Multifunctional engineering probiotic preparation, preparation method thereof and application of multifunctional engineering probiotic preparation in preparation of medicine for treating clostridium difficile infection

Abstract

The invention belongs to the technical field of biological medicines, and relates to a multifunctional engineering probiotic preparation, a preparation method thereof and application of the multifunctional engineering probiotic preparation in preparation of medicines for treating clostridium difficile infection. The preparation comprises probiotics, and the surface of the probiotics is sequentially coated with a phenylboronic acid chitosan compound and hyaluronic acid; the probiotics are bifidobacterium bifidum, and the phenylboronic acid chitosan compound is formed by compounding phenylboronic acid chitosan and beta-hydroxybutyric acid. The invention further discloses a preparation method of the feed additive. The multifunctional engineering probiotic preparation provided by the invention integrates immune regulation, targeted colonization and metabolic reprogramming functions, not only can enhance intestinal colonization, relieve colon injury and inflammation and reduce the recurrence rate of clostridium difficile infection, but also can recover microbial diversity and metabolic steady state and enhance the host protection effect, so that the preparation has a good application prospect. Therefore, an excellent CDI treatment effect is shown.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a multifunctional engineered probiotic preparation, its preparation method, and its application in the preparation of drugs for treating Clostridium difficile infection. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Clostridium difficile ( Clostridioides difficile Clostridium difficile is a Gram-positive anaerobic bacillus widely found in soil, water, and the intestines of humans and animals. Under normal circumstances, the gut microbiota inhibits Clostridium difficile colonization and pathogenicity through colonization resistance. However, in hospitalized patients undergoing long-term antibiotic or proton pump inhibitor treatment, gut microbiota dysbiosis often occurs, promoting excessive proliferation and toxin production of Clostridium difficile, thus inducing Clostridium difficile infection. Clostridioides difficile CDI (Coronary Infection) is mainly characterized by self-limiting diarrhea and fever, but in severe cases it can lead to dehydration, hypoproteinemia, and even death. Currently, antibiotics such as metronidazole, fenamimycin, and vancomycin (VAN) are commonly used to treat CDI. However, antibiotic treatment can further disrupt the gut microbiota, leading to recurrent CDI and thus limiting its effectiveness.

[0004] Probiotic therapy is considered a promising strategy. Studies have shown that Saccharomyces boulardii (Saccharomyces boulardii)... Saccharomyces boulardii ), Lactobacillus ( Lactobacillus Probiotics and probiotic blends can alleviate chronic gut microbiota infections (CDIs) by modulating host immunity and restoring the gut microbiota. However, gastrointestinal factors such as gastric acid, pepsin, and pancreatic enzymes can impair probiotic activity, and rapid intestinal transit can limit their colonization, thus reducing the stability of probiotic therapy. Therefore, to achieve effective treatment of CDIs, an innovative strategy is needed that can simultaneously regulate the gut microbiota, modulate host immunity, and improve the intestinal colonization efficiency of bacterial strains. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a multifunctional engineered probiotic preparation, its preparation method, and its application in the treatment of Clostridium difficile infection (CDI). The multifunctional engineered probiotic preparation provided by this invention integrates immune regulation, targeted colonization, and metabolic reprogramming functions. It not only enhances intestinal colonization, reduces colonic damage and inflammation, and lowers the recurrence rate of CDI, but also restores microbial diversity and metabolic homeostasis, enhancing host protection and thus exhibiting excellent therapeutic effects for CDI.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, a multifunctional engineered probiotic preparation includes probiotics, wherein the surface of the probiotics is sequentially coated with a phenylboronic acid-chitosan complex (PCB) and hyaluronic acid (HA); the probiotics are Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum (abbreviated as BB), the chitosan complex is formed by combining phenylboronic acid chitosan (PC) and β-hydroxybutyric acid (BHB).

[0007] This invention first demonstrates, through bioinformatics analysis of 16S rRNA data from CDI patients, that Bifidobacterium (Bifidobacterium) Bifidobacterium Bifidobacterium abundance decreased significantly. KEGG analysis showed upregulation of the host immunity-related NOD-like receptor signaling pathway, which was negatively correlated with Bifidobacterium abundance. Cross-species data validation using mouse colon RNA-seq data indicated that the NOD-like receptor pathway is a key target. Supplementation with Bifidobacterium may regulate this pathway. Furthermore, enhancing Bifidobacterium's ability to regulate the NOD-like receptor signaling pathway while supplementing with Bifidobacterium is expected to improve CDI. BHB can inhibit NLRP3 inflammasome activation and alleviate inflammatory diseases, and is a targeted inhibitor of NLRP3; moreover, BHB has good biocompatibility. Therefore, the combined application of BHB and Bifidobacterium can synergistically regulate the gut microbiota and host immunity.

[0008] The genus Bifidobacterium includes many strains, such as Bifidobacterium adolescentis (… Bifidobacterium adolescents is Bifidobacterium bifidum ( Bifidobacterium bifidum Bifidobacterium longum ( Bifidobacterium longum ) and Bifidobacterium animalis ( Bifidobacterium animalis ), etc.; This invention has found that Bifidobacterium bifidum ( Bifidobacterium bifidum Bifidobacterium bifidum is selected in this invention because it not only has good compatibility with BHB (BHB does not inhibit the growth of the strain), but also has good colonic adhesion ability.

[0009] Furthermore, this invention encapsulates probiotics by forming a dynamically reversible complex through the conjugation of PC and BHB, followed by HA encapsulation as a "protective shield" to enhance delivery. HA not only protects the activity of probiotics but also degrades in the colon, achieving targeted release.

[0010] The multifunctional engineered probiotic preparation provided by this invention can maintain the activity of probiotics, enhance intestinal colonization, and promote the release of probiotics and BHB, thereby achieving a synergistic therapeutic effect.

[0011] On the other hand, a method for preparing an engineered probiotic preparation according to the first aspect of the present invention includes the following steps: PC and BHB are mixed in solution to form PCB; Bifidobacterium bifidum was incubated with PCB, so that the PCB coated the surface of Bifidobacterium bifidum to prepare a formulation precursor (BB@PCB). The formulation precursor is incubated with HA, allowing HA to self-assemble and coat the surface of the formulation precursor, thus obtaining the engineered probiotic formulation (BB@PCB-HA).

[0012] Thirdly, the use of an engineered probiotic preparation as described in the first aspect of the present invention in the preparation of a medicament for treating Clostridium difficile infection.

[0013] Fourthly, a method for treating Clostridium difficile infection includes: An effective amount of the engineered probiotic preparation of the first aspect of the present invention is administered to a subject.

[0014] The beneficial effects of this invention are as follows: The engineered probiotic formulation provided by this invention can scavenge ROS and regulate NLRP3 signaling in vitro. Oral administration enhances intestinal colonization of probiotics, reduces colonic damage and inflammation, and lowers the recurrence rate, showing better efficacy than vancomycin treatment. Multi-omics analysis shows that this engineered probiotic formulation restores microbial diversity and metabolic homeostasis, particularly significantly improving tryptophan and nucleotide metabolism-related pathways, thereby enhancing host defense capabilities.

