Development and research of mare's milk casein antioxidant peptide based on computer and experiment

Abstract

The application relates to development and application of horse milk casein antioxidant peptides. Antioxidant peptides are screened and prepared from horse milk casein, and function verification is carried out. Through virtual enzymolysis and computer simulation, polypeptide sequences possibly generated after horse milk casein hydrolysis are predicted, and polypeptides with potential antioxidant activity are screened out by combining with molecular docking analysis of Keap1 protein. The function of the screened antioxidant peptides is verified by simulating the gastrointestinal digestion process. High-purity antioxidant peptides are synthesized by chemical synthesis, and in-vitro chemical antioxidant experiments are carried out, and it is found that the antioxidant peptides have a significant free radical scavenging capacity. In the cell experiment, the biological activity is verified by using an H2O2-induced oxidative damage model, and the results show that the antioxidant peptides can significantly improve the activity of intracellular antioxidant enzymes and effectively resist oxidative damage. The method provides an efficient path for the development of natural antioxidant peptides, and the screened functional polypeptides have a wide application prospect in the food field.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polypeptide products and molecular biology, and particularly relates to development and function research of a mare milk casein-derived antioxidant peptide. BACKGROUND

[0002] Oxidation process is inevitable in organisms, and oxidative metabolism produces free radicals and reactive oxygen species (ROS). Oxidation reactions that occur during food storage can lead to nutrient loss, quality deterioration, 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] Antioxidant peptides, as a natural antioxidant, 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 for use as food additives to extend the shelf life of food and improve nutritional value. Studies have 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 influences on their antioxidant activity. Therefore, in this study, we first screened potential high-antioxidant peptides from mare milk casein using a computer screening method, then verified their free radical scavenging and metal ion chelating effects in vitro, and finally verified their antioxidant activity at the cellular level, demonstrating their potential application prospects in the food industry. SUMMARY

[0004] The purpose of the present application is to provide a mare milk casein-derived antioxidant peptide with antioxidant function, which has good antioxidant activity in vitro and at the cellular level and can be used for preparing food.

[0005] The purpose of the present application is achieved by the following technical solutions.

[0006] A mare milk casein-derived antioxidant peptide with antioxidant function, which has an amino acid sequence of HPCPHPSF, and the sequence is shown as SEQ ID NO. 17.

[0007] The present application provides a method for preparing the above-mentioned antioxidant peptide, which comprises the following steps: centrifuging 3000 g of fresh mare milk for 20 min to remove milk fat, adjusting the pH of defatted mare milk to 4.6 with acetic acid, precipitating casein, centrifuging at 12000 g for 20 min at 4 DEG C, removing the supernatant, washing twice with water and centrifuging, and freeze-drying the precipitated casein for standby use.

[0008] The casein is weighed, wetted with a small amount of NaOH solution, and dissolved in distilled water to prepare a 50 mg / ml solution, and ultrasonic treatment is performed for 15 min to accelerate the dissolution. First, 1M HCL is used to adjust the pH to 2.0, and then pepsin is added. Then, the pH is adjusted to 7.6, and then trypsin is added. Finally, the pH is adjusted to 8.0, and then chymotrypsin is added. The amount of each enzyme added is 6000 U / g. The magnetic stirring water bath is used for hydrolysis at 37 DEG C for 2 h. After hydrolysis is completed, inactivation is performed at 95 DEG C for 15 min. After cooling to room temperature, the pH is adjusted to neutral. Centrifugation is performed at 12000 g, 4 DEG C for 15 min. First, the solution is filtered through a 0.45 mu m filter membrane, and then filtered through a 0.22 mu m filter membrane. The filtrate after filtration is freeze-dried to obtain a crude polypeptide powder for later use. The crude polypeptide powder is dissolved and subjected to ultrafiltration through a 3KDa ultrafiltration tube to obtain two components with a molecular weight greater than 3KDa and less than 3KDa. Since the predicted antioxidant peptide molecular weight is less than 1500KDa, the component with a molecular weight less than 3KDa is used to identify the peptide segment information.

