Alpha-lactalbumin multifunctional peptide with antioxidant, anticoagulant and tyrosinase inhibitory activities and application thereof

Abstract

This invention discloses a multifunctional α-lactalbumin peptide with antioxidant, anticoagulant, and tyrosinase inhibitory activities and its applications, belonging to the fields of food, biopharmaceutical, and cosmetic technologies. The multifunctional peptide SEKLDQWLCEKL obtained through screening exhibits the following characteristics: a DPPH scavenging rate of 62.32% and an ABTS scavenging rate of 88.32%, a hydroxyl radical scavenging rate of 78.89%, a PT and TT prolongation of 3.4 s and TT, respectively, and a tyrosinase inhibition rate of 89.07%. This overcomes the limitations of existing single-functional peptides in application.

Description

Technical Field

[0001] This invention relates to a multifunctional α-lactalbumin peptide with antioxidant, anticoagulant and tyrosinase inhibitory activities and its applications, belonging to the fields of food, biopharmaceutical and cosmetic technologies. Background Technology

[0002] Whey protein, a major byproduct of bovine milk casein production, is hailed as the "king of proteins" due to its balanced amino acid composition and high nutritional value. It is composed of various functional proteins, including α-lactalbumin and lactoferrin. In recent years, with the maturation of enzymatic hydrolysis technology, releasing bioactive peptides with specific physiological functions from whey proteins (such as α-lactalbumin) has become a research hotspot in food science and biomedicine. These whey protein peptides exhibit enormous application potential due to their small molecular weight, high absorption efficiency, and diverse biological activities. Known activities include immune enhancement, blood pressure reduction, antibacterial activity, and antioxidant activity.

[0003] Among numerous bioactivities, antioxidant and anticoagulant activities have garnered significant attention due to their importance in maintaining bodily health, particularly in preventing cardiovascular disease. Oxidative stress is a key initiating factor leading to cell damage, inflammation, and atherosclerosis; while a hypercoagulable state of blood is a direct cause of thrombosis, subsequently inducing acute events such as myocardial infarction and cerebral infarction. Simultaneously, abnormally elevated activity of tyrosinase, a key rate-limiting enzyme in melanin production, is a major cause of skin pigmentation and age spots. Theoretically, active ingredients possessing multiple functions—antioxidant, anticoagulant, and tyrosinase inhibitory—can synergistically intervene at different pathological or physiological stages, providing more comprehensive protection for cardiovascular health and playing a role in improving skin pigmentation. Their comprehensive application value is significantly higher than that of single-function ingredients.

[0004] However, current research and applications of whey protein peptides have significant limitations. Most studies focus on developing and characterizing peptides with single activities, such as obtaining peptides with antioxidant or anticoagulant functions through conventional enzymatic hydrolysis. These peptides are mostly single-function and cannot meet the practical needs of multi-target intervention for complex health problems (such as atherosclerosis). Therefore, there is an urgent need in this field for a method to efficiently and purposefully prepare whey protein peptides with significant antioxidant, anticoagulant, and tyrosinase-inhibiting multiple bioactive properties, and to expand their application in practical products, thereby filling the current technological gap in the development of comprehensive functional peptides. Summary of the Invention

[0005] [Technical Issues] Existing technologies mostly focus on improving the hydrolysis degree or general biological activity (such as antioxidation) of bioactive peptides, but lack multifunctional peptides that simultaneously possess antioxidant, anticoagulant, and tyrosinase inhibitory activities.

[0006] [Technical Solution] To address the aforementioned problems, the present invention aims to provide a whey protein peptide with antioxidant and anticoagulant activities, and its preparation method. The method involves hydrolyzing whey protein using enzymatic co-fermentation technology. The hydrolysate is then freeze-dried to obtain peptide powder rich in antioxidant and anticoagulant activities. LC-MS / MS technology is then used to identify the peptide fragments contained in the peptide powder, screening for active peptides with multiple functions including antioxidant, anticoagulant, and tyrosinase inhibitory activities. These active peptides are then added to a product to obtain a functional product with antioxidant, anticoagulant, and tyrosinase inhibitory activities.

