Anti-inflammatory active peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of protein, and relates to an anti-inflammatory active peptide as well as a preparation method and application thereof. The amino acid sequence of the anti-inflammatory peptide is LLVK. The anti-inflammatory peptide fragment is spontaneously combined with I kappa B protein through hydrogen bonds and hydrophobic interaction to form a more stable compound, phosphorylation of the I kappa B protein is inhibited, secretion of inflammatory factors IL-1beta, IL-6 and TNF-alpha and a chemotactic factor MCP-1 is reduced, and therefore inflammatory reaction is prevented. The invention also provides a preparation method and application of the anti-inflammatory active peptide.

Description

Technical Field

[0001] The invention belongs to the technical field of proteins and relates to an anti-inflammatory active peptide and a preparation method and application thereof. Background Art

[0002] Inflammation is a complex physiological response. Excessive inflammation can lead to tissue damage and organ dysfunction. Studies have shown that injuries including obesity, inflammatory bowel disease, heat stress injury and neurodegenerative diseases are all related to inflammation. Inflammatory diseases have become one of the major threats to global human health in the 21st century.

[0003] At present, the treatment of inflammation mainly includes taking non-steroidal anti-inflammatory drugs such as aspirin and dexamethasone and corticosteroids. In the post-epidemic era, people have been repeatedly affected by diseases that cause inflammatory responses, such as influenza A. However, as the number of illnesses increases, the efficacy of anti-inflammatory drugs decreases, and long-term use will produce side effects such as dry mouth, nausea, and headaches. Anti-inflammatory drugs can only treat, and cannot effectively prevent inflammation. Therefore, finding new food-borne substitutes is an effective means of preventing inflammation.

[0004] Soy peptides are enzymatic hydrolysis products of soy protein isolates. They have a low molecular weight and usually contain 3-20 amino acids. They retain the advantages of protein nutrition and amino acid balance, and are easily soluble and absorbed. Therefore, we use soy peptide raw materials for separation to explore the key effective ingredients for preventing inflammation and provide green and healthy food-borne anti-inflammatory drugs. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a peptide with anti-inflammatory activity, a preparation method and application thereof, and to solve the problem that traditional drugs cannot prevent inflammation and avoid the side effects caused by long-term use of anti-inflammatory drugs by screening and preparing peptides with anti-inflammatory activity, so as to effectively help patients with inflammation-related diseases.

[0006] The purpose of the present invention and the solution to its technical problem can be achieved through the following technical solutions.

[0007] In one aspect, the present invention provides an anti-inflammatory active peptide, the amino acid sequence of the peptide is Leu-Leu-Val-Lys, or represented by LLVK, and the molecular weight is 472.35 Da. The peptide is hydrophobic and stable, and contains leucine and lysine.

[0008] On the other hand, the present invention provides a method for preparing the anti-inflammatory active peptide, comprising the following steps:

[0009] 1) using bromelain and flavor protease to enzymatically hydrolyze soy protein isolate to obtain soy peptides;

[0010] 2) Using pepsin and pancreatin to simulate in vitro digestion of soybean peptides to obtain soybean peptide digestion products;

[0011] 3) simulating the absorption of soybean peptide digestion products in vitro and isolating them using a Caco-2 monolayer cell model to obtain absorbed soybean peptides; and

[0012] 4) Identifying the absorbed soybean peptide to obtain the anti-inflammatory active peptide LLVK.

[0013] In a third aspect, the present invention provides an anti-inflammatory composition comprising the anti-inflammatory active peptide of the present invention.

[0014] In a fourth aspect, the present invention provides use of the anti-inflammatory active peptide of the present invention in the preparation of anti-inflammatory drugs.

[0015] The anti-inflammatory active peptide of the present invention can improve lipopolysaccharide-induced RAW264.7 cell inflammation, spontaneously bind to IκB protein through hydrophobic interaction and hydrogen bonding to form a more stable complex, thereby inhibiting the occurrence of inflammatory response, and thus has good anti-inflammatory activity. In addition, the sources for preparing the anti-inflammatory active peptide of the present invention are wide, the preparation process is simple, and it is easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The resistance value change of the Caco-2 monolayer cell model after 21 days of culture;

[0017] Figure 2 The effects of different concentrations of digested soybean peptides on the survival rate of Caco-2 cells;

