Mucin-tannic acid packaged probiotic preparation as well as preparation method and application thereof

Abstract

The invention belongs to the field of biotechnology and medical configuration products, and provides a mucoprotein-tannic acid packaged probiotic preparation as well as a preparation method and application thereof. According to the preparation method disclosed by the invention, tannic acid and mucoprotein are encapsulated in escherichia coli Nissle 1917 and lactobacillus plantarum NC8, so that the mucoprotein-tannic acid encapsulated probiotic preparation is obtained. The probiotic microbial agent prepared by the invention has excellent resistance to severe environment of gastrointestinal tracts, and the adhesiveness of the probiotic microbial agent in intestinal tracts is enhanced, so that the colonization and growth of the probiotics in a mucous layer are enhanced. Meanwhile, bacterial enteritis, especially enteritis caused by enterotoxigenic escherichia coli (ETEC), can be effectively treated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medical preparations, and particularly relates to a mucin-tannic acid encapsulated probiotic preparation and a preparation method and application thereof. Background Art

[0002] Bacterial enteritis is one of the major diseases that affect the healthy development of the livestock breeding industry. The most prevalent bacterial enteric pathogens include Escherichia coli, Campylobacter jejuni, Pseudomonas aeruginosa, Salmonella, and Salmonella typhi serotypes. Infection with enteric bacterial pathogens activates pathogen recognition receptors, leading to an innate response that promotes host defense.

[0003] Escherichia coli is an important pathogen, and nine pathological variations of E. coli strains isolated from humans that cause diarrhea and extraintestinal diseases have been revealed. Among them, seven pathological types have been described as enteropathogenic E. coli, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusible adherent E. coli (DAEC), and a new pathological type, adherent-invasive E. coli (AIEC), which mainly cause diarrhea and intestinal diseases.

[0004] Enterotoxigenic Escherichia coli (ETEC) is a common pathogen of swine colibacillosis, which can cause a variety of diseases and bring serious economic losses to the livestock industry. Currently, probiotic intervention strategy is a potential prevention and treatment method for intestinal diseases. A study has disclosed a super probiotic (EcN@TA-Ca 2+ @Mucin), which can significantly improve inflammation by removing reactive oxygen species, reduce the side effects of bacterial translocation in inflammatory bowel diseases, and increase the diversity of intestinal flora. However, it has not been involved in the treatment of bacterial enteritis, especially enteritis caused by enterotoxigenic Escherichia coli (ETEC).

[0005] Therefore, although there are many studies on the combination of traditional Chinese medicine and probiotics, the industry has not yet found a probiotic agent that can effectively treat bacterial enteritis, especially enteritis caused by enterotoxigenic Escherichia coli (ETEC). Summary of the invention

[0006] In order to solve the above problems, the present invention provides a mucin-tannic acid encapsulated probiotic preparation and its preparation method and application. The present invention is inspired by microbiome-based therapy. After screening, it is found that: Escherichia coli Nissle 1917 (hereinafter referred to as EcN) and Lactobacillus plantarum NC8 (hereinafter referred to as Lp) are used as composite bacterial agents, and mucin (hereinafter referred to as Mucin) and tannic acid (hereinafter referred to as TA) are used for encapsulation to construct an encapsulated probiotic system that can spontaneously regulate the pathological microenvironment of inflammatory diseases. Among them, there is no antagonism between Escherichia coli Nissle 1917 and Lactobacillus plantarum NC8, and the mucin and tannic acid shells have no effect on the normal growth of EcN and Lp. The obtained probiotic agent has excellent resistance to the harsh environment of the gastrointestinal tract and enhances its adhesion in the intestine, thereby strengthening the colonization and growth of probiotics in the mucus layer. At the same time, it can effectively treat bacterial enteritis, especially enteritis caused by enterotoxigenic Escherichia coli (ETEC).

[0007] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for preparing a mucin-tannic acid encapsulated probiotic preparation, comprising: Tannic acid and mucin were encapsulated in Escherichia coli Nissle 1917 and Lactobacillus plantarum NC8 to obtain mucin-tannic acid encapsulated probiotic preparations.

[0008] Preferably, the specific method of encapsulation includes: mixing Escherichia coli Nissle 1917 bacterial solution and Lactobacillus plantarum NC8 bacterial solution to obtain a mixed bacterial solution; The mixed bacterial solution is mixed with a tannic acid solution and a calcium chloride solution to react. After the reaction is completed, the solution is washed and centrifuged, the supernatant is discarded, and mucin is added and continuously rotated to obtain the product.

[0009] Preferably, the mass ratio of the mixed bacterial liquid to tannic acid, calcium chloride and mucin is 2:3:1:1-1.1.

[0010] Preferably, the method for preparing the Escherichia coli Nissle 1917 bacterial solution comprises: The Escherichia coli Nissle 1917 was revived and streaked, and a single colony was picked for culture in LB broth. Then, the Escherichia coli Nissle 1917 was inoculated into the LB broth at a ratio of 1 wt%-1.1 wt% and continued to be cultured. Finally, the culture was centrifuged, the supernatant was discarded, and the culture was washed and the supernatant was discarded.

[0011] Preferably, the method for preparing the Lactobacillus plantarum NC8 bacterial liquid comprises: The Lactobacillus plantarum NC8 was revived and streaked, and a single colony was picked for culture in MRS broth. Then, the Lactobacillus plantarum NC8 was inoculated into the MRS broth at a ratio of 1 wt%-1.1 wt% and continued to be cultured. Finally, the strain was centrifuged, the supernatant was discarded, and the strain was washed and the supernatant was discarded to obtain the strain.

[0012] The second aspect of the present invention provides a mucin-tannic acid encapsulated probiotic preparation prepared by the above method.

[0013] The third aspect of the present invention provides the use of the above-mentioned mucin-tannic acid encapsulated probiotic preparation in the preparation of a medicament for treating bacterial enteritis.

[0014] Preferably, the pathogen is enterotoxigenic Escherichia coli K88.

[0015] Preferably, the mucin-tannic acid encapsulated probiotic preparation has at least one of the following immune effects: (1) Promote CD80 + CD86 + CD11c + DCs, CD3 + CD4 + CD25 + Foxp3 + Treg proliferation and activation promote B220 in the mouse intestine + IgA + B cell proliferation; (2) Reduce the transcription levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-17, and IL-33 genes; (3) Increase the transcription levels of anti-inflammatory factors IL-10 and IL-22, and increase the expression of intestinal tight junction proteins ZO-1 and Occludin; (4) Improve weight loss; (5) Reduce the degree of damage to the small intestine and colon; (6) Improvement of Lactobacillus spp. in the ELTCM group ( Lactobacillus ) and Zürichbacillus ( Turicibacter ) abundance.

