A probiotic preparation for preventing and improving infantile intestinal pathogenic bacterial infection

Abstract

This invention discloses a postbiotic preparation that can prevent and improve intestinal pathogen infection in infants and young children, belonging to the field of pharmaceutical and functional food technology. The active ingredient of the postbiotic preparation prepared in this invention is *Ackermannia myxotropicis* extracellular vesicles (EVs). This study systematically evaluated various infection-related indicators in a pathogen infection model, elucidating the intrinsic regulatory mechanism of *Ackermannia myxotropicis* EVs on host intestinal immunity. Compared with the model infection group, the EVs intervention group showed a significant increase in the weight of young mice and a significant reduction in intestinal tissue pathological damage. Mechanistic studies confirmed that *Ackermannia myxotropicis* EVs can regulate M1 macrophage immune training, promote TNF-α secretion, and thereby upregulate the expression level of the intestinal antimicrobial peptide Reg3, ultimately achieving the effect of resisting intestinal pathogen infection. This invention has significant effects on improving infectious diarrhea and has good prospects for consumption and application, providing a suitable health care and diarrhea-relieving product for infants and young children.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and functional food technology, specifically relating to a postbiotic preparation for preventing and improving intestinal pathogen infection in infants and young children. Background Technology

[0002] Infectious diarrhea, especially diarrhea induced by pathogens, remains a global public health challenge and a significant contributing factor to high mortality rates in developing countries. In early life, the intestinal physiology of infants is not yet fully developed. The body primarily relies on the symbiotic gut microbiota of infants, dominated by bacteria such as Bifidobacteria, and the immune protective effect of secretory IgA from breast milk on the intestinal mucosa to jointly construct and maintain intestinal barrier homeostasis and ensure the body's health. However, common intestinal pathogens such as pathogenic Escherichia coli, Salmonella typhi, and Shigella dysenteriae can easily invade the intestines of infants, disrupting the structure of the intestinal symbiotic microbiota, damaging the integrity of the intestinal barrier, and thus inducing intestinal infections and diarrhea.

[0003] Akkermansia myxophilus, an important intestinal commensal bacterium, can colonize the intestinal epithelial surface, thicken the intestinal mucus layer, repair damaged intestinal barriers, and decompose mucin to produce short-chain fatty acids (SCFAs), thereby improving intestinal integrity and alleviating metabolic inflammation. Studies have confirmed that supplementing with this bacterium in Clostridium difficile and Salmonella infection models can reduce host susceptibility to pathogenic bacteria, regulate intestinal flora structure and metabolic profile, and studies on diet-induced non-alcoholic fatty liver disease have found that inactivated Akkermansia myxophilus can also improve intestinal permeability. Therefore, both live and inactivated Akkermansia myxophilus have good development prospects and are highly promising probiotic candidates.

[0004] Extracellular vesicles (EVs) are nanoscale, bilayered membrane vesicles secreted by bacteria, containing bioactive components such as proteins, lipids, and nucleic acids. They can mediate intercellular communication, substance transport, and immune regulation, and possess excellent biocompatibility, strong stability, and prominent targeting capabilities, making them highly promising for the prevention and treatment of diseases such as infection, inflammation, metabolic disorders, and tumors. Compared with live bacterial preparations, bacterial EVs do not pose a risk of live bacterial colonization and are therefore safer, making them a research hotspot for novel functional factors in the biomedical field. EVs secreted by intestinal symbiotic bacteria can maintain intestinal microecological homeostasis, strengthen the mucosal barrier, and regulate the body's immunity, opening up new avenues for the intervention of intestinal diseases. Akkermansia myxophilus, as a key beneficial intestinal bacterium, secretes EVs that can target the intestinal epithelium, upregulate tight junction protein expression, repair the intestinal barrier to alleviate intestinal inflammatory damage, and also have the potential to regulate intestinal flora structure and antagonize pathogen invasion, demonstrating unique roles and significant research value in intestinal protection and damage repair.

