Camel lactoferrin-derived antioxidant peptides, their preparation methods, and applications

Abstract

This invention discloses a camel lactoferrin-derived antioxidant peptide, its preparation method, and its applications, belonging to the field of biotechnology. To address the limitations of existing bovine lactoferrin hydrolysates in antioxidant activity and the unknown sequences of highly active peptides derived from camel lactoferrin, this invention provides a camel lactoferrin-derived antioxidant peptide with the amino acid sequence DWTGPPEPL or RPFLDWTGPPEPLQK. The preparation method includes in vitro simulated gastrointestinal sequential digestion of camel lactoferrin as a raw material, followed by centrifugation, ultrafiltration, desalting, and drying. The peptide sequence is identified using LC-MS / MS, and the target peptide is obtained by combining Peptid Ranker activity prediction, ToxinPred 3 toxicity assessment, Innovagen water solubility assessment, and AutoDock 4.2 molecular docking screening. The antioxidant peptide of this invention exhibits high activity and good safety, and can be used to prepare functional foods, health products, or cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. More specifically, this invention relates to a camel lactoferrin-derived antioxidant peptide, its preparation method, and its applications. Background Technology

[0002] Lactoferrin is an important functional glycoprotein. Its bioactive peptides, obtained through enzymatic hydrolysis, possess various physiological functions, including inhibiting lipid oxidation and scavenging free radicals. Currently, research on antioxidant peptides derived from lactoferrin mainly focuses on bovine lactoferrin (BLF). Schaaf JM et al. investigated the proteolytic behavior of BLF in an in vitro gastrointestinal digestion model of infants. The results showed that although BLF can be hydrolyzed to produce peptides, the antioxidant activity of its hydrolysates is limited (Cited reference 1: Schaaf JM, Goulding DA, O'Regan J, et al. Proteolysis of lactoferrin and β-casein in complex coacervate and uncomplexed forms during invitro infant gastrointestinal digestion [J]. Journal of Functional Foods, 2024, 116: 106141).

[0003] Camel lactoferrin (CLF), a core functional protein in camel milk, differs significantly from black lactoferrin (BLF) in its glycosylation level, amino acid sequence, and spatial structure, theoretically possessing unique functional potential. However, current research focuses only on the raw material characteristics of CLF and has not yet explored the specific sequences of the antioxidant peptides produced after gastrointestinal digestion, resulting in the underutilization of CLF's application potential in the field of antioxidant peptides.

[0004] Besides lactoferrin, other milk-derived proteins have also reported peptides with antioxidant activity. For example, E. VG et al. identified the whey protein-derived antioxidant peptide WYSLAMAASDI from sweet whey beverages (Cited Reference 2: E. VG, Isabel S, Zaira P, et al. Antioxidant Potential of the Sweet Whey-Based Beverage Colada after the Digestive Process and Relationships with the Lipid and Protein Fractions[J]. Antioxidants, 2022, 11(9): 1827). However, the mechanism of action of this peptide is still unclear, and there is a lack of systematic research comparing its activity with antioxidant peptides derived from lactoferrin from different sources.

[0005] Therefore, there is a lack of an antioxidant peptide with a well-defined sequence, a clear mechanism of action, and derived from camel lactoferrin in the existing technology to meet the demand for novel natural antioxidants in functional foods, health products and other fields. Summary of the Invention

[0006] The present invention aims to solve the above-mentioned problems existing in the prior art, and to provide a camel lactoferrin-derived antioxidant peptide with high antioxidant activity, low toxicity and good water solubility, and to clarify its screening and preparation method and mechanism of action.

[0007] To achieve these objectives and other advantages according to the present invention, a camel lactoferrin-derived antioxidant peptide is provided, the amino acid sequence of which is DWTGPPEPL or RPFLDWTGPPEPLQK.

[0008] A method for preparing camel lactoferrin-derived antioxidant peptides is provided, comprising the following steps: S1. Using camel lactoferrin as raw material, simulated gastrointestinal sequential digestion was performed in vitro. S2. Centrifuge the digested products, take the supernatant and use an ultrafiltration membrane to perform ultrafiltration, and collect the ultrafiltration filtrate.