[0016] The fourth aspect of the present invention provides the use of the above-mentioned mucin-tannic acid encapsulated probiotic preparation in the preparation of a drug for resisting ETEC infection.

[0017] Beneficial effects of the present invention (1) The present invention uses mucin and tannic acid to encapsulate probiotics and develops a new type of encapsulated probiotics. The outer shell retains the function of probiotics, that is, it can adhere to the intestinal mucus layer and has an immune adjuvant effect. 2+@Mucin can effectively regulate the gut microbiome to restore intestinal homeostasis.

[0018] (2) The present invention found that the CD80 of mice immunized with encapsulated probiotics + CD86 + CD11c + DCs, B220 + IgA + B. CD3 + CD4 + CD25 + Foxp3 + The number of Tregs was significantly higher than that of the control group, indicating that encapsulated probiotics can promote the proliferation and differentiation of the above-mentioned immune-related cells. The mucin tannic acid encapsulated probiotics prepared by the present invention can increase the content of sIgA (secretory immunoglobulin A) in intestinal mucus after immunization of mice, and can improve the survival rate of mice after ETEC K88 infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 Antagonism detection between EcN and Lp, (A) EcN was evenly spread on LB agar plate, and the liquid in the Oxford cup was the Lp culture supernatant, (B) Lp was evenly spread on MRS agar plate, and the liquid in the Oxford cup was the EcN culture supernatant.

[0021] Figure 2 Growth curves of EcN wild strain and encapsulated bacteria, Lp wild strain and encapsulated bacteria, (A) OD values ​​of EcN wild strain and encapsulated bacteria; E is EcN wild strain, ETC is EcN@TA-Ca 2+ ETCM is EcN@TA-Ca 2+ @Mucin, (B) OD values ​​of wild-type Lp and encapsulated bacteria; Lp is the wild-type Lp, and LpTC is Lp@TA-Ca 2+ , LpTCM is Lp@TA-Ca 2+ @Mucin.

[0022] Figure 3 Observation of fluorescence signals of Lp alone and EcN strains carrying pPG1-EGFP plasmid, (A) blue fluorescence detection of Lp alone, (B) green fluorescence detection of EcN strains carrying pPG1-EGFP plasmid.

[0023] Figure 4Observation results of fluorescence signals of tannic acid stained with rhodamine B, mucin stained with FITC, and Lp. (A) Red fluorescence of tannic acid stained with rhodamine B, (B) green fluorescence of mucin stained with FITC, and (C) blue fluorescence of Lp.

[0024] Figure 5 Bacterial growth curves after simulating the harsh environment of the gastrointestinal tract. (A) Growth curve in LB broth after simulating the harsh environment of the gastrointestinal tract. (B) Growth curve in MRS broth after simulating the harsh environment of the gastrointestinal tract. EL is a mixed bacterial solution of EcN and Lp, and ELTC is EcN-Lp@TA-Ca 2+ 、ELTCM is EcN-Lp@TA-Ca 2+ @Mucin.

[0025] Figure 6 Effects of EL, ELTC, ELTCM, and ETEC K88 treatment on the survival rate of Caco-2 cells at different time periods, (A) 2 h (B) 4 h (C) 6 h (D) 8 h. Among them, control is Caco-2 cells, EL is EL and ETEC K88 co-cultured, ELTC is ELTC and ETEC K88 co-cultured, ELTCM is ELTCM and ETEC K88 co-cultured, and K88 is ETEC K88 cultured.

[0026] Figure 7 Macrophage uptake of EL, ELTC, ELTCM, and ETEC K88, (A) LB agar plate counting results, (B) MRS agar plate counting results.

[0027] Figure 8 Adhesion of Caco-2 cells to EL, ELTC, and ELTCM. (A) LB agar plate counting results (B) MRS agar plate counting results.

[0028] Fig. 9 Results of flow cytometry (FACS) detection of dendritic cells in spleen (SP), mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) of mice after immunization. A: CD11c in SP + Cell number; B: CD11c in MLNs + Cell number; C: CD11c in PPs + Cell number.

[0029] Fig.10 FACS detection results of dendritic cells in SP, MLNs and PPs after the last immunization of mice, A: CD11c in SP + Cell number; B: CD11c in MLNs +Cell number; C: CD11c in PPs + Cell number.

[0030] Fig.11 FACS detection results of B cell numbers in SP, MLN and PPs of mice after one immunization, A: IgA in SP + Cell number; B: IgA in MLNs + Cell number; C: IgA in PPs + Cell number.

[0031] Fig.12 FACS detection results of B cell numbers in SP, MLN and PPs after the last immunization of mice, A: IgA in SP + Cell number; B: IgA in MLNs + Cell number; C: IgA in PPs + Cell number.

[0032] Fig.13 FACS detection results of Treg cell numbers in SP and MLNs of mice after one immunization. A: FOXP3 in SP + Cell number; B: FOXP3 in MLNs + Cell number.

[0033] Fig.14 FACS detection results of Treg cell numbers in SP and MLNs after the last immunization of mice, A: FOXP3 in SP + Cell number; B: FOXP3 in MLNs + Cell number.

[0034] Fig.15 sIgA levels in mouse feces (A) and IgG levels in mouse serum (B). Fig.16 ELISA was used to detect the levels of TNF-α (A), IL-1β (B) and IL-10 (C) in the mouse serum after the last immunization.

[0035] Fig.17 RT-PCR was used to detect the transcription levels of TNF-α (A), IL-1β (B), IL-33 (C), IL-17 (D), IL-10 (E), and IL-22 (F) genes in the small intestine of mice.

[0036] Fig.18 RT-PCR was used to detect the transcription levels of TNF-α (A), IL-1β (B), IL-33 (C), IL-17 (D), IL-10 (E), and IL-22 (F) genes in mouse colon.

[0037] Fig.19Effects of encapsulated probiotics on the transcriptional levels of intestinal tight junction protein-related genes ZO-1 (A) and Occludin (B) in the small intestine of mice.