[0009] Further, polypeptidomics analysis and detection are performed on the milk casein protease hydrolysate to identify the peptide segment sequence information after enzymolysis.

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

[0011] The high-activity polypeptide sequence is chemically synthesized to obtain a milk casein antioxidant peptide with a purity of more than 98%, and antioxidant activity verification is performed.

[0012] The application also provides an application of the screening and preparation method in the screening and preparation of antioxidant peptides, and specifically in the screening and preparation of milk casein antioxidant peptides.

[0013] Further, the application also provides an application of the antioxidant peptide in the food for preventing oxidative damage.

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

[0015] 1. The application obtains a new type of peptide with high antioxidant activity through simulation hydrolysis and computer screening. By using an effective database and molecular docking, the main action target of the antioxidant activity peptide is simulated, and rapid and efficient antioxidant peptide identification is realized.

[0016] 2. The antioxidant peptide screened by the application has strong comprehensive antioxidant activity, has the characteristics of high efficiency, simple preparation method and good repeatability, and has a broad prospect in the fields of food, medicine and cosmetics, and provides an efficient way for the development of milk casein functional polypeptide products. The specific advantages are as follows:

[0017] (1) HPCPHPSF showed good DPPH· radical scavenging activity and ABTS·+ radical scavenging capacity, better O - 2 superoxide anion scavenging activity and ferrous ion reducing capacity.

[0018] (2) The results of cell level antioxidant activity showed that HPCPHPSF peptide had protective effect on H2O2-induced HepG2 cell damage. Compared with the model group, HPCPHPSF peptide showed different intensity of antioxidant activity in cells, which could significantly reduce the production of malondialdehyde (MDA), significantly increase the enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and significantly reduce the content of reactive oxygen species (ROS) in cells. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

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

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

[0025] Figure 10 is the effect of different H2O2 concentrations on the survival rate of HepG2 cells. Note: marked with **** indicates extremely significant difference (P<0.0001).

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

[0027] Figure 12 is the protective effect of the peptide on H2O2-induced oxidative damage of HepG2 cells. Note: 100 in the figure indicates 100 μg / ml peptide solution + H2O2; 200 indicates 200 μg / ml peptide solution + H2O2. ns indicates no significant difference; * is the significant level P<0.05; ** is the significant level P<0.01.

[0028] Figure 13 Effect of the peptide on MDA content in HepG2 cells. Note: 200 in the figure represents 200 μg / ml peptide solution + H2O2; ** represents significant level P<0.01; *** represents significant level P<0.001.

[0029] Figure 14 Effect of the peptide on SOD activity in HepG2 cells. Note: 200 in the figure represents 200 μg / ml peptide solution + H2O2; ** represents significant level P<0.01; **** represents extremely significant difference (P<0.0001).

[0030] Figure 15 Effect of the peptide on CAT activity in HepG2 cells. Note: 200 in the figure represents 200 μg / ml peptide solution + H2O2; * represents significant level P<0.05; **** represents extremely significant difference (P<0.0001).

[0031] Figure 16 Effect of the peptide on GSH-Px activity in HepG2 cells. Note: 200 in the figure represents 200 μg / ml peptide solution + H2O2; * represents significant level P<0.05; *** represents significant level P<0.001.

[0032] Figure 17 Effect of the peptide on ROS content in HepG2 cells; (a) ROS fluorescence result map of HepG2 cells; (b) ROS fluorescence result quantification map of HepG2 cells. DETAILED DESCRIPTION

[0033] The concept and the generated technical effects of the present application are further described below in combination with specific embodiments, so as to fully understand the purposes, features and effects of the present application. The methods are all conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present application and the descriptions thereof are used to explain the present application, and do not constitute an improper limitation on the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0034] Example 1: Virtual enzymolysis and screening of mare milk casein-derived antioxidant peptides

[0035] 1. Experimental method

[0036] (1) A total of 16 horse whey protein sequences were screened according to the relative abundance in the UniProt database (https: / / www.uniprot.org / ), which showed information about their entries, protein names, amino acid sequence lengths, and molecular weights (see Table 1). In silico simulation of the gastrointestinal tract digestion, the PeptideCutter program in BIOPEP

[0037] (https: / / www.expasy.org / resources / peptidecutter) selected pepsin (pH <2), trypsin, and chymotrypsin (high specificity) for virtual gastrointestinal hydrolysis of all screened proteins. From the hydrolysates produced by the three enzymes, dipeptides, tripeptides, tetrapeptides, pentapeptides, and polypeptides were screened to form a peptide library.

