A multifunctional lactoferrin peptide prepared by combined microbial fermentation and enzymatic hydrolysis and its application

Abstract

This invention discloses a multifunctional lactoferrin peptide prepared by a combined microbial fermentation and enzymatic hydrolysis process, and its applications, belonging to the fields of food, biopharmaceuticals, and cosmetics. This invention directionally releases and identifies a novel lactoferrin peptide, VDGKEDLIWKL, from whey protein. The lactoferrin peptide VDGKEDLIWKL exhibits excellent antioxidant activity, achieving a 60.35% ABTS scavenging rate, a 71.31% DPPH free radical scavenging rate, and a 26.12% hydroxyl free radical scavenging rate. It also demonstrates significant anticoagulant effects and tyrosinase inhibition capabilities. This invention overcomes the limitations of existing single-function active peptides, providing a new core raw material and solution for developing pharmaceuticals and cosmetics with synergistic antioxidant, anticoagulant, and skin-brightening functions.

Description

Technical Field

[0001] This invention relates to a multifunctional lactoferrin peptide prepared by a combination of microbial fermentation and enzymatic hydrolysis and its applications, belonging to the fields of food, biopharmaceuticals and cosmetics. Background Technology

[0002] Oxidative stress and thrombosis are common key pathological mechanisms in many major diseases, including atherosclerosis, cardiovascular and cerebrovascular diseases, diabetic complications, and neurodegenerative diseases. Oxidative stress stems from an imbalance between oxidants and antioxidants in the body, leading to the excessive production of free radicals that damage cell structure and function. Thrombosis, on the other hand, results from the overactivation of the coagulation system, leading to abnormal thrombus formation in blood vessels, obstructing blood flow, and causing tissue ischemia and necrosis. Furthermore, in the field of skin health and beauty, the overactivation of tyrosinase, a key enzyme in melanin synthesis, and its resulting pigmentation problems are of great concern. Traditional treatment strategies often target single pathological processes, such as using antioxidants to scavenge free radicals, using anticoagulants to inhibit coagulation, or using tyrosinase inhibitors to improve pigmentation, but their effectiveness is limited and may be accompanied by side effects.

[0003] In recent years, multi-target intervention strategies have received increasing attention. Studies have shown that certain naturally derived bioactive molecules, such as polyphenols and certain peptides, may possess multiple biological activities simultaneously, thereby intervening in disease processes at multiple levels. However, the bioavailability of natural polyphenols is generally low, and the discovery and development of peptides that can synergistically exert multiple effects such as antioxidation, anticoagulation, and even tyrosinase inhibition still face challenges.

[0004] Whey protein, a byproduct of the dairy industry, is rich in various proteins with potential biological activity (such as lactoferrin). Through enzymatic hydrolysis and microbial fermentation, peptides with specific sequences and functions can be released from whey protein. Reported whey protein peptides, especially those derived from lactoferrin, possess a variety of physiological activities, including antioxidant, antihypertensive, antibacterial, immunomodulatory, and tyrosinase inhibitory activities. However, current research on whey protein peptides mainly focuses on single activities; reports of whey protein peptides exhibiting synergistic and significant antioxidant and anticoagulant activities are rare, and multifunctional peptides possessing antioxidant, anticoagulant, and tyrosinase inhibitory activities simultaneously have not been reported.

[0005] Therefore, developing a novel lactoferrin-derived pure peptide with synergistic antioxidant, anticoagulant, and tyrosinase inhibitory activities, and elucidating its mechanism of action, will provide new ideas and options for the development of novel functional foods, health products, drugs, and cosmetics. Summary of the Invention

[0006] [Technical Issues] Existing technologies mostly focus on improving the hydrolysis degree or general biological activity (such as antioxidation) of bioactive peptides, lacking targeted release strategies for peptides with multiple functions such as antioxidation and anticoagulation.

[0007] [Technical Solution] To address the aforementioned problems, the present invention aims to provide a whey protein peptide with high antioxidant, anticoagulant, and tyrosinase inhibitory activities, and its preparation method. This involves fermenting a whey protein solution with *Lactobacillus plantarum* and hydrolyzing it with bromelain, followed by freeze-drying the hydrolysate and subsequent activity determination and mass spectrometry identification. By using the above process on the whey protein solution, the present invention yields lactoferrin peptide VDGKEDLIWKL, which possesses antioxidant, anticoagulant, and tyrosinase inhibitory activities.