[0038] Fig. 20 Effects of encapsulated probiotics on the transcriptional levels of intestinal tight junction protein-related genes (A) and Occludin (B) in mouse colon.

[0039] Fig.21 Body weight changes of mice after ETEC K88 challenge, (A) Body weight changes of mice in the probiotic group within 72 hours after challenge, (B) Body weight difference of mice in the probiotic group at 72 hours after challenge, (C) Body weight changes of mice in the dirty cage group within 72 hours after challenge, (D) Body weight difference of mice in the dirty cage group at 72 hours after challenge.

[0040] Fig. 22 Colon tissue lesions and length detection, (A) Colon pathological changes, (from left to right: control, EL, ELTC, ELTCM); (B) Colon length.

[0041] Fig.23 Histopathological images of small intestine of mice in each group.

[0042] Fig.24 Colon histopathological images of mice in each group.

[0043] Fig.25 Species composition of the intestinal flora of mice in each group, (A) phylum level (B) genus level.

[0044] Fig.26 Alpha diversity analysis.

[0045] Fig. 27 Beta diversity analysis, (A) distance matrix and PCoA analysis (B) Hierarchical clustering tree diagram on the left and stacked bar chart of the top 10 genera in abundance on the right.

[0046] Fig.28 ASV / OTU Venn diagram represents the shared OTUs among different groups.

[0047] Fig.29 Species composition heat map.

[0048] Note: Figure 9-Figure 24 In the table, control refers to the PBS (phosphate buffered saline) treatment group, EL refers to the EL treatment group, ELTC refers to the ELTC treatment group, ELTCM refers to the ELTCM treatment group, D-EL refers to the dirty cage shared with the EL group, D-ELTC refers to the dirty cage shared with the ELTC group, and D-ELTCM refers to the dirty cage shared with the ELTCM group.

[0049] Figure 25-29 In the figure, ctrl is the PBS treatment group, EL is the EL treatment group, ELTC is the ELTC treatment group, ELTCM is the ELTCM treatment group, DEL is the dirty cage shared with the EL group, DELTC is the dirty cage shared with the ELTC group, and DELTCM is the dirty cage shared with the ELTCM group. DETAILED DESCRIPTION

[0050] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0051] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0052] In the following embodiments, E. coli Nissle 1917 (EcN), L. plantarum NC8 (Lp) and ETEC K88 (enterotoxigenic Escherichia coli) were purchased from BNCC; All reagents and culture media were commercially available.

[0053] Example 1 Detection of antagonistic effects of encapsulated strains Prepare LB agar plates, MRS agar plates and two Oxford cups respectively, take 100 μL of EcN and spread it evenly on the LB agar plate with a spreading stick, place the Oxford cup in the center of the LB agar plate, add Lp to the Oxford cup and incubate it in a 37 ℃ incubator for 14 h, take 100 μL of Lp, spread it evenly on the MRS agar plate with a spreading stick, place the Oxford cup in the center of the MRS agar plate, add EcN to the Oxford cup and incubate it in an anaerobic jar at 37 ℃ incubator for 14 h.

[0054] Example 2 Detection of whether mucin and tannic acid shells affect the normal growth of probiotics (1) EcN encapsulation alone: ​​Take 2 mL of EcN bacterial solution as the control group. Take 2 mL of EcN solution, centrifuge and discard the supernatant, wash twice with NaCl (sodium hydroxide), centrifuge and discard the supernatant, add 3 mL of tannic acid (TA) (1 mg / mL) and 1 mL of calcium chloride (CaCl2) (1 mg / mL), shake for 30 min, wash once with NaCl, centrifuge at 4000 rpm at 4 °C for 10 min, discard the supernatant, and then add 1 mL of PBS (phosphate buffer) to resuspend the resulting solution as EcN@TA-Ca 2+Take 2 mL of EcN, centrifuge and discard the supernatant, wash twice with NaCl, centrifuge and discard the supernatant, add 3 mL of tannic acid (TA) (1 mg / mL) and 1 mL of calcium chloride (CaCl2) (1 mg / mL), shake for 30 min, wash once with NaCl, centrifuge at 4000 rpm at 4 °C for 10 min, discard the supernatant, then add 1 mL of mucin and rotate continuously at 4 °C. The resulting solution is EcN@TA-Ca 2+ @Mucin.

[0055] (2) Encapsulation of Lp alone: ​​Take 2 mL of Lp bacterial solution as the control group. Take 2 mL of Lp solution and centrifuge to discard the supernatant, wash twice with NaCl, centrifuge to discard the supernatant, add 3 mL of tannic acid (1 mg / mL) and 1 mL of calcium chloride (1 mg / mL), shake for 30 minutes, wash once with NaCl, centrifuge at 4000 rpm for 10 minutes at 4 °C, discard the supernatant, and then add 1 mL of PBS to resuspend the resulting solution as Lp@TA-Ca 2+ Take 2 mL of Lp, centrifuge and discard the supernatant, wash twice with NaCl, centrifuge and discard the supernatant, add 3 mL of tannic acid (TA) (1 mg / mL) and 1 mL of calcium chloride (CaCl2) (1 mg / mL), shake for 30 min, wash once with NaCl, centrifuge at 4000 rpm at 4 °C for 10 min, discard the supernatant, then add 1 mL of mucin and rotate continuously at 4 °C. The resulting solution is Lp@TA-Ca 2+ @Mucin.

[0056] (3) Prepared bacterial solution EcN, EcN@TA-Ca 2+ 、EcN@TA-Ca 2+ @Mucin were added to LB broth, Lp, Lp@TA-Ca 2+ 、Lp@TA-Ca 2+ @Mucin was added to MRS broth and the OD value was measured every hour for a total of 12 hours to observe whether the mucin and tannic acid shell affected the normal growth of probiotics.

[0057] Example 3 Preparation of Encapsulated Probiotics (1) In a clean bench, resuscitate the EcN and Lp stored in the laboratory and incubate them at 37 °C for 14 h to obtain a plateau bacterial solution; (2) In a clean bench, streak the revived bacterial suspension on the surface of LB agar for EcN and on the surface of MRS agar for Lp and incubate at 37 °C for 14 h. (3) The next day, in a clean bench, pick a single colony and place it in 10 mL LB broth and 10 mL MRS broth, and shake at 37 °C overnight (14 h); (4) In a clean bench, add EcN and Lp to 10 mL LB broth and 10 mL MRS broth at a ratio of 1%, respectively, and incubate at 37 °C for 14 h. (5) In a clean bench, take 2 mL each of EcN and Lp, centrifuge and discard the supernatant, add NaCl to wash twice, and centrifuge and discard the supernatant.