[0005] Currently, antibiotics are routinely used in clinical practice to treat intestinal pathogen infections in infants and young children. However, the overuse of antibiotics can easily lead to a series of problems such as intestinal flora imbalance and the development of multidrug resistance, especially adversely affecting the immature intestinal tracts of infants and young children. Therefore, developing a low-drug-resistance drug or health food for infant intestinal pathogen infections has become one of the most important issues that urgently needs to be addressed. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a postbiotic preparation for preventing and improving intestinal pathogen infection in infants and young children. Based on the oral intervention of Akkermansia myxophilus EVs, it enhances the host's resistance to intestinal pathogens in infants and young children, and strengthens the intestinal barrier and intestinal immune function, thereby achieving the safe and effective effect of reducing intestinal pathogen infection and protecting intestinal health in infants and young children.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a postbiotic preparation for preventing and improving intestinal pathogen infection in infants and young children, the postbiotic preparation being composed of Akkermansia myxophilus exovesicles (EVs) and pharmaceutically acceptable carriers or excipients.

[0008] Based on the above technical solution, furthermore, the only active ingredient in the postbiotic preparation is the Akkermansia muciniphila exovesicles (EVs).

[0009] Based on the above technical solution, the pharmaceutically acceptable carrier or excipient includes, but is not limited to, fillers, diluents, binders, disintegrants, lubricants, coating materials or enteric materials.

[0010] Secondly, the present invention provides a method for preparing Akkermansia myxophilus exovesicles (EVs) in the aforementioned postbiotic preparation, comprising the following steps: (1) The bacteria were cultured in brain heart broth medium, the bacterial solution was collected and the supernatant was obtained by centrifugation and filtered through a microporous membrane to obtain the filtrate. (2) Add extracellular vesicle extraction reagent to the filtrate, incubate at 2-8℃, and collect the precipitate by centrifugation; (3) The precipitate was resuspended in sterile PBS, centrifuged and the supernatant was collected. The supernatant was filtered through an exosome purification column and the filtrate was collected to obtain the extracellular vesicle suspension of Akkermansia muciniphila.

[0011] Thirdly, the present invention provides the use of the postbiotic preparation or the Akkermansia myxophilus extravesicles (EVs) in the preparation of a medicament for treating Escherichia coli infection.

[0012] Based on the above technical solution, the Akkermansia muciniphila EVs further induce intestinal M1 macrophages to train immunity.

[0013] Based on the above technical solution, the Akkermansia muciniphila EVs further induce M1 macrophages to secrete TNF-α, and TNF-α continuously stimulates the colon to secrete regenerating islet-derived protein 3 (Reg3) intestinal antimicrobial peptides.

[0014] Based on the above technical solution, the Escherichia coli is further selected from enterotoxigenic Escherichia coli, enteroinvasive Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, or enteroaggregative Escherichia coli.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention establishes a three-week-old C57BL / 6 mouse model of infection, compares and analyzes various infection-related indicators between the infected model group and the *Ackermannia myxotropicis* EV intervention group, and further explores the intrinsic molecular mechanism by which *Ackermannia myxotropicis* EVs regulate intestinal immunity through in vitro experiments. The study confirms that *Ackermannia myxotropicis* EVs can induce host intestinal M1 macrophages to produce training immunity, promoting the secretion of large amounts of tumor necrosis factor-alpha (TNF-α); subsequently, it continuously activates the colon to secrete and regenerate pancreatic islet-derived protein 3 (Reg3) and other intestinal antimicrobial peptides, thereby effectively alleviating intestinal pathological damage induced by *Escherichia coli* O157. This postbiotic preparation has a good effect on improving infectious diarrhea and has no obvious toxic side effects, showing good prospects for research and development and practical application. Attached Figure Description

[0016] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0017] Figure 1 The results of Akkermansia muciniphila EVs alleviating host intestinal infection damage induced by Escherichia coli O157 in Example 2 of the present invention are shown in (A) the results of changes in the weight of offspring mice; (B) a comparison of the length of offspring mouse colon samples; (C) the results of colon tissue pathological sections; and (D) the expression level of Reg3β in the colon of offspring mice.

[0018] Figure 2The results of M1 macrophage immunity induced by Akkermansia myxophilus EVs in Example 3 of the present invention are shown in (A) transmission electron microscopy image of Akkermansia myxophilus EVs; (B) particle size distribution of Akkermansia myxophilus EVs; (C) polarization level of M1 macrophages induced by Akkermansia myxophilus EVs; and (D) diagram of M1 macrophages and TNF-α secretion.