[0008] In one embodiment of the present invention, the amino acid sequence of the lactoferrin peptide VDGKEDLIWKL is Val-Asp-Gly-Lys-Glu-Asp-Leu-Ile-Trp-Lys-Leu.

[0009] In one embodiment of the present invention, the lactoferrin peptide VDGKEDLIWKL is derived from lactoferrin in whey protein.

[0010] In one embodiment of the present invention, the preparation method of the lactoferrin peptide VDGKEDLIWKL includes the following steps: (1) Dissolve whey protein in deionized water containing glucose, adjust the pH, and obtain a whey protein aqueous solution; (2) Ferment the whey protein aqueous solution obtained in step (1) using Lactobacillus plantarum; (3) Heat-treat the fermentation broth obtained in step (2) to inactivate the enzymes; (4) Hydrolyze the solution after step (3) using bromelain; (5) After centrifuging the hydrolysis product, the supernatant was freeze-dried to obtain whey protein mixed peptides; (6) The lyophilized mixture from step (5) was identified by LC-MS / MS to obtain a series of peptide sequences; (7) After synthesis by solid phase method, the activity was verified and a multifunctional peptide with both antioxidant and anticoagulant activities was screened out and named VDGKEDLIWKL. Its tyrosinase inhibitory activity was measured.

[0011] In one embodiment of the present invention, the whey protein aqueous solution in step (1) has a concentration of 3% (w / v), a pH of 6.8, and a glucose content of 0.5% (w / v).

[0012] In one embodiment of the present invention, the fermentation conditions of *Lactobacillus plantarum* in step (2) are as follows: inoculum size 6% (v / v, with OD500). 600 =1 bacterial culture (the fermentation time was 18 h, the shaking speed was 130 rpm, the fermentation temperature was 30℃, and the fermentation time was 18 h).

[0013] In one embodiment of the present invention, the heat treatment conditions in step (3) are 90°C for 10 min.

[0014] In one embodiment of the present invention, the enzymatic hydrolysis conditions for bromelain in step (4) are: temperature 55℃, pH 7.0, enzyme dosage 2500 U / g substrate protein, and hydrolysis time 2 h. After the enzymatic hydrolysis reaction is completed, the protein hydrolysate is placed in a boiling water bath and heated for 10 min to inactivate the protease. After cooling to room temperature, the pH of the solution is adjusted to 7. The pH-adjusted protein hydrolysate is dispensed into 50 mL centrifuge tubes and centrifuged at 8000 rpm for 15 min.

[0015] In one embodiment of the present invention, the freeze-drying in step (5) 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.

[0016] In one embodiment of the present invention, the detection conditions of LC-MS / MS in step (6) are as follows: gradient separation of sample for 60 min, column flow rate controlled at 300 nL / min, chromatographic column is C18 reversed phase column (20 cm × 75 μm id, 1.9 μm), column temperature is 40°C, electrospray voltage is 2 kV, gradient starts from 5% B phase (80% acetonitrile, 0.1% formic acid), increases to 72% within 57 min, then increases to 99% within 1 min and maintains it for 2 min; 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] The present invention also provides a product containing the lactoferrin peptide VDGKEDLIWKL.

[0018] In one embodiment of the present invention, the product includes pharmaceuticals or daily chemical products.

[0019] In one embodiment of the present invention, the daily chemical products include cosmetics or skin care products.

[0020] 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.

[0021] In one embodiment of the present invention, the skin care product includes serum, lotion, cream or soluble essence powder.

[0022] In one embodiment of the present invention, the essence further comprises one or more of dipropylene glycol, niacinamide, 3-O-ethyl ascorbic acid, panthenol, p-hydroxyacetophenone, and hexanediol.

[0023] In one embodiment of the present invention, the dosage form of the product includes liquid, solid, paste, granules or powder formulation.

[0024] In one embodiment of the present invention, the pharmaceutical product further comprises conventional pharmaceutical carriers and / or pharmaceutical excipients.

[0025] In one embodiment of the present invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; the pharmaceutical excipient comprises excipients, disintegrants, lubricants or flavoring agents.

[0026] The present invention also provides the application of the lactoferrin peptide VDGKEDLIWKL in the preparation of anticoagulant drugs.

[0027] The present invention also provides the application of the lactoferrin peptide VDGKEDLIWKL in the preparation of antioxidant pharmaceuticals.