[0009] S3. The ultrafiltration filtrate is desalted and then vacuum dried to obtain a peptide mixture, wherein the peptide mixture contains the antioxidant peptide described in claim 1.

[0010] Preferably, the sequential digestion in step S1 specifically involves first hydrolyzing with pepsin at a final enzyme activity of 400 U / mL and at 37°C for 2 hours, and then hydrolyzing with trypsin at a final enzyme activity of 100 U / mL and at 37°C for 2 hours.

[0011] Preferably, the digested product in step S2 is centrifuged at 20,000g for 5 minutes; The supernatant was ultrafiltered by centrifugation at 12000g for 15 minutes using a 10kDa ultrafiltration membrane.

[0012] Preferably, it also includes a screening step, including: S4. The peptide mixture was identified by LC-MS / MS to obtain the amino acid sequences of multiple peptides. S5. The Peptid Ranker tool was used to predict the antioxidant activity of the amino acid sequences of multiple peptides, and peptides with a score of not less than 0.5 were screened out. S6. The ToxinPred 3 tool was used to evaluate the toxicity of the screened peptides, and the Innovagen tool was used to evaluate the water solubility of the screened peptides. Peptides that were predicted to be non-toxic by ToxinPred 3 and to be water-soluble by Innovagen were selected. S7. Using AutoDock 4.2 software, the screened non-toxic and water-soluble peptides were subjected to molecular docking simulation with ABTS free radicals. The minimum binding energy of each peptide with ABTS free radicals was calculated. With the minimum binding energy below -5.69 kcal / mol as the standard, peptides with a minimum binding energy not lower than the standard were screened, and the antioxidant peptides were obtained.

[0013] Preferably, the sequential digestion in step S1 includes: first, hydrolysis with pepsin at a final enzyme activity of 400 U / mL and at 37°C for 2 hours; then, simultaneous hydrolysis with trypsin and α-chymotrypsin, wherein the final enzyme activity of trypsin is 150 U / mL and the final enzyme activity of α-chymotrypsin is 50 U / mL, and the hydrolysis time is 2.5 hours.

[0014] Preferably, the ultrafiltration in step S2 includes: firstly, ultrafiltration is performed using a 30kDa ultrafiltration membrane to collect the permeate; then, ultrafiltration is performed using a 5kDa ultrafiltration membrane to collect the retentate.

[0015] This invention provides an application of camel lactoferrin-derived antioxidant peptides in the preparation of functional foods, health products, or cosmetics.

[0016] Preferably, the application is for preparing antioxidant products with free radical scavenging function.

[0017] Preferably, the application is the preparation of food supplements for improving the body's antioxidant capacity.

[0018] The present invention has at least the following beneficial effects: First, experimental data show that the total antioxidant capacity of camel lactoferrin hydrolysate containing the antioxidant peptides described in this invention is 1.29 µmol / mL, which is significantly higher than the total antioxidant capacity of bovine lactoferrin hydrolysate (1.03 µmol / mL) under the same conditions, as shown in the attached figure. Figure 1 There was no significant difference in the ABTS and DPPH free radical scavenging rates between undigested camel lactoferrin and bovine lactoferrin. However, after digestion, the ABTS free radical scavenging rate of camel lactoferrin was significantly higher than that of bovine lactoferrin, as shown in the attached figure. Figure 2 3. This indicates that the peptide described in this invention contributes stronger antioxidant activity. Furthermore, molecular docking analysis demonstrated that the minimum binding energy between the antioxidant peptide DWTGPPEPL screened in this invention and the ABTS free radical is -6.30 kcal / mol. The molecular docking simulation results are attached. Figure 4 The minimum binding energy between the antioxidant peptide RPFLDWTGPPEPLQK and the ABTS free radical is -5.91 kcal / mol. Molecular docking simulation results are attached. Figure 5 Both are lower than the binding energy (-5.69 kcal / mol) of the whey protein-derived antioxidant peptide WYSLAMAASDI to ABTS free radicals reported in existing technologies. Molecular docking simulation results are attached. Figure 6 This data indicates that the antioxidant peptides described in this invention have a stronger free radical binding capacity, thus exhibiting superior antioxidant activity.