[0015] The engineered probiotic formulation provided by this invention integrates immune regulation, targeted colonization, and metabolic reprogramming functions. In preclinical models, its efficacy is superior to conventional antibiotic treatment, and it establishes a translatable clinical framework for precise microbial intervention in CDI and related gastrointestinal diseases. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1This invention employs cross-species bioinformatics analysis to determine probiotic screening and material design strategies. (A) Schematic diagram of public clinical data mining and cross-species validation. (B) Schematic diagram of 16S rRNA sequencing of human fecal samples. (C) Principal coordinate analysis (PCoA) of the gut microbiota. (D) Alpha diversity index of the gut microbiota. (E) Composition of the gut microbiota at the phylum level. (F) Heatmap of the gut microbiota at the genus level. (G) Relative abundance of differentially expressed microorganisms. (H) Schematic diagram of KEGG primary pathway classification. (I) Differential analysis of immune system-related signaling pathways. (J) Heatmap of the correlation between differentially expressed gut microbiota and signaling pathways. (K) Partial least squares discriminant analysis (PLS-DA) of differentially expressed genes (DEGs). (L) Volcano plot of differentially expressed genes. (M) Gene set enrichment analysis (GSEA) of the NOD-like receptor signaling pathway.

[0018] Figure 2 The following are examples of the design and characterization of engineered probiotic formulations in this invention. (A) Schematic diagram of the construction of the engineered probiotic formulation. (B) Particle size analysis of the engineered probiotic formulation. (C) Transmission electron microscopy (TEM) image of the engineered probiotic formulation. (D) Zeta potential of the engineered probiotic formulation. (E) Fourier transform infrared spectroscopy (FTIR) analysis of the engineered probiotic formulation. (F) Quantitative fluorescence analysis of FITC-chitosan. (G) Surface roughness analysis of the engineered probiotic formulation. (H) Morphological characteristics of the engineered probiotic formulation. (I) Confocal laser scanning microscopy (CLSM) analysis of the engineered probiotic formulation. (J) Representative atomic force microscopy (AFM) image of the engineered probiotic. (K) Scanning analysis of representative atomic force microscopy (AFM) images of the engineered probiotic.

[0019] Figure 3 This invention illustrates the gastrointestinal protective effect and targeted delivery performance of the engineered probiotic formulation. (A) Schematic diagram of in vitro and in vivo analysis of the targeting and adhesion characteristics of the engineered probiotic formulation. (B) Survival analysis of probiotics after co-incubation with artificial gastric juice (SGF) and artificial intestinal juice (SIF). (C) Survival images of probiotics after co-incubation with artificial gastric juice (SGF) and artificial intestinal juice (SIF). (D) Survival rate curves of probiotics after co-incubation with artificial gastric juice (SGF) and artificial intestinal juice (SIF). (E) Fluorescence imaging of the engineered probiotic formulation in mice at different time points. (F) In vitro fluorescence imaging of the engineered probiotic formulation after 72 hours. (G) In vitro fluorescence imaging of normal mice and CDI mice after 12 hours. (H) FITC staining images of corresponding colon tissues.

[0020] Figure 4This invention evaluates the therapeutic effect of engineered probiotic preparations on a mouse model infected with Clostridium difficile. (A) Schematic diagram of the experimental procedure. (B) Mouse body weight change curve. (C) Mouse survival rate change curve. (D) Representative image of the colon. (E) Statistical analysis of the colon. (F) Representative HE-stained images and histological scores of colon tissue. (G) Level of TNF-α in the colon. (H) Level of IL-1β in the colon. (I) Level of IL-6 in the colon. (J) Level of IL-18 in the colon. (L) Level of IL-10 in the colon. (M) Representative immunofluorescence images and quantitative analysis of NLRP3, IL-1β, Occludin, Claudin-1, and ZO-1.

[0021] Figure 5 This invention evaluates the therapeutic effect of engineered probiotic preparations on a mouse model of recurrent Clostridium difficile infection. (A) Schematic diagram of the experimental procedure. (B) Mouse body weight change curve. (C) Mouse survival rate curve. (D) Representative image of the colon. (E) Statistical analysis of the colon. (F) Representative image and histological score of colon tissue stained with HE (scale bar 20 μm). (G) TNF-α content in the colon. (H) IL-1β content in the colon. (I) IL-6 content in the colon. (J) IL-18 content in the colon. (L) IL-10 content in the colon. (M) Representative immunofluorescence images and quantitative analysis of NLRP3, IL-1β, Occludin, Claudin-1, and ZO-1.

[0022] Figure 6 This is an evaluation of the effect of engineered probiotic preparations on the restoration of intestinal flora in mice infected with Clostridium difficile in this invention. (A) Alpha diversity index of intestinal microbiota. (B) Non-metric multidimensional scaling (NMDS) plot of intestinal microbiota. (C) Composition diagram of intestinal microbiota. (D) LEfSe analysis results. (E) Relative abundance of differentially expressed microorganisms. (F) Heatmap of correlation analysis between differentially expressed intestinal microorganisms and various indicators.

[0023] Figure 7This is an evaluation of the effect of engineered probiotic preparations on the recovery of intestinal metabolism in mice infected with Clostridium difficile in this invention. (A) Venn diagram of differential intestinal metabolites in the CON, CDI, and BB@PCB-HA groups. (B) Venn diagram of differential intestinal metabolites in the BB, BB@PCB, and BB@PCB-HA groups. (C) Partial least squares discriminant analysis plot of intestinal metabolites. (D) Heatmap of intestinal metabolites. (E) Volcano plot of differential intestinal metabolites between the CON and CDI groups. (F) Volcano plot of differential intestinal metabolites between the BB@PCB-HA and CDI groups. (G) KEGG compound classification analysis of differential intestinal metabolites between the CON and CDI groups. (H) KEGG compound classification analysis of differential intestinal metabolites between the BB@PCB-HA and CDI groups. (I) KEGG pathway analysis of upregulated intestinal metabolites in the CON and CDI groups. (J) KEGG pathway analysis of downregulated intestinal metabolites in the CON and CDI groups. (K) KEGG pathway analysis of upregulated gut metabolites in the BB@PCB-HA and CDI groups. (L) KEGG pathway analysis of downregulated gut metabolites in the BB@PCB-HA and CDI groups. (M) Heatmap of Mantel analysis of gut microbiota, gut metabolites, and phenotypic indicators. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0026] In view of the need for an effective strategy that can simultaneously regulate the gut microbiota, modulate host immunity, and improve the gut colonization efficiency of bacterial strains to achieve effective treatment of CDI, this invention proposes a multifunctional engineered probiotic preparation, its preparation method, and its application in the treatment of Clostridium difficile infection.

