Preparation method of egg yolk protein peptide and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a preparation method of egg yolk protein peptide and application thereof. The preparation method is obtained by step-by-step enzymatic hydrolysis combined with macroporous resin, ultrafiltration and gel chromatography separation and purification, and further 10 oligopeptides with specific sequences are identified, and then GQILPDKFV, RPLDSGFNH and LFDSPCCTI with the most optimal antioxidant activity are further screened out. GQILPDKFV can be used in cosmetics and further compounded with lariciresin to achieve synergistic effect, which greatly expands the application potential of egg yolk peptide in cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing egg yolk protein peptides and their applications. Background Technology

[0002] Egg yolk protein is an important protein component of eggs, rich in various functional proteins, including phosphoprotein, low-density lipoprotein, high-density lipoprotein, and vitellin. In recent years, with the development of enzymatic hydrolysis technology, egg yolk protein has attracted much attention due to its unique biological activity. Its enzymatic hydrolysis product, egg yolk peptides, shows broad application prospects in functional foods and biomedicine. Studies have shown that egg yolk protein hydrolysates possess various physiological activities, including antioxidant, blood pressure-lowering, osteogenic promotion, and immunomodulatory effects. Among these, antioxidant activity is one of the most fundamental and widely studied functional properties of egg yolk peptides.

[0003] In the preparation of egg yolk peptides through enzymatic hydrolysis, Qin Jiayan et al. ("Preparation of Egg Yolk Protein Peptides and Study on Their Antioxidant Activity") used alkaline protease and complex flavor protease to stepwise hydrolyze egg yolk protein. After separation by gradient ultrafiltration and Sephadex G-25 gel chromatography, they obtained peptide components with antioxidant activity and determined their scavenging abilities against DPPH free radicals, ABTS free radicals, and hydroxyl free radicals. However, this study only focused on the activity evaluation at the crude component level, without identifying specific peptide sequences or exploring the application of egg yolk peptides in broader functional areas such as skin whitening.

[0004] Furthermore, CN110101084B discloses an algal oil nano-pickerlin emulsion stabilized by egg yolk protein peptide particles. This emulsion uses trypsin to enzymatically deliquinate egg yolk protein powder to prepare the egg yolk protein peptide particles, primarily utilizing the interfacial emulsification properties of the egg yolk peptides rather than their biological activity. CN119033614A discloses a naturally derived whitening composition containing raspberry glycoside (raspberry ketone glucoside), ellagic acid, and polypeptides (EAPSTNHESDN and YPSDNRAWEAPQ) with tyrosinase inhibitory activity prepared from mussels. This patent combines whitening ingredients with polypeptides, but the polypeptides used are derived from mussels rather than egg yolks, and it does not address the synergistic mechanism between antioxidant peptides and whitening ingredients.

[0005] In summary, the existing technology has the following shortcomings: on the one hand, the sequences of antioxidant peptides prepared from egg yolk protein have not been fully identified and clarified; on the other hand, the synergistic effect between egg yolk antioxidant peptides and natural whitening ingredients has not been revealed, which limits the application potential of egg yolk peptides in the field of whitening cosmetics and skin care products.

[0006] Therefore, how to efficiently prepare antioxidant oligopeptides with specific sequences from egg yolk protein and explore their synergistic combination with natural whitening ingredients is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing egg yolk protein peptides and their applications, in order to solve the problem of unclear egg yolk protein peptide sequences and efficacy in the prior art, and to expand their application prospects.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an egg yolk protein peptide, wherein the amino acid sequence of the egg yolk protein peptide is selected from at least one of the following sequences: GQILPDKFV, RPLDSGFNH, LFDSPCCTI, AYVKN, VEDQPMKGA, KICTDQ, MTFQNRLDA, YHPDLSECK, NESYAKFT, DLPEMRQHS.

[0009] As a preferred technical solution, the amino acid sequence of the egg yolk protein peptide is selected from any one of RPLLDSGFNH, LFDSPCCTI, or GQILPDKFV.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned egg yolk protein peptide, which is carried out by solid-phase synthesis or liquid-phase synthesis.

[0011] Thirdly, the present invention provides a composition comprising the above-mentioned egg yolk protein peptide and a whitening ingredient, wherein the whitening ingredient is selected from at least one of pine resin, ellagic acid, tetrahydromagnoliol, and raspberry ketone glucoside.

