Ovalbumin Anti-inflammatory peptide and application thereof

Abstract

To provide an ovalbumin anti-inflammatory active peptide, and the preparation and application of a Maillard reaction product thereof.SOLUTION: An anti-inflammatory peptide is FGPH(Phe-Gly-Pro-His). A preparation method for FGPH-MRPs, the Maillard reaction product of FGPH, includes the following steps: weighing and preparing FGPH into an aqueous solution, adding a dextran according to a peptide to dextran mass ratio of 1.5:1, mixing thoroughly, reacting at 80°C and pH 9.0 for 10 hours, and cooling to room temperature after the reacting is completed, to obtain the Maillard product of FGPH, FGPH-MRPs.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to the technical field of active peptides, and in particular to the preparation and use of ovalbumin anti-inflammatory active peptides and their Maillard reaction products. [Background technology]

[0002] Bioactive peptides are biologically active fragments derived from proteins. They have unique biological activities beyond basic nutritional requirements, and are attracting more and more attention due to their contribution to human health. The complex structure of peptides hidden in the parent protein makes it difficult to effectively exert their biological activity. After undergoing enzymatic degradation by external proteases, biochemical reactions such as fermentation, or digestion in the gastrointestinal tract, they can generate multiple physiological functions, which have a positive impact on human health.

[0003] In recent years, many studies have been conducted to identify novel egg white protein peptides, and the publication of the chicken genome has greatly improved the ability to identify proteins and polypeptides from complex samples. Research has shown that egg white proteins can be identified after enzymatic digestion with specific proteases to obtain different types of bioactive peptides.

[0004] The Maillard reaction refers to a chemical reaction that occurs between amino compounds, such as proteins, polypeptides, and amino acids, and carbonyl compounds, such as reducing sugars, aldehydes, and ketones. A controlled Maillard reaction can progress to form desirable sensory attributes, including color and flavor, and can also affect biological activity.

[0005] At present, bioactive peptides have been used in prevention and treatment. China, as a major country in the egg industry, has the world's largest egg production and consumption. Egg white contains abundant protein and a large amount of bioactive substances, so it has physiological functions such as disease prevention and treatment, immune regulation, etc. At present, due to the lack of in-depth development and utilization of egg white, a large amount of high-quality protein resources are wasted, and the in-depth processing and utilization of anti-inflammatory active peptides of egg white protein are lacking. Therefore, it has good prospects and significance to research and develop high-quality egg white polypeptide products using egg white protein as raw material. Summary of the Invention [Problem to be solved by the invention]

[0006] Egg white of chicken eggs contains abundant protein and is an important source for producing bioactive peptides. After the protein / peptide undergoes Maillard reaction, the activity is further improved and the flavor is improved. Therefore, the present invention investigates the activity and flavor changes of polypeptides produced by enzymatic hydrolysis of egg white protein and undergoing Maillard reaction. [Means for solving the problem]

[0007] The technical solution 1 provided in the present invention is an egg white anti-inflammatory peptide, the amino acid sequence of the anti-inflammatory peptide is Phe-Gly-Pro-His, hereinafter referred to as FGPH; The anti-inflammatory peptide FGPH can be obtained by enzymatic digestion and screening of egg white, and can also be obtained by artificial synthesis.

[0008] The technical solution 2 provided in the present invention is the use of said active peptide FGPH in anti-inflammatory reaction, especially in the production of anti-inflammatory drugs, foods or other products.

[0009] The technical solution 3 provided in the present invention is FGPH-MRPs, Maillard reaction products of FGPH.

[0010] Furthermore, the method for producing the FGPH-MRPs is as follows: FGPH is weighed and prepared into an aqueous solution, dextran is added according to a peptide-sugar mass ratio of 1.5:1, the solution is thoroughly mixed, and reacted at 80°C and pH 9.0 for 10 hours. After the reaction is completed, the solution is cooled to room temperature to obtain the FGPH-MRPs, a Maillard product of FGPH.

[0011] The technical solution 4 provided in the present invention is the use of the Maillard reaction products FGPH-MRPs of FGPH described in technical solution 3, in particular in anti-inflammatory reactions, especially in the manufacture of anti-inflammatory drugs, foods or other products. Effect of the Invention

[0012] In this study, anti-inflammatory polypeptides are produced from egg white by enzymatic hydrolysis, and after a series of separation, purification, identification, and synthesis, a polypeptide with high anti-inflammatory activity, FGPH, is obtained. By verifying the anti-inflammatory activity after artificially synthesizing FGPH, it was found that at a concentration of 500μg / mL, the inhibition rates of FGPH against inflammatory mediators (NO) and inflammatory factors (TNF-α, IL-6, IL-1β) induced by LPS in RAW264.7 were 64.41±0.47%, 28.07±2.10%, 41.72±1.83%, and 52.43±2.29%, respectively.

