process monitoring module, an elution process analysis module, an ion exchange column monitoring module, an ion exchange column analysis module, a comprehensive evaluation module, and a storage database. The storage database is connected to the eluent analysis module, the ion exchange column analysis module, and the comprehensive evaluation module, respectively. The comprehensive evaluation module is connected to the elution process analysis module, the eluent monitoring module is connected to the eluent analysis module, the elution process monitoring module is connected to the elution process analysis module, and the ion exchange column monitoring module is connected to the ion exchange column analysis module.

[0054] The storage database is used to store various standard parameters during the elution process.

[0055] The eluent monitoring module is used to acquire images of the initial condition of the eluent to obtain an initial image of the eluent; the high-definition imaging unit in the eluent monitoring module of this invention can acquire the initial image of the eluent.

[0056] The eluent analysis module reads the initial image of the eluent and uses image processing to obtain the amount of floating matter and precipitate in the eluent. When the amount of floating matter and precipitate in the eluent is less than the preset value, the eluent is qualified and can proceed to the next elution operation. When the amount of floating matter and precipitate in the eluent is greater than or equal to the preset value, the eluent is unqualified. The corresponding unqualified eluent should be replaced or reprocessed immediately. The next elution operation can only be carried out when the eluent analysis shows that it is qualified.

[0057] The ion exchange column monitoring module is used to collect defect data of the ion exchange column before elution.

[0058] Defect data of the ion exchange column are collected in high definition by a high-definition monitor within the ion exchange column monitoring module. The contamination area and wear area of ​​the ion exchange column are analyzed through image processing and denoted as S1. i S2 i .

[0059] The ion exchange column analysis module is used to analyze the defect data of the ion exchange column to obtain the pass / fail coefficient of the ion exchange column, which is denoted as θ. When the pass coefficient of the ion exchange column is 0, the ion exchange column needs to be cleaned or replaced so that the pass coefficient of the treated ion exchange column is 1 before elution can be performed.

[0060] The elution process monitoring module is used to collect elution parameters during elution processes with water, 30% ethanol, and 60% ethanol. These parameters include elution temperature, elution flow rate, and pH value during elution. For water elution, the module collects elution temperature, flow rate, and pH value at each preset time point during the elution process, and records the pH value at each preset time point as pH1. i Let i represent the i-th preset time point, i = 1, 2, ..., m. The average elution temperature, average elution flow rate, and average pH value during water elution are obtained by averaging, and are denoted as follows: Simultaneously, the maximum and minimum elution temperatures, maximum and minimum elution flow rates for water elution were screened and denoted as T1. max T1 min V1 max V1 min Similarly, the average elution temperature, average elution flow rate, and average pH value during elution in the 30% ethanol elution treatment are denoted as follows: The maximum and minimum elution temperatures and the maximum and minimum elution flow rates for 30% ethanol elution are denoted as T2. max T2 min V2 max V2 min The average elution temperature, average elution flow rate, and average pH value during elution of 60% ethanol were denoted as follows: The maximum and minimum elution temperatures and the maximum and minimum elution flow rates for 30% ethanol elution are denoted as T3. max T3 min V3 max V3 min Meanwhile, the elution process monitoring module can also monitor the ion concentration at each preset time point during the elution process of water, 30% ethanol, and 60% ethanol. The average ion concentration during the elution process of water, 30% ethanol, and 60% ethanol is obtained by averaging and recorded as υ1, υ2, and υ3, respectively.

[0061] The elution process analysis module is used to read elution parameters, analyze them, and obtain the elution process pass coefficient, which is then denoted as... The formula for calculating the pass / fail coefficient of the elution process is: Let κ1, κ2, and κ3 represent the pass coefficients for the water elution process, the 30% ethanol elution process, and the 60% ethanol elution process, respectively. Let κ1, κ2, and κ3 represent the preset weights of the pass coefficients for the water elution process, the 30% ethanol elution process, and the 60% ethanol elution process, respectively, with κ2 > κ3 > κ1. 标 υ2 标υ3 标 These represent the standard ion concentrations during elution with water, 30% ethanol, and 60% ethanol, respectively; the formula for calculating the pass / fail coefficient of the water elution process is as follows: Where e represents a constant, T1 标 Indicates the optimal temperature for water washing and desorption, V1 标 The optimal flow rate for water washing and dehydration is This indicates that the pH value during the water washing process is not within the range of 3-6. This indicates that the pH value during water washing is in the range of 3-6.

