A method for screening and preparing antioxidant peptides derived from horse milk whey protein and its application

Abstract

The present invention relates to a method for screening and preparing antioxidant peptides derived from mare's milk whey protein and its application. Through virtual enzymolysis and computer prediction of polypeptide information, molecular docking with keap1 protein is performed to screen out polypeptides with potential antioxidant activity. The feasibility of obtaining antioxidant peptides is verified by simulating gastrointestinal digestion with pepsin, trypsin and chymotrypsin. Finally, in vitro chemical antioxidant activity is verified by chemically synthesizing high-purity antioxidant peptides, and it is found that the antioxidant peptides of the present invention have good free radical scavenging activity. An oxidative damage model is induced on cells using H2O2, and it is found that the antioxidant peptides can resist cellular oxidative damage by stimulating the activity of a series of antioxidant enzymes. The natural antioxidant peptides screened out by the present invention have broad application prospects in the fields of food, medicine and cosmetics, and provide an efficient way for the development of functional polypeptide products derived from mare's milk whey protein.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide products and molecular biology, and specifically relates to a method for screening and preparing a horse milk whey protein-derived antioxidant peptide and application thereof. Background Art

[0002] Oxidation is an inevitable process in all organisms. Free radicals and reactive oxygen species are formed during oxidative metabolism. The imbalance between free radical generation and antioxidant levels leads to dysfunction of the antioxidant system, resulting in harmful and irreversible chemical modifications such as cell death, apoptosis, lipid oxidation and degradation of cellular components, which ultimately lead to a variety of human diseases, including cancer, inflammation, cardiovascular disease and neurodegenerative diseases. On the other hand, oxidation of food during storage can lead to reduced nutrition, decay, and odor, resulting in quality loss. Therefore, it is necessary to develop more antioxidants to scavenge free radicals and prevent food oxidation. Compared with natural antioxidants, synthetic antioxidants have potential risks in the application process. The development of natural antioxidants from food can be used to replace synthetic antioxidants with side effects as anti-oxidative food additives.

[0003] Antioxidant peptides are natural antioxidants that can prevent the formation of free radicals, scavenge free radicals and reactive oxygen species after entering the human body. Studies have shown that mare's milk has high nutritional value and can be used as a substitute for human milk because it has similar ingredients and low allergen content. It has been found to contain a variety of peptides that can prevent diabetes, lower blood pressure, and improve immune function, but there are few studies on antioxidant peptides. This study first screened out peptides with high antioxidant activity in mare's milk whey protein through computer screening methods, indicating that mare's milk whey protein hydrolysate has potential antioxidant biological activity. At present, ABTS free radical scavenging ability and DPPH free radical scavenging ability are commonly used in vitro detection methods to evaluate the efficiency of antioxidant peptides in different systems, and verifying its antioxidant activity at the cellular level can more intuitively reflect its value in application in organisms. Summary of the invention

[0004] The invention aims to provide a mare's milk whey protein-derived antioxidant peptide with antioxidant function, which has good antioxidant activity both in vitro and at the cellular level and can be used for preparing food, medicine, cosmetics and other products.

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

[0006] A mare milk whey protein-derived antioxidant peptide with antioxidant function, whose amino acid sequence is ALQPLPGR, and whose sequence is shown in SEQ ID NO.11.

[0007] The present invention also provides: a method for screening and preparing horse milk whey protein-derived antioxidant peptides, wherein the method for screening the horse milk whey protein-derived antioxidant peptides comprises the following steps:

[0008] (1) Screening of mare milk whey protein sequences based on their relative abundance in the UniProt database;

[0009] Preferably, a total of 2 sequences of horse milk whey proteins were screened according to their relative abundance in the UniProt database (https: / / www.uniprot.org / ), and information about their entries, protein names, amino acid sequence lengths, and molecular weights were displayed.

[0010] (2) Perform virtual gastrointestinal digestion in a computer and screen dipeptides, tripeptides, tetrapeptides, pentapeptides, and polypeptides from the hydrolyzates produced by the selected enzymes to form a peptide library;

[0011] Preferably, the PeptideCutter program in BIOPEP (https: / / www.expasy.org / resources / peptidecutter) was used to select pepsin (pH < 2), trypsin, and chymotrypsin (high specificity) for virtual gastrointestinal hydrolysis of all screened proteins, and dipeptides, tripeptides, tetrapeptides, pentapeptides, and polypeptides were screened from the hydrolysates produced by the three enzymes to form a peptide library.

[0012] (3) Import the peptide sequences in the peptide library into the peptide biological activity prediction website to predict the activity of peptide segments and screen out new peptides;

[0013] Preferably, PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) is used to predict the probability of each peptide in the peptide library having biological activity, and peptides with scores higher than 0.5 are screened. Peptides with scores higher than 0.5 are considered to have great potential for high biological activity. The BIOPEP-UWM bioactive peptide library (https: / / biochemia.uwm.edu.pl / en / biopep-uwm-2 / ) is used to screen out novel peptides from the above candidate peptides.

[0014] (4) Predict the antioxidant activity of new peptides through the peptide antioxidant activity prediction website;

[0015] Preferably, AnOxPePred 1.0 / prediction tool (https: / / services.healthtech.dtu.dk / services / ) is used to predict whether a novel peptide has free radical scavenging ability.

[0016] (5) Select peptide sequences with higher scores and use toxicity prediction tools to predict peptide toxicity, isoelectric point, and hydrophilicity;

[0017] Preferably, the prediction tool of the toxicity prediction website Toixinpred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html4) is used to screen non-toxic and highly active peptides. ADMET (https: / / admetmesh.scbdd.com / ) is used for property prediction, which can predict the effects of peptides including blood-brain barrier, human intestinal absorption, carcinogenicity and acute oral toxicity.