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

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

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

[0041] (5) The above-mentioned novel peptides screened for free radical scavenging ability were evaluated for toxicity using the Toxinpred prediction tool (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html4) of the toxicity prediction website, and their basic properties such as isoelectric point and hydrophilicity were evaluated.

[0042] (6) The ADMET tool was used for property prediction (https: / / admetmesh.scbdd.com / ), which can predict the effects of polypeptides on the blood-brain barrier, human intestinal absorption, carcinogenicity, and acute oral toxicity.

[0043] (7) The final several polypeptides screened are subjected to molecular docking with the Keap1 protein. The kelch region (PDB ID: 1U6D) protein structure of the Keap1 protein is downloaded from the PDB database (https: / / www.rcsb.org / ), and the peptide chain is modeled by using Maestro 11.0 software. The peptide chain is designated as a ligand, and the kelch region of the Keap1 protein is designated as a 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 charge checking. The polypeptide is subjected to energy minimization processing, and H++3 online server is used for protonation processing under neutral conditions (pH = 7). Then, UCSF Chimera software is used to assign Amber14SB charges. Finally, the conformation with the first docking score is selected for subsequent analysis, and 3D mapping analysis is performed by using Discovery Studio. The docking energy is generally negative, and the smaller the value, the higher the docking result score, that is, the tighter the binding between the ligand and the receptor protein, the strongest the interaction, and the more likely to have antioxidant activity.

[0044] 2. Experimental results

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

[0046] Table 1 Sequence of mare milk casein

[0047]

[0048]

[0049]

[0050]

[0051] (2) The following table is the peptide segment with a score higher than 0.8 screened from the peptide library

[0052] Table 2 Casein peptide segment with a biological activity score higher than 0.8

[0053]

[0054] After the biological activity peptide library is queried, NF, MY, SF, and QW are the biological activity peptides that have been found. Considering that new active peptides are to be screened, the four are removed, and the other peptide segments are subjected to subsequent activity prediction.

[0055] (3) The following table is the free radical scavenging capacity of the new peptide​

[0056] Table 3 New type of peptide free radical scavenging effect score

[0057]

[0058] Table 4 New type of peptide chelation score

[0059]

[0060] In summary, from the above table we can see that the above polypeptides can have free radical scavenging activity, among them the polypeptide HPCPHPSF (SEQ ID NO. 17) has better free radical scavenging activity in general, the following toxicity evaluation of the above polypeptides confirms that they have no biological toxicity.

[0061] (4) The following table is the polypeptide toxicity and other basic attribute prediction

[0062] Table 5 New type of peptide toxicity prediction and basic attributes

[0063]

[0064] (5) The following table is the docking binding energy of the screened candidate peptide and Keap1 protein molecule

[0065] Table 6 New type of peptide and Keap1 protein molecule docking binding energy

[0066]

[0067] Table 6 is the docking binding energy of the 3 new type of peptides screened and the Kelch region of Keap1, the molecular docking score is from -8.7 kcal / mol (MNW) to -9.3 kcal / mol (HPCPHPSF), in other studies, the absolute value of the molecular docking score is positively correlated with the antioxidant activity, and the binding energy score < -8.0 kcal / mol belongs to a relatively strong binding force, the lower the negative value, the stronger the binding force. The score of HPCPHPSF is -9.3 kcal / mol, which is the most closely combined with the Kelch region of Keap1 protein, indicating that it is most likely an antioxidant active polypeptide. Figures 1-4 The figure of the molecular docking results of the polypeptide HPCPHPSF (SEQ ID NO. 17) and the Kelch region of Keap1 includes 3D and local detail figures. It can be seen that the polypeptide HPCPHPSF is combined in the Kelch region of Keap1 through hydrogen bonds VAL514, VAL561, VAL467, VAL608, VAL369, which is the antioxidant peptide we finally screened.

