Active peptides for modulating the function of the intestinal epithelial mucus barrier and methods of making and using the same
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
- Filing Date
- 2026-05-21
- Publication Date
- 2026-06-19
Smart Images
Figure CN122234147A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology. More specifically, this invention relates to an active peptide that regulates the function of the intestinal epithelial mucus barrier, its preparation method, and its applications. Background Technology
[0002] The intestinal mucus barrier is a core component of the intestinal mucosal defense system, mainly composed of mucin secreted by goblet cells, a continuous mucus layer, and related regulatory factors. Mucin MUC2 is a key structural protein in constructing the gel-like mucus barrier of the intestine, effectively blocking pathogens and toxins from direct contact with intestinal epithelial cells and maintaining the integrity of the intestinal barrier and the homeostasis of the intestinal microenvironment. Under external stresses such as inflammation, oxidative stress, pathogen-related molecules, and bile acid disorders, the intestinal epithelium is prone to damage, including goblet cell dysfunction, thinning of the mucus layer, downregulation of MUC2 expression, and increased mucus barrier permeability, which in turn induces intestinal dysfunction, exacerbates inflammatory responses, and reduces mucosal defense capabilities.
[0003] Current research on the intestinal barrier largely focuses on epithelial tight junctions, transepithelial electrical resistance, and permeability regulation. Targeted intervention studies on the specific functional unit of the intestinal epithelial mucus barrier remain scarce. Animal processing byproducts, rich in collagen and structural proteins, are high-quality raw materials for preparing bioactive peptides and possess significant potential for high-value utilization. While some intestinal protective bioactive peptides have been reported in existing technologies, these primarily focus on generalized intestinal anti-inflammatory effects and improvements in epithelial integrity. Specific bioactive peptides using duck skin as a raw material that target and regulate intestinal epithelial mucus barrier function and clearly act on mucin secretion and MUC2 expression have yet to be systematically disclosed or technically proven.
[0004] The existing technology has the following obvious limitations: it lacks specific active peptides targeting the intestinal epithelial mucus barrier function; most peptides only achieve generalized intestinal protection without targeting mucin secretion, goblet cell function, and mucus layer repair; the enzymatic hydrolysate products made from duck skin have not established a targeted screening system for mucus barrier function, and their functions and structures are unclear; the synergistic regulatory effects of active peptides on total mucin secretion, MUC2 gene / protein expression, and goblet cell function have not been systematically verified, and the mechanism of action is unclear.
[0005] Therefore, developing a bioactive peptide with a clear source, stable process, and well-defined structure that can target and repair the intestinal epithelial mucus barrier, promote mucin secretion and MUC2 expression, and maintain goblet cell function is of great value for the high-value utilization of livestock and poultry by-products and the development of intestinal health products. Summary of the Invention
[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.
[0007] Another objective of this invention is to provide an active peptide that regulates the function of the intestinal epithelial mucus barrier, its preparation method, and its application, which solves the technical problem of the lack of specific active peptides derived from duck skin that target the intestinal epithelial mucus barrier, promote mucin secretion and MUC2 expression, and can promote mucin secretion and MUC2 expression in the prior art.
[0008] To achieve these objectives and other advantages according to the present invention, an active peptide for regulating the function of the intestinal epithelial mucus barrier is provided, the amino acid sequence of which is shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.
[0009] This invention also provides a method for preparing the aforementioned active peptide that regulates the intestinal epithelial mucus barrier function, comprising the following steps: Step 1: Take duck skin, and after washing, defatting, cutting and homogenizing pretreatment, use ultrasonic-assisted extraction or enzyme-assisted extraction to obtain duck skin protein extract; Step 2: Adjust the duck skin protein extract to a suitable pH, add a compound special enzyme for targeted enzymatic hydrolysis, heat to inactivate the enzyme after enzymatic hydrolysis, centrifuge to collect the supernatant, and obtain duck skin protein hydrolysate. Step 3: The duck skin protein hydrolysate is subjected to ultrafiltration fractionation to collect low molecular weight peptide components, which are then freeze-dried to obtain crude active peptide powder; the crude active peptide powder contains multiple candidate active peptides; Step 4: Apply the multiple candidate active peptides to the intestinal epithelial mucus barrier function damage model, detect and compare the effects of each candidate active peptide on total mucin secretion, MUC2 gene or protein expression, and goblet cell secretion function, select the peptides that perform well in the indicators, and obtain the target active peptides after separation and purification. The amino acid sequence of the target active peptides is shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.