[0018] (6) Molecular docking of the above-screened peptides with Keap1 protein to find active peptides with strong binding ability;

[0019] Preferably, the final screened peptides are molecularly docked with Keap1 protein. The protein structure of the kelch region of Keap1 protein (PDB ID: 1U6D) was downloaded from the PDB database (https: / / www.rcsb.org / ), and the peptide chain was modeled using Maestro 11.0 software. The peptide chain was designated as a ligand, and the kelch region of Keap1 protein was designated as a receptor for molecular docking. PyMOL (version 4.3.0) software (https: / / pymol.org / ) was used for dehydration and removal of organic matter, and AutodockTools (http: / / mgltools.scripps.edu / downloads) was used for hydrogenation and charge checking. The peptide was energy minimized, and the protonation treatment under neutral conditions (pH=7) was performed using the H++3 online server. Then, the Amber14SB charge was assigned using the UCSF Chimera software. Finally, the conformation with the first docking score was selected for subsequent analysis, and 3D mapping analysis was performed using Schrödinger and Discovery Studio. Docking energy is generally negative, and the smaller the value, the higher the score of the docking result, that is, the tighter the binding between the ligand and the receptor protein, the stronger the interaction, and the more likely it is to have antioxidant activity.

[0020] (7) Verify whether the active peptide segments obtained by virtual screening can actually be obtained from mare milk whey protein;

[0021] Preferably, fresh mare's milk 3000 g The milk fat was removed by centrifugation for 20 min, the pH of skimmed mare's milk was adjusted to 4.6 with acetic acid, casein was precipitated, and then g , 20 min, 4°C to remove casein, take the supernatant as mare milk whey protein, and freeze-dry for later use.

[0022] Weigh whey protein to make a 50 mg / ml solution, ultrasonicate for 15 min to accelerate its dissolution, first adjust the pH to 2.0 with 1 M HCL, add pepsin, then adjust the pH to 7.6, add trypsin, and finally adjust the pH to 8.0, add chymotrypsin, each enzyme addition amount is 6000 U / g, hydrolyze in sequence at 37℃ in a magnetic stirring water bath for 2 h, inactivate at 95℃ for 15 min after hydrolysis, cool to room temperature, adjust the pH to neutral, and incubate at 12000 g , 4°C, centrifuged for 15 min, first passed through a 0.45 μm filter membrane, then passed through a 0.22 μm filter membrane, and the filtrate after passing through the membrane was lyophilized to obtain a crude polypeptide powder for use, which was recorded as M1. The crude polypeptide powder was dissolved and ultrafiltered under a 3 KDa ultrafiltration tube to obtain two components >3 KDa and <3 KDa, which were lyophilized separately. The >3 KDa component was recorded as M2, and the <3 KDa component was recorded as M3. Because the molecular weight of the antioxidant peptides we predicted was less than 1500 KDa, the <3 KDa component was used to identify its peptide information, and the antioxidant properties of these three components were also determined.

[0023] (8) performing peptidomics analysis and detection on the mare's milk whey enzymatic hydrolysate to identify the peptide sequence information after enzymatic hydrolysis;

[0024] Preferably, a Vanquish Neo / Orbitrap Exploris 480 liquid chromatography-mass spectrometer is used to analyze peptide sequence information.

[0025] (9) The highly active peptide sequence was chemically synthesized to obtain horse milk whey protein antioxidant peptides with a purity of more than 98%, and the antioxidant activity was verified.

[0026] The present invention also provides an application of a screening and preparation method in screening and preparation of antioxidant peptides; specifically, an application in screening and preparation of antioxidant peptides derived from horse milk whey protein.

[0027] Furthermore, the present invention also provides an application of an antioxidant peptide in foods, medicines and cosmetics for protecting against oxidative damage.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 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 targets of antioxidant active peptides, rapid and efficient identification of antioxidant peptides is achieved. Compared with the conventional production procedures of bioactive peptides, this bioinformatics technology has the advantages of time saving, accuracy and efficiency, but the availability of predicted peptides in actual production is a major problem. Therefore, we carried out conventional hydrolysis and characterization to confirm the feasibility of this computer simulation method, and experimentally proved that the candidate peptides screened by the computer method are also present in horse milk whey protein hydrolysate. These data show that two effective new peptides can be isolated from the hydrolysate by experiment.

[0030] 2. The antioxidant peptides screened by the present invention have strong comprehensive antioxidant activity, are highly efficient, have simple preparation methods and good repeatability, and have broad prospects in the fields of food, medicine and cosmetics, and provide an efficient way for the development of functional polypeptide products derived from mare's milk whey protein. Its specific advantages are:

[0031] (1) The scavenging rate of ALQPLPGR was comparable to that of GSH, showing good DPPH· free radical scavenging activity and O - 2Superoxide anion scavenging activity, good ABTS·+ free radical scavenging ability, ·OH free radical activity and ferrous ion reducing ability.

[0032] (2) Antioxidant activity at the cellular level The results showed that ALQPLPGR peptide had a protective effect on H2O2-induced HepG2 cell damage. Compared with the model group, ALQPLPGR peptide showed different levels 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. RT-PCR analysis also showed that it could enhance the antioxidant capacity of HepG2 cells by activating the Nrf2 pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the 3D diagram of the docking of peptide ALQPLPGR and Keap-1 protein molecule.

[0034] Figure 2 This is a 3D perspective diagram of the docking of peptide ALQPLPGR and Keap-1 protein molecule.

[0035] Figure 3 This is the 2D diagram of the docking of peptide ALQPLPGR and Keap-1 protein molecule.

[0036] Figure 4This is a local plane detail diagram of the docking of peptide ALQPLPGR and Keap-1 protein molecule.

[0037] Figure 5 This is the total ion current chromatogram of HPLC MS / MS analysis of the M3 component peptide.

