Coated tannic acid for relieving escherichia coli induced intestinal injury and application of coated tannic acid

Abstract

The invention discloses coated tannic acid (ETA) for relieving Escherichia coli induced intestinal injury and application thereof.The coated tannic acid (ETA) is prepared by coating tannic acid with glycerin monostearate through a microcapsule technology, the prepared ETA is in the shape of creamy white oval or oblate particles with a complete structure, FTIR analysis proves that the spectrum of the ETA is the superposition characteristic of tannic acid (TA) and MG, and the Escherichia coli induced intestinal injury can be relieved. The structure can effectively avoid excessive digestion of gastric juice and directionally release TA in intestinal juice; animal experiments show that the ETA can effectively reverse ETEC-induced weight loss of mice, relieve red and swollen small intestines, improve the form of intestinal tissues and reduce the spleen index, and is non-toxic to the liver, the kidney and the heart; in gene and protein regulation and control, ETA can inhibit overexpression of inflammatory factors such as TNF-alpha and TLR4 / NF-KB signal channel key genes in intestinal tissues and up-regulate expression of tight junction proteins such as ZO-1 and nutrition transporter related genes such as SGLT1 at the same time. Therefore, the ETA provided by the invention can effectively relieve escherichia coli lipopolysaccharide induced intestinal injury, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to a feed additive and an application thereof, and relates to a coated tannic acid for alleviating intestinal damage induced by Escherichia coli and an application thereof. Background Art

[0002] The intestine is the body's first line of defense against external pathogens and toxins, and the integrity of its epithelial barrier is crucial for maintaining the body's health. Lipopolysaccharide (LPS), the main component of the cell wall of Gram-negative bacteria, is composed of three parts: lipid A, core polysaccharide, and O antigen. Lipid A, as the basic skeleton of LPS, is composed of phospholipids and fatty acids; O antigen is the main antigenic determinant of LPS, composed of repeating oligosaccharide units and has a high degree of diversity; core polysaccharide is located between O antigen and core lipid, connecting O antigen and core lipid, and has less antigenicity and diversity. Among them, lipid A is a conserved region responsible for the recognition of LPS by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). Numerous studies have demonstrated that LPS can specifically bind to toll-like receptor 4 (TLR4) on the surface of intestinal epithelial cells, activating the nuclear factor-κ-gene binding (NF-κB) signaling pathway and triggering the release of numerous proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin (IL-1β). This, in turn, damages the intestinal epithelial barrier, triggering an intestinal inflammatory response and leading to symptoms such as diarrhea, depression, and poor growth. Enterotoxigenic Escherichia coli (ETEC) is a common pathogen in animal production. ETEC colonizes the intestines and produces enterotoxins, disrupting the integrity of the intestinal epithelium and impairing intestinal barrier function. In pig farming, E. coli-induced intestinal damage can lead to reduced feed utilization, impaired growth performance, and reduced meat quality, posing a potential threat to human health through bioaccumulation.

[0003] Tannic acid (TA) is a widely used food preservative and feed additive with antioxidant, anti-inflammatory, hemostatic, and antidiarrheal properties. It is primarily digested, absorbed, and transported in the small intestine. Studies have shown that TA can inhibit the growth of harmful intestinal bacteria, enhance the immune response of piglets, improve intestinal barrier function, and promote the digestion and absorption of nutrients. However, due to its strong reducing properties, TA is unstable in the presence of gastric acid and is easily degraded, resulting in a reduced effective dose reaching the small intestine, thus limiting its effectiveness in the prevention and treatment of intestinal diseases.

[0004] To address these issues, various measures have been implemented to improve intestinal health and address E. coli-induced intestinal damage. For example, antibiotics are used to alleviate enteritis, but their overuse and misuse have led to the emergence of drug-resistant bacteria, posing a significant threat to public health. Consequently, many countries have banned the non-therapeutic use of antibiotics in animal feed. Other studies have attempted to develop alternative therapies, such as organic acids and probiotics, but some have limited effectiveness or safety concerns and have yet to fully meet practical needs.

[0005] Microencapsulation technology is a cutting-edge technique that encapsulates active ingredients within tiny capsules, effectively protecting them from environmental influences and enabling targeted release at specific locations. Monoglyceride (MG), a commonly used microencapsulation material, exhibits excellent biocompatibility, biodegradability, and cost-effectiveness. Microencapsulating TA to form encapsulated tannic acid (ETA) is expected to improve TA's stability in the gastrointestinal tract, achieve targeted release, and enhance its protective effects against intestinal damage, thereby enhancing its antibacterial and anti-inflammatory properties in the intestine. Current research aims to determine whether ETA is superior to TA in reducing cellular inflammation and damage to intestinal epithelial integrity, thereby providing a scientific basis for its application in production. However, research on ETA's ability to mitigate Escherichia coli-induced intestinal damage by inhibiting the NF-κB pathway is still under investigation, and its mechanism of action, optimal dosage, and efficacy require further in-depth study to enable its application in livestock farming and other fields to improve animal health and production efficiency. Summary of the Invention

[0006] Purpose of the invention: The purpose of the present invention is to provide an encapsulated tannic acid (ETA) capable of alleviating intestinal damage induced by Escherichia coli lipopolysaccharide and its application.

[0007] Technical solution: The coated tannic acid of the present invention is prepared by coating tannic acid with glyceryl monostearate using microencapsulation technology.

[0008] The coated tannic acid has full coated tannic acid particles, a smooth surface, a clear outline, and is oval or oblate, and is off-white.

[0009] The coated tannic acid has a particle size of 400-1600 μm.

[0010] The method for preparing the coated tannic acid comprises the following steps:

[0011] (1) Tannic acid, a binder and water are mixed, extruded into a shape, and sieved to obtain granules.

[0012] (2) Dissolving glyceryl monostearate in ethanol to obtain an encapsulation solution; preferably, the concentration of glyceryl monostearate in the encapsulation solution is 10 to 25 g / L.

[0013] (3) placing the particles obtained in step (1) in a fluidized bed coating machine and dynamically coating them with the encapsulation solution of step (2); drying to obtain the particles coated with tannic acid. Preferably, when the fluidized bed coating machine is dynamically coating, the inlet air temperature is 75±2°C, the outlet air temperature is 22±2°C; the atomizing pressure is 0.1-0.4 MPa, and the spraying rate is 0.05-0.15 L / min; more preferably, the fluidizing air volume is 10-30 m 3 / h.