[0019] Figure 3 This is an immunomodulatory model of HIF-1-driven M1 macrophage training in Example 3 of the present invention, where (A) is the M1 macrophage training immunomodulatory model of Akkermansia myxophilus EVs; (B) is the β-type macrophage. NO content in the supernatant of glucan-induced cells; (C) represents β-cells. glucan training treatment induced TNF in cell supernatant (A) α content; (D) Fluorescent labeling results of Akkermansia myxophilus EVs and M1 marker CD86; (E) Polarization and distribution ratio of M1 macrophages, and TNF-α secretion map.

[0020] Figure 4 This is the result of Akkermansia myxophilus EVs regulating Reg3 secretion in intestinal epithelial cells via the TNF-α-mediated TNFR1 / STAT3 pathway in Example 4 of the present invention. Among them, (A) is the immunoblotting detection of Reg3α protein in HT-29 cells treated with TNF-α; (B) the concentration of Reg3α in the cell supernatant of each inhibitor treatment group (pg / mL); (C) the immunoblotting detection of Reg3α protein in HT-29 cells treated with STAT3 inhibitor; (D) the immunoblotting detection of STAT3 / p-STAT3 protein in HT-29 cells treated with TNF-α. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0022] This invention provides the application of Akkermansia myxophilus EVs in modulating host intestinal immunity and resistance to intestinal pathogens. In this invention, the intestinal pathogen is preferably induced by enterohemorrhagic Escherichia coli (EHEC) O157. This invention does not specifically limit the Escherichia coli O157 infection model; those skilled in the art can follow conventional procedures.

[0023] Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0024] Example 1: Preparation of Akkermansia myxophilus EVs Akkermansia myxophilus was cultured in brain heart broth for 72 h. After thorough mixing, 40 mL of the culture was transferred to a 50 mL EP tube and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected and filtered through a 0.22 μm sterile filter. Exosome extraction reagent was added at a ratio of 4:1 according to the extraction method of the extracellular vesicle extraction and purification kit. The mixture was thoroughly shaken and incubated overnight at 4°C. After incubation, the precipitate was centrifuged at 10,000×g for 60 min at 4°C. The precipitate was resuspended in an appropriate amount of sterile PBS and centrifuged at 12,000×g for 3 min at 4°C. The supernatant was transferred to the upper chamber of an Exosome Purification Filter (EPF column) and centrifuged at 3,000 rpm for 10 min at 4°C. The liquid collected at the bottom of the tube was the Akkermansia myxophilus EV suspension, which was stored at -80°C.

[0025] Transmission Electron Microscopy (TEM) Imaging of Akkermansia myxophilus EVs: The Akkermansia myxophilus EVs prepared in this example were fixed with 2% paraformaldehyde at 4°C for 30 min. 10 μL of the suspension was added dropwise to a carbon-coated Formvar copper mesh and incubated at room temperature for 20 min. After washing three times with PBS, the EVs were fixed with 2.5% glutaraldehyde at room temperature for 15 min, rinsed with double-distilled water, negatively stained with 2% uranium acetate for 60 s, blotted dry with filter paper, and air-dried at room temperature. Imaging was performed using a Tecnai G2 SpiritBioTWIN TEM (accelerating voltage 80 kV). After positioning under low magnification (100-700×), images were acquired under high magnification (20000-50000×), with ≥3 non-overlapping fields of view captured for each sample. The diameter of ≥100 EVs was measured using ImageJ software, and the size distribution and typical morphological characteristics were statistically analyzed.

[0026] Nanoflow cytometry particle size distribution tracking of Akkermansia myxophilus EVs: EV samples prepared in this example were treated with 0.1% Triton X-100, and purity was assessed by changes in particle concentration. EVs were labeled with PKH67 fluorescent dye (final concentration 2 μM) at 37°C in the dark for 15 min, and resuspended after removing free dye. A standard curve of scattered light intensity versus particle size was established using a series of silica nanoparticles, and refractive index differences were corrected using Michaelis-Menten theory. Detection was performed on a nanoflow cytometer (NanoAnalyzer) with an excitation wavelength of 488 nm triggered by side scattering (SS) signals. Samples were diluted 1:200 and loaded at a flow rate of 10 μL / min, with at least 50,000 samples collected per sample. Background was excluded using SS gating, and labeling efficiency was calculated when the PKH67 fluorescence intensity exceeded three times the standard deviation of the negative control. Particle size distribution was obtained by fitting the standard curve.