[0062] The calculation methods for the pass coefficients of the 30% ethanol elution process and the 60% ethanol elution process are the same as those for the water elution process, and will not be elaborated further here.

[0063] It is understandable that the data on elution temperature, elution flow rate, pH value during elution, and ion concentration can all be collected using existing sensors, and will not be discussed further here.

[0064] The comprehensive evaluation module is used to analyze the quality of hydrolyzed fish collagen based on the elution process pass coefficient. Specifically, it compares the elution process pass coefficient with the elution process pass coefficient threshold. If the elution process pass coefficient is greater than the elution process pass coefficient threshold, it means that the elution process operation is qualified; otherwise, it is unqualified.

[0065] Example

[0066] S1. Preparation of hydrolyzed fish collagen.

[0067] S11. Enzymatic hydrolysis of fish skin collagen: Take 10g of fish skin collagen powder, add 100g of water, stir to dissolve, and adjust the pH to 4.5. Add 0.22g of papain. Stir and hydrolyze at 45℃ for 4 hours. After inactivating the enzyme solution at 85℃ for 30 minutes, filter through a 0.22-micron polypropylene membrane.

[0068] S12. Ion exchange resin purification: The enzymatic hydrolysate was loaded onto a D941 weak anion exchange resin column (200g resin), and eluted sequentially with 200ml water, 600ml 30% ethanol, and 600ml 60% ethanol. The 30% ethanol eluent was collected, and the solvent was recovered to obtain hydrolyzed fish collagen.

[0069] S2. Preparation of peptide composition: The peptide composition consists of the following components in parts by weight: 0.5 parts hydrolyzed fish collagen, 0.1 parts palmitoyl tripeptide-5, 89.3 parts water, 10 parts 1,2-hexanediol, and 0.1 parts ethylhexylglycerin.

[0070] The peptide composition was prepared into a 5% aqueous solution and left at room temperature, 4°C, and 45°C for 3 months. The solution was clear and transparent.

[0071] Comparative Example

[0072] Hydrolyzed fish collagen was prepared into a peptide composition without enzymatic hydrolysis or ion exchange resin purification. The peptide composition consisted of the following components in parts by weight: 0.5 parts hydrolyzed fish collagen, 0.1 parts palmitoyl tripeptide-5, 89.3 parts water, 10 parts 1,2-hexanediol, and 0.1 parts ethylhexylglycerol.

[0073] When the peptide composition is prepared into a 5% aqueous solution and left at room temperature and 4°C for 2 days, flocculent matter is observed to form.

[0074] like Figure 3 As shown, the left figure is the comparative sample, and the right figure is the example sample.

[0075] Evaluation of anti-wrinkle efficacy in the human body

[0076] (I) Experimental Basis

[0077] Anti-wrinkle efficacy refers to the ability to slow down the formation of wrinkles or make them less noticeable. Based on literature and the T / ZHCA006-2019 Test Method for Anti-wrinkle Efficacy of Cosmetics, the efficacy of the sample in this example was tested using the human real-time anti-wrinkle efficacy evaluation test method.

[0078] This experiment used the Visioscan VC98 imaging system to capture images of crow's feet and analyze wrinkle parameters and smoothness changes to evaluate the efficacy of the samples.

[0079] The VC98 uses a CCD skin imaging system with an ultraviolet light source within a special probe to test the skin surface and obtain an image of the skin's activity state. After digital processing and calculation, various skin parameters that reflect the anti-wrinkle effect are obtained, such as skin metallic roughness R3, skin wrinkle parameter SEw, skin smoothness parameter SEsm, and angular density CornerD. The smaller the values ​​of R3, SEw, and SEsm, and the larger the CornerD, the less noticeable the wrinkles.

[0080] (II) Test Methods

[0081] Samples: A base serum containing 5% of the example sample and a matrix control, the main components of which include water, 0.3% U21, 0.3% methylparaben, and 0.3% triethanolamine.

[0082] The left and right corners of the eyes were selected for evaluation using an instrumental method. Participants were aged 25–50 years, and 15 participants were included. On the day of the test, the faces were washed, patted dry with tissues, and dried and equilibrated in a constant temperature and humidity chamber (temperature 20±2℃, relative humidity 30%–60%) for 20–30 minutes. The left and right corners of the eyes were marked, and the wrinkles at the corners of the eyes before the test were photographed and analyzed using a VC98 skin microscope and a SELS active skin surface analysis system.