[0014] In the preparation method of the coated tannic acid, the adhesive comprises starch, cyclodextrin, sodium carboxymethyl starch and lactose.

[0015] The coated tannic acid is used in the preparation of a medicine for preventing and / or treating intestinal damage induced by Escherichia coli.

[0016] The application and the medicine include human medicine and veterinary medicine.

[0017] The coated tannic acid is used in feed additives.

[0018] The application, the coated tannic acid is used as a feed additive to prevent and / or treat intestinal damage induced by Escherichia coli.

[0019] In the application, the Escherichia coli includes enterotoxigenic Escherichia coli.

[0020] Furthermore, to achieve the objectives of the invention, ETA was first prepared by coating TA with MG. ETA was characterized and analyzed using optical microscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy (FTIR). In vitro simulated digestion experiments were conducted to determine its release characteristics in simulated porcine gastric fluid (SGF) and simulated porcine intestinal fluid (SIF). In a cellular assay, porcine intestinal epithelial cell line J2 (IPEC-J2) was treated with ETA digestion supernatant at varying concentrations. The effect on cell viability was evaluated to determine an optimal concentration. This concentration was then used to treat ETEC-induced inflammation cells. Gene expression was measured to assess the effects of ETA on cellular inflammation and barrier function. Oxidative stress-related markers were also measured in cells from the different treatment groups to explore the effects of TA and ETA on oxidative damage. In terms of animal experiments, an ETEC-stimulated ICR mouse enteritis model was constructed, and the mice were divided into groups for treatment. Changes in body weight and organ indexes were observed, the small intestine was morphologically analyzed, and the expression of related genes and proteins in the small intestinal tissue was detected. At the same time, oxidative stress-related indicators in the serum and intestine of mice in different treatment groups were detected to explore the effects of TA and ETA on the oxidative stress status of mice and verify the improvement effect of ETA on E. coli-induced intestinal inflammation and barrier function damage in mice.

[0021] The ETA provided by the present invention has the following characteristics: relatively complete structure, plump particles, smooth surface, clear outline, oval or oblate shape, uniform off-white particles, and the main particles are concentrated around 800-1000 mm. FTIR confirmed that its spectrum is a superposition of TA and MG. In simulated digestive fluid, the release of ETA in SGF was very low in the first two hours, but after the addition of SIF, the TA concentration in the solution increased rapidly, indicating that ETA can effectively avoid excessive digestion in gastric juice and achieve targeted decomposition and release of TA in intestinal fluid. Animal experiments have shown that ETA can significantly improve intestinal morphology. After ICR mice with ETEC-induced inflammation were fed a feed containing ETA, the mice's weight loss trend was reversed, the redness and swelling of the small intestine were significantly alleviated, and there was no toxicity to the liver, kidneys, and heart. At the gene and protein regulatory levels, ETA effectively inhibited the mRNA and protein expression levels of inflammatory factors TNF-α, IL-1β, interleukin-8 (IL-8) and key genes of the NF-κ signaling pathway, including TLR4, myeloid differentiation primary response 88 (MYD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), and transforming growth factor-β-activated kinase 1 (TAK1) in intestinal tissue, while upregulating the tight junction proteins zonula occludens-1 (ZO-1), occludins (OCLN), claudin-1 (CLDN1), claudin-3 (CLDN3), and the nutrient transporter sodium-glucose co-transporter 1 (Sodium-glucose co-transporter 1). It can inhibit the expression of genes related to ETA (e.g., SGLT1), excitatory amino acid transporter 1 (EAAC1), and alanine-serine-cysteine transporter 2 (ASCT2), thereby effectively reducing intestinal inflammation, protecting the intestinal barrier, and promoting nutrient transport. Molecular mechanism studies have shown that TA, as a core component of ETA, strongly interacts with the nuclear factor erythroid 2-related factor 2 (NRF2) signaling complex.ETA treatment significantly upregulated the mRNA and protein expression levels of NRF2, heme oxygenase-1 (HO-1), superoxide dismutase (SOD), and catalase (CAT), activating the NRF2 signaling pathway. The significantly increased expression of SOD and CAT, key antioxidant enzymes, enhanced the antioxidant capacity of intestinal cells and reduced intestinal damage caused by oxidative stress. Dose-effect studies demonstrated that 500 mg / kg of ETA had the best overall effect, significantly reversing weight loss, restoring intestinal morphology, and optimizing the expression of inflammatory factors, tight junction proteins, and antioxidant genes. Compared with uncoated TA and other doses, it was more effective in maintaining intestinal health and enhancing antioxidant capacity. IPEC-J2 cell and mouse studies demonstrated that SOD activity in the ETA-treated group was higher than that in the uncoated TA-treated group, while the increase in malondialdehyde (MDA) content was significantly lower, confirming that ETA is more efficient in scavenging free radicals and reducing intestinal damage caused by oxidative stress. This shows that compared with uncoated TA, ETA plays a more active role and has better results in maintaining pig intestinal health and improving antioxidant performance.

[0022] The present invention addresses the problem that LPS, a component of the cell wall of Gram-negative bacteria, activates the NF-KB pathway of pig intestinal epithelial cells, induces the release of inflammatory factors such as TNF-α and IL-1β, and destroys the intestinal barrier. TA with antibacterial, anti-inflammatory and antioxidant activities is used to alleviate intestinal inflammation. However, TA is easily decomposed and destroyed by gastric acid in the gastric juice environment, resulting in a significant decrease in its effective concentration in the intestine, which restricts the full exertion of its function.

[0023] The present invention uses microencapsulation technology, using MG as the coating material to encapsulate TA to form ETA. This technology can effectively prevent TA from being overdigested in gastric juice, ensuring its precise release and effectiveness in the intestine.

[0024] Morphological images of ETA particles were captured using an optical microscope. Surface features of ETA and TA particles treated with gold ion spraying were observed using a scanning electron microscope, and particle size distribution was determined using measurement software. The particles were then ground into a powder, and the ETA was characterized using Fourier transform infrared spectroscopy.

