Preparation method and application of goat milk-derived antioxidant peptide

Abstract

The invention discloses a preparation method and application of goat milk-derived antioxidant peptide. Firstly, goat milk protein is subjected to enzymolysis through compound protease, then the antioxidative peptide is obtained through separation and purification from an enzymolysis product, and the antioxidative peptide is novel in structure and has a strong protection effect on oxidative damage of cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of antioxidant peptides, and in particular relates to a preparation method and application of a goat milk-derived antioxidant peptide. Background Art

[0002] Under normal physiological conditions, the body's endogenous enzyme systems and non-enzymatic defense systems work synergistically to effectively eliminate free radicals and reactive substances produced during metabolism. However, when the body is stimulated by harmful factors such as inflammation and smoking, the oxidative-antioxidant balance is disrupted, and the antioxidant system is unable to promptly eliminate excessive free radicals, leading to their accumulation. This imbalance in free radical metabolism is defined as oxidative stress. Oxidative stress is associated with a variety of non-communicable diseases, including cancer and neurodegenerative disorders. Synthetic drugs can be used for these diseases, but their adverse reactions limit their long-term use. Therefore, the development of natural antioxidants has become a multidisciplinary research hotspot.

[0003] Since Marcuse first reported the antioxidant activity of amino acids, the antioxidant properties of food-derived peptides have garnered increasing attention. Milk, a nutritious food source, is rich in essential amino acids, short- and medium-chain fatty acids, and minerals such as potassium, phosphorus, and calcium, all essential for human growth and development. In recent years, goat milk has garnered significant attention due to its nutritional value, which more closely resembles that of breast milk. Research has shown that goat milk casein is a highly nutritious, complete protein, and that whey protein has multiple physiological functions, including lowering blood pressure, providing antioxidant benefits, and inhibiting bacteria.

[0004] Currently, the use of goat milk to prepare antioxidant peptides has become a research focus. Invention patents such as "A method for preparing goat milk active beverages using rhamnosus Lactobacillus protease" (patent application number: 201710963850.5) and "A method for preparing an antioxidant goat milk beverage" (patent application number: 201511024060.8), as well as published papers such as "Screening of probiotic lactobacilli for producing antioxidant peptides from fermented goat milk," "Study on fermented goat milk and its antioxidant active peptides," "Response surface methodology optimization of enzymatic hydrolysis of goat milk casein to prepare antioxidant peptides," and "Effect of proteases on the production of antioxidant peptides from defatted goat milk fermentation" all report that enzymatic hydrolysis products with antioxidant activity can be prepared using lactic acid bacteria fermentation or protease hydrolysis. However, the above studies are limited to the preparation of fermentation products or enzymatic hydrolysis products with antioxidant activity, and no antioxidant peptides with novel structures have been isolated. Summary of the Invention

[0005] The present invention aims to overcome the deficiencies of the prior art and provides a preparation method and application of goat milk-derived antioxidant peptides.

[0006] In order to achieve the above object, the technical solution provided by the present invention is:

[0007] The preparation method of the goat milk-derived antioxidant peptide comprises the following steps:

[0008] (1) reconstituted goat milk by mixing skimmed goat milk powder and distilled water in a mass-to-volume ratio of 1:8, wherein the unit of the mass-to-volume ratio is g / mL;

[0009] (2) adding alkaline protease and neutral protease to the reconstituted goat milk, performing enzymolysis in a constant temperature water bath to obtain an enzymolyzate; the mass ratio of the alkaline protease to the neutral protease is 1:1 to 1:2, the amount of the two enzymes added is 10,000 to 15,000 U / g, the enzymolysis pH is 8.5 to 9.5, and the enzymolysis time is 50 to 100 min;

[0010] (3) Separate and purify the enzymatic hydrolysate using Sephadex G-25 chromatography, monitor the elution process by absorbance at a wavelength of 280 nm, collect the components and freeze-dry them for later use, determine the antioxidant activity of each component, and determine the fraction with the strongest antioxidant activity;

[0011] (4) The antioxidant peptides were isolated and identified from the fractions with the strongest antioxidant activity using LC-MS / MS mass spectrometry, and the biological activities of the peptides were scored using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), thereby screening out antioxidant peptides with high scores and novel structures; the amino acid sequences of the antioxidant peptides are shown in SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4. The polypeptide with the amino acid sequence shown in SEQ ID NO. 4 can be used to prepare a cell antioxidant damage protectant.

