Metal polyphenol coating for coprophilous fungi, armor coprophilous fungi and preparation method and application of armor coprophilous fungi

Abstract

The invention discloses a metal polyphenol coating for coprophilous fungi, armor coprophilous fungi and a preparation method and application of the armor coprophilous fungi, the metal polyphenol coating comprises a metal polyphenol network formed on the surface of the coprophilous fungi, the metal polyphenol network is formed by reaction of metal ions and polyphenols, cerium oxide nanoparticles are connected to the metal polyphenol network, and the metal polyphenol network is formed by reaction of metal ions and polyphenols. The cerium oxide nanoparticles comprise trivalent cerium ions and tetravalent cerium ions, and the content of the trivalent cerium ions is greater than the content of the tetravalent cerium ions; according to the preparation method, the coprophilous fungi can be targeted to an intestinal inflammation area by utilizing a metal polyphenol network, the colonization rate of the armor coprophilous fungi is remarkably improved, meanwhile, through cerium oxide nanoparticles connected to polyphenol, active oxygen can be effectively removed, meanwhile, an Nrf2 / Keap1 pathway is activated to inhibit inflammatory cell infiltration, and the survival rate of the armor coprophilous fungi is increased. Therefore, the recovery of intestinal barriers and the reestablishment of microbial homeostasis are effectively promoted, and the local repair and regeneration of inflammatory intestinal tissues at pathological damage parts are effectively promoted.

Description

Technical Field

[0001] The present invention relates to the field of microbial medicine technology, and in particular to a metal polyphenol coating for fecal bacteria, armored fecal bacteria, and a preparation method and application thereof. Background Art

[0002] The intestinal microbiota plays a crucial role in maintaining tissue homeostasis and microecological balance. Disturbances in the microbiota balance may lead to metabolic and immune disorders and are closely related to the occurrence and development of various diseases, especially digestive, metabolic, neurological, and immune-related diseases.

[0003] Fecal microbiota transplantation (FMT) has become an attractive approach to modulating the gut microbiome. It involves transferring fecal microbes from a healthy donor into a patient to reestablish gut microbial diversity disrupted by dysbiosis. This treatment has demonstrated a nearly 90% success rate in managing recurrent Clostridium difficile infection and has been approved for clinical use.

[0004] The main routes of fecal microbiota transplantation include oral fecal microbiota capsules and enema administration. Among them, oral fecal microbiota capsules need to face the influence of the low pH environment in the stomach, a variety of digestive enzymes in the intestines, and bile salts. For example, patent CN118903059A discloses a fecal microbiota capsule based on fecal microbiota, which uses polydopamine and gelatin attached to the surface of the microcapsule to reduce the influence of gastric acid and digestive enzymes on fecal microbiota colonization. However, in order to ensure a sufficient colonization rate, the number of full-dose fecal microbiota capsules required for ingestion in clinical practice is usually large, which may cause gastrointestinal discomfort symptoms such as nausea, vomiting and abdominal distension. Enema administration allows probiotics to enter the colon directly, avoiding the adverse environment of the upper digestive tract, thereby increasing the possibility of successful colonization at the target location. At the same time, enema administration is favored in clinical settings because of its low discomfort, high patient compliance and simple operation.

[0005] The success of fecal microbiota transplantation is closely related to the colonization rate of fecal microbes and the dose ingested. In existing technologies, enema administration still faces the problems of low colonization rate and poor therapeutic effect, especially for inflammatory bowel disease (IBD) with a highly dysregulated intestinal environment. In IBD patients, immune disorders and changes in the intestinal microenvironment, including changes in pH, transit time, and enzyme activity, can affect the survival and implantation of fecal microbes. In addition, the colonic mucus of IBD patients undergoes significant changes in composition, physical properties, and function, which further hinders the attachment of fecal microbes in the inflamed colon, resulting in reduced effectiveness of bacterial therapy. Summary of the Invention

[0006] One purpose of the present invention is to provide a metal polyphenol coating for fecal bacteria, which can target positively charged proteins in the intestine by forming a metal polyphenolic network (MPN) on the surface of fecal bacteria, thereby increasing the colonization rate of fecal bacteria in the colon. At the same time, the cerium oxide nanoparticles connected to the metal polyphenolic network can also effectively remove reactive oxygen species, effectively regulate oxidative stress, and activate Nrf2 / Keap1 pathway to inhibit inflammatory cell infiltration, protect the intestinal barrier and microbial environment, and promote the restoration of intestinal homeostasis.

