Nutritional metal ion modified anaerobic probiotics, and preparation method and application thereof

Abstract

The present application relates to a kind of operating nutrient metal ion modified anaerobic probiotics and its preparation method and application, and the surface of anaerobic probiotics is sequentially deposited with polyphenols and sodium alginate Nutrient metal ion is obtained.The present application can supplement nutrient element while improving the oxygen tolerance and gastrointestinal environment tolerance of anaerobic probiotics, increase the intestinal adhesion retention capacity of probiotics;Modified probiotics can repair intestinal barrier, regulate intestinal mucosal immune balance, reshape intestinal flora structure, and improve systemic inflammatory response through intestine-multiple organ interaction axis, and has significant therapeutic effect on extraintestinal immune-mediated inflammatory diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to an anaerobic probiotic modified with nutrient metal ions, its preparation method, and its application. Background Technology

[0002] Immune-mediated inflammatory diseases (IMIDs) are severe chronic inflammatory diseases affecting multiple organs, including rheumatoid arthritis (RA), multiple sclerosis, psoriasis, ankylosing spondylitis, and many other conditions. Currently, there is no cure, and mainstream treatments often involve broad-spectrum immunomodulators and some immunomodulators targeting specific organs. However, long-term use of these drugs often causes serious infections, increases the risk of tumors, or induces IMIDs in other organs. These inflammatory diseases share both common and specific immune pathways, with a typical pathological feature being the local infiltration of pro-inflammatory immune cells. Recent studies have found that the pathogenesis and progression of IMIDs are closely related to early intestinal barrier damage and gut microbiota imbalance. Intestinal barrier damage, gut microbiota dysbiosis, and abnormal mucosal immune activation, through multi-organ interactions such as the gut-liver axis, gut-joint axis, and gut-brain axis, mediate the migration of immune cells to extraintestinal target sites, thereby inducing a systemic inflammatory response. This is an important pathogenesis and a potential therapeutic target.

[0003] Probiotics, given their biosafety and bioactive functions, have become promising therapeutic agents and delivery carriers. In particular, *Bacteroides fragilis* has been reported to alleviate extraintestinal intraepithelial neoplasia-associated diseases (IMIDs) through its anti-inflammatory metabolites or by targeting the intestinal barrier and modulating intestinal mucosal immunity, laying the foundation for developing novel oral probiotic systems for treating IMIDs. However, probiotics face many challenges in practical application, especially anaerobic bacteria. Besides requiring a strictly anaerobic environment for growth, they are also susceptible to processing conditions. The harsh gastrointestinal physicochemical environment during oral administration can destroy the bacteria, resulting in low survival rates and ultimately, limited colonization and short retention time in the diseased gut, rendering them ineffective. Simply increasing the oral dose may lead to excessive proliferation or ectopic bacteria in the small intestine, causing adverse reactions. Furthermore, many patients with IMIDs, including those with rheumatoid arthritis (RA) and multiple sclerosis, suffer from zinc deficiency. Zinc deficiency drives Th17 cell differentiation, causes Treg cell dysfunction, leads to immune imbalance, and exacerbates inflammatory responses. Meanwhile, zinc ions have been reported to promote intestinal mucosal repair, further improving nutrient absorption efficiency and aiding in the treatment of IMIDs. However, traditional probiotic preparations cannot simultaneously address the issues of bacterial protection and key trace element supplementation.

[0004] Micronutrient imbalance is a key pathological feature of intraluminal microinfections (IMIDs), and relying solely on bacterial delivery is insufficient to achieve synergistic therapeutic effects of nutritional supplementation and immune regulation. While existing technologies can utilize coordination structures to protect the surface of probiotics, most lack targeted loading and controlled release design for disease-related micronutrient deficiencies, failing to simultaneously improve nutritional status, repair the intestinal barrier, and regulate intestinal and systemic immune imbalances. Therefore, a novel probiotic formulation needs to be developed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of existing anaerobic probiotics in the treatment of extraintestinal immune-mediated inflammatory diseases (IMIDs) and the urgent need for new and safe therapies, by providing an anaerobic probiotic modified with nutrient metal ions, its preparation method and application.

