Active peptide for enhancing intestinal barrier function and preparation method and application thereof

Abstract

The present invention belongs to the field of protein technology, and relates to an active peptide for enhancing intestinal barrier function, a preparation method and an application thereof, wherein the amino acid sequence of the peptide is LIPR. The active peptide enhances the intestinal barrier function by promoting the expression of claudin, occludin and ZO-1 protein, thereby reducing toxin invasion and maintaining intestinal health. The present invention also provides a preparation method and an application of the active peptide for enhancing intestinal barrier function.

Description

Technical Field

[0001] The invention belongs to the technical field of proteins, and in particular relates to an active peptide for enhancing intestinal barrier function and a preparation method and application thereof. Background Art

[0002] Intestinal diseases are an important public health issue worldwide. Their prevalence and impact are increasing year by year, seriously affecting patients' quality of life and socioeconomic development. The intestinal barrier is a protective layer on the intestinal lining, and its main function is to prevent harmful substances (such as pathogens and toxins) from entering the blood and other parts of the body. Impaired intestinal barrier function can lead to increased intestinal permeability, and harmful substances entering the blood can cause systemic health problems.

[0003] The current treatment for intestinal barrier damage is a combination of drug therapy and lifestyle changes. Among them, drug therapy mainly chooses to take azathioprine, cyclosporine, etc. to protect the intestinal barrier by suppressing the excessive response of the immune system. However, long-term use is accompanied by potential side effects, such as nausea, vomiting, abnormal liver and kidney function, etc., so finding new safe alternatives is an effective way to improve intestinal barrier damage.

[0004] Soy peptide is the enzymatic hydrolysis product of soy protein isolate. It has a low molecular weight and usually contains 3 to 20 amino acids. It not only retains the advantages of protein nutrition and amino acid balance, but is also easy to dissolve and absorb. Soy peptide has been proven by domestic and foreign scholars to regulate intestinal barrier function. Therefore, using soy peptide raw materials for separation and exploring key effective ingredients to enhance intestinal barrier function has great practical application value. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an active peptide with enhanced intestinal barrier function, as well as a preparation method and application thereof. By screening and preparing active peptides with enhanced intestinal barrier function, the intestinal protection ability is improved, the damage of external toxins to the intestinal barrier and the invasion of toxins into the human body through the intestine are reduced, and the side effects of taking drugs such as azathioprine and cyclosporine are avoided, so as to effectively help patients with damaged intestinal barrier.

[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 active peptide for enhancing intestinal barrier function, wherein the amino acid sequence of the peptide is Leu-Ile-Pro-Arg, or represented by LIPR, and the molecular weight is 497.63 Da. The peptide is hydrophobic and stable, and contains leucine, isoleucine and arginine.

[0008] On the other hand, the present invention provides a method for preparing the above-mentioned 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) performing simulated absorption in vitro on the soybean peptide digestion product, separating it using a Caco-2 monolayer cell model to obtain absorbed soybean peptides; and

[0012] 4) Identifying the absorbed soybean peptide to obtain the active peptide LIPR.

[0013] In a third aspect, the present invention provides a composition for enhancing intestinal barrier function, the composition comprising the active peptide of the present invention.

[0014] In a fourth aspect, the present invention provides use of the active peptide of the present invention in the preparation of a medicament for enhancing intestinal barrier function.

[0015] The active peptide of the present invention can increase the expression of claudin, occludin and tight junction protein-1 (ZO-1 protein) in Caco-2 cell monolayer, increase the transepithelial cell resistance value, enhance the intestinal barrier function, thereby reducing toxin invasion and maintaining intestinal health. In addition, the active peptide of the present invention is prepared by enzymatic hydrolysis, separation and identification of soy protein isolate, so the raw material source is wide, and the preparation process is easy to operate and realize. 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 effect of digested soybean peptides at different concentrations on the survival rate of Caco-2 cells;

[0018] Figure 3 To study the effect of soybean peptides on the electrical resistance of Caco-2 monolayer cells during absorption.

[0019] Figure 4 The effect of different concentrations of LIPR on the survival rate of Caco-2 cells;

[0020] Figure 5 The effect of LIPR on the expression of claudin, occludin and ZO-1 proteins. DETAILED DESCRIPTION

[0021] 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.