[0007] The present invention is achieved through the following technical solutions: The metal polyphenol coating for fecal bacteria includes a metal polyphenol network formed on the surface of the fecal bacteria, wherein the metal polyphenol network is formed by the reaction of metal ions and polyphenols, and cerium oxide nanoparticles are connected to the metal polyphenol network. The cerium oxide nanoparticles include trivalent cerium ions and tetravalent cerium ions, and the content of the trivalent cerium ions is greater than the content of the tetravalent cerium ions.

[0008] In this technical solution, through the self-assembly reaction of metal ions and polyphenols, multiple interactions can occur with proteins on the surface of fecal bacteria, forming a nanocoating on the surface of fecal bacteria single cells, namely a metal polyphenol network. The metal polyphenol network covering the surface of fecal bacteria is negatively charged, which can carry fecal bacteria to target positively charged proteins in the inflamed colon, such as transferrin overexpressed in the inflammatory site. At the same time, the multiple phenolic hydroxyl groups contained in polyphenols can form hydrogen bonds, covalent bonds, electrostatic interactions and π-π stacking interactions with intestinal mucosal proteins, which can significantly enhance the colonization rate of fecal bacteria.

[0009] In some preferred embodiments, the polyphenol can be at least one of proanthocyanidins, tara tannins, bayberry tannins, black wattle bark tannins, larch tannins, tannic acid, ellagic acid, epigallocatechin gallate, catechin gallate, or catechin. More preferably, the polyphenol is tannic acid or epigallocatechin gallate.

[0010] In some preferred embodiments, the metal ion can be at least one of zinc ion, iron ion, aluminum ion, and copper ion. In more preferred embodiments, the metal ion is copper ion or iron ion.

[0011] In this technical solution, cerium oxide nanoparticles are also connected to the metal polyphenol network. More specifically, the cerium oxide nanoparticles are connected to the polyphenols in the metal polyphenol network. x Including trivalent cerium ions Ce 3+ and tetravalent cerium ions Ce 4+ , and in cerium oxide nanoparticles, trivalent cerium ions Ce3+ The content is higher than Ce 4+ For example, in some preferred embodiments, cerium oxide nanoparticles CeO x Contains 61.2% Ce 3+ and 39.8% of Ce 4+ When the fecal bacteria encapsulated by the metal polyphenol coating arrive at the inflamed intestine, cerium oxide nanoparticles CeO x Ce in 3+ and Ce 4+ The rapid redox cycle between the two can effectively remove reactive oxygen species (ROS) and activate Nrf2 / Keap1 The study found that fecal bacteria coated with metal polyphenols can protect the intestinal barrier and microbial environment, reshape the immune response, and have broad application prospects in the precise and effective treatment of IBD.

[0012] In this technical solution, the metal polyphenol network can not only target cerium oxide nanoparticles to the inflamed intestine to scavenge reactive oxygen species, but also, through experiments, it was found that compared with directly adding cerium oxide nanoparticles to the inflamed intestine, the reactive oxygen species scavenging activity of cerium oxide nanoparticles connected to the metal polyphenol network and the protective effect on cells damaged by ROS will be further enhanced. This may be due to the fact that the metal polyphenol network stably implants cerium oxide nanoparticles in the inflammatory site to better inhibit the infiltration of inflammatory cells.

[0013] Furthermore, the mass ratio of the metal ions and polyphenols is 1:1~1:4. The mass ratio of metal ions to polyphenols determines whether the metal polyphenol network covering the surface of the fecal bacteria can maintain a stable state for a long time, and the amount of cerium oxide nanoparticles that can be loaded by the metal polyphenol coating. For example, when the content of metal ions is too low, it is difficult for the metal ions to fully coordinate, resulting in a decrease in the stability of the metal polyphenol network and an impact on the colonization rate of the armored fecal bacteria; and when the content of metal ions is too high, more metal ions occupy the connection sites of the polyphenols, resulting in a decrease in the connection sites of cerium oxide nanoparticles and IL-10, and the number of cerium oxide nanoparticles and IL-10 carried by the armored fecal bacteria is even smaller, ultimately resulting in a decrease in the therapeutic effect of the armored fecal bacteria. Therefore, in this technical solution, the effects of the connection sites of polyphenols on the colonization rate and therapeutic effect of the armored fecal bacteria are balanced, and the mass ratio of metal ions to polyphenols is 1:1~1:4. Furthermore, in some preferred embodiments, the mass ratio of metal ions to polyphenols is 1:2~1:3.