[0013] The effect of the Maillard reaction on the activity and flavor of the anti-inflammatory peptide FGPH was further investigated. By investigating the Maillard products FGPH-MRPs using Fourier infrared spectroscopy and scanning electron microscopy, it was found that both the structure and morphology changed compared to before the reaction. By measuring the change in activity before and after the Maillard reaction, the inhibition rate of NO production in RAW264.7 cells induced by LPS was improved from 64.41±0.47% to 68.16±0.48%, and the clearance rates EC of DPPH, ABTS, and hydroxyl radicals were improved. 50The values ​​were found to decrease from 4.96±0.05mg / mL to 4.07±0.03mg / mL, from 0.81±0.01mg / mL to 0.61±0.01mg / mL, and from 2.01±0.03mg / mL to 1.56±0.02mg / mL, respectively, and the taste of FGPH was improved according to the sensory evaluation and electronic tongue measurement evaluation, and the characteristic odor response value was obviously improved according to the electronic nose measurement evaluation. This explains that the NO inhibition rate, antioxidant activity, and flavor of FGPH are improved by the Maillard reaction. [Brief description of the drawings]

[0014] [Figure 1] Effect of polypeptides of different molecular weights (>10 kDa, 5-10 kDa, 3-5 kDa, <3 kDa) on cell viability. [Diagram 2] Effect of polypeptides with molecular weights <3 kDa on NO production. [Diagram 3] This is a total ion chromatogram of polypeptide components with molecular weights of <3 kDa. [Figure 4] The chemical structure of FGPH. [Diagram 5] FIG. 1 is a diagram showing the interaction between FGPH and iNOS. [Figure 6] Effect of FGPH on cell viability. [Figure 7] Effects of different concentrations of FGPH on the amount of cellular NO (A), TNF-α (B), IL-6 (C), and IL-1β (D) secreted. [Figure 8] FIG. 1 shows infrared spectrum analysis of FGPH and FGPH-MRPs. [Figure 9] Scanning electron microscope images of FGPH (A) and FGPH-MRPs (B). [Figure 10] Effects of FGPH and FGPH-MRPs on cell viability. [Figure 11] Effects of FGPH and FGPH-MRPs on cellular NO production. [Figure 12]1 is a radar chart of the sensory evaluation of FGPH and FGPH-MRPs. [Figure 13] 1 is a radar chart of electronic tongue taste indices of FGPH and FGPH-MRPs. [Figure 14] 1 is a radar chart of electronic nose odor indexes of FGPH and FGPH-MRPs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail with reference to specific examples. The specific examples described in this specification are only for the purpose of illustrating the present invention, and are not intended to limit the present invention.

[0016] The anti-inflammatory peptide FGPH described in the present invention can be obtained by enzymatic degradation and screening of egg white, and can also be obtained by artificial synthesis.

[0017] Some of the test methods adopted in the present invention are as follows:

[0018] 1. Measurement of Anti-inflammatory Activity 1.1 Cultivation of RAW264.7 cells Prepare DMEM medium containing 10% fetal bovine serum, activate mouse macrophage cells RAW264.7, and quickly add them to the medium. Observe the morphology of the cells under a microscope, then transfer them to an incubator for culture. Set the incubator temperature to 37°C and incubate in CO 2 The concentration is set to 5%. Observe the cell morphology every 24 h, perform subculture, digest the cells by adding 0.25% pancreatin (containing 0.01% EDTA), wash the cells with DMEM medium, centrifuge the cells, and finally wash with PBS, centrifuge the cells, and inoculate the cells on a culture dish for subsequent experimental procedures.

[0019] 1.2 Effects on RAW264.7 cell viability Measure the cell viability of RAW264.7 cells using the MTT method. RAW264.7 cells in logarithmic growth phase were seeded into 96-well plates at a density of 4 × 10 4 The outermost layer of the 96-well plate was sealed with PBS solution, and the 96-well plate was transferred to an incubator and cultured for 12 to 24 h at 37 °C and CO 2 The concentration is 5%. After the cells adhere to the wall, the discarded supernatant in the 96-well plate is aspirated. Three groups were set up: a blank group (100 μL of culture medium was added), an LPS group (100 μL of LPS with a final mass concentration of 1 μg / mL was added), and a sample group (100 μL of peptides with different or the same mass concentrations were added, reacted for 2 h, and then LPS with a final mass concentration of 1 μg / mL was added). Three duplicate wells were designed for each group. After 24 h of culture, 20 μL of MTT solution with a concentration of 5 mg / mL was taken and added to each well, and the wells were further cultured in an incubator for 4 h, and then 100 μL of dimethyl sulfoxide (DMSO) was added and gently shaken for 10 min. The absorbance value at 570 nm was measured by a microplate reader, and the cell viability (%) was calculated by the following formula (1).

number

[0020] where V represents cell viability (%) and A 1 is the absorbance value at 570 nm of the blank group, and A 2 is the absorbance value at 570 nm of the sample group.

[0021] 1.3 Effect on NO production in RAW264.7 cells The amount of NO produced by the cells is measured by the Griess method. The grouping, treatment and culture methods are all the same as in 1.2. After 24 hours of culture, the supernatant is collected and the amount of NO produced is measured using a NO detection kit. 50 μL of cell supernatant is mixed with 50 μL of Griess I reagent and 50 μL of Griess II reagent evenly, and after standing for 5 minutes, the absorbance value at 540 nm is measured. The amount of NO produced by each sample is calculated according to the instructions of the NO kit, and the inhibition rate of NO production is calculated by formula (2).