[0007] To achieve the above objectives, the present invention first provides a method for preparing whey protein peptides with antioxidant, anticoagulant, and tyrosinase-inhibiting effects, comprising the following steps: (1) Dissolve whey protein in deionized water and sterilize it to obtain a whey protein aqueous solution; (2) Ferment the whey protein aqueous solution obtained in step (1) using Lactobacillus plantarum; (3) Sterilize the fermentation broth from step (2), and then hydrolyze it with bromelain. (4) Freeze-dry the supernatant obtained in step (3); (5) The lyophilized mixture from step (4) was identified by LC-MS / MS to obtain a series of peptide sequences; (6) After synthesis by solid phase method, the antioxidant activity was verified and pure peptide with both antioxidant and anticoagulant activities was screened out and denoted as SEKLDQWLCEKL.

[0008] In one embodiment of the present invention, the inoculation amount of Lactobacillus plantarum in step (2) is 6%.

[0009] In one embodiment of the present invention, the fermentation temperature in step (2) is 30 °C.

[0010] In one embodiment of the present invention, the fermentation time in step (2) is 18 h.

[0011] In one embodiment of the present invention, the amount of enzyme added in step (3) is 2500 U / g.

[0012] In one embodiment of the present invention, the enzymatic hydrolysis temperature in step (3) is 55 °C.

[0013] In one embodiment of the present invention, the enzymatic hydrolysis time in step (3) is 2 h.

[0014] In one embodiment of the present invention, the freeze-drying in step (4) is performed by freezing under conditions where the cold trap temperature is below -50 °C, the vacuum degree is 0.0 mbar, and the freeze-drying time is 48 h.

[0015] In one embodiment of the present invention, the LC-MS / MS detection conditions in step (5) are as follows: the sample is separated by a gradient of 60 min, the column flow rate is controlled at 300 nL / min, the column temperature is 40 °C, the electrospray voltage is 2 kV, the gradient starts from 5% B phase, increases to 72% within 57 min, increases to 99% within 1 min, and is maintained for 2 min.

[0016] The mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows: (1) MS: Scan range (m / z): 200-1550; Resolution: 120,000; Normalized AGC target: 300%; Maximum injection time: 20 ms; (2) HCD-MS / MS: Resolution: 15,000; Normalized AGC target: 200%; Maximum injection time: 22 ms; Collision energy: 32%; Dynamic exclusion time: 35 s.

[0017] This invention provides an α-lactalbumin multifunctional peptide with antioxidant, anticoagulant, and tyrosinase inhibitory activities. The amino acid sequence of the α-lactalbumin multifunctional peptide is Ser-Glu-Lys-Leu-Asp-Gln-Trp-Leu-Cys-Glu-Lys-Leu, denoted as SEKLDQWLCEKL.

[0018] In one embodiment of the present invention, the α-lactalbumin multifunctional peptide is isolated from α-lactalbumin in whey protein.

[0019] The present invention also provides products containing the α-lactalbumin multifunctional peptide, wherein the products are pharmaceuticals or daily chemical products.

[0020] In one embodiment of the present invention, the daily chemical products include cosmetics or skin care products.

[0021] In one embodiment of the present invention, the dosage form of the daily chemical product includes solution, emulsion, cream, gel, powder, aerosol, wax-based agent or suspension.

[0022] In one embodiment of the present invention, the daily chemical products include sheet masks, cream masks, peel-off masks, toners, lotions, gels, makeup removers, serums, or massage oils.

[0023] In one embodiment of the present invention, the drug further comprises conventional pharmaceutical carriers and / or pharmaceutical excipients.

[0024] In one embodiment of the present invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0025] In one embodiment of the present invention, the pharmaceutical excipients include excipients, disintegrants, lubricants, or flavoring agents.

[0026] In one embodiment of the present invention, the medicine contains one or more of the following: vitamin C, grape seed extract, DHA powder, beetroot powder, mixed berry powder, maltodextrin, and fructooligosaccharides.