[0012] As a preferred technical solution, the egg yolk protein peptide is GQILPDKFV, and the whitening ingredient is pine bark extract.

[0013] As a preferred technical solution, the mass ratio of GQILPDKFV to pine resin is 1:3.

[0014] Fourthly, the present invention provides the use of the above composition in the preparation of cosmetics or skin care products having antioxidant and / or whitening effects.

[0015] As a preferred technical solution, the dosage form of the cosmetic or skin care product is a lotion, cream, serum, or mask.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: Firstly, this invention is the first to identify 10 oligopeptides with specific sequences from egg yolk protein hydrolysates. In vitro antioxidant activity screening confirmed that GQILPDKFV, RPLDSGFNH, and LFDSPCCTI possess significant antioxidant activity. In different antioxidant assay systems, each peptide exhibited differentiated advantages: RPLDSGFNH showed the strongest scavenging ability against DPPH and ABTS free radicals; while GQILPDKFV showed the highest activity in hydroxyl radical scavenging experiments.

[0017] Secondly, this invention is the first to discover and verify a synergistic effect between the egg yolk antioxidant peptide GQILPDKFV and pine resin. In vitro experimental data show that although GQILPDKFV, when used alone, has weaker tyrosinase inhibitory activity and some antioxidant indicators than RPLDSGFNH and LFDSPCCTI, when combined with pine resin at a 1:3 mass ratio, it exhibits a synergistic effect on two key whitening indicators: tyrosinase inhibition and inhibition of melanin synthesis in B16 melanoma cells. Notably, the measured inhibition rate of this combination is not only higher than that of GQILPDKFV and pine resin used alone, but also higher than that of the more potent single-use RPLDSGFNH or LFDSPCCTI combined with pine resin under the same conditions. This result suggests that the specific sequence structure of GQILPDKFV may endow it with a unique ability to produce a highly efficient synergistic interaction with pine resin, thus overcoming the activity limitations of single egg yolk peptides. This discovery provides new scientific evidence for the application of egg yolk peptides in cosmetics or skincare products with synergistic whitening effects. Detailed Implementation

[0018] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and are therefore only examples and should not be used to limit the scope of protection of the present invention.

[0019] Example 1 This embodiment provides a method for preparing egg yolk protein peptides, as follows: (1) Take 100g of fresh egg yolks and add 900mL of distilled water to prepare a 10% suspension. Stir well. First, add 0.8% alkaline protease (based on substrate mass), adjust the pH to 8.5 with 1mol / L NaOH, and hydrolyze in a 50℃ constant temperature water bath for 2h. During the hydrolysis process, keep stirring and maintain a constant pH. Then, add 0.4% complex flavor protease (based on substrate mass), adjust the pH to 6.0 with 1mol / L HCl, and continue hydrolyzing in a 50℃ constant temperature water bath for 2h. After the enzymatic hydrolysis is completed, place the hydrolysate in a 90℃ water bath for 10min to inactivate the enzyme, cool, and centrifuge at 8000r / min for 20min. Take the supernatant.

[0020] (2) The supernatant was loaded onto a DA201-CII macroporous resin column (column volume 200 mL) at a flow rate of 1.5 BV / h. First, it was eluted with 3 BV of distilled water to remove salts and macromolecular impurities, and then eluted with 3 BV of 20% ethanol solution at a flow rate of 1.5 BV / h. The 20% ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried.

[0021] (3) Dissolve the macroporous resin eluent in distilled water and pass it through ultrafiltration membranes with molecular weight cutoffs of 5kD and 3kD in sequence at an operating pressure of 0.2MPa. Collect the filtrate that has passed through the 3kD membrane and freeze-dry it.

[0022] (4) The ultrafiltration filtrate was dissolved in distilled water and loaded onto a Sephadex G-25 gel chromatography column (2.6cm×60cm). It was eluted with an aqueous solution containing 0.15mol / L NaCl at a flow rate of 0.5mL / min. The detection wavelength was 280nm. The main elution peak was collected, concentrated and desalted under reduced pressure, and then freeze-dried to obtain the egg yolk protein peptide component (F component).

[0023] Example 2 This embodiment describes the peptide sequence identification of component F prepared in Example 1, specifically as follows: Take an appropriate amount of the freeze-dried component F from Example 1, dissolve it in 0.1% formic acid aqueous solution to prepare a sample solution with a concentration of 1 μg / μL. After centrifuging at 12000 r / min for 10 min to remove insoluble impurities, take the supernatant for testing.