[0014] Furthermore, the mass ratio of polyphenols to cerium oxide nanoparticles in the metal polyphenol network is 1:1~1:2.5. The mass ratio of cerium oxide nanoparticles to polyphenols will also affect the therapeutic effect and colonization rate of armored fecal bacteria. Too many cerium oxide nanoparticles will cause a decrease in the number of metal ions and IL-10, while too few cerium oxide nanoparticles will cause a decrease in the scavenging activity of reactive oxygen species and a decrease in the ability to inhibit inflammatory cell infiltration. Therefore, in the present technical solution, the mass ratio of polyphenols to cerium oxide nanoparticles in the metal polyphenol network is 1:1~1:2.5. In a more preferred embodiment, the mass ratio of polyphenols to cerium oxide nanoparticles is 1:1~1:2.

[0015] As a preferred embodiment of the present invention, IL-10 is also connected to the metal polyphenol network. In this technical solution, the polyphenols of the metal polyphenol network are also connected to the anti-inflammatory cytokine IL-10. IL-10 is mainly used to maintain immune homeostasis, control inflammatory response and promote tissue repair. Its synergistic effect with cerium oxide nanoparticles can effectively regulate oxidative stress after the cerium oxide nanoparticles clear the reactive oxygen species in the inflamed intestine, and further promote the recovery of intestinal homeostasis. Through experiments, it was found that compared with armored fecal bacteria that are not connected to IL-10, armored fecal bacteria that are connected to cerium oxide nanoparticles and IL-10 at the same time showed a stronger ability to promote intestinal homeostasis and can more effectively inhibit the typical symptoms of colon shortening in DSS treatment.

[0016] Furthermore, the mass ratio of polyphenols to IL-10 in the metal polyphenol network is 1:1 to 1:2. IL-10 also needs to be connected to the polyphenols in the metal polyphenol network, so the amount of IL-10 used will also affect the stability of colonization and the therapeutic effect of armored fecal bacteria. Therefore, in this technical solution, the mass ratio of polyphenols to IL-10 is limited to 1:1 to 1:2. In some preferred embodiments, the mass ratio of polyphenols to IL-10 is limited to 1:1 to 1:1.5.

[0017] Another object of the present invention is to provide an armored fecal bacteria with a surface covered with a metal polyphenol coating based on any of the aforementioned metal polyphenol coatings. The armored fecal bacteria include fecal bacteria, and the surface of the fecal bacteria is covered with a metal polyphenol coating. The armored fecal bacteria can be targeted and delivered to the inflamed colon site and increase the colonization rate of microorganisms, and can stably exert the function of fecal bacteria for a long time. At the same time, the combination of cerium oxide nanoparticles and the anti-inflammatory cytokine IL-10 can effectively remove reactive oxygen, promote intestinal homeostasis, and effectively improve intestinal microbial balance and overall health.

[0018] In some preferred embodiments, the fecal bacteria may be at least one of Escherichia coli, Bifidobacterium, and Lactobacillus.

[0019] Another object of the present invention is to provide a method for preparing any of the aforementioned armored feces fungi, specifically comprising the following steps: Adding metal ions and polyphenols to a cell suspension containing fecal bacteria to obtain fecal bacteria with a metal-polyphenol network covering the surface; Cerium oxide nanoparticles are added to the bacterial liquid of fecal bacteria whose surface is covered with a metal polyphenol network, and mixed evenly to obtain armored fecal bacteria with cerium oxide nanoparticles connected to the metal polyphenol network.

[0020] In some preferred embodiments, after washing the fecal bacteria solution with PBS, the sample is resuspended in a PBS solution to obtain a cell suspension. Subsequently, a metal ion solution and a polyphenol solution are added to the cell suspension, followed by the addition of a PBS solution. In one or more embodiments, vortex mixing is required after each addition to obtain a cell suspension in which the surface of the fecal bacteria is covered with a metal polyphenol network.

[0021] In some preferred embodiments, cerium oxide nanoparticles are further added to the cell suspension, mixed evenly, and then washed to obtain armored fecal bacteria with cerium oxide nanoparticles connected to the metal polyphenol network.

[0022] In a more preferred embodiment, IL-10 is added to the bacterial liquid of armored feces fungi with cerium oxide nanoparticles connected to the metal polyphenol network, and mixed evenly to obtain armored feces fungi with cerium oxide nanoparticles and IL-10 connected to the metal polyphenol network.

[0023] In one or more embodiments, the fecal microbial fluid is obtained by immediately embedding the fecal sample in glycerol, homogenizing, filtering, and transferring it to -80°C for storage after collection, thawing the sample 30 minutes before the enema operation and placing it in a centrifuge tube to prepare the fecal microbial fluid.