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[0022] In the formula, B represents the NO inhibition rate (%), and B 0 is the amount of NO produced in the blank group, and B 1 is the amount of NO produced in the LPS group, and B 2 is the amount of NO produced in the sample group.

[0023] 1.4 Measurement of cytokines TNF-α, IL-6, and IL-1β Seed RAW264.7 cells in logarithmic growth phase into 96-well plates at a density of 4 × 10 4 / well, and three duplicates were designed for each well. A blank group (100 μL of culture medium was added), an LPS group (100 μL of LPS with a final mass concentration of 1 μg / mL was added), and a sample group (100 μL of peptides with different concentrations were added, reacted for 2 h, and then LPS with a final mass concentration of 1 μg / mL was added) were set up, and the three groups were cultured in an incubator for 24 h, respectively, and the supernatants were collected. The absorbance value at 450 nm was measured according to the instructions of the ELISA kit, and the secretion amount of the inflammatory factors TNF-α, IL-6, and IL-1β was calculated, and the inhibition rate of the three kinds of inflammatory factors was calculated by formula (3).

number

[0024] In the formula, C represents the inhibition rate (%) of the inflammatory factor, and C 0 is the amount of inflammatory factors secreted in the blank group, and C 1 is the amount of inflammatory factors secreted in the LPS group, and C 2 is the amount of inflammatory factor secreted in the sample group.

[0025] 2. Measurement of Antioxidant Activity 2.1 Clearance rate of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical DPPH is measured with reference to GB / T39100-2020 polypeptide antioxidant properties. Preparation of DPPH solution: 5mg of DPPH is fully dissolved in an appropriate amount of anhydrous ethanol by the method of light shielding and ultrasonication, and the volume is adjusted to 100mL to prepare a DPPH solution with a concentration of 50μg / mL, which is prepared and used at the time of use. Preparation of polypeptide solution: 10mg of polypeptide is fully dissolved in an appropriate amount of distilled water, and the volume is adjusted to 1mL to prepare a polypeptide solution with a concentration of 10mg / mL, and then diluted according to a certain ratio to obtain polypeptide solutions with different concentrations. Take three test tubes numbered 1, 2, and 3, add the reagents to the test tubes according to Table 1, measure the absorbance value at 517nm, and calculate the clearance rate of DPPH free radicals according to formula (4). The natural logarithm of the polypeptide solution concentration is taken as the abscissa and the clearance rate of the DPPH free radical is taken as the ordinate, and a linear equation is obtained between the natural logarithm of the polypeptide concentration and the clearance rate, and the half-maximal effective concentration EC 50 is calculated.

number

[0026] In the formula, P1 represents the clearance rate (%) of DPPH free radical, A s is the absorbance value at 517 nm after mixing the DPPH solution with the polypeptide solution, and A c is the absorbance value at 517 nm after mixing the polypeptide solution with the ethanol solution, and A bis the absorbance value at 517 nm after mixing the DPPH solution with the sample reagent solution.

[0027] [Table 1] Reagent addition amounts for DPPH free radical clearance experiments JPEG2025014104000006.jpg44148

[0028] 2.2 Clearance rate of 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radical ABTS is measured with reference to GB / T39100-2020 polypeptide antioxidant properties. Preparation of ABTS solution: 200mg of ABTS reagent and 34.4mg of potassium persulfate are taken, dissolved thoroughly in 50mL of distilled water, shaken evenly, and stored in the dark at room temperature for 24 hours. The ABTS test solution is prepared by diluting with 95% ethanol to obtain an absorbance value of 0.70±0.02 at 734nm. The solution is prepared and used at the time of use. 10mg of polypeptide is completely dissolved in an appropriate amount of distilled water, and the volume is adjusted to 1mL to obtain a polypeptide solution with a concentration of 10mg / mL. The solution is diluted with 95% ethanol to obtain polypeptide solutions with different concentrations. Two test tubes numbered 1 and 2 are taken, and the reagents are added to the test tubes according to Table 2, and the absorbance value at 734nm is measured. The clearance rate of ABTS free radicals is calculated by formula (5), EC 50 The calculation method is the same as in 2.1.

number

[0029] In the formula, P2 represents the clearance rate (%) of ABTS free radical, and A b is the absorbance value at 734 nm after mixing the ABTS solution with the sample reagent solution, and A s is the absorbance value at 734 nm after mixing the ABTS solution with the polypeptide solution.