[0083] After cleansing morning and evening, apply the sample. Apply a 5% sample (from the example) to the left corner of the eye and a matrix control sample to the right corner of the eye. Apply a pea-sized amount evenly to the test area and keep it dry. At 2, 4, 6, and 8 weeks after use, after cleansing and balancing on the test day, photograph the wrinkles around the eyes again using a VC98 skin microscope and a SELS active skin surface analysis system to analyze changes in SEw and SEsm.

[0084] (III) Test Results

[0085] Based on the pre-use test values, the matrix control test values ​​at each time point were calculated as 100%, and the effect of the samples on skin wrinkles and related parameters was calculated. The TTest method was used to calculate the significance of the difference between the samples before and after use, *P<0.05, **P<0.01. The calculation formula is as follows:

[0086] a) Change rate % = (Value after use / Value before use – 1) × 100%

[0087] b) Relative change rate % = (Sample group change rate / Matrix group change rate) × 100%

[0088] First, we analyzed the crow's feet wrinkles of subjects aged 25 to 50.

[0089] like Figure 4 As shown, * represents PTC < 0.05 and ** represents PTC < 0.01 compared to before use (0). The evaluation results of VC98 on skin wrinkle parameters and smoothness parameters show that, compared to before use (0), after 2 weeks of use of the serum containing 5% of the example sample, the skin wrinkle parameter SEw began to decrease, and a significant difference was observed after 6 weeks of use. After 8 weeks of continuous use, skin wrinkles were reduced, with a maximum reduction rate of 86.3%. This indicates that long-term use of the serum containing 5% of the example sample can improve skin wrinkles.

[0090] An analysis was conducted on crow's feet wrinkles in subjects aged 50-65. In a group of 10 subjects, compared to before use, the degree of wrinkle reduction was observed after 2 weeks of using the serum containing 5% PTC, and the reduction in wrinkle severity increased after 6 weeks of use. Figure 5The image shows the anti-wrinkle effect of two typical subjects.

[0091] It should be noted that the above-mentioned PTC is the peptide composition prepared in the example, and the peptide composition will be referred to as PTC in the following text.

[0092] Fibroblast anti-aging test

[0093] (I) MTT Testing

[0094] HDF-a cells were seeded at 5*10³ cells / well in 96-well plates and cultured in complete medium for 24 h until the cells adhered. PTC was prepared at concentrations of 0.125%–1%, and positive controls were treated with medium containing 10% FBS. The original medium in the 96-well plates was discarded, and the plates were washed once with PBS. 200 μl of the prepared samples at different concentrations were added to each well, and the plates were incubated at 37°C with 5% CO2 for 24, 48, and 72 h. Negative and positive control groups were included in each plate, with at least three replicates for each concentration group. After incubation, 200 μl of pre-warmed staining solution (0.5 mg / ml) was added to each well, and the plates were incubated for 4 h. The culture medium was discarded, and 150 μl of dimethyl sulfoxide was added. After gentle shaking at room temperature for 10 min, the absorbance was measured at 492 nm using a microplate reader.

[0095] (II) ELISA Testing

[0096] HDF-a cells were seeded at a rate of 5*10⁴ cells / well in 12-well plates. After incubation, the cell supernatant was collected, and 200 μL of 1% Triton-X cell lysis buffer was added to each well of the lower cell layer. The cells were lysed on ice for 30 min. The expression levels of COLI and COLIII in both the cell supernatant and cell lysis buffer were measured according to the kit instructions. The expression levels of FN and TGF-β1 in the cell supernatant were also measured, and their relative contents were calculated based on the standard curve.

[0097] (III) Immunofluorescence detection

[0098] HDF-a cells were seeded at a rate of 5*10⁴ cells / well in 12-well plates. After incubation, the culture medium was discarded, and the cells were washed twice with PBS. 1 ml of cold methanol was added to each well for fixation at 4°C for 10 min, followed by 1 ml of 0.5% Triton-X cell lysis buffer and lysis at room temperature for 15 min. Then, 3% BSA, primary antibody solution, and secondary antibody solution were added for fluorescent staining. The primary antibody was incubated overnight, and DAPI staining solution was added simultaneously with the secondary antibody. The staining was observed and photographed under a fluorescence microscope.