[0024] Furthermore, the preparation method of the cerium oxide nanoparticles comprises the following steps: Cerium acetate hydrate and oleylamine are dissolved in a solvent, mixed evenly and then heated for reaction to prepare cerium oxide nanoparticles containing trivalent cerium ions and tetravalent cerium ions.

[0025] In some preferred embodiments, cerium acetate hydrate and oleylamine are dissolved in a solvent, the mixture is sonicated, and heated to 80-100°C. The reaction is confirmed when the solution changes color from white to yellow. After the reaction is complete, the solution is aged for a predetermined period of time, cooled to room temperature, and then centrifuged and washed to obtain cerium oxide nanoparticles.

[0026] Another object of the present invention is to provide an application of any of the aforementioned armored fecal bacteria, specifically, the armored fecal bacteria is used to prepare fecal microbiota transplantation enema preparations. This cell surface engineering strategy based on metal polyphenol networks provides a powerful solution for overcoming colonization barriers in fecal microbiota transplantation, demonstrating a promising enema preparation in the precise and effective treatment of IBD.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The metal polyphenol coating for fecal bacteria disclosed in the present invention can target fecal bacteria to the intestinal inflammation area by using the metal polyphenol network, significantly improving the colonization rate of armored fecal bacteria. At the same time, the cerium oxide nanoparticles connected to the polyphenol can effectively remove active oxygen and activate Nrf2 / Keap1 pathways to inhibit inflammatory cell infiltration, thereby effectively promoting the restoration of the intestinal barrier and the re-establishment of microbial homeostasis, as well as the local repair and regeneration of inflamed intestinal tissue at the site of pathological damage; 2. The metal polyphenol network of the present invention is also connected to the anti-inflammatory cytokine IL-10, which is used to maintain immune homeostasis, control inflammatory responses and promote tissue repair. Its synergistic effect with cerium oxide nanoparticles can effectively regulate oxidative stress after the cerium oxide nanoparticles clear reactive oxygen species in the inflamed intestine, further promoting the restoration of intestinal homeostasis. 3. By optimizing the mass ratio of polyphenols to metal ions, cerium oxide nanoparticles, and IL-10, the present invention can rationally distribute the attachment sites of polyphenols, maximize the colonization rate, effectively exert the effects of cerium oxide nanoparticles and IL-10, effectively regulate oxidative stress, and promote the restoration of intestinal homeostasis; 4. The armored fecal bacteria of the present invention can enter the inflamed intestine through enema administration, target the inflamed intestine, and after efficient colonization, remove reactive oxygen species, inhibit inflammatory cell infiltration, and effectively protect the intestinal barrier and microbial environment; 5. The armored fecal bacteria of the present invention can effectively increase the abundance of beneficial bacteria such as lactic acid bacteria and Bacteroides, while reducing the abundance of pathogenic Erysipelothrix and Enterobacter, increasing the abundance and diversity of microorganisms, and helping to reshape the intestinal flora; 6. The preparation method of the armored fecal fungus of the present invention has a simple process and mild reaction conditions, which is conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings: Figure 1 It is a flowchart of the preparation process in a specific embodiment of the present invention; Figure 2It is the armored fecal fungus EcN / FecalMason in the specific embodiment of the present invention. Pro Schematic diagram of the preparation mechanism; Figure 3 shows the characterization of cerium oxide nanoparticles prepared in a specific embodiment of the present invention; Figure 4 The cerium oxide nanoparticles CeO in the specific embodiment of the present invention are shown. x , Metal polyphenol coating material FecalMason, Metal polyphenol coating material FecalMason pro ROS activity detection, where the horizontal axis is CeO x The concentration of CeO in the coating material x The concentrations of the samples were analyzed. The vertical axes are A: CAT enzyme activity (U / mL), B: SOD anion resistance (U / L), and C: hydrogen peroxide clearance rate (%). Figure 5 The figure shows the comparison of free radical content in the cell protection test of metal polyphenol coating materials against ROS-induced damage in specific embodiments of the present invention; Figure 6 Shows the armored fecal fungus EcN / FecalMason in a specific embodiment of the present invention pro representation of; Figure 7 The figure shows the colonization of armored fecal bacteria in the intestines of mice after a certain period of enema in a specific embodiment of the present invention; Figure 8 The figure shows the therapeutic effect of Armored Faecalis in a DSS-induced colitis mouse model according to a specific embodiment of the present invention; Figure 9 The results show the therapeutic effect of F. armatum on reshaping the intestinal flora in a DSS-induced colitis mouse model in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with examples and drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0030] All raw materials of the present invention are not particularly limited in their sources and can be purchased on the market or prepared according to conventional methods well known to those skilled in the art. All raw materials of the present invention are not particularly limited in their purity. The present invention preferably adopts analytically pure or conventional purity requirements in the field of microbial medicine. All raw materials of the present invention, their brands and abbreviations are conventional brands and abbreviations in the field, and each brand and abbreviation is clear and unambiguous in the field of its related use. Those skilled in the art can purchase them from the market or prepare them by conventional methods based on the brand, abbreviation and corresponding use.