[0058] Then, according to the strain (the number of viable bacteria was 1.0×10 9 CFU / mL), tannic acid, calcium chloride, and mucin were mixed in a volume ratio of 2:3:1:1. After the bacterial solution was mixed, 6 mL of tannic acid (1 mg / mL) and 2 mL of calcium chloride (1 mg / mL) were added, and the mixture was shaken for 30 min, washed once with NaCl, centrifuged at 4000 rpm at 4°C for 10 min, the supernatant was discarded, and 2 mL of mucin was added and continuously rotated at 4°C.

[0059] Example 4 Detection of Encapsulated Probiotics 1. Detection of encapsulated probiotic properties 1.1 Scanning electron microscope detection The prepared EcN, EcN@TA-Ca 2+ 、EcN@TA-Ca 2+ @Mucin performed scanning electron microscopy.

[0060] 1.2 Fluorescence microscopy After encapsulating the EcN carrying the pPG1-EGFP plasmid, the intensity of its green fluorescence signal was detected by fluorescence microscopy. Lp was stained with (10 μg / mL) DAPI (4',6-diamidino-2-phenylindole), 2 mL of Lp was centrifuged and the supernatant was discarded, the bacterial solution was gently resuspended with DAPI, and the supernatant was discarded after standing for 30-40 min, resuspended with PBS, and then dropped on the slide. Fluorescent Lp can be observed under a blue fluorescence microscope. Then, the strain containing the pPG1-EGFP plasmid and the Lp stained with DAPI were encapsulated using EcN. Take 2 mL of the bacterial solution EcN containing the pPG1-EGFP plasmid and 2 mL of Lp stained by DAPI in the stationary phase, wash twice with NaCl, discard the supernatant, mix the two bacteria, add 6 mL of tannic acid stained with rhodamine B (0.25 mol / L) and 2 mL of calcium chloride, oscillate for 30 min, wash once with NaCl, centrifuge at 4 ℃ 4000 rpm for 10 min, discard the supernatant, add 2 mL of mucin stained with FITC (fluorescein isothiocyanate, 1 mg / mL), and rotate continuously at 4 ℃. Under a fluorescence microscope, the autofluorescent EcN containing the pPG1-EGFP plasmid, tannic acid stained with rhodamine B under red light, mucin stained with FITC (1 mg / mL) under green light, and Lp stained with DAPI under blue light were observed. The strains overlapped under the four colors of light, proving that they were successfully encapsulated.

[0061] 2 Encapsulated probiotics tolerance test (1) Preparation of bacterial culture (a) Take 2 mL each of EcN and Lp at the stationary phase, centrifuge and discard the supernatant, wash twice with NaCl, then add 2 mL of PBS to resuspend, mix the two bacterial solutions and record them as EL.

[0062] (b) Preparation of EcN-Lp@TA-Ca 2+ (ELTC), take 2 mL of the bacterial solution EcN and Lp in the stationary phase, centrifuge and discard the supernatant, wash twice with NaCl, add 6 mL of tannic acid (1 mg / mL) and 2 mL of calcium chloride (1 mg / mL), shake for 30 min, wash once with NaCl, centrifuge at 4000 rpm at 4 ℃ for 10 min, discard the supernatant, and then add 2 mL of PBS.

[0063] (c) Preparation of encapsulated bacterial solution, and the prepared EcN-Lp@TA-Ca 2+ @Mucin (ELTCM) was dropped onto the plate for counting, and then adjusted to a concentration of 2.5×10 7 CFU / mL, to be used for incubation with cells.

[0064] (2) Probiotic stability testing The prepared bacterial solution EL, EcN-Lp@TA-Ca 2+ (ELTC), EcN-Lp@TA-Ca 2+ @Mucin (ELTCM) Take 1 mL of each bacterial solution as a group, and take two groups of bacterial solutions, group A and group B. Then add 4 mL of gastric juice to each bacterial solution in each group for suspension. Group A is incubated in a shaker and group B is incubated under anaerobic conditions. After 30 minutes, centrifuge at 4 ℃ and 1000 rpm for 10 minutes and discard the supernatant. Add 3 mL of intestinal juice to each bacterial solution of group A and group B, incubate for 1 hour, centrifuge at 4 ℃ and 1000 rpm for 10 minutes, discard the supernatant, add 3 mL of bile acid to each bacterial solution of group A and group B, incubate for 1 hour, centrifuge at 4 ℃ and 1000 rpm for 10 minutes, discard the supernatant, and resuspend with PBS. Then add the bacterial solution obtained from group A to three bottles of LB broth and culture in a 37 ℃ incubator, and add the bacterial solution obtained from group B to three bottles of MRS broth and culture under anaerobic conditions at 37 ℃. Then, each bacterial solution is tested for OD every hour. 600 The absorbance at 600 nm was measured for 40 h.

[0065] 3 Effects of encapsulated probiotics on Caco-2 cell activity The present invention adopts trypan blue staining method to detect cell activity, and the specific steps are as follows: (1) Cell recovery (2) Cell passaging (3) Cell culture When the passaged cells were fully spread in the cell bottle, the complete medium was discarded, and the cells were washed twice with pre-warmed PBS. Then, 2 mL of pre-warmed trypsin digestion solution containing 0.25 wt% EDTA was added to digest the adherent cells. The cells were placed in a 37°C, 5% CO2 incubator for 5 min. When the cells were completely detached, an equal volume of complete medium was added to terminate the digestion. The liquid in the cell bottle was extracted into two 2 mL centrifuge tubes, centrifuged at 4°C and 1000 rpm for 5 min, and the supernatant was discarded. The cells in the two centrifuge tubes were resuspended with 1 mL of complete medium, and the obtained cells were counted. The number of Caco-2 cells was about 1×10 5The cells were inoculated into a 24-well plate at a concentration of 100 / mL, and 45 μL-50 μL of cell suspension was added to each well. After that, 1 mL of complete medium was added to each well, and the mixture was gently shaken and placed in a 37 ℃, 5% CO2 incubator for about 36 h. After the cells were fully confluent, they were washed once with pre-warmed PBS. The above-prepared bacterial solution was co-cultured with Caco-2 cells at an infection multiplicity (MOI) of 100:1. Three replicates were performed in each group. The cells that were not co-cultured with bacteria in the experiment were used as controls. After that, the well plate was placed in a 37 ℃, 5% CO2 incubator for 2 h, 4 h, 6 h, and 8 h. After the co-culture, the medium was discarded, the cells were washed with pre-warmed PBS, and the adherent cells were digested with trypsin containing 0.25 wt% EDTA. The single cell suspension was appropriately diluted and mixed evenly with trypan blue dye at a ratio of 9:1. The mixture was added to the red blood cell counting plate and quickly inverted on a microscope to detect cell survival rate. Living cells are transparent and colorless, while dead cells appear distinctly blue.