[0024] Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using a Waters Acquity UPLC BEH C18 reversed-phase column (2.1 mm × 100 mm, 1.7 μm). The column temperature was set at 40 °C, and the flow rate was controlled at 0.3 mL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The elution gradient was set as follows: 0–2 min to maintain 5% B, 2–35 min to linearly increase to 35% B, 35–38 min to increase to 80% B and maintain it, and then rapidly return to 5% B equilibrium.

[0025] Mass spectrometry was performed in electrospray ionization (ESI+) mode with a full scan range of m / z 200-2000. Data-dependent acquisition (DDA) mode was used for automatic secondary fragmentation of precursor ions with high signal intensity. The acquired raw mass spectrometry data were imported into PEAKS Studio software, and the secondary mass spectra were analyzed using the De novo sequencing algorithm. During the analysis, peptide confidence scores (ALC scores) and peak intensities were used for screening. Ultimately, 10 major peptide sequences were identified from fraction F: AYVKN, LFDSPCCTI, VEDQPMKGA, RPLDSGFNH, KICTDQ, MTFQNRLDA, YHPDLSECK, GQILPDKFV, NESYAKFT, and DLPEMRQHS.

[0026] Example 3 This embodiment further screens the peptides obtained in Example 2 above for antioxidant activity: The 10 peptides identified in Example 2 were chemically synthesized by a peptide synthesis company using solid-phase synthesis, achieving a purity ≥95%. 1 mg / mL peptide solutions were prepared for the following antioxidant activity tests: (1) Determination of DPPH free radical scavenging rate: Take 2 mL of 0.1 mmol / L DPPH-ethanol solution, add 2 mL of peptide solutions of different concentrations, mix well, react in the dark for 30 min, measure the absorbance at 517 nm, and calculate the scavenging rate and IC50. 50 value.

[0027] (2) Determination of ABTS free radical scavenging rate: Mix 7 mmol / L ABTS solution with an equal volume of 2.45 mmol / L potassium persulfate solution, and incubate in the dark for 12-16 h. Dilute with PBS to an absorbance of 0.70 ± 0.02. Add 2 mL of ABTS working solution to 0.1 mL of peptide solution, react for 6 min, and measure the absorbance at 734 nm. Calculate the scavenging rate and IC50. 50 value.

[0028] (3) Determination of hydroxyl radical scavenging rate: The Fenton reaction system was used. 0.1 mL of peptide solution, 0.6 mL of 6 mmol / L FeSO4, 0.6 mL of 6 mmol / L salicylic acid and 0.6 mL of 6 mmol / L H2O2 were added to 1 mL of the reaction system. After reacting at 37℃ for 30 min, the absorbance was measured at 510 nm, and the scavenging rate and IC50 were calculated. 50 value.

[0029] The experimental results are shown in Table 1.

[0030] Table 1. Antioxidant activity (IC50) of 10 egg yolk protein peptides 50Value (μg / mL)

[0031] Table 1 lists the IC50 values ​​of 10 chemically synthesized peptides in three free radical scavenging systems. 50 The results showed that the antioxidant activities of different peptides varied significantly. Specifically, RPLDSGFNH showed the highest IC50 values ​​in both DPPH and ABTS systems. 50 The values ​​were the lowest (87.3 and 62.6 μg / mL, respectively), indicating the strongest DPPH and ABTS radical scavenging abilities. LFDSPCCTI showed the second highest ABTS scavenging ability. GQILPDKFV, on the other hand, exhibited the highest activity (IC50) in the hydroxyl radical scavenging experiment. 50 (148.6 μg / mL), and its DPPH and ABTS scavenging abilities also remained at a high level.

[0032] Based on the three indicators, RPLDSGFNH, GQILPDKFV, and LFDSPCCTI were identified as the three peptides with relatively strong antioxidant activity in this batch. The remaining seven peptides showed varying IC50 values ​​in their respective systems. 50 The values ​​are all relatively high, indicating weak antioxidant activity.

[0033] Example 4 The top-ranking antioxidant peptide GQILPDKFV, selected in Example 3, was combined with pine bark extract at a mass ratio of 1:3 to prepare a composition. Specifically, 10 mg of GQILPDKFV and 30 mg of pine bark extract were weighed and dissolved in 10 mL of PBS buffer (pH 6.8) containing 5% DMSO. The mixture was thoroughly mixed to obtain a stock solution of the composition at a concentration of 4 mg / mL. Subsequent experiments required dilution to the corresponding test concentration.