[0027] A typical embodiment of the present invention provides a multifunctional engineered probiotic preparation comprising probiotics, wherein the surface of the probiotics is sequentially coated with PCB and HA; the probiotics are Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidumThe PCB is formed by combining PC and BHB.

[0028] In some embodiments, PC is composed of carboxyphenylboronic acid and chitosan through the formation of amide bonds. The carboxyphenylboronic acid can be 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid, etc.

[0029] In some embodiments, the mass ratio of PC to BHB is 1:18~22, g / mmol.

[0030] In some embodiments, the ratio of PC to probiotics is 1: [(2~3) × 10 9 ], g / CFU.

[0031] In some embodiments, the ratio of HA to probiotics is 10: [(0.5~1.5) × 10⁻⁶]. 9 ], mg / CFU.

[0032] Secondly, a method for preparing the above-mentioned engineered probiotic preparation includes the following steps: PC and BHB were mixed in solution to prepare a chitosan complex (PCB). Bifidobacterium bifidum was incubated with PCBs, allowing the chitosan complex to coat the Bifidobacterium bifidum. Bifidobacterium bifidum On the surface of the PCB, a formulation precursor (BB@PCB) is prepared. The formulation precursor (BB@PCB) is incubated with HA, allowing HA to self-assemble and coat the surface of the formulation precursor, thus obtaining the engineered probiotic formulation (BB@PCB-HA).

[0033] In some embodiments, the feed ratio of PC to BHB is 1:18~22, g / mmol.

[0034] In some embodiments, the temperature at which PC and BHB are mixed in the solution is 35-40 °C. Specifically, the mixing time is 3.5-4.5 hours.

[0035] In some embodiments, Bifidobacterium bifidum ( Bifidobacterium bifidum The temperature for incubating PC with PCB is 35-40℃. Specifically, the incubation time is 1.5-2.5 hours. Specifically, PC and Bifidobacterium bifidum (… Bifidobacterium bifidum The ratio is 1: [(2~3) × 10 9 ], g / CFU.

[0036] In some embodiments, the formulation precursor is incubated with HA at a temperature of 35–40 °C. Specifically, the incubation time is 0.8–1.2 hours. Specifically, HA is incubated with Bifidobacterium bifidum (… Bifidobacterium bifidum The ratio is 10: [(0.5~1.5) × 10 9 ], mg / CFU.

[0037] Thirdly, the application of the aforementioned engineered probiotic preparation in the preparation of a drug for treating Clostridium difficile infection.

[0038] In some embodiments, the drug is a composition comprising an active ingredient and pharmaceutical excipients, wherein the active ingredient is the engineered probiotic preparation. The pharmaceutical excipients are pharmaceutical carriers and / or excipients. The pharmaceutical carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, etc. The excipients include, but are not limited to, binders, fillers, stabilizers, prebiotics, vitamins, minerals, preservatives, sweeteners, etc. The content of the pharmaceutical excipients in the drug composition can be 1% to 98% by weight.

[0039] In some embodiments, the dosage form of the drug is tablets, capsules, granules, drops, oral liquids, powders, etc.

[0040] Fourthly, a method for treating Clostridium difficile infection includes: The above-mentioned engineered probiotic preparation was administered to the subjects in an effective amount.

[0041] The "effective dose" as used in this invention refers to a dose that can achieve treatment and / or prevention of the disease or condition described in this invention in a subject.

[0042] The term "subject" in this invention may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the engineered probiotic preparation described in this invention to treat and / or prevent the diseases or conditions described in this invention.

[0043] In some embodiments, the administration to the subject may be by oral, topical (skin, mouth, etc.), inhalation, rectal, or intravenous injection.

[0044] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0045] Example Materials and Methods 1. Materials Phenylated chitosan (PC) was purchased from Meiluo Technology Co., Ltd. BHB (purity ≥98%) was purchased from Aladdin Biotechnology. HA (purity ≥99%) was from Maclean Biotechnology. Antibiotics including kanamycin, colistin, metronidazole, gentamicin, clindamycin, vancomycin, and cefoxitin were purchased from Reagent Biotechnology. FITC and FITC-WGA were purchased from Xi'an Qiyue Biotechnology; DiR was purchased from Beijing Fluorescent Biotechnology. ELISA kits were purchased from Enzyme-Linked Biotechnology. Antibodies were from Wuhan Sanying Biotechnology Co., Ltd. All other reagents were of analytical grade.

[0046] Clostridium difficile ( Clostridioides difficile The strain (ATCC accession number 43593) was purchased from the American Type Culture Collection (ATCC). *Clostridium difficile* was cultured in brain heart extract medium. After reaching the optimal concentration (OD600 = 0.8), the culture was diluted 10⁻⁶ times. 7 The culture was inoculated onto a Clostridium difficile selective medium (CCFA agar, 2.5% D-cyclic serine, 2.5% egg yolk emulsion, 1.6 mg cefoxitin) to obtain single colonies of Clostridium difficile. These single colonies were re-inoculated onto brain and heart infusion medium, cultured to an appropriate concentration, centrifuged, and resuspended in sterile physiological saline. Bifidobacterium bifidum (… Bifidobacterium bifidum (Strain preservation number CICC 10395), Bifidobacterium adolescentis ( Bifidobacterium adolescents is The strain preservation number is CICC 6070), Bifidobacterium longum ( Bifidobacterium longum The strain preservation number is CICC 6192), Bifidobacterium animalis ( Bifidobacterium animalis The strain (CICC 6250) was purchased from the China Industrial Microbial Culture Collection Center (CICC). All Bifidobacterium strains were cultured in Clostridium-enriched medium.

[0047] 2. Bioinformatics analysis of public datasets A microbiome dataset containing 514 fecal 16S rRNA samples (PRJNA419097, PRJNA493204, PRJNA307992, PRJNA386260) was processed using QIIME 2. After filtering out low-quality samples (<20 genera), 473 samples remained: 221 healthy volunteers and 252 CDI patients. Community composition and β-diversity were analyzed using PCoA. Functional profiles were predicted by mapping taxonomic units to KEGG pathways and categorized into six higher-order categories and 39 lower-order modules, with a focus on immune-related functions. Pathway differences were tested using limma's rankSumTestWithCorrelation and Bonferroni correction; the correlation between bacterial genera and immune pathways was assessed using Spearman analysis.

[0048] To perform cross-species validation, mouse colon RNA-seq datasets (PRJNA995763, PRJNA896182, PRJNA897872, PRJNA767246, PRJNA630214, PRJNA754089, PRJNA517543, PRJNA598958, PRJNA706375, RJNA563499, PRJNA1142690, 108 samples) were retrieved. After quality control (fastp, Q30 ≥80%), 107 samples remained (76 healthy, 31 CDI). Transcriptome differences were examined using PLS-DA (mixOmics), and differentially expressed genes (DEGs) (log2FC>1, after correction) were identified. P <0.05). GSEA highlights the NOD-like receptor signaling pathway.