[0010] Preferably, in the preparation method of the active peptide, the enzymatic hydrolysis conditions in step two are: pH value of 7.5, temperature of 55℃, and enzymatic hydrolysis time of 3.5h.
[0011] Preferably, in the preparation method of the active peptide, step three involves ultrafiltration fractionation to separate components with a molecular weight cutoff of less than 1 kDa.
[0012] Preferably, in the method for preparing the active peptide, the complex enzyme is a neutral protease and a flavor protease, and the ratio of the complex enzyme to the duck skin protein extract is 1500 U / g, wherein the activity ratio of the neutral protease to the flavor protease is 1.5:1.
[0013] This invention provides the application of the aforementioned active peptide in the preparation of products for regulating the function of the intestinal epithelial mucus barrier.
[0014] Preferably, the application modulates the intestinal epithelial mucus barrier function by improving mucin secretion levels, promoting MUC2 expression, maintaining goblet cell secretion function, or restoring the integrity of the mucus layer.
[0015] Preferably, in the application described, the product improves the mucus barrier function of the intestinal epithelium damaged by inflammatory stimulation, oxidative stress, or pathogen-related factors, thereby blocking direct contact between pathogenic microorganisms and their toxins and epithelial cells.
[0016] The present invention has at least the following beneficial effects: This invention uses duck skin as raw material, and through extraction, targeted enzymatic hydrolysis, ultrafiltration grading and functional screening, obtains active peptides with clear amino acid sequences, realizing the high-value utilization of livestock and poultry processing by-products; The active peptides of this invention specifically target the intestinal epithelial mucus barrier, unlike generalized intestinal protective peptides. They can significantly increase total mucin secretion, upregulate MUC2 expression, maintain goblet cell function, and repair the damaged mucus layer. The mechanism of action of this invention is clear: by improving the mucus barrier function under inflammation / oxidative stress, it blocks the invasion of pathogens and toxins, providing a new functional molecule and technological basis for the development of intestinal health-related products.
[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description
[0018] Figure 1 This is the mass spectrum of the active peptide prepared in Example 1 of the present invention. Detailed Implementation
[0019] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it based on the description.
[0020] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.
[0021] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.
[0022] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.
[0023] Example 1: Preparation and Screening of Active Peptides from Duck Skin for Regulating Intestinal Epithelial Mucus Barrier Function Step 1: Raw material pretreatment and protein extraction Take fresh duck skin, remove residual fat and connective tissue, rinse it clean with running water, cut it into small pieces, add deionized water at a material-to-liquid ratio of 1:4 (g / mL) to homogenize, extract with ultrasonic assistance (power 400W, time 20min, interval 2s / 3s), centrifuge (10000rpm, 20min) and collect the supernatant to obtain duck skin protein extract.
[0024] Step 2: Targeted enzymatic hydrolysis Adjust the pH of the duck skin protein extract, add a compound enzyme, and hydrolyze at a constant temperature. After hydrolysis, inactivate the enzyme by boiling in a water bath for 10 minutes, cool, centrifuge, and collect the supernatant to obtain the duck skin protein hydrolysate.
[0025] Step 3: Ultrafiltration Fractionation and Coarse Powder Preparation The enzymatic hydrolysate was fractionated by ultrafiltration membrane, and low molecular weight peptide fractions with a molecular weight cutoff of less than 1 kDa were collected and freeze-dried to obtain crude active peptide powder.
[0026] Step 4: Screening and purification of active peptides An intestinal epithelial mucus barrier injury model (LPS / TNF-α / hydrogen peroxide induction) was constructed using HT29-MTX or LS174T cells. Multiple candidate peptides isolated from crude bioactive peptide powder were applied to the injury model, and the results were analyzed. Total mucin secretion; MUC2 gene and protein expression levels; PAS / AB staining of goblet cells and their secretory function.
[0027] The target active peptide was obtained by separation and purification using liquid chromatography-mass spectrometry, with the optimal index as the standard.
[0028] Optimization of extraction and enzymatic hydrolysis processes Single-factor experiment Using the increase in total mucin secretion rate as an indicator, single-factor experiments were conducted to investigate the effects of four factors—the amount of compound enzyme added, pH value, enzymatic hydrolysis time, and enzymatic hydrolysis temperature—on the activity of the enzymatic hydrolysis products. Each experiment was repeated three times, and the average value was taken. The results are shown in Table 1.