[0027] In one embodiment of the present invention, the dosage form of the drug includes tablets, capsules, granules, injections, liposome nanoparticles, sustained-release agents, or dispersible tablets.

[0028] The present invention also provides the application of the α-lactalbumin multifunctional peptide in the preparation of antioxidant and / or whitening and spot-fading products.

[0029] In one embodiment of the present invention, the product includes daily chemical products.

[0030] The present invention also provides the use of the α-lactalbumin multifunctional peptide in the preparation of antioxidant and / or anticoagulant pharmaceutical products.

[0031] Beneficial effects This invention obtains whey protein mixed peptides through enzymatic hydrolysis and co-fermentation. Using a combination of activity verification and mass spectrometry identification, a novel multifunctional peptide with a defined sequence (SEKLDQWLCEKL) was screened and verified. At a concentration of 5 mg / mL, the α-lactalbumin multifunctional peptide SEKLDQWLCEKL achieved maximum DPPH and ABTS scavenging rates of 62.32% and 88.32%, respectively. At a concentration of 2.5 mg / mL, it achieved a maximum hydroxyl radical scavenging rate of 78.89%. At a concentration of 7.5 mg / mL, SEKLDQWLCEKL prolonged PT and TT by 3.4 s and 4.7 s, respectively. At a concentration of 5 mg / mL, it achieved an inhibition rate of 89.07% against tyrosinase, overcoming the limitations of existing single-functional peptides in application. Attached Figure Description

[0032] Figure 1 The antioxidant activity of whey protein mixed peptides. Figure 1 In the figure, (A) represents the DPPH scavenging rate of whey protein mixed peptides produced by fermentation and enzymatic hydrolysis; Figure 1 (B) represents the scavenging rate of hydroxyl radicals by the whey protein mixed peptides produced by fermentation and enzymatic hydrolysis; Figure 1 (C) represents the scavenging rate of ABTS free radicals by the whey protein mixed peptides produced by fermentation and enzymatic hydrolysis.

[0033] Figure 2 This describes the anticoagulant activity of whey protein peptides. Figure 2 (A) in the figure represents the APTT of whey protein mixed peptides produced by fermentation and enzymatic hydrolysis; Figure 2 (B) in the figure represents the PT of whey protein mixed peptides produced by fermentation and enzymatic hydrolysis; Figure 2 (C) in the figure represents the TT of whey protein mixed peptides produced by fermentation and enzymatic hydrolysis.

[0034] Figure 3 LC-MS / MS identification results of peptides in a mixture of whey protein hydrolysates.

[0035] Figure 4 The antioxidant activity of the pure peptide SEKLDQWLCEKL. Figure 4 In the figure, (A) represents the scavenging rate of the pure peptide SEKLDQWLCEKL against DPPH free radicals; Figure 4 (B) represents the scavenging rate of the pure peptide SEKLDQWLCEKL against ABTS free radicals; Figure 4 In the figure, (C) represents the scavenging rate of hydroxyl radicals by the pure peptide SEKLDQWLCEKL.

[0036] Figure 5 The anticoagulant activity of the pure peptide SEKLDQWLCEKL. Figure 5 (A) in the text represents the PT of the pure peptide SEKLDQWLCEKL; Figure 5 (B) in the text represents the TT of the pure peptide SEKLDQWLCEKL.

[0037] Figure 6 The tyrosinase inhibitory activity of the pure peptide SEKLDQWLCEKL. Detailed Implementation

[0038] The specific embodiments of the present invention will be further described below, but the scope of protection of the present invention is not limited to the scope described in the embodiments.

[0039] Raw material sources: The bromelain used in the examples and comparative examples of this invention had an enzyme activity of 300 U / mg and was purchased from Shanghai Yuanye Co., Ltd. The whey protein used in the examples and comparative examples of this invention was also purchased from Shanghai Yuanye Co., Ltd. APTT, PT, and TT kits were purchased from Wuhan Zhongtai Biotechnology Co., Ltd.