[0028] This invention also provides the application of the lactoferrin peptide VDGKEDLIWKL in the preparation of daily chemical products with antioxidant and / or skin whitening and brightening functions.

[0029] Beneficial effects: This invention screened and verified a novel multifunctional lactoferrin peptide, VDGKEDLIWKL, with a defined sequence. VDGKEDLIWKL exhibits a maximum ABTS scavenging rate of 60.35%, a maximum DPPH free radical scavenging rate of 71.31%, and a maximum hydroxyl free radical scavenging rate of 26.12%. It also demonstrates significant anticoagulant effects (TT prolongation up to 3.37 s at the tested concentration) and tyrosinase inhibition capabilities, overcoming the limitations of existing single-functional peptides in application. Attached Figure Description

[0030] Figure 1 The in vitro anticoagulant activity (TT) of whey protein mixed peptides.

[0031] Figure 2 The in vitro anticoagulant activity (TT) of the pure peptide VDGKEDLIWKL.

[0032] Figure 3 The in vitro antioxidant activity of the pure peptide VDGKEDLIWKL. Figure 3 In the figure, (A) represents the DPPH removal rate; Figure 3 (B) in the figure represents the clearance rate of ABTS; Figure 3 (C) in the figure represents the scavenging rate of hydroxyl radicals.

[0033] Figure 4 The in vitro tyrosinase inhibitory activity of the pure peptide VDGKEDLIWKL was measured. Detailed Implementation

[0034] 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.

[0035] Raw material sources: The *Lactobacillus plantarum* used in the following examples were strains screened and preserved in the laboratory. Whey protein was derived from commercially available products.

[0036] Example 1: Isolation and Functional Verification of Multifunctional Hybrid Peptides from Whey Protein Whey protein powder was dissolved in deionized water containing 0.5% (w / v) glucose, and the pH was adjusted to 6.8 with hydrochloric acid or sodium hydroxide solution to obtain a 3% (w / v) whey protein solution. Second-generation bacterial culture (OD) of *Lactobacillus plantarum*, screened and preserved in the laboratory, after activation, was used. 600 =1), and inoculated into the above solution at a 6% (v / v) inoculum, fermented at 30℃ for 18 h. After fermentation, the filtrate was heat-treated at 90℃ for 10 min to inactivate the enzyme. Subsequently, the fermentation broth was further enzymatically hydrolyzed using bromelain under the following conditions: temperature 55℃, pH 7.0, enzyme dosage 2500 U / g substrate protein, and hydrolysis time 2 h. After the enzymatic hydrolysis reaction, the protease hydrolysate was heated in a boiling water bath for 10 min to inactivate the protease. After cooling to room temperature, the pH of the solution was adjusted to 7. The pH-adjusted protease hydrolysate was aliquoted into 50 mL centrifuge tubes and centrifuged at 8000 rpm for 15 min. Finally, it was frozen at a cold trap temperature below -50℃ and freeze-dried under a vacuum of 0.0 mbar for 48 h to obtain a whey protein mixed peptide lyophilized powder, which was stored at -40℃ for further use.

[0037] Validation of anticoagulant activity of multifunctional mixed peptides of whey protein (TT): 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 whey protein hydrolysate solutions of 2.5, 5, 10, and 20 mg / mL with 50 mM imidazole buffer (containing 150 mM sodium chloride, pH 7.4), using imidazole buffer as a blank control.

[0038] TT was determined according to the method provided in the kit. The TT reagent was reconstituted with 1 mL of reconstitution solution and allowed to stand at room temperature for 15 min before use. 50 μL of peptide samples of different concentrations (with imidazole buffer as a blank control) and 50 μL of plasma were added to a coagulation cup equipped with magnetic beads, mixed well, and incubated at 37°C for 3 min. Then, 100 μL of TT reagent was added, and the mixture was immediately mixed and the timing was recorded.

[0039] In vitro anticoagulation test such as Figure 1 As shown, the mixed peptides significantly prolonged thrombin time (TT). At all tested concentrations, TT was significantly prolonged, with a maximum delay of 9.74 s (P<0.0001), indicating that the mixture can strongly inhibit thrombin activity or interfere with the conversion of fibrinogen to fibrin.