[0034] Comparative Example 1-1: The only difference from Example 4 is that the mass ratio of GQILPDKFV to pine resin is 1:1.

[0035] Comparative Examples 1-2: The only difference from Example 4 is that the mass ratio of GQILPDKFV to pine resin is 1:5.

[0036] Comparative Examples 1-3: The only difference from Example 4 is that RPLLDSGFNH is used instead of GQILPDKFV and is compounded with pine syrup at a mass ratio of 1:3.

[0037] Comparative Examples 1-4: The only difference from Example 4 is that LFDSPCCTI is used instead of GQILPDKFV and is compounded with pine syrup at a mass ratio of 1:3.

[0038] Comparative Examples 1-5: The only difference from Example 4 is that ellagic acid is used instead of pine oleate and is compounded with GQILPDKFV at a mass ratio of 1:3.

[0039] Comparative Examples 1-6: The only difference from Example 4 is that tetrahydromagnoliol is used instead of pine syrup, and it is compounded with GQILPDKFV at a mass ratio of 1:3.

[0040] Comparative Examples 1-7: The only difference from Example 4 is that raspberry ketone glucoside is used instead of pine syrup, and it is compounded with GQILPDKFV at a mass ratio of 1:3.

[0041] Experimental Example 1

[0042] 1.1 Effect of the combination of antioxidant peptides and whitening ingredients on tyrosinase inhibition rate

[0043] Tyrosinase is a key rate-limiting enzyme in melanin biosynthesis, and inhibiting its activity is an important in vitro indicator for evaluating the skin-whitening efficacy of substances. This experiment aims to investigate the effect of egg yolk antioxidant peptides combined with different plant-derived skin-whitening ingredients on tyrosinase activity, thereby observing whether the two have a synergistic effect and determining the optimal combination ratio and specific combination.

[0044] In this experiment, L-tyrosine was used as the substrate. Tyrosinase solution and different combination solutions were added to PBS buffer (pH 6.8). After reacting at 37℃ for 15 min, the absorbance was measured at 475 nm to calculate the tyrosinase inhibition rate. The total test concentration of all single control groups and compound experimental groups was set to 100 μg / mL. The measured inhibition rates of each group were directly compared to evaluate the compound effect. The results are shown in Table 2.

[0045] Table 2. Effect of the combination of antioxidant peptides and whitening ingredients on tyrosinase inhibition rate.

[0046] Table 2 shows the inhibition rates of tyrosinase in each experimental group at a total concentration of 100 μg / mL. In the single-peptide control group, the inhibition rates of the three peptides were ranked as follows: LFDSPCCTI (32.5%) > RPLDSGFNH (28.6%) > GQILPDKFV (23.4%), all lower than that of pine pine extract alone (52.3%). In the compound group, Example 4 (GQILPDKFV:pine pine extract = 1:3) showed the highest inhibition rate, reaching 75.8%, significantly higher than the values ​​of either peptide alone. After changing the ratio, the inhibition rates of Comparative Example 1-1 (1:1) and Comparative Example 1-2 (1:5) were 63.5% and 67.3%, respectively, both lower than the 1:3 ratio group, indicating that under these experimental conditions, 1:3 is the optimal compound ratio.

[0047] It is noteworthy that when GQILPDKFV was replaced with RPLDSGFNH or LFDSPCCTI (comparative Examples 1-3 and 1-4), which have higher single-use activity, the inhibition rates at the same 1:3 ratio were 62.1% and 53.8%, respectively, both lower than in Example 4. This result indicates that there is no positive correlation between the single-use activity and the synergistic potential of the combination; GQILPDKFV exhibits a stronger synergistic inhibitory effect when combined with pine bark extract. Furthermore, when pine bark extract was replaced with other whitening ingredients (comparative Examples 1-5 to 1-7), the inhibition rates significantly decreased, indicating that this synergistic effect is combination-specific.

[0048] 1.2 Effects of the combination of antioxidant peptides and whitening ingredients on melanin content in B16 cells

[0049] This study aims to observe the optimal composition obtained from in vitro screening at the B16 melanocyte level.