[0025] The present invention verifies the performance and mechanism of ETA through in vitro release tests, cell tests, and animal tests. In the in vitro release test, SGF with a pH of 3.0 and SIF with a pH of 7.0 were prepared. 0.1g of ETA was placed in a centrifuge tube for preheating. 5mL of SGF was first added. The supernatant was removed and the TA concentration was tested at 30 minutes, 60 minutes, 90 minutes, and 120 minutes. After 2 hours, 5mL of SIF was added and the pH was adjusted to 7.0 to continue digestion. The supernatant was removed at 180 minutes, 240 minutes, 300 minutes, and 360 minutes. The absorbance was measured at 225nm using an ultraviolet spectrophotometer. An in vitro digestion release curve was drawn based on the TA content, and the digestibility was calculated to verify the release characteristics of ETA in a simulated gastrointestinal environment and the effect of microcapsule preparation.

[0026] In the cell experiment, after IPEC-J2 cells were cultured and passaged, TA and ETA were subjected to simulated gastrointestinal digestion (digested in SGF for 2 hours and then transferred to SIF for further digestion for 4 hours). The ETA digestion supernatant was diluted to concentrations of 0.2%, 0.4%, 0.6%, 0.8% and 1.0%. After preliminary screening, 0.6% was selected as the subsequent test concentration and compared with the concentration of 1×10 6 CFU / mL of ETEC were co-treated with IPEC-J2 cells for 24 hours to establish an in vitro inflammation and barrier damage model; IPEC-J2 cells were cultured at 5×10 3 Cells were seeded at a density of 100 cells / well in 96-well plates. After treatment, CCK-8 reagent was added to detect cell viability. qPCR was used to detect the mRNA expression levels of inflammatory factors (TNF-α, IL-1β, and IL-8), tight junction protein-related genes (ZO-1, OCLN, CLDN1, and CLDN3), and key genes in the NF-KB pathway (TLR4, MYD88, TRAF6, and TAK1). Western blot was used to detect related protein expression. Immunofluorescence staining was used to visualize p-p65 nuclear translocation and the distribution of ZO-1 and OCLN proteins. The regulatory effect of ETA on NRF2 and its downstream genes (HO-1, SOD, and CAT) was verified. Intracellular SOD activity and MDA content were measured using biochemical detection kits to analyze the mechanism of ETA's effect on intestinal epithelial cell inflammation, barrier function, and oxidative damage.

[0027] The animal experiment was divided into two parts. In the ETEC-induced mouse enteritis experiment, 32 ICR mice were randomly divided into a control group (CON), an ETEC model group, an ETEC+TA treatment group (8 g / kg TA), and an ETEC+ETA treatment group (equivalent ETA). After 8 days of dietary intervention, the ETEC model group and the treatment group were intraperitoneally injected with 10 mg / kg body weight of ETEC, and the CON group was injected with normal saline. After 24 hours, the mice were weighed, and heart, liver, spleen, and kidney tissues were collected and weighed. The small intestine was resected and photographed, and the intestinal tissue was fixed in 4% paraformaldehyde for H&E staining to evaluate intestinal morphology, measure villus height and crypt depth, and the jejunum tissue was frozen in liquid nitrogen for molecular biology research to detect lactate dehydrogenase (LDH), alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN). Serum biochemical indicators of nitrogen (BUN) and creatinine (CRE) and oxidative stress indicators (SOD activity, MDA content) were analyzed by qPCR and western blot. Blot technology was used to detect the expression of inflammatory factors, tight junction protein-related genes, key genes of the NF-KB pathway, and nutrient transport-related genes (SGLT1, EAAC1, and ASCT2) in small intestinal tissue. Molecular docking technology was used to analyze the interaction between TA and the NRF2 signaling complex, verifying the regulatory effect of ETA on NRF2 and its downstream genes (HO-1, SOD, and CAT). In the dose-effect relationship experiment of ETA, 40 mice were randomly divided into CON group, ETEC group, and ETEC+ETA low / medium / high dose groups (250 / 500 / 750 mg / kg). After 8 days of dietary intervention, the ETEC group and the ETA-treated group were intraperitoneally injected with 10 mg / kg body weight of ETEC, and the CON group was injected with normal saline. After 24 hours, the mice were weighed and their organs were collected. qPCR technology was used to detect the mRNA expression levels of inflammatory factors, tight junction protein-related genes, key genes of the NF-KB pathway, and downstream genes of the NRF2 pathway in small intestinal tissue after intervention with different doses of ETA to explore the dose-effect relationship of ETA.

[0028] The present invention achieves intestinal targeted release of tannic acid through microencapsulation technology, effectively solving the problem of its easy decomposition in gastric juice. Cell and animal experiments have verified the significant effects of ETA in improving intestinal inflammation, repairing the intestinal barrier and regulating oxidative stress, providing a safe and effective solution for maintaining animal intestinal health.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the ETA prepared by the present invention is in the form of off-white oval or oblate particles with complete structure and uniform size. FTIR confirms that its spectrum is a superposition of TA and MG. The release amount in simulated gastric fluid is low in the first 2 hours, and the TA release amount increases sharply after adding simulated intestinal fluid, which can effectively avoid excessive digestion of gastric fluid and release TA in a targeted manner in intestinal fluid. Animal experiments show that ETA can reverse ETEC-induced weight loss in mice, relieve small intestinal congestion, improve intestinal morphology, reduce spleen index and has no toxicity to the liver, kidney and heart. In terms of gene and protein regulation, ETA can inhibit the expression of TNF-α and IL- It inhibits the expression of inflammatory factors such as IL-1β and IL-8, and key genes of NF-KB pathway such as TLR4, MYD88, TRAF6, and TAK1, and upregulates the expression of tight junction proteins such as ZO-1, OCLN, CLDN1, and CLDN3, and nutrient transporter-related genes such as SGLT1, EAAC1, and ASCT2. In terms of molecular mechanism, TA, the core component of ETA, interacts strongly with the NRF2 signaling complex, upregulates the expression of NRF2, HO-1, SOD, and CAT, activates the antioxidant pathway, increases SOD activity, reduces MDA content, and enhances antioxidant capacity. Dose-effect studies have shown that 500 mg / kg ETA has the best comprehensive effect, which can significantly reverse weight loss, repair intestinal morphology, and optimize the expression of inflammatory factors, tight junction proteins, and antioxidant genes. It is better than uncoated TA and other doses in maintaining intestinal health and improving antioxidant performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Microscope images of lipid particles and schematic diagram of Fourier transform infrared spectroscopy (FTIR) characterization;

[0031] Figure 2 is the in vitro digestion release curve of ETA;

[0032] Figure 3 Flowchart of experimental design for the ETEC-induced IPEC-J2 cell inflammation and barrier damage model;