[0040] Example 1: Isolation and Functional Verification of Multifunctional Hybrid Peptides from Whey Protein 1. Isolation of multifunctional mixed peptides of whey protein (1) Fermentation by Lactobacillus plantarum Second-generation Lactobacillus plantarum was inoculated at a rate of 6% into a sterilized 3% (w / v) whey protein solution (containing 0.5% glucose) at pH 6.8. Fermentation was carried out for 18 h at a shaker speed of 130 rpm and a temperature of 30 °C.

[0041] (2) Enzymatic hydrolysis of bromelain After fermentation, the fermentation broth was subjected to enzyme inactivation at 90 °C for 10 min. Then, whey protein was hydrolyzed using bromelain under the following conditions: temperature 55 °C, pH 7, enzyme dosage 2500 U / g, and hydrolysis time 2 h. After hydrolysis, the hydrolysate was heated in a boiling water bath for 10 min to inactivate the protease. After cooling to room temperature, the pH was adjusted to 7. The pH-adjusted hydrolysate was aliquoted into 50 mL centrifuge tubes and centrifuged at 8000 rpm for 15 min. The supernatant obtained after centrifugation was lyophilized and used as a sample for subsequent analysis.

[0042] 2. Functional validation of whey protein multifunctional mixed peptides (1) Antioxidant activity DPPH free radical scavenging activity: Equal volumes of sample solutions of different concentrations (10 mg / mL, 20 mg / mL, 30 mg / mL) and DPPH (dissolved in anhydrous ethanol) were thoroughly mixed and incubated at 37 °C in the dark for 30 min. The absorbance was then measured at 517 nm. Pure water was used as a blank control instead of the sample solution.

[0043] DPPH free radical scavenging rate (%) = (A0-A1) / A0 × 100%; Where A1 is the absorbance value of the experimental group and A0 is the absorbance value of the blank group.

[0044] Hydroxyl radical scavenging activity: 100 μL of samples at different concentrations (2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, and 30 mg / mL) were mixed with equal volumes of FeSO4 (9 mM) and H2O2 (8 mM), respectively. After mixing, the solutions were incubated at 37 ℃ in the dark for 10 min. Subsequently, 100 μL of a salicylic acid ethanol solution (9 mM) was added, and the mixture was incubated at 37 ℃ in the dark for another 30 min. Finally, the absorbance was measured at 510 nm. Pure water was used as a blank control instead of the sample solution.

[0045] Hydroxyl radical scavenging rate (%) = (A0 - A1) / A0 × 100%; Where A1 is the absorbance value of the experimental group and A0 is the absorbance value of the blank group.

[0046] ABTS free radical scavenging activity: The ABTS stock solution was diluted with PB buffer (50 mM, pH 7.4) to an absorbance of 0.7 ± 0.02 at 734 nm to obtain the ABTS working solution. 10 µL of samples at different concentrations (5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL) and 990 µL of ABTS working solution were thoroughly mixed and incubated at room temperature in the dark for 6 min. The absorbance was measured at 734 nm. Pure water was used as a blank control instead of the sample solution.

[0047] ABTS radical scavenging rate (%) = (A0 - A1) / A0 × 100%; Where A1 is the absorbance value of the experimental group and A0 is the absorbance value of the blank group.

[0048] Conclusion: Figure 1 As shown in (A) to (C), the whey protein multifunctional mixed peptide exhibits certain scavenging abilities against DPPH, hydroxyl radicals, and ABTS. At a hydrolysate concentration of 30 mg / mL, the scavenging rates for DPPH, hydroxyl radicals, and ABTS were 52.73%, 93.79%, and 89.12%, respectively. In conclusion, the whey protein multifunctional mixed peptide possesses good antioxidant activity.

[0049] (2) Anticoagulant activity Sample preparation: Preheat the semi-automatic coagulation analyzer to 37 ℃. Centrifuge sterile anticoagulated rabbit blood containing sodium citrate (sodium citrate to blood ratio of 1:9) at 3000 rpm for 15 min, and separate the supernatant plasma for later use. Prepare a whey protein hydrolysis product solution by reconstituted the sample (lyophilized supernatant obtained in step 1) with 50 mM imidazole buffer (containing 150 mM sodium chloride, pH 7.4), using imidazole buffer as a blank control.