[0022] On the one hand, the present invention provides an active peptide for enhancing intestinal barrier function, wherein the amino acid sequence of the peptide is Leu-Ile-Pro-Arg, or expressed as LIPR, and the molecular weight is 497.63 Da.

[0023] In an embodiment of the present invention, the active peptide can increase the expression of tight junction proteins such as claudin, occludin and ZO-1 protein (tight junction protein-1) in the Caco-2 cell monolayer, increase the transepithelial cell resistance, enhance the intestinal barrier capacity, and strengthen the intestinal barrier function, thereby reducing toxin invasion and maintaining intestinal health.

[0024] On the other hand, the present invention provides a method for preparing the above-mentioned active peptide, comprising the following steps:

[0025] 1) using bromelain and flavor protease to enzymatically hydrolyze soy protein isolate to obtain soy peptides;

[0026] 2) Using pepsin and pancreatin to simulate in vitro digestion of soybean peptides to obtain soybean peptide digestion products;

[0027] 3) performing simulated absorption in vitro on the soybean peptide digestion product, separating it using a Caco-2 monolayer cell model to obtain absorbed soybean peptides; and

[0028] 4) Identifying the absorbed soybean peptide to obtain the active peptide LIPR.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] In an embodiment of the present invention, step 2) specifically comprises the following steps:

[0033] 2.1) Dissolve the soybean peptide in water, add simulated oral fluid, and shake to obtain the soybean peptide oral digestive fluid.

[0034] 2.2) Add simulated gastric fluid to the soybean peptide oral digestive fluid, adjust the pH to 3.0, then add pepsin, and oscillate to react to obtain soybean peptide gastric digestive fluid.

[0035] 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

[0036] 2.4) freeze-drying the soybean peptide digestion solution to obtain digested soybean peptide powder, i.e., soybean peptide digestion product.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The reason why CaCl2(H2O)2 needs to be added separately before use in the above-mentioned simulation liquid is that 1) the ionic strength of calcium ions in the simulated gastrointestinal fluid is simulated; 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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 cells are cultured for 21 days to stabilize their resistance value to 720-770Ω, thereby constructing a Caco-2 monolayer cell model, and then after 1-3 hours of absorption, the digested soybean peptides are separated into absorbed soybean peptides and unabsorbed soybean peptides. In a specific embodiment, the resistance value of the Caco-2 monolayer cells can be 720, 730, 740, 750, 760, 770Ω, etc.

[0046] In a third aspect, the present invention provides a composition for enhancing intestinal barrier function, comprising the above-mentioned active peptide. Those skilled in the art will appreciate that the composition may also include other active substances that enhance intestinal barrier function, or active substances that synergistically enhance intestinal barrier function, and pharmaceutically acceptable carriers or excipients.

[0047] In a fourth aspect, the present invention provides the use of the above active peptide in the preparation of a drug for enhancing intestinal barrier function.

[0048] Compared with the prior art, the beneficial effect of the present invention is that the present invention provides an active peptide for enhancing the intestinal barrier function, which can enhance the intestinal barrier function and maintain intestinal health by regulating the expression of tight junction proteins, thus having a good effect of improving the intestinal barrier function.

[0049] The present invention also provides a method for preparing the active peptide, which is prepared by subjecting soybean protein isolate to enzymatic hydrolysis, separation and identification. The raw material sources of the method of the present invention are wide, and the preparation process is easy to operate and implement.

[0050] 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.

[0051] Example 1

[0052] This embodiment provides an active peptide for enhancing intestinal barrier function, whose amino acid sequence is Leu-Ile-Pro-Arg, or represented by LIPR, and whose molecular weight is 497.63Da. The preparation process of the active peptide for enhancing intestinal barrier function is as follows:

[0053] (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.

[0054] (2) Prepare simulated gastric fluid and simulated intestinal fluid, as shown in Table 1.

[0055] Table 1. Preparation of simulated gastric fluid and simulated intestinal fluid

[0056]

[0057] 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.

[0058] 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, 1 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.

[0059] 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 Caco-2 monolayer cell model was constructed after culturing for 21 days and the resistance value was >300Ω.