[0038] Figure 6 This is the secondary mass spectrum of ALQPLPGR detected in the M3 component.

[0039] Figure 7 This is the molecular weight distribution of the peptide segment of the M3 component.

[0040] Figure 8 The amino acid composition of the M3 component.

[0041] Fig. 9 These are the DPPH clearance results of different molecular weight components of horse milk whey protein hydrolysate.

[0042] Fig.10 (a) HPLC graph of the peptide ALQPLPGR after chemical solid phase synthesis; (b) MS graph of the peptide ALQPLPGR after chemical solid phase synthesis.

[0043] Fig.11 is the DPPH clearance rate of the peptide at different concentrations.

[0044] Fig.12 ABTS·+ clearance rate at different peptide concentrations.

[0045] Fig.13 is the O of peptide at different concentrations - 2Superoxide anion clearance rate.

[0046] Fig.14 The effect of different H2O2 concentrations on the survival rate of HepG2 cells. Indicates extremely significant difference (P<0.0001).

[0047] Fig.15 The effect of peptides on the survival rate of HepG2 cells.

[0048] Fig.16 The protective effect of peptide on H2O2-induced oxidative damage in HepG2 cells. Note: In the figure, 100 means 100 μg / ml peptide solution + H2O2; 200 means 200 μg / ml peptide solution + H2O2. Compared with the model group, The significance level is P<0.05; compared with the model group, ### is the significance level P<0.001, and #### is the significance level P<0.0001.

[0049] Fig.17 The effect of peptide on MDA content in HepG2 cells. Note: 200 in the figure means 200 μg / ml peptide solution + H2O2; compared with the model group, The significance level is P<0.05; ## is the significance level P<0.01 when comparing the sample group with the model group.

[0050] Fig.18 The effect of peptide on SOD activity in HepG2 cells. Note: 200 in the figure means 200 μg / ml peptide solution + H2O2; compared with the model group, The significance level is P<0.01; compared with the model group, #### is the significance level P<0.0001.

[0051] Fig.19 The effect of peptide on CAT activity in HepG2 cells. Note: 200 in the figure means 200 μg / ml peptide solution + H2O2; compared with the model group, The significance level is P<0.0001; ## is the significance level P<0.01 when the sample group is compared with the model group.

[0052] Fig. 20 The effect of peptide on GSH-Px activity in HepG2 cells. Note: 200 in the figure means 200 μg / ml peptide solution + H2O2; compared with the model group, The significance level is P<0.05; compared with the model group, ### is the significance level P<0.001.

[0053] Fig.21 The effect of peptides on the expression of genes related to the Keap-1 / Nrf2 pathway in HepG2 cells. The significance level was P < 0.05. The significance level is P < 0.01. The significance level is P < 0.001. The significance level was P < 0.0001.

[0054] Fig. 22 Effects of peptides on ROS content in HepG2 cells; (a) ROS fluorescence results of HepG2 cells; (b) quantification of ROS fluorescence results of HepG2 cells. DETAILED DESCRIPTION

[0055] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0056] Example 1: Virtual enzymatic hydrolysis and screening of antioxidant peptides derived from horse milk whey protein

[0057] 1. Experimental methods

[0058] (1) According to the relative abundance in the UniProt database (https: / / www.uniprot.org / ), a total of 2 sequences of horse milk whey proteins were screened (see Table 1), showing information about their entries, protein names, amino acid sequence lengths, and molecular weights. Gastrointestinal digestion was simulated in silico, and the PeptideCutter program in BIOPEP (https: / / www.expasy.org / resources / peptidecutter) selected pepsin (pH < 2), trypsin, and chymotrypsin (high specificity) for virtual gastrointestinal hydrolysis of all screened proteins. Dipeptides, tripeptides, tetrapeptides, pentapeptides, and polypeptides were screened from the hydrolyzates produced by the three enzymes to form a peptide library.

[0059] (2) PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the probability of each peptide in the peptide library having biological activity, and peptides with scores higher than 0.5 were screened. Peptides with scores higher than 0.5 were considered to have great potential for high biological activity.

[0060] (3) The BIOPEP-UWM bioactive peptide library (https: / / biochemia.uwm.edu.pl / en / biopep-uwm-2 / ) was used to screen out novel peptides from the above candidate peptides.

[0061] (4) AnOxPePred 1.0 / prediction tool (https: / / services.healthtech.dtu.dk / services / ) was then used to predict whether the novel peptide had free radical scavenging ability.

[0062] (5) The toxicity of the above-screened novel peptides with free radical scavenging ability was evaluated using the toxicity prediction tool of the Toixinpred website (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html4), and their basic properties such as isoelectric point and hydrophilicity were evaluated.

[0063] (6) Use ADMET tools for property prediction (https: / / admetmesh.scbdd.com / ), which can predict the effects of peptides including blood-brain barrier, human intestinal absorption, carcinogenicity and acute oral toxicity.

[0064] (7) The final selected peptides were molecularly docked with Keap1 protein. The protein structure of the kelch region of Keap1 protein (PDB ID: 1U6D) was downloaded from the PDB database (https: / / www.rcsb.org / ), and the peptide chain was modeled using Maestro 11.0 software. The peptide chain was designated as the ligand, and the Kelch region of Keap1 protein was designated as the receptor for molecular docking. PyMOL (version 4.3.0) software (https: / / pymol.org / ) was used for dehydration and removal of organic matter, and AutodockTools (http: / / mgltools.scripps.edu / downloads) was used for hydrogenation and charge checking. The peptide was energy minimized, and the protonation treatment under neutral conditions (pH=7) was performed using the H++3 online server. Then, the Amber14SB charge was assigned using the UCSF Chimera software. Finally, the conformation with the highest docking score was selected for subsequent analysis, and 3D mapping analysis was performed using Schrödinger and Discovery Studio. Docking energy is generally negative, and the smaller the value, the higher the score of the docking result, that is, the tighter the binding between the ligand and the receptor protein, the stronger the interaction, and the more likely it is to have antioxidant activity.