[0049] 3. Synthesis and Characterization of BB@PCB-HA 20 mmol BHB and 2 g PC were dissolved in 5 mL of deionized water and stirred at 37 °C for 4 hours to obtain PCB stock solution; HA was dissolved alone at 10 mg / mL. BB cells were collected by centrifugation (3000 rpm, 5 min, 4 °C), washed twice with PBS, and resuspended to 1 × 10⁻⁶. 9 CFU / mL. 5 mL of the suspension was incubated with the entire PCB stock solution at 37°C with shaking for 2 hours, followed by centrifugation (6000 rpm, 5 min, 4°C) and washing to remove excess PCB. The resulting BB@PCB precipitate was resuspended in deionized water, mixed with 5 mL of HA solution, and incubated with shaking at 37°C for 1 hour. Finally, unbound HA was washed to remove the BB@PCB-HA engineered probiotic formulation. The BHB concentration in the final formulation was determined using a commercial assay kit (MA-0132, Akrivisbio, USA). Encapsulation efficiency (EE) and loading (LC) were calculated using the following formulas: EE (%) = [(Total mass of BHB - Mass of free BHB) / Total mass of BHB] × 100 (%) LC (%) = [(Total mass of BHB - Mass of free BHB) / Mass of carrier BHB] × 100 (%) The morphology of probiotics was examined by TEM at an accelerating voltage of 200 kV. Zeta potential and particle size were measured using a Malvern Zetasizer Nano ZS (Malvern Panalytical Ltd., UK). Sample turbidity was assessed using UV-Vis absorbance at 600 nm and digital imaging. BB cells were stained with Hoechst 33342, and the coating layer was visualized using FITC-chitosan. To verify successful coating, confocal laser scanning microscopy (CLSM; WD03040303, Oxford Instruments, Shanghai, China) was used, and further verification and characterization of surface morphology and roughness were performed using atomic force microscopy (ACST-AFM, ACST, USA).

[0050] 4. Therapeutic effect in CDI-induced colitis model Thirty-six male C57BL / 6J mice (6 weeks old) were used for the experiment after a one-week acclimatization period. At week 7, a CDI model was induced. Except for the control group, mice received antibiotics (kanamycin, gentamicin, colistin, metronidazole, vancomycin) in their drinking water for 3 days, followed by intraperitoneal injection of clindamycin to further disrupt the gut microbiota. After a 24-hour fast, mice were orally administered a Clostridium difficile suspension (10... 8 (CFU / mL, 0.2 mL); the control group received a sterile vector. During treatment, each group received either a sterile vector, BB, BB@PCB, BB@PCB-HA, or vancomycin (20 mg / kg / day). Body weight and disease index were monitored. Feces were collected for 16S sequencing and metabolomics analysis; colons were collected for histological observation, cytokine detection, and immunofluorescence analysis.

[0051] 5. Preventive effect in rCDI-induced colitis model Sixty male C57BL / 6J mice (5 weeks old) were used for the experiment after a one-week acclimatization period. Following initial CDI induction, the mice were treated with vancomycin (20 mg / kg / day), and then re-challenged with Clostridium difficile (10 mg / kg / day) after antibiotic discontinuation. 8 A rCDI model was established using CFU / mL (0.2 mL). The control group received a sterile vector. During treatment, the CON and CDI groups received a sterile vector, while the BB, BB@PCB, and BB@PCB-HA groups were administered the corresponding formulations by gavage. Body weight and disease activity index were monitored daily. At the end of the study, mice were sacrificed, and colons were collected for histological observation, cytokine detection, and immunofluorescence analysis.

[0052] 6. Gut microbiome analysis Fecal samples were collected (n = 6 per group) and stored at -80 °C. DNA was extracted and sequenced, and raw reads were stored in NCBI SRA. Sequences were processed using DADA2 in QIIME2 (v2024) to generate ASV and species annotation tables. Alpha and Beta diversity were analyzed in QIIME2 and visualized using ggplot2. NMDS analysis was performed based on Bray-Curtis distance to assess community structure differences among groups. Microeco and LEfSe (LDA score > 2) were used. P <0.05) Differential taxa were identified. Taxa were associated with pathways using KEGG mapping, and Spearman correlation heatmaps showing their correlation with cytokines, body weight, and colon length were visualized. All analyses were performed using the MegaBio cloud platform.

[0053] 7. Intestinal metabolomics analysis Fecal samples were collected (n = 6 per group) and stored at -80 °C. Metabolomics analysis based on LC / MS was performed by Megibio. Common metabolites were quantified, and PLS-DA, volcano plots, and cluster heatmaps were generated using mixOmics and ggplot2 in R. Differential metabolites were defined as VIP > 1 and FDR < 0.05. Identified metabolites were mapped to KEGG pathways, and enrichment analysis was performed using hypergeometric distribution. P <0.05). KEGG enrichment results were visualized using a lollipop plot. The Mantel test was used to assess the correlation between bacterial phyla and metabolites, while a pheatmap was used to assess their association with phenotypic indicators.

[0054] 8. Animal Research Ethics C57BL / 6J mice (purchased from Chongqing Tengxin Biotechnology Co., Ltd.) were housed in a controlled environment with a temperature maintained at 22 ℃, a relative humidity of 50%, and a light / dark cycle of 12 h / 12 ​​h. Mice had free access to sterile feed and sterilized drinking water. Water was sterilized at 132 ℃ and 205.8 kPa, and sterile feed was purchased from the same supplier.

[0055] 9. Statistical Analysis Data are expressed as mean ± standard error. Statistical analysis was performed using GraphPad Prism v.8 (GraphPad Software Inc., San Diego, USA). The statistical methods used were one-way ANOVA with Bonferroni correction. Tukey's test was also used for multiple comparisons to compare significant differences between the two groups. P A value <0.05 is considered statistically significant. * indicates P<0.05, ** indicates P <0.01.

[0056] result 1. Public clinical data mining and cross-species validation To explore the key pathogenic factors and potential therapeutic interventions of CDI, bioinformatics analysis was performed on publicly available sequencing datasets, and cross-species validation was conducted. Figure 1 A). After removing low-quality samples, 221 stool samples from healthy individuals and 252 stool samples from CDI patients were retained. Figure 1 B). Principal coordinate analysis (PCoA) showed significant differences in gut microbiota composition between healthy individuals and patients with CDI (Brain Disorders of Diagnosis). Figure 1 C). Compared with healthy controls, the ACE index, Chao index, and Shannon index were significantly decreased, while the Simpson index was increased, indicating a significant decrease in microbial diversity and uneven community distribution in CDI patients. Figure 1 D). Phylum-level analysis showed a decrease in the abundance of Actinobacteria and an increase in the abundance of Proteobacteria. Figure 1 E). In the genus-level analysis, Bacteroides ( Bacteroides Brucella ( ) Blautia ), genus *Faecalibacterium* Faecalibacterium Bifidobacterium spp. Bifidobacterium ) and Pseudomycium spp. Pseudobutyrivibrio — Significant differences exist between healthy individuals and patients with CDI (the top five genera with the lowest FDR). Figure 1 F, G). KEGG functional pathway analysis categorizes microbial activity into six modules (F, G). Figure 1 H). Immune-related pathway analysis showed that the NOD-like receptor signaling pathway was significantly enriched in CDI patients compared to healthy controls. Figure 1 I, and was negatively correlated with the abundance of Bifidobacteria ( Figure 1 These results support the rationale for using Bifidobacterium as a probiotic to regulate the gut microbiota and NOD-like receptor signaling pathway in patients with CDI.