[0040] Example 2: LC-MS / MS identification and target peptide screening of whey protein hydrolysate mixtures The detection conditions for LC-MS / MS were as follows: a gradient separation of whey protein mixed peptide lyophilized powder solution for 60 min, using a C18 reversed-phase column (20 cm × 75 μm id, 1.9 μm particle size), column flow rate controlled at 300 nL / min, column temperature at 40℃, electrospray voltage at 2 kV, gradient starting from 5% B phase (80% acetonitrile, 0.1% formic acid), increasing to 72% within 57 min, then increasing to 99% within 1 min and maintaining for 2 min; the mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition; the mass spectrometry parameters were 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. Mass spectrometry data were analyzed using PEAKS Studio software. Bovine (Bos taurus) specific protein databases and the SwissProt database were searched. Non-specific enzyme digestion was set, considering methionine oxidation and asparagine / glutamine deamidation modification. 277 peptides derived from whey proteins were identified, ranging in length from 3 to 36 amino acids. The target peptide, with the sequence Val-Asp-Gly-Lys-Glu-Asp-Leu-Ile-Trp-Lys-Leu (VDGKEDLIWKL), derived from bovine lactoferrin, was finally screened from the identified peptides. High-purity (>95%) VDGKEDLIWKL peptides were synthesized using solid-phase synthesis for subsequent activity validation.

[0041] Example 3: In vitro anticoagulant activity of pure peptide VDGKEDLIWKL Based on the identification results of Example 2, the solid-phase synthesized peptide VDGKEDLIWKL was used to prepare imidazole solutions of different concentrations of VDGKEDLIWKL. The in vitro anticoagulant activity of the peptide VDGKEDLIWKL was detected by measuring thrombin time (TT). The test method is as follows.

[0042] The experimental method was the same as in Example 1, except that the lactoferrin peptide VDGKEDLIWKL solution (50 mM imidazole buffer containing 150 mM sodium chloride, pH 7.4) was diluted to 2.5, 5 and 7.5 mg / mL for functional verification.

[0043] Conclusion: From Figure 2It was found that the pure peptide VDGKEDLIWKL could prolong thrombin time (TT). At a concentration of 7.5 mg / mL, TT was significantly prolonged to 14.6 s (p<0.01). TT directly reflects the ability of thrombin to convert fibrinogen into fibrin, and this result strongly suggests that the mixture contains an active ingredient that can directly inhibit thrombin activity or effectively interfere with the conversion of fibrinogen to fibrin. The results indicate that the pure peptide VDGKEDLIWKL has definite in vitro anticoagulant activity.

[0044] Example 4: In vitro antioxidant activity of pure peptide VDGKEDLIWKL Based on the identification results of Example 2, the solid-phase synthesized peptide VDGKEDLIWKL was used to prepare aqueous solutions of different concentrations of VDGKEDLIWKL. The in vitro antioxidant activity of the peptide VDGKEDLIWKL was detected by DPPH free radical scavenging rate, ABTS free radical scavenging rate and hydroxyl free radical scavenging ability. The test methods are as follows.

[0045] (1) DPPH free radical scavenging rate of pure peptide VDGKEDLIWKL The DPPH scavenging rate was determined as follows: 0.5 mL of 0.1, 0.5, 1, 2.5, and 5 mg / mL lactoferrin peptide VDGKEDLIWKL (dissolved in pure water) was thoroughly mixed with 0.5 mL of DPPH solution (0.1 mM, dissolved in anhydrous ethanol), 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.

[0046] 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 control group.

[0047] like Figure 3 As shown in (A), the pure peptide VDGKEDLIWKL exhibits a strong scavenging ability against DPPH free radicals in a concentration-dependent manner. At a concentration of 5 mg / mL, the scavenging rate reaches approximately 71.31%.

[0048] (2) ABTS free radical scavenging rate of pure peptide VDGKEDLIWKL The ABTS clearance rate was determined as follows: The ABTS stock solution was diluted with PBS (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 0.5, 1, 2.5, and 5 mg / mL lactoferrin peptide VDGKEDLIWKL was thoroughly mixed with 990 µL of the ABTS working solution 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.

[0049] ABTS 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.

[0050] like Figure 3 As shown in (B), the pure peptide also showed a significant scavenging effect on ABTS free radicals, which was also concentration-dependent. At a concentration of 5 mg / mL, the scavenging rate reached 60.35%.