[0065] 2. Experimental results

[0066] (1) The following table shows the sequence information of the screened whey proteins

[0067] Table 1 Sequences of horse milk whey proteins

[0068]

[0069] (2) The following table shows the peptides with scores higher than 0.5 screened from the peptide library.

[0070] Table 2 Whey peptides with bioactivity scores higher than 0.5

[0071]

[0072] After searching the bioactive peptide library, MF, IF, LL, and LR are discovered bioactive peptides. Considering the need to screen new active peptides, we removed these four and performed subsequent activity predictions on other peptides.

[0073] (3) The following table shows the free radical scavenging ability of the new peptides

[0074] Table 3 Free radical scavenging effect scores of novel peptides

[0075]

[0076] Table 4 Chelation scores of novel peptides

[0077]

[0078] In summary, from the table we can see that the above polypeptides may have free radical scavenging activity, among which the polypeptide ALQPLPGR (SEQ ID NO.11) has good free radical scavenging activity overall, and it is believed that it has great free radical scavenging potential. The toxicity of the above polypeptides is evaluated below to confirm that they have no biological toxicity.

[0079] (4) The following table shows the prediction of peptide toxicity and other basic properties

[0080] Table 5 Toxicity prediction and basic properties of novel peptides

[0081]

[0082] (5) The following table shows the docking binding energy of the screened candidate peptides and Keap1 protein molecules

[0083] Table 6 Docking binding energy of novel peptides and Keap1 protein molecules

[0084]

[0085] Table 6 shows the molecular docking binding energy of the 6 novel peptides screened and the Kelch region of Keap1. The molecular docking scores range from -6.2 kcal / mol (CMK) to -8.1 kcal / mol (ALQPLPGR). In other studies, the absolute value of the molecular docking score is positively correlated with antioxidant activity, and the binding energy score <-8.0 kcal / mol is considered to have a relatively strong binding force. The score of ALQPLPGR is -8.1 kcal / mol, which binds tightly to the Kelch region of Keap1 protein, indicating that it is very likely to be an antioxidant active peptide. Figure 1-4The molecular docking result diagram of the peptide ALQPLPGR (SEQ ID NO.11) and the Kelch region of Keap1, including 3D diagram and local detail diagram. It can be seen that the peptide ALQPLPGR binds to the Kelch region of keap1 through hydrogen bonds VAL369, LEU557, GLN563, and VAL604, which is the antioxidant peptide we finally screened.

[0086] Example 2: Preparation, identification and solid phase synthesis of horse milk whey protein antioxidant peptides

[0087] 1. Experimental methods and contents

[0088] (1) Preparation of antioxidant peptides from horse milk whey protein

[0089] Fresh horse milk 3000 g The milk fat was removed by centrifugation for 20 min, the pH of skimmed mare's milk was adjusted to 4.6 with acetic acid, casein was precipitated, and then g , 20 min, 4°C to remove casein, take the supernatant as mare milk whey protein, and freeze-dry for later use.

[0090] Weigh whey protein to make a 50 mg / ml solution, ultrasonicate for 15 min to accelerate its dissolution, first adjust the pH to 2.0 with 1 M HCL, add pepsin, then adjust the pH to 7.6, add trypsin, and finally adjust the pH to 8.0, add chymotrypsin, each enzyme addition amount is 6000 U / g, hydrolyze in sequence at 37℃ in a magnetic stirring water bath for 2 h, inactivate at 95℃ for 15 min after hydrolysis, cool to room temperature, adjust the pH to neutral, and incubate at 12000 g , 4°C, centrifuged for 15 min, first passed through a 0.45 μm filter membrane, then passed through a 0.22 μm filter membrane, and the filtrate after passing through the membrane was lyophilized to obtain a crude polypeptide powder for use, which was recorded as M1. The crude polypeptide powder was dissolved and ultrafiltered under a 3 KDa ultrafiltration tube to obtain two components >3 KDa and <3 KDa, which were lyophilized separately. The >3 KDa component was recorded as M2, and the <3 KDa component was recorded as M3. Because the molecular weight of the antioxidant peptides we predicted was less than 1500 KDa, the <3 KDa component was used to identify its peptide information, and the antioxidant properties of these three components were also determined.

[0091] (2) Peptide composition analysis

[0092] a) Pre-treatment method

[0093] Sample dissolution: weigh 1 mg of sample and add 0.1 mL 50 mM NH4HCO3 to dissolve;

[0094] C 18Desalting (Stage-Tip): Activation: Activate the desalting column twice with 300 μL 100% ACN, Equilibration: Equilibrate the desalting column twice with 200 μL 0.1% TFA, Loading: Load twice, Desalting: Desalt three times with 200 μL 0.1% TFA, Elution: Elute once with 300 μL 80% ACN-20% 0.1% TFA, and dry in vacuo at 45°C.

[0095] Peptide quantification: Use a pipette to accurately transfer 25 µL of sample into a new 1.5 mL EP tube, use a pipette to accurately transfer 20 µL of standard and sample into a microplate, and add 180 µL of working reagent to each well. Mix on a microplate shaker for 30 s and 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.

