Egg white peptide sequence qvplw having skin damage alleviating effect and application thereof

Abstract

The application belongs to the field of bioactive peptides, and particularly relates to an egg white peptide sequence QVPLW having a skin damage alleviating effect and application thereof. Specifically, the egg white peptide is prepared by alkaline protease enzymolysis, and after in vitro simulation of gastrointestinal digestion, the sequence thereof is determined by mass spectrometry. The egg white peptide QVPLW can significantly alleviate mechanical damage of skin fibroblasts (HSF) caused by cell scratching. Meanwhile, the egg white peptide QVPLW can also promote the proliferation of HSF cells and the migration of HSF cells in a Transwell chamber. The application provides a new idea for deep processing of egg products and targeted preparation of high-activity egg white peptides, and provides a new perspective for the development of functional foods applied in the auxiliary treatment of skin damage.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptides, specifically relating to an egg white peptide sequence QVPLW with a skin damage relief effect and its applications. Background Technology

[0002] Skin is frequently damaged by various acute and chronic traumas, which disrupt its barrier function and alter the body's perception of temperature, touch, and pain. However, skin wound repair is complex, often involving interactions between cells and the extracellular matrix, as well as the coordinated efforts of multiple tissue cells. Therefore, accelerating wound healing remains a global challenge. Research has found that nutrition is a key factor influencing wound healing; appropriate supplementation with nutrients such as protein and peptides can effectively improve the area and depth of wounds, helping to alleviate skin damage and accelerate healing. Therefore, there is an urgent need to develop a safe and highly effective functional food for skin damage repair to accelerate the skin damage repair process.

[0003] Developing and preparing egg white-derived bioactive peptides is of great significance for improving the economic benefits of egg products in my country. Egg white-derived bioactive peptides obtained through enzymatic hydrolysis of egg white protein have also been shown to alleviate skin trauma. Based on this, this invention utilizes an alkaline protease hydrolysis method to efficiently prepare egg white peptides, and after simulated digestion in vitro, a single egg white peptide with the sequence QVPLW was detected by mass spectrometry. The results indicate that the egg white peptide QVPLW can assist in alleviating skin damage by promoting the proliferation and migration of HSF cells, and it holds promise as a functional food ingredient in the skin damage repair process. This invention provides a new perspective and strategy for the efficient and stable preparation of egg white-derived bioactive peptides and their auxiliary repair of skin damage. Summary of the Invention

[0004] The purpose of this invention is to provide an egg white peptide sequence QVPLW (Gln-Val-Pro-Leu-Trp) with the effect of alleviating skin damage and its application, so as to solve the problems related to the development of functional foods with the effect of alleviating skin damage.

[0005] This invention provides a method for preparing an egg white peptide sequence QVPLW that has the effect of alleviating skin damage. It can be obtained by alkaline protease digestion and in vitro simulated gastrointestinal digestion. Its skin damage alleviating effect was confirmed by a skin cell mechanical damage model.

[0006] The egg white peptide described in this invention, which alleviates skin damage, can promote the proliferation and migration of HSF cells. It includes the following steps:

[0007] Step 1: Preparation of Egg White Peptide QVPLW

[0008] Weigh 50-100g of egg white protein powder and add distilled water to prepare an egg white protein solution with a mass fraction of 5-10%. Incubate the egg white protein solution in a water bath at 90℃ for 10-15 min, then cool to 50℃. Adjust the pH of the system to 10 using 1mol / L NaOH solution. Under conditions of 50℃ and pH 10, enzymatically hydrolyze the egg white protein solution with alkaline protease (3-6% mass fraction) for 2-4 h. After enzymatic hydrolysis, inactivate the enzyme by boiling in a water bath for 8-12 min, and then cool to room temperature. Centrifuge the hydrolysate at 4℃ at 8000-12000 r / min for 10 min to obtain an egg white peptide solution. After in vitro simulated intestinal digestion, purify the egg white peptide fraction with a molecular weight less than 1 kDa by ultrafiltration. Identify the structure using LC-MS / MS, and based on the mass spectrum information, combined with software analysis and protein databases, finally determine the amino acid sequence of the egg white peptide.

[0009] Step 2: Egg white peptide QVPLW has the effect of relieving skin damage.