[0031] The term "connection" used in the present invention may refer to direct connection or indirect connection via other groups unless otherwise specified.

[0032] 1. Preparation and testing of metal polyphenol coating materials [Example 1] Metal polyphenol coating material FecalMason (a) Cerium oxide (CeO x ) Cerium acetate hydrate (1 mM) and oleylamine (12 mM) were dissolved in 15 mL of xylene. The mixture was sonicated for 40 min and heated at 2 °C min -1 The reaction solution was heated to 90 °C at a heating rate of 100 ℃. Then, 1 mL of deionized water was injected into the reaction solution. The color of the solution changed from white to yellow, indicating that the reaction had occurred. After aging at 90 °C for 3 h, the resulting solution was cooled to room temperature and washed with acetone several times using centrifugation (30,000 g). The obtained cerium oxide nanoparticles (CeO x ) was redispersed in water to obtain CeO x spare.

[0033] Figure 3 The characterization of cerium oxide nanoparticles is shown in the figure. x The size of the synthesized CeO is about 3 nm, the lattice spacing is 0.31 mm, and the quantitative analysis of X-ray photoelectron spectroscopy shows that x Contains 61.2% Ce 3+ and 39.8% of Ce 4+ .

[0034] (b) Preparation of FecalMason metal polyphenol coating material 20 μL of CuSO4 (2 mg / mL) and 20 μL of tannic acid TA (2 mg / mL) were added to 640 μL of PBS solution, and then 360 μL of PBS was added. The mixture was vortexed for 30 s between each addition to obtain a metal polyphenol network (MPN) solution. The metal polyphenol network solution was washed by centrifugation three times (10000 g, 5 min), and 20 μL of CeO was added. x The metal polyphenol coating material FecalMason was prepared by vortexing for 30 s and washing by centrifugation for 3 times (10000 g, 5 min).

[0035] [Example 2] Metal polyphenol coating material FecalMason pro Based on Example 1, 20 μL of IL-10 (1 mg / mL) was added to the solution of the metal polyphenol coating material FecalMason, vortexed for 30 s, and centrifuged and washed three times (10000 g, 5 min) to prepare the metal polyphenol coating material FecalMason. pro .

[0036] [Example 3] Reactive oxygen species (ROS) scavenging activity Using the main reactive oxygen species (ROS) • O2 – and H2O2 analysis of CeO x All experiments were performed according to the instructions of each kit. – ) inhibition and generation detection kits (Nanjing Jiancheng Bioengineering Institute). Hydrogen peroxide scavenging activity: Catalase (CAT) activity detection kit (Solarbio) and H2O2 content detection kit (Solarbio) were used.

[0037] The experimental results are as follows Figure 4 As shown, only CeO x The experimental group showed some activity in simulating superoxide dismutase (SOD) and catalase (CAT) activities. However, after being connected to the metal polyphenol network, the FecalMason group and the FecalMason pro The activity of the group was significantly improved, and FecalMason pro The activity of the group was higher than that of the FecalMason group. pro The group had the best free radical scavenging rate at each concentration. In addition, FecalMason pro The ability of the group to scavenge ROS increases with the CeO xIncreased with increasing concentration.

[0038] [Example 4] Test on cell protection against ROS-induced damage In this example, HCT116 cells (8 × 10³ cells per well) were seeded in a 96-well plate and incubated for 24 hours. The culture medium was removed, and 100 μL of fresh culture medium (with or without 500 μM H₂O₂) was added to each well. After a 2-hour incubation, the cells were washed with culture medium, and the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, 5 μM) was added to each well. After 30 minutes, intracellular ROS levels were observed under a confocal microscope.

[0039] Figure 5 The free radical contents of the five experimental groups are shown in the figure. It can be seen from the figure that the free radical content of the H2O2+PBS experimental group with the addition of PBS and H2O2 is higher. x , Metal polyphenol coating material FecalMason, Metal polyphenol coating material FecalMason pro After the treatment, the number of free radicals decreased significantly, especially the coating with additional IL-10, which further reduced the free radical content and effectively protected the cells from oxidative stress damage.