[0019] Secondly, through molecular docking simulation and visualization analysis, this invention elucidates the interaction mode between the antioxidant peptide DWTGPPEPL and ABTS free radicals. This peptide binds tightly to ABTS free radicals through hydrogen bonding (involving amino acid residues such as Asp-1 and Trp-2) and hydrophobic interactions. This multi-faceted binding mode explains the structural basis of its highly efficient antioxidant activity at the molecular level, providing a theoretical basis for subsequent structural optimization and application development.

[0020] Third, the antioxidant peptides described in this invention are derived from natural camel milk and belong to the category of bioactive peptides from dietary protein sources. According to ToxinPred 3, all peptides are non-toxic; according to Innovagen, all peptides exhibit good water solubility. This ensures their safe application in functional foods, health products, and cosmetics.

[0021] Fourth, the antioxidant peptides described in this invention were obtained through screening by simulating the gastrointestinal digestive process, demonstrating their tolerance to hydrolysis by pepsin and trypsin, and their stability under both acidic (pH 2.0) and near-neutral (pH 7.0) conditions. This characteristic allows them to maintain good activity during food processing and oral formulation preparation, making them suitable for addition to various water-based or emulsion-based products.

[0022] Fifth, cell experiments confirmed that the antioxidant peptides described in this invention have good intracellular antioxidant activity, and when used in combination with vitamin C, they have a significant synergistic effect (synergistic indices of 0.69 and 0.71, respectively, showing a strong synergistic antioxidant effect (judgment criterion is CI<0.9)), indicating that they have broad application prospects in compound antioxidant products.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1 The graph shows the results of the total antioxidant capacity determination in Example 1 of the present invention.

[0025] Figure 2 The graph shows the results of ABTS free radical scavenging rate determination in Example 1 of the present invention.

[0026] Figure 3 The graph shows the results of the DPPH free radical scavenging rate measurement in Example 1 of the present invention.

[0027] Figure 4 This is a schematic diagram of the interaction between the polypeptide DWTGPPEPL and the ABTS free radical molecule in Example 1 of the present invention.

[0028] Figure 5 This is a schematic diagram of the interaction between the polypeptide RPFLDWTGPPEPLQK and the ABTS free radical molecule in Example 1 of the present invention.

[0029] Figure 6 This is a schematic diagram of the interaction between the polypeptide WYSLAMAASDI (an existing bovine whey antioxidant peptide) and the ABTS free radical molecule in Example 1 of the present invention.

[0030] Figure 7 The mass spectrometry peptide distribution histograms in Example 1 of this invention are shown below (a. length distribution histogram, b. molecular weight distribution histogram). Detailed Implementation

[0031] The present invention will now be described in further detail with reference to examples, so that those skilled in the art can implement it based on the description.

[0032] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0033] Example 1 The preparation method of camel lactoferrin-derived antioxidant peptides includes the following steps: 1. Raw material preparation Camel lactoferrin (CLF) was extracted from camel whey protein at Baotuo Camel Professional Breeding Base (E87°44'13", N43°48'54") in Shuimogou District, Urumqi, Xinjiang, and its purity was ≥90% after purification.

[0034] 2. In vitro simulation of sequential gastrointestinal digestion (1) Gastric digestion stage: Take 2 mL of 2 mg / mL CLF solution and mix it with 1.9 mL of simulated gastric digestion fluid (SGF, containing 0.129 g KCl, 0.525 g NaHCO3, and 0.69 g NaCl, dissolved in 250 mL distilled water, adjusted to pH 2.0 with hydrochloric acid, and passed through a 0.45 μm microporous membrane). Add 100 μL of 53 mg / mL pepsin solution (final enzyme activity 400 U / mL) and incubate at 37℃ and 150 r / min for 2 hours. After the reaction is completed, adjust the pH to neutral with 1 M NaOH to terminate the reaction.

[0035] (2) Enteric digestion stage: Add 1.4 mL of simulated enteric digestion solution (SIF, containing 0.127 g KCl, 1.785 g NaHCO3, and 0.56 g NaCl, dissolved in 250 mL distilled water, pH 7.0) and 1.2 mg trypsin (final enzyme activity 100 U / mL) to the above gastric digestion products (2.8 mL), and incubate at 37 °C and 150 r / min for 2 hours. After the reaction is completed, heat at 100 °C for 10 minutes to terminate the enzyme reaction.