[0066] 4. Detection of the effect of encapsulated probiotics on macrophage uptake The cultured cells were cultured to a cell number of 2.5 × 10 5 cell / well, and pass them to 24-well plates for subsequent experiments. Wash gently with PBS, discard the supernatant, and add 200 μL and 800 μL of the prepared bacterial solution EL, ELTC, ELTCM, and ETEC K88 to each well, with 3 replicates for each bacterial solution. Discard the uningested bacteria after 2 h of co-culture. Wash the cells twice with PBS, add 1 mL of RPMI 1640 incomplete medium containing 100 μg / mL gentamicin to each well, incubate for 1 h, discard the supernatant, add 500 μL of 1% Triton X-100 to each well, and place it in a 37°C, 5% CO2 incubator for 20 min -30 min, and then drop it onto MRS and LB agar plates for counting. Three replicates per group to reduce errors.

[0067] 4.5 Detection of the adhesion effect of encapsulated probiotics on Caco-2 cells The prepared bacterial suspensions EL, ELTC, and ELTCM were centrifuged at 5000 rpm for 10 min at 4 °C, the bacteria were collected, washed three times with pre-cooled PBS, and resuspended in MEM complete medium. The bacterial suspension was inoculated into a monolayer of Caco-2 cells (2.5×10 7 bacteria and 2.5×10 5Cells were cultured in a 37°C incubator with a volume concentration of 5% CO2. After 1 h, the complete medium was discarded and the cells were washed three times with pre-warmed PBS to remove the non-adherent bacteria. The washing was gentle and the cells should not fall off. After washing, 200 μL of 1% Triton X-100 was added to each well and the cells were cultured in a 37°C incubator for 30 min. The lysate was diluted with PBS at a ratio of 1:10 and counted on a drop plate. The cells were cultured in a 37°C incubator overnight. Three replicates were performed in each group to reduce errors.

[0068] Example 5 Study on the immunomodulatory function of encapsulated probiotics In the following implementation, the immunization dose (mouse / time) is 1.0×10 9 CFU / mL (0.2 mL).

[0069] 1. Immunization of mice with encapsulated probiotics 1.1 Preparation of encapsulated probiotic suspension The EcN and Lp strains were cultured at 37 °C for 12 h and then the colonies were counted. The average value was taken after three counts. The strains cultured at the same time were used to prepare EcN-Lp@TA-Ca 2+ @Mucin encapsulated probiotics for oral inoculation in mice.

[0070] 1.2 Experimental animal grouping and immunization scheme (1) Animal grouping: The healthy mice were randomly divided into 7 groups, namely, PBS group, EL group, ELTC group, and ELTCM group, with 12 mice in each group, and D-EL group, D-ELTC group, and D-ELTCM dirty cage group, with 10 mice in each group. The PBS group served as the control group.

[0071] (2) Immunization scheme: After 7 days of pre-feeding, oral immunization with probiotics was performed during the formal trial period. The mice were fasted for 2 hours before immunization and resumed normal feeding 1 hour after immunization. The mice in the Control group, EL group, ELTC group, and ELTCM group were orally immunized according to the above scheme for 7 consecutive days. After an interval of 7 days, the mice were given a booster immunization for 7 days (2×10 8 CFU / mL). The D-EL, D-ELTC, and D-ELTCM groups ate normally and shared dirty cages with the EL, ELTC, and ELTCM groups, respectively, and the bedding was changed every 3 days.

[0072] During the formal test period (0-7 days), one immunization was performed, and the immunization lasted for 7 days; the first flow cytometry test was performed on the 13th day. The last immunization was performed on the 15th day to the 21st day, and the flow cytometry test was performed on the 27th day. After the last immunization, 5 mice were randomly selected from each group to collect feces for 16S rRNA flora diversity detection.

[0073] 2. Serum Collection On the 0th, 8th and 22nd days (i.e. before the first immunization, after the first immunization and after the second immunization), three mice were randomly selected from each group for blood collection from the fundus venous plexus. The skin between the two ears was grasped, the mouse head was fixed to make the eyeball bulge outward, and a blood collection capillary was used to pierce the back of the eyeball from the inner canthus of the orbit. The blood was collected in a 1.5 mL centrifuge tube, incubated in a 37°C incubator for 1 h, in a 4°C refrigerator for 6 h, and centrifuged at 5000 rpm for 15 min. The obtained serum was stored at -40°C for later use.

[0074] 3. Fecal supernatant collection On the 0th, 8th and 22nd days (i.e. before the first immunization, after the first immunization and after the second immunization), three mice from each group were randomly selected, and the feces were collected and weighed, and stored in a sterile centrifuge tube. Then, sterile sodium chloride 3 times the weight of the feces was added and the feces were crushed and dissolved with the tail end of a 1 mL syringe. Then, protease inhibitors were added, and the tube was centrifuged at 5000 rpm for 15 min. The supernatant was collected and stored at -40 ℃ for later use.

[0075] 4. Flow cytometry (FACS) detection Three mice were randomly selected from the Control group, EL group, ELTC group, and ELTCM group, and blood was taken from the eyeballs of the mice. The mice were then killed by cervical dislocation. The spleen (SP), mesenteric lymph nodes (MLNs), and Peyer's lymph nodes (PPs) of the mice were collected in a clean bench. At the same time, different intestinal segments of the mice were taken and preserved in formaldehyde fixative and liquid nitrogen, respectively.