[0030] [Table 2] ABTS free radical clearance experiment Reagent addition amount JPEG2025014104000008.jpg37119

[0031] 2.3 Hydroxyl radical clearance rate The hydroxyl radical clearance rate is measured with reference to the method of Chen et al. Preparation of reaction solution: Dissolve appropriate amounts of ferrous sulfate, hydrogen peroxide and salicylic acid thoroughly in distilled water to prepare 9mmol / L ferrous sulfate solution, 8.8mmol / L hydrogen peroxide solution and 9mmol / L salicylic acid solution. Preparation of polypeptide solution: Put 10mg of polypeptide into appropriate amount of distilled water and dissolve it completely, adjust to 1mL, prepare a polypeptide solution with a concentration of 10mg / mL, and dilute according to a certain ratio to obtain polypeptide solutions of different concentrations. Put 1mL of polypeptide solution of different concentrations into a test tube, mix it evenly with 1mL of ferrous sulfate and 1mL of salicylic acid, react for 10min, then put 1mL of hydrogen peroxide solution into a test tube, react at 37℃ for 30min, and measure the absorbance value at 510nm. Take 1mL of distilled water instead of the polypeptide solution, and perform the same operation as above to measure the absorbance value at 510nm. Instead of the hydrogen peroxide solution, 1 mL of distilled water was used, and the procedure was repeated. The absorbance value at 510 nm was measured, and the clearance rate of hydroxyl radicals was calculated by the formula (6), EC 50 The calculation method is the same as in 2.1.

number

[0032] In the formula, P3 represents the clearance rate (%) of hydroxyl radicals, and A i is the absorbance value of the polypeptide solution at 510 nm, and A j is the absorbance value at 510 nm when distilled water is used instead of hydrogen peroxide solution, and A 0 is the absorbance value at 510 nm when distilled water was used instead of the polypeptide solution.

[0033] 3.LC-MS / MS separation identification The amino acid sequence and molecular weight of the polypeptide components are measured by LC-MS / MS. Before placing the sample in the machine, reduction, alkylation and desalting are performed first. The precolumn of the capillary liquid chromatography column used is a 300μm×5mm Acclaim PepMap RPLC C18 analytical column (5μm, 100Å), and the analytical column is a 150μm×150mm Acclaim PepMap RPLC C18 analytical column (1.9μm, 100Å). Mobile phase A is a 0.1% (v / v) formic acid solution and a 2% (v / v) acetonitrile solution, and mobile phase B is a 0.1% (v / v) formic acid solution and a 80% (v / v) acetonitrile solution, the flow rate is 600nL / min, and the analysis time per component is 60min.

[0034] The MS and MS / MS parameters are as follows: (1) MS parameters: the resolution is 70,000, the maximum injection time is 40 ms, and the scan range is 300-1,400 m / z. (2) MS / MS parameters: the resolution is 175,000, the maximum injection time is 60 ms, the scan range is 300-1,400 m / z, Top N=20, and NCE / steeped NCE=27.

[0035] The original MS / MS files were analyzed based on the Mascot software of the sample type, and matched with the searched Uniprot database (https: / / www.uniprot.org / ) Gallus gallus (Chicken), thus determining the amino acid sequence of the peptide.

[0036] The present invention will be further explained and described below with reference to specific embodiments.

[0037] Example 1 Enzymatic degradation of egg white protein peptides 1. Preheating and preparation of polypeptide Egg white protein was dissolved in distilled water at a substrate concentration of 4% (w / v) and primed in a water bath at 90°C for 20 min. After priming, the temperature was lowered to 45°C. The pH value of the protein solution is adjusted to 7.5, the temperature is 45℃, 3000U / g of composite protease (C8800, Solarbio) is added according to the mass of the substrate, and the enzyme is hydrolyzed for 1.5h, the pH value is adjusted to 10.0, the temperature is 40℃, 3000U / g of alkaline protease is added according to the mass of the substrate, and the enzyme is hydrolyzed for 1.5h, the pH value is adjusted to 7.5, the temperature is 50℃, 3000U / g of flavor protease is added according to the mass of the substrate, and the enzyme is hydrolyzed for 1.5h, after the enzyme hydrolysis is completed, the enzyme is inactivated by boiling for 10min, and then the enzyme hydrolysis solution is centrifuged at 6000r / min for 20min to remove the residual protein, and the supernatant is obtained, the pH is adjusted to 7.0, and the solution is freeze-dried for further use.

[0038] The polypeptide yield was measured using the Biuret method, and the result was 42.08±0.48%.

[0039] 2. Ultrafiltration separation of polypeptides The egg white protein enzymatic digest was centrifuged at 6000 r / min for 20 min to obtain the supernatant. First, it was filtered through a 0.45 μm filter membrane, and the filtrate was collected and subjected to the following operations. The filtrate was separated through 10 kDa, 5 kDa, and 3 kDa ultrafiltration membranes to collect polypeptides with different molecular weights (>10 kDa, 5-10 kDa, 3-5 kDa, and <3 kDa). The four polypeptide solutions with different molecular weights were vacuum freeze-dried and stored at -80°C.

[0040] 3. Measurement of Anti-inflammatory Activity 3.1 Effect on RAW264.7 cell viability The final concentration of the enzyme digestion solution with molecular weight >10kDa, 5-10kDa, 3-5kDa, and <3kDa was set to 1mg / mL, and the cell viability of four kinds of samples was measured. As shown in Figure 1, after the addition of the four kinds of samples, the LPS-induced RAW264.7 cell viability was all above 100%, without any toxic effect. Here, the cell viability was the highest after the action of the molecular weight <3kDa, reaching 103.62±0.84%.

