Development and research of horse cheese protein antioxidant peptide based on computer and experiment

Abstract

The invention relates to development and application of a horse cheese protein antioxidant peptide. The antioxidant peptide is screened and prepared from horse milk casein, and functional verification is carried out. Through virtual enzymolysis and computer simulation, a polypeptide sequence possibly generated after hydrolysis of horse milk casein is predicted, and polypeptide with potential antioxidant activity is screened out by combining molecular docking analysis with Keap1 protein. The function of the screened antioxidant peptide is verified by simulating the gastrointestinal tract digestion process. The high-purity antioxidant peptide is chemically synthesized, an in-vitro chemical antioxidant experiment is carried out, and it is found that the antioxidant peptide has the remarkable free radical scavenging capacity. In a cell experiment, the biological activity of the antioxidant peptide is verified by an H2O2-induced oxidative damage model, and a result shows that the antioxidant peptide can remarkably improve the activity of antioxidant enzymes in cells and effectively resist oxidative damage. The method provides an efficient path for development of natural antioxidant peptide, and the screened functional polypeptide has a wide application prospect in the field of food.

Description

Technical Field

[0001] The present invention belongs to the technical fields of polypeptide products and molecular biology, and particularly relates to the development and functional research of an antioxidant peptide derived from horse milk casein. Background Art

[0002] The oxidation process is inevitable in living organisms, and oxidative metabolism generates free radicals and reactive oxygen species (ROS). The oxidation reaction that occurs during food storage can lead to the loss of nutrients, deterioration of quality, and the generation of off-flavors, affecting food quality and safety. Therefore, it is crucial to develop effective antioxidants to scavenge free radicals and prevent food spoilage.

[0003] As a natural antioxidant, antioxidant peptides have shown great application potential in the fields of food, health products, medicine, and cosmetics due to their high efficiency and safety. Compared with synthetic antioxidants, natural antioxidant peptides have lower toxicity and are more suitable as food additives to extend the shelf life of food and improve its nutritional value. Research has shown that antioxidant peptides can exert their antioxidant effects through mechanisms such as scavenging free radicals, chelating metal ions, and inhibiting lipid peroxidation. The amino acid composition, sequence, and molecular weight of antioxidant peptides also have important effects on their antioxidant activity. Therefore, in this study, peptides with potential high antioxidant activity were screened from horse milk casein through a computer screening method, and then their effects of scavenging free radicals and chelating metal ions were verified through in vitro experiments, and their antioxidant activity was verified at the cellular level, demonstrating their potential application prospects in the food field. Summary of the Invention

[0004] The purpose of the present invention is to provide an antioxidant peptide derived from horse milk casein with antioxidant function, which has good antioxidant activity both in vitro and at the cellular level and can be used for the preparation of food.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] An antioxidant peptide derived from horse milk casein with antioxidant function, its amino acid sequence is HPCPHPSF, and its sequence is as shown in SEQ ID NO.17.

[0007] The present invention provides a method for preparing the above antioxidant peptide, including centrifuging 3000 g of fresh horse milk for 20 min to remove milk fat, adjusting the pH of defatted horse milk to 4.6 with acetic acid to precipitate casein, then centrifuging at 12000 g for 20 min at 4 °C, removing the supernatant, washing twice with water and centrifuging, and freeze-drying the precipitated casein for standby.

[0008] Weigh casein, moisten it with a small amount of NaOH solution, add distilled water to make a 50 mg / ml solution, and ultrasonicate for 15 min to accelerate its dissolution. First, adjust the pH to 2.0 with 1M HCl, add pepsin, then adjust the pH to 7.6, add trypsin, and finally adjust the pH to 8.0, add chymotrypsin. The addition amount of each enzyme is 6000 U / g. Hydrolyze successively at 37 °C for 2 h with a magnetic stirring water bath. After hydrolysis, inactivate at 95 °C for 15 min, cool to room temperature, adjust the pH to neutral, centrifuge at 12000 g, 4 °C for 15 min, first pass through a 0.45 μm filter membrane, and then pass through a 0.22 μm filter membrane. Lyophilize the filtrate after passing through the membrane to obtain a crude polypeptide powder for standby. Dissolve the crude polypeptide powder and ultrafilter it with a 3 KDa ultrafiltration tube to obtain two fractions of >3 KDa and <3 KDa, and lyophilize them respectively. Since the molecular weights of the antioxidant peptides we predicted are all less than 1500 KDa, the <3 KDa fraction is used to identify its peptide segment information.

[0009] It also includes performing polypeptide group analysis and detection on the horse casein protease hydrolysate to identify the peptide segment sequence information after enzymatic hydrolysis;

[0010] Preferably, a Vanquish Neo / Orbitrap Exploris 480 liquid chromatography - mass spectrometer is used for analyzing the peptide segment sequence information.