[0096] b) Liquid quality testing

[0097] A Vanquish Neo / Orbitrap Exploris 480 liquid chromatography-mass spectrometer was used, and the liquid chromatography conditions were:

[0098] 1) Analytical column: 75 μm id×25 cm, NanoViper C18 1.9 μm, 100 A

[0099] 2) Mobile phase A: 0.1% FA;

[0100] 3) Mobile phase B: 0.1% FA, 80% ACN;

[0101] 4) Flow rate: 300 nL / min;

[0102] 5) Analysis time for each component: 66 min;

[0103] 6) The specific chromatographic conditions are:

[0104]

[0105] The full mass spectrometry scan range was 100-1500 m / z, the primary mass spectrometry resolution was set to 120000, AGC was Standard, MaximumIT: 20 ms; the secondary mass spectrometry resolution was set to Resolution: 15000, AGC was Standard, MaximumIT: 22msCycle time: 1.3s, and the peptide fragmentation collision energy was set to 30 to generate mass spectrometry detection raw data. The mass spectrometry raw file was used to search the target protein database using the software, and the search parameters were as follows:

[0106] 1) Fixed modifications: Carbamidomethyl (C).

[0107] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0108] 3) Enzyme: Non specific.

[0109] 4) Database: uniprotkb_proteome_Equus_caballus_2024_06_24.

[0110] 5) Peptide Mass Tolerance: 20 ppm

[0111] 6) Fragment Mass Tolerance: 0.02 Da

[0112] The components separated by chromatography continuously enter the mass spectrometer, which continuously scans for data collection. Each scan produces a mass spectrum, and ultimately the total ion current chromatogram and secondary spectra of different peptide segments can be obtained.

[0113] c) In order to obtain a higher purity ALQPLPGR peptide for subsequent antioxidant testing, we performed chemical solid phase synthesis of the peptide and determined its purity to be over 98% by HPLC.

[0114] 2. Experimental results

[0115] (1) The crude peptides obtained by actual simulated gastrointestinal digestion were tested by peptideomics. Figure 5 HPLC-MS / MS total ion chromatogram and Figure 6 The secondary mass spectrum of ALQPLPGR shows that the computer-screened polypeptide ALQPLPGR (SEQ ID NO.11) can be obtained from horse milk whey protein through actual enzymatic digestion.

[0116] (2) A total of 1,463 peptides were detected by peptideomics. Figure 7The molecular weight distribution of peptides in the M3 component is shown, showing that the molecular weight of all detected peptides is below 3 KDa, which is consistent with our actual extraction situation, among which peptides <1 KDa account for the largest proportion. Since shorter peptides may have higher antioxidant properties, it is speculated that the M3 component may have stronger antioxidant properties based on the molecular weight distribution. Figure 8 The amino acid composition of the M3 component is shown, among which leucine (L) and proline (P) are the most abundant. Both of them are hydrophobic amino acids. Hydrophobic amino acids stabilize free radicals by providing protons and are effective antioxidants. Fig. 9 The DPPH free radical scavenging results of crude peptides extracted from mare's milk whey protein tested by us showed that the M3 component had a higher free radical scavenging activity.

[0117] (3) Fig.10 (a) HPLC graph of peptide ALQPLPGR after chemical solid phase synthesis, showing that its purity is 99.216%; (b) MS graph of peptide ALQPLPGR after chemical solid phase synthesis.

[0118] Example 3: In vitro chemical antioxidant activity of horse milk whey protein antioxidant peptides

[0119] 1. Experimental content and methods

[0120] (1) DPPH clearance rate determination

[0121] Weigh 19.7 mg of 2,2-biphenyl-1-picrylhydrazyl (DPPH) and dissolve it in anhydrous ethanol, dilute to 250 mL, prepare a DPPH solution with a concentration of 0.2 mmol / L, and prepare peptide solutions of different concentrations. According to the experimental group As: 100 μL peptide solution + 100 μL DPPH solution; control group Ac: 100 μL peptide solution + 100 μL anhydrous ethanol; blank group Ab: 100 μL distilled water + 100 μL DPPH solution, mix the reaction system thoroughly, place it at room temperature and avoid light for 30 min, measure the absorbance at 517 nm, and calculate the DPPH clearance rate according to formula 1. (1)

[0122] (2) Determination of ABTS·+ clearance rate

[0123] Refer to the instructions of the total antioxidant capacity detection kit (ABTS rapid method) for ABTS·+ scavenging activity determination. Prepare ABTS working solution first, use it immediately, store it at room temperature away from light, use it within 30 minutes, and prepare peptide solutions of different concentrations. The antioxidant capacity of antioxidants is expressed by the relative total antioxidant capacity of Trolox. Therefore, it is necessary to dilute Trolox in a gradient to prepare a standard curve. According to the experimental group: 20 μL peroxidase working solution + 10 μL peptide solution + 170 μL ABTS working solution; standard curve group: 20 μL peroxidase working solution + 10 μL Trolox standard solution + 170 μL ABTS working solution, blank group: 20 μL peroxidase working solution + 10 μL distilled water + 170 μL ABTS working solution. After the reaction system was gently mixed, it was incubated at room temperature in the dark for 6 min, and its absorbance was measured at 405 nm. Its ABTS·+ scavenging ability was expressed by Trolox relative antioxidant capacity (TEAC), that is, expressed in μmol TE / g.