[0006] This invention provides an anaerobic probiotic modified with nutrient metal ions, which is obtained by sequentially depositing nutrient metal ions on the surface of the anaerobic probiotic with polyphenolic substances and sodium alginate.

[0007] Preferably, the anaerobic probiotic is selected from one of the following families: Bifidobacterium, Lactobacillus, Bacteroidetes, Clostridium, Verrucous, Veillonellae, and Eubacteraceae. More preferably, the anaerobic probiotic is selected from one of the following genera: Bifidobacterium, Bacteroidetes, Lactobacillus, Faecalibacterium, Clostridium, Akkermansia, Veillonella, Eubacterium, and Butycoccus. Most preferably, the anaerobic probiotic is selected from one of the following: Bifidobacterium, Lactobacillus rhamnosus GG, Bacteroidetes polymorpha, Clostridium butyricum, and Akkermansia myxophilia.

[0008] Preferably, the polyphenolic substance is tannic acid. The polyphenolic substance and sodium alginate cross-link through intermolecular forces, electrostatic adsorption, or coordination to form a coating that encapsulates the surface of the anaerobic probiotics.

[0009] Preferably, the nutrient metal ion includes one of zinc ions, copper ions, selenium ions, magnesium ions, manganese ions, and chromium ions. More preferably, the nutrient metal ion is zinc ions, which have immune regulation and repair-promoting functions.

[0010] This invention also provides a method for preparing anaerobic probiotics modified with nutrient metal ions, comprising the following steps:

[0011] (1) Add polyphenolic substance solution and nutrient metal ion solution to anaerobic probiotic liquid. Polyphenolic substance and nutrient metal ion form metal polyphenol network (MPNs) through coordination and encapsulate the surface of anaerobic probiotics.

[0012] (2) Add nutrient metal ion solution and sodium alginate solution to the anaerobic probiotic solution modified with MPNs to obtain nutrient metal ion modified anaerobic probiotics.

[0013] The present invention also provides the application of anaerobic probiotics modified with nutrient metal ions in the preparation of biological agents for treating extraintestinal immune-mediated inflammatory diseases.

[0014] Preferably, the parenteral immune-mediated inflammatory disease includes one of the following disease spectrums: rheumatoid arthritis, primary sclerosing cholangitis, multiple sclerosis, psoriasis, atopic dermatitis, connective tissue diseases, and spondyloarthritis.

[0015] Specifically, the anaerobic probiotic modified with nutrient metal ions is an oral biological agent with excellent resistance to oxygen, gastrointestinal environment, and targeted colonization and long-term retention in damaged intestines. It can supplement nutrient metal ions, promote intestinal barrier immune homeostasis, and regulate systemic immune response.

[0016] Specifically, the alginate-nutrient metal ion gel layer enhances the bacteria's resistance to gastric acid, enabling the stable delivery of sufficient, highly active bacteria to the damaged gut; the MPNs layer improves their oxygen resistance and targeted adhesion to the damaged gut, while the cross-linked nutrient metal ions provide nutritional supplementation and synergistic immune regulation. Anaerobic probiotics can produce short-chain fatty acids and maintain gut microbiota homeostasis, thereby regulating immunity.

[0017] Beneficial effects

[0018] This invention can improve the oxygen tolerance and gastrointestinal environment tolerance of anaerobic probiotics while supplementing nutritional elements, and increase the intestinal adhesion and retention capacity of probiotics. The modified probiotics can repair the intestinal barrier, regulate the immune balance of the intestinal mucosa, reshape the intestinal flora structure, and improve the systemic inflammatory response through the gut-multi-organ interaction axis, and have significant therapeutic effects on extraintestinal immune-mediated inflammatory diseases. Attached Figure Description

[0019] Figure 1The flowchart for preparing anaerobic probiotics modified with nutrient metal ions and the characterization of the modified bacteria are shown. Specifically, a is a schematic diagram of the preparation method in Example 1; b is the particle size of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA measured by DLS (mean ± SD, n=3); c is a representative TEM image of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA, with a scale bar of 1 μm for Bt and Bt@TA-Zn and 500 nm for Bt@TA-Zn@SA; d is the zeta potential of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA measured by DLS (mean ± SD, n=10); e is a confocal image of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA, where TA is labeled with Rhodamine B, sodium alginate is labeled with FITC, green and red represent SA and TA respectively, and the scale bar is 5 μm.