[0018] Figure 3 The effect of absorbed soy peptides on inflammation prevention;

[0019] Figure 4 The effect of different concentrations of LLVK on the survival rate of RAW264.7 cells;

[0020] Figure 5 Visualization of molecular docking between LLVK and IκB protein;

[0021] Figure 6 The effect of LLVK on IκB protein expression;

[0022] Figure 7 is the expression level of IL-1β, IL-6, TNF-α and MCP-1 by LLVK. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but those skilled in the art should understand that the embodiments described below are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] It has been proven that soybean peptides can prevent inflammatory diseases, but there is a lack of research on the dedicated sequences of food-borne anti-inflammatory peptides. The inventors enzymatically hydrolyze and separate soybean protein isolate raw materials to finally obtain peptides with anti-inflammatory activity, thereby proposing the present invention.

[0025] On the one hand, the present invention provides an anti-inflammatory active peptide, the amino acid sequence of the peptide is Leu-Leu-Val-Lys, or represented by LLVK, and the molecular weight is 472.35Da.

[0026] The anti-inflammatory active peptide of the present invention spontaneously combines with the IκB protein through hydrophobic interaction and hydrogen bonding to form a more stable complex, thereby improving lipopolysaccharide-induced RAW264.7 cell inflammation and inhibiting the occurrence of inflammatory response.

[0027] In an embodiment of the present invention, the peptide segment can reduce the levels of IL-1β, IL-6, TNF-α and MCP-1, thereby alleviating the inflammatory symptoms of RAW264.7 cells.

[0028] On the other hand, the present invention provides a method for preparing the anti-inflammatory active peptide, comprising the following steps:

[0029] 1) using bromelain and flavor protease to enzymatically hydrolyze soy protein isolate to obtain soy peptides;

[0030] 2) Using pepsin and pancreatin to simulate in vitro digestion of soybean peptides to obtain soybean peptide digestion products;

[0031] 3) simulating the absorption of soybean peptide digestion products in vitro and isolating them using a Caco-2 monolayer cell model to obtain absorbed soybean peptides; and

[0032] 4) The absorbed soybean peptides were identified and the anti-inflammatory active peptide LLVK was obtained.

[0033] In an embodiment of the present invention, in step 1), the amount of bromelain added is 1.2-1.4% of the soy protein isolate by mass fraction, the enzymolysis time is 2.5-3.5h, the enzymolysis temperature is 45-55°C, and the pH is 7.0-7.5. In a specific embodiment, the amount of bromelain added can be 1.2%, 1.3%, 1.4% of the soy protein isolate by mass fraction, the enzymolysis time can be 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3.0h, 3.1h, 3.2h, 3.3h, 3.4h, 3.5h, the enzymolysis temperature can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 51°C, 54°C, 55°C, and the pH can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5. In a preferred embodiment, the added amount of bromelain is 1.3% of the soy protein isolate by mass fraction, the enzymolysis time is 3 hours, the enzymolysis temperature is 50° C., and the pH is 7.0.

[0034] In an embodiment of the present invention, in step 1), the amount of flavor protease added can be 9.0-11.0% of the soy protein isolate by mass fraction, the enzymolysis time can be 2.5-3.5h, the enzymolysis temperature can be 50-60°C, and the pH value can be 7.0-7.5. In a specific embodiment, the amount of flavor protease added can be 9.0%, 9.1%, 9.2%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10.0%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 11.0% of the soy protein isolate by mass fraction, The enzymolysis time can be 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3.0h, 3.1h, 3.2h, 3.3h, 3.4h, 3.5h, the enzymolysis temperature can be 50°C, 51°C, 52°C, 51°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, and the pH can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5. In a preferred embodiment, the amount of flavor protease added is 10.0% of the soy protein isolate by mass fraction, the enzymolysis time is 3h, the enzymolysis temperature is 55°C, and the pH is 7.0.

[0035] In an embodiment of the method of the present invention, in step 1), after the enzymatic hydrolysis is completed, the enzyme may be inactivated by placing in a 95° C. water bath for 10 min.

[0036] In an embodiment of the present invention, step 2) specifically comprises the following steps:

[0037] 2.1) Dissolve the soybean peptide in water, add simulated oral fluid, and shake to obtain the soybean peptide oral digestive fluid.