[0057] Although 16S rRNA sequencing can predict microbial composition and the KEGG pathway, it cannot directly assess host gene expression. Given the scarcity of CDI patient biopsy samples, RNA-seq data from colon tissue of a CDI mouse model were analyzed for cross-species validation. After quality control, colon samples from 76 healthy mice and 31 CDI mice were retained for analysis. PLS-DA showed significant differences in gene expression between healthy and CDI mice. Figure 1K). Differential expression analysis identified 535 upregulated genes and 2,900 downregulated genes in CDI colon tissue. Figure 1 L). Gene set enrichment analysis (GSEA) showed that the NOD-like receptor signaling pathway was significantly enriched in CDI mice. Figure 1 (M), consistent with observations from human microbiome data. These results suggest that restoring Bifidobacterium abundance and modulating the NOD-like receptor signaling pathway may be helpful in treating CDI. Current evidence supports that Bifidobacterium supplementation can modulate host NOD-like receptor signaling, reduce the severity of CDI, and further enhancing this regulatory effect may improve its therapeutic efficacy.

[0058] 2. Rational design and characterization of BB@PCB-HA Based on the above findings, further development of probiotic formulations for the treatment of CDI is planned, aiming to achieve effective co-supplementation of Bifidobacteria and NOD-like receptor signaling pathway inhibitors in the colon. NLRP3 is a core inflammation-related receptor in the NOD-like receptor signaling pathway and a key effector of inflammation activation. C. difficile Toxins (TcdA and TcdB) mediate intestinal inflammation by activating NLRP3 and ASC. β-hydroxybutyrate (BHB), as an endogenous metabolite and an inhibitor targeting NLRP3, exhibits good biocompatibility. This embodiment systematically evaluated the compatibility of BHB with multiple Bifidobacterium strains: physiological concentrations of BHB inhibited Bifidobacterium adolescentis (… Bifidobacterium adolescentis ), but for BB ( Bifidobacterium bifidum Bifidobacterium longum ( Bifidobacterium longum ) and Bifidobacterium animalis ( Bifidobacterium animali No significant impact.

[0059] The colonic colonization and adhesion ability of probiotics is a key factor determining therapeutic efficacy and helps eliminate pathogens. Colonic adhesion assessment showed that BB exhibited the strongest intestinal adhesion, possibly attributed to its extracellular neuraminidase SiaBb2. Therefore, BB was selected as the engineered probiotic strain. PC binds to BHB via reversible borate bonds and carboxyl-assisted coordination to form a dynamic phenylboronic acid-BHB complex (PBA-BHB, PCB). Subsequently, PCB coats BB through specific recognition of surface polysaccharides and reversible covalent interactions, forming BB@PCB. Then, HA assembles onto BB@PCB through layer-by-layer self-assembly, yielding BB@PCB-HA. Figure 2A). The average particle size of BB was 2.63 μm, which increased to 3.76 μm after coating BB@PCB, and further to 4.17 μm for BB@PCB-HA. The zeta potential decreased from +38.7 mV to -13.51 mV after coating, demonstrating stronger stability. The average BHB encapsulation efficiency of BB@PCB-HA was 55.21%, corresponding to a dosage of 0.109 mg per serving. Transmission electron microscopy (TEM) showed that the coating structure resembled cell membrane armor, confirming successful HA encapsulation. HA interacts with the probiotic surface through electrostatic interactions, thereby enhancing its functional properties. Fourier transform infrared spectroscopy (FTIR) was also performed. Figure 2 E) shows that the -OH stretching vibration peak changes from 3408 cm⁻¹ in BB. -1 To BB@PCB and BB@PCB-HA 3289 cm -1 The shift occurred, while the COC stretching vibration peak changed from 1038 cm⁻¹. -1 Towards 1070cm -1 The offset indicates an electrostatic interaction between the PCB and HA. Macroscopic observation shows increased turbidity of the coating probiotics, possibly due to enhanced light scattering caused by uniform bacterial distribution. Figure 2 H). Confocal laser scanning microscopy (CLSM) confirmed tight co-positioning of the PCB and BB, promoting BHB internalization. Figure 2 I). The fluorescence intensity of BB@PCB-HA is 2.58 times that of BB@PCB, consistent with its gel-like surface morphology. Figure 2 F, 2H). Atomic force microscopy (AFM) analysis showed that the PCB formed a dense and uniform coating on the BB surface, while subsequent HA modification produced an irregular and heterogeneous surface, increasing the surface roughness from 87.32 μm to 117.52 μm. Figure 2 (G, 2J-K). In summary, BB@PCB-HA enhances the regulation of the NLRP3 signaling pathway and improves structural stability, thus supporting its potential application value in the treatment of CDI.

[0060] 3. Protective effect of BB@PCB–HA against H2O2 and TcdB-induced cell damage. CDI can induce oxidative stress and inflammation, leading to excessive ROS accumulation in colonic tissue. Since ROS is a potent activator of NLRP3, the ROS scavenging capacity of BB@PCB-HA was assessed. A H2O2-induced cell damage model was established, exhibiting a dose-dependent decrease in cell viability, with 2 mM chosen as the experimental concentration. Compared to the PBS control, both BB@PCB and BB@PCB-HA significantly improved cell viability and reduced the number of ROS-positive cells. Furthermore, malondialdehyde (MDA) levels, a marker of oxidative stress, were also significantly reduced by BB@PCB and BB@PCB-HA. These results suggest that BB@PCB and BB@PCB-HA have a more effective protective effect against oxidative stress-induced cell damage compared to BB alone, which may be attributed to the antioxidant properties of BHB.