[0099] (iv) Analytical Methods

[0100] All test data were first subtracted from the blank control, and then the negative control was set at 100% to calculate the changes in cell viability and protein content for each sample. Intracellular COLI and COLIII expression levels were photographed and displayed. The significance of the difference between the samples and the blank control was calculated using the TTest method, where *PTC<0.05, **PTC<0.01, and ***PTC<0.001. The calculation formula is as follows:

[0101] a) Cell viability (%) = OD value of sample group / OD value of negative control group × 100%

[0102] b) Change rate (%) = (Expression level sample group - Expression level negative control group) / Expression level negative control group × 100%

[0103] (V) Evaluation of Cell Proliferation Results

[0104] See the appendix for cell viability results. Figure 6 Compared with the negative control group (no FBS treatment), the cell viability of the positive control group (FBS treatment) increased from 100% to 120.0%, 136.4%, and 144.6% at 24, 48, and 72 hours, respectively, indicating that under serum-free conditions, cell proliferation slowed down with increasing culture time, showing signs of senescence. Treatment with 0.125%–1% PTC significantly improved cell viability at 24–72 hours, with the strongest promoting effect at 72 hours. Under 0.125% PTC treatment, compared with the control group, cell viability increased to 114.5%, 133.0%, and 149.2% at 24, 48, and 72 hours, respectively, close to that of the FBS treatment group. The effect of 1% PTC at 48 and 72 hours was slightly weaker than that of 0.125%, which is related to the sensitivity of the cell model, but it still had an effect on improving cell viability.

[0105] Microscopic images of cells stained with MTT at 72 hours are attached. Figure 7 As shown, compared with the control group, the number of cells increased significantly after treatment with 0.25% and 0.5% PTC (peptide composition), which is consistent with the test results.

[0106] (VI) Evaluation of Collagen Results

[0107] The content of intracellular and extracellular collagen was detected, and the results of the ELISA method are as follows: Figure 8As shown in the figure, the expression levels of COLI and COLIII in the cell lysate were 0.54 ng / ml and 117.0 pg / ml, respectively; while in the cell supernatant, the expression levels were 224.27 ng / ml and 658.5 pg / ml, respectively. This indicates that the expression level of COLI was significantly higher than that of COLIII, and the expression level of collagen secreted into the cell supernatant was also significantly higher than that within the cells, suggesting that most of the synthesized collagen is secreted into the intercellular matrix to exert its function. PTC treatment significantly promoted both intracellular collagen synthesis and intercellular collagen secretion.

[0108] It should be noted that, Figure 8 Compared with the negative control group (CK), ** represents PTC < 0.01, and *** represents PTC < 0.001.

[0109] See Figure 8 Regarding COLI, compared with the negative control group (CK), the positive control group (TGF-β1) showed increased intracellular and extracellular protein expression levels of 1.12 and 299.4 ng / ml, respectively, at 48 h, indicating that TGF-β1 can promote the synthesis and secretion of type I collagen and increase its total content. Treatment with 0.125%–0.5% PTC also significantly increased intracellular and intercellular COLI expression levels, with maximum increases of 121% and 48%, respectively. Its promoting effect on intracellularly synthesized COLI was stronger than that on COLI secreted into the intercellular space, indicating that PTC can promote collagen synthesis more effectively from the source. For COLIII, TGF-β1 did not show a promoting effect, while 0.125%–0.5% PTC still had a significant promoting effect, increasing intracellular and intercellular COLIII expression levels to over 117.8 pg / ml and 1300 pg / ml, respectively, with maximum increases of 107% and 99%, respectively.

[0110] The results of immunofluorescence are as follows Figure 9 and Figure 10 As shown, compared with the negative control (CK), the green fluorescence intensity was significantly upregulated after treatment with 0.25% and 0.5% PTC, indicating that the expression levels of COLI and COLIII synthesized in the cells were significantly increased, which is consistent with the results of ELISA.

[0111] Figure 9 and Figure 10 The blue fluorescence represents the cell nucleus after DAPI staining, and the green fluorescence represents collagen.

[0112] Evaluation of Type VII Collagen Results

[0113] Type VII collagen can be found in the basement membrane region beneath lamellar squamous epithelium and is a component that anchors fibrils. The content of intracellular and extracellular COL VII was measured, and the results of the ELISA method are as follows: Figure 11 As shown in the figure, after 48 hours of incubation, the expression levels of COL VII in the cell lysate and cell supernatant were 0.3 ng / ml and 3.7 ng / ml, respectively. This indicates that the expression level of COL VII secreted into the cell supernatant is much higher than that intracellularly, suggesting that most of the synthesized COL VII is secreted into the intercellular space to exert its function.