[0038] 2.2) Add simulated gastric fluid to the soybean peptide oral digestive fluid, adjust the pH to 3.0, then add pepsin, and oscillate the reaction to obtain the soybean peptide gastric digestive fluid,

[0039] 2.3) adding simulated intestinal fluid to the soybean peptide gastric digestion fluid, adjusting the pH to 7.0, and then adding pancreatic enzyme, shaking the reaction, to obtain soybean peptide intestinal digestion fluid, and

[0040] 2.4) freeze-drying the soybean peptide digestion solution to obtain digested soybean peptide powder, i.e., soybean peptide digestion product.

[0041] Wherein, based on 400 mL, the simulated oral fluid contains 15.1 mL 15.1 mM KCl, 3.7 mL 3.7 mM KH2PO4, 13.6 mL 6.8 mM NaHCO3, 0.5 mL 0.15 mM MgCl2(H2O)6, 0.06 mL 0.06 mM (NH4)2CO3, 0.09 mL 1.1 mM HCl, dH2O is added to make up, and 2.5 mL 1.5 mM CaCl2(H2O)2 is added separately before use.

[0042] Based on 400 mL, the simulated gastric fluid contains 6.9 mL 6.9 mM KCl, 0.9 mL 0.9 mM KH2PO4, 12.5 mL 25.0 mM NaHCO3, 11.8 mL 47.2 mM NaCl, 0.4 mL 0.12 mM MgCl2 (H2O)6, 0.5 mL 0.5 mM (NH4)2CO3, 1.3 mL 15.6 mM HCl, and dH2O is added to make it complete. Before use, 0.25 mL 0.15 mM CaCl2 (H2O)2 is added separately.

[0043] The simulated intestinal fluid contained 6.8 mL 6.8 mM KCl, 0.8 mL 0.8 mM KH2PO4, 42.5 mL 85.0 mM NaHCO3, 9.6 mL 38.4 mM NaCl, 1.1 mL 0.33 mM MgCl2 (H2O)6, 0.7 mL 8.4 mM HCl, and was made up with dH2O. 2 mL 0.6 mM CaCl2 (H2O)2 was added separately before use.

[0044] The reason why CaCl2(H2O)2 needs to be added separately before use in the above-mentioned simulated liquid is that 1) the ionic strength of calcium ions in the simulated gastrointestinal fluid; 2) calcium ions in the solution can stabilize and activate enzymes; 3) calcium ions are involved in biological processes such as muscle contraction, nerve conduction and blood coagulation. The reason for adding CaCl2(H2O)2 separately before use is that: 1) calcium ions have high reactivity and are easy to react with other components or form precipitation; 2) calcium ions will affect the solubility of other components.

[0045] In an embodiment of the method of the present invention, in step 2), the amount of pepsin added is 0.2-0.4% by mass of the soybean peptide, the enzymolysis time is 1-3h, the enzymolysis temperature is 35-40°C, and the pH is 2.5-3.5. In a specific embodiment, the amount of pepsin added can be 0.2%, 0.25%, 0.3%, 0.35% or 0.4% by mass of the soybean peptide, the enzymolysis time can be 1h, 1.5h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, the enzymolysis temperature can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, the pH can be 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, and the pH can be 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5. In a preferred embodiment, the amount of pepsin added can be 0.3% of the soybean peptide by mass fraction, the enzymolysis time is 2 hours, the enzymolysis temperature is 37° C., and the pH is 3.0.

[0046] In the embodiment of the method of the present invention, in step 2), the amount of pancreatin added can be 0.02-0.04% of the soybean peptide by mass fraction, the enzymolysis time can be 1-3h, the enzymolysis temperature can be 35-40°C, and the pH is 7.0-7.5. In the specific scheme of the present invention, the amount of pancreatin added can be 0.02%, 0.03%, 0.04% of the soybean peptide by mass fraction, the enzymolysis time can be 1h, 1.5h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, the enzymolysis temperature can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, and the pH can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5. In a preferred embodiment, the amount of pancreatin added can be 0.03% of the soybean peptide by mass fraction, the enzymolysis time is 2h, the enzymolysis temperature is 37°C, and the pH is 7.0.

[0047] In an embodiment of the method of the present invention, in step 2), after the enzymatic hydrolysis is completed, the enzyme is inactivated by placing in a water bath at 95° C. for 10 min.