[0040] 2. Preparation of Armored Fecal Bacteria like Figure 1 As shown, the preparation method of armored fecal fungus includes the following steps: Adding metal ions and polyphenols to a cell suspension containing fecal bacteria to obtain fecal bacteria with a metal-polyphenol network covering the surface; Adding cerium oxide nanoparticles to the bacterial liquid of the fecal bacteria with the metal polyphenol network on the surface, mixing them evenly, to obtain armored fecal bacteria with cerium oxide nanoparticles connected to the metal polyphenol network; IL-10 was added to the bacterial liquid of armored feces bacteria with cerium oxide nanoparticles connected to the metal polyphenol network, and the mixture was evenly mixed to obtain armored feces bacteria with cerium oxide nanoparticles and IL-10 connected to the metal polyphenol network.

[0041] Figure 2 A preferred armored fecal bacteria is shown, which uses tannic acid and copper ions to self-assemble on the surface of Escherichia coli Nissle1917 (EcN) to form a metal polyphenol network, and then uses the adhesion properties of the metal polyphenol network to attach cerium oxide nanoparticles CeO x and anti-inflammatory cytokine IL-10 adhere to the surface of fecal bacteria to produce armored fecal bacteria EcN / FecalMason pro .

[0042] [Example 5] Take 1 mL of EcN bacterial solution and wash it three times with PBS by centrifugation (3000 g, 3 min). Then resuspend the sample in 600 μL of PBS solution to obtain a cell suspension. Add 20 μL of CuSO4 (2 mg / mL), 40 μL of tannic acid (2 mg / mL), and then add 340 μL of PBS to the cell suspension. Vortex for 30 seconds between each addition. Wash it three times with PBS by centrifugation (3000 g, 3 min) to remove the residual Cu in the solution. 2+ and TA, and then resuspend the bacterial solution in 1 mL PBS.

[0043] Then, 40 μL of CeO was added to the cell suspension. x (2.5 mg / mL), and similarly, vortex mixing was performed for 30 seconds between each addition, followed by centrifugation washing three times (10,000 g, 5 min) to prepare armored fecal bacteria EcN / FecalMason.

[0044] [Example 6] Take 1 mL of EcN bacterial solution and wash it three times with PBS by centrifugation (3000 g, 3 min). Then resuspend the sample in 600 μL of PBS solution to obtain a cell suspension. Add 20 μL of CuSO4 (2 mg / mL), 40 μL of tannic acid (2 mg / mL), and then add 340 μL of PBS to the cell suspension. Vortex for 30 seconds between each addition. Wash it three times with PBS by centrifugation (3000 g, 3 min) to remove the residual Cu in the solution. 2+ and TA, and then resuspend the bacterial solution in 1 mL PBS.

[0045] Then, 40 μL of CeO was added to the cell suspension. x (2.5 mg / mL) and 20 μL of IL-10 (1 mg / mL), vortexed for 30 s, and then centrifuged and washed three times (10000 g, 5 min) to prepare armored fecal bacteria EcN / FecalMason pro .

[0046] Figure 6 Shown is the armored fecal fungus EcN / FecalMason proAs shown in the figure, scanning electron microscopy (SEM) images show that a dense metal polyphenol coating is attached to the surface of EcN. IL-10 and bovine serum albumin (BSA) were labeled with Cyanine 7 (Cy7) and fluorescein isothiocyanate (FITC), respectively. Confocal fluorescence images show that the surface of EcN has obvious Cy7 and FITC fluorescent shells, which together prove that EcN / FecalMason Pro Successful preparation.

[0047] [Example 7] In this example, the preparation method of armored fecal fungi is similar to that in Example 6, except that, in this example, 20 μL of CuSO4 (2 mg / mL) and 20 μL of tannic acid (2 mg / mL) are added to the cell suspension in sequence.

[0048] [Example 8] In this example, the preparation method of armored fecal fungi is similar to that in Example 6, except that, in this example, 20 μL of FeCl3 (2 mg / mL) and 60 μL of tannic acid (2 mg / mL) are added to the cell suspension in sequence.

[0049] [Example 9] In this example, the preparation method of armored fecal fungi is similar to that in Example 6, except that, in this example, 20 μL of FeCl3 (2 mg / mL) and 60 μL of tannic acid (2 mg / mL) are added to the cell suspension in sequence.

[0050] [Example 10] In this example, the preparation method of armored fecal bacteria is similar to that in Example 6, except that, in this example, 20 μL of FeCl3 (2 mg / mL) and 60 μL of epigallocatechin gallate (EGCG, 2 mg / mL) are added to the cell suspension in sequence.

[0051] [Example 11] In this example, the preparation method of armored fecal bacteria is similar to that in Example 6, except that, in this example, 20 μL of CuSO4 (2 mg / mL) and 20 μL of epigallocatechin gallate (EGCG, 2 mg / mL) are added to the cell suspension in sequence.