[0011] Chemically synthesize the high - activity polypeptide sequence to obtain horse casein antioxidant peptides with a purity of over 98%, and verify their antioxidant activities.

[0012] The present invention also provides an application of the screening and preparation method in the screening and preparation of antioxidant peptides; specifically, an application in the screening and preparation of horse casein - derived antioxidant peptides.

[0013] Furthermore, the present invention also provides an application of an antioxidant peptide in foods for defending against oxidative damage.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] 1. The present invention obtains a novel peptide with high antioxidant activity through simulated hydrolysis and computer screening. By using an effective database and molecular docking to simulate the main action targets of antioxidant peptides, rapid and efficient identification of antioxidant peptides is achieved.

[0016] 2. The antioxidant peptides screened by the present invention have strong comprehensive antioxidant activities, and have the characteristics of high efficiency, simple preparation method and good repeatability. They have broad prospects in the fields of food, medicine and cosmetics, providing an efficient way for the development of horse casein - derived functional polypeptide products. Its specific advantages are as follows:

[0017] (1) HPCPHPSF exhibits good DPPH· radical scavenging activity and ABTS·+ radical scavenging ability, as well as good superoxide anion scavenging activity and ferrous ion reducing ability. - 2

[0018] (2) The results of antioxidant activity at the cellular level show that the HPCPHPSF peptide has a protective effect on H 2 O 2 -induced HepG2 cell damage. Compared with the model group, the HPCPHPSF peptide exhibits different intensities of antioxidant activity in cells, significantly reducing the production of malondialdehyde (MDA), significantly increasing the enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and significantly reducing the content of intracellular reactive oxygen species (ROS). Description of the Drawings

[0019] Figures 1-4 They are the 3D, 2D diagrams and local planar detail diagrams of the molecular docking results of the peptide HPCPHPSF with the Keap-1 protein, respectively.

[0020] Figure 5 It is the secondary mass spectrum diagram of HPCPHPSF detected in the <3KDa fraction.

[0021] Figure 6 It is the detection diagram of the polypeptide HPCPHPSF after chemical solid-phase synthesis. a is the HPLC diagram; b is the MS diagram.

[0022] Figure 7 It is the DPPH· scavenging rate of the peptide at different concentrations.

[0023] Figure 8 It is the ABTS·+ scavenging rate of the peptide at different concentrations.

[0024] Figure 9 It is the superoxide anion scavenging rate of the peptide at different concentrations. - 2

[0025] Figure 10 It is the effect of different H 2 O 2 concentrations on the survival rate of HepG2 cells. Note: Marked with **** indicates extremely significant difference (P < 0.0001).

[0026] Figure 11 It is the effect of the peptide on the survival rate of HepG2 cells.

[0027] Figure 12 It is the effect of the peptide on H 2 O 2Protective effect on inducing oxidative damage of HepG2 cells. Note: 100 in the figure represents 100 μg / ml peptide solution + H 2 O 2 ; 200 represents 200 μg / ml peptide solution + H 2 O 2 . ns indicates no significant difference; * represents a significant level of P < 0.05; ** represents a significant level of P < 0.01.

[0028] Figure 13 Effect of peptide on MDA content in HepG2 cells. Note: 200 in the figure represents 200 μg / ml peptide solution + H 2 O 2 ; ** represents a significant level of P < 0.01; *** represents a significant level of P < 0.001.

[0029] Figure 14 Effect of peptide on SOD activity in HepG2 cells. Note: 200 in the figure represents 200 μg / ml peptide solution + H 2 O 2 ; ** represents a significant level of P < 0.01; **** indicates extremely significant difference (P < 0.0001).

[0030] Figure 15 Effect of peptide on CAT activity in HepG2 cells. Note: 200 in the figure represents 200 μg / ml peptide solution + H 2 O 2 ; * represents a significant level of P < 0.05; **** indicates extremely significant difference (P < 0.0001).

[0031] Figure 16 Effect of peptide on GSH-Px activity in HepG2 cells. Note: 200 in the figure represents 200 μg / ml peptide solution + H 2 O 2 ; * represents a significant level of P < 0.05; *** represents a significant level of P < 0.001.

[0032] Figure 17 Effect of peptide on intracellular ROS content in HepG2 cells; (a) ROS fluorescence result map of HepG2 cells; (b) Quantification map of ROS fluorescence results of HepG2 cells. Detailed implementation method

[0033] The concept of the present invention and the resulting technical effects will be further elaborated below in conjunction with specific embodiments to fully understand the purpose, features, and effects of the present invention. Unless otherwise specified, the methods are conventional methods. Unless otherwise specified, the materials can be obtained from public commercial channels. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0034] Example 1: In silico enzymatic hydrolysis and screening of antioxidant peptides from horse milk proteins

[0035] 1. Experimental methods

[0036] (1) According to the relative abundances in the UniProt database (https: / / www.uniprot.org / ), a total of 16 horse milk protein sequences were screened (see Table 1), showing information about their entries, protein names, amino acid sequence lengths, and molecular weights. In silico gastrointestinal digestion was simulated in a computer, and the PeptideCutter program in BIOPEP

[0037] (https: / / www.expasy.org / resources / peptidecutter) was used to select pepsin (pH < 2), trypsin, and chymotrypsin (high specificity) to perform in silico gastrointestinal hydrolysis on all the screened proteins. Dipeptides, tripeptides, tetrapeptides, pentapeptides, and polypeptides were screened from the hydrolysis products generated by the three enzymes to form a peptide library.