[0068] Example 2: Preparation, identification and solid phase synthesis of mare cheese protein antioxidant peptides 1. Experimental methods and contents

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

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

[0071] The casein was weighed and moistened with a small amount of NaOH solution, and distilled water was added to prepare a 50mg / ml solution. The solution was ultrasonicated for 15 min to accelerate dissolution. First, 1M HCL was used to adjust the pH to 2.0, and then pepsin was added. The pH was adjusted to 7.6, and then trypsin was added. Finally, the pH was adjusted to 8.0, and then chymotrypsin was added. The amount of each enzyme added was 6000U / g. The mixture was stirred in a magnetic stirring water bath at 37°C for 2h. After hydrolysis was completed, the mixture was inactivated at 95°C for 15 min, cooled to room temperature, and the pH was adjusted to neutral. The mixture was centrifuged at 12000g at 4°C for 15 min. The supernatant was first filtered through a 0.45μm filter, and then through a 0.22μm filter. The filtrate was lyophilized to obtain a crude polypeptide powder, which was stored for use and was designated as M1. The crude polypeptide powder was dissolved and ultrafiltered through a 3KDa ultrafiltration tube to obtain two components, >3KDa and <3KDa. Since the predicted molecular weight of the antioxidant peptides was less than 1500KDa, the <3KDa component was used to identify the peptide segment information.

[0072] (2) Polypeptide composition analysis

[0073] a) Pretreatment method

[0074] Sample dissolution: 1mg of sample was weighed and dissolved in 0.1mL of 50mM NH4HCO3 solution;

[0075] C 18 Desalting (Stage-Tip): Activation: The desalting column was activated twice with 300μL of 100% ACN, Equilibration: The desalting column was equilibrated twice with 200μL of 0.1% TFA, Loading: Loaded twice, Desalting: Desalted three times with 200μL of 0.1% TFA, Elution: Eluted once with 300μL of 80% ACN-20% 0.1% TFA, and dried at 45°C under vacuum.

[0076] Peptide segment quantification: 25μL of sample was accurately pipetted into a new 1.5mL EP tube, and 20μL of standard and sample was accurately pipetted into a microplate. 180μL of working reagent was added to each well. After mixing on a microplate shaker for 30s, incubation was performed at 37°C for 15 min. After cooling to room temperature, the 96-well plate was placed on a full-wavelength microplate reader, and the absorbance at 480nm was measured. A standard curve was prepared, and the sample concentration was calculated.

[0077] b) Liquid chromatography-mass spectrometry

[0078] Using Vanquish Neo / Orbitrap Exploris 480 liquid chromatograph-mass spectrometer, the liquid chromatograph conditions are as follows:

[0079] 1) Analysis column: 75 μm i.d. x 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 of each component: 66 min;

[0084] 6) The specific chromatographic conditions are as follows:

[0085]

[0086] The mass spectrometry full scan range is 100-1500 m / z, the first-order mass spectrometry resolution is set to 120000, the AGC is Standard, the Maximum IT is 20 ms; the second-order mass spectrometry resolution is set to Resolution: 15000, the AGC is Standard, the Maximum IT is 22 ms Cycle time: 1.3 s, and the peptide fragment collision energy is set to 30. The mass spectrometry raw data is detected to generate a mass spectrum. The mass spectrometry raw file is searched using software to search a target protein database, and the search parameters are as follows:

[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) First-order mass spectrometry deviation (Peptide Mass Tolerance): 20 ppm

[0092] 6) Fragment Mass Tolerance: 0.02 Da

[0093] The components separated by chromatography continuously enter the mass spectrometer, and the mass spectrometer continuously scans to collect data. Each scan obtains a mass spectrum, and finally the total ion flow chromatogram and the secondary spectrum of different peptides can be obtained.

[0094] c) In order to obtain HPCPHPSF polypeptides with higher purity for subsequent antioxidant detection, we carried out chemical solid-phase synthesis of polypeptides, and determined the purity of more than 98% by HPLC detection.