[0052] [Example 12] In this example, the preparation method of armored fecal bacteria is similar to that in Example 6, except that in this example, 40 μL of CeO was added to the cell suspension. x (2.5 mg / mL) and 40 μL of IL-10 (1 mg / mL).

[0053] [Example 13] In this embodiment, the preparation method of armored fecal bacteria is similar to that of Example 6, except that in this embodiment, 30 μL of CeO was added to the cell suspension. x (2.5 mg / mL) and 20 μL of IL-10 (1 mg / mL).

[0054] Although the above embodiments illustrate metal polyphenol networks composed of certain polyphenols and metal ions, those skilled in the art will appreciate that other polyphenols and metal ions can also constitute metal polyphenol networks of fecal bacteria. In some embodiments, the polyphenol may also be selected from at least one of proanthocyanidins, tara tannins, bayberry tannins, black wattle bark tannins, larch tannins, tannic acid, ellagic acid, catechin gallate, or catechins. In some embodiments, the metal ion may also be an aluminum ion or a zinc ion.

[0055] Similarly, in addition to using Escherichia coli EcN, the metal polyphenol coating can also be applied to other beneficial fecal bacteria. For example, in one or more embodiments, the fecal bacteria can be selected from Bifidobacterium, Lactobacillus, etc.

[0056] 3. Performance test of armored fecal bacteria [Example 14] In this example, the ability of Armored Faecalis to colonize the mouse intestine was tested. Specifically, Escherichia coli Nissle 1917 carrying the pGEN-luxCDABE plasmid was provided by Zhao Hui's laboratory at Sichuan University. This strain was used for in vivo imaging (IVIS) imaging to monitor the distribution of the probiotic in the intestine.

[0057] Before the experiment, mice were fasted for 18 h but had free access to water. Then, 200 μL of uncoated EcN, the armored fecal bacteria EcN / FecalMason prepared in Example 5, and the armored fecal bacteria EcN / FecalMason prepared in Example 6 were administered to ICR mice by enema. Pro (10 9 CFU). Mice were sacrificed at various time points after dosing, and their intestines were harvested for fluorescence imaging. The harvested intestines were further divided into different sections, including the cecum and colon. The harvested tissues were individually ground, suspended in 1 mL of PBS, and CFU counts were performed on selective plates.

[0058] The experimental results are as follows Figure 7As shown in the figure, the fluorescence intensity of the uncoated EcN (No treatment) dropped sharply 12 hours after administration, indicating that its residence time in the mouse intestine was about 12 hours. Pro The fluorescence of the 24 h-expressing bacteria was still detectable even after 24 h. These findings suggest that armored bacteria have a higher survival and colonization rate in the intestine.

[0059] Furthermore, in order to quantitatively determine the viable cells in the intestine, the number of active EcN cells in the colon and cecum was determined by counting on a plate containing kanamycin. Figure 7 As shown, 24 hours after enema, the armored fecal bacteria EcN / FecalMason Pro The content in the cecum region (Caecum) was about 4 times that of the uncoated EcN group, and the content in the colon region (Colon) was about 44 times that of the uncoated EcN group. There were almost no living cells in the colon region.

[0060] [Example 15] In this example, the therapeutic effect of Faecalis spp. in a dextran sulfate (DSS)-induced colitis mouse model was tested.

[0061] Specifically, in order to prepare FM / FecalMason Pro , fecal samples from healthy mice were collected and immediately embedded in glycerol, homogenized, filtered, and transferred to -80°C for storage. Thaw the sample 30 minutes before the enema operation and place it in a centrifuge tube. Take 1 mL of the bacterial solution and wash it three times with PBS by centrifugation (3000 g, 3 min). Then, the fecal bacteria (Fecal microbiota, FM) sample was resuspended in 600 μL of PBS solution. Following steps similar to those in Example 2, a metal polyphenol coating material FecalMason was formed on the surface of the fecal bacteria to prepare armored fecal bacteria FM / FecalMason; following steps similar to those in Example 3, a metal polyphenol coating material FecalMason was formed on the surface of the fecal bacteria. pro , prepare armored fecal bacteria FM / FecalMason Pro .

[0062] To establish a DSS-induced colitis mouse model, 6- to 8-week-old male ICR mice were administered sterile drinking water containing 2.5% DSS (molecular weight 36,000 to 50,000 kDa) for 7 days. The mice were then randomly divided into groups, with untreated healthy mice serving as controls. Visible stool consistency, weight change, and fecal bleeding were recorded throughout the treatment period. At the conclusion of the experiment, the mice were anesthetized and sacrificed, and their colons were collected.