[0027] Example 2: Akkermansia myxophilus EVs alleviate host intestinal damage induced by Escherichia coli O157 infection. This embodiment uses SPF-grade, 3-week-old C57BL / 6J mice, with 12 hours of lighting daily, ample water and food, and a room temperature maintained at 24±1℃. The offspring are randomly divided into three groups (n=5): a blank control group (Con), a model group (EHEC), and an intervention group (AkkEVs). On day 20, offspring in each group are administered 50 mg / kg streptomycin sulfate via gavage. Starting on day 21, the intervention group is administered Akkella myxophilia EVs (prepared in Example 1) at a concentration of 10 μg / mL via gavage every morning for 10 days, at a dose of 0.2 mL / mouse / day. Offspring in the EHEC and AkkEVs groups are given 2 × 10 ml of the solution every evening. 9 CFU / mL Escherichia coli O157 was administered via gavage at a dose of 0.2 mL / mouse / day. After 10 days of continuous infection, the offspring mice were euthanized by cervical dislocation and their colons were harvested. Changes in the weight and fecal shape of the offspring mice were recorded daily.

[0028] Histopathological analysis of the colon tissue of offspring mice: After euthanizing the mice by cervical dislocation, the colon of each group was harvested. A clean mid-colon segment of about 1 cm was cut out, and 1 mL of 4% histiocytogenetic fixative was added. The tissue was then dehydrated and embedded in paraffin for fixation. The embedded sample was cut into 4 μm sections. Subsequently, the sections were stained with hematoxylin and eosin and observed under an optical microscope at 10× and 40× magnification. Pathological scores were calculated based on the tissue sections.

[0029] By comparing the weight changes of offspring mice in each group during the infection period, it was found that compared with the control group, the weight gain of offspring mice in the Escherichia coli infection group was slow, while the weight gain of offspring mice in the intervention group recovered. Figure 1A). Comparison of colon length among groups revealed that, compared with the control group, the colon of offspring mice in the Escherichia coli infection group was significantly shortened, while the shortening of the colon in offspring mice supplemented with Akkermansia muciniphilis EVs was significantly alleviated. Figure 1 B) p<0.05; p<0.01). Colonic H&E sections showed that in the Escherichia coli-infected group, the colonic villi of offspring mice were significantly shortened, crypts were severely atrophied with metaplasia, and the serosa and muscularis were significantly separated, with mild to moderate inflammation; while in the group supplemented with Akkermansia myxophilus, the colonic crypts of offspring mice showed significant recovery, and the pathological score was significantly reduced. Figure 1 C) p<0.05; p<0.01).

[0030] Detection of Reg3β levels in the colon of offspring mice: To further verify whether Akkermansia muciniphila EVs resist infection by increasing antimicrobial peptides, approximately 0.1 g of colon from each group of mice was taken, 1 mL of pre-cooled sterile PBS was added, and the mixture was thoroughly homogenized on a tissue homogenizer. According to the manufacturer's instructions, the level of Reg3β in the intestinal tissue supernatant was quantitatively determined using a mouse regenerating family member 3β (Reg3β) ELISA kit.

[0031] ELISA results showed that, compared with the control group, the Reg3β level in the intestines of offspring mice infected with Escherichia coli was significantly decreased; while in the supplementation group, the Reg3β level was significantly increased. These results suggest that supplementation with Akkermansia myxophilus EVs can effectively increase the expression level of the intestinal antimicrobial peptide Reg3. Figure 1 D) p<0.001) Example 3: Akkermansia myxophilus EVs induce M1 macrophage training for immunity Immunization training of M1 macrophages was induced using Akkermansia muciniphilis EVs prepared in Example 1. Raw264.7 mouse mononuclear macrophage cells were transferred from liquid nitrogen to a -80°C freezer and kept for 24 h. After being removed from the freezer, the cells were quickly placed in a 37°C water bath for 1 min until the cryopreservation solution was completely thawed. The cells were then sterilized and placed in a clean bench in the cell culture room. The liquid was transferred to a 5 mL EP tube, and 2 mL of prepared complete cell culture medium (DMEM medium + 10% fetal bovine serum + 1% P / S) was added. The cells were mixed by pipetting and centrifugation (1200 rpm, 3 min). The supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium and added to a cell culture dish. Another 7 mL of complete cell culture medium was added, and the cells were shaken well using the cross-hatching method. The cell state was observed under an inverted microscope, and the cells were incubated in a cell culture incubator containing 5% CO2 for 1-2 days. Once the cells have grown to approximately 70%-80% confluence on the plate, they can be passaged. After aspirating the culture medium, wash twice with sterile PBS, add 1 mL of trypsin, and incubate for 3 min. Remove the plate and gently shake until the cells detach. Observe under a microscope that the cells have almost completely detached. Add 1 mL of culture medium to stop the digestion, transfer to a new 5 mL EP tube, add 2 mL of complete culture medium to resuspend, and centrifuge. During this process, add 7 mL of complete culture medium to the new cell culture dish for later use. After centrifugation, collect the precipitate, add 1 mL of complete culture medium to resuspend, and evenly add it to the culture dish. Shake well using the cross method and examine under a microscope. Incubate in a 5% CO2 cell culture incubator for 1-2 days.