[0061] TcdA primarily mediates enterotoxicity, while TcdB primarily induces cytotoxicity. To investigate the effect of BB@PCB-HA on NLRP3 signaling, a TcdB-induced cell damage model was used. Immunofluorescence analysis showed that BB, BB@PCB, and BB@PCB-HA all significantly reduced the expression of NLRP3 and C-Caspase-1, with BB@PCB and BB@PCB-HA showing stronger regulatory effects than BB alone. This may be due to the BHB-mediated inhibition, thereby mitigating TcdB-induced cytotoxicity. Mechanistically, NLRP3 acts as a core inflammasome sensor, and ASC connects NLRP3 to pro-caspase-1, promoting its cleavage into C-Caspase-1. Subsequently, C-Caspase-1 cleaves Gasdermin D (GSDMD) into GSDMD-N, inducing pyroptosis and processing pro-IL-1β and pro-IL-18 into mature cytokines. Western blot analysis showed that TcdB upregulated the levels of NLRP3, C-Caspase-1, GSDMD-N, and mature IL-1β / IL-18 compared to the control group (CON). BB, BB@PCB, and BB@PCB–HA inhibited the expression of these proteins to varying degrees, with BB@PCB–HA showing the most significant inhibitory effect. Quantitative analysis showed that the levels of NLRP3, C-Caspase-1, GSDMD-N, and mature IL-1β / IL-18 in the BB@PCB and BB@PCB–HA groups were significantly lower than those in the BB group, indicating that the addition of BHB enhanced the anti-inflammatory effect by targeting and regulating NLRP3 signaling.

[0062] In summary, BB@PCB-HA inhibits inflammasome activation through a dual mechanism: relying on the probiotic's own NLRP3 regulatory capacity on the one hand, and achieving targeted inhibition through BHB on the other. Simultaneously, BB@PCB-HA alleviates NLRP3-mediated cellular inflammation and oxidative damage by scavenging ROS, highlighting its potent protective effect against CDI-induced cytotoxicity and inflammatory damage.

[0063] 4. BB@PCB-HA enhances resistance to gastrointestinal stress. Efficient delivery of probiotics to the site of colonic lesions is crucial for achieving therapeutic efficacy. A major challenge in oral probiotic therapy is maintaining adequate therapeutic concentrations during passage through the gastrointestinal tract. Gastrointestinal tolerability of BB, BB@PCB, and BB@PCB-HA was assessed using simulated gastric juice (SGF) and simulated intestinal juice (SIF). Figure 3 A). After 1 hour of incubation in SGF, the number of viable bacteria in both BB@PCB and BB@PCB-HA was higher than that in BB, with BB@PCB-HA showing the highest number. After 2 hours of incubation, almost no viable bacteria were detected in BB, while viable bacteria were still detectable in both BB@PCB and BB@PCB-HA, with BB@PCB-HA showing a survival rate 2.14 times higher than BB@PCB. In SIF with added trypsin, BB@PCB and BB@PCB-HA remained stable after 2 hours of incubation, and decreased slightly after 4 hours, but the survival rate of BB@PCB-HA was still higher than that of BB@PCB. Figure 3 C). Growth curve analysis showed that most BB cells were inactivated, while BB@PCB cells survived but experienced impaired growth. These results indicate that BB@PCB–HA can effectively resist gastrointestinal stress, thereby enhancing the survival rate of probiotics, which is crucial for subsequent persistence and colonization.

[0064] 5. BB@PCB–HA enhances the body's ability to retain and target inflammation. Stable colonization of probiotics in the gut is crucial for niche occupation, restoration of colonization resistance, and effective treatment of chronic obstructive inflammatory disease (CDI). Considering the presence of the intestinal mucus layer, the HA on the BB@PCB–HA surface is hypothesized to interact with mucins via hydrogen bonding and electrostatic adsorption, thereby enhancing adhesion and colonization. In vivo retention and colonization capacity were assessed using IVIS and FITC staining. Figure 3 A). Mice were orally administered 1×10⁻⁶ gavage. 9 CFU BB@PCB-HA. DiR labeling combined with IVIS analysis showed that oral BB alone had limited retention, with almost no fluorescence signal after 48 hours, indicating limited colonization in vivo. BB@PCB and BB@PCB-HA showed prolonged intestinal retention, with BB@PCB-HA showing higher fluorescence intensity at 48 and 72 hours. Figure 3E). Quantitative analysis confirmed that the PCB coating enhances probiotic survival, while HA, acting as an outer "armor," further strengthens its structural stability in the gastrointestinal environment. In vitro IVIS imaging showed that after 72 hours, BB@PCB-HA was mainly localized in the colon, and its fluorescence was significantly higher than that of BB@PCB, indicating enhanced colonic colonization and retention capacity. Figure 3 F). HA modification may achieve local enrichment through degradation by colonic hyaluronidase, thereby avoiding premature release of BHB and maintaining probiotic activity.

[0065] To assess adhesion ability in the inflamed colon, BB colonization in CDI mice was monitored. The fluorescence intensity in the CDI+BB@PCB-HA group was significantly higher than that in the CON+BB@PCB-HA group, indicating enhanced retention of the engineered probiotic formulation in an inflammatory environment. Ex vivo intestinal imaging showed higher and more uniform fluorescence in the colon and cecum of CDI mice. Figure 3 These results indicate that BB@PCB-HA can rapidly localize to the inflamed colon, thereby promoting probiotic colonization. Enhanced colonization is partly attributed to accelerated gastrointestinal transit in CDI-associated diarrhea and inflammation-activated CD44 (Cluster of Differentiation 44). HA on the surface of BB@PCB-HA specifically recognizes CD44 and adheres to the site of inflammation, thus enhancing colonization in the lesion area. FITC staining of colon tissue sections further validated the colonization. Compared with the BB and BB@PCB groups, BB@PCB-HA colonization in colon tissue was significantly enhanced. In CDI mice, the fluorescence intensity of BB@PCB-HA was significantly higher than that of healthy controls and was mainly distributed in the inflamed area (…). Figure 3 In summary, these results directly demonstrate that BB@PCB-HA enhances intestinal colonization, targets and enriches bacteria in inflamed areas, and protects probiotic activity, thus supporting its efficacy in the treatment of CDI.

[0066] 6. The interventional efficacy of BB@PCB-HA on CDI-induced colitis The therapeutic potential of BB@PCB-HA was evaluated using a mouse CDI model. The model was established according to a standard protocol: the gut microbiota was disrupted by administration of an antibiotic mixture via drinking water, followed by oral gavage. C. difficile Mice were randomly assigned to five treatment groups, receiving PBS, BB, BB@PCB, BB@PCB-HA, or vancomycin (VAN) for five days, respectively. Figure 4 A). Healthy mice served as the control group.

[0067] Following CDI induction, both the CDI and BB groups exhibited significant weight loss and higher mortality rates. In contrast, BB@PCB and BB@PCB-HA significantly reduced weight loss and decreased mortality, with BB@PCB-HA showing the most significant therapeutic effect. Figure 4 B-4C). Quantitative analysis of colon length showed that, compared with the CDI group, the colon length of the BB@PCB, BB@PCB-HA, and VAN groups was significantly restored (B-4C). Figure 4 D-4E). H&E staining and comprehensive histological scoring (including loss of structural integrity, crypt and goblet cell damage, inflammatory infiltration, and edema) showed extensive inflammation and structural damage in the intestines of CDI mice. BB and BB@PCB could alleviate tissue damage, while the BB@PCB–HA and VAN groups showed more significant improvement, with BB@PCB–HA exhibiting the most significant repair effect. Figure 4 F). CDI can cause intestinal epithelial damage and activate the host immune response, triggering significant inflammation. Imbalance of inflammatory factors disrupts intestinal homeostasis, leading to tissue damage and exacerbating inflammation. ELISA analysis showed that BB treatment increased the level of the anti-inflammatory factor IL-10, while BB@PCB, BB@PCB-HA, and VAN treatments not only inhibited the expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-18, TNF-α) but also enhanced IL-10 expression, with BB@PCB-HA exhibiting the strongest anti-inflammatory effect. Overall, BB@PCB-HA was more effective than BB, BB@PCB, and VAN in alleviating CDI-induced inflammation. Figure 4 Studies have shown that BB, BHB, and HA each have anti-inflammatory effects, while BB@PCB–HA integrates these components to achieve the best therapeutic effect.