[0076] 4.1 Preparation of single cell suspension (1) Place a 200-mesh copper grid in a 35 mm sterile dish and add 1 mL FACS (flow cytometry) buffer (containing 2 wt% FBS (fetal bovine serum), EDTA (ethylenediaminetetraacetic acid), and RPMI 1640 culture medium). Place the tissue on the copper grid and place on ice. (2) Grind the tissue with the end of a 1 mL syringe, and filter through a copper mesh to obtain the ground tissue fluid; (3) Place the tissue grinding solution in a 1.5 mL centrifuge tube and place it on ice. Then centrifuge it at 1000 rpm for 10 min in a horizontal centrifuge precooled to 4 °C, and slowly aspirate and discard the supernatant. (4) Add 1 mL of pre-cooled red blood cell lysis buffer to the spleen cells, gently pipette to mix, place on ice for 5 min, then directly add an appropriate amount of FACS buffer, centrifuge at 1000 rpm for 5 min at 4 °C, and discard the supernatant; (5) Wash SP, MLNs, and PPs cell pellets once with 1 mL FACS buffer, centrifuge at 1000 rpm for 5 min at 4 °C, discard the supernatant, and resuspend the cells with 1 mL FACS buffer; (6) The cells of each group were diluted with PBS and counted. Approximately 1.0×10 7 cells; MLNs collected about 1.0×10 7 cells; PPs collected about 5.0×10 6 cells; (7) Wash the collected cells once, discard the supernatant, add 100 μL FACS buffer to resuspend, and store at 4°C for later use.

[0077] 4.2 DCs (Dendritic Cells) Detection (1) Add CD11c-percp-cy5.5 (40-fold dilution), CD80-PE (35-fold dilution), and CD86-FITC (35-fold dilution) antibodies to 100 μL of single-cell suspension. After mixing the four antibodies, add 40 μL (10 μL of each antibody working solution) to each sample and gently pipette to mix. Incubate at 4 °C in the dark for 30 min. (2) After incubation, add 150 μL FACS buffer, centrifuge at 1000 rpm for 5 min at 4 °C, and discard the supernatant to collect the cell pellet; (3) Add 200 μL FACS buffer to resuspend the cells, filter through a nylon mesh into a flow cytometer, add 100 μL FACS buffer and mix well. Store at 4°C until ready for testing.

[0078] 4.3 B lymphocyte detection (1) Add 10 μL of CD45R / B220-percp-cy5.5 (35-fold dilution) antibody to 100 μL of single-cell suspension, gently pipette to mix, and incubate at 4 °C in the dark for 30 min; (2) After incubation, add 150 μL FACS buffer, centrifuge at 1000 rpm for 5 min at 4 °C, and discard the supernatant to collect the cell pellet; (3) Add 80 μL of Fixation / Permeabilization working solution to resuspend the cells and incubate at 4 °C in the dark for 40 min. (4) After incubation, add 150 μL of permeabilization solution, centrifuge at 1000 rpm for 5 min at 4 °C, and discard the supernatant to collect the cell pellet; (5) Add 10 μL of IgA-FITC antibody (40-fold dilution), gently pipette to mix, and incubate at 4 °C in the dark for 30 min; (6) After the incubation, add 200 μL of permeabilization solution, centrifuge at 1000 rpm for 5 min at 4 °C, and discard the supernatant to collect the cell pellet; (7) Add 200 μL FACS buffer to resuspend the cells, filter through a nylon mesh into a flow cytometer, add 100 μL FACS buffer and mix well. Store at 4 °C until ready for testing.

[0079] 4.4 Treg (regulatory T cells) cell detection (1) Add 10 μL of CD25-PE (35-fold dilution) and CD4-PE-cy5 (35-fold dilution) antibodies to 100 μL of single-cell suspension, gently pipette to mix, and incubate at 4 °C in the dark for 30 min; (2) After incubation, add 150 μL FACS buffer, centrifuge at 2000 rpm at 4 °C for 5 min, remove the supernatant, and collect the cell pellet; (3) Add 80 μL of Fixation / Permeabilization working solution to resuspend the cells and incubate at 4 °C in the dark for 40 min; centrifuge at 4 °C, 1850 rpm, 6 min, and discard the supernatant; (4) After incubation, add 150 μL of permeabilization solution, centrifuge at 1850 rpm for 6 min at 4 °C, remove the supernatant, and collect the cell pellet; (5) Add 10 μL of Foxp3-FITC antibody (35-fold dilution), gently pipette to mix, and incubate at 4 °C in the dark for 30 min; (6) After the incubation, add 200 μL of permeabilization solution, centrifuge at 1850 rpm for 6 min at 4 °C, remove the supernatant, and collect the cell pellet; (7) Add 200 μL FACS buffer to resuspend the cells, filter through a nylon mesh into a flow cytometer, add 100 μL FACS buffer, mix well, and store at 4 °C until ready for testing.

[0080] 5 ELISA (enzyme-linked immunosorbent assay) test 5.1 Detection of sIgA (secretory immunoglobulin A) levels in mouse feces Take the mouse feces supernatant stored at -40°C in step 3, and use ELISA to detect the content of sIgA in mouse feces. The operating steps refer to the instructions of the kit.

[0081] 5.2 Detection of IgG (immunoglobulin G) levels in mouse serum Take the mouse serum stored at -40°C in step 2 and use ELISA to detect the content of mouse serum IgG. The operating steps refer to the instructions of the kit.

[0082] 5.3 Detection of inflammatory cytokine levels in mouse serum The mouse serum stored at -40°C in step 2 was taken and the levels of TNF-α, IL-1β and IL-10 in mouse serum were detected by ELISA. The operation steps were referred to the instructions of the kit.

[0083] 5.4 Detection of cytokine mRNA (messenger RNA) expression levels 5.4.1 Extraction of tissue RNA (ribonucleic acid) Take the small intestinal tissue stored in liquid nitrogen and extract its total RNA 5.4.2 Reverse transcription of total RNA from small intestinal tissue The extracted RNA was reverse transcribed, and the reverse transcription products were stored at -40°C for subsequent RT-PCR detection of the differences in transcription levels of intestinal inflammatory cytokines in different groups.

[0084] 5.4.3 RT-PCR detection Using the mouse Actin gene as the internal reference gene, Real-Time PCR primers were designed according to the DNA sequences of mouse inflammatory factors IL-1β, IL-10, IL-17, IL-22, IL-33, TNF-α and Actin on NCBI for RT-PCR detection.