[0020] Figure 2 The activity (a) and growth curves (b) of Bt, Bt@TA-Zn and Bt@TA-Zn@SA are shown.

[0021] Figure 3 The in vitro stability, metabolic activity, and adhesion to Caco-2 cells of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA are shown. Specifically, a) represents the survival rate of Bt and Bt@TA-Zn in aerobic and anaerobic environments under nutrient-free culture conditions; b) represents the growth of Bt and Bt@TA-Zn on BHIS nutrient medium plates after culture in aerobic and anaerobic environments; c) represents the survival rate of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA at different time points in simulated gastric juice; d) represents the propionic acid concentration in the supernatant fermentation broth of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA after 3 days of culture in a simulated inflammatory gut microecological environment; e) represents the concentration of 1×10⁻⁶ ions / mL. 7 The number of CFU Bt and Bt@TA-Zn cells adsorbed after incubation and washing with Caco-2 cells.

[0022] Figure 4The adhesion and in vivo retention capacity of Bt@TA-Zn@SA to the inflamed gut are shown. Specifically, a) shows fluorescence images of Cy7-labeled Bt, Bt@TA-Zn, and Bt@TA-Zn@SA before and after direct incubation and washing with the intestinal mucosa of mice with enteritis; b) shows the quantitative fluorescence statistics of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA before and after direct incubation and washing with the intestinal mucosa of mice with enteritis; c) shows the in vivo fluorescence retention rate of mice with colitis treated with Bt and Bt@TA-Zn@SA for 8 h; d) shows in vivo fluorescence imaging images of mice with colitis treated with Bt and Bt@TA-Zn@SA for 4, 8, and 12 h; and e) shows the in vivo fluorescence retention rate of mice with colitis treated with Bt and Bt@TA-Zn@SA for 12 h (with the average fluorescence intensity at 4 h as the reference).

[0023] Figure 5 This study demonstrated the repair effect of Bt@TA-Zn@SA on severely damaged intestines (administered bacterial count: 2 × 10⁻⁶). 7 CFU); where a is a representative image of H&E staining of colon tissue from each group of mice, scale bar: 100 μm; b is a representative image of AB-PAS staining, scale bar: 100 μm.

[0024] Figure 6 The study demonstrates the repair effect of Bt@TA-Zn@SA on the intestinal barrier; where a represents the relative expression level of the MUC2 gene; b represents the relative expression level of the intestinal barrier-related gene ZO-1 in vivo; c represents the relative expression level of Occludin; d represents the relative expression level of CB1R; and e represents the immunofluorescence representation of ZO-1 and Occludin in the colon tissue of mice in each group. Scale bar: 100 μm.

[0025] Figure 7 The study illustrates the regulatory effect of Bt@TA-Zn@SA on intestinal mucosal immunity; where a is a representative graph of the proportion of Th17 cells in the intestinal parietal nodes of mice in each group; b is a representative graph of the proportion of Treg cells in the intestinal parietal nodes of mice in each group; c is a histogram of the proportion of Th17 cells in the intestinal parietal nodes of mice in each group; and d is a histogram of the proportion of Treg cells in the intestinal parietal nodes of mice in each group.

[0026] Figure 8 The regulatory effect of Bt@TA-Zn@SA on the gut microbiota of a damaged gut is shown; where a is the number of observable species; b is the Chao1 index; and c is the Venn diagram.

[0027] Figure 9The study illustrates the alleviating effect of Bt@TA-Zn@SA on arthritis in RA mice. Specifically, a) shows the arthritis score curves for each group of mice; b) shows a gross representation of the mouse paws and corresponding micro-CT images for each group; c) shows representative images of H&E staining (scale bar: 300 μm), Safranin-Fix-Green staining (scale bar: 300 μm), and TRAP staining (scale bar: 100 μm) for each group of mouse paws; d) shows the quantitative statistical histogram of TNF-α immunohistochemical staining in each group of mouse joints; and e) shows a representative image of TNF-α immunohistochemical staining in each group of mouse joints (scale bar: 50 μm).