[0036] 3. Purification treatment The digestion product was centrifuged at 20,000 g for 5 minutes, and the supernatant was transferred to a 10 kDa ultrafiltration centrifuge tube. The tube was centrifuged at 12,000 g for 15 minutes, and the ultrafiltration filtrate was collected. 200 μL of 0.1% TFA was added to the ultrafiltration filtrate, and the tube was centrifuged at 12,000 g for 15 minutes to desalt the peptides. This process was repeated twice. The product was then further desalted using a C18 StageTip centrifuge and vacuum dried to obtain a peptide mixture.

[0037] 4. Peptide identification and screening (1) Mass spectrometry identification: The peptide mixture was reconstituted with 0.1% formic acid and identified by LC-MS / MS. Liquid phase separation conditions: flow rate 300 nL / min, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution program: 0-2 min, B solution 2%-5%; 2-44 min, B solution 5%-28%; 44-51 min, B solution 28%-40%; 51-53 min, B solution 40%-100%; 53-60 min, B solution maintained at 100%. Mass spectrometry data were obtained by database retrieval, as shown in the appendix. Figure 7 The histogram of peptide distribution in camel lactoferrin digest and the amino acid sequences of 144 peptides are shown in Table 1.

[0038] (2) Bioinformatics screening: The antioxidant activity of the identified peptides was predicted using the Peptid Ranker tool, and 14 peptides with a score ≥0.5 were screened out, as shown in Table 2. The toxicity of these 14 peptides was predicted using the ToxinPred 3 tool, and the water solubility was predicted using the Innovagen tool. Nine peptides with predicted results of non-toxicity and good water solubility were screened out, including DWTGPPEPL (P1) and RPFLDWTGPPEPLQK (P2). Among them, 5 peptides were excluded: LRPFL, LGLLR, WTGPPEPL (toxic or poorly water-soluble); TAGWNIPMGLL, LGPQY (non-toxic but poorly water-soluble).

[0039] (3) Molecular docking verification: The nine selected peptides and the control peptide P3 were simulated with ABTS free radical (CID: 5360881) using AutoDock 4.2 software. The whey protein-derived antioxidant peptide WYSLAMAASDI (P3), which has been reported in the literature, was used as a control (hereinafter referred to as the control peptide), and the minimum binding energy of each peptide with ABTS was calculated. The results are shown in Table 3. The binding energy of P1 was -6.30 kcal / mol, and the binding energy of P2 was -5.91 kcal / mol, both of which were lower than the control P3's -5.69 kcal / mol; the binding energies of two other peptides, P4 and P5, were close to those of P3. P1 and P2 were identified as highly active antioxidant peptides due to their lower binding energies.

[0040] surface Mass spectrometry identification results of camel lactoferrin in vitro hydrolysis Table 2. Activity, toxicity, and water solubility assessment of CLF hydrolysates Table 3. Binding energies of the screened antioxidant peptides to ABTS free radical molecules Example 2 Antioxidant activity verification, including: 1. Determination of total antioxidant capacity The total antioxidant capacity of the peptide mixture prepared in Example 1 and the bovine lactoferrin digestion product was determined using the potassium ferricyanide method. The absorbance was measured at 700 nm after mixing the sample with the reaction solution, and the absorbance value was used to express the antioxidant capacity. The results are as follows: Figure 1 As shown, the absorbance of the camel lactoferrin digestion product was 1.29 µmol / mL, significantly higher than that of the bovine lactoferrin digestion product (1.03 µmol / mL). p <0.05).

[0041] 2. Determination of ABTS free radical scavenging ability The sample was mixed with an equal volume of ABTS working solution and reacted in the dark for 6 minutes. The absorbance was measured at 405 nm, and the scavenging rate was calculated. The results showed that the scavenging rate of the peptide mixture containing P1 and P2 on ABTS free radicals was concentration-dependent and superior to that of bovine lactoferrin digestion products at the same concentration.

[0042] 3. DPPH free radical scavenging capacity determination The sample was mixed with an equal volume of 0.2 mmol / L DPPH ethanol solution and reacted at room temperature in the dark for 30 minutes. The absorbance was measured at 517 nm, and the scavenging rate was calculated. The results showed that the peptide mixture had significant DPPH free radical scavenging activity.