[0068] To elucidate the repair mechanism, the NLRP3 signaling pathway was detected by immunofluorescence. Compared with the CDI group, the fluorescence signals of NLRP3 and IL-1β in the colon tissue of the BB@PCB, BB@PCB–HA, and VAN groups were significantly reduced, with the most significant inhibition observed in BB@PCB–HA. Epithelial barrier integrity was assessed using tight junction proteins (ZO-1, Claudin-1, and Occludin). The colonic barrier of mice in the BB@PCB-HA group showed significant recovery, while the BB@PCB and VAN groups showed partial improvement. Figure 4 These results highlight the powerful role of BB@PCB–HA in anti-inflammation and barrier repair, consistent with the synergistic function of BB, BHB, and HA. The superior repair capacity of BB@PCB–HA compared to VAN may be due to VAN's ability to clear [inflammatory lesions / barriers]. C. difficile It also disrupts the gut microbiota, thereby exacerbating inflammation and barrier damage.

[0069] 7. The preventive efficacy of BB@PCB–HA against recurrent CDI-induced colitis (rCDI). Conventional VAN treatment for CDI often leads to rCDI. To address this issue, this embodiment establishes a VAN-induced rCDI mouse model and makes slight modifications to the method to more accurately simulate clinical relapse. CDI mice were treated with VAN 20 mg / kg on days 1 and 2, followed by drug withdrawal to allow for recurrent infection. Simultaneously, BB, BB@PCB, and BB@PCB-HA were administered for prophylactic and therapeutic interventions to evaluate their efficacy in preventing and treating rCDI. Figure 5 A).

[0070] Following rCDI induction, mice in both the rCDI and BB groups exhibited significant weight loss and a 50% mortality rate, indicating rapid disease progression. In contrast, BB@PCB and BB@PCB-HA significantly improved body weight and reduced mortality, with BB@PCB-HA showing a faster recovery rate than BB@PCB. Figure 5 B-5C). Colon length measurement and H&E staining showed that the colon in rCDI mice was significantly shortened and had histopathological damage, comparable to primary CDI. BB partially alleviated the tissue damage but did not significantly restore colon length. BB@PCB and BB@PCB-HA significantly reduced colonic lesions, with BB@PCB–HA showing the mildest colon shortening and the lowest histopathological score, close to the level of healthy controls. Figure 5 D–F), highlighting its therapeutic advantages. ELISA analysis showed that pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-18) were elevated in rCDI mice, consistent with primary CDI. BB treatment did not significantly increase the anti-inflammatory factor IL-10, indicating its limited preventive effect. In contrast, BB@PCB and BB@PCB-HA significantly inhibited pro-inflammatory factors and enhanced IL-10 expression, with BB@PCB-HA exhibiting the strongest anti-inflammatory effect ( Figure 5 Immunofluorescence showed that NLRP3 inflammasomes were significantly activated in the colonic tissue of rCDI mice, as evidenced by elevated NLRP3 and IL-1β signaling. BB had limited alleviating effect, while BB@PCB, especially BB@PCB-HA, significantly inhibited inflammasome activation. Immunostaining with tight junction proteins ZO-1, Claudin-1, and Occludin showed significant damage to the epithelial barrier in rCDI mice. BB slightly improved the intestinal barrier, BB@PCB significantly restored the intestinal barrier, and BB@PCB-HA almost restored the intestinal barrier to the level of healthy controls, indicating effective intestinal barrier repair. Figure 5 In summary, BB@PCB-HA effectively prevents and treats rCDI by enhancing survival, reducing colonic damage, regulating inflammatory factors, inhibiting NLRP3 activation, and restoring epithelial barrier integrity.

[0071] 8. Regulation of gut microbiota in CDI-induced colitis mice by BB@PCB–HA Alpha-diversity analysis showed that, compared with the CON group, the CDI group had significantly decreased Shannon and ACE indices, while the Simpson index increased, reflecting the decrease in microbial richness and evenness caused by the antibiotic regimen used to establish the CDI model. BB alone did not significantly alter alpha-diversity, while BB@PCB and BB@PCB-HA significantly increased the Shannon index and decreased the Simpson index, restoring microbial diversity to near-healthy control levels. Although VAN... C. difficile It is effective, but it significantly reduces Shannon and ACE indices, exacerbates gut microbiota dysbiosis, and may increase the risk of rCDI. Figure 6 A).

[0072] NMDS analysis showed that the gut microbiota of CDI mice was significantly disrupted. The microbiota of the BB, BB@PCB, and BB@PCB–HA groups were closer to those of healthy controls, while the VAN group remained far from normal levels, highlighting the advantage of BB@PCB–HA in restoring gut structure. Figure 6 B). Community bar charts showed that CON mice were predominantly Bacteroidota, Bacillota, and Verrucomicrobiota, while Pseudomonadota appeared in other groups, which may be a potential marker of CDI. At the genus level, BB@PCB-HA shifted the microbial composition towards that of healthy controls, and its effect was stronger than BB or BB@PCB. Figure 6 C). LEfSe analysis (LDA>3, FDR<0.05) showed that... Bifidobacterium It was significantly enriched in the CON group mice, while Escherichia- Shigella , Thomasclavelia and Clostridioides It was significantly enriched in CDI mice. VAN treatment led to... Enterococcus The dominance of BB@PCB–HA may promote rCDI, while BB@PCB–HA enrichment... Parabacteroides and Blautia Beneficial bacteria ( ) Figure 6 DE). Relative abundance analysis further confirmed that BB@PCB–HA most effectively enhances Bifidobacterium Increase the abundance of beneficial bacteria (such as...) norank_f_Muribaculaceae The abundance of ) while inhibiting pathogenic bacteria (such as Escherichia- Shigella The abundance of BB@PCB–HA and VAN can be reduced. ClostridioidesAbundance, but BB@PCB–HA achieved effective clearance without antibiotics. Correlation analysis showed that gut health indicators (body weight, colon length, Claudin-1, Occludin, ZO-1) and the anti-inflammatory factor IL-10 were positively correlated with probiotics, while pathogenic bacteria were positively correlated with pro-inflammatory factors (IL-1β, IL-6, IL-18, TNF-α, NLRP3) and higher histological scores. Bifidobacterium It showed the strongest positive correlation with host health indicators and a negative correlation with pro-inflammatory factors, highlighting its central role in reducing inflammation. Overall, BB@PCB-HA remodels the composition and function of the gut microbiota, enriches beneficial bacteria and inhibits pathogenic bacteria, and is more effective than VAN in restoring gut homeostasis. Figure 6 F).