[0012] The present invention will be further described below:

[0013] The present invention includes:

[0014] 1. Add skimmed goat milk powder to distilled water at a ratio of 1:8 (w / v) to prepare reconstituted goat milk;

[0015] 2. The enzymatic hydrolysis method of the goat milk is as follows: alkaline protease and neutral protease are added to the reconstituted goat milk obtained by the treatment in 1, and enzymatic hydrolysis is carried out in a constant temperature water bath; the mass ratio of the alkaline protease to the neutral protease is 1:1 to 1:2, the enzyme addition amount is 10000-15000 U / g, the enzymatic hydrolysis pH is 8.5-9.5, and the enzymatic hydrolysis time is 50-100 min;

[0016] 3. The enzymatic hydrolysate obtained in 2 was separated and purified using Sephadex G-25 chromatography, with a sample concentration of 40 mg / mL and a sample volume of 4 mL. Elution was performed with ultrapure water at a rate of 2 mL / min. The elution process was monitored by absorbance at a wavelength of 280 nm. The fractions were collected to obtain three components, named F1, F2, and F3. The antioxidant activity of each component was determined and the product was lyophilized for later use.

[0017] 4. The antioxidant peptides in the F2 fraction with the strongest antioxidant activity were separated and identified using LC-MS / MS mass spectrometry. The biological activities of the peptides were scored using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), and the antioxidant peptides RTGLAVAPAWCLLQVAG, KYLP, and HLISCI with the highest scores and novel structures were screened out.

[0018] 5. Calculate the isoelectric point, net charge, and hydrophobicity of the peptide using the PepDraw (https: / / www2.tulane.edu / —biochem / WW / PepDraw / ) online tool;

[0019] 6. Use the ProtParam tool (https: / / web.expasy.org / protparam / ) on the ExPASy server to calculate the instability index, aliphatic index, average hydrophilicity coefficient and other parameters of the peptide;

[0020] 7. In addition, the water solubility of the peptide was predicted using the Innovagen tool (http: / / www.innovagen.com / proteomics-tools);

[0021] 8. The three peptides screened in step 5 were synthesized, among which the peptide HLISCI had good DPPH and ABTS free radical scavenging activity;

[0022] 9. Using t-BHP to induce a cell oxidative damage model, different concentrations of the peptide HLISCI can increase the GSH content, SOD and CAT activities in oxidatively damaged cells, indicating that the peptide can effectively protect cells from damage caused by oxidative stress.

[0023] The present invention uses skimmed goat milk powder as a raw material and adds distilled water to prepare reconstituted goat milk. Alkaline protease and neutral protease are added for enzymatic hydrolysis, and the enzymes are inactivated by heating and then centrifuged. Antioxidant peptides in the goat milk enzymatic hydrolysate are separated and purified by gel chromatography to obtain three components. The amino acid sequence of the F2 component with the best antioxidant activity is identified by LC-MS / MS, and three peptide segments with potential antioxidant activity are screened through bioinformatics prediction. The amino acid sequences are RTGLAVAPAWCLLQVAG (SEQ ID NO.2), KYLP (SEQ ID NO.3), and HLISCI (SEQ ID NO.4). Among them, the polypeptide HLISCI has the strongest overall antioxidant activity and can effectively scavenge free radicals. An oxidative stress model is established by inducing HepG2 cells using t-BHP. The synthesized antioxidant polypeptide HLISCI can significantly improve the cell survival rate after oxidative damage, and increase SOD activity, CAT activity and GSH content (p < 0.05).

[0024] In summary, the present invention firstly utilizes a composite protease to enzymatically hydrolyze goat milk protein, and then separates and purifies the enzymatic hydrolysis product to obtain an antioxidant peptide. The antioxidant peptide has a novel structure and can inhibit oxidative damage to cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of compound protease on DH (a) and antioxidant activity (b) of goat milk enzymatic hydrolysate;

[0026] Figure 2 This is the gel chromatography separation spectrum of goat milk protein hydrolysate;

[0027] Figure 3 is the antioxidant activity graph of each eluted fraction;

[0028] Figure 4 is the total ion current of component F2;

[0029] Figure 5 It is to identify the antioxidant activity of peptides;