[0095] 2、Experimental results

[0096] (1) After actual simulation of gastrointestinal digestion, the crude peptide obtained by polypeptidomics detection obtained the secondary mass spectrum of HPCPHPSF Figure 5 , which shows that the computer screened polypeptide HPCPHPSF (SEQ ID NO. 17) can be obtained from mare casein by actual enzymatic digestion.

[0097] (2) Figure 6 a The HPLC chart of polypeptide HPCPHPSF synthesized by chemical solid-phase synthesis can be seen that its purity is 98%; Figure 6 b MS chart of polypeptide HPCPHPSF synthesized by chemical solid-phase synthesis.

[0098] Example 3: In vitro chemical antioxidant activity of mare casein antioxidant peptide

[0099] 1、Experimental content and method

[0100] (1) DPPH· clearance rate determination

[0101] Weigh 19.7 mg of 2,2-diphenyl-1-picrylhydrazyl (DPPH) into anhydrous ethanol, dilute to 250 mL, and prepare a DPPH solution with a concentration of 0.2 mmol / L. Prepare different concentrations of peptide solution. According to the experimental group As: 100 μL of peptide solution + 100 μL of DPPH solution; control group Ac: 100 μL of peptide solution + 100 μL of anhydrous ethanol; blank group Ab: 100 μL of distilled water + 100 μL of DPPH solution, mix the reaction system thoroughly, and place it in the dark at room temperature for 30 min. Measure the absorbance value at 517 nm, and calculate the DPPH· clearance rate according to formula 1.

[0102]

[0103] (2) ABTS·+ clearance rate determination

[0104] ABTS·+ scavenging activity assay was performed according to the instruction of total antioxidant capacity assay kit (ABTS fast method). ABTS working solution was prepared, which was used immediately and stored at room temperature in the dark, and used up within 30 min. Peptide solution with different concentrations was prepared. The antioxidant capacity of antioxidants was expressed by the relative Trolox total antioxidant capacity. Therefore, gradient dilution of Trolox was needed to make a standard curve. According to the experimental group: 20 μL peroxide enzyme working solution + 10 μL peptide solution + 170 μL ABTS working solution; the standard curve group: 20 μL peroxide enzyme working solution + 10 μL Trolox standard solution + 170 μL ABTS working solution, the blank group: 20 μL peroxide enzyme working solution + 10 μL distilled water + 170 μL ABTS working solution. The reaction system was mixed gently, and then incubated at room temperature in the dark for 6 min. The absorbance value was measured at 405 nm, and the ABTS·+ scavenging capacity was expressed by the relative antioxidant capacity of Trolox (TEAC), that is, μmol Trolox eq.

[0105] (3) Superoxide anion (O - 2) Scavenging rate determination

[0106] According to the instruction of superoxide anion scavenging capacity assay kit. Working Reagent was prepared, which was used immediately, and peptide solution with different concentrations was prepared. According to the experimental group: 20 μL Working Xanthine Oxidase working solution + 20 μL peptide solution + 80 μL Working Reagent; the control group: 20 μL Working Xanthine Oxidase working solution + 20 μL distilled water + 80 μL Working Reagent; the blank group: 40 μL distilled water + 80 μL Working Reagent. The reaction system was mixed, and the absorbance A0 at 450 nm was immediately read. After incubation at room temperature in the dark for 60 min, the absorbance value at 450 nm was read again. 60 60 空 对 测 对 对 空 实 实 空 Superoxide anion (O - 2) Scavenging rate was calculated according to formula 2:

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

[0108] (4)Fe 3+ Reducing power assay

[0109] Reference to the total antioxidant capacity assay kit (FRAP method) instructions Fe 3+ Reducing power assay, first configuration FRAP working solution, incubated after preparation, present. The antioxidant capacity of antioxidant is expressed by the relative total antioxidant capacity of ferrous sulfate. Therefore, the gradient dilution of FeSO4·7H2O is required to make a standard curve. According to the experimental group: 5 μL peptide solution + 180 μL FRAP working solution; standard curve group: 5 μL FeSO4·7H2O standard solution + 180 μL FRAP working solution, blank group: 5 μL distilled water + 180 μL FRAP working solution. After mixing the reaction system gently, incubate at 37℃ for 5 min, then measure A 593 , Fe 3+ Reducing power is expressed by the relative antioxidant capacity of FeSO4·7H2O, that is, μmol FeSO4·7H2O eq / g.