[0029] Table 1 Results of the single-factor experiment Orthogonal experiment: Based on the single-factor experiment, a 4-factor, 3-level L9(34) orthogonal experiment was designed with the amount of compound enzyme added, pH value, enzymatic hydrolysis time and enzymatic hydrolysis temperature as factors and the total mucin secretion enhancement rate as the indicator. The levels of each factor are shown in Table 2. The orthogonal experiment was designed and variance analysis was performed using IBM SPSS Statistics V20 to determine the optimal conditions for the preparation process. The results are shown in Table 3.
[0030] Table 2 Orthogonal Experimental Design result: Table 3 Results of the orthogonal experiment The optimal group is A2B2C2D2, and the order of influence is: C>D>B>A.
[0031] Table 3 shows that different levels of each factor significantly affect the ability of duck skin-derived active peptides to promote total mucin secretion. Range analysis reveals that the order of influence of the four factors on the increase in total mucin secretion rate is: hydrolysis temperature (C) > enzyme dosage (D) > hydrolysis time (B) > pH (A). The optimal levels for each factor are A2, B2, C2, and D2, respectively; therefore, the optimal process combination is determined to be A2B2C2D2. Further, the preferred parameters for the preparation process of the active peptides of this invention are determined to be: hydrolysis temperature 55℃, pH 7.5, hydrolysis time 3.5h, and compound enzyme dosage 1500U / g protein. The compound enzyme is a mixture of neutral protease and flavor protease (activity ratio 1.5:1).
[0032] Mass spectrometry analysis was performed on the crude active peptide powder prepared under optimized conditions, such as... Figure 1 As shown; the mass spectrometry method is as follows: mass spectrometry data are acquired using a QExactive HF mass spectrometer connected in series with an UltiMate 3000 RSLC nano liquid chromatography system. The active peptide sample is dissolved in the loading buffer, aspirated by the autosampler, and separated by the analytical column. The analytical column specifications are:
[75] μm ×
[25] cm, C18, [2] μm,
[100] Å. An analytical gradient is established using two mobile phases: mobile phase A is 0.1% formic acid aqueous solution (containing 2% dimethyl sulfoxide), and mobile phase B is 0.1% formic acid-80% acetonitrile (containing 2% dimethyl sulfoxide). The flow rate of the liquid phase is set to
[300] nL / min. Mass spectrometry data is acquired in DDA mode, and each scan cycle includes one full MS scan (resolution =
[60] K, automatic gain control target value = [3] e 6 Maximum ion implantation time =
[50] ms, scan range =
[300] –[1800 m / z), and subsequent MS / MS scan (resolution =
[15] K, automatic gain control target value = [1] e 5 Maximum ion implantation time =
[45] ms. High-energy collision dissociation collision energy is set to [28 eV]. Quadrupole screening window is set to [1.6] Da. Dynamic exclusion time for repeated ion acquisition is set to
[30] s.
[0033] Mass spectrometry data were retrieved using MaxQuant (V[1.6.17.0]) software, and the database retrieval algorithm used was Andromeda. The main retrieval parameters were as follows: the item type was selected as label-free quantification; the variable modification was selected as methionine oxidation and protein N-terminal acetylation; the fixed modification was selected as cysteine carboxymethylation; and the enzyme digestion was selected as non-specific enzyme digestion. The search results were screened based on the protein and peptide level [1]% false discovery rate. Finally, several candidate peptides with repair effects on the intestinal epithelial mucus barrier injury model were obtained. Among them, the amino acid sequences of the peptides with excellent performance were SEQ ID NO: 1, 2, 3, 4, 5, 6.
[0034] The above six amino acid sequences were used to synthesize a single active peptide, which was numbered as follows: Peptide 1: SEQ ID NO: 1 is FLWRWTWTYKDQ; Peptide 2: SEQ ID NO: 2 is YRTHKFLGSWDA; Peptide 3: SEQ ID NO: 3 is KEERWLTGQTF; Peptide 4: SEQ ID NO: 4 is FLADYGTRWHS; Peptide 5: SEQ ID NO: 5 is RYFKLGWQTHA; Peptide 6: SEQ ID NO: 6 is YSWKYSHRQTA.