[0124] (3) Superoxide anion (O - 2) Clearance determination

[0125] Refer to the instructions of the superoxide anion scavenging ability test kit. You need to prepare the working solution (Working Reagent) first, prepare it for immediate use, and prepare peptide solutions of different concentrations. According to the experimental group: 20 μL Working Xanthine Oxidase working solution + 20 μL peptide solution + 80 μL Working Reagent; control group: 20 μL Working Xanthine Oxidase working solution + 20 μL distilled water + 80 μL Working Reagent; blank group: 40 μL distilled water + 80 μL Working Reagent. Mix the reaction system, immediately read the absorbance A0 at 450 nm, incubate at room temperature in the dark for 60 min, and read the absorbance value A at 450 nm again. 60 ,calculate A=A 60 -A0, denoted as A 空 , A 对 , A 测 ,calculate A 对 = A 对 - A 空 , A实 = A 实 - A 空 . Superoxide anion (O - 2) The clearance rate is calculated according to Formula 2:

[0126] (2)

[0127] (4) Determination of hydroxyl radical (·OH) scavenging rate

[0128] First, weigh 0.2502 g of FeSO4 and dissolve it in 100 mL of ultrapure water to make a 9 mmol / L FeSO4 solution; weigh 0.1243 g of salicylic acid and dissolve it in 100 mL of anhydrous ethanol to make a 9 mmol / L salicylic acid solution; take 0.1 mL of 30% H2O2 and dilute it to 100 mL with ultrapure water. Take 1 mL of FeSO4 solution, 1 mL of salicylic acid solution, 1 mL of H2O2 solution, and 1 mL of sample solution, and react them in the dark for 30 min. Determine its absorbance at a wavelength of 510 nm, which is recorded as A1; replace the sample solution with ultrapure water, measure the absorbance after the reaction, and record it as A0; replace the H2O2 solution with ultrapure water, measure the absorbance after the reaction, and record it as A2. The scavenging rate of hydroxyl radicals (·OH) is calculated according to formula 3:

[0129] (3)

[0130] (5) Fe 3+ Reducing power determination

[0131] Refer to the instructions of the total antioxidant capacity test kit (FRAP method) for Fe 3+ For reducing power determination, prepare the FRAP working solution first, incubate at 37ºC after preparation, and use it immediately. The antioxidant capacity of antioxidants is expressed by the relative total antioxidant capacity of ferrous salts. Therefore, it is necessary to gradient dilute FeSO4•7H2O to prepare 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 gently mixing the reaction system, incubate at 37ºC for 5 min and then measure A 593 , its Fe 3+ The reducing power is expressed by the relative antioxidant capacity of FeSO4•7H2O, that is, μmol FeSO4•7H2O eq / g.

[0132] 2. Experimental results

[0133] (1) Results and analysis of DPPH free radical scavenging activity

[0134] DPPH· free radical scavenging activity of antioxidant peptides at different concentrations Fig.11 As shown in the figure, it can be seen that the higher the concentration of antioxidant peptides, the more steadily the clearance rate increases. At a concentration of 1 mg / mL, the clearance rate can reach more than 90%, which is equivalent to 1 mg / mL GSH, showing strong DPPH· free radical scavenging activity.

[0135] (2) ABTS·+ scavenging activity results and analysis

[0136] The free radical scavenging activity of antioxidant peptides at different concentrations of ABTS·+ Fig.12 As shown in the figure, it can be seen that at 0.5 mg / mL, its antioxidant capacity is 0.255 mM TE / g. Thereafter, as the concentration increases, its antioxidant capacity stabilizes within this range, showing good and stable DPPH· free radical scavenging activity.

[0137] (3) Superoxide anion (O - 2) Clearance activity results and analysis

[0138] Antioxidant peptides in the presence of different concentrations of superoxide anion (O - 2) Clearing activity such as Fig.13 As shown in Figure 2, with the increase of peptide concentration, O - 2 The stronger the scavenging activity, the positive correlation is present. At 5 mg / mL, the clearance rate reached 73.9%, indicating that it has good O - 2. Clearing ability.

[0139] (4) Results and analysis of hydroxyl radical (·OH) scavenging activity

[0140] The hydroxyl radical (·OH) scavenging activity of the antioxidant peptide is shown in Table 7. In this experiment, the hydroxyl radical (·OH) scavenging activity was only detected at 5 mg / mL, which was 16.55%. The scavenging effect was not good at lower concentrations, indicating that it can have hydroxyl radical (·OH) scavenging activity when the concentration increases.

[0141] Table 7 Hydroxyl radical (·OH) scavenging activity

[0142]

[0143] (5) Fe 3+ Reducing power determination results and analysis

[0144] Antioxidant peptide Fe 3+ The reducing power is shown in Table 8 below, which is 35.79 μmol FeSO4•7H2O eq / g, indicating that it has Fe3+ Reducing power, but the reducing power is not as strong as the free radical scavenging effect.

[0145] Table 8 Fe 3+ Reduction power

[0146]

[0147] Example 4: Protective effect of horse milk whey protein antioxidant peptides on H2O2-induced oxidative damage in HepG2 cells

[0148] 1. Experimental content and methods

[0149] (1) Cell culture

[0150] Cell recovery: Take the cryopreserved tube containing HepG2 cells out of the liquid nitrogen tank, put it in a 37°C water bath, and thaw it quickly within 1 min. Add 2 mL of culture medium and heat at 1500 °C. g Centrifuge for 5 min, discard the supernatant, add 2 mL of culture medium, pipette to resuspend, and then inoculate into a cell culture dish. Tilt the dish 30°, gently pipette 10 times, and then culture in a constant temperature cell culture incubator at 37°C and 5% CO2.

[0151] Cell passaging: When the cell density reaches 80-90% and the cells are in good condition, they can be passaged. Aspirate the original culture medium, add PBS along the wall to wash, aspirate the PBS, add 2 mL of 0.25% trypsin to a 10 cm culture dish, digest for 3 min, add 4 mL of culture medium containing serum to terminate digestion, blow to remove the cells, and collect them in a 10 mL centrifuge tube, centrifuge at 1500 g After centrifugation for 5 min, discard the supernatant, add 3 mL of culture medium to resuspend, subculture the cells into a new culture dish, shake well, let the cells settle for 15 min, and culture them in an incubator.

[0152] (2) CCK8 cell viability assay

[0153] HepG2 cells were cultured at 10 4 Cells were inoculated at a density of 100 cells / well in a 96-well plate. When the cell density reached 70-80%, they were treated with the corresponding drugs for a certain period of time. After the drug administration time, 100 μL of culture medium containing 10% CCK8 reagent was added to each well. After incubation at 37°C for 45 min, the absorbance at 450 nm was measured, and the cell viability was calculated according to Formula 4. Six technical replicates were set for each group.