[0033] Figure 4 This is a graph showing the effects of different concentrations of ETA digestion solutions on IPEC-J2 cell viability;

[0034] Figure 5 The figure shows the detection of mRNA and protein expression levels of inflammatory factors (TNF-α, IL-1β and IL-8) in IPEC-J2 cells;

[0035] Figure 6 Detection of mRNA and protein expression levels of genes related to the NF-KB signaling pathway (TLR4, MYD88, TRAF6, and TAK1) in IPEC-J2 cells;

[0036] Figure 7 This is an immunofluorescence staining image of the nuclear localization of p-p65 protein in IPEC-J2 cells;

[0037] Figure 8 Detection of mRNA and protein expression levels of tight junction proteins (ZO-1, OCLN, CLDN1, and CLDN3) in IPEC-J2 cells;

[0038] Figure 9 Immunofluorescence staining images of the distribution of ZO-1 and OCLN proteins in IPEC-J2 cells (scale bar: 50 μm);

[0039] Figure 10 The graph shows the mRNA expression levels of antioxidant-related genes (NRF2, HO-1, SOD, and CAT) and enzyme activities (SOD and MDA) in IPEC-J2 cells;

[0040] Figure 11 Flowchart for experimental design of ETEC-induced enteritis model in mice;

[0041] Figure 12 Schematic diagram of statistical detection of body weight changes and organ weight index of mice in different treatment groups;

[0042] Figure 13 Schematic diagram of the observation of small intestine morphology of mice in different treatment groups;

[0043] Figure 14 H&E staining images of the duodenal tissue morphology of mice in different treatment groups and schematic diagrams of the measurement of villus height (VH) and crypt depth (CD);

[0044] Figure 15 H&E staining images of jejunum tissue morphology of mice in different treatment groups and schematic diagram of VH and CD measurements;

[0045] Figure 16 H&E staining images of ileum tissue morphology of mice in different treatment groups and schematic diagram of VH and CD measurements;

[0046] Figure 17 Schematic diagram of H&E staining observation of mouse heart, liver, and kidney tissues;

[0047] Figure 18 Schematic diagram of the detection of serum biochemical indicators (LDH, ALT, AST, BUN and CRE) in mice treated with ETA;

[0048] Figure 19 The figure shows the detection of mRNA and protein expression levels of inflammatory factors (TNF-α, IL-1β and IL-8) in mouse intestinal tissue;

[0049] Figure 20 This is a graph showing the mRNA and protein expression levels of genes related to the NF-κB signaling pathway (TLR4, MYD88, TRAF6, and TAK1) in the mouse intestine;

[0050] Figure 21 The figure shows the detection of mRNA and protein expression levels of tight junction proteins (ZO-1, OCLN, CLDN1 and CLDN3) in the mouse intestine;

[0051] Figure 22 This is a graph showing the mRNA expression levels of antioxidant-related genes (NRF2, HO-1, SOD, and CAT) in the mouse intestine;

[0052] Figure 23 This is a graph showing the mRNA expression level of mouse intestinal nutrient transporter genes (SGLT1, EAAC1, and ASCT2);

[0053] Figure 24 Schematic diagram of the detection of SOD activity and MDA content in the serum and intestinal tissues of mice in different treatment groups;

[0054] Figure 25 Flow chart of experimental design for ETA dose-effect relationship study;

[0055] Figure 26 Schematic diagram of the effect of different doses of ETA on the body weight and organ weight index of mice;

[0056] Figure 27 Schematic diagram of the observation of small intestine morphology of mice in groups treated with different doses of ETA;

[0057] Figure 28 The graph shows the mRNA expression levels of intestinal inflammatory factors (TNF-α, IL-1β and IL-8) in mice treated with different doses of ETA;

[0058] Figure 29 The graph shows the mRNA expression levels of intestinal tight junction protein genes (ZO-1, OCLN, CLDN1 and CLDN3) in mice treated with different doses of ETA;

[0059] Figure 30 The graph shows the mRNA expression levels of antioxidant-related genes (NRF2, HO-1, SOD, and CAT) in the intestine of mice treated with different doses of ETA;

[0060] Figure 31This is a schematic diagram of the process by which ETA inhibits the Gram-negative bacterial cell wall component LPS from activating the NF-κB pathway in intestinal epithelial cells, promoting the release of inflammatory factors such as TNF-α and IL-1β, and inhibiting the release of tight junction proteins such as ZO-1 and OCLN, thereby destroying the intestinal barrier. DETAILED DESCRIPTION

[0061] Example 1: Preparation of ETA

[0062] Accurately weigh 600g of tannic acid (TA, purity ≥99%) and 400g of corn starch, add 250mL of distilled water (water-to-solid ratio 1:4), and mix using an IKAT25 digital disperser at room temperature (22±2°C) for 15 minutes (2000rpm). Transfer the mixture to a GHL-6 laboratory spherical granulator, control the extrusion pressure at 0.5MPa, and granulate through a 30-mesh standard sieve (pore size 600μm) to obtain granules for use. Accurately weigh 300g of glyceryl monostearate (MG) and dissolve it in 18L of ethanol to obtain an encapsulation solution. Weigh 700g of the granules obtained in the previous step and place them in a FLUIZ-5 laboratory fluidized bed coater for dynamic coating with the encapsulation solution. Parameters are: inlet air temperature 75±2°C, outlet air temperature 22±2°C, atomization pressure 0.2MPa, spray rate 0.1L / min, and fluidizing air volume 20m3 / s. 3 / h, and the coating time was controlled at 210±10min. After the coating was completed, fluidized drying was continued for 0.5h to finally obtain ETA granules, which were sealed and stored in a desiccator at 25°C (relative humidity ≤30%).

[0063] Example 2: Characterization and in vitro release test of ETA

[0064] 1. ETA Observation

[0065] An optical microscope was used to observe the color, morphology and particle size of ETA and collect images. The conductive glue was pasted on the electron microscope sample stage, and a thin layer of TA microparticle sample and ETA microparticle sample were evenly spread on the conductive glue. The two particles were sprayed with gold ions, and the floating powder was blown off after 30 seconds to give them conductivity. Subsequently, the surface characteristics of the lipid particles were observed with the help of a scanning electron microscope. The image was manually measured using Image Pro Plus software to obtain the particle diameter, and the particle size distribution image was drawn using GraphPad Prism 8.0 software. Afterwards, the particles were ground into powder and analyzed using a Nicolet iS5 Fourier transform infrared spectrometer at 400-4000cm -1 Wavenumber range, spectral resolution 4cm -1 Thermo Scientific OMNIC software was used to identify and compare characteristic peaks.