[0035] Example 2: Stability Test of Active Peptides The target active peptide was dissolved in sterile PBS (pH 7.4), aliquoted into sealed, light-protected EP tubes, and stored at constant temperatures of 40℃, 25℃, 4℃, and -20℃, with three replicates for each group. Samples were collected at 0, 7, 14, and 28 days. The retention rate of total mucin secretion-promoting activity in the HT29-MTX cell mucus barrier damage model was used as the evaluation index to detect the storage stability of the active peptide. The experimental results are shown in Table 4.
[0036] The experimental results are shown in Table 4.
[0037] Table 4 Results of Stability Tests for Active Peptides As shown in Table 4, the bioactive peptides provided by this invention exhibited a certain time-dependent stability trend under different storage temperatures. Overall, the activity retention rate of each bioactive peptide gradually decreased with increasing storage temperature and duration, with the most significant decrease observed at 40℃, while the changes were smaller at 4℃ and -20℃. Further comparison of the different bioactive peptides revealed some differences in stability under the same conditions. Peptide 1 and peptide 2 showed better stability overall, while the remaining bioactive peptides also exhibited good storage stability. Especially under low-temperature conditions (4℃ and -20℃), each bioactive peptide maintained high activity after 28 days of storage, indicating that low temperature is beneficial for maintaining the structural stability and functional integrity of the bioactive peptides.
[0038] In summary, the active peptides obtained by this invention through optimized preparation processes exhibit both high efficiency and long-lasting function in solution, overcoming the technical bottlenecks of traditional active peptides being prone to inactivation and requiring stringent storage conditions. The active peptides provided by this invention have excellent storage stability, especially maintaining high activity for extended periods at 4°C and below; even after storage at 40°C for 28 days, they still retain high mucoprotein secretion activity, indicating that these active peptides possess both good thermal stability and application adaptability, which can support their development and application in functional foods, intestinal health preparations, and related products.
[0039] Example 3: The repairing effect of active peptides on the damaged intestinal epithelial mucus barrier function Experimental materials Target active peptides (SEQ ID NO: 1, 2, 3, 4, 5, 6); HT29-MTX / LS174T cells; LPS / TNF-α; mucin detection kit, qPCR reagent, WB reagent, PAS / AB staining kit.
[0040] Experimental Groups Blank control group, model group (LPS / TNF-α induced damage), low-dose active peptide group, medium-dose active peptide group, and high-dose active peptide group.
[0041] detection indicators Cell viability, total mucin secretion, MUC2 mRNA and protein expression, goblet cell secretory function, and mucus layer integrity.
[0042] Experimental results Compared with the model group, the intervention of active peptides significantly increased total mucin secretion, upregulated MUC2 expression, improved goblet cell function, and repaired the mucus barrier in a dose-dependent manner (P<0.05), as shown in Table 5.
[0043] Table 5. Effects of bioactive peptides on intestinal epithelial mucus barrier function indicators (mean ± SD, n=3) Group Cell viability (%) Total mucin secretion relative expression level of MUC2 mRNA relative expression level of MUC2 protein Goblet cell positivity rate (%) Blank control group 100.0±3.2 1.00±0.05 1.00±0.06 1.00±0.04 32.5±2.1 Model group 68.4±4.1 0.58±0.04 0.52±0.05 0.49±0.03 15.3±1.8 Low-dose group of active peptide 1 78.9±3.6 0.72±0.05 0.76±0.06 0.71±0.05 21.7±1.9 Medium dose group of active peptide 1 87.6±3.8 0.88±0.06 0.94±0.07 0.89±0.06 27.9±2.3 High-dose group of active peptide 1 94.2±3.1 1.05±0.07 1.18±0.08 1.12±0.07 33.8±2.5 Low-dose group of active peptide 2 77.2±3.5 0.70±0.05 0.73±0.06 0.69±0.05 21.0±1.8 Medium dose group of active peptide 2 85.9±3.7 0.85±0.06 0.90±0.07 0.86±0.06 27.1±2.2 High-dose group of active peptide 2 92.8±3.2 1.02±0.07 1.12±0.08 1.07±0.07 32.6±2.4 Low-dose group of active peptide 3 75.6±3.4 