[0048] In an embodiment of the method of the present invention, in step 3), the soybean peptide digestion product is dissolved in HBSS buffer and inoculated into Caco-2 monolayer cells, wherein the concentration of the soybean peptide digestion product in HBSS buffer is 4 mg / mL.

[0049] In an embodiment of the method of the present invention, in step 3), the soybean peptide digestion product is subjected to in vitro simulated absorption using a Caco-2 monolayer cell model, and the soybean peptide digestion product is separated into absorbed soybean peptides and unabsorbed soybean peptides. In a specific embodiment, the Caco-2 cell monolayer model is constructed by culturing the Caco-2 cells for 21 days to stabilize their resistance value at 600-700Ω, and then the digested soybean peptides are separated into absorbed soybean peptides and unabsorbed soybean peptides after 1-3 hours of absorption. In a specific embodiment, the resistance value of the Caco-2 monolayer cell can be 610, 620, 630, 640, 650, 660, 670, 680, 690, 700Ω.

[0050] In a third aspect, the present invention provides an anti-inflammatory composition comprising the above anti-inflammatory peptides. Those skilled in the art will appreciate that the composition may also comprise other anti-inflammatory active substances, or active substances that synergistically increase anti-inflammatory activity, and pharmaceutically acceptable carriers or excipients.

[0051] In a fourth aspect, the present invention provides use of the anti-inflammatory active peptide of the present invention in the preparation of anti-inflammatory drugs.

[0052] Compared with the prior art, the beneficial effect of the present invention is that the present invention provides a peptide with anti-inflammatory activity, which has good anti-inflammatory activity.

[0053] The present invention also provides a method for preparing anti-inflammatory active peptides, which are prepared by enzymatically hydrolyzing, separating and identifying soy protein isolates. The method of the present invention has a wide range of raw material sources and the preparation process is easy to operate and implement.

[0054] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0055] Example 1

[0056] This embodiment provides an anti-inflammatory active peptide, whose amino acid sequence is Leu-Leu-Val-Lys, or represented by LLVK, and whose molecular weight is 472.35Da. The preparation process of the above anti-inflammatory active peptide is as follows:

[0057] (1) A 5% soy protein isolate solution was prepared and sequentially enzymolyzed with bromelain (Shanghai Yuanye Biotechnology Co., Ltd., S10009, the amount of bromelain added was 1.3% of the soy protein isolate by mass fraction) and flavor protease (Shanghai Yuanye Biotechnology Co., Ltd., S10153, the amount of flavor protease added was 10.0% of the soy protein isolate by mass fraction), the bromelain enzymolysis time was 3 hours, the enzymolysis conditions were: enzymolysis temperature was 50°C, pH 7.0; the flavor protease enzymolysis time was 3 hours, the enzymolysis conditions were: enzymolysis temperature was 55°C, pH 7.0; after the enzymolysis was completed, the enzyme was inactivated in a 95°C water bath for 10 minutes to obtain a soy peptide solution. The soy peptide solution was stored at -40°C and then freeze-dried for 48 hours to obtain a soy peptide powder.

[0058] (2) Prepare simulated oral fluid, simulated gastric fluid and simulated intestinal fluid, as shown in Table 1.

[0059] Table 1 Preparation of simulated oral fluid, simulated gastric fluid and simulated intestinal fluid

[0060]

[0061] 1) Dissolve 10g of soybean peptide in 10mL of distilled water and mix well, add 8mL of simulated oral fluid and 0.05mL of 1.5mM CaCl2(H2O)2 solution, add 1.95mL of distilled water to make the system 20mL, then oscillate and react at 37°C for 2min to obtain soybean peptide oral digestion solution. Then add 16mL of simulated gastric fluid and 0.01mL of 0.15mM CaCl2(H2O)2 solution to the soybean peptide oral digestion solution, adjust the pH to 3.0 with HCl, then add 1mL of pepsin to make its final activity in the solution 2000U / mL, add distilled water to make the system 40mL, then oscillate and react at 37°C for 2h to obtain soybean peptide gastric digestion solution. Finally, 16 mL of simulated intestinal fluid and 0.08 mL of 0.6 mM CaCl2 (H2O) 2 solution were added to the soybean peptide gastric digestion solution, and the pH was adjusted to 7.0 using NaOH, followed by the addition of 10 mL of pancreatic enzyme solution to make its final activity in the solution 100 U / mL, and distilled water was added to make the system 80 mL, followed by an oscillation reaction at 37 ° C for 2 h to obtain soybean peptide intestinal digestion solution, namely soybean peptide digestion solution. The soybean peptide digestion solution was stored at -40 ° C, and then freeze-dried for 48 h to obtain digested soybean peptide powder.