[0010] A cell mechanical injury model was constructed using an HSF cell scratch assay, and the mechanically injured cells were treated with egg white peptide QVPLW. The results showed that, compared with the control group, QVPLW significantly accelerated the closure of HSF cell scratches, indicating that QVPLW promotes skin injury repair. MTS cell proliferation assays showed that egg white peptide QVPLW promoted HSF cell proliferation. Furthermore, Transwell cell migration assays showed that egg white peptide QVPLW promoted HSF cell migration within Transwell chambers. In conclusion, egg white peptide QVPLW can alleviate skin injury by promoting HSF cell proliferation and migration.

[0011] The present invention has the following beneficial effects:

[0012] 1. Egg white protein powder is used as raw material, and it is prepared by alkaline protease hydrolysis. The final product is obtained by ultrafiltration separation and purification.

[0013] 2. Egg white peptide QVPLW can promote the closure of HSF cell scratches and alleviate skin damage in the cell mechanical injury model; it can significantly promote HSF cell proliferation in the MTS cell proliferation experiment; and it can promote the migration of HSF cells in the chamber in the Transwell cell migration experiment.

[0014] 3. This invention not only provides new ideas for developing safe and efficient egg white peptide damage repair products, expanding egg product processing categories and improving processing levels, but also provides a scientific basis for the functional development of egg white peptide active substances and the creation of high-value products. Attached Figure Description

[0015] Figure 1 HSF cell relative scratch closure rate

[0016] Figure 2 HSF cell proliferation activity

[0017] Figure 3 HSF cell migration ability Detailed Implementation

[0018] The present invention will now be described with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0019] Example 1: Preparation, isolation and identification of egg white peptide QVPLW

[0020] (1) Weigh 50-100g of egg white protein powder, add distilled water, and prepare an egg white protein solution with a mass fraction of 5-10%. Place the egg white protein powder solution in a water bath at 90℃ for 10-15 minutes, and then cool it down to 50℃.

[0021] (2) Adjust the pH of the system to 10 using 1 mol / L NaOH solution. Under the conditions of 50℃ and pH 10, enzymatically hydrolyze the egg white protein solution with alkaline protease (Alcalase) for 2-4 hours, with an Alcalase mass fraction of 3-6%.

[0022] (3) After enzymatic hydrolysis, the enzyme was inactivated by boiling in a water bath for 8-12 minutes, and then cooled to room temperature. The hydrolysate was centrifuged at 8000-12000 r / min for 9-12 minutes at 4℃ to obtain an egg white peptide solution. After in vitro simulated intestinal digestion, the egg white peptide components with a molecular weight of less than 1 kDa were separated and purified by ultrafiltration.

[0023] (4) The structure was identified by LC-MS / MS. Based on the mass spectrum information, combined with software analysis and protein database, the amino acid sequence of the egg white peptide was finally determined.

[0024] Example 2: Construction of a Cellular Mechanical Damage Model

[0025] A cell mechanical damage model was constructed using a cell scratch assay. HSF cells were injected at a rate of 3-6 × 10⁻⁶. 5 Cells were seeded per well in six-well plates with a streaked bottom and cultured for 18-24 hours. A sterile 200 μL pipette tip was used to make a straight cut on the plate vertically, followed by rinsing three times with PBS to remove the cut cells, thus constructing a skin cell mechanical damage model.

[0026] Example 3: Application of 50μM protein peptides in alleviating skin damage

[0027] (1) Cells were treated with different concentrations of QVPLW solution (0 μM, 50 μM), and photographed and recorded under an inverted microscope. The changes in the distance between cells at the same location were compared at 0 h, 24 h and 48 h. The cell migration speed was quantitatively compared, and the relative scratch closure rate was calculated to verify the effect of egg white peptide QVPLW on the migration ability of HSF cells.

[0028] (2) Cell proliferation activity assay: using 3-6 × 10⁻⁶ cells / year. 3 HSF cells were seeded into 96-well black culture plates at a culture density of cells / well. 100 μL of culture medium was added, and the cells were cultured for 12–18 h. The culture medium was then discarded, and the cells were treated with different concentrations of QVPLW solution (0 μM, 50 μM). After incubating the cells with peptides for 18–24 h, 20 μL of LMTS was added and incubated for 1–2 h. The fluorescence intensity at 490 nm was measured using a fluorescent plate reader, and the cell proliferation activity was calculated.