[0050] The experimental method is briefly described as follows: B16 cells were cultured in DMEM medium containing 10% FBS and seeded in 6-well plates. After cell adhesion, medium containing different compositions was added to each group. The total test concentration for each group was 100 μg / mL. A control group was also set up. After culturing for another 48 h, cell lysis was collected and absorbance was measured. The melanin content inhibition rate was calculated. The experimental results are shown in Table 3.

[0051] Table 3. Effects of the combination of antioxidant peptides and whitening ingredients on melanin content in B16 cells.

[0052] Table 3 shows the inhibition rate of melanin synthesis in the B16 cell model for each group, with a total test concentration of 100 μg / mL. When used alone, the inhibition rate of GQILPDKFV (24.8%) was lower than that of RPLDSGFNH (30.5%) and LFDSPCCTI (35.2%), while the inhibition rate of pine resin alone was 38.5%. The inhibition rate of the composition in Example 4 (GQILPDKFV:pine resin = 1:3) reached 65.2%, which was higher than that of each single-use group and other compound groups.

[0053] After changing the ratio, the inhibition rates of comparative ratios 1-1 (1:1) and 1-2 (1:5) decreased to 42.6% and 51.3%, respectively, indicating a weakened synergistic effect and further supporting 1:3 as the optimal ratio. The groups that replaced GQILPDKFV with RPLDSGFNH or LFDSPCCTI (comparative ratios 1-3 and 1-4) showed inhibition rates of 53.4% ​​and 45.7%, respectively, neither reaching the level of Example 4. The above cellular experimental results are consistent with the trends in in vitro enzymatic experiments, indicating that when GQILPDKFV and pine resin are combined in a 1:3 ratio, they also exhibit superior melanin synthesis inhibition at the cellular level.

[0054] 1.3 Comparison with existing technology in CN119033614A

[0055] Based on CN119033614A, the following comparative example is set: Comparative Example 2-1: Following CN119033614A, mussel polypeptide EAPSTNHESDN was synthesized and compounded with pine syrup at a mass ratio of 1:3. The sample was recorded as Comparative Example 2-1.

[0056] Comparative Example 2-2: Following CN119033614A, the mussel polypeptide YPSDNRAWEAPQ was synthesized and compounded with pine syrup at a mass ratio of 1:3. The sample was recorded as Comparative Example 2-2.

[0057] This test aims to compare the antioxidant and whitening effects of the composition of Example 4 with those of Comparative Examples 2-1 and 2-2. The total concentration of the test samples was set at 100 μg / mL, and the DPPH free radical scavenging rate and tyrosinase inhibition rate were measured. The results are shown in Table 4.

[0058] Table 4 Comparison of the effects of the composition with existing technology compositions

[0059] Table 4 compares the effects of the composition of Example 4 with the mussel polypeptide-pine bark extract combination in the prior art (CN119033614A) at the same total concentration. Regarding DPPH free radical scavenging rate, Example 4 achieved 87.3%, which is 23.8 and 28.2 percentage points higher than Comparative Examples 2-1 (63.5%) and 2-2 (59.1%), respectively. Regarding tyrosinase inhibition rate, Example 4 achieved 76.4%, which is 9.2 and 11.0 percentage points higher than Comparative Examples 2-1 (67.2%) and 2-2 (65.4%), respectively. These comparative results indicate that, under the experimental conditions, the combination of egg yolk peptide GQILPDKFV and pine bark extract provided by this invention is superior to the mussel polypeptide combination scheme used in the prior art in both antioxidant and tyrosinase inhibition indicators.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A yolk protein peptide, characterized in that, The amino acid sequence of the egg yolk protein peptide is GQILPDKFV.

2. A method for preparing the egg yolk protein peptide of claim 1, characterized in that, Artificial chemical synthesis is carried out using solid-phase synthesis or liquid-phase synthesis.

3. A composition, characterized in that, It contains the egg yolk protein peptide as described in claim 1 and a whitening ingredient, wherein the whitening ingredient is pine bark extract.

4. The composition according to claim 3, characterized in that, The mass ratio of GQILPDKFV to pine resin is 1:

3.

5. The use of the composition according to any one of claims 3 or 4 in the preparation of cosmetics or skin care products having antioxidant and / or whitening effects.

6. The application according to claim 5, characterized in that, The cosmetics or skincare products mentioned are in the form of lotion, cream, serum, or mask.

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