[0066] 2. In vitro release test of ETA

[0067] (1) Configuration of SGF and SIF

[0068] The configuration standards are shown in Table 1. The pH of SGF and SIF were adjusted to 3.0 or 7.0 respectively.

[0069] Table 1 Composition of SGF and SIF

[0070] Table 1 The components of SGF and SIF

[0071]

[0072] (2) In vitro release test

[0073] 0.1 g of ETA was placed in a 15 mL centrifuge tube and preheated in a 37°C waterbath. The tube was then shaken on a shaker at the desired frequency to simulate the gastrointestinal environment. To test ETA release in SGF, 5 mL of SGF was added to the preheated tube. Appropriate amounts of the digestion supernatant were collected at 30, 60, 90, and 120 minutes. After 2 hours, 5 mL of SIF was added and the pH was adjusted to 7.0. Digestion was continued for 4 hours to test ETA release in SIF. Appropriate amounts of the digestion supernatant were collected at 180, 240, 300, and 360 minutes. TA concentration was measured using a spectrophotometer at a wavelength of 225 nm. The in vitro release spectroscopy assay was repeated three times.

[0074] Take seven 2mL EP tubes, add TA standard stock solution and water to each tube, shake and mix thoroughly, then add 0.2mL of ammonia solution to each tube. Measure the absorbance at 225nm using a microplate reader to construct a TA standard curve. Plot the curve using the TA content (mg / mL) in the TA standard solution series as the horizontal axis and the measured absorbance as the vertical axis. Place 0.2mL of TA supernatant from digestion at different time points into a 2mL EP tube. Pipette 200μL of the mixed solution into a 96-well plate, let it stand, and measure the absorbance at 225nm using a UV spectrophotometer. If samples are measured on different days than the standard curve, a blank control should be included when testing the samples. TA content is measured using a spectrophotometer. Based on the TA content at different time points, plot the in vitro digestion and release curve of ETA and calculate the digestibility.

[0075] 3. Implementation Results

[0076] (1) Characterization of ETA

[0077] Under a microscope, TA and ETA have significant differences in appearance. TA is yellow or brown, with an uneven surface covered with small and dense holes. The particle size and shape are irregular, forming an irregular block structure; ETA is off-white, with a complete structure, full particles, a smooth surface without depressions, no impurities, clear outlines, and is oval or oblate, and uniform in size (see Figure 1 -A). The particle size distribution of ETA particles shows that the main particles are concentrated in 800-1000μm (median particle size), a small number of large particles are close to 1600μm, and a small number of small particles are about 400μm (boundary particle size) (see Figure 1 -B). FTIR analysis showed that the characteristic spectrum of ETA was the superposition of TA and monoglyceride spectra (see Figure 1 -C).

[0078] (2) Release of ETA in simulated gastric and intestinal fluids

[0079] Within the specified time, the amount of ETA released in SGF was extremely low; after adding SIF, the amount of TA released from ETA increased sharply within only 30 minutes, and the TA concentration in the solution increased rapidly. This result shows that using MG to coat TA can effectively prevent it from being over-digested in gastric juice, and achieve decomposition and release of TA mainly in intestinal fluid (see Figure 2 ).

[0080] Example 3: ETA alleviates ETEC-induced IPEC-J2

[0081] The schematic diagram of ETA inhibiting LPS from activating the NF-KB pathway in intestinal epithelial cells, promoting the release of inflammatory factors such as TNF-α and IL-1β, and inhibiting the release of tight junction proteins such as ZO-1 and OCLN, thereby destroying the intestinal barrier is shown in Figure 31 We conducted the following targeted research:

[0082] 1. Cell culture

[0083] IPEC-J2 cells were seeded in T25 culture flasks containing DMEM complete medium (supplemented with 10% fetal bovine serum and 1% double antibody) at a density of 5×10 4 Culture cells at a density of 10 cells / flask until near confluence (approximately 2 days) and then passage. During passage, trypsinize cells until the intercellular spaces are wide but not detached. Immediately add complete medium containing serum to terminate digestion. Gently pipette the cells to create a suspension, avoiding air bubbles. Centrifuge at 1000 rpm for 3-5 minutes and discard the supernatant. Resuspend the cells in fresh DMEM medium and inoculate them into new culture flasks at a 1:2 ratio.

[0084] 2. Construction of in vitro inflammation and barrier damage model

[0085] TA and ETA solids were subjected to simulated gastrointestinal digestion: first digested in SGF for 2 hours, then transferred to SIF for further digestion for 4 hours. The ETA digest supernatant was diluted to a concentration gradient of 0.2%, 0.4%, 0.6%, 0.8% and 1.0%, and incubated with IPEC-J2 cells for 24 hours to screen the optimal concentration. Based on the results of the preliminary test, 0.6% was selected as the subsequent test concentration. The TA and ETA digestion solutions were diluted to this concentration and mixed with 1×10 6 IPEC-J2 cells were co-treated with ETEC at 500 CFU / mL for 24 hours to establish an in vitro inflammation and barrier damage model.

[0086] 3. CCK-8 assay for cell viability

[0087] IPEC-J2 cells were cultured at 5 × 10 3 Cells were seeded at a density of 100 μl / well in a 96-well plate. After complete attachment, cells were treated with various concentrations of ETA digestion solution (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) or co-treated with ETEC solution (10 μL / mL). After 24 hours of incubation, the culture medium containing CCK-8 reagent was replaced and incubation continued for another 3 hours. Absorbance at 450 nm was measured using a microplate reader. The test was repeated three times.

[0088] 4. qPCR detection of gene mRNA expression

[0089] IPEC-J2 cells were seeded on 12-well plates and cultured to 80% confluency, then treated with ETA digestion solution and ETEC. After 24 hours of incubation, TRIzol TM Total RNA was extracted with a 500 μg PCR agarose gel electrophoresis kit (ELISA) and the RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer. 1 μg of total RNA was reverse transcribed into cDNA and qPCR was performed on an ABIPRISM 7300HT system using TransStart Green qPCR SuperMix to detect the mRNA expression levels of genes such as TNF-α, IL-1β, IL-8, ZO-1, OCLN, CLDN1, CLDN3, TLR4, MYD88, TRAF6, and TAK1. β-actin was used as an internal reference and 2 -ΔΔCT The relative expression levels were analyzed by the PCR method, and the experiment was repeated three times. The primer sequences are shown in Table 2 (designed by Primer 5.0 software).