[0050] Measurement of APTT, PT, and TT APTT, PT, and TT were measured according to the methods provided in the kit.

[0051] APTT assay: Redissolve the APTT reagent in 1 mL of deionized water and let it stand at room temperature for 15 min before use. Preheat the calcium chloride solution to 37 °C for at least 10 min but no more than 30 min before use. Add 25 μL of peptide solution samples of different concentrations (with imidazole buffer as a blank control), 25 μL of plasma, and 50 μL of APTT reagent to a coagulation cup equipped with magnetic beads, mix well, and incubate at 37 °C for 3 min. Then add 50 μL of preheated calcium chloride solution, mix immediately upon addition, and record the blood clotting time. PT assay: Redissolve the PT reagent in 1 mL of reconstituted solution and let it stand at room temperature for 15 min before use. Preheat the PT reagent to 37 °C for at least 10 min but no more than 30 min before use. Add 25 μL of peptide sample samples of different concentrations (with imidazole buffer as a blank control) and 25 μL of plasma to a coagulation cup equipped with magnetic beads, mix well, and incubate at 37 °C for 3 min. Then add 100 μL of pre-warmed PT reagent solution, mixing immediately upon addition and timing the blood clotting time. For TT determination: Reconstitute the TT reagent with 1 mL of reconstitution solution, let it stand at room temperature for 15 min before use. Add 50 μL of peptide samples of different concentrations (with imidazole buffer as a blank control) and 50 μL of plasma to a coagulation cup equipped with magnetic beads, mix well, and incubate at 37 ℃ for 3 min. Then add 100 μL of TT reagent, mixing immediately upon addition and timing the blood clotting time.

[0052] Conclusion: Figure 2 The (A) to (C) multifunctional whey protein peptides in the sample prolonged APTT, PT, and TT, and this prolongation was concentration-dependent. At 20 mg / mL, APTT, PT, and TT were prolonged by approximately 6.7 s, 2.3 s, and 8.4 s, respectively. This indicates that the multifunctional whey protein peptides prepared by fermentation-assisted enzymatic hydrolysis possess certain anticoagulant functions.

[0053] Example 2: Identification and Synthesis of Multifunctional Whey Protein Peptides 1. Identification of active components by LC-MS / MS The lyophilized supernatant of whey protein hydrolysate, which was verified to have antioxidant and anticoagulant activities in Example 1, was identified by LC-MS / MS, yielding a series of peptide sequences.

[0054] The sample was analyzed by LC-MS / MS equipped with an online nanospray ionization source. The entire system was an Orbitrap FusionLumos mass spectrometer (Thermo Fisher Scientific, MA, USA) with a Vanquish NeoUHPLC ultra-high performance liquid chromatography system in tandem. A total of 5 μL of sample (C18 column: 20 cm × 75 μm id, 1.9 μm particle size) was loaded, and the sample was separated by a gradient of 60 min. The column flow rate was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 5% B phase, increased to 72% within 57 min, increased to 99% within 1 min, and was maintained for 2 min.

[0055] The mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows: (1) MS: Scan range (m / z): 200-1550; Resolution: 120,000; Normalized AGC target: 300%; Maximum injection time: 20 ms; (2) HCD-MS / MS: Resolution: 15,000; Normalized AGC target: 200%; Maximum injection time: 22 ms; Collision energy: 32%; Dynamic exclusion time: 35 s.

[0056] 278 peptides were identified from whey protein, with lengths ranging from 3 to 36 amino acids. Figure 3 This finding indicates that the sample contains a rich polypeptide composition and may possess both antioxidant and anticoagulant activities.