[0062] 2) Caco-2 cells (from the Institute of Basic Medicine, Chinese Academy of Medical Sciences) were cultured in a complete medium of MEM + 20% FBS + 1% NEAA + 1% PS, at a culture temperature of 37°C and a culture humidity of 5% CO2 / 95% air. Every three days, when the cell density reached 80%, the cells were passaged using 0.25% TE at a ratio of 1:3. The CCK-8 method (see Liu, W., Yu, S., Han, Yu., Chen, L., An, J., Li, He., Liu, X, 2024, Systematic sequence characterization of enzymatic-derived soybean peptides for precision enhancement of anti-inflammatory properties. Food Bioscience, 60, 104292) was used to determine the effects of different concentrations of digested soybean peptides (0 mg / mL, 3 mg / mL and 4 mg / mL) on the viability of Caco-2 cells. The results are shown in the table below. Figure 2 In order to exclude cytotoxic effects, the maximum concentration that did not affect the viability of Caco-2 cells, i.e. 4 mg / mL, was selected for subsequent experiments.

[0063] 3) Caco-2 cells were inoculated into 12-well Transwell chambers, the culture medium was changed every two days, and transepithelial resistance was measured using a resistor meter (Millipore, USA, model MERS00002). The results are shown in Figure 1 As shown, the construction of the Caco-2 monolayer cell model was completed after culturing for 21 days and the resistance value was >300Ω.

[0064] 4) Caco-2 cells were inoculated into a 6-well Transwell chamber, the culture medium was changed every two days, and the transepithelial resistance was measured using a resistance meter. The Caco-2 monolayer cell model was constructed after 21 days of culture and the resistance value was >300Ω.

[0065] 5) Cultivate RAW264.7 cells (from the Institute of Basic Medicine, Chinese Academy of Medical Sciences) in a complete medium of DMEM + 10% FBS + 1% NEAA + 1% PS, at a culture temperature of 37°C and a culture humidity of 5% CO2 / 95% air. Every two days, when the cell density is 80%, the cells are scraped and passaged at a ratio of 1:3. Add RAW264.7 cells with a cell density of 80% to the BL side of the Transwell chamber, and construct a Caco-2 / RAW264.7 cell co-culture model with the Caco-2 monolayer cells differentiated for 21 days in step 4).

[0066] 6) The digested soybean peptide powder obtained in step 1) was dissolved in DMEM serum-free basal medium at a concentration of 4 mg / mL obtained in step 2), and 1.5 mL was inoculated into the Caco-2 / RAW264.7 cell co-culture model constructed in step 5), and 2.6 mL of DMEM serum-free basal medium was inoculated into the BL side. After 2 hours of absorption, the BL side solution and cells were taken for determination of inflammatory factors and protein expression. It was found that the absorbed soybean peptide inhibited the occurrence of inflammatory response by inhibiting IκB protein, reducing the expression of downstream IL-1β, IL-6, TNF-α and MCP-1, and effectively preventing the occurrence of inflammatory response.