[0032] When the Raw264.7 cells reached 80% confluence, the cells were seeded into 6-well plates with cell spreaders to achieve a cell density of 102 cells per well. 5 Cell / well. Akkermansia myxophilus was grown in brain heart broth for 72 h. Extracellular vesicles (EVs) were extracted using an extracellular vesicle extraction and purification kit and diluted to a protein concentration of 10 μg / mL. EV suspension and staining solution were prepared using the PKH26 red cell membrane staining kit and incubated in the dark for 5 min. Excess dye was neutralized with FBS, and 100 μL of red fluorescently labeled EVs was added to each well for 0, 6, and 12 h. At different time points, the smears were removed, cells were fixed with 4% paraformaldehyde for 1 h, washed three times with PBS, and incubated overnight at 4°C with CD86 primary antibody (1:200). Then, Alexa Fluor 488 (1:200) was added and incubated for 1 h. The cells were then washed five times with PBS, stained with DAPI for 3 min, washed three times with PBS, and mounted. The red and green fluorescence of Raw264.7 cells was observed under a fluorescence microscope.

[0033] Log-phase Raw264.7 macrophages were seeded at an appropriate density in cell culture plates and cultured in a 37°C, 5% CO2 incubator for 12 h. When the cell confluence reached 70%-80%, the cells were divided into groups to establish a macrophage training immune model. Akkermansia myxophilus EVs, a training immunopositive control group (β-glucan, and an equal volume of empty medium) were added to the cells. The Akkermansia myxophilus EVs treatment group received Akkermansia myxophilus EVs at a predetermined concentration and incubated in a cell culture incubator for 12 h to complete the training phase. After training, the medium was discarded, cells were washed with sterile PBS and replaced with fresh complete medium, and the cells were placed in an incubator for 3 days of rest to simulate the resting phase after in vivo immune cell training, allowing the cells to establish a stable trained immunophenotype. After rest, lipopolysaccharide (LPS) (a classic M1 macrophage polarization inducer) at a final concentration of 100 ng / mL was added to each well to simulate the immune stimulation scenario of pathogen reinvasion in vivo. Six hours after the second stimulation, the cell supernatant was collected from each group. The secretion level of nitric oxide (NO) in the supernatant was detected using the nitrate reductase method, and the expression level of TNF-α was quantitatively detected using enzyme-linked immunosorbent assay (ELISA). The NO levels among the three groups were compared. The study investigated the differences in TNF-α secretion. Cells were treated with *Ackermannia myxophilus* EVs at protein concentrations of 10 μg / mL and 100 μg / mL for 24 h. After incubation, cell supernatants were collected, and TNF-α concentration was detected using a TNF-α ELISA kit. The cell pellet obtained after centrifugation was incubated in a dark room with 1 μL of CD16 / 32 blocking antibody and CD86 flow cytometry antibody for 30 min, and M1 cell polarization levels were detected by flow cytometry. The induced effect of *Ackermannia myxophilus* EVs on the training immunity of Raw264.7 cells was evaluated.

[0034] Akkermansia myxophilus EVs were obtained using a bacterial EV extraction kit. Their morphology and size were observed using transmission electron microscopy. The results showed that the Akkermansia myxophilus EVs exhibited a classic double-membrane vesicle spherical structure, varying in size. Figure 2 A). The size distribution of Akkermansia myxophilus EVs was determined by nanoflow cytometry. The results showed that the particle size distribution of Akkermansia myxophilus EVs was between 50 and 100 nm. Figure 2 B). Compared with the control group, the proportion of M1 macrophages was significantly increased after LPS treatment, and the proportion of M1 macrophages was significantly increased after stimulation with both high and low doses of Akkermansia myxophilus EVs. P<0.01, P<0.001) Figure 2 C). TNF-α levels in cell supernatants were simultaneously detected using ELISA. The results showed that, compared with the control group, the TNF-α levels in the cell supernatants of both the LPS group and the Akkermansia xylophilus EVs group were significantly increased. P<0.01, P<0.001) Figure 2 D).