[0060] 4) 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 3), and 1.5 mL of HBSS buffer was inoculated into the BL side. The resistance value was measured 2 hours after absorption, and it was found that during the absorption process of the digested soybean peptide, the resistance value of the Caco-2 monolayer cell increased ( Figure 3 ), the intestinal barrier function is increased.

[0061] 5) 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. The first step is sample pretreatment. The soybean peptide absorbed for 2 hours was dissolved in ultrapure water, and dithiothreitol solution was added to make the final concentration of 10mmol / L. It was reduced in a 56°C water bath for 1 hour. Iodoacetamide solution was added to make the final concentration of 50mmol / 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, LC-MS detection was performed using a nanoliter ultra-high performance liquid chromatograph Easy-nLC 1200 (Thermo Fisher Scientific QExactive TM HybridQuadrupole-Orbitrap TM Mass Spectrometer) was used for detection, and the capillary liquid chromatography pre-column was AcclaimPepMap RPLC C18 (300μm id×5mm, 5μm, ), the analytical column was Acclaim PepMap RPLC C18 (50 μm id×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 HybridQuadrupole-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 3×10 6 , 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 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.

[0062] The peptide sequence information of soybean peptides after 2 hours of absorption is shown in Table 2. According to the high-activity bioactive peptides with low molecular weight and ultraviolet absorption capacity (A 280 ), hydrophobic, stable, and rich in hydrophobic and basic amino acids. Soybean peptides with the potential to enhance intestinal barrier function were selected. LIPR has a low molecular weight, UV absorption ability, stability, and contains four key amino acids: leucine, isoleucine, proline, and arginine. Therefore, this peptide was selected for subsequent experiments.

[0063] Table 2. Peptide analysis of soybean peptides after 2 hours of absorption

[0064]

[0065]

[0066] Experimental Example 1

[0067] 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 LIPR concentrations (0 mg / mL, 0.5 mg / mL and 1 mg / mL) on Caco-2 cell viability. Figure 4As shown, the maximum concentration that does not affect the viability of Caco-2 cells, i.e., 1 mg / mL, was selected for subsequent cell experiments. Caco-2 cells were inoculated in a 6-well plate of a Transwell chamber and differentiated for 21 days. Subsequent experiments were performed when the resistance value was greater than 300Ω. 1 mg / mL of LIPR was added to the AP side, and the Caco-2 monolayer cells were taken after 2 hours of transport to determine the expression of tight junction proteins. 50 μL of lysis solution was added to each well on the AP side, and the cells were lysed on ice for 30 minutes, with the six-well plate shaken every 5 minutes to fully lyse. The protein concentration in the lysate was determined according to the instructions of the BCA kit of Beijing Solebold Technology Co., Ltd., and the diluent was added to unify the protein concentration of all samples. The samples were centrifuged at 9184g for 2 minutes, and the supernatant was taken for determination.

[0068] The integrity of the intestinal barrier depends on the expression of tight junction proteins such as claudin, occludin and ZO-1 proteins. The claudin, occludin and ZO-1 protein kits (human claudin-4 ELISA research kit; human occludin ELISA research kit; human tight junction protein-1 (ZO-1) ELISA research kit) of Jiangsu Enzyme Immunity Industry Co., Ltd. were used to measure the content of claudin, occludin and ZO-1 proteins in Caco-2 monolayer cells according to the instructions. Figure 5 As shown in the figure, LIPR significantly increased the expression of claudin, occludin and ZO-1 proteins, indicating that LIPR can enhance intestinal barrier function and maintain intestinal health by regulating the expression of tight junction proteins.

[0069] 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. Active peptides for enhancing intestinal barrier function, characterized in that: The amino acid sequence of the peptide is LIPR, and the molecular weight is 497.63 Da.

2. A method for preparing the 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) performing in vitro simulated absorption of the soybean peptide digestion product, and separating it using a Caco-2 monolayer cell model to obtain absorbed soybean peptides; and 4) Identifying the absorbed soybean peptide to obtain the active peptide LIPR.

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 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 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 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. A composition for enhancing intestinal barrier function, comprising the active peptide according to claim 1.

10. Use of the active peptide according to claim 1 in the preparation of a drug for enhancing intestinal barrier function.

Images