[0067] 7) The digested soybean peptide obtained in step 1) was dissolved in HBSS buffer at a concentration of 4 mg / mL obtained in step 2), and 0.5 mL was inoculated into the AP side of the Caco-2 monolayer cell model constructed in step 4), and 1.5 mL of HBSS buffer was inoculated into the BL side. After absorbing for 2 hours, the soybean peptide absorbed on the BL side of the Transwell chamber was taken for peptide identification using liquid chromatography-mass spectrometry. First, the sample was pretreated. The soybean peptide absorbed for 2 hours was dissolved in ultrapure water, and dithiothreitol solution was added to make the final concentration of 10 mmol / L, and it was reduced in a 56°C water bath for 1 hour. Iodoacetamide solution was added to make the final concentration of 50 mmol / L, and the reaction was protected from light for 40 minutes. Desalting was performed using a desalting column, and the solvent was evaporated in a vacuum centrifugal concentrator at 45°C. Then, liquid chromatography-mass spectrometry was performed, and the nanoliter ultra-high performance liquid chromatograph Easy-nLC 1200 was used for detection, and the capillary liquid chromatography pre-column was Acclaim PepMap RPLC C18 (300μm id×5mm, 5μm, ), the analytical column was Acclaim PepMap RPLC C18 (50 μm i.d. × 150 mm, 1.9 μm, ). Mobile phase A was 0.1% formic acid in water, and mobile phase B was 80% acetonitrile / 0.1% formic acid in water. Gradient elution, elution program: 0-5min, 4%-10% B; 5-85min, 10%-22% B; 85-110min, 22%-40% B; 110-115min, 40%-95% B; 115-120min, 95% B. Flow rate 600nL / min, injection volume 5μL. Electrospray-combined ion trap Orbitrap mass spectrometer (Thermo Fisher Scientific Q Exactive TM Hybrid Quadrupole-Orbitrap TM Mass Spectrometer) was used for detection, with a spray voltage of 2.2 kV and a capillary temperature of 270 °C. Primary mass spectrometer parameters: resolution 70000, automatic gain control target 3×106 , MaximumIT 100ms, scanning range 300 to 1800m / z. Secondary mass spectrometry parameters: resolution 17500, automatic gain control target 1×10 5 , maximum IT 50ms, TopN 20, collision energy 28. Finally, the database search, the mass spectrum raw file uses Byonic to search the soybean protein isolate database (https: / / www.uniprot.org / ), protein modification is Carbamidomethyl (C) (fixed), Oxidation (M) (variable); enzyme is nonspecific; missed enzyme cleavage site is 3; primary mass spectrometry error is 20ppm, secondary mass spectrometry error is 0.02Da.

[0068] The peptide sequence information of soybean peptides after 2 hours of absorption is shown in Table 2. 280 ), hydrophobic, stable, containing branched-chain amino acids at the N-terminus and basic amino acids at the C-terminus, soy peptides with potential anti-inflammatory activity were selected. LLVK has a low molecular weight, UV absorption ability, stability, contains four key amino acids: leucine, valine and lysine, is rich in branched-chain amino acids at the N-terminus, and is rich in basic amino acids at the C-terminus. Therefore, this peptide was selected for subsequent experiments.

[0069] Table 2. Peptide analysis of soybean peptides after 2 hours of absorption

[0070]

[0071]

[0072] Experimental Example 1

[0073] In this experimental example, the CCK-8 method was used (see Liu, W., Yu, S., Han, Yu., Chen, L., An, J., Li, He., Liu, X, 2024, Systematic sequence characterization of enzymatic-derived soybean peptides for precision enhancement of anti-inflammatory properties. Food Bioscience, 60, 104292) to determine the effect of different LLVK concentrations (0 mg / mL, 0.5 mg / mL and 1 mg / mL) on the viability of RAW264.7 cells.

[0074] like Figure 4As shown, the maximum concentration that does not affect the viability of RAW264.7 cells, i.e., 1 mg / mL, was selected for subsequent cell experiments. RAW264.7 cells were inoculated in 6-well plates, and after overnight attachment, they were treated with 1 mg / mL of LLVK for 2 h. Then, 10 μg / mL of lipopolysaccharide was selected for stimulation for 24 h to establish a cell inflammation model. The expression of IκB protein in cells was measured after inflammatory damage. 100 μL of lysis buffer was added to each well of the six-well plate, and the lysis was carried out on ice for 30 min, and the six-well plate was shaken every 5 min to fully lyse it. BCA was used to determine the protein concentration in the lysate, and the diluent was added to unify the protein concentration of all samples, and then 5× loading buffer was added, and the cells were placed in a 100°C water bath for heating and denaturation for 5 min, cooled, centrifuged at 9184g for 2 min, and the supernatant was taken for determination. The proteins were separated in 10% SAS-PAGE and transferred to PVDF membranes, incubated with primary antibody (IκB antibody 1:1000) at 4°C overnight, washed off unbound primary antibody, incubated with secondary antibody (IgG (H+L), 1:1000) at room temperature for 1 hour, and protein expression was standardized with α-tubulin, and displayed by ECL chemiluminescence. Band intensity was measured using Image J software.