[0110] 2、Experimental results

[0111] (1) DPPH·radical scavenging activity results and analysis

[0112] The DPPH·radical scavenging activity of antioxidant peptides at different concentrations is shown in Figure 7 , the higher the concentration of antioxidant peptides, the higher the clearance rate, the clearance rate is about 42% at a concentration of 0.1 mg / mL, and the DPPH scavenging activity reaches 86% when the concentration increases to 5 mg / ml, showing a good dose-DPPH·radical scavenging activity effect.

[0113] (2) ABTS·+ scavenging activity results and analysis

[0114] The ABTS·+ radical scavenging activity of antioxidant peptides at different concentrations is shown in Figure 8 , the ABTS clearance rate of antioxidant peptides gradually increases with the increase of concentration, and reaches the highest when the concentration is 5 mg / ml, the absorbance is about 0.1, that is, the active ABTS·radicals in the detection system are almost completely cleared, and it shows good ABTS·radical scavenging activity.

[0115] (3) Superoxide anion (O - 2) scavenging activity results and analysis

[0116] The superoxide anion (O- 2) scavenging activity as shown in Table 2, with the increase of the concentration of the polypeptide, the O Figure 9 - 2) The scavenging activity is stabilized at about 16%, which shows that it has O - 2) scavenging ability, but is lower than the DPPH scavenging rate.

[0117] (4) Fe 3+ reducing power determination results and analysis

[0118] antioxidant peptide Fe 3+ reducing power is shown in Table 7 as 40.72 μmol FeSO4·7H2O eq / g, which shows 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 mare milk casein antioxidant peptide on H2O2-induced oxidative damage of HepG2 cells

[0122] 1. Experimental content and method

[0123] (1) Cell culture

[0124] Cell recovery: Take the cryopreservation tube containing HepG2 cells from the liquid nitrogen tank and place it in a 37°C water bath pot. Melt quickly within 1 min, add 2 mL of culture medium, centrifuge at 1500g for 5 min, discard the supernatant, add 2 mL of culture medium and resuspend by blowing, then connect to the cell culture dish. Tilt the culture dish at 30°, gently blow 10 times, and then place it in a 37°C 5% CO2 incubator.

[0125] Cell passage: When the cell density reaches 80-90% and the state is good, it can be passaged. Remove the original culture medium, wash along the wall with PBS, remove the PBS, add 2 mL of 0.25% trypsin to a 10 cm culture dish for 3 min, then add 4 mL of culture medium containing serum to terminate digestion, blow to make the cells fall off, and collect into a 10 mL centrifuge tube. Centrifuge at 1500g for 5 min, discard the supernatant, add 3 mL of culture medium, resuspend, and pass the cells to a new culture dish, shake well, and let the cells settle for 15 min. Place it in the incubator.

[0126] (2) CCK8 cell viability determination

[0127] HepG2 cells were seeded in 96-well plates at a density of 10 4 ​Cells were seeded at a density of 1,000 cells / well in 96-well plates, and when the cell density reached 70-80%, the cells were treated with the corresponding drugs for a certain period of time. After the drug administration time ended, 100 μL of culture medium containing 10% CCK8 reagent was added to each well, and the absorbance at 450 nm was measured after incubation at 37°C for 45 min. The cell viability was calculated according to formula 4, and 6 technical replicates were set for each group.