[0029] (3) Transwell cell migration assay: After HSF cells reached 70%-80% confluence, the complete culture medium was replaced with serum-free DMEM, and the cells were starved for 18-24 hours. After washing the cells with PBS, 2 mL of trypsin was added to digest the cells and the cells were counted. Medium containing 10% FBS was added to 24-well plates, and Transwell chambers were placed in the 24-well plates. 3-6 × 10⁶ cells were added to each chamber. 4 Cells were cultured in an incubator with the same concentration of QVPLW solution (0 μM and 50 μM) in both the upper and lower chambers. After 48 h of culture, the culture medium in the upper chamber was discarded, and the cells were washed twice with PBS. The chambers were then inverted and air-dried on a table. Simultaneously, 400-600 μL of methanol was added to a 24-well plate, and the chambers were placed in the plate for fixation. After 15-20 min, the chambers were inverted and air-dried again. The methanol was discarded, and the cells were stained with 0.1% crystal violet solution for 10-15 min. The staining was then stopped, and the chambers were washed three times with PBS until no obvious purple color remained. The cells were then photographed under a microscope.

[0030] (4) Statistical analysis: Data are expressed as mean ± standard deviation (Mean ± SD) of three independent experiments. SPSS 22.0 software was used to analyze differences between samples. One-way ANOVA was used to compare the significance of differences between samples, with Duncan's test selected for significant difference analysis. Statistical significance was set at p < 0.05. * indicates a significant difference between the 0 μM peptide group and other groups (p < 0.05), and ** indicates an extremely significant difference between the 0 μM peptide group and other groups (p < 0.01).

[0031] (5) Experimental results:

[0032] Figure 1 HSF cell relative scratch closure rate

[0033] A cell scratch assay was used to construct a model of cellular mechanical damage. The results showed that the closure rate of HSF cell scratches increased with prolonged treatment time with 50 μM protein peptide QVPLW. Furthermore, the closure rate of cell scratches treated with 50 μM peptide for 24 h was significantly lower than that of the untreated group; however, at 48 h, there was no significant difference in the closure rate between the 50 μM peptide group and the peptide-treated group. This indicates that 50 μM protein peptide can promote the closure of HSF cell scratches, alleviate cellular mechanical damage, and may be an adjunct treatment for skin injuries.

[0034] Figure 2 HSF cell proliferation activity

[0035] The ability of egg white peptide (QVPLW) to promote HSF cell proliferation was evaluated using the MTS cell proliferation assay kit. The results showed that, compared with the untreated HSF (0 μM), QVPLW significantly promoted HSF cell proliferation at a concentration of 50 μM, indicating that QVPLW can alleviate skin damage by promoting HSF cell proliferation.

[0036] Figure 3 HSF cell migration activity

[0037] The cell migration-promoting activity of egg white peptide QVPLW was evaluated using a Transwell cell migration assay. The results showed that, compared to the untreated case (0 μM), QVPLW at a concentration of 50 μM promoted the migration of HSF cells in the Transwell chambers. Furthermore, with increasing QVPLW concentration, the number of cells crossing the bottom of the Transwell increased, indicating an increased cell migration-promoting ability of QVPLW. These results suggest that QVPLW can alleviate skin damage by promoting the migration of HSF cells.

[0038] Example 4: Application of 100-200μM protein peptides in alleviating skin damage

[0039] (1) Cells were treated with different concentrations of QVPLW solution (0 μM, 100 μM, 200 μM), and photographed under an inverted microscope. The changes in the distance between cells at the same location were compared at 0 h, 24 h and 48 h. The cell migration speed was quantitatively compared and the relative scratch closure rate was calculated to verify the effect of egg white peptide QVPLW on the migration ability of HSF cells.

[0040] (2) Cell proliferation activity assay: using 3-6 × 10⁻⁶ cells / year. 3HSF cells were seeded into 96-well black culture plates at a culture density of cells / well. 100 μL of culture medium was added, and the cells were cultured for 12–18 h. The culture medium was then discarded, and the cells were treated with different concentrations of QVPLW solution (0 μM, 100 μM, 200 μM). After incubating the cells with peptides for 18–24 h, 20 μL of MTS was added and incubated for 1–2 h. The fluorescence intensity at 490 nm was measured using a fluorescent plate reader, and the cell proliferation activity was calculated.