[0038] (2) PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the probability of each peptide segment in the peptide library having biological activity, and peptide segments with scores higher than 0.8 were screened. A score higher than 0.8 was considered to have a high probability of having high biological activity.

[0039] (3) The BIOPEP-UWM bioactive peptide library (https: / / biochemia.uwm.edu.pl / en / biopep-uwm-2 / ) was used to screen for novel peptides among the above-mentioned candidate peptide segments.

[0040] (4) The AnOxPePred 1.0 / prediction tool (https: / / services.healthtech.dtu.dk / services / ) was used to predict whether the novel peptides have free radical scavenging ability.

[0041] (5) Use the toxicity prediction website Toixinpred prediction tool (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html4) to evaluate the toxicity of the above-mentioned newly discovered peptides with free radical scavenging ability, and evaluate their basic properties such as isoelectric point and hydrophilicity.

[0042] (6) Use the ADMET tool for property prediction (https: / / admetmesh.scbdd.com / ), which can predict polypeptide properties including blood-brain barrier, human intestinal absorption, carcinogenicity, and acute oral toxicity effects.

[0043] (7) Perform molecular docking of the finally selected several polypeptides with the Keap1 protein. Download the protein structure of the kelch region of the Keap1 protein (PDB ID: 1U6D) from the PDB database (https: / / www.rcsb.org / ), and use Maestro 11.0 software to model the peptide chain. Designate the peptide chain as the ligand and the Kelch region of the Keap1 protein as the receptor for molecular docking. PyMOL (version 4.3.0) software (https: / / pymol.org / ) is used for dehydration and removal of organic matter, and AutodockTools (http: / / mgltools.scripps.edu / downloads) is used for hydrogenation and checking charges. The polypeptide is subjected to energy minimization, and protonation is carried out under neutral conditions (pH = 7) using the H++3 online server. Then use UCSF Chimera software to assign Amber14SB charges. Finally, select the conformation with the highest docking score for subsequent analysis, and use and Discovery Studio for 3D mapping analysis. The docking energy is generally negative, and the smaller the value, the higher the docking score, indicating that the binding between the ligand and the receptor protein is tighter, the interaction is stronger, and it is more likely to have antioxidant activity.

[0044] 2. Experimental Results

[0045] (1) The following table shows the casein sequence information screened

[0046] Table 1 Horse casein sequence

[0047]

[0048]

[0049]

[0050]

[0051] (2) The following table shows the peptide segments with scores higher than 0.8 screened from the peptide library

[0052] Table 2 Casein peptide segments with bioactivity scores higher than 0.8

[0053]

[0054] After querying the bioactive peptide library, NF, MY, SF, and QW are bioactive peptides that have been discovered. Considering the need to screen for novel active peptides, these four are removed, and subsequent activity predictions are performed on other peptide segments

[0055] (3) The following table shows the free radical scavenging ability of the novel peptides

[0056] Table 3 Scores of free radical scavenging effects of novel peptides

[0057]

[0058] Table 4 Chelation scores of novel peptides

[0059]

[0060] In summary, from the table, we can see that the above polypeptides may all have free radical scavenging activity. Among them, the polypeptide HPCPHPSF (SEQ ID NO.17) has better free radical scavenging activity overall. Next, the toxicity of the above polypeptides will be evaluated to confirm that they have no biological toxicity

[0061] (4) The following table shows the prediction of polypeptide toxicity and other basic properties

[0062] Table 5 Toxicity prediction and basic properties of novel peptides

[0063]

[0064] (5) The following table shows the docking binding energy of the screened candidate peptides with the Keap1 protein molecule

[0065] Table 6 Docking binding energy of novel peptides with the Keap1 protein molecule

[0066]

[0067] Table 6 shows the molecular docking binding energies of the three newly screened peptides with the Kelch region of Keap1. The molecular docking scores range from -8.7 kcal / mol (MNW) to -9.3 kcal / mol (HPCPHPSF). In other studies, the absolute value of the molecular docking score has a positive correlation with antioxidant activity. A binding energy score < -8.0 kcal / mol indicates a relatively strong binding force, and the lower the negative value, the stronger the binding force. The score of HPCPHPSF is -9.3 kcal / mol, which binds most tightly to the Kelch region of the Keap1 protein, indicating that it is very likely to be an antioxidant active polypeptide. Figures 1-4 Figure 2 shows the molecular docking results of polypeptide HPCPHPSF (SEQ ID NO.17) with the Kelch region of Keap1, including a 3D map and local detail maps. It can be seen that polypeptide HPCPHPSF binds to the Kelch region of keap1 through hydrogen bonds VAL514, VAL561, VAL467, VAL608, VAL369, and it is the antioxidant peptide finally screened by us.