[0114] PTC treatment significantly promoted both intracellularly synthesized COL VII and COL VII secreted into the intercellular space. Compared with the negative control group (0), treatment with 0.063%–1% PTC increased intracellular and intercellular COL VII expression levels to over 0.97 ng / ml and 6.26 ng / ml, respectively, with the increase rate increasing with time and dosage. At 1% treatment for 72 hours, the maximum increase rates in intracellular and extracellular expression reached 713.4% and 278.0%, respectively.

[0115] Figure 11 Compared with the negative control group (0), * represents PTC<0.05, and ** represents PTC<0.01.

[0116] PTC at concentrations of 0.063% to 1% can significantly promote keratinocyte proliferation, increase the expression of type IV collagen, type VII collagen, and integrin β1, and at the same time promote the expression of transforming growth factor TGF-β1, thus having potential anti-wrinkle effects.

[0117] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a peptide composition, characterized in that, Includes the following steps: S1. Preparation of hydrolyzed fish collagen, the raw material for peptide compositions; S11. Enzymatic hydrolysis of fish skin collagen: Fish skin collagen powder is reconstituted with water, then enzymatic hydrolysis is performed by adding protease. After inactivation of the hydrolysate, it is filtered through a 0.22-micron polypropylene membrane to obtain the enzymatic hydrolysate. In step S11, the mass ratio of fish skin collagen powder to water is 1:5-30, and the protease is papain. The amount of protease used is 0.1-0.5% of the weight of the fish skin collagen powder aqueous solution. Enzymatic hydrolysis temperature: 25–50℃, enzymatic hydrolysis pH: 4–9, enzymatic hydrolysis time: 4–8h; S12. Ion exchange resin purification: Adjust the pH of the enzymatic hydrolysis filtrate to 3-6, load the sample onto a weak anion exchange resin column, and elute sequentially with water, 30% ethanol, and 60% ethanol; collect the 30% ethanol eluent and recover the solvent to obtain hydrolyzed fish collagen. S2. Preparation of the peptide composition: The peptide composition consists of the following components in parts by weight: 0.3-0.7 parts hydrolyzed fish collagen, 0.01-0.2 parts palmitoyl tripeptide-5, 98.9-99.6 parts solvent, and 0.08-0.12 parts moisturizer.

2. The method for preparing a peptide composition according to claim 1, characterized in that, The weak anion exchange resins in step S12 include: D941, D301, D380, D392, LX-94, LX-T5, and LXD-762.

3. The method for preparing a peptide composition according to claim 1, characterized in that, The moisturizer is ethylhexylglycerin; the solvent is a mixture of water and polyol, with a mixing ratio of 70-90:10-30; the polyol is selected from at least one of 1,2-hexanediol, 1,3-butanediol, and glycerol.

4. A method for preparing a peptide composition according to claims 1-3, characterized in that, In step S12, the ion exchange resin purification is intelligently monitored using an ion exchange resin purification monitoring and analysis system to analyze the quality of the hydrolyzed fish collagen.

5. The method for preparing a peptide composition according to claim 4, characterized in that, The ion exchange resin purification monitoring and analysis system includes an eluent monitoring module, an eluent analysis module, an elution process monitoring module, an elution process analysis module, an ion exchange column monitoring module, an ion exchange column analysis module, a comprehensive evaluation module, and a storage database. The eluent monitoring module is used to acquire images of the initial condition of the eluent and obtain the initial image of the eluent. The eluent analysis module reads the initial image of the eluent and uses image processing to obtain the amount of floating matter and precipitate in the eluent, thereby determining whether the eluent is qualified. Unqualified eluent should be replaced or reprocessed immediately. The ion exchange column monitoring module is used to collect defect data of the ion exchange column before elution. The ion exchange column analysis module is used to analyze the defect data of the ion exchange column to obtain the pass coefficient of the ion exchange column. The elution process monitoring module is used to collect elution parameters during the elution process of the eluent; The elution process analysis module is used to read elution parameters and analyze them to obtain the elution process pass coefficient; The comprehensive evaluation module is used to analyze the quality of hydrolyzed fish collagen based on the pass coefficient of the elution process; The storage database is used to store various standard parameters during the elution process.

6. The method for preparing a peptide composition according to claim 5, characterized in that, The elution parameters include elution temperature, elution flow rate, and pH value during elution.

7. The method for preparing a peptide composition according to claim 5, characterized in that, The defect data of the ion exchange column is obtained by acquiring high-definition images of the ion exchange column and analyzing the contamination area and wear area of ​​the ion exchange column through image processing.

Images