[0085] 6. Statistical analysis Adoption 2 -ΔΔCt Methods Analysis Relative quantitative data analysis. Graphical Prism 8.3 software was used for data statistics and analysis. One-way ANOVA was used to test the results between the experimental groups. The differences in the analysis data were significant and the graphs were drawn (*, P < 0.05; **, P < 0.01;***, P < 0.001; ****, P < 0.0001) Example 6 Study on the prevention of intestinal barrier damage by encapsulated probiotics 1. Attack plan One week after the last immunization, i.e., on day 28, 6 mice were selected from each group and the ETEC K88 strain was cultured to the stationary phase. The bacterial solution was centrifuged and the supernatant was discarded to collect the precipitate, which was washed once with PBS and then resuspended in PBS to a concentration of 2×10 7 The mice were challenged with the virus by intraperitoneal injection for 3 days, and their body weights were measured every 12 hours to detect pathological changes including feces, blood in stool, and weight changes of mice.

[0086] 2. Histopathological examination 2.1 Tissue embedding and sectioning The duodenum, jejunum, ileum and colorectum of each group were embedded and sectioned.

[0087] 2.2 HE (Hematoxylin-eosin) staining The dried sections were placed in xylene for 8 min and in different concentrations of alcohol for gradient dehydration for 1 min each, and washed twice with water; stained with hematoxylin for 5 min and washed twice with water; differentiated with 1% hydrochloric acid alcohol for 5 s and washed twice with water; stained with eosin for 2 min and washed twice with water; sealed with neutral gum and examined under a microscope.

[0088] 3. Detection of intestinal flora and metabolites 3.1 Collection of mouse feces Take 5 mice from each group that have been immunized for the last time and place them in sterile cages. Wait for 10 minutes. Use tweezers to pick up the feces in the cages or boxes and put them in cryotubes. Take two tubes from each group and collect 4-6 feces in each tube. Immediately put the cryotubes containing feces on ice to prevent DNA degradation in the feces. After each group has taken all the feces, put the cryotubes in liquid nitrogen for storage.

[0089] 3.2 Extraction and PCR amplification of fecal microbial genomic DNA (deoxyribonucleic acid) The genomic DNA of mouse fecal microorganisms was extracted using a magnetic bead kit, and the purity and concentration of the DNA were detected by 1% agarose gel electrophoresis, and then the sample concentration was diluted to 20 ng / μL with sterile water. A pair of universal primers was used to amplify the V3-V4 hypervariable region of the 16S rRNA gene.

[0090] The reaction procedure was as follows: pre-denaturation at 98 °C for 2 min, denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and extension at 72 °C for another 5 min after 25-30 cycles.

[0091] 3.3 Metagenomic library construction, library testing and machine testing Paired-end sequencing of community DNA fragments was performed using the Illumina platform. The raw sequencing data was saved in FASTQ format (R1.fastq and R2.fastq, Read 1 and Read 2 sequences were paired one by one). After denoising or clustering, the sequences were compared with the reference sequence database (Greengenes) and species taxonomic annotation analysis was performed.

[0092] 4. Statistical analysis Data were processed using R and Python software. Statistical analysis methods such as LEfSe were used to test the species composition and community of the grouped samples and draw correlation diagrams. SPSS18.0 and Prism 8.3 were used to analyze the significant differences in data and draw maps (*, P <0.05; **, P <0.01;***, P <0.001; ****, P <0.0001).

[0093] Depend on Figure 1 It can be seen that EcN and Lp strains have no antagonistic effect and can be encapsulated at the same time.

[0094] Depend on Figure 2 It can be seen that there is no significant difference in the growth curves represented by the three colors. The results show that mucin and tannic acid shells have no effect on the normal growth of EcN or Lp.

[0095] The SEM results show that the surface of the EL strain is smooth, the surface of the ELTC has a small amount of wrinkles and membrane structure, and the ELTCM has a large number of wrinkles and an obvious shell structure. The above results show that EcN-Lp is successfully encapsulated.

[0096] Since the EcN strain carries the pPG1-EGFP plasmid, the EGFP (enhanced green fluorescent protein) it expresses exhibits green fluorescence ( Figure 4 B, while tannic acid stained with rhodamine B can be observed under red light ( Figure 4 In center A, mucin stained with FITC (fluorescein isothiocyanate) can be observed under green light ( Figure 4 In B, Lp stained with DAPI (4',6-diamidino-2-phenylindole) can be observed under blue light ( Figure 3 In the middle (A), the light spots at the same position overlap under different colors of fluorescence ( Figure 4 (C). This result indicates that EcN and Lp were successfully encapsulated.

[0097] As shown in Figure 5 (A) and (B), EL (EcN-Lp), ELTC (EcN-Lp@TA-Ca 2+)、ELTCM(EcN-Lp@TA-Ca 2+ @Mucin) After passing through the harsh environment of the gastrointestinal tract, the three groups of probiotics were added to LB and MRS agar media and cultured for 40 hours. The results showed that the ELTCM group of probiotics had stronger tolerance in both LB and MRS.

[0098] like Figure 6 As shown in the figure, compared with the Control group, when the probiotic MOI = 100 and the treatment time ≤ 6h, there was no significant difference in cell survival rate at each time point. When the challenge time was extended to 8 h, the cell survival rate of the K88 group was significantly lower than that of the Control group ( P <0.05), which was significantly lower than that of the ELTCM group ( P <0.01). As the cell infection time increases, the cell survival rate decreases. Therefore, based on the test results of the present invention, it is determined that the optimal time for K88 strain to infect cells is within 6 h.

[0099] like Figure 7 As shown in the LB agar plate count results, there was no significant statistical difference in the macrophage uptake ability of the ELTCM group compared with the ELTC group, EL group, and ETEC K88 group ( P >0.05). In the MRS agar plate count results, the ability of the ELTCM group to promote macrophage uptake was significantly different from that of the EL group ( P <0.05, but there was no significant difference in the ability to promote macrophage uptake compared with the ELTC group and the ETEC K88 group ( P >0.05).