[0068] Example 2: Preparation, identification and solid-phase synthesis of antioxidant peptides from horse milk casein 1. Experimental methods and content

[0069] (1) Preparation of antioxidant peptides from horse milk casein

[0070] 3000 g of fresh horse milk was centrifuged at 20 min to remove milk fat. The pH of the defatted horse milk was adjusted to 4.6 with acetic acid to precipitate casein, and then centrifuged at 12000 g for 20 min at 4 °C. The supernatant was removed, washed twice with water and centrifuged. The precipitated casein was freeze-dried for later use.

[0071] Weighed casein was moistened with a small amount of NaOH solution and dissolved in distilled water to form a 50 mg / ml solution. Ultrasonic treatment for 15 min was used to accelerate its dissolution. First, the pH was adjusted to 2.0 with 1M HCL, pepsin was added, then the pH was adjusted to 7.6, trypsin was added, and finally the pH was adjusted to 8.0, and chymotrypsin was added. The addition amount of each enzyme was 6000 U / g. Hydrolysis was carried out successively at 37 °C for 2 h with a magnetic stirring water bath. After hydrolysis was completed, it was inactivated at 95 °C for 15 min, cooled to room temperature, the pH was adjusted to neutral, centrifuged at 12000 g, 4 °C for 15 min, first passed through a 0.45 μm filter membrane, and then through a 0.22 μm filter membrane. The filtrate after passing through the membrane was freeze-dried to obtain a crude polypeptide powder for later use, denoted as M1. The crude polypeptide powder was dissolved and ultrafiltered under a 3KDa ultrafiltration tube to obtain two components of >3KDa and <3KDa, which were freeze-dried respectively. Since the predicted molecular weight of the antioxidant peptide is less than 1500KDa, the <3KDa component was used to identify its peptide segment information.

[0072] (2) Polypeptide composition analysis

[0073] a) Pretreatment method

[0074] Sample dissolution: Weigh 1 mg of the sample and add 0.1 mL of 50 mM NH 4 HCO 3 for dissolution;

[0075] C 18 Desalting (Stage-Tip): Activation: Activate the desalting column twice with 300 μL of 100% ACN, Equilibration: Equilibrate the desalting column twice with 200 μL of 0.1% TFA, Loading: Load the sample twice, Desalting: Desalt with 200 μL of 0.1% TFA three times, Elution: Elute once with 300 μL of 80% ACN - 20% 0.1% TFA, and dry under vacuum at 45°C.

[0076] Peptide quantification: Accurately pipette 25 μL of the sample into a new 1.5 mL EP tube, accurately pipette 20 μL of the standard and the sample into a microplate, and add 180 μL of the working reagent to each well. After mixing on a microplate shaker for 30 s, incubate at 37°C for 15 min. After cooling to room temperature, place the 96-well plate in a full-wavelength microplate reader and measure the absorbance at 480 nm. Prepare a standard curve and calculate the sample concentration.

[0077] b) LC-MS detection

[0078] Use a Vanquish Neo / Orbitrap Exploris 480 liquid chromatography - mass spectrometer. The liquid chromatography conditions are as follows:

[0079] 1) Analytical column: 75 μm i.d. × 25 cm, NanoViper C18 1.9 μm, 100A

[0080] 2) Mobile phase A: 0.1% FA;

[0081] 3) Mobile phase B: 0.1% FA, 80% ACN;

[0082] 4) Flow rate: 300 nL / min;

[0083] 5) Analysis time for each component: 66 min;

[0084] 6) Specific chromatographic conditions are as follows:

[0085]

[0086] The full scan range of the mass spectrometry is 100 - 1500 m / z. The resolution of the first-level mass spectrometry is set to 120000, AGC is Standard, Maximum IT: 20 ms; the resolution of the second-level mass spectrometry is set to Resolution: 15000, AGC is Standard, Maximum IT: 22 ms, Cycle time: 1.3 s, and the peptide fragmentation collision energy is set to 30 to generate the original mass spectrometry detection data. The original mass spectrometry file is used to retrieve the target protein database with the following retrieval parameters:

[0087] 1) Fixed modifications: Carbamidomethyl(C).

[0088] 2) Variable modifications: Oxidation(M), Acetyl(Peptide N-term).

[0089] 3) Enzyme: Non specific.

[0090] 4) Database: uniprotkb_proteome_Equus_caballus_2024_06_24.