[0090] Table 2 Primers used for porcine qPCR analysis

[0091] Table 2 Primers Used for qPCR Analysis in Pigs

[0092]

[0093]

[0094] 5. Western blot detection of protein expression

[0095] ETA and TA solids (equal amounts of TA) were digested with SGF for 2 hours and SIF for 4 hours, and the supernatant was diluted to 1% for later use. IPEC-J2 cells were seeded in culture dishes and cultured to 80% confluency. They were then divided into four groups: CON, ETEC, ETEC + TA, and ETEC + ETA. Each group was treated for 24 hours. Total protein was extracted using RIPA buffer, and protein concentration was determined by the BCA assay. Equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.6) for 2 hours at room temperature. Primary antibodies (TNF-α, IL-8, IL-1β, claudin-3, TLR4, IKBα, 1:200; ZO-1, occludin, 1:1000; claudin-1, 1:500; NRF2, p65, p-p65, 1:2000; β-actin, 1:1000) were then added and incubated overnight at 4°C. The next day, HRP-conjugated secondary antibodies (1:2000) were added and incubated at 37°C for 1 hour. After washing with TBST, signals were detected using a Bio-Rad Gel Doc 2000 system, and band intensity was analyzed with ImageJ software.

[0096] 6. Immunofluorescence staining to detect intracellular protein localization

[0097] IPEC-J2 cells were seeded onto culture plates and, after reaching full confluence, treated for 24 hours according to the CON, ETEC, ETEC+ETA, and ETEC+TA groups. They were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.25% Triton X-100 for 15 minutes at room temperature. Primary antibodies (ZO-1, Occludin, NRF2, 1:100; p-p65, 1:200) were added and incubated overnight at 4°C. Alexa Fluor 488 / 584-conjugated secondary antibodies were then added (1:200) and incubated at 37°C for 1 hour. After washing with PBS, cell nuclei were stained with DAPI. Immunofluorescence images were acquired using the Invitrogen EVOS FL automated cell imaging system.

[0098] 7. Oxidative stress index detection

[0099] Biochemical detection kits were used to quantitatively measure the intracellular SOD activity and MDA content to evaluate the cellular oxidative stress level and antioxidant capacity.

[0100] 8. Implementation Results

[0101] (1) IPEC-J2 cell culture and model construction

[0102] After IPEC-J2 cells were expanded through standardized culture procedures, an in vitro model of ETEC-induced inflammation and barrier damage was successfully established (see Figure 3 ), providing a stable cell carrier for subsequent mechanism studies.

[0103] (2) Analysis of the dose effect of ETA on cell activity

[0104] ETA digestion solutions at concentrations of 0.2%, 0.4%, 0.6% and 0.8% had no significant effect on cell viability, while the cell viability of the 1.0% concentration group was significantly reduced compared with the control group (see Figure 4 -A), suggesting that high concentrations may be potentially toxic. In the ETEC injury model, the above concentrations of ETA digestive fluid can significantly improve the viability of damaged cells (see Figure 4 -B), where 0.6% concentration showed the best protective effect, combining both safety and effectiveness.

[0105] (3) ETA regulates ETEC-induced inflammatory response and barrier function

[0106] ① Regulation of inflammatory factor expression

[0107] Compared with the ETEC group, all concentrations of ETA digestive fluid significantly downregulated the mRNA expression levels of TNF-α, IL-1β, and IL-8, and the ETA group showed a better inhibitory effect than the TA group (see Figure 5 -A). Western blot results showed that ETA could continuously reduce the protein expression of the above inflammatory factors (see Figure 5 -B, 5-C), confirming its ability to inhibit inflammatory responses at both the transcriptional and translational levels.

[0108] ②Intervention of key genes in the NF-κB pathway

[0109] ETEC treatment alone significantly upregulated the mRNA expression of TLR4, MYD88, TRAF6, TAK1 and other pathway genes (see Figure 6 -A), while both ETA and TA groups could significantly reverse this trend. Western blot detected consistent downregulation of protein expression levels, and the ETA group showed a better inhibitory effect than the TA group (see Figure 6-B, 6-C). In addition, immunofluorescence staining showed that ETA effectively inhibited the nuclear accumulation of p-p65 protein (see Figure 7 ), revealing its molecular mechanism of alleviating inflammation by blocking the activation of the NF-κB pathway.

[0110] ③Tight junction protein repair effect

[0111] Compared with the ETEC group, ETA treatment significantly upregulated the mRNA expression of tight junction genes such as ZO-1, OCLN, CLDN1, and CLDN3, and showed stronger repair ability than the TA group (see Figure 8 -A). Protein level detection showed that ETA could significantly increase the expression of ZO-1, OCLN and CLDN3 (see Figure 8 -B, 8-C). Immunofluorescence imaging showed that ETEC caused the structural disorder of ZO-1 and OCLN cytoskeleton, however, both TA and ETA treatments effectively alleviated this damage, and ETA showed better efficacy compared with TA (see Figure 9 -A, 9-B), indicating its efficient repair effect on intestinal barrier function.

[0112] (4) Bidirectional regulatory mechanism of ETA on oxidative stress

[0113] Analysis of key genes in the NRF2 signaling pathway showed that both TA and ETA treatments significantly upregulated the mRNA levels of NRF2, HO-1, SOD, and CAT compared with the ETEC group (see Figure 10 -A).

[0114] Antioxidant enzyme activity detection showed that the SOD activity in the ETEC group was significantly reduced, while that in the ETA group was restored to the level of the control group and was better than that in the TA treatment (see Figure 10 -B); The lipid peroxidation index MDA decreased significantly in the ETA group, approaching the normal physiological level (see Figure 10 These results indicate that ETA can effectively reverse ETEC-induced oxidative damage by activating the NRF2 pathway and enhancing the activity of the antioxidant enzyme system.