[0154] Cell survival rate = (A 加药 -A 空白 ) / (A 对照 -A 空白 )(4)

[0155] A 加药 : Contains cells, culture medium, CCK-8 solution and drug solution

[0156] A 空白 :Cell-free and drug-free culture medium

[0157] A 对照 : Contains cells, culture medium, CCK-8 solution, no drugs

[0158] (3) Establishment of HepG2 cell oxidative damage model

[0159] When the cells grow to 80-90% of the culture dish, perform the same subculture and pre-freezing treatments, discard the supernatant, add 1 mL or 2 mL of complete culture medium, and use a pipette to fully aspirate. Take 10-20 μL of cell fluid, add it to a hemocytometer, and observe and count the cells under a microscope. Calculate the total number of cells in the cell fluid and dilute the cells to 1×10 4 / mL concentration, take a 96-well cell culture plate and add 100 μL / well diluted cell solution, put it in the incubator and culture it for 12~24 hours, until the cells are attached to the plate and the density is moderate, discard the supernatant, add 100 μL of a series of H2O2 (200, 400, 600, 800, 1000, 1200, 1400, 1600 μM) prepared with complete culture medium, set 6 parallels for each concentration, and culture it in the incubator for 12 hours. The survival rate was determined by CCK8 method, and the H2O2 concentration with a cell survival rate of about 50% was selected as the subsequent cell injury model.

[0160] (4) Effect of antioxidant peptides on the survival rate of HepG2 cells

[0161] Take cells in the logarithmic growth phase and perform the same operation as (3). Discard the supernatant and add 1 mL or 2 mL of complete culture medium. Aspirate thoroughly and evenly. Count the cells and calculate the total number of cells in the cell fluid. Dilute the cells to 1×10 4 / mL concentration, add 100 μL of diluted cell solution to a 96-well cell culture plate, place incubator and culture for 12-24 h, until the cells adhere to the plate and the density is moderate, discard the supernatant, 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 the incubator for 24 h. The survival rate was determined by CCK8 method.

[0162] (5) Protective effect of antioxidant peptides on H2O2-induced oxidative damage in HepG2 cells

[0163] The cells were inoculated into a 96-well cell culture plate as in (3). After the cells grew well, the supernatant was discarded, and a series of peptide solutions (100, 200 μg / mL) prepared with complete culture medium and 800 μM H2O2 mixed solution were added, and the cells were cultured in an incubator for 12 h. The supernatant was discarded, the cells were washed once with PBS, and the cell survival rate was determined by CCK8 method.

[0164] (6) Determination of cell-related antioxidant indicators

[0165] When the cells grow to 80-90% of the culture dish, the operation is the same as the subculture and pre-freezing treatment. After resuspending the cells, take a 6-well cell culture plate and add the diluted cell suspension at 2 mL / well, mix thoroughly, and place in the incubator for 12-24 h. When the cells are in the logarithmic growth phase density, the supernatant is discarded, and the cells are washed twice with PBS. The samples prepared with culture medium are added and divided into control group, sample group and model group. The control group is added with complete culture medium, the model group is added with 800 μM H2O2, and the sample group is added with 200 μg / mL peptide solution + 800 μM H2O2, and incubated in the incubator for 12 h. The supernatant is discarded, the cells are washed twice with PBS, digested with 500 μL of trypsin for 2 min, and 1 mL of culture medium is added to terminate the digestion. The PBS is resuspended and transferred to a 1.5 mL centrifuge tube, and 1500 g After centrifugation for 5 min, the supernatant was discarded, 300 μL PBS was added to the cell pellet, and the pellet was placed on ice. The cells were ultrasonically disrupted at a power of 300 W, with each disruption for 2 s, an interval of 10 s, and a disruption for 1 min. Finally, the supernatant of the disrupted cells was collected for the determination of various indicators (malondialdehyde MDA, superoxide dismutase SOD, catalase CAT, and 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.

[0166] (7) Determination of reactive oxygen species (ROS)

[0167] The initial cell culture, administration, and induction were the same as the steps for determining the relevant antioxidant index values ​​in (6). DCFH-DA fluorescent probe was prepared in serum-free medium at a ratio of 1:1000 according to the instructions of the ROS kit for use. After the cell induction was completed, the supernatant was discarded, and 1 mL of the prepared 10 μM DCFH-DA fluorescent probe was added to a 12-well plate in a light-proof environment, and cultured in a cell culture incubator for 30 min. Take out (all subsequent operations were performed in a light-proof environment) and wash with PBS 4 times to fully wash away the background excess probe. Under a laser confocal microscope, use an excitation wavelength of 488 nm and an emission wavelength of 525 nm to observe the cell ROS fluorescence.

[0168] (8) Total RNA extraction and qRT-PCR

[0169] The cell pre-culture, modeling, administration, and induction were the same as the steps for determining the relevant antioxidant index values ​​in (6). After 12 hours of modeling and administration, the 6-well plate was removed, the supernatant was discarded, and it was washed twice with PBS; 1 mL of Trizol was added to each well, and the cells were lysed on ice for 15 min. The liquid in the 6-well plate was collected into 1.5 mL centrifuge tubes without RNAse by repeated pipetting. 200 μL of chloroform (Trizol: chloroform = 5:1) was added to each tube, and the mixture was mixed by vigorous vortexing. Incubate at 4°C at 11000 g Centrifuge for 15 min. Collect the supernatant and add an equal volume of isopropanol, mix by inversion, and place in a -20℃ refrigerator for 2 h. g The supernatant was discarded after centrifugation for 15 min. 500 μL of 75% alcohol prepared with 0.1% DEPC water was added. The precipitate was washed by vortexing and incubated at 4°C at 11000 g Centrifuge for 10 min, remove the supernatant, and the precipitate is the extracted RNA. Dry the precipitate at room temperature and dissolve it in 0.1% DEPC water preheated to 60°C. Use NanoDrop 2000 to measure the RNA concentration, and then use the cDNA synthesis kit for reverse transcription. The specific steps are carried out according to the instructions. After the reversal, use the fluorescence quantitative premix kit and the fluorescence quantitative PCR instrument for real-time quantitative PCR (RT-qPCR). The reaction program and primer sequence used (primer sequence is from the primer bank and verified by Blast) are shown in Tables 9 and 10 below.