[0051] (3) Hydroxyl radical scavenging ability of pure peptide VDGKEDLIWKL The method for determining hydroxyl radical scavenging ability is as follows: 0.5 mL of 0.5 and 1 mg / mL lactoferrin peptide VDGKEDLIWKL (dissolved in pure water) was thoroughly mixed with 0.5 mL of FeSO4 solution (9 mM) and 0.5 mL of H2O2 (8.8 mM); the mixture was incubated at 37°C in the dark for 10 min; then 0.5 mL of salicylic acid-ethanol solution (9 mM) was added; the mixture was incubated at 37°C in the dark for another 30 min; finally, the absorbance was measured at a wavelength of 510 nm. Pure water was used instead of the sample solution, and the remaining procedures were exactly the same as described above, serving as a blank control.

[0052] Hydroxyl radical scavenging capacity (%) = (A0 - A1) / A0 × 100%; where A1 is the absorbance value of the experimental group and A0 is the absorbance value of the blank group.

[0053] like Figure 3 As shown in (C), the scavenging ability of this pure peptide against hydroxyl radicals exhibits a non-linear concentration-dependent relationship, initially increasing and then slightly decreasing. Within the tested concentration range, the scavenging rate peaked at 1 mg / mL, reaching 26.12%.

[0054] Conclusion: From Figure 3It was found that, within the tested concentration range, the pure peptide VDGKEDLIWKL exhibited certain scavenging effects against DPPH, ABTS, and hydroxyl radicals, with the highest scavenging activities reaching 71.31%, 60.35%, and 26.12% for DPPH, ABTS, and hydroxyl radicals, respectively. Its antioxidant activity may be attributed to the fact that the amino acid residues such as tyrosine (Y) and tryptophan (W) in the peptide can efficiently scavenge DPPH· and ABTS· radicals, primarily through single-electron transfer (SET) mechanisms, by donating hydrogen atoms or electrons. + .

[0055] Example 5: In vitro tyrosinase inhibitory activity of pure peptide VDGKEDLIWKL Based on the identification results of Example 2, the peptide VDGKEDLIWKL was synthesized in a solid phase, and phosphate-buffered saline (PBS) solutions of VDGKEDLIWKL with different concentrations were prepared. The in vitro tyrosinase inhibitory activity of the peptide VDGKEDLIWKL was detected by measuring the tyrosinase inhibition rate.

[0056] The purified peptide VDGKEDLIWKL was prepared into solutions of 1, 2.5, and 5 mg / mL using phosphate-buffered saline (PBS, 50 mM, pH 6.8) for use as experimental samples. A 0.1 mM kojic acid solution was also prepared as a positive control sample.

[0057] 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 (or PBS buffer as a control) and 50 μL of tyrosinase solution dissolved in PBS (enzyme activity 400 U / mL) were added sequentially, and incubated at 37°C 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°C 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.

[0058] Sample blank group: Same as the sample group, except that PBS buffer is used instead of tyrosinase solution.

[0059] Enzyme reaction control group: Same as the sample group, except that PBS buffer is used instead of the sample solution.

[0060] Reagent blank group: Same as the sample group, except that PBS buffer is used instead of sample solution and tyrosinase solution.

[0061] The formula for calculating the tyrosinase inhibition rate (%) is: R = [1 - (OD)] D - ODC ) / (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.

[0062] Conclusion: From Figure 4 It was found that the pure peptide VDGKEDLIWKL exhibited a significant concentration-dependent inhibitory activity against tyrosinase within the concentration range of 1-5 mg / mL, with the inhibition rate increasing with increasing peptide concentration. At the highest tested concentration of 5 mg / mL, the inhibition rate reached 13.41%, exceeding the effect of 0.1 mM kojic acid (positive control, 10.17%). These results indicate that the pure peptide VDGKEDLIWKL possesses certain in vitro tyrosinase inhibitory activity, while the whey protein multifunctional mixed peptide in Example 1 did not show corresponding activity.

[0063] Example 6: Molecular docking simulation of pure peptide VDGKEDLIWKL (Thrombin) To elucidate the potential molecular mechanism of its anticoagulant effect, molecular docking technology was used to simulate the interaction between the pure peptide VDGKEDLIWKL and the active pocket of the key coagulation factor thrombin. The docking results showed that the peptide can stably intercalate into the active region of thrombin, forming a high-affinity complex.