[0041] 3.2 Effect of polypeptides with molecular weights <3 kDa on NO production The effect of polypeptide components with molecular weights <3 kDa in the concentration range of 250-1000 μg / mL on the NO production of RAW264.7 macrophage cells induced by LPS. As shown in Figure 2, after induction by LPS with a concentration of 1 μg / mL, the NO production of RAW264.7 cells increased significantly (P<0.05), about 5.08 times that of the blank group, which explains that the inflammation model of RAW264.7 macrophage cells induced by LPS was successfully established. When the concentration of polypeptide components with molecular weights <3 kDa was 1000 μg / mL, the NO production was the lowest, 21.57 ± 0.47 μM.

[0042] 3.3 Antioxidant activity of polypeptides with molecular weights <3 kDa The EC50 value of the antioxidant capacity of polypeptide components with molecular weights <3 kDa is shown in Table 3. Polypeptide components with molecular weights <3 kDa have the ability to clear all three types of free radicals, DPPH, ABTS, and hydroxyl radicals, and therefore have a certain antioxidant activity. For example, free radicals such as hydroxyl radicals, superoxide anion free radicals, singlet oxygen, etc. are interrelated with inflammatory responses, and inflammation is related to the excessive production and release of reactive oxygen, so polypeptides with good antioxidant activity usually have good anti-inflammatory potential. In addition, it is necessary to simultaneously investigate the antioxidant and anti-inflammatory activities of bioactive peptides.

[0043] [Table 3] Antioxidant capacity of polypeptide components with molecular weights <3 kDa JPEG2025014104000010.jpg30138

[0044] Example 2 Isolation and identification of egg white protein peptides LC-MS / MS liquid chromatography mass spectrometry was performed on polypeptide components with a molecular weight of <3 kDa, and the total ion chromatogram is shown in Figure 3. A total of 790 polypeptide sequences were obtained by LC-MS / MS analysis and database search and comparison.

[0045] 2. Virtual Screening and Molecular Docking 2.1 Virtual screening The peptide sequences obtained by LC / MS-MS were plotted into 2D peptide structure diagrams using ChemDraw 2D software, and then the 3D peptide structure diagrams were plotted using Chem3D Ultra 14.0 software, and the energy was set to be the minimum structure (pdbqt format). The x-ray crystal structure (PDB format) of inducible nitric oxide synthase (iNOS, PDB ID: 3e6t) was downloaded from the RCSB Protein Data Bank database, and operations such as water molecule removal, hydrogen addition, and charge calculation were performed on the receptor iNOS using Autodock tools1.5.6 software. All peptide fragments were docked to the receptor protein respectively using PyRx0.8 software, and it was explained that in general circumstances, the docking energy is less than 0, and the smaller the value, the tighter the docking and the higher the anti-inflammatory potential; therefore, the peptide fragments were ranked according to the absolute magnitude of the docking energy, and the peptide fragments with the previous ranking in Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) were used to predict the biological activity value they may have.

[0046] Inducible nitric oxide synthase (iNOS) is a nervous system enzyme closely related to inflammatory responses. When stimulated by various inflammatory conditions, LPS or cytokines induce the expression of iNOS in large quantities, which then produces NO continuously, which may cause tissue damage at the site of inflammation. The control of iNOS is one of the important policies for controlling inflammatory responses in various types of pathological conditions, and iNOS has already become an important target for treating inflammatory diseases. PyRx software can simulate docking of receptors and different ligands in batches, and the strength of their interaction strength can be judged according to the magnitude of the docking energy, thus realizing the virtual screening effect. The docking energy scoring can indicate the binding potential between the receptor and the ligand, and generally speaking, a lower score represents a stronger binding ability between the two. As shown in Table 4, the peptide fragments obtained by LC-MS / MS went through simulated scoring and docking energy ranking, and the docking energy of the six peptide fragments with the docking energy ranking at the front was ≦-8.5 kcalmol -1 This explains the strong binding affinity with iNOS. When the six peptide fragments were collated with the BIOPEP-UWM database, WINPIGT and WNIP were synthesized and verified as antioxidant peptides, and RADHPFL was synthesized and verified as an angiotensin-converting enzyme inhibitor peptide. All six peptide fragments are bioactive peptides derived from egg white, where the peptide fragment phenylalanine-glycine-proline-histidine (FGPH) did not match any of the known bioactive peptide sequences in the BIOPEP-UWM database, and the docking energy of the unstudied peptide fragment was the lowest, −9.3 kcal mol -1 This explains that FGPH may have a strong iNOS inhibitory effect and is a potential new type of anti-inflammatory peptide.