0.68±0.05 0.71±0.06 0.67±0.05 20.3±1.8 Medium dose group of active peptide 3 83.7±3.6 0.82±0.06 0.87±0.07 0.83±0.06 26.0±2.1 High-dose group of active peptide 3 90.9±3.1 0.98±0.07 1.08±0.08 1.03±0.07 31.4±2.3 Low-dose group of active peptide 4 74.1±3.3 0.66±0.05 0.69±0.06 0.65±0.05 19.6±1.7 Medium dose group of active peptide 4 82.0±3.5 0.79±0.06 0.84±0.07 0.80±0.06 24.9±2.0 High-dose group of active peptide 4 89.2±3.0 0.95±0.07 1.04±0.08 0.99±0.07 30.1±2.2 Low-dose group of active peptide 5 72.8±3.2 0.64±0.05 0.67±0.06 0.63±0.05 18.9±1.7 Medium dose group of active peptide 5 80.5±3.4 0.76±0.06 0.81±0.07 0.77±0.06 23.8±2.0 High-dose group of active peptide 5 87.6±2.9 0.92±0.07 1.00±0.08 0.95±0.07 28.9±2.1 Low-dose group of active peptide 6 71.2±3.1 0.62±0.05 0.65±0.06 0.61±0.05 18.1±1.6 Medium dose group of active peptide 6 79.0±3.3 0.73±0.06 0.78±0.07 0.74±0.06 22.7±1.9 High-dose group of active peptide 6 85.9±2.8 0.88±0.07 0.96±0.08 0.91±0.07 27.6±2.0 As shown in Table 5, compared with the blank control group, the model group exhibited significantly decreased cell viability, significantly reduced total mucin secretion, significantly downregulated MUC2 mRNA and protein expression levels, and significantly reduced goblet cell positivity rate under LPS / TNF-α induction (P<0.05), indicating significant damage to the intestinal epithelial mucus barrier function. After intervention with the active peptide, each dose group showed varying degrees of repair effects. The active peptide significantly improved cell viability, significantly promoted the recovery of total mucin secretion levels, upregulated MUC2 gene and protein expression, increased goblet cell positivity rate, restored secretory function, and thus alleviated inflammatory damage. Furthermore, the high-dose group approached or exceeded the levels of the blank control group in several indicators, indicating that the active peptide mainly exerts a protective effect at the mucus layer level by promoting mucin secretion and MUC2 expression, rather than simply achieving intestinal barrier repair through tight junction protein regulation.
[0044] Explanation of the structural characteristics and functional relationship of active peptides The amino acid sequences of the active peptides described in this invention are shown in SEQ ID NO: 1-6, specifically as follows: Peptide 1 (SEQ ID NO: 1): FLWRWTWTYKDQ; Peptide 2 (SEQ ID NO: 2): YRTHKFLGSWDA; Peptide 3 (SEQ ID NO: 3): KEERWLTGQTF; Peptide 4 (SEQ ID NO: 4): FLADYGTRWHS; Peptide 5 (SEQ ID NO: 5): RYFKLGWQTHA; Peptide 6 (SEQ ID NO: 6): YSWKYSHRQTA; The above-mentioned bioactive peptides are all oligopeptides with a molecular weight of less than 1 kDa, and they share the following common structural features: Rich in hydrophobic amino acids: Each sequence contains a high proportion of hydrophobic residues such as tryptophan (W), phenylalanine (F), tyrosine (Y), and leucine (L). The hydrophobic regions facilitate hydrophobic interactions between the bioactive peptide and receptors on the surface of intestinal epithelial cells or goblet cells, promoting cellular uptake and signal transduction, thereby upregulating MUC2 gene expression and mucin secretion.
[0045] It contains basic amino acid residues such as arginine (R), lysine (K), and histidine (H). These positively charged residues can electrostatically interact with negatively charged glycosylated regions (such as sialic acid residues) in mucin molecules in a neutral intestinal environment, enhancing the binding ability of active peptides to the mucus layer, prolonging their local action time, and possibly promoting goblet cell differentiation and secretory function by activating specific signaling pathways (such as the EGFR or ERK pathway).
[0046] Possessing specific sequence motifs: Some peptide segments (such as "WTWTY" in peptide 1, "KFLGSW" in peptide 2, and "YGTRW" in peptide 4) exhibit alternating aromatic-polar residue arrangements. This type of structure is conducive to forming β-turn or amphiphilic conformations, enhancing the stability of peptide molecules and their binding specificity to target proteins. In particular, the indole ring of tryptophan residues can interact with the core domain of mucin through π-π stacking, directly stabilizing the mucogel network.