[0064] The specific quantitative binding parameters are as follows: the binding energy (CDOCKER_ENERGY) is -133.592 kcal / mol, and the interaction energy (CDOCKER_INTERACTION_ENERGY) is -75.2741 kcal / mol. These negative values ​​indicate that the binding process can proceed spontaneously and is relatively stable, suggesting a strong interaction between VDGKEDLIWKL and thrombin.

[0065] Table 1. Energy of VDGKEDLIWKL peptide docking with Thrombin

[0066] Docking results showed that the peptide VDGKEDLIWKL stably binds to the thrombin active pocket through multiple interactions, including hydrogen bonds, hydrophobic interactions, and van der Waals forces. VDGKEDLIWKL forms a tight hydrogen bond network with key arginine and tyrosine residues within the thrombin pocket; simultaneously, the hydrophobic residues in the peptide interact with phenylalanine and methionine residues within the thrombin pocket, enhancing the stability of the complex. These interactions explain the molecular mechanism by which the peptide VDGKEDLIWKL prolongs thrombin time (TT).

[0067] Example 7: Molecular docking simulation of pure peptide VDGKEDLIWKL (Keap1) To elucidate the structural basis of its antioxidant activity, molecular docking technology was used to simulate the interaction between the pure peptide VDGKEDLIWKL and the Kelch domain of Keap1, a core regulatory protein for antioxidant activity. The docking results showed that the peptide could bind to the active pocket of Keap1 with high affinity, demonstrating its great potential as a competitive inhibitor of Keap1.

[0068] The specific quantitative binding parameters are as follows: the binding energy (CDOCKER_ENERGY) is -177.992 kcal / mol, and the interaction energy (CDOCKER_INTERACTION_ENERGY) is -119.772 kcal / mol. These values ​​are significantly higher than the reference values ​​for common antioxidant peptides, indicating that the binding of VDGKEDLIWKL to Keap1 protein has exceptional stability and specificity.

[0069] Table 2 Energy of VDGKEDLIWKL peptide docking with Keap1

[0070] The docking results show that VDGKEDLIWKL can bind to Keap1 through the formation of a dense hydrogen bond network (mainly binding to three arginine residues in the Kelch domain of Keap1), good electrostatic interactions (binding to arginine, tyrosine and other residues in Keap1), and extensive hydrophobic contacts (binding to phenylalanine, tyrosine and other residues in Keap1), thereby regulating the Keap1-Nrf2 pathway to exert antioxidant effects.

[0071] Example 8: Preparation of a solid sports drink containing lactoferrin peptides and probiotics Lactoferrin peptide powder (VDGKEDLIWKL) was premixed with maltodextrin, resistant dextrin, fructooligosaccharides, taurine, vitamin C, B vitamins, citric acid, malic acid, steviol glycosides, silicon dioxide, and grapefruit / lemon flavoring. The premixing method employed an incremental, equal-volume approach: first, VDGKEDLIWKL was mixed with an equal volume of maltodextrin for 5 minutes. Then, taurine, vitamin C, B vitamins, steviol glycosides, citric acid, and malic acid were added sequentially, mixing for 5-10 minutes after each addition to ensure uniform distribution. Subsequently, resistant dextrin, fructooligosaccharides, and the remaining maltodextrin were added and mixed for 10 minutes. Finally, under conditions of ambient temperature below 25°C and relative humidity ≤45%, the lyophilized probiotic powder was slowly added to the premix, and the mixture was stirred using a three-dimensional motion mixer for 20-30 minutes until the color and texture were completely homogeneous. The uniformly mixed powder is sieved through an 80-mesh sieve to obtain a homogeneous product with good flowability. The sieved powder is then nitrogen-filled and sealed using an automatic powder packaging machine in an environment with a relative humidity not exceeding 45%, using high-barrier packaging materials such as aluminum foil bags to prevent moisture and oxidation, and to maximize the activity of probiotics.

[0072] In the powder, the lactoferrin peptide VDGKEDLIWKL accounts for 0.01%-8% of the total mass of the mixture (added according to the reconstitution ratio), and the probiotic freeze-dried powder (such as Lactobacillus plantarum LP-ONLLY, with a viable count ≥1 × 10⁻⁶) contains... 11 The composition is as follows: 1.0%-10.0% CFU / g, with maltodextrin as the balance; 10.0%-30.0% resistant dextrin; 5.0%-15.0% fructooligosaccharides (prebiotics); 0.1%-2.0% taurine; 0.1%-3.0% vitamin C; 0.01%-1% B vitamins; 0.1%-3.0% citric acid; 0.2%-1.5% malic acid; 0.01%-0.1% steviol glycosides; 0.1%-1.5% silicon dioxide; 0.1%-1.0% grapefruit flavor; and 0.1%-1.0% lemon flavor. All percentages refer to mass percentages.