[0047] [Table 4] Results of virtual screening and matching with the BIOPEP-UWM peptide fragment database JPEG2025014104000011.jpg58158

[0048] The potential biological activities of the six peptides were ranked using Peptide Ranker, and the ToxinPred program was used to evaluate the toxicity and physicochemical properties of the polypeptides. The results are shown in Table 5. All six peptides were predicted to be non-toxic, and the biological activity score of FGPH in Peptide Ranker was 0.93, which indicates that FGPH may have good biological activity.

[0049] [Table 5] Prediction of biological activity scores and physicochemical properties of peptide fragments JPEG2025014104000012.jpg51162

[0050] The chemical structure of the potential anti-inflammatory peptide Phe-Gly-Pro-His (FGPH) is shown in FIG.

[0051] 2.2 Molecular docking The docking software was Autodock Vina, and semi-flexible docking was performed between the peptide fragments in pdbqt format obtained by Chem 3D as the ligand and the iNOS in PDB format downloaded from the RCSB Protein Data Bank as the receptor. Visualization analysis of the interactions between single small molecules and the ligand was performed using Discovery studio 2.5 software.

[0052] The anti-inflammatory mechanism of FGPH was studied by molecular docking. FGPH was docked into the receptor protein iNOS. FGPH was docked into the cavity of iNOS, and hydrogen bonds, carbon-hydrogen bonds, and cation-π interactions were the main forces between the peptide and iNOS. The docking results showed that the absolute docking energy between FGPH and iNOS was 9.3 kcal mol -1 It was found that FGPH binds tightly to the receptor protein.

[0053] The interactions between FGPH and iNOS are shown in Figure 5. Gln257 of iNOS forms one hydrogen bond with the carbonyl oxygen atom of Gly of FGPH and one carbon-hydrogen bond with Pro. Arg260 forms one hydrogen bond with the carboxyl group of His. Arg382 forms two hydrogen bonds with the nitrogen atom of His and the carbonyl oxygen atom of Pro. Pro344 forms a π-alkyl group with the benzene ring of Phe. Arg375 forms two cation-π interactions with the imidazole ring of His.

[0054] Example 3 Polypeptide synthesis and artificial validation The peptide fragments were synthesized by solid-phase synthesis using Nanjing Yuan Peptide Biological Co., Ltd., with a purity of ≧95%, which met the experimental requirements for activity detection.

[0055] 1. Analysis of anti-inflammatory activity of FGPH 1.1 Cell viability The effect of FGPH on cell viability was investigated when the concentration was different. As shown in Figure 6, when the concentration was within the range of 125-1000 μg / mL, the effect of FGPH on LPS-induced RAW264.7 cell viability tended to increase first and then decrease with increasing peptide concentration. When the concentration of FGPH was 125, 250, or 500 μg / mL, the cell viability was higher than 109%, indicating a cell proliferation-promoting effect, and when the concentration of FGPH was 1000 μg / mL, the cell viability was lower than 97%, indicating an inhibition of cell viability. Therefore, FGPH has a certain cell proliferation-promoting ability, and when the concentration of FGPH was selected to be 125-500 μg / mL, the effect on the NO production and inflammatory factor secretion of LPS-induced RAW264.7 cells was subsequently measured.

[0056] 1.2 Anti-inflammatory activity of FGPH We investigated the effects of FGPH at concentrations ranging from 125 to 500 μg / mL on the amount of NO production and the amount of inflammatory factors TNF-α, IL-1β, and IL-6 secreted by LPS-induced RAW264.7 cells.

[0057] As shown in Figure 7A, after being induced by LPS at a concentration of 1 μg / mL, the NO production of RAW264.7 cells increased significantly (P<0.05), about 4.94 times that of the blank group, which illustrates the successful establishment of the inflammation model of LPS-induced RAW264.7 macrophage cells. Compared with the LPS group, FGPH had a significant inhibitory effect on the NO production of LPS-induced RAW264.7 cells at concentrations of 125, 250, and 500 μg / mL (P<0.05), and under the action of these three concentrations of FGPH, the cellular NO production was 18.07±0.31, 16.23±0.31, and 13.82±0.31 μM, respectively. As can be seen, FGPH significantly inhibited the LPS-induced NO production in RAW264.7 cells in a concentration-dependent manner. With increasing FGPH concentration, the inhibitory effect on cellular NO production became more obvious. When the FGPH concentration was 500 μg / mL, the NO production was the lowest and the inhibition rate of NO production was the highest, reaching 64.41 ± 0.47%.

[0058] As shown in Figure 7B, after being induced by LPS at a concentration of 1 μg / mL, the amount of TNF-α secreted by RAW264.7 cells increased significantly (P<0.05), about 11.03 times that of the blank group. All three concentrations of FGPH could significantly inhibit the secretion of cellular TNF-α (P<0.05), exhibiting a concentration-dependent characteristic, and under the action of FGPH at a concentration of 500 μg / mL, it exhibited the highest inhibition rate, reaching 28.07±2.10%.