[0091] 5) Peptide Mass Tolerance of the first-level mass spectrometry: 20 ppm

[0092] 6) Fragment Mass Tolerance of the second-level mass spectrometry: 0.02 Da

[0093] The components eluted through chromatographic separation continuously enter the mass spectrometry, and the mass spectrometry continuously scans for data acquisition. Each scan obtains a mass spectrometry diagram, and finally, the total ion current chromatogram and the second-level spectra of different peptides can be obtained.

[0094] c) To obtain a higher-purity HPCPHPSF polypeptide for subsequent antioxidant detection, we carried out chemical solid-phase synthesis of the polypeptide and determined its purity to be over 98% through HPLC detection.

[0095] 2. Experimental results

[0096] (1) The crude peptides obtained through actual simulated gastrointestinal digestion were detected by polypeptideomics, and the second-level mass spectrometry diagram of Figure 5 HPCPHPSF was obtained, indicating that the polypeptide HPCPHPSF (SEQ ID NO.17) screened by computer can be obtained from horse casein through actual enzymatic digestion.

[0097] (2) Figure 6 The HPLC chromatogram of the polypeptide HPCPHPSF synthesized by chemical solid-phase synthesis shows that its purity is 98%; Figure 6 b The MS chromatogram of the polypeptide HPCPHPSF synthesized by chemical solid-phase synthesis.

[0098] Example 3: In vitro chemical antioxidant activity of horse milk casein antioxidant peptides

[0099] 1. Experimental content and methods

[0100] (1) Determination of DPPH· scavenging rate

[0101] Weigh 19.7 mg of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and dissolve it in absolute ethanol, then make up the volume to 250 mL to prepare a DPPH solution with a concentration of 0.2 mmol / L, and prepare peptide solutions with different concentrations. According to the experimental group As: 100 μL of peptide solution + 100 μL of DPPH solution; the control group Ac: 100 μL of peptide solution + 100 μL of absolute ethanol; the blank group Ab: 100 μL of distilled water + 100 μL of DPPH solution. After thoroughly mixing the reaction system, place it in the dark at room temperature for a full reaction of 30 min, measure the absorbance value at 517 nm, and calculate the DPPH· scavenging rate according to formula 1.

[0102]

[0103] (2) Determination of ABTS·+ scavenging rate

[0104] Refer to the instruction manual of the total antioxidant capacity detection kit (ABTS rapid method) to determine the ABTS·+ scavenging activity. First, prepare the ABTS working solution, which should be prepared immediately before use, stored in the dark at room temperature, and used up within 30 min, and prepare peptide solutions with different concentrations. The antioxidant capacity of the antioxidant is expressed by the relative total antioxidant capacity of Trolox. Therefore, it is necessary to perform gradient dilution of Trolox to make a standard curve. According to the experimental group: 20 μL of peroxidase working solution + 10 μL of peptide solution + 170 μL of ABTS working solution; the standard curve group: 20 μL of peroxidase working solution + 10 μL of Trolox standard solution + 170 μL of ABTS working solution, the blank group: 20 μL of peroxidase working solution + 10 μL of distilled water + 170 μL of ABTS working solution. After gently mixing the reaction system, place it in the dark at room temperature for incubation for 6 min, measure the absorbance value at 405 nm, and the ABTS·+ scavenging ability is expressed by the relative antioxidant capacity of Trolox (TEAC), that is, expressed in μmol Ttolox eq.

[0105] (3) Determination of superoxide anion (O - 2 ) scavenging rate

[0106] It is carried out according to the instruction manual of the superoxide anion scavenging ability detection kit. It is necessary to first prepare the working solution (Working Reagent), which should be prepared and used immediately, and prepare peptide solutions with different concentrations. According to the experimental group: 20 μL of Working Xanthine Oxidase working solution + 20 μL of peptide solution + 80 μL of Working Reagent; control group: 20 μL of Working Xanthine Oxidase working solution + 20 μL of distilled water + 80 μL of Working Reagent; blank group: 40 μL of distilled water + 80 μL of Working Reagent. Mix the reaction system evenly and immediately read the absorbance A at 450 nm 0 , incubate in the dark at room temperature for 60 min, and read the absorbance value A at 450 nm again 60 , calculate ΔA = A 60 - A 0 , and record it as ΔA 空 , ΔA 对 , ΔA 测 , calculate ΔΔA 对 = ΔA 对 - ΔA 空 , ΔΔA 实 = ΔA 实 - ΔA 空 . The scavenging rate of superoxide anion (O - 2 ) is calculated according to formula (2):

[0107] O - 2 Scavenging rate (%) = (ΔΔA 对 _ΔΔA 实 ) / ΔΔA 对 × 100% (2)