[0035] HIF-1α-regulated aerobic glycolysis reprogramming constitutes a key metabolic feature of monocyte-macrophage training for immunity. β-glucan induction leads to a significant upregulation of HIF-1α expression in macrophages, promoting glucose metabolism towards aerobic glycolysis, thereby enhancing the macrophage's inflammatory response to secondary stimuli (such as LPS). This invention establishes an M1 macrophage training and immunity model of Akkermansia myxophilus EVs (…). Figure 3 A). The results showed that, compared with the control group, β Glucan training treatment significantly induced NO and TNF levels in cell supernatant. Treatment with Akkermansia myxophilus EVs can produce similar regulatory effects on α-secretion, including NO and TNF. The secretion level of α was also significantly increased. P<0.01, P<0.001) Figure 3 B, C). Fluorescent dyes were used to label *Ackermannia myxophilus* EVs and the M1 marker CD86, respectively. The results showed that the intensity of both red and green fluorescence signals increased with incubation time, suggesting that *Ackermannia myxophilus* EVs can be gradually phagocytosed and internalized by Raw264.7 cells over time, thereby mediating subsequent biological effects. Figure 3 D). Raw264.7 cells were pretreated with the HIF-1α specific inhibitor PX-478 (10 μM), followed by induction of M1 macrophage polarization by Akkermansia myxophilus EVs to investigate the regulatory role of this signaling pathway. Results showed that the proportion of M1 macrophages was significantly increased in the Akkermansia myxophilus EV treatment group compared to the control group; this induction effect was significantly reversed after pretreatment with the HIF-1α inhibitor PX-478, and M1 macrophage polarization was significantly inhibited. P<0.001) Figure 3E). The above results demonstrate that Akkermansia myxophilus EVs can exert an immune-training effect on M1 macrophages in a HIF-1α-dependent manner.

[0036] Example 4: Akkermansia myxophilus EVs indirectly activate Reg3 secretion in intestinal cells via TNF-α: Human colon cancer cell HT-29 cryopreservation tubes were transferred from liquid nitrogen to a -80°C freezer. After removal from the freezer, the tubes were quickly placed in a 37°C water bath for 1 min until the cryopreservation solution was completely thawed. The tubes were then sterilized and placed in a clean bench in the cell culture room. The liquid was transferred to 5 mL EP tubes, and 2 mL of prepared complete cell culture medium (McCoy's 5A medium + 10% fetal bovine serum + 1% P / S) was added. The mixture was pipetted and centrifuged (1200 rpm, 3 min). The supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium and added to a cell culture dish. Another 7 mL of complete cell culture medium was added, and the cells were vortexed using the cross-hatching method to mix. The cell condition was observed under an inverted microscope, and the cells were incubated in a 5% CO2 cell culture incubator for 1-2 days. When the cells reached approximately 70%-80% confluence, they were passaged. The culture medium was aspirated, and the cells were washed twice with sterile PBS. 1 mL of trypsin was added, and the cells were incubated for 3 min. After removing the cells, shake the culture dish evenly until they detach. Under a microscope, observe that the cells have almost completely detached. Add 1 mL of culture medium to stop the digestion, transfer the cells to a new 5 mL EP tube, add 2 mL of complete culture medium to resuspend the cells, and centrifuge. During this process, add 7 mL of complete culture medium to the new cell culture dish for later use. After centrifugation, take the precipitate, add 1 mL of complete culture medium to resuspend the cells, and add the precipitate evenly to the culture dish. Shake well using the cross method and examine under a microscope. Incubate in a 5% CO2 cell culture incubator for 1-2 days.

[0037] When HT-29 cells reached 80% confluence, they were digested and seeded into 6-well plates (102). 5 TNF-α was diluted to 0, 125, 250, and 500 ng / mL using 5% FBSMcCoy's 5A medium and added to 6-well plates for incubation for 12 h. Cells and cell supernatants were then collected. Rag3α concentration was detected using an ELISA kit. Collected cells were lysed with protein lysis buffer (PMSF 1:100) to prepare protein loading buffer, and Rag3α protein levels were detected by Western blotting.