[0059] As shown in Figure 7C, after induction by LPS at a concentration of 1 μg / mL, the amount of IL-6 secreted by the cells increased significantly (P<0.05), about 8.05 times that of the blank group. Compared with the LPS group, FGPH had a significant inhibitory effect on the amount of IL-6 secreted by macrophage cells induced by LPS at concentrations of 125, 250, and 500 μg / mL (P<0.05). FGPH significantly inhibited the amount of IL-6 secreted by RAW264.7 cells induced by LPS in a concentration-dependent manner, and the inhibitory effect on the amount of IL-6 secreted by the cells improved with increasing FGPH concentration, reaching 41.72±1.83% at a concentration of 500 μg / mL.

[0060] As shown in Figure 7D, after induction by LPS at a concentration of 1 μg / mL, the amount of IL-1β secreted by cells increased significantly (P<0.05), about 5.68 times that of the blank group. Compared with the LPS group, FGPH had a significant inhibitory effect on the amount of IL-1β secreted by cells induced by LPS at concentrations of 125, 250, and 50 μg / mL (P<0.05). FGPH significantly inhibited the amount of IL-1β secreted by RAW264.7 cells induced by LPS in a concentration-dependent manner, and the higher the concentration of FGPH, the better the inhibitory effect on the amount of IL-1β secreted by cells, reaching 52.43±2.29% when the concentration of FGPH was 500 μg / mL.

[0061] 2. Analysis of FGPH antioxidant activity EC of antioxidant capacity of FGPH 50 The values ​​are shown in Table 6. FGPH has the ability to clear all three types of free radicals, DPPH, ABTS, and hydroxyl radicals, and therefore has a certain degree of antioxidant activity.

[0062] [Table 6] Antioxidant capacity of FGPH JPEG2025014104000013.jpg30138

[0063] Example 4 Maillard reaction of FGPH 1. Manufacturing method A certain amount of FGPH was weighed out and prepared into a 20 mg / mL solution. Dextran was added according to a peptide-sugar mass ratio of 1.5:1, mixed thoroughly, and reacted at a temperature of 80°C and a pH value of 9.0 for 10 hours. After the reaction was completed, the solution was cooled to room temperature to obtain the FGPH Maillard product FGPH-MRPs.

[0064] Using the absorbance value at 294 nm as a measure of the amount of Maillard intermediate products produced and the absorbance value at 420 nm as a measure of the degree of browning as indicators, the absorbance value at 294 nm of the above Maillard product FGPH-MRPs was 0.736±0.010, and the absorbance value at 420 nm was 0.696±0.010.

[0065] 2. Measurement of physicochemical properties 2.1 Fourier Infrared (FTIR) Analysis The Fourier infrared spectrograms of FGPH and FGPH-MRPs are shown in Figure 8. Fourier infrared spectroscopy is often used to study the interactions between proteins and carbohydrates and the changes that characterize protein structure. Proteins have multiple characteristic absorption peaks in the infrared spectrum, among which the amide I band, amide II band, and amide III band are located at 1700-1600 cm, respectively. -1 , 1600~1500cm -1 , 1450~1240cm -1 Compared with proteins, peptides, as small molecular substances derived from proteins, have most of the secondary structure of proteins disappeared, and the absorption peak on the amide II band is not clear. After undergoing the Maillard reaction, proteins / peptides show two typical features in the infrared spectrum. One is the tensile vibration of free hydroxyl groups, which causes a peak at 3500-3000 cm in the infrared spectrum. -1 The first is the appearance of a broad peak in the range of 1260–1000 cm due to the tensile vibration of the C–O bond. -1 As shown in Figure 8, a clear absorption peak appears in the range of 3500 to 3000 cm -1 and 1260~1000cm-1 The absorption at 1000 ng / mL was higher for FGPH-MRPs than for FGPH, demonstrating that the Maillard reaction had already occurred.

[0066] The peak in the amide I band of FGPH is at 1668 cm -1 The peak in the amide I band of FGPH-MRPs is located at 1683 cm -1 The blue shift in the peak position is mainly due to the reaction of -NH and -C=O, which reduces the carbonyl electron cloud density. In the aromatic amino acid phenylalanine structure, the CH plane is bent outward, so FGPH-MRPs has a blue shift in the peak position of the amide III band at 1435 cm. -1 At this position, a clearer absorption peak appeared than in FGPH, which also demonstrated the occurrence of the Maillard reaction.

[0067] 2.2 Scanning Electron Microscope (SEM) Analysis FGPH and FGPH-MRPs were placed under a scanning electron microscope and magnified to the same magnification, and the SEM image of FGPH was shown in Figure 9A, and the image of FGPH-MRPs was shown in Figure 9B. After undergoing the Maillard reaction, the micro-morphology of FGPH changed obviously, and FGPH-MRPs exhibited an irregular sheet-like structure and had pores.

[0068] 2.3 Effect on RAW264.7 cell viability The effect of FGPH and FGPH-MRPs at 500μg / mL on cell viability was investigated. As shown in Figure 10, under the action of the two samples at a concentration of 500μg / mL, the cell viability of LPS-induced RAW264.7 was both above 114%, indicating a certain cell proliferation ability, and there was no significant difference between the cell viability of the two samples (P>0.05).