[0170] Table 9 RT-qPCR reaction procedure

[0171]

[0172] Table 10 Primer sequences

[0173]

[0174] 2. Experimental results

[0175] (1) Establishment of H2O2-induced oxidative damage model of HepG2 cells

[0176] Fig.14 Figure 2 shows the cell survival rate of HepG2 cells induced by H2O2 for 12 h in the concentration range of 200-1600 μmol / L. As the H2O2 concentration increased, the survival rate of HepG2 cells decreased significantly ( P<0.05), at a concentration of 800 μmol / L, the cell survival rate dropped to 71.97%, and at 1.0 mmol / mL, its cell lethality reached 47.72%. Since the damage of H2O2 to cells may be irreversible at a lower survival rate, a H2O2 concentration of 800 μmol / L was selected as the subsequent cell injury model.

[0177] (2) Effect of antioxidant peptides on the survival rate of HepG2 cells

[0178] Effects of antioxidant peptides on the survival rate of HepG2 cells Fig.15 As shown. When the peptide concentration was 12.5-800 μg / mL, the cell survival rate was greater than 100%, which shows that it was not only non-toxic to HepG2 cells, but also had a certain protective effect on them. In order to avoid the waste of peptides, 100 and 200 μg / mL peptide concentrations were selected for subsequent experiments.

[0179] (3) Protective effect of antioxidant peptides on H2O2-induced oxidative damage in HepG2 cells

[0180] The protective effect of adding antioxidant peptides and H2O2 to HepG2 cells after culturing for 12 h Fig.16 Compared with the model group, the cell survival rate of antioxidant peptides at concentrations of 100 and 200 μg / mL increased significantly, and the effect of 200 μg / mL was extremely significant, so 200 μg / mL was selected for subsequent experiments.

[0181] (4) Analysis of related antioxidant enzyme activities and damage markers

[0182] a) Malondialdehyde (MDA)

[0183] Malondialdehyde is a product of cellular lipid peroxidation. The higher the MDA content, the more serious the cellular oxidative stress damage. Fig.17 As shown, compared with the model group, antioxidant peptides can significantly reduce the cellular MDA content from 17.81 μmol / mg to 12.14 μmol / mg.

[0184] b) Total superoxide dismutase T-SOD

[0185] Total superoxide dismutase T-SOD is an important antioxidant enzyme in the body that participates in resisting oxidative stress. Fig.18 As shown, compared with the model group, antioxidant peptides can significantly increase the activity of cellular T-SOD enzyme from 49.88 to 135.31 U / mg.

[0186] c) Catalase CAT

[0187] CAT can convert excess H2O2 in cells into H2O and O2, reducing cell oxidative stress damage. Fig.19 As shown in the figure, compared with the model group, antioxidant peptides can significantly increase the activity of cell CAT enzyme, and the enzyme activity increased from 2.26 U / mg to 2.95 U / mg.

[0188] d) Glutathione peroxidase GSH-Px

[0189] like Fig. 20 As shown in the figure, compared with the model group, antioxidant peptides can significantly increase the activity of cellular glutathione peroxidase GSH-Px, and the enzyme activity increased from 0.54 U / mg to 1.47 U / mg.

[0190] (5) Expression of genes related to oxidative stress pathway

[0191] The Keap1 / Nrf2 signaling pathway plays a key role in the occurrence and development of oxidative stress. This study detected the expression of genes related to this pathway. Fig.21 As shown in the figure, the expressions of Nrf2 and its downstream antioxidant genes HO-1, NQO1, and SOD1 were significantly decreased in the H2O2 group ( P <0.01), while after treatment with antioxidant peptides, the expression of these genes was significantly upregulated ( P <0.05). The above results suggest that antioxidant peptides protect HepG2 cells from oxidative damage by regulating the Keap1 / Nrf2 signaling pathway.

[0192] (6) Reactive oxygen species (ROS)

[0193] Depend on Fig. 22 It can be clearly seen in (a) that the fluorescence intensity of the antioxidant peptide treatment group is significantly lower than that of the model group, indicating that the peptide can reduce the oxidative stress damage to cells by removing ROS in cells. Fig. 22 As shown in the fluorescence quantification diagram in (b), compared with the model group, each peptide can significantly reduce the relative content of cellular ROS ( P <0.0001).

[0194] In summary, the antioxidant peptide ALQPLPGR (SEQ ID NO.1) has no toxicity to HepG2 cells and has a protective effect on H2O2-induced HepG2 cell damage. Analysis of antioxidant indicators at the cellular level showed that antioxidant peptides can regulate the balance between antioxidant defense systems in HepG2 cells.

[0195] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A mare milk whey protein-derived antioxidant peptide having antioxidant function, characterized in that: The amino acid sequence of the horse milk whey protein-derived antioxidant peptide is shown in SEQ ID NO.11, specifically: ALQPLPGR.

2. The horse milk whey protein-derived antioxidant peptide according to claim 1, characterized in that: Possesses free radical scavenging activity.

3. Use of the antioxidant peptide according to any one of claims 1-2 in the preparation of food for protecting against oxidative damage.

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