[0041] (3) Transwell cell migration assay: After HSF cells reached 70%-80% confluence, the complete culture medium was replaced with serum-free DMEM, and the cells were starved for 18-24 hours. After washing the cells with PBS, 2 mL of trypsin was added to digest the cells and the cells were counted. Medium containing 10% FBS was added to 24-well plates, and Transwell chambers were placed in the 24-well plates. 3-6 × 10⁶ cells were added to each chamber. 4 Cells were cultured in an incubator with equal concentrations of QVPLW solution (0 μM, 100 μM, 200 μM) added to both the upper and lower chambers. After 48 hours of culture, the culture medium in the upper chamber was discarded, and the cells were washed twice with PBS. The chambers were then inverted and air-dried on a table. Simultaneously, 400-600 μL of methanol was added to a 24-well plate, and the chambers were placed in the plate for fixation. After 15-20 minutes, the chambers were inverted and air-dried again. The methanol was discarded, and the cells were stained with 0.1% crystal violet solution for 10-15 minutes. The staining was then stopped, and the chambers were washed three times with PBS until no obvious purple color remained. The cells were then photographed under a microscope.

[0042] (4) Statistical analysis: Data are expressed as mean ± standard deviation (Mean ± SD) of three independent experiments. SPSS 22.0 software was used to analyze differences between samples. One-way ANOVA was used to compare the significance of differences between samples, with Duncan's test selected for significant difference analysis. Statistical significance was set at p < 0.05. * indicates a significant difference between the 0 μM peptide group and other groups (p < 0.05), and ** indicates an extremely significant difference between the 0 μM peptide group and other groups (p < 0.01).

[0043] (5) Experimental results:

[0044] Figure 1 HSF cell relative scratch closure rate

[0045] A cell scratch assay was used to construct a model of cellular mechanical damage. The results showed that the ability of QVPLW to promote cell scratch closure increased with increasing concentration. Compared to the control group without egg white peptide (0 μM), HSF cells cultured with egg white peptide QVPLW for 24 h and 48 h showed that both 100 μM and 200 μM QVPLW significantly promoted scratch closure. Compared to the control group without egg white peptide, treatment with 100-200 μM QVPLW for 48 h promoted HSF cell scratch closure by 150%-250%. These results demonstrate that egg white peptide QVPLW can promote HSF cell scratch closure, alleviate cellular mechanical damage, and may be used as an adjunct treatment for skin injuries.

[0046] Figure 2 HSF cell proliferation activity

[0047] The ability of egg white peptide (QVPLW) to promote HSF cell proliferation was evaluated using the MTS cell proliferation assay kit. The results showed that, compared with the untreated HSF (0 μM), QVPLW significantly promoted HSF cell proliferation at concentrations of 100 μM and 200 μM, indicating that QVPLW can alleviate skin damage by promoting HSF cell proliferation.

[0048] Figure 3 HSF cell migration activity

[0049] The cell migration-promoting activity of egg white peptide QVPLW was evaluated using a Transwell cell migration assay. The results showed that, compared to the untreated case (0 μM), QVPLW at concentrations of 100 μM and 200 μM promoted the migration of HSF cells in the Transwell chambers. Furthermore, with increasing QVPLW concentration, the number of cells crossing the bottom of the Transwell increased, indicating an increased cell migration-promoting capacity of QVPLW. These results suggest that QVPLW can alleviate skin damage by promoting the migration of HSF cells.

[0050] The above description is only a preferred embodiment of the present invention. For those skilled in the art, various improvements and modifications can be made to the above embodiments without departing from the principles and spirit of the present invention, and such improvements and modifications all fall within the protection scope of the present invention.

Claims

1. Use of an egg white peptide QVPLW, characterized in that: The egg white peptide QVPLW is used for preparing a medicine for relieving skin injury. The HSF cell scratch experiment is used to construct a cell mechanical injury model, and the egg white peptide QVPLW is used to treat the mechanical injury cells; the MTS cell proliferation experiment and the Transwell cell migration experiment are used to prove the effect of relieving skin injury.

2. Use according to claim 1, characterized in that, Compared with the blank group without the egg white peptide, the group containing the egg white peptide QVPLW promotes the closure of the human skin fibroblast scratch by 150%-250% when the cells are treated with 100-200 μM of QVPLW for 48 h.

3. Use according to claim 1, characterized in that, The egg white peptide QVPLW relieves the human skin fibroblast injury caused by the cell scratch experiment, and increases the proliferation of the human skin fibroblast and the migration of the human skin fibroblast in the Transwell chamber.

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