[0043] Example 3 Molecular mechanism analysis, including: The molecular docking results of P1 (DWTGPPEPL) and ABTS radicals were visualized and analyzed using PyMOL software. For example... Figure 4 As shown, P1 binds tightly to ABTS radicals through hydrogen bonding (involving residues such as Asp-1 and Trp-2) and hydrophobic interactions. This multi-binding mode is the molecular basis for its highly efficient antioxidant activity.

[0044] This invention successfully screened out two peptides, DWTGPPEPL and RPFLDWTGPPEPLQK, with high antioxidant activity from camel lactoferrin, and verified their superior antioxidant properties through experiments, clarifying their mechanism of action, and providing an important foundation for the development of novel natural antioxidants.

[0045] The meanings of the English and Chinese terms in the attached figures are shown in the table below: Table 4. Explanation of English Terminology in the Appendix Example 4 Targeted enzymatic hydrolysis and large-scale purification of high-purity target antioxidant peptides, including: While the method described in Example 1 can obtain a mixture containing the target peptide, the content of the target peptide is low, and it is difficult to scale up production. This example provides an optimized preparation method that can significantly improve the yield and purity of the target antioxidant peptide, meeting the needs of industrial production.

[0046] 1. Optimization of raw material pretreatment and enzymatic hydrolysis conditions Take 10g of camel lactoferrin (CLF) with a purity ≥95%, dissolve it in 500mL of phosphate buffer (50mM, pH 2.5) to prepare a protein solution of 20mg / mL. Use a multi-enzyme stepwise hydrolysis method: Step 1 (Targeted Hydrolysis with Pepsin): Pepsin (enzyme activity 3000 U / mg) was added to the protein solution to achieve a final enzyme activity of 800 U / mL (higher than 400 U / mL in Example 1). Hydrolysis was carried out at 37°C for 3 hours (extended by 1 hour). After the reaction was completed, the pH was adjusted to 7.0 with 1M NaOH to terminate the reaction. Increasing the enzyme concentration and extending the hydrolysis time allows for more complete degradation of lactoferrin, releasing more intermediate fragments containing the target sequence.

[0047] Step 2 (synergistic hydrolysis by trypsin and chymotrypsin): Trypsin (final enzyme activity 150 U / mL) and α-chymotrypsin (final enzyme activity 50 U / mL) were added to the above solution, and hydrolysis was carried out at 37°C for 2.5 hours. After the reaction was completed, the enzymes were inactivated by heating at 100°C for 10 minutes. The introduction of chymotrypsin can specifically cleave the carboxyl terminus of aromatic amino acids (such as Phe and Trp), which helps to release the P1 sequence (containing Trp) and P2 sequence (containing Phe and Trp) of this invention and improves the yield of the target peptide.

[0048] 2. Multistage membrane separation and enrichment Primary separation (impurity removal): The enzymatic hydrolysate is centrifuged at 4°C and 15000g for 10 min to remove unhydrolyzed macromolecular precipitates. The supernatant is first passed through a 30kDa ultrafiltration membrane (tangential flow filtration system) to remove incompletely hydrolyzed macromolecular proteins, and the permeate is collected.

[0049] Secondary enrichment (target peptide enrichment): The 30kDa permeate is passed through a 5kDa ultrafiltration membrane and circulated at an operating pressure of 0.2MPa. During this process, peptides with molecular weights between 0.5kDa and 5kDa (containing the target sequence of this invention) are retained and enriched, and the retentate is collected. In this invention, P1 has a molecular weight of 1.0kDa, and P2 has a molecular weight of 1.8kDa. Using a 5kDa membrane can effectively remove smaller impurity peptides, achieving the enrichment of the target peptide.

[0050] 3. Preparative high-performance liquid chromatography purification The enriched solution was concentrated under vacuum and redissolved in 0.1% trifluoroacetic acid (TFA) solution. Purification was then performed using preparative RP-HPLC. Chromatographic column: C18 preparative column (250 mm × 21.2 mm, 10 μm) Mobile phases: Phase A: 0.1% TFA / water; Phase B: 0.1% TFA / acetonitrile Elution gradient: 0-10 min, 5% B; 10-40 min, 5%-35% B; 40-45 min, 35%-80% B; flow rate 10 mL / min; detection wavelength 214 nm.