[0030] Figure 6 It is the interaction between the peptide FVFLFLFLFFCPVFVR and the Keap1 protein;

[0031] Figure 7 It is the interaction between the peptide RTGLAVAPAWCLLQVAG and the Keap1 protein;

[0032] Figure 8 It is the interaction between the peptide KYLP and the Keap1 protein;

[0033] Figure 9 It is the interaction between the peptide HLISCI and the Keap1 protein;

[0034] Figure 10 is the effect of different concentrations of peptides on the survival rate of HepG2 cells;

[0035] Figure 11 It is the protective effect of peptides on oxidative damage of HepG2 cells;

[0036] Figure 12 is the effect of peptides on GSH content in HepG2 cells induced by t-BHP;

[0037] Figure 13 Effects of peptides on t-BHP-induced CAT activity in HepG2 cells;

[0038] Figure 14 It is the effect of polypeptide on t-BHP-induced SOD activity of HepG2 cells. DETAILED DESCRIPTION

[0039] 1. Preparation of goat milk hydrolysate

[0040] (1) Adding skimmed goat milk powder to distilled water at a ratio of 1:8 (w / v) to prepare reconstituted goat milk;

[0041] (2) adding alkaline protease and neutral protease to the reconstituted goat milk obtained in (1), and performing enzymolysis in a constant temperature water bath; the mass ratio of the alkaline protease to the neutral protease is 1:1.87, the amount of enzyme added is 10338U / g, the enzymolysis pH is 9.5, the enzymolysis time is 83min, and after the enzymolysis is completed, the enzyme is quickly inactivated in a boiling water bath for 10min, and the milk is freeze-dried for later use;

[0042] The antioxidant activity of the hydrolyzate of goat milk by compound protease is shown in Figure 1 The antioxidant activity of the hydrolysis products of alkaline protease and neutral protease was significantly higher than that of other combinations and better than that of single enzyme hydrolysis (p<0.05).

[0043] 2. Gel filtration chromatography purification:

[0044] The enzymatic hydrolysate of goat milk was prepared into a 40 mg / mL solution and loaded onto a Sephadex G-25 gel filtration column (1.6 cm × 40 cm). The solution was eluted with ultrapure water at a rate of 2 mL / min. The elution process was monitored by absorbance at a wavelength of 280 nm. The fractions were collected and lyophilized for later use. The antioxidant activity of each fraction was determined.

[0045] 3. Antioxidant activity analysis

[0046] (1) Determination of DPPH free radical scavenging rate

[0047] Pipette 0.5 mL of the sample to be tested into a test tube, add 0.5 mL of 0.1 mmol / L DPPH-ethanol solution and mix thoroughly. React in the dark at room temperature for 30 min, and finally measure the absorbance at 517 nm.

[0048]

[0049] Wherein: A1 is the absorbance value of the sample group, A2 is the absorbance value of the ethanol-substituted DPPH group, and A3 is the absorbance value of the ethanol-substituted sample group.

[0050] (2) Determination of hydroxyl radical scavenging ability

[0051] Pipette 200 μL of sample, place the sample solution to be tested in a test tube, add 200 μL of 6 mmol / L FeSO4 solution and 200 μL of 6 mmol / L H2O2 solution in sequence, mix well, incubate at 37°C for 10 minutes, then add 200 μL of 6 mmol / L salicylic acid solution, mix well, incubate at 37°C for 30 minutes, and finally measure the absorbance at 510 nm.

[0052]

[0053] Where: A1 is the absorbance value of the sample group, and A2 is the absorbance value of the sample group replaced by distilled water.

[0054] (3) Determination of ABTS free radical scavenging rate

[0055] Mix 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution in equal proportions. Incubate in the dark for 12 hours at room temperature to generate ABTS radical cations. Immediately before use, dilute the ABTS solution to an absorbance of 0.70 ± 0.02 with anhydrous ethanol. Combine 50 μL of sample with 1.5 mL of ABTS solution, incubate in the dark for 6 minutes, and measure absorbance at 734 nm.

[0056]

[0057] Where: A1 is the absorbance value of the sample group, and A2 is the absorbance value of the sample group replaced by distilled water.