[0108] (4) Determination of Fe 3+ Reducing power

[0109] The determination of Fe 3+ reducing power is carried out according to the instruction manual of the total antioxidant capacity detection kit (FRAP method). First, prepare the FRAP working solution, incubate it at 37 °C after preparation, and use it immediately after preparation. The antioxidant capacity of the antioxidant is expressed by the relative total antioxidant capacity of ferrous salt. Therefore, it is necessary to dilute FeSO 4 ·7H 2 O in gradient to make a standard curve. According to the experimental group: 5 μL of peptide solution + 180 μL of FRAP working solution; standard curve group: 5 μL of FeSO 4 ·7H 2O standard solution + 180 μL of FRAP working solution, blank group: 5 μL of distilled water + 180 μL of FRAP working solution. After gently mixing the reaction system, incubate at 37 °C for 5 min and then measure A 593 , its Fe 3+ reducing power is expressed by the relative antioxidant capacity of FeSO 4 ·7H 2 O, that is, expressed in μmol FeSO 4 ·7H 2 Oeq / g.

[0110] 2. Experimental results

[0111] (1) Results and analysis of the scavenging activity of DPPH· free radicals

[0112] The scavenging activity of antioxidant peptides against DPPH· free radicals at different concentrations is as Figure 7 shown. The higher the concentration of antioxidant peptides, the gradually increasing scavenging rate. At a concentration of 0.1 mg / mL, the scavenging rate is about 42%. When the concentration increases to 5 mg / ml, the DPPH scavenging activity reaches 86%, showing a good dose-DPPH· free radical scavenging activity effect.

[0113] (2) Results and analysis of the scavenging activity of ABTS·+

[0114] The scavenging activity of antioxidant peptides against ABTS·+ free radicals at different concentrations is as Figure 8 shown. The ABTS scavenging rate of antioxidant peptides gradually increases with the increase of concentration. When it reaches 5 mg / ml, the scavenging rate reaches the highest, and the absorbance is about 0.1, that is, almost all the active ABTS· free radicals in the detection system are scavenged at this time, showing good ABTS· free radical scavenging activity.

[0115] (3) Results and analysis of the scavenging activity of superoxide anion (O - 2 )

[0116] The scavenging activity of antioxidant peptides against superoxide anion (O - 2 ) at different concentrations is as Figure 9 shown. With the increase of the polypeptide concentration, the O - 2 scavenging activity is stable at about 16%, indicating that it has the ability to scavenge O - 2 , but the scavenging rate is not as high as that of DPPH.

[0117] (4) Results and analysis of the determination of Fe 3+ reducing power

[0118] The Fe 3+The reducing power is 40.72 μmol FeSO 4 ·7H 2 O eq / g as shown in Table 7 below, indicating that it has Fe 3+ reducing power, but the reducing power effect is not as strong as the free radical scavenging effect.

[0119] Table 7 Fe 3+ Reducing power

[0120]

[0121] Example 4: Protective effect of horse casein antioxidant peptides on H 2 O 2 2-induced oxidative damage of HepG2 cells

[0122] 1. Experimental content and methods

[0123] (1) Cell culture

[0124] Cell resuscitation: Take out the cryopreservation tube containing HepG2 cells from the liquid nitrogen tank, put it into a 37 °C water bath, quickly melt it within 1 min, add 2 mL of medium, centrifuge at 1500 g for 5 min, discard the supernatant, add 2 mL of medium, resuspend by pipetting, and transfer it into a cell culture dish. Tilt the culture dish at 30 °, gently pipette 10 times, and then place it in a 37 °C 5% CO 2 2 incubator for culture.

[0125] Cell passage: When the cell density reaches 80 - 90% and the cell state is good, passage can be carried out. Aspirate the original medium, add PBS along the wall for washing, aspirate the PBS, add 2 mL of 0.25% trypsin to a 10 cm culture dish for digestion for 3 min, then add 4 mL of medium containing serum to terminate the digestion, pipette to make the cells detached, and collect them into a 10 mL centrifuge tube. After centrifuging at 1500 g for 5 min, discard the supernatant, add 3 mL of medium to resuspend, transfer the cells to a new culture dish, shake well, let the cells settle for 15 min, and then place them in the incubator for culture.

[0126] (2) CCK8 cell viability assay

[0127] Seed HepG2 cells into a 96-well plate at a density of 10 4 cells / well. When the cell density reaches 70 - 80%, treat with the corresponding drug for a certain time. After the drug treatment time ends, add 100 μL of medium containing 10% CCK8 reagent to each well, incubate at 37 °C for 45 min, then measure the absorbance at 450 nm, and calculate the cell viability according to formula 4. Set 6 technical replicates for each group.