[0069] 2.4 Effect on NO production in RAW264.7 cells As shown in Figure 11, after being induced by LPS at a concentration of 1 μg / mL, the NO production of RAW264.7 cells increased significantly (P<0.05), about 4.94 times that of the blank group, which explains the successful establishment of the inflammation model of LPS-induced RAW264.7 cells. Compared with the LPS group, FGPH and FGPH-MRPs both significantly inhibited the NO production of LPS-induced RAW264.7 cells at a concentration of 500 μg / mL (P<0.05). The inhibition rate of NO production after the Maillard reaction was improved from 64.41±0.47% to 68.16±0.48%.

[0070] 2.5 Effect of the Maillard reaction on antioxidant activity The antioxidant capacity of FGPH and FGPH-MRPs is shown in Table 7. FGPH reduced the EC of the clearance rate of DPPH, ABTS, and hydroxyl radicals after undergoing the Maillard reaction. 50 The value decreased to some extent (P<0.05), which explains that the Maillard reaction is helpful in improving the antioxidant activity of FGPH.

[0071] [Table 7] Antioxidant capacity of FGPH and FGPH-MRPs JPEG2025014104000014.jpg58164

[0072] 2.6 Investigation of FGPH flavor changes before and after the Maillard reaction using sensory evaluation, electronic tongue, and electronic nose The flavors of FGPH and FGPH-MRPs before and after the Maillard reaction were tested using sensory evaluation, electronic tongue, and electronic nose. The taste of FGPH-MRPs was significantly better than that of FGPH, with the bitterness, astringency, and saltiness covered to a certain extent, and the overall acceptability was higher than that of FGPH. It also acquired the mellow flavor that is characteristic of Maillard, and the smell also changed obviously. The electronic tongue and electronic nose were able to clearly distinguish FGPH from FGPH-MRPs.

[0073] 2.6.1 Sensory evaluation Sensory evaluation plays an important role in the study of the taste characteristics of food. As shown in Figure 12, there was a large difference in the sensory evaluation scores of the two samples. The bitterness, astringency, and saltiness scores of FGPH-MRPs were lower than those of FGPH, indicating that the Maillard reaction helped to cover up the bad taste. The mellowness score and overall acceptability score of FGPH-MRPs were higher than that of FGPH, indicating that the taste of the sample after the Maillard reaction was obviously improved, and the mellowness, which is a characteristic taste of Maillard, was clearly produced.

[0074] 2.6.2 Measurement evaluation using electronic tongue The taste values ​​of FGPH and FGPH-MRPs were compared by electronic tongue, and a taste radar chart was created. No sour or bitter aftertaste was detected in either of the two samples. As shown in Figure 13, the bitter, astringent, and salty tastes of the Maillard products FGPH-MRPs were lower than those of FGPH, which explains that the Maillard reaction can cover the poor taste to a certain extent. FGPH itself has a certain umami taste and richness, and after the Maillard reaction, the umami taste is further improved, which explains that the Maillard reaction helps to highlight the characteristic taste of peptides.

[0075] 2.6.3 Measurement evaluation using electronic nose The odor radar chart of the electronic nose sensor of FGPH and FGPH-MRPs is shown in Figure 14, and the odors before and after the Maillard reaction were obviously changed. The response values ​​of FGPH-MRPs on the LY2 / gCT, T30 / 1, P10 / 1, P40 / 1, T70 / 2, PA / 2, and P30 / 1 sensors were all obviously higher than that of FGPH, and the free amino group of the peptide FGPH in the present invention was located at phenylalanine, and the electronic nose response value of the volatile flavor substances of the phenylalanine Maillard products was high, resulting in the characteristic odor of Maillard.

[0076] The overall taste of FGPH that underwent the Maillard reaction was improved and the odor became clearer.

[0077] The above-described examples merely represent some embodiments of the present invention, and the description is specific and detailed, but should not be understood as limiting the scope of the patent. It should be understood that a person skilled in the art can make some further modifications, combinations and improvements to the above-described embodiments without departing from the concept of the present invention, and all of these fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be governed by the claims.

Claims

1. 1. An anti-inflammatory peptide having FGPH, the amino acid sequence of which is Phe-Gly-Pro-His.

2. 2. Use of the FGPH according to claim 1 in anti-inflammatory responses.

3. 3. The use according to claim 2, characterized in that it is the use of FGPH in the manufacture of anti-inflammatory drugs, food or other products.

4. Maillard reaction products of FGPH, FGPH-MRPs.

5. The method for producing the Maillard reaction product FGPH-MRPs of claim 4, comprising the steps of: weighing FGPH to prepare it into an aqueous solution; adding dextran according to a peptide-sugar mass ratio of 1.5:1; mixing thoroughly; reacting at 80°C and pH 9.0 for 10 hours; and cooling to room temperature after the reaction is completed to obtain the Maillard reaction product FGPH-MRPs.

6. Use of the Maillard reaction products FGPH-MRPs of FGPH according to claim 4 in anti-inflammatory responses.

7. 7. Use according to claim 6, characterized in that it is a use in the manufacture of anti-inflammatory drugs, food or other products.

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