[0057] 2. Screening of antioxidant and anticoagulant peptides and their solid-phase synthesis: Whey protein peptides with potential antioxidant and anticoagulant functions were screened using bioactive peptide docking technology. To further verify the activity of the isolated and purified peptides, α-lactalbumin peptide SEKLDQWLCEKL, derived from whey protein, was synthesized by Jier Biochemical (Shanghai) Co., Ltd. using solid-phase chromatography. High-performance liquid chromatography confirmed that the purity of the synthesized peptide was 95%. The amino acid sequence of the SEKLDQWLCEKL peptide is Ser-Glu-Lys-Leu-Asp-Gln-Trp-Leu-Cys-Glu-Lys-Leu.

[0058] Example 3: Functional validation of α-lactalbumin peptide SEKLDQWLCEKL with antioxidant and anticoagulant activities 1. Antioxidant activity verification The experimental method was the same as in Example 1. The peptide SEKLDQWLCEKL was diluted with deionized water to 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL and 5 mg / mL for functional verification.

[0059] Conclusion: Figure 4 As shown in (A) to (C), the SEKLDQWLCEKL peptide exhibited good antioxidant activity in the antioxidant experiments. The DPPH scavenging rate was 62.32%, and the hydroxyl radical scavenging rate was 78.89%, further confirming its antioxidant effect. The ABTS scavenging rate showed a concentration-dependent effect, reaching a maximum of 88.32%. Overall, the SEKLDQWLCEKL peptide demonstrated good antioxidant properties and possesses value as a potential antioxidant.

[0060] 2. Verification of anticoagulant activity The experimental method was the same as in Example 1. The peptide SEKLDQWLCEKL was diluted to 2.5 mg / mL, 5 mg / mL and 7.5 mg / mL for functional verification.

[0061] Conclusion: Figure 5 As shown in (A) to (B), the SEKLDQWLCEKL peptide prolonged both PT and TT in the anticoagulation assay. Furthermore, the prolongation of PT and TT was concentration-dependent. At a concentration of 7.5 mg / mL, PT was prolonged by approximately 3.4 s, and TT by approximately 4.7 s. The fact that this peptide can simultaneously prolong both PT and TT indicates that it interferes with both the extrinsic pathway and the final common pathway of the coagulation process to achieve its anticoagulation effect.

[0062] Example 4: In vitro tyrosinase inhibitory activity assay of pure peptide SEKLDQWLCEKL Based on the identification results of Example 2, the artificially synthesized peptide SEKLDQWLCEKL was used to prepare SEKLDQWLCEKL solutions of different concentrations (dissolved in PBS), and the in vitro tyrosinase inhibitory activity of the peptide SEKLDQWLCEKL was detected by measuring the tyrosinase inhibition rate.

[0063] The purified peptide SEKLDQWLCEKL was prepared into solutions of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL, and 5 mg / mL using phosphate-buffered saline (PBS, 50 mM, pH 6.8) for use as experimental samples. A 5 mM kojic acid solution was also prepared as a positive control. The specific experimental steps are as follows: Sample group: In a 96-well plate, 100 μL of sample solutions of different concentrations dissolved in PBS and 50 μL of tyrosinase solution dissolved in PBS (enzyme activity 400 U / mL) were added sequentially, and incubated at 37 ℃ in the dark for 10 min. Subsequently, 200 μL of levodopa solution (0.5 mM, dissolved in 50 mM PBS, pH 6.8) was added to each well, mixed well, and incubated at 37 ℃ in the dark for another 15 min. After the reaction, the absorbance (OD) value of each well was immediately measured at 475 nm using a microplate reader.

[0064] Sample blank group: Same as the sample group, except that PBS buffer is used instead of tyrosinase solution.

[0065] Enzyme reaction control group: Same as the sample group, except that PBS buffer is used instead of the sample solution.

[0066] Reagent blank group: Same as the sample group, except that PBS buffer is used instead of sample solution and tyrosinase solution.