[0073] Example 9: Preparation of tablets containing lactoferrin peptides Lactoferrin peptide powder (VDGKEDLIWKL), nattokinase powder, red yeast rice extract, microcrystalline cellulose, and pregelatinized starch were premixed. The premixing method employed an equal-incremental mixing technique: first, VDGKEDLIWKL was initially mixed with an equal amount of microcrystalline cellulose in a mixer; then, nattokinase powder, red yeast rice extract, and the remaining main ingredients were gradually added, mixing for 25 minutes until homogeneous. A suitable amount of purified water was added to the homogeneous powder as a binder, and wet granulation was performed, passing the wet granules through a 20-mesh sieve. The wet granules were dried at 50-60℃ until the moisture content was below 5.0%, and then granulated through a 20-mesh sieve. The granulated granules were mixed with lubricants and flow aids such as magnesium stearate and silica for 5 minutes. The mixed granules were then compressed into tablet cores using a rotary tablet press. The tablet cores were coated with a gastrointestinal film-coating premix. The coated tablets were then packaged.

[0074] In the tablets, lactoferrin peptide (VDGKEDLIWKL) accounts for 0.01%-5% of the tablet core mass, nattokinase powder accounts for 1.0%-10.0%, red yeast rice extract accounts for 5.0%-15.0%, microcrystalline cellulose accounts for 20.0%-70.0%, pregelatinized starch accounts for 5.0%-20.0%, magnesium stearate accounts for 0.1%-1.0%, and silica accounts for 0.1%-1.0%. The film coating weight gain is approximately 1.0%-10.0% of the tablet core mass. All percentages above refer to mass percentages.

[0075] Example 10: Preparation of Brightening Ampoule Essence Containing Lactoferrin Peptide Deionized water, dipropylene glycol, nicotinamide, and 3-O-ethyl ascorbic acid (vitamin C ethyl ether) were accurately weighed into the main preparation vessel, stirred, and appropriately heated to 40-45°C until completely dissolved, serving as the aqueous phase matrix. After the system cooled to below 30°C, lactoferrin peptide (VDGKEDLIWKL) was added and stirred until completely dissolved. Then, panthenol, p-hydroxyacetophenone, and hexanediol (preservative) were added, and an appropriate amount of citric acid solution was added to adjust the pH of the system to 5.5-6.0. The resulting clear solution was aseptically filtered through a 0.22 μm microporous membrane. Under aseptic conditions, the filtrate was quantitatively dispensed into pre-sterilized glass ampoules or disposable plastic capsules, and immediately sealed or capped to obtain the brightening ampoule essence.

[0076] The brightening ampoule essence contains the following components: deionized water (balance), dipropylene glycol (1.0%-5.0%), niacinamide (1.0%-5.0%), 3-O-ethyl ascorbic acid (1.0%-3.0%), lactoferrin peptide (VDGKEDLIWKL) (0.01%-8%), panthenol (0.1%-5.0%), p-hydroxyacetophenone (0.1%-1.0%), and hexanediol (0.1%-1.5%). All percentages refer to mass percentages.

[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 lactoferrin peptide, VDGKEDLIWKL, characterized in that, The amino acid sequence of the lactoferrin peptide VDGKEDLIWKL is Val-Asp-Gly-Lys-Glu-Asp-Leu-Ile-Trp-Lys-Leu.

2. A pharmaceutical product containing the lactoferrin peptide VDGKEDLIWKL as described in claim 1.

3. The medicine as described in claim 2, characterized in that, The drug also includes a conventional pharmaceutical carrier.

4. The medicine as described in claim 3, characterized in that, The pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; 5. The medicine as described in claim 2, characterized in that, The medicine also contains pharmaceutical excipients.

6. The medicine as described in claim 5, characterized in that, The pharmaceutical excipients include excipients, disintegrants, lubricants, or flavoring agents.

7. The use of the lactoferrin peptide VDGKEDLIWKL according to claim 1 in the preparation of anticoagulant drugs.

8. The use of the lactoferrin peptide VDGKEDLIWKL according to claim 1 in the preparation of antioxidant pharmaceuticals.

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