[0128] Cell survival rate = (A 加药 - A空白 ) / (A 对照 -A 空白 )(4)

[0129] A 加药 : Containing cells, culture medium, CCK-8 solution and drug solution

[0130] A 空白 : Culture medium without cells and drugs

[0131] A 对照 : Containing cells, culture medium, CCK-8 solution, without drugs

[0132] (3) Establish a HepG2 cell oxidative damage model

[0133] When the cells grow to 80-90% of the culture dish, the operation is the same as that before passage and cryopreservation. Discard the supernatant, add 1 mL or 2 mL of complete culture medium, and aspirate and mix well with a pipette. Take 10-20 μL of cell suspension, add it to a hemocytometer, and observe and count the cells under a microscope. Calculate the total number of cells in the cell suspension, and dilute the cells to a concentration of 1×10 4 / mL. Take a 96-well cell culture plate and add 100 μL / well of the diluted cell suspension, and place it in an incubator for 12-24 h until the cells adhere to the plate and the density is appropriate. Discard the supernatant, and add 100 μL of a series of H 2 O 2 (200, 400, 600, 800, 1000, 1200, 1400, 1600 μM) prepared with complete culture medium. Set 6 parallels for each concentration and culture in an incubator for 12 h. Use the CCK8 method to measure its survival rate, and select the H 2 O 2 concentration with a cell survival rate of about 50% as the subsequent cell damage model.

[0134] (4) Effect of antioxidant peptides on the survival rate of HepG2 cells

[0135] Take cells in the logarithmic growth phase, and the operation is the same as (3). Discard the supernatant, add 1 mL or 2 mL of complete culture medium, and aspirate and mix well. Count the cells, calculate the total number of cells in the cell suspension, and dilute the cells to a concentration of 1×10 4 / mL. Take a 96-well cell culture plate and add 100 μL of the diluted cell suspension, and place it in an incubator for 12-24 h until the cells adhere to the plate and the density is appropriate. Discard the supernatant, and add 100 μL of a series of peptide solutions (12.5, 25, 50, 100, 200, 400, 600, 800 μg / mL) prepared with complete culture medium. Set 6 parallels for each concentration and culture in an incubator for 24 h. Use the CCK8 method to measure its survival rate.

[0136] (5) Protective effect of antioxidant peptides on H 2 O 2 induced oxidative damage of HepG2 cells

[0137] Same as the operation of inoculating cells into a 96-well cell culture plate in (3). After the cells grew well, the supernatant was discarded, and a series of peptide solutions (100, 200 μg / mL) prepared with complete medium and 800 μM H 2 O 2 mixture were added, and the cells were further cultured in an incubator for 12 h. The supernatant was discarded, the cells were washed once with PBS, and the cell viability was determined by the CCK8 method.

[0138] (6) Determination of cell-related antioxidant indexes

[0139] When the cells grew to 80 - 90% confluence in the culture dish, the operation was the same as that before passage and cryopreservation. After resuspending the cells, 6-well cell culture plates were taken and the diluted cell suspension was added at 2 mL / well, mixed well, and placed in an incubator for 12 - 24 h. When the cells were at the logarithmic growth phase density, the supernatant was discarded, the cells were washed twice with PBS, and the samples prepared with medium were added. They were divided into a control group, a sample group, and a model group. The control group was added with complete medium, the model group was added with 800 μM H 2 O 2 , the sample group was added with 200 μg / mL peptide solution + 800 μM H 2 O 2 , and they were placed in an incubator for incubation for 12 h. The supernatant was discarded, the cells were washed twice with PBS, digested with 500 μL of trypsin for 2 min, then 1 mL of medium was added to terminate the digestion, resuspended with PBS and transferred to a 1.5 mL centrifuge tube, centrifuged at 1500 g for 5 min, the supernatant was discarded, 300 μL of PBS was added to the cell pellet, placed on ice, and the cells were ultrasonically disrupted under the conditions of a power of 300 W, 2 s of disruption each time, an interval of 10 s, and a total disruption time of 1 min. Finally, the supernatant of the disrupted cells was collected for the determination of various indexes (malondialdehyde MDA, superoxide dismutase SOD, catalase CAT, glutathione peroxidase GSH-Px). The activities of MDA, SOD, CAT, and GSH-Px were determined strictly according to the relevant steps in the kit instructions. Protein was quantitatively determined by the BCA method.

[0140] (7) Determination of reactive oxygen species ROS

[0141] The steps of culturing cells in the prophase, administering drugs, and inducing are the same as those for measuring relevant antioxidant index values in (6). According to the ROS kit instructions, prepare the DCFH-DA fluorescent probe with serum-free medium at a ratio of 1:1000 for standby. After the cell induction is completed, discard the supernatant. In a light-proof environment, add 1 mL of the prepared 10 μM DCFH-DA fluorescent probe to the 12-well plate and continue culturing in the cell incubator for 30 min. Take it out (subsequent operations are all carried out under light-proof conditions), wash it 4 times with PBS to thoroughly wash away the excess probe in the background. Under the laser confocal microscope, use an excitation wavelength of 488 nm and an emission wavelength of 525 nm to observe the ROS fluorescence of the cells.