[0115] Example 4: Alleviating effect of ETA on ETEC-induced intestinal epithelial cell inflammation in mice

[0116] 1. Implement animal and group design

[0117] To establish an ETEC-induced enteritis model in ICR mice, 32 mice were randomly divided into four groups (n=8): CON, ETEC, ETEC+TA, and ETEC+ETA. The CON and ETEC groups were fed a standard diet, while the ETEC+TA group received a diet supplemented with 8g / kg of TA, and the ETEC+ETA group received a diet containing an equal dose of TA and ETA. After 7 days of dietary intervention, ETEC (10mg / kg body weight) was intraperitoneally injected in the CON group, except for the CON group, which received saline. Twenty-four hours after injection, body weights were measured, and tissue samples (heart, liver, spleen, kidney, and small intestine) were collected and fixed (4% paraformaldehyde for histological analysis and liquid nitrogen-frozen jejunum for molecular biology studies).

[0118] In a dose-response study, 40 mice were randomly divided into five groups (n=8): a CON group, an ETEC model group, and ETEC+ETA low-, medium-, and high-dose groups (ETA supplemented in the diet at 250 mg / kg, 500 mg / kg, and 750 mg / kg, respectively). After 8 days of dietary intervention, the ETEC and ETA groups were injected with ETEC, while the CON group was injected with saline. Sampling procedures were consistent with those previously described, focusing on dose-dependent changes in small intestinal morphology and gene expression.

[0119] 2. Construction and evaluation of ETEC-induced enteritis model in mice

[0120] (1) Morphological observation of small intestine

[0121] The small intestines of mice were fixed and the four most representative groups of small intestines were selected to compare the morphology of the small intestines of the three groups of mice and the blank control group.

[0122] (2) H&E staining and histological analysis

[0123] Duodenum, jejunum, and ileum tissue (1-2 cm) isolated from different groups of mice were fixed in pre-cooled 4% paraformaldehyde for 24 hours, dehydrated in 25% and then 30% sucrose solutions, embedded in paraffin, and sectioned at 4 μm. The sections were then dewaxed and stained with hematoxylin-eosin (hematoxylin stains cell nuclei blue and eosin stains cytoplasm red), then dehydrated and mounted. The prepared small intestinal tissue sections were photographed using Nikon microscope imaging software to observe the villus height (VH) and crypt depth (CD) of the small intestine of the different groups of mice.

[0124] The heart, liver, and kidney tissues of the CON and ETA groups were fixed, stained with H&E, and observed and photographed under a microscope.

[0125] (3) Serum biochemical index detection

[0126] Blood was collected from the retroorbital venous plexus to detect liver and kidney function markers such as LDH, ALT, AST, BUN and CRE.

[0127] (4) Assessment of oxidative stress status

[0128] Biochemical detection kits were used to determine the SOD activity and MDA content in the serum and intestinal tissues of mice in different groups to analyze the oxidative stress status of mice induced by ETEC.

[0129] (5) qPCR analysis of inflammation and barrier-related gene expression

[0130] The qPCR method for detecting related gene expression has been described in detail above (see Example 3). This method was used to evaluate the mRNA expression levels of TNF-α, IL-1β, IL-8, ZO-1, OCLN, CLDN1, CLDN3, TLR4, MYD88, TRAF6, TAK1, HO-1, SOD, and CAT. The primer sequences are shown in Table 3 below. β-actin was used as the housekeeping gene as the internal reference, and 2 -ΔΔCT Methods Relative gene expression was analyzed.

[0131] Table 3 Primers used for mouse qPCR analysis

[0132] Table 3 Primers Used for qPCR Analysis in Mice

[0133]

[0134]

[0135] (6) Western blot verification of protein expression

[0136] The protein expression detection method of Western blot has been described in detail above (see Example 3). This method was used to detect the protein expression of the above genes.

[0137] (7) Molecular docking verification of the interaction between TA and NRF2 signaling complex

[0138] The structure of the NRF2 signaling complex (PDB ID: 7K2F) was obtained from the Protein Data Bank (PDB), and a 3D model of TA was constructed using Chem3D. The protein was preprocessed using PyMOL (to remove solvent and ligands), and molecular docking was performed using the AutoDock tool and AutoDockVina to calculate the binding energy between TA and the NRF2 signaling complex.

[0139] 3. Explore the dose-effect relationship of ETA

[0140] (1) Morphological observation of small intestine

[0141] The small intestines of mice were fixed and the five most representative groups of small intestines were selected to compare the morphology of the small intestines of the four groups of mice and the blank control group.

[0142] (5) qPCR analysis of inflammation and barrier-related gene expression

[0143] Following the process described in Example 3, the mRNA expression levels of inflammation-related genes such as TNF-a, IL-1β, IL-8 and barrier function genes such as ZO-1, OCLN, CLDN1, CLDN3, as well as antioxidant genes such as HO-1, SOD and CAT were evaluated. All primers were designed using Primer 5.0 software (sequences are shown in Table 3 above), with β-actin as the internal reference gene, and 2 -ΔΔCT The relative expression level was analyzed by the method to achieve quantitative analysis of gene transcription level.

[0144] 4. Implementation Results

[0145] (1) ETEC-induced enteritis model in mice

[0146] ① Growth status and organ index changes

[0147] Mice were divided into groups and experimental design (see Figure 11 Compared with the control group, ETEC treatment significantly reduced the body weight of mice, while ETA treatment effectively reversed this trend (see Figure 12 -A). There were no significant differences in liver, kidney or cardiac indices between the different groups (see Figure 12 -B, 12-C, 12-D); It is worth noting that the spleen index in the ETEC group was significantly increased, and decreased after treatment with TA and ETA, with the regulatory effect of ETA being more prominent (see Figure 12 -E), highlighting its protective efficacy on immune organs.

[0148] ② Morphological changes of the small intestine

[0149] The appearance of the small intestine showed that the ETEC group had severe redness and swelling, followed by the TA group, while the ETA group was most similar to the CON group (see Figure 13 This result directly confirms that TA has a certain anti-inflammatory effect, and ETA has a more significant targeted therapeutic effect on ETEC-induced enteritis, suggesting its highly efficient local biological activity in the intestine.

[0150] ③H&E staining observation

[0151] Duodenal section analysis showed that the villi in the ETEC group were disorganized, with significantly reduced height, increased crypt depth, damaged goblet cells, and infiltration of inflammatory cells. After intervention with TA and ETA, intestinal morphology was significantly improved, especially in the ETA group, with microvilli arranged tightly and regularly, and villus integrity restored (see Figure 14 -A, 14-B). Jejunum and ileum tissues showed similar trends (see Figure 15 -A, 15-B, 16-A, 16-B), further verifying the protective advantage of ETA on the whole intestine. In addition, H&E staining of heart, liver and kidney tissues showed that there was no significant difference between the ETA group and the control group (see Figure 17 ), which strongly proves the safety of the substance in vivo.