[0073] 9. Regulation of intestinal metabolites by BB@PCB-HA To elucidate the intestinal microbiota metabolic mechanism of BB@PCB-HA treatment for CDI-induced colitis in mice, this study performed a non-targeted metabolomics analysis of colonic contents. Venn diagram analysis showed that the CON group and the BB@PCB-HA group shared 170 metabolites, which was higher than the 142 metabolites shared by the CON group and the CDI group. Figure 7 AB). PLS-DA showed that the metabolic profiles of each group were significantly different, with the CON group and the BB@PCB–HA group showing similar clustering ( ). Figure 7 C). Heatmap and volcano plot analyses showed that, compared to the CON group, the CDI group had 792 upregulated metabolites and 667 downregulated metabolites, while BB@PCB–HA treatment regulated 478 upregulated metabolites and 227 downregulated metabolites, respectively. Figure 7 DF). Differential metabolites are mainly enriched in carboxylic acids and amino acids (DF). Figure 7 CDI disrupts the correlation network between metabolites, generating a denser but less specific network, while BB@PCB–HA restores network balance.

[0074] KEGG enrichment analysis showed that nucleotide metabolism, tryptophan metabolism, and ABC transport pathways were downregulated in the CDI group, while BB@PCB-HA could restore these pathways. Figure 7 IL). Nucleotide metabolism supports microbial proliferation and intestinal barrier repair, while tryptophan metabolites can protect the mucosa and limit inflammation. Upregulation of the ABC transport pathway suggests... Bifidobacterium It may promote colonization by enhancing oligosaccharide uptake. Spearman correlation-based co-occurrence network analysis associated significantly altered microbiota with metabolites (LDA>3, VIP>1.8, FDR<0.05). Figure 7 M). Seven metabolites and Bifidobacterium Significant correlation (Mantel) P<0.05): 4-Hydroxyestradiol sulfate, Thr-Ala-Met, and Parylene C showed a positive correlation, while Lys-Asp-Phe, 3-hydroxywheat acid, 5-hydroxytryptamine, and asparagine showed a negative correlation. BB@PCB–HA enhances… Bifidobacterium Colonization promotes the accumulation of beneficial metabolites and regulates the overall metabolic network. These results indicate that BB@PCB–HA can restore gut microbiota metabolic homeostasis and achieve therapeutic effects on both new and recurrent CDI without the need for antibiotics.

[0075] 10. Biosafety assessment of BB@PCB–HA Before assessing the in vivo therapeutic efficacy, the biocompatibility of BB, BB@PCB, and BB@PCB-HA was evaluated in an antibiotic-induced gut microbiota imbalance mouse model. Administration of BB, BB@PCB, or BB@PCB-HA did not significantly affect body weight compared to the control group. No histopathological abnormalities were observed in major organs or the colon. Hematological parameters (white blood cell count, red blood cell count), liver function (AST, ALT, ALP, ALB), and kidney function (creatinine, blood urea nitrogen, urea) showed no significant differences among the groups. BB@PCB-HA and its components were well-tolerated and exhibited excellent in vivo biocompatibility, providing a basis for their therapeutic use.

[0076] In summary, this embodiment designed BB@PCB-HA based on cross-species bioinformatics analysis. By co-assembling the NOD-like receptor inhibitor BHB with BB and modifying its surface with hyaluronic acid (HA), this platform achieves integrated functions of anti-inflammatory regulation, targeted intestinal colonization, and metabolic reprogramming. In vitro experiments showed that BB@PCB-HA can efficiently scavenge reactive oxygen species (ROS), regulate the NLRP3 signaling pathway, and exhibit excellent gastrointestinal stability and colonic adhesion. It promotes the colonization and long-term proliferation of Bifidobacteria in the CDI-damaged intestine, thereby improving the therapeutic effect. In a CDI mouse model, oral administration of BB@PCB-HA significantly reduced colonic damage and inflammation and effectively prevented recurrence, with a therapeutic effect superior to vancomycin.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multifunctional engineered probiotic preparation, characterized in that, The product includes probiotics, the surface of which is sequentially coated with a phenylboronic acid-chitosan complex and hyaluronic acid; the probiotics are Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum The phenylboronic acid-chitosan complex is formed by combining phenylboronic acid-chitosan with β-hydroxybutyric acid.

2. The engineered probiotic preparation as described in claim 1, characterized in that, Benzyl borate chitosan is composed of carboxyphenylboronic acid and chitosan through the formation of amide bonds; Alternatively, the mass ratio of PC to BHB is 1:18~22, g / mmol; Alternatively, the ratio of PC to probiotics is 1:[(2~3) × 10⁻⁶]. 9 ], g / CFU; Alternatively, the ratio of HA to probiotics can be 10: [(0.5~1.5) × 10⁻⁶]. 9 ], mg / CFU.

3. A method for preparing the engineered probiotic preparation according to claim 1, characterized in that, Includes the following steps: PC and BHB were mixed in solution to prepare a chitosan complex; Bifidobacterium bifidum ( Bifidobacterium bifidum The PCB was incubated with Bifidobacterium bifidum, causing the PCB to be coated with Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum On the surface, a formulation precursor is obtained; The formulation precursor is incubated with HA, so that HA self-assembles and coats the surface of the formulation precursor, thus obtaining the engineered probiotic formulation.

4. The preparation method according to claim 3, characterized in that, The feed ratio of BHB to PC is 1:18~22, g / mmol; Or, PC and Bifidobacterium bifidum ( Bifidobacterium bifidum The ratio is 1: [(2~3) × 10 9 ], g / CFU; Or, HA and Bifidobacterium bifidum ( Bifidobacterium bifidum The ratio is 10: [(0.5~1.5) ×10 9 ], mg / CFU.

5. The preparation method according to claim 3, characterized in that, The temperature at which PC and BHB are mixed in the solution is 35~40℃.

6. The preparation method according to claim 3, characterized in that, Bifidobacterium bifidum ( Bifidobacterium bifidum The temperature for incubating the chitosan complex with the chitosan complex is 35~40 ℃.

7. The preparation method according to claim 3, characterized in that, The incubation temperature for the formulation precursor and HA is 35~40 ℃.

8. The use of the engineered probiotic preparation according to claim 1 or 2 in the preparation of a medicament for treating Clostridium difficile infection.

9. The application as described in claim 8, characterized in that, The drug is a composition comprising an active ingredient and pharmaceutical excipients, wherein the active ingredient is the engineered probiotic preparation.

10. The application as described in claim 8, characterized in that, The dosage form of the drug is tablets, capsules, granules, drops, oral liquid, or powder.

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