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

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

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

[0131] A 对照 : cells, culture medium, CCK-8 solution, and no drugs

[0132] (3) Establishing an oxidative damage model of HepG2 cells

[0133] When the cells grew to 80-90% in the culture dish, the operation was the same as the pre-freezing and subculturing treatment. The supernatant was discarded, and 1 mL or 2 mL of complete culture medium was added and fully aspirated to be uniform. 10-20 μL of cell solution was taken and added to a hemocytometer plate, and the cells were observed and counted under a microscope. The total number of cells in the cell solution was calculated, and the cells were diluted to a concentration of 1×10 4 / mL. 100 μL of the diluted cell solution was added to a 96-well cell culture plate, and the plate was placed in an incubator for 12-24 h until the cells adhered to the plate and the density was moderate. The supernatant was discarded, and 100 μL of a series of H2O2 (200, 400, 600, 800, 1000, 1200, 1400, 1600 μM) prepared with complete culture medium was added. Each concentration was set in 6 parallel wells, and the plate was incubated in the incubator for 12 h. The survival rate was determined by the CCK8 method, and the H2O2 concentration at which the cell survival rate was about 50% was selected as the subsequent cell damage model.

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

[0135] Cells in the logarithmic growth phase were taken, and the operation was the same as in (3). The supernatant was discarded, and 1 mL or 2 mL of complete culture medium was added and fully aspirated to be uniform. The cells were counted, and the total number of cells in the cell solution was calculated. The cells were diluted to a concentration of 1×10 4 / mL, 100 μL of the diluted cell solution was added to a 96-well cell culture plate, and the plate was placed in an incubator for 12-24 h until the cells were well attached to the plate and had a moderate density. The supernatant was discarded, and 100 μL of a series of peptide solutions (12.5, 25, 50, 100, 200, 400, 600, 800 μg / mL) prepared with complete medium was added. Six replicates were set up for each concentration, and the plate was incubated in the incubator for 24 h. The survival rate was determined by the CCK8 method.

[0136] (5) Protective effect of antioxidant peptides on H2O2-induced oxidative damage to HepG2 cells

[0137] The cells were seeded into a 96-well cell culture plate as in (3), and after the cells grew well, the supernatant was discarded, and a mixture of a series of peptide solutions (100, 200 μg / mL) prepared with complete medium and 800 μM H2O2 was added. The plate was incubated in the incubator for 12 h. The supernatant was discarded, the cells were washed once with PBS, and the cell survival rate was determined by the CCK8 method.

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

[0139] When the cells grew to 80-90% of the culture dish, the operation was the same as the pre-freezing and subculturing operation. After the cells were resuspended, 6-well cell culture plates were prepared with 2 mL of the diluted cell suspension per well, mixed thoroughly, and placed in an incubator for 12-24 h. When the cells were in the logarithmic growth phase, the supernatant was discarded, the cells were washed twice with PBS, and the samples prepared with the medium were added. The samples 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 H2O2, and the sample group was added with 200 μg / mL peptide solution + 800 μM H2O2. The plate was incubated in the incubator for 12 h. The supernatant was discarded, the cells were washed twice with PBS, and the cells were digested with 500 μL of trypsin for 2 min. The digestion was terminated by adding 1 mL of medium, and the cells were resuspended with PBS and transferred to a 1.5 mL centrifuge tube. The tube was centrifuged at 1500 g for 5 min, the supernatant was discarded, 300 μL of PBS was added to the cell pellet, and the tube was placed on ice. The cells were ultrasonically broken under the following conditions: power 300 W, 2 s each time, 10 s interval, and 1 min of breaking. The supernatant of the broken cells was collected for determination of the indicators (malondialdehyde MDA, superoxide dismutase SOD, catalase CAT, and glutathione peroxidase GSH-Px). The activities of MDA, SOD, CAT, and GSH-Px were determined according to the relevant procedures in the kit instructions. The protein was quantitatively determined by the BCA method.

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

[0141] Cell pre-culture, drug administration, induction and determination of antioxidant index values in (6). According to the ROS kit instructions, DCFH-DA fluorescent probe was prepared with serum-free medium at a ratio of 1:1000. After the induction of cells, the supernatant was discarded, and 1 mL of prepared 10 μM DCFH-DA fluorescent probe was added in a 12-well plate in a light-proof environment. The cells were incubated in a cell culture box for 30 min. After taking out (subsequent operations were all in a light-proof condition), the cells were washed with PBS for 4 times to wash away the excess probe. Under the laser confocal microscope, the ROS fluorescence of the cells was observed using 488 nm excitation wavelength and 525 nm emission wavelength.