[0067] The formula for calculating the tyrosinase inhibition rate (%) is: R = [1 - (OD)] D - OD C ) / (OD B - OD A ) ] ×100%. The experiment consisted of four groups to calculate the inhibition rate: OD A (Reagent blank): Buffer solution + levodopa, OD B (Enzyme reaction control): Buffer (in place of sample) + Tyrosinase + L-DOPA, OD C (Sample blank): Sample + buffer (instead of enzyme) + L-DOPA, OD D (Sample group): Sample + Tyrosinase + Levodopa.

[0068] Conclusion: From Figure 6 The peptide SEKLDQWLCEKL exhibits a good inhibitory effect on tyrosinase, and this inhibitory effect is concentration-dependent. Especially at higher concentrations (e.g., 2.5 mg / mL and 5 mg / mL), the inhibitory capacity of this active peptide on tyrosinase surpasses that of kojic acid (at a concentration of 5 mM), reaching 89.07% at the highest tested concentration. This indicates that this active peptide has great potential for development as a whitening or anti-pigmentation product. It was verified that the whey protein multifunctional mixed peptide in Example 1 did not show tyrosinase inhibitory activity.

[0069] Example 5: Molecular docking experiment of SEKLDQWLCEKL peptide with thrombin and Keap1 The potential binding sites between the peptide SEKLDQWLCEKL and thrombin and the intracellular signal transduction protein Keap1 were explored using Discovery Studio. X-ray crystal structures of thrombin (PDB code: 2BVR) and Keap1 (PDB code: 2FLU) were downloaded from the Protein Structure Database (https: / / rcsb.org / ). Water and ligands were removed to obtain a stable SEKLDQWLCEKL peptide receptor. The interaction between the receptor and ligand was visualized using Discovery Studio visualization tools, yielding parameters such as binding energy and bond distance.

[0070] Table 1. Energy of SEKLDQWLCEKL peptide docking with thrombin and Keap1

[0071] Conclusion: Docking results indicate that the peptide SEKLDQWLCEKL binds to thrombin through multiple interactions, including hydrogen bonds and hydrophobic interactions. Specifically, the peptide forms an extensive hydrogen bond network with amino acid residues such as arginine (Arg) and glutamine (Gln) of thrombin. The peptide interacts with tyrosine (Tyr) residues of thrombin through Pi-Pi stacking and Pi-alkyl formation. The peptide also forms alkyl interactions with arginine (Arg) and lysine (Lys) residues of thrombin. In summary, this peptide achieves specific binding to thrombin through multiple molecular interactions.

[0072] Docking results showed that the peptide SEKLDQWLCEKL binds to the Keap1 protein through multiple forces, including hydrogen bonds, electrostatic attraction, and hydrophobic interactions. The hydrogen bonds formed between SEKLDQWLCEKL and Keap1 involve residues such as serine (Ser), asparagine (Asn), and valine (Val) in Keap1. SEKLDQWLCEKL also forms stable Pi-Pi stacks with phenylalanine (Phe) in Keap1 and forms alkyl and electrostatic interactions with arginine (Arg). These results indicate a strong binding force between the peptide and the Keap1 protein.

[0073] Example 6: Preparation of a multi-effect whitening and anti-aging facial mask containing α-lactalbumin peptide SEKLDQWLCEKL According to the formula ratio, accurately weigh all raw materials. (1) Aqueous phase preparation. Add 60-70% deionized water to the main mixing tank, heat to 60-70°C, and add chelating agent (disodium EDTA), humectant (glycerin and butylene glycol), and thickener (xanthan gum) in sequence. Stir at high speed to prevent clumping, and stir until completely dispersed and dissolved to form a uniform gel base. (2) Dissolution of active peptides and other active ingredients. In another clean tank, take a small amount of deionized water (about 5%), add sodium hyaluronate (large and small molecules), and stir slowly to fully hydrate and swell to dissolve. At below 40°C, slowly add the active peptides to the premixing tank A and stir gently until completely dissolved. Add nicotinamide, VC derivative, resveratrol, etc. in sequence and stir until uniformly dissolved. (3) Premixing of preservative system. Premix the preservatives (phenoxyethanol, 1,2-hexanediol, etc.) in a small amount of deionized water (about 2-3%) evenly. When the temperature of the main tank drops below 40 °C, slowly pump the mixture of active ingredient solution and preservative system into the aqueous phase tank and stir until homogeneous. After pH adjustment, filtration, and degassing (using a 0.2-0.45 μm microporous membrane for fine filtration) of the mask essence, fill the essence into pre-sterilized mask bags. Quickly heat-seal to ensure airtightness and prevent secondary contamination.