[0142] 2. Experimental results

[0143] (1) H 2 O 2 Establishment of an oxidative damage model of HepG2 cells induced by H

[0144] Figure 10 To determine the cell viability of HepG2 cells after being damaged by H 2 O 2 for 12 h within the concentration range of 200 - 1600 μmol / L. As the concentration of H 2 O 2 increased, the cell viability of HepG2 cells decreased significantly (P < 0.05). When the concentration was 800 μmol / L, the cell viability decreased to 71.97%, and when it was 1.0 mmol / mL, the lethality rate to the cells reached 47.72%. Since the damage to the cells by H 2 O 2 might be irreversible at a relatively low cell viability, the concentration of H 2 O 2 at 800 μmol / L was selected as the subsequent cell damage model.

[0145] (2) Effect of antioxidant peptides on the viability of HepG2 cells

[0146] The effect of antioxidant peptides on the viability of HepG2 cells is as Figure 11 shown. When the peptide concentration was in the range of 12.5 - 800 μg / mL, the cell viability was greater than 80%. A cell viability of more than 80% indicates that it has no toxicity to HepG2 cells. To avoid waste of the polypeptide, peptide concentrations of 100 and 200 μg / mL were selected for subsequent experiments.

[0147] (3) Protective effect of antioxidant peptides on oxidative damage of HepG2 cells induced by H 2 O 2

[0148] HepG2 cells were added with antioxidant peptides and H 2 ​O 2 The protective effect on cells after 12 h of culture is as Figure 12 shown. Compared with the model group, the cell survival rate at the concentration of 100 μg / mL of the antioxidant peptide increased but not significantly, and the cell survival rate at the concentration of 200 μg / mL increased significantly. Therefore, 200 μg / mL was selected for subsequent experiments.

[0149] (4) Analysis of related antioxidant enzyme activities and damage markers

[0150] a) Malondialdehyde MDA

[0151] Malondialdehyde is the product of cell lipid peroxidation. The higher the MDA content, the more serious the oxidative stress damage of the cell. As Figure 13 shown, compared with the model group, the antioxidant peptide could significantly reduce the MDA content in cells, from 17.81 μmol / mg to 11.50 μmol / mg.

[0152] b) Total superoxide dismutase T-SOD

[0153] Total superoxide dismutase T-SOD is an important antioxidant enzyme in the body that participates in resisting oxidative stress. As Figure 14 shown, compared with the model group, the antioxidant peptide could significantly increase the T-SOD enzyme activity in cells, from 49.88 to 115.79 U / mg.

[0154] c) Catalase CAT

[0155] CAT can convert excessive H2O2 in cells into H2O and O2, reducing cell oxidative stress damage. As Figure 15 shown, compared with the model group, the antioxidant peptide could significantly increase the CAT enzyme activity in cells, and the enzyme activity increased from 2.26 U / mg to 2.66 U / mg.

[0156] d) Glutathione peroxidase GSH-Px

[0157] As Figure 16 shown, compared with the model group, the antioxidant peptide could significantly increase the glutathione peroxidase GSH-Px enzyme activity in cells, and the enzyme activity increased from 0.54 U / mg to 1.13 U / mg.

[0158] (5) Reactive oxygen species ROS

[0159] It can be clearly seen from Figure 17 (a) that the fluorescence intensity of the antioxidant peptide treatment group was significantly lower than that of the model group, indicating that the peptide could reduce the oxidative stress damage of cells by scavenging intracellular ROS. As Figure 17As shown in the fluorescence quantification graph in (b), compared with the model group, each peptide could significantly reduce the relative content of cellular ROS (P < 0.0001).

[0160] In summary, the antioxidant peptide HPCPHPSF (SEQ ID NO.17) has no toxicity to HepG2 cells and has a protective effect on the damage of HepG2 cells induced by H2O2. The analysis of antioxidant indexes at the cellular level shows that the antioxidant peptide can regulate the balance among the antioxidant defense systems in HepG2 cells.

[0161] The embodiments described above are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A horse milk casein-derived antioxidant peptide having antioxidant function, characterized in that: The amino acid sequence of the horse milk casein-derived antioxidant peptide is shown in SEQ ID NO.17, specifically: HPCPHPSF.

2. A composition with antioxidant function, characterized in that: Contains the horse milk casein-derived antioxidant peptide as claimed in claim 1.

3. Use of the horse milk casein-derived antioxidant peptide as claimed in claim 1 in antioxidant products.

4. Use of the antioxidant peptide according to claim 1 in preparing a composition with antioxidant efficacy, wherein the composition is a cosmetic composition.

5. Use of the antioxidant peptide according to claim 1 in preparing a composition with antioxidant efficacy, wherein the composition is a food or a food composition.

6. A composition comprising the horse milk casein-derived antioxidant peptide according to claim 1, and optionally comprising auxiliary materials; The composition is a food composition or a cosmetic composition.

7. Use of the horse milk casein-derived antioxidant peptide according to claim 1 in foods for protecting against oxidative damage.

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