[0038] Inoculate HT-29 into a 6-well plate (10 5After cell / well incubation, solutions containing the TNF-α receptor inhibitor TNFR1 Ab (5 μg / mL), the NF-κB inhibitor PDTC (Pyrrolidinedithiocarbamate) (10 μM), the MAPK inhibitor Adz (Adezmapimod) (10 μM), and the STAT3 inhibitor S31-201 (NSC 74859) (100 μM) were added to 6-well cell plates and incubated for 6 h. Each well was then treated with 500 ng / mL TNF-α stimulant for 12 h. The Rag3α concentration and protein level were measured in the cell supernatants.

[0039] Reg3β is a key component of the C-type lectin superfamily and one of the major antimicrobial peptides in mice. Reg3α (also known as HIP / PAP), encoded in the human genome, is a highly conserved homolog of Reg3β; the two not only share significant conserved amino acid sequences but also possess highly similar biological functions. This invention uses Western blotting to detect high concentrations of TNF. HT after alpha stimulation The expression level of Reg3α in 29 cells was analyzed, and the results confirmed that high concentrations of TNF... α can significantly induce HT The large-scale synthesis and expression of Reg3α in 29 cells ( P<0.001) Figure 4 A). The results showed that only the STAT3 inhibitor S31 Treatment with 201 significantly reduced Reg3α levels compared to TNF alone. There were no significant differences in the alpha stimulation group and the other inhibitor treatment groups. P<0.001) Figure 4 B). Immunoblot analysis also showed that, via S31 After 201 processing, HT The expression level of Reg3α protein in 29 cells was significantly reduced. P<0.01, P<0.001) Figure 4 C). Furthermore, immunoblotting results showed that TNF... Alpha stimulation can significantly upregulate HT Phosphorylated STAT3 (p) in 29 cells The ratio of STAT3 to total STAT3 suggests the presence of TNF. α can induce phosphorylation and activation of STAT3 ( P<0.001) Figure 4 D). The above results suggest that HT The secretion of Reg3α in cells 29 is not directly regulated by A. muciniphila EVs, but rather through TNF. α-activation indirectly regulates the TNFR1 / STAT3 signaling pathway.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A postbiotic preparation for preventing and improving intestinal pathogen infection in infants and young children, characterized in that, The postbiotic formulation consists of Akkermansia myxophilus exovesicles (EVs) and pharmaceutically acceptable carriers or excipients.

2. The post-biotic preparation according to claim 1, characterized in that, The metabiotic preparation contains Akkermansia muciniphila exovesicles (EVs) as the sole active ingredient.

3. The post-biotic preparation according to claim 2, characterized in that, The pharmaceutically acceptable carriers or excipients include, but are not limited to, fillers, diluents, binders, disintegrants, lubricants, coating materials, or enteric materials.

4. The post-biotic preparation according to claim 1, characterized in that, The method for preparing the Akkermansia muciniphila extravesicles (EVs) includes the following steps: (1) The bacteria were cultured in brain heart broth medium, the bacterial solution was collected and the supernatant was obtained by centrifugation and filtered through a microporous membrane to obtain the filtrate. (2) Add extracellular vesicle extraction reagent to the filtrate, incubate at 2-8℃, and collect the precipitate by centrifugation; (3) The precipitate was resuspended in sterile PBS, centrifuged and the supernatant was collected. The supernatant was filtered through an exosome purification column and the filtrate was collected to obtain the extracellular vesicle suspension of Akkermansia muciniphila.

5. The use of the metabiotic preparation according to any one of claims 1 to 4 or the Akkermansia muciniphila exovesicles (EVs) in the preparation of a medicament for treating Escherichia coli infection.

6. The application according to claim 5, characterized in that, The Acetophilic Akkermansia EVs induce intestinal M1 macrophages to train immunity.

7. The application according to claim 5, characterized in that, The described Akkermansia muciniphila EVs induce M1 macrophages to secrete TNF-α, and TNF-α continuously stimulates the colon to secrete regenerated islet-derived protein 3 intestinal antimicrobial peptides.

8. The application according to claim 5, characterized in that, The Escherichia coli is selected from enterotoxigenic Escherichia coli, enteroinvasive Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, or enteroaggregative Escherichia coli.

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