[0100] like Figure 8 As shown in (A), in the LB agar plate counting results, the ELTC group and the EL group had significant differences in their ability to adhere to intestinal epithelial cells ( P <0.05), there was no significant difference between the ELTCM group and the EL group ( P >0.05). There was a significant difference in the adhesion ability of ELTCM group and ELTC group to intestinal epithelial cells ( P <0.01). Figure 8 As shown in (B), there was no significant difference in the adhesion ability of ELTC group and EL group to intestinal epithelial cells in the MRS agar plate counting results ( P >0.05), there was a significant difference in the adhesion ability of ELTCM group and EL group to intestinal epithelial cells ( P <0.01), which was significantly different from the ELTC group ( P <0.001). The results show that the encapsulated probiotic ELTCM group has a stronger adhesion ability to cells.

[0101] The results of the study on the immunomodulatory function of encapsulated probiotics are as follows Figure 10-Figure 20 As shown, it can be seen that: (1) EcN-Lp@TA-Ca 2+ @Mucin can promote the levels of fecal sIgA and serum IgG in mice, upregulate the gene expression levels of IL-10 and IL-22, and downregulate the gene expression levels of TNF-α, IL-1β, IL-33, and IL-17; (2) EcN-Lp@TA-Ca 2+ @Mucin can enhance the gene transcription levels of tight junction proteins ZO-1 and Occludin; EcN-Lp@TA-Ca 2+ @Mucin can promote CD80 + CD86 + CD11c + DCs, B220 + IgA + B. CD3 + CD4 + CD25 + Foxp3 + Proliferation and differentiation of Treg cells.

[0102] The results of the study on the effect of encapsulated probiotics on preventing intestinal barrier damage are as follows Figure 21-Figure 29As shown in Figure 27, the differences in the richness of samples in each group at the genus level are shown. The right side of the figure shows the information of the 10 most abundant bacterial communities, namely Lactobacillus, Desulfovibrio, Allobaculum, Turicibacter, Oscillospira, Adlercreutzia, Prevotella, X. Prevotella, Staphylococcus, and Bifidobacterium. Figure 29 shows the abundance of the top 20 genera, which are Coprococcus, Staphylococcus, Corynebacterium, Adlercreutzia, Ruminococcus, Helicobacter pylori, Oscillospira, Akkermansia, Desulfovibrio, Ruminococcus, Lactobacillus, Parabacteroides, Prevotella, [Prevotella], Clostridium, Bacteroides, Sutterella, Allobaculum, Turicibacter, Bifidobacterium. From this we can see that: (1) EcN-Lp@TA-Ca 2+ The colon length of mice in the @Mucin group did not change significantly, and the intestinal pathological damage caused by ETEC infection was reduced; (2) EcN-Lp@TA-Ca 2+ @Mucin can promote the relative abundance of Firmicutes and Bacteroidetes and reduce pathogenic bacteria Desulfovibrio (Desulfovibrio spp.) abundance.

[0103] (3) The bacterial flora environment that dirty cage mice are exposed to makes them resistant to ETEC infection and exerts its anti-disease or immunomodulatory effects.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a mucin-tannic acid encapsulated probiotic preparation, characterized in that: include: Tannic acid and mucin were encapsulated in Escherichia coli Nissle 1917 and Lactobacillus plantarum NC8 to obtain mucin-tannic acid encapsulated probiotic preparations.

2. The method for preparing the mucin-tannic acid encapsulated probiotic preparation according to claim 1, characterized in that: The specific encapsulation method comprises: mixing Escherichia coli Nissle 1917 bacterial solution and Lactobacillus plantarum NC8 bacterial solution to obtain a mixed bacterial solution; The mixed bacterial solution is mixed with a tannic acid solution and a calcium chloride solution to react. After the reaction is completed, the solution is washed and centrifuged, the supernatant is discarded, and mucin is added and continuously rotated to obtain the product.

3. The method for preparing the mucin-tannic acid encapsulated probiotic preparation according to claim 1, characterized in that: The mass ratio of the mixed bacterial liquid to tannic acid, calcium chloride and mucin is 2:3:1:1-1.

1.

4. The method for preparing the mucin-tannic acid encapsulated probiotic preparation according to claim 1, characterized in that: The preparation method of the Escherichia coli Nissle 1917 bacterial liquid comprises: The Escherichia coli Nissle 1917 was revived and streaked, and a single colony was picked for culture in LB broth. Then, the Escherichia coli Nissle 1917 was inoculated into the LB broth at a ratio of 1 wt%-1.1 wt% and continued to be cultured. Finally, the culture was centrifuged, the supernatant was discarded, and the culture was washed and the supernatant was discarded.

5. The method for preparing the mucin-tannic acid encapsulated probiotic preparation according to claim 1, characterized in that: The preparation method of the Lactobacillus plantarum NC8 bacterial liquid comprises: The Lactobacillus plantarum NC8 was revived and streaked, and a single colony was picked for culture in MRS broth. Then, the Lactobacillus plantarum NC8 was inoculated into the MRS broth at a ratio of 1 wt%-1.1 wt% and continued to be cultured. Finally, the Lactobacillus plantarum NC8 was centrifuged, the supernatant was discarded, and the supernatant was washed and discarded to obtain the product.

6. A mucin-tannic acid encapsulated probiotic preparation prepared by the method according to any one of claims 1 to 5.

7. Use of the mucin-tannic acid encapsulated probiotic preparation according to claim 6 in the preparation of a medicament for treating bacterial enteritis.

8. Use of the mucin-tannic acid encapsulated probiotic preparation according to claim 6 in the preparation of a drug for treating bacterial enteritis, characterized in that: The pathogen is enterotoxigenic Escherichia coli K88.

9. Use of the mucin-tannic acid encapsulated probiotic preparation according to claim 6 in the preparation of a drug for treating bacterial enteritis, characterized in that: The mucin-tannic acid encapsulated probiotic preparation has at least one of the following immune effects: (1) Promote CD80 + CD86 + CD11c + DCs, CD3 + CD4 + CD25 + Foxp3 + Treg proliferation and activation promote B220 in the mouse intestine + IgA + B cell proliferation; (2) Reduce the transcription levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-17, and IL-33 genes; (3) Increase the transcription levels of anti-inflammatory factors IL-10 and IL-22, and increase the expression of intestinal tight junction proteins ZO-1 and Occludin; (4) Improve weight loss; (5) Reduce the degree of damage to the small intestine and colon; (6) Improvement of Lactobacillus spp. in the ELTCM group ( Lactobacillus ) and Zürichbacillus ( Turicibacter ) abundance.

10. Use of the mucin-tannic acid encapsulated probiotic preparation according to claim 6 in the preparation of a drug for resisting ETEC infection.

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