[0074] The composition includes 70-80% deionized water / purified water, 1-3% glycerin, 2% butylene glycol, 0.05-0.1% metal chelating agent, 0.5-6% α-lactalbumin peptide (SEKLDQWLCEKL), 0.5-8% other whitening synergistic ingredients (niacinamide, VC derivatives, resveratrol), 0.1-3% thickener (xanthan gum), and 0.1-2% preservatives.

[0075] Example 7: Preparation of a solid beverage containing α-lactalbumin peptide SEKLDQWLCEKL with anti-aging and cardiovascular protective effects According to the formula ratio, all raw materials are accurately weighed using a high-precision electronic scale. Moisture-absorbing raw materials must be handled in a dry environment. Active peptides, sweeteners, citric acid, silica, and a portion of maltodextrin are premixed. The premix, hydrolyzed collagen peptides, vitamin C, grape seed extract, DHA powder, beetroot powder, mixed berry powder, the remaining maltodextrin, and fructooligosaccharides are mixed at room temperature and in a dry environment for 30-45 minutes to ensure that all components are highly uniformly distributed. The mixed powder is then passed through a vibrating sieve (80-100 mesh) to further break up any soft lumps and ensure a fine powder texture. The uniformly mixed powder is then dispensed into individual aluminum foil composite bags according to the preset single-serving weight (2 g) using an automated dispensing machine.

[0076] The composition includes α-lactalbumin peptide SEKLDQWLCEKL (0.05-17%), hydrolyzed fish collagen peptide (0.05-20%), vitamin C (0.1-8%), DHA powder (0.5-3%), mixed berry powder (5-20%), beetroot powder (1-7%), maltodextrin (3-23%), fructooligosaccharides (0.2-2%), and silicon dioxide (0.05-0.6%).

[0077] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A multifunctional α-lactalbumin peptide, characterized in that, The amino acid sequence of the α-lactalbumin multifunctional peptide is Ser-Glu-Lys-Leu-Asp-Gln-Trp-Leu-Cys-Glu-Lys-Leu or SEKLDQWLCEKL.

2. A product comprising the alpha-lactalbumin multifunctional peptide according to claim 1, characterized in that, The product in question is a pharmaceutical or daily chemical product.

3. The product of claim 2, wherein, The daily chemical products include cosmetics; the dosage forms of the daily chemical products include solutions, emulsions, creams, gels, powders, aerosols, wax-based agents, or suspensions.

4. The product of claim 2, wherein, The daily chemical products mentioned include skin care products.

5. The product as described in claim 2, characterized in that, The daily chemical products include sheet masks, cream masks, peel-off masks, toners, lotions, gels, makeup removers, serums, or massage oils.

6. The product of claim 2, wherein, The drug also includes a conventional pharmaceutical carrier.

7. The product of claim 2, wherein, The medicine also contains pharmaceutical excipients.

8. The product according to claim 6 or 7, characterized in that The pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; the pharmaceutical excipient comprises excipients.

9. The product of claim 8, wherein, The medicine contains one or more of the following: vitamin C, grape seed extract, DHA powder, beetroot powder, mixed berry powder, maltodextrin, and fructooligosaccharides.

10. The product as described in claim 9, characterized in that, The dosage forms of the medicine include tablets, capsules, granules, injections, liposome nanoparticles, sustained-release formulations, or dispersible tablets.

11. The use of the α-lactalbumin multifunctional peptide of claim 1 in the preparation of daily chemical products with antioxidant and / or whitening and spot-fading properties.

12. The use of the α-lactalbumin multifunctional peptide of claim 1 in the preparation of an antioxidant and / or anticoagulant pharmaceutical product.

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