[0051] Collection and identification: Based on the retention time of the standards (which can be predetermined by LC-MS / MS), the chromatographic peaks corresponding to P1 and P2 are collected separately. The collected solution is subjected to rotary evaporation to remove acetonitrile, and then freeze-dried to obtain P1 and P2 monomeric peptides with a purity ≥95%.

[0052] From 10g of camel lactoferrin, P1 can be prepared at 85mg and P2 at 120mg, with a total yield of 2.05%, which is significantly higher than the mixture yield of Example 1 (0.5%-1%).

[0053] HPLC analysis showed that the purities of P1 and P2 monomers were 96.3% and 95.8%, respectively, which meet the standards for functional food ingredients.

[0054] This embodiment achieves efficient and high-purity preparation of target antioxidant peptides by optimizing enzyme combinations, adjusting enzymatic hydrolysis parameters, and introducing multi-stage membrane separation combined with preparative chromatography. This method overcomes the shortcomings of the basic approach, such as low target peptide content and difficulty in separation, providing a feasible production process for subsequent commercial applications.

[0055] Example 5 Chemical methods (ABTS / DPPH) can only reflect free radical scavenging capacity in vitro and cannot simulate the complex environment in vivo. This embodiment aims to verify the actual antioxidant efficacy of the antioxidant peptides described in this invention in vivo using a cell model, and to explore their synergistic effect with vitamin C, including: 1. Assay of cellular antioxidant activity (CAA) Cell culture: The human hepatocellular carcinoma cell line HepG2 was used and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0056] Fluorescent probe loading: HepG2 cells were loaded with 6 × 10⁻⁶ fluorescent probes. 4 Cells were seeded at a density of / well in a 96-well black plate and cultured for 24 h. The culture medium was discarded, and after washing with PBS, 100 μL of medium containing 25 μM DCFH-DA fluorescent probe and different concentrations of the target peptides (P1, P2) was added to each well. Vitamin C was used as a positive control, and cells without samples were used as a negative control. Cells were incubated at 37°C in the dark for 1 h.

[0057] Oxidation induction and detection: Discard the supernatant, wash with PBS, and add 100 μL of 600 μM ABAP (free radical initiator) to each well. Immediately place the 96-well plate into a fluorescence microplate reader and measure the fluorescence intensity every 5 min at 37°C with an excitation wavelength of 485 nm and an emission wavelength of 538 nm for 1 h.

[0058] Calculate the area under the fluorescence intensity-time curve (AUC) for each sample group. Using the AUC of the negative control group as a benchmark, calculate the CAA value (cellular antioxidant activity value). The CAA value is calculated using the following formula: The CAA value is measured in micromolar quercetin equivalents per 100 micromolar samples (μmol QE / 100 μmol). A higher CAA value indicates stronger intracellular antioxidant activity.

[0059] 2. Study on synergistic antioxidant effects Combine P1 or P2 with vitamin C (V c The samples were mixed at a 1:1 molar ratio, and the ABTS radical scavenging rates of individual samples and the mixed samples were determined. The synergistic effect was assessed using the synergistic index (CI). Mixed-sample theory CI < 0.9 indicates a synergistic effect; CI 0.9–1.1 indicates an additive effect; and CI > 1.1 indicates an antagonistic effect. IC50 determination: The sample was diluted to different concentration gradients, and the scavenging rate was determined according to the ABTS free radical scavenging assay. The sample concentration was plotted on the x-axis, and the scavenging rate on the y-axis. The fitted curve was used to calculate the sample concentration at which the scavenging rate was 50%, which is the IC50 value.

[0060] 3. Experimental Results Table 5. Results of the cellular antioxidant activity (CAA) of the antioxidant peptides of the present invention. Relative activity was calculated with the CAA value of vitamin C at 100%; data are expressed as mean ± standard deviation (n=3), compared with the vitamin C group. p <0.05.

[0061] At the cellular level, the CAA values ​​of P1 and P2 of this invention are 1.78 times and 1.51 times that of vitamin C, respectively, and are significantly higher than those of existing peptides. This indicates that the peptides can not only scavenge free radicals in in vitro chemical systems, but also penetrate cell membranes to exert antioxidant protective effects within cells.