[0058] During gel chromatography purification, the three components eluted were ranked in ascending order of molecular weight: F1>F2>F3 (see Figure 2 ). The antioxidant results of the three separated components are as follows Figure 3As shown, in terms of DPPH free radical scavenging rate, F3 > F2 > F1; in terms of hydroxyl free radical scavenging rate, F1 > F2 > F3, with no significant difference between F1 and F2 (p>0.05). F2 had the strongest ABTS free radical scavenging ability, significantly higher than both F1 and F3 (p<0.05). Overall, F2 exhibited superior antioxidant activity and was ultimately selected for subsequent mass spectrometry identification.

[0059] 4. Mass spectrometry identification of peptides

[0060] (1) Liquid chromatography conditions

[0061] Precolumn: PEPMAP NEO C18 300 μm × 5 mm; Analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120C18-AQ 1.9 μm; Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% acetonitrile; Flow rate: 600 nL / min, gradient elution program, see Table 1.

[0062] Table 1 Liquid chromatography gradient elution program

[0063]

[0064] (2) Mass spectrometry conditions

[0065] The separated peptides were detected by mass spectrometry. The primary mass spectrometry parameters were: resolution: 120,000; AGC target: Standard; maximum injection time: 20 ms; scan range: 100-1500 m / z; and the secondary mass spectrometry parameters were: resolution: 15,000; AGC target: 15,000; maximum injection time: 22 ms; cycle time: 2 s; collision energy: 30. After mass spectrometry, the raw files were searched against the target protein database using Byonic software. The search parameters are shown in Table 2.

[0066] Table 2 Database search parameters

[0067]

[0068]

[0069] 5. Scoring of biological activity and antioxidant activity of peptides

[0070] (1) The biological activity of the peptides was scored using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ). The antioxidant scoring scale is shown in Table 3.

[0071] Table 3 Antioxidant score scale

[0072]

[0073] The F2 component was analyzed by LC-MS / MS, and the target database was searched and compared using Bynioc software, resulting in the identification of 162 peptides. Figure 4 The total ion map of the F2 component is shown in Figure 4. Among all peptides, 10 peptides with potential antioxidant activity were identified based on their peak area greater than 0 and peptide length less than 20 ng, as determined by screening using the BIOPEP database. The results are shown in Table 4. These 10 peptides with potential antioxidant activity were scored using PeptideRanker, and the scoring results are shown in Table 5.

[0074] Table 4 Identification of antioxidant peptides

[0075]

[0076] Table 5 Biological activity scores and antioxidant activity scores of peptides

[0077]

[0078]

[0079] (2) Physicochemical properties of antioxidant peptides

[0080] The isoelectric point, net charge, and hydrophobicity of the peptide were calculated using the PepDraw tool (https: / / www2.tulane.edu / —biochem / WW / PepDraw / ). The instability index, aliphatic index, average hydrophilicity coefficient, and other parameters of the peptide were calculated using the ProtParam tool (https: / / web.expasy.org / protparam / ) on the ExPASy server. In addition, the water solubility of the peptide was predicted using the Innovagen tool (http: / / www.innovagen.com / proteomics-tools).

[0081] The physicochemical properties of the peptides are shown in Table 6. The isoelectric points of the four peptides ranged from 7.16 to 10.10, including one neutral peptide (theoretical isoelectric point 6.01-8.00) and three basic peptides (theoretical isoelectric points >8.00). Net charges ranged from 0 to +1, and hydrophobicity values ​​ranged from -7.27 to +8.88. The instability index, an important metric for assessing peptide stability, was less than 40 for the peptides FVFLFLFLFFCPVFVR and KYLP, indicating stability in vitro. However, the instability indexes of RTGLAVAPAWCLLQVAG and HLISCI were greater than 40, suggesting that chemical modification may be necessary to improve stability. Analysis of the average hydrophilicity coefficient revealed that KYLP had a hydrophilicity coefficient of -0.775, less than 0, indicating good hydrophilicity and favorable in vivo delivery. The hydrophilicity coefficients of the other three peptides were all greater than 0, indicating weaker hydrophilicity. Based on the combined instability index and average hydrophilicity coefficient, FVFLFLFLFFCPVFVR is a stable hydrophobic peptide, KYLP is a stable hydrophilic peptide, and RTGLAVAPAWCLLQVAG and HLISCI are unstable hydrophobic peptides. The aliphatic index is a key parameter for assessing peptide thermal stability. Of the four peptides, KYLP has an aliphatic index of 70, while the remaining three peptides, FVFLFLFLFFCPVFVR, HLISCI, and PLDLVYVILHCRVDVQ, all have aliphatic indices greater than 120, indicating that all four peptides possess good thermal stability and are suitable for use in high-temperature environments. Water solubility prediction results show that KYLP exhibits good solubility in water, while FVFLFLFLFFCPVFVR, RTGLAVAPAWCLLQVAG, and HLISCI exhibit poor water solubility.