[0075] like Figure 3 As shown, the absorption of soybean peptides mainly inhibits the inflammatory response by inhibiting the phosphorylation of IκB protein and reducing the expression of inflammatory factors downstream of the PI3K / Akt / NF-κB pathway. IκB protein is a specific regulatory site.

[0076] Experimental Example 2

[0077] This experimental example is based on molecular docking technology (see Nguyen, N., Nguyen, T., Pham, T., Huy, N., Bay, M., Pham, M., Nam, P., Vu, V., Ngo, S., 2020, Autodock Vina adopts more accurate binding poses but Autodock4 forms better binding affinity. Journal of Chemical Information and Modeling, 60(1): 204-211) to explore the inflammation prevention mechanism of LLVK.

[0078] The crystal structure of the Kelch domain of IκB was selected from the PDB database (PDB ID: 1NFI). The structure of LLVK was simulated by Chem Draw and Chem 3D, and the structure with the lowest energy was selected for docking. Before docking, the water molecules on IκB were deleted and charges and hydrogen atoms were added using AutoDockTools. The docking simulation was calculated using AutoDockVina, and the structure with the best docking effect was determined based on the scoring results. The results of AutoDockVina were then drawn using Pymol software.

[0079] Figure 5 The visualization of LLVK binding to IκB protein was shown. The experimental results showed that the binding energy between LLVK and IκB protein was -6.3 kcal / mol, indicating that the binding between the peptide and IκB protein was spontaneous. The action sites of LLVK and IκB protein include: ARG50, GLU225, PHE239, TRP258, PRO275, GLN241, GLY259, GLN266. LLVK mainly interacts with IκB protein through hydrophobic interactions and hydrogen bonds. From the perspective of computer simulation, it can be shown that LLVK can form a more stable complex with IκB protein, thereby reducing the binding of IκB protein to phosphate groups and inhibiting inflammation.

[0080] Experimental Example 3

[0081] Inflammatory factors and chemokines play a key role in inflammatory reactions, and their detection is of great significance for the diagnosis and management of various inflammatory diseases. In this example, IL-1β, IL-6, TNF-α and MCP-1 kits (Hangzhou Lianke Biotechnology Co., Ltd., Mouse IL-1β ELISA Kit detection kit; Mouse IL-6 ELISA Kit detection kit; Mouse TNF-α ELISA Kit detection kit; Mouse CCL2 / MCP-1 ELISA Kit detection kit) were used to determine the content of inflammatory factors IL-1β, IL-6, TNF-α and chemokine MCP-1 secreted by RAW264.7 cells according to the manufacturer's instructions.

[0082] The results are as follows Figure 7 As shown, lipopolysaccharide stimulation increases the levels of IL-1β, IL-6, TNF-α and MCP-1, and inflammatory response occurs. LLVK can significantly reduce the levels of IL-1β, IL-6 and MCP-1. The experimental results show that LLVK can effectively prevent the occurrence of inflammatory response.

[0083] Soy peptides with specific sequences are gradually entering people's lives as a nutritionally comprehensive inflammation prevention product. The present invention prepares the anti-inflammatory active peptide LLVK by a proteolysis screening method, and finds that it spontaneously combines with IκB protein through hydrogen bonds and hydrophobic interactions to form a more stable complex, inhibits IκB protein phosphorylation, and reduces the secretion of inflammatory factors IL-1β, IL-6, TNF-α and chemokine MCP-1, thereby preventing the occurrence of inflammatory reactions. The present invention also provides a preparation method and application of the anti-inflammatory active peptide. Theoretical support is provided for the use of anti-inflammatory active peptides in related foods, health products and medicines.

[0084] The experimental results are as follows Figure 6 As shown, LLVK can significantly upregulate the expression of IκB protein, and the cell experiment results are consistent with the molecular docking results.

[0085] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. An anti-inflammatory active peptide, characterized in that: The amino acid sequence of the peptide is LLVK and the molecular weight is 472.35 Da.

2. A method for preparing the anti-inflammatory active peptide according to claim 1, characterized in that: The following steps are involved: 1) using bromelain and flavor protease to enzymatically hydrolyze soy protein isolate to obtain soy peptides; 2) Using pepsin and pancreatin to simulate in vitro digestion of soybean peptides to obtain soybean peptide digestion products; 3) using a Caco-2 monolayer cell model to simulate in vitro absorption of the soybean peptide digestion product to obtain absorbed soybean peptide; and 4) Identifying the absorbed soybean peptide to obtain the anti-inflammatory active peptide LLVK.