[0152] ④ Serum biochemical index detection

[0153] The levels of LDH, ALT, AST, BUN and CRE in the CON and ETA groups were within the standard range and had no significant differences, indicating that ETA had no damaging effect on important organs at therapeutic doses (see Figure 18 -A~E).

[0154] ⑤Effects of ETA on ETEC-induced inflammation and barrier function damage

[0155] A. Inhibition of inflammatory cytokine expression

[0156] The ETA group significantly downregulated the mRNA and protein expression levels of TNF-α, IL-1β, and IL-8, and showed stronger anti-inflammatory activity than the TA group (see Figure 19 -A, 19-B, 19-C).

[0157] B. NF-κB pathway intervention

[0158] ETEC infection activated the mRNA expression levels of TLR4, MYD88, TRAF6, and TAK1 (see Figure 20 -A). However, both ETEC+TA and ETEC+ETA treatments significantly reduced the expression of these inflammatory marker genes compared to the ETEC group. Western blot analysis supported these results, showing consistent changes in protein expression levels (see Figure 20 -B, 20-C).

[0159] C. Tight junction protein repair

[0160] Compared with the ETEC group, ETA treatment significantly upregulated the expression of genes and proteins such as ZO-1 and OCLN, strengthened the intestinal barrier function, and the effect was better than that of TA (see Figure 21 -A, 21-B, 21-C).

[0161] D. Enhanced transporter function

[0162] Compared with the ETEC group, ETA treatment significantly upregulated the expression of transporter-related genes SGLT1, EAAC1, and ASCT2, providing molecular support for nutrient absorption (see Figure 23 ).

[0163] ⑥ Oxidative stress regulation mechanism

[0164] The expression of key genes in the NRF2 pathway (NRF2, HO-1, SOD, and CAT) was significantly upregulated after ETA treatment, and the protein levels were consistent with the transcriptional trends (see Figure 22 Functional tests showed that the SOD activity in serum and intestinal tissues of the ETA group returned to normal levels, and the MDA content was significantly reduced (see Figure 24 -A, 24-B, 24-C, and 24-D), confirming their high antioxidant capacity.

[0165] (2) Study on the dose-effect relationship of ETA

[0166] ①Dose dependence of growth and organ indices

[0167] Mice were divided into groups and experimental design (see Figure 25 Compared with the control group, ETEC treatment significantly reduced the body weight of mice, while ETA treatment effectively reversed this trend, with 500 mg / kg ETA having the strongest reversal effect (see Figure 26 -A). There were no significant differences in liver, kidney or cardiac indices between the different groups (see Figure 26 -B, 26-C, 26-E). Compared with the control group, ETEC treatment significantly increased the spleen index, while 500 mg / kg ETA was the most effective in reducing the spleen index (see Figure 26 -D).

[0168] ② Morphological changes of the small intestine

[0169] Intestinal appearance evaluation showed that although the inflammation of the 750 mg / kg ETA group was more significantly relieved than that of the 250 mg / kg group, the small intestinal morphology of the 500 mg / kg ETA group was closest to that of the control group (see Figure 27 ), suggesting that this dose has the best targeted therapeutic effect on enteritis.

[0170] ③Dose response of ETA on ETEC-induced inflammation and barrier function

[0171] A. Inflammation regulation

[0172] 500mg / kg and 750mg / kg ETA were equally effective in inhibiting the expression of TNF-α, IL-1β and IL-8, and were significantly better than the 250mg / kg dose (see Figure 28 ), indicating that these two higher concentrations of ETA can effectively inhibit the expression of inflammatory factor genes induced by ETEC and alleviate the inflammatory response.

[0173] B. Barrier repair

[0174] The up-regulation effect of 500mg / kg ETA on tight junction protein genes such as ZO-1 and OCLN was slightly stronger than that of 750mg / kg group (see Figure 29 ), indicating that moderate concentration is more conducive to intestinal barrier repair.

[0175] ④Dose optimization of antioxidant pathways

[0176] Analysis of NRF2 and its downstream gene expression showed that 500 mg / kg ETA performed best in activating antioxidant enzymes such as HO-1, SOD, and CAT (see Figure 30 ), proving that this dose can effectively activate the body's antioxidant defense system.

[0177] Overall, in the process of alleviating ETEC-induced enteritis in mice, 500 mg / kg ETA demonstrated comprehensive advantages in inhibiting inflammation, repairing barriers, and activating antioxidant pathways. It is the key dosage node for ETA to exert its biological effects and provides important parameter basis for subsequent applications.

Claims

1. A coated tannic acid, characterized in that: It is prepared by using microencapsulation technology to encapsulate tannic acid with glyceryl monostearate.

2. The coated tannic acid according to claim 1, characterized in that The prepared coated tannic acid particles are full, smooth in surface, clear in outline, oval or oblate in shape, and off-white in color.

3. The coated tannic acid according to claim 1, characterized in that The size of the prepared coated tannic acid particles is 400-1600 μm.

4. A method for preparing the coated tannic acid according to claim 1, characterized in that: The following steps are involved: (1) mixing tannic acid, a binder and water, extruding and forming, and sieving to obtain granules; (2) dissolving glyceryl monostearate in ethanol to obtain an encapsulation solution; (3) placing the particles obtained in step (1) in a fluidized bed coating machine and dynamically coating them with the encapsulation solution of step (2); and drying to obtain the tannic acid-coated particles.

5. The method for preparing coated tannic acid according to claim 4, wherein The adhesive comprises starch, cyclodextrin, sodium carboxymethyl starch and lactose.

6. Use of the coated tannic acid according to claim 1 in the preparation of a medicament for preventing and / or treating intestinal damage induced by Escherichia coli.

7. The use according to claim 6, characterized in that The medicines include human medicines and veterinary medicines.

8. Use of the coated tannic acid according to claim 1 in feed additives.

9. The use according to claim 8, characterized in that The coated tannic acid is used as a feed additive for preventing and / or treating intestinal damage induced by Escherichia coli.

10. The use according to claim 6 or 9, characterized in that: The Escherichia coli includes enterotoxigenic Escherichia coli.

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