[0082] Table 6 Physicochemical characteristics of peptides

[0083]

[0084] In the actual synthesis process, the peptide FVFLFLFFCPVFVR was difficult to dissolve in water, which made it impossible to purify it. Therefore, the three peptides RTGLAVAPAWCLLQVAG (P1), KYLP (P2), and HLISCI (P3) were finally selected for synthesis. The antioxidant activity test results of the synthesized peptides are shown in Figure 2. Figure 5 As shown, at a concentration of 1 mg / mL, the antioxidant activities of the three synthetic peptides were significantly higher than those of the crude enzymatic hydrolysate and the isolated and purified fraction F2. Among the three synthetic peptides, peptide KYLP (P2) exhibited the highest activity in the hydroxyl radical scavenging system. Peptide HLISCI (P3) exhibited the best DPPH and ABTS radical scavenging ability, significantly outperforming the other two peptides. Comprehensive multi-index analysis revealed that peptide HLISCI (P3) exhibited the most outstanding antioxidant properties and is suitable for subsequent cellular antioxidant assays.

[0085] 6. Molecular docking

[0086] The peptide two-dimensional structure was constructed using ChemBioDraw Ultra 20.0 and minimized with ChemBio3D (Minimum RMS Gradient parameter was 0.001). The peptide structure was then converted to mol2 format and imported into AutoDockTools 1.5.6 for preprocessing. The Keap1 crystal structure (PDB ID: 4L7B) was obtained from the PDB database, processed with PyMol 2.3.0, and exported as a pdbqt file. Finally, semi-flexible docking was performed using AutoDock Vina 1.1.7, with the docking box center coordinates set to (x = -3.685, y = 9.038, z = -39.826) and the search space dimensions set to size_x = 59.85, size_y = 59.85, size_z = 59.85 (grid spacing: Number of spatial structural changes: 9). All docking results were visualized using PyMol and LigPlot software.

[0087] It is generally believed that a binding energy below -5.0 kcal / mol indicates good intermolecular binding; a binding energy below -7.0 kcal / mol indicates a significant strong binding effect. The molecular docking analysis results for the four peptides in Table 6 are shown in Table 7. The binding energies of the four peptides with the Keap1 protein ranged from -7.4 kcal / mol to -8.3 kcal / mol, all below -7.0 kcal / mol. This indicates that the molecular docking results are reliable and that the four peptides can form stable complexes with the Keap1 protein.

[0088] Table 7 Molecular docking analysis of 4 antioxidant peptides and Keap1 protein

[0089]

[0090]

[0091] Figure 6 The docking results showed that the peptide FVFLFLFLFFCPVFVR could form four hydrogen bonds with Val465, His516, Arg326, and Leu610, and generate hydrophobic interactions with 25 amino acid residues including Ala466, Cys513, Cys368, and Thr560. Figure 7The docking results showed that the peptide RTGLAVAPAWCLLQVAG formed 11 hydrogen bonds with Asp422, Arg326, Thr560, Asn517, etc., and formed hydrophobic interactions with 21 amino acid residues such as Gln563, Val608, Arg326, and Ieu515. Figure 8 The docking results showed that the peptide KYLP formed two hydrogen bonds with the amino acid residues Ser555 and Ser508 of the Keap1 protein and formed hydrophobic interactions with 14 amino acid residues including Ser555 and Ser508. Figure 9 Docking results revealed that the peptide HLISCI forms four hydrogen bonds with Keap1 residues Leu365, Ile559, Val561, and Thr560, as well as hydrophobic interactions with 15 other residues, including Gly367, Ala466, Cys368, and Cys513. Studies have shown that hydrogen bonding not only maintains conformational stability in the complex but also actively regulates the inhibitory or activating effects between substrate and receptor. Molecular docking revealed that the four peptides competitively bind to Keap1 through hydrogen bonds or hydrophobic interactions, leading to the release of Nrf2. These peptides then exhibit antioxidant activity in vivo by activating the Keap1-Nrf2-ARE pathway.