[0047] Source Description: The above-mentioned bioactive peptides were obtained from duck skin proteins through targeted enzymatic digestion with a complex enzyme (neutral protease and flavor protease), ultrafiltration fractionation (<1 kDa), and functional screening using an intestinal epithelial mucus barrier damage model. Mass spectrometry identification results showed that these peptides originated from structural or glycoproteins in duck skin, and their unique sequences endow them with specific biological activities that target and regulate the mucus barrier.
[0048] Based on the results of Example 3, peptides 1-6 all dose-dependently increased total mucin secretion, upregulated MUC2 mRNA and protein expression, and increased goblet cell positivity. Peptides 1 and 2 showed the best effects at high doses (relative MUC2 mRNA expression levels of 1.18 and 1.12, respectively, and goblet cell positivity rates of 33.8% and 32.6%, respectively), consistent with their high hydrophobic amino acid content and aromatic residue density. Peptide 6 had relatively lower activity but was still significantly better than the model group. The above structure-activity relationship indicates that the active peptides of this invention mainly promote mucin secretion and goblet cell function synergistically through hydrophobic interactions and electrostatic binding, thereby directionally repairing the intestinal epithelial mucus barrier, rather than exerting their effects through non-specific anti-inflammatory or tight junction regulation.
[0049] In summary, this invention, based on targeted enzymatic hydrolysis and functional screening of duck skin protein, yielded a series of bioactive oligopeptides with well-defined structures and novel sequences. These bioactive peptides, through their unique hydrophobic / cationic amino acid composition and spatial conformation, effectively promote MUC2 expression and mucin secretion, restoring the integrity of the mucus layer. They provide ideal functional molecules for the high-value utilization of livestock and poultry by-products and the development of intestinal health products.
[0050] Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.
[0051] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.
Claims
1. Active peptide modulating the function of the mucus barrier of the intestinal epithelium, characterized in that, The amino acid sequences of the active peptides are shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.
2. The method for preparing an active peptide for regulating the function of intestinal epithelial mucus barrier according to claim 1, wherein, Includes the following steps: Step 1: Take duck skin, and after washing, defatting, cutting and homogenizing pretreatment, use ultrasonic-assisted extraction or enzyme-assisted extraction to obtain duck skin protein extract; Step 2: Adjust the pH of the duck skin protein extract to 7.5, add the compound enzyme for targeted enzymatic hydrolysis, heat to inactivate the enzyme after hydrolysis, centrifuge and collect the supernatant to obtain duck skin protein hydrolysate; Step 3: The duck skin protein hydrolysate is subjected to ultrafiltration fractionation to collect low molecular weight peptide components, which are then freeze-dried to obtain crude active peptide powder; the crude active peptide powder contains multiple candidate active peptides; Step 4: Apply multiple candidate active peptides to the intestinal epithelial mucus barrier function damage model, detect and compare the effects of each candidate active peptide on total mucin secretion, MUC2 gene or protein expression, and goblet cell secretion function, select peptides that perform well in the indicators, and obtain the target active peptide after separation and purification. The amino acid sequence of the target active peptide is shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.
3. The method for preparing the active peptide as described in claim 2, characterized in that, In step two, the enzymatic hydrolysis conditions are: pH 7.5, temperature 55℃, and hydrolysis time 3.5h.
4. The method for preparing the active peptide as described in claim 3, characterized in that, In step three, ultrafiltration fractionation is performed to retain components with a molecular weight cutoff of less than 1 kDa.
5. The method for preparing the active peptide as described in claim 2, characterized in that, The compound enzyme consists of a neutral protease and a flavor protease. The ratio of the compound enzyme to the duck skin protein extract is 1500 U / g, and the activity ratio of the neutral protease to the flavor protease is 1.5:
1.
6. The use of the active peptide as described in claim 1 or the active peptide prepared according to any one of claims 2 to 5 in the preparation of products for regulating the function of the intestinal epithelial mucus barrier.
7. The application as described in claim 6, characterized in that, Regulating the intestinal epithelial mucus barrier function includes improving mucin secretion levels, promoting MUC2 expression, maintaining goblet cell secretion function, or restoring the integrity of the mucus layer.
8. The application as described in claim 6, characterized in that, The product improves the mucus barrier function of the intestinal epithelium, which is damaged by inflammatory stimulation, oxidative stress, or pathogen-related factors, thereby blocking direct contact between pathogenic microorganisms and their toxins and epithelial cells.