[0062] Table 6 Synergistic antioxidant effect of the antioxidant peptides and vitamin C of the present invention Data are expressed as mean ± standard deviation (n=3). All combinations were significantly different from the vitamin C-only group. p <0.05) When P1 and P2 are used in combination with vitamin C, the synergistic indices are 0.69 and 0.71, respectively, exhibiting a strong synergistic antioxidant effect. This means that a smaller amount of antioxidant can be used in a compound formulation to achieve better results, which is of great value for developing low-cost, high-efficiency compound antioxidant products.

[0063] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A camel lactoferrin-derived antioxidant peptide, characterized in that, The amino acid sequence of the antioxidant peptide is DWTGPPEPL or RPFLDWTGPPEPLQK.

2. The method for preparing camel lactoferrin-derived antioxidant peptides as described in claim 1, characterized in that, Includes the following steps: S1. Using camel lactoferrin as raw material, simulated gastrointestinal sequential digestion was performed in vitro. S2. Centrifuge the digested product, take the supernatant and use an ultrafiltration membrane to ultrafilter it, and collect the ultrafiltration filtrate. S3. The ultrafiltration filtrate is desalted and then vacuum dried to obtain a peptide mixture, wherein the peptide mixture contains the antioxidant peptide described in claim 1.

3. The method for preparing camel lactoferrin-derived antioxidant peptides as described in claim 2, characterized in that, In step S1, the sequential digestion specifically involves first hydrolyzing with pepsin at a final enzyme activity of 400 U / mL and at 37°C for 2 hours, and then hydrolyzing with trypsin at a final enzyme activity of 100 U / mL and at 37°C for 2 hours.

4. The method for preparing camel lactoferrin-derived antioxidant peptides as described in claim 2, characterized in that, The digested product in step S2 was centrifuged at 20,000g for 5 minutes; The supernatant was ultrafiltered by centrifugation at 12000g for 15 minutes using a 10kDa ultrafiltration membrane.

5. The method for preparing camel lactoferrin-derived antioxidant peptides as described in claim 2, characterized in that, It also includes a screening step, including: S4. The peptide mixture was identified by LC-MS / MS to obtain the amino acid sequences of multiple peptides. S5. The Peptid Ranker tool was used to predict the antioxidant activity of the amino acid sequences of multiple peptides, and peptides with a score of not less than 0.5 were screened out. S6. The ToxinPred 3 tool was used to evaluate the toxicity of the screened peptides, and the Innovagen tool was used to evaluate the water solubility of the screened peptides. Peptides that were predicted to be non-toxic by ToxinPred 3 and to be water-soluble by Innovagen were selected. S7. Using AutoDock 4.2 software, the screened non-toxic and water-soluble peptides were subjected to molecular docking simulation with ABTS free radicals. The minimum binding energy of each peptide with ABTS free radicals was calculated. With the minimum binding energy below -5.69 kcal / mol as the standard, peptides with a minimum binding energy not lower than the standard were screened, and the antioxidant peptides were obtained.

6. The method for preparing camel lactoferrin-derived antioxidant peptides as described in claim 2, characterized in that, The sequential digestion in step S1 includes: first, hydrolysis with pepsin at 37°C for 2 hours with a final enzyme activity of 400 U / mL; then, simultaneous hydrolysis with trypsin and α-chymotrypsin, wherein the final enzyme activity of trypsin is 150 U / mL and the final enzyme activity of α-chymotrypsin is 50 U / mL, and the hydrolysis time is 2.5 hours.

7. The method for preparing camel lactoferrin-derived antioxidant peptides as described in claim 6, characterized in that, Step S2 ultrafiltration includes: first, using a 30kDa ultrafiltration membrane to perform ultrafiltration and collecting the permeate; then, using a 5kDa ultrafiltration membrane to perform ultrafiltration on the permeate and collecting the retentate.

8. The application of the camel lactoferrin-derived antioxidant peptide as described in claim 1 in the preparation of functional foods, health products, or cosmetics.

9. The application as described in claim 8, characterized in that, The application is to prepare antioxidant products with free radical scavenging functions.

10. The application as described in claim 8, characterized in that, The application is to prepare food supplements to enhance the body's antioxidant capacity.

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