3. The preparation method according to claim 2, characterized in that: In step 1), the amount of bromelain added is 1.2-1.4% by mass of the soy protein isolate, and the amount of flavor protease added is 9.0-11.0% by mass of the soy protein isolate.

4. The preparation method according to claim 2, characterized in that: In step 1), the bromelain enzymolysis time is 2.5-3.5 hours, the enzymolysis temperature is 45-55° C., and the pH is 7.0-7.5; the flavor protease enzymolysis time is 2.5-3.5 hours, the enzymolysis temperature is 50-60° C., and the pH is 7.0-7.

5. After the enzymolysis is completed, the enzyme is inactivated in a 95° C. water bath for 10 minutes.

5. The preparation method according to claim 2, step 2) specifically comprises the following steps: 2.1) dissolving the soybean peptide in water, adding simulated oral fluid, and shaking the reaction to obtain a soybean peptide oral digestive fluid, 2.2) adding simulated gastric fluid to the soybean peptide oral digestive fluid, adjusting the pH to 3.0, then adding pepsin, and oscillating the reaction to obtain soybean peptide gastric digestive fluid, 2.3) adding simulated intestinal fluid to the soybean peptide gastric digestion fluid, adjusting the pH to 7.0, and then adding pancreatic enzyme, shaking the reaction, to obtain soybean peptide intestinal digestion fluid, and 2.4) freeze-drying the soybean peptide digestion solution to obtain digested soybean peptide powder, i.e., the soybean peptide digestion product. Wherein, based on 400 mL, the simulated oral fluid contains 15.1 mL 15.1 mM KCl, 3.7 mL 3.7 mM KH2PO4, 13.6 mL 6.8 mM NaHCO3, 0.5 mL 0.15 mM MgCl2(H2O)6, 0.06 mL 0.06 mM (NH4)2CO3, 0.09 mL 1.1 mM HCl, dH2O is added to make up, and 2.5 mL 1.5 mM CaCl2(H2O)2 is added separately before use. Based on 400 mL, the simulated gastric fluid contains 6.9 mL 6.9 mM KCl, 0.9 mL 0.9 mM KH2PO4, 12.5 mL 25.0 mM NaHCO3, 11.8 mL 47.2 mM NaCl, 0.4 mL 0.12 mM MgCl2 (H2O)6, 0.5 mL 0.5 mM (NH4)2CO3, 1.3 mL 15.6 mM HCl, and dH2O is added to make it complete. Before use, 0.25 mL 0.15 mM CaCl2 (H2O)2 is added separately. The simulated intestinal fluid contained 6.8 mL 6.8 mM KCl, 0.8 mL 0.8 mM KH2PO4, 42.5 mL 85.0 mM NaHCO3, 9.6 mL 38.4 mM NaCl, 1.1 mL 0.33 mM MgCl2 (H2O)6, 0.7 mL 8.4 mM HCl, and was made up with dH2O. 2 mL 0.6 mM CaCl2 (H2O)2 was added separately before use.

6. The method for preparing the anti-inflammatory active peptide according to claim 2, characterized in that: In step 2), the amount of pepsin added is 0.2-0.4% by mass of the soybean peptide, and the amount of pancreatin added is 0.02-0.04% by mass of the soybean peptide.

7. The method for preparing the anti-inflammatory active peptide according to claim 2, characterized in that: In step 2), the pepsin hydrolysis time is 1-3h, the hydrolysis temperature is 35-40°C, and the pH is 2.5-3.5; the pancreatin hydrolysis time is 1-3h, the hydrolysis temperature is 35-40°C, and the pH is 7.0-7.

5. After the hydrolysis is completed, the enzyme is inactivated in a 95°C water bath for 10 minutes.

8. The method for preparing the anti-inflammatory active peptide according to claim 2, characterized in that: In step 3), the soybean peptide digestion product is dissolved in HBSS buffer and inoculated into Caco-2 monolayer cells, wherein the concentration of the soybean peptide digestion product in HBSS buffer is 4 mg / mL.

9. An anti-inflammatory composition comprising the anti-inflammatory active peptide according to claim 1.

10. Use of the anti-inflammatory active peptide according to claim 1 in the preparation of anti-inflammatory drugs.

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