[0063] The experimental results are as follows Figure 8 As shown, compared with the uncoated FM group and the FecalMason FM / FecalMason group, the FecalMason FM / FecalMason Pro The group was able to effectively inhibit the weight loss of IBD mice and downregulate the disease activity index (DAI). The colon of the mice was collected on day 14 for photographing and measurement. The results showed that the Fecal Mason FM / Fecal Mason group and the Fecal Mason FM / Fecal Mason group had a significant effect on the body weight of IBD mice. Pro The colon length of the group was significantly longer than that of the other groups, indicating that Fecal Mason can inhibit the typical colon shortening in DSS treatment. At the same time, based on the synergistic effect of cerium oxide nanoparticles and anti-inflammatory cytokine IL-10, Fecal Mason FM / Fecal Mason Pro The colon length of the group was longer than that of the FecalMason FM / FecalMason group, reflecting that the combination of cerium oxide nanoparticles and anti-inflammatory cytokine IL-10 can promote intestinal homeostasis and effectively improve intestinal microbial balance and overall health status.

[0064] Furthermore, it was found through experiments that Figure 9 As shown, compared with the uncoated FM group, the armored fecal bacteria FM / FecalMason and armored fecal bacteria FM / FecalMason Pro The group can increase the relative abundance of beneficial bacteria such as Lactobacillus and Bacteroidaceae, and significantly reduce the abundance of pathogenic Erysipelotrichaceae and Enterobacteriaceae, indicating that armored fecal bacteria can effectively increase the abundance and diversity of microorganisms and reshape the intestinal flora. This may be due to the CeO x and IL-10 can effectively remove ROS in the intestinal microenvironment, thereby effectively alleviating the inflammatory symptoms of IBD and then effectively reshaping the intestinal flora homeostasis.

[0065] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A metal polyphenol coating for fecal bacteria, characterized in that: It includes a metal polyphenol network formed on the surface of fecal bacteria, which is formed by the reaction of metal ions and polyphenols. Cerium oxide nanoparticles are connected to the metal polyphenol network, and the cerium oxide nanoparticles include trivalent cerium ions and tetravalent cerium ions, and the content of the trivalent cerium ions is greater than the content of the tetravalent cerium ions.

2. The metal polyphenol coating for fecal bacteria according to claim 1, characterized in that The mass ratio of the metal ions to the polyphenols is 1:1 to 1:

4.

3. The metal polyphenol coating for fecal bacteria according to claim 1, characterized in that The mass ratio of polyphenols to cerium oxide nanoparticles in the metal polyphenol network is 1:1 to 1:2.

5.

4. The metal polyphenol coating for fecal bacteria according to any one of claims 1 to 3, characterized in that IL-10 is also connected to the metal polyphenol network.

5. The metal polyphenol coating for fecal bacteria according to claim 4, characterized in that The mass ratio of polyphenols to IL-10 in the metal polyphenol network is 1:1 to 1:

2.

6. An armored fecal fungus, characterized in that: It includes fecal bacteria, the surface of which is covered with a metal polyphenol coating, and the metal polyphenol coating is the metal polyphenol coating for fecal bacteria as described in any one of claims 1 to 5.

7. A method for preparing armored fecal fungus, characterized in that: For preparing the armored feces fungus according to claim 6, the preparation method comprises the following steps: Adding metal ions and polyphenols to a cell suspension containing fecal bacteria to obtain fecal bacteria with a metal-polyphenol network covering the surface; Cerium oxide nanoparticles are added to the bacterial liquid of fecal bacteria whose surface is covered with a metal polyphenol network, and mixed evenly to obtain armored fecal bacteria with cerium oxide nanoparticles connected to the metal polyphenol network.

8. The method for preparing armored feces fungus according to claim 7, characterized in that: The following steps are also included: IL-10 was added to the bacterial liquid of armored feces bacteria with cerium oxide nanoparticles connected to the metal polyphenol network, and the mixture was evenly mixed to obtain armored feces bacteria with cerium oxide nanoparticles and IL-10 connected to the metal polyphenol network.

9. The method for preparing armored fecal fungus according to claim 7 or 8, characterized in that: The preparation method of the cerium oxide nanoparticles comprises the following steps: Cerium acetate hydrate and oleylamine are dissolved in a solvent, mixed evenly and then heated for reaction to prepare cerium oxide nanoparticles containing trivalent cerium ions and tetravalent cerium ions.

10. A fecal microbiota transplant enema preparation, characterized in that: Including an armored fecal fungus as described in claim 6.

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