[0092] 7. Study on the protective effect of antioxidant peptides on cellular oxidative damage

[0093] (1) Cell culture

[0094] HepG2 cells were placed in complete culture medium containing 10% fetal bovine serum and 1% double-antibody, and cultured in a saturated humidity incubator at 37°C and 5% CO2. When the cell count reached 80% to 90%, the culture medium was aspirated, the cells were rinsed twice with PBS, and 1 mL of trypsin was added for digestion for 3 minutes before subculture.

[0095] (2) Establishment of t-BHP-induced oxidative stress injury model in HepG2 cells

[0096] Cells in the logarithmic growth phase were collected at a rate of 1.5×10 4 Cells were seeded into 96-well plates at 100 μL per well. 100 μL of cell suspension was added to each well and incubated at 37°C in a 5% CO2 saturated humidity for 24 hours. The culture medium was discarded and various concentrations of t-BHP solution were added for a further 2 hours. Then, 10 μL of CCK-8 solution was added to each well and incubated for 1 hour. Cell viability was then determined by measuring absorbance at 450 nm using a microplate reader. The t-BHP concentration that resulted in a cell viability of nearly 50% was defined as the optimal concentration for oxidative damage.

[0097] Calculation formula (IV):

[0098]

[0099] OD dosing: absorbance value of the wells with cells, culture medium, CCK-8 solution, and drug solution;

[0100] OD blank: absorbance value of wells with culture medium, CCK-8 solution, and no cells;

[0101] OD control: absorbance value of wells containing cells, culture medium, CCK-8 solution, but no drug solution;

[0102] (3) Toxicity test of peptides on HepG2 cells

[0103] Take 100 μL of cell suspension and add 1.5×10 4 The cells were seeded into 96-well plates. After the cells adhered to the wall, different concentrations of peptide (HLISCI) solution were added and cultured for 24 hours. The cell activity was then determined using a CCK-8 kit to determine whether the peptide had a toxic effect on HepG2 cells. The cell viability calculation formula was the same as above.

[0104] Figure 10 The effect of different concentrations of peptide (HLISCI) on cell survival rate. Figure 10 It can be seen that after treating HepG2 cells with different concentrations of peptide (HLISCI) for 24 hours, the cell survival rate was above 90%, and the cell survival rate was not significantly lower than that of the blank group (p>0.05). In addition, the peptide (HLISCI) had a certain promoting effect on the cells in the concentration range of 0.1-0.4 mg / mL, indicating that the peptide HLISCI had no obvious toxic effect on the cells in the concentration range of 0.05-0.4 mg / mL.

[0105] (4) Protective effect of peptides on oxidative damage in HepG2 cells

[0106] Take 100 μL of cell suspension and add 1.5×10 4 100 μL of 400 μmoL / L t-BHP solution were added and cultured for 2 h. Cell viability was then determined using a CCK-8 kit to determine the protective effect of the peptide on t-BHP-induced HepG2 cells. The cell viability calculation formula was the same as above.

[0107] The effects of different concentrations of peptide HLISCI on the survival rate of damaged HepG2 cells are shown in Figure 11 .Depend on Figure 11After 2 hours of treatment with 0.4 mmol / L t-BHP, the cell viability was 49.58% ± 1.24, a significant decrease compared to the control group (p < 0.05), indicating that the t-BHP-induced oxidative damage model in HepG2 cells was successfully established. Cell viability was significantly increased compared to the oxidative damage group at 0.05, 0.1, 0.2, and 0.4 mg / mL of the peptide (HLISCI) (p < 0.05). Cell viability increased in a dose-dependent manner with increasing peptide concentrations. Cell viability at the four concentration gradients was 56.08% ± 2.79, 66.76% ± 2.9, 71.80% ± 2.55, and 75.82% ± 2.15, respectively. This suggests that the peptide can alleviate t-BHP-induced oxidative stress in HepG2 cells and has a protective effect against oxidatively damaged cells.