[0142] 2. Experimental results

[0143] (1) Establishment of H2O2-induced oxidative damage model of HepG2 cells

[0144] Figure 10 The survival rate of cells was determined after H2O2-induced damage to HepG2 cells for 12 h in the concentration range of 200-1600 μmol / L. As the concentration of H2O2 increased, the survival rate of HepG2 cells decreased significantly (P<0.05). At a concentration of 800 μmol / L, the survival rate of cells decreased to 71.97%, and at a concentration of 1.0 mmol / mL, the lethality rate of cells reached 47.72%. Since the damage to cells by H2O2 at a lower survival rate may be irreversible, the concentration of H2O2 was selected as 800 μmol / L for the subsequent cell damage model.

[0145] (2) Effect of antioxidant peptide on the survival rate of HepG2 cells

[0146] The effect of antioxidant peptide on the survival rate of HepG2 cells is shown in Figure 11 . When the concentration of peptide was 12.5-800 μg / mL, the survival rate of cells was greater than 80%, and the survival rate of more than 80% indicated that the peptide had no toxicity to HepG2 cells. In order to avoid the waste of polypeptide, 100 and 200 μg / mL peptide concentrations were selected for subsequent experiments.

[0147] (3) Protective effect of antioxidant peptide on H2O2-induced oxidative damage of HepG2 cells

[0148] The protective effect of antioxidant peptide on HepG2 cells after adding antioxidant peptide and H2O2 for 12 h is shown in Figure 12 . Compared with the model group, the survival rate of cells with antioxidant peptide at a concentration of 100 μg / mL increased but was not significant, and the survival rate of cells with antioxidant peptide at a concentration of 200 μg / mL increased significantly, so 200 μg / mL was selected for subsequent experiments.

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

[0150] a) Malondialdehyde (MDA)

[0151] Malondialdehyde (MDA) is a product of cell lipid peroxidation, and a higher MDA content indicates more severe cell oxidative stress damage. As shown in Table 4, the antioxidant peptide significantly reduced the cell MDA content from 17.81 μmol / mg to 11.50 μmol / mg compared to the model group. Figure 13

[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 shown in Table 5, the antioxidant peptide significantly increased the cell T-SOD enzyme activity from 49.88 to 115.79 U / mg compared to the model group. Figure 14

[0154] c) Catalase (CAT)

[0155] CAT can convert excess H2O2 in cells to H2O and O2, reducing cell oxidative stress damage. As shown in Table 6, the antioxidant peptide significantly increased the cell CAT enzyme activity from 2.26 U / mg to 2.66 U / mg compared to the model group. Figure 15

[0156] d) Glutathione peroxidase (GSH-Px)

[0157] As shown in Table 7, the antioxidant peptide significantly increased the cell glutathione peroxidase (GSH-Px) enzyme activity from 0.54 U / mg to 1.13 U / mg compared to the model group. Figure 16

[0158] (5) Reactive oxygen species (ROS)

[0159] As shown in Figure 2(a), the fluorescence intensity of the antioxidant peptide treatment group was significantly lower than that of the model group, indicating that the peptide can clear ROS in cells and reduce oxidative stress damage to cells. As shown in Figure 2(b), the relative content of ROS in the cells was significantly reduced by each peptide compared to the model group (P<0.0001). Figure 17 Figure 17

[0160] In summary, the antioxidant peptide HPCPHPSF (SEQ ID NO. 17) has no toxicity to HepG2 cells and has a protective effect on H2O2-induced HepG2 cell damage. Cell-level antioxidant index analysis shows that the antioxidant peptide can regulate the balance between the antioxidant defense system in HepG2 cells. ​​​​​​

[0161] The above-described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative effort shall fall within the scope of the present application.

Claims

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

2. A cosmetic composition having an antioxidant function, characterized by, 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 the preparation of an antioxidant drug.

4. Use of the antioxidant peptide as claimed in claim 1 in the preparation of a composition with antioxidant efficacy, which is a cosmetic composition.

5. A composition comprising the horse milk casein-derived antioxidant peptide as claimed in claim 1, and further comprising an adjuvant; The composition is a cosmetic composition.

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