[0108] (5) Determination of GSH, CAT, and SOD levels in HepG2 cells induced by t-BHP by peptides

[0109] HepG2 cells (6.0×10 5 cells / well) were inoculated into 6-well plates and cultured for 24 hours. After the cells adhered, the old culture medium was aspirated, and after washing twice with PBS, the 6-well plates were divided into a control group, a damaged group, and a sample group. 1 mL of complete culture medium was added to the control and damaged groups; 1 mL of peptide solution of different concentrations (0.05, 0.1, 0.2, and 0.4 mg / mL) was added to the sample group, and the cells were incubated for 24 hours and damaged with 1 mL of 400 μmoL / L t-BHP for 2 hours (1 mL of complete culture medium was added to the control group). The old culture medium was then removed, washed twice with PBS, and the cells were digested with cell lysis buffer RIPA (ice bath) for 30 minutes. The cells were scraped and centrifuged at 1000g for 5 minutes. The total protein content in the supernatant was determined using a BCA protein assay kit. The levels of GSH, CAT, and SOD in the cells were detected according to the kit instructions.

[0110] After pretreatment with peptides at different concentrations, the GSH content of HepG2 cells was significantly increased compared with the damaged group (p<0.05) (see Figure 12), increased by 4.15μmoL / g prot, 7.78μmoL / g prot, 14.73μmoL / g prot, 17.79μmoL / g prot, indicating that the peptide has good antioxidant activity and can increase the antioxidant activity of glutathione peroxidase; in the concentration range of 0.05-0.4mg / mL, the activity of CAT in the cells increased by 6.63U / mgprot, 11.25U / mgprot, 14.03U / mgprot, 18.69U / mgprot, respectively, indicating that the peptide can significantly increase the activity of CAT enzyme in damaged cells (see Figure 13 ); In the groups treated with different concentrations of peptide, the activity of SOD in the cells increased to 30.17U / mgprot, 33.47U / mgprot, 36.74U / mgprot and 39.83U / mgprot, which were 47.27%, 58.29%, 64.67% and 76.94% of the control group, respectively, and were significantly higher than those in the oxidative damage group (p<0.05) (see Figure 14 In summary, antioxidant peptides (HLISCI) can stimulate the intracellular antioxidant system by increasing GSH content and antioxidant enzyme (SOD, CAT) activity, thereby reducing t-BHP-induced cellular oxidative damage.

[0111] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing goat milk-derived antioxidant peptides, characterized in that: The method comprises the following steps: (1) Reconstituted goat milk by mixing skimmed goat milk powder and distilled water in a mass-to-volume ratio of 1:8, where the unit of the mass-to-volume ratio is g / mL; (2) adding alkaline protease and neutral protease to the reconstituted goat milk, performing enzymolysis in a constant temperature water bath to obtain an enzymolyzate; the mass ratio of the alkaline protease to the neutral protease is 1:1 to 1:2, the amount of the two enzymes added is 10,000 to 15,000 U / g, the enzymolysis pH is 8.5 to 9.5, and the enzymolysis time is 50 to 100 min; (3) Separate and purify the enzymatic hydrolysate using Sephadex G-25 chromatography, monitor the elution process by absorbance at a wavelength of 280 nm, collect the components and freeze-dry them for later use, determine the antioxidant activity of each component, and determine the fraction with the strongest antioxidant activity; (4) The antioxidant peptides were separated and identified from the fractions with the strongest antioxidant activity using LC-MS / MS mass spectrometry, and the biological activities of the peptides were scored using PeptideRanker to screen out antioxidant peptides with high scores and novel structures; the amino acid sequences of the antioxidant peptides are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.

4.

2. The antioxidant peptide prepared by the method for preparing goat milk-derived antioxidant peptide according to claim 1, characterized in that: The antioxidant peptides include polypeptides with amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.

4.

3. The antioxidant peptide prepared by the method for preparing goat milk-derived antioxidant peptide according to claim 1, characterized in that: The binding energy of the antioxidant peptides to the Keap1 protein is lower than -7.0 kcal / mol, showing strong binding ability. They can competitively bind to the Keap1 protein through hydrogen bonds or hydrophobic interactions, thereby triggering the release of the Nrf2 transcription factor and activating the Keap1-Nrf2-ARE antioxidant signaling pathway.

4. The polypeptide prepared by the method for preparing goat milk-derived antioxidant peptides according to claim 2, characterized in that: The antioxidant peptide is a polypeptide with an amino acid sequence as shown in SEQ ID NO.

4.

5. Use of a polypeptide having an amino acid sequence as shown in SEQ ID NO. 4 in the preparation of a cell antioxidant.

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