[0028] Figure 10 The study demonstrates the repair and immunomodulatory effects of Bt@TA-Zn@SA on the damaged intestines of arthritic mice. Specifically, a) is a histogram showing the colon length of each group of mice; b) is a histogram showing the MUC2 expression level in the colon tissue of each group of mice; c) is a histogram showing the Claudin-1 expression level; d) is a histogram showing the Claudin-2 expression level; e) is a histogram showing the CB1R expression level; f) is a histogram showing the spleen weight of each group of mice; g) is a statistical graph showing the proportion of Th17 cells in the spleen; and h) is a statistical graph showing the proportion of Treg cells in the spleen.

[0029] Figure 11 Cellular biocompatibility of Bt@TA-Zn@SA is shown; where a is a histogram of cell survival rates after co-incubation of Bt, Bt@TA-Zn and Bt@TA-Zn@SA with Caco-2 cells; b is a histogram of cell survival rates after co-incubation of Bt, Bt@TA-Zn and Bt@TA-Zn@SA with RAW264.7 cells.

[0030] Figure 12 The H&E staining of heart, liver, spleen, lung, and kidney tissues from each group of mice is shown. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0032] Example 1

[0033] In this embodiment, an anaerobic probiotic modified with nutrient metal ions was prepared, and its performance was tested.

[0034] Nutrient metal ions were modified onto the surface of anaerobic probiotics using a layer-by-layer encapsulation technique with MPNs and sodium alginate gel, thus constructing nutrient metal ion-modified anaerobic probiotics. Figure 1a) The specific method includes the following steps:

[0035] For the preparation of nutrient metal ion modified anaerobic probiotics (Bt@TA-Zn@SA), logarithmic growth phase Bacteroides polymorpha (Bt) was resuspended in physiological saline. A 30 mg / mL tannic acid (TA) solution, 15 mg / mL and 41.3 mg / mL zinc nitrate hexahydrate solutions, and a 10 wt% sodium alginate solution were prepared using ultrapure water. After diluting the Bt bacterial suspension with physiological saline, 15 mg / mL zinc nitrate hexahydrate solution was added at a 1:100 (v / v) ratio. The mixture was vortexed for 10 s, followed by the addition of 30 mg / mL TA solution at a 1:100 (v / v) ratio, and then vortexed for another 10 s. An equal volume of pre-cooled 2× MOPS buffer was added to stabilize the MPNs. After vortexing for 10 s, the mixture was centrifuged at 4000 g for 10 min and washed once with sterile PBS to obtain Bt@TA-Zn. After resuspending Bt@TA-Zn in PBS, add 41.3 mg / mL zinc nitrate hexahydrate solution at a ratio of 1:9 (v / v), vortex for 3 min, then add 10 wt% sodium alginate solution at a ratio of 1:20 (v / v), vortex for 5 min, then adjust the pH to 5 with 10% hydrochloric acid to stabilize the gel crosslinking, centrifuge at 4000 g for 10 min to collect the precipitate, wash twice with sterile PBS at pH=5 to obtain zinc ion-loaded Bt@TA-Zn@SA.

[0036] Bacterial viability assays showed no significant difference in bacterial activity between unmodified Bt and Bt@TA-Zn and Bt@TA-Zn@SA. Figure 2 a) The growth curves of Bt and Bt@TA-Zn basically overlap, while Bt@TA-Zn@SA lags behind the former two in the logarithmic growth phase, but still reaches the plateau phase at 12 h at the same time as Bt and Bt@TA-Zn. Figure 2 (b) indicates that the surface modification method is safe and feasible, and its impact on bacterial viability is negligible. Dynamic light scattering (DLS) results show that after bilayer modification, the particle size of Bt increased from 1129 ± 14.57 nm to 1284 ± 26.06 nm and 2408 ± 135.7 nm, respectively. Figure 1 b); Simultaneously, the zeta potential of Bt increased from -12.59 ± 1.18 mV to -14.99 ± 1.43 mV ( Figure 1 d). Comparing the transmission electron microscopy (TEM) images of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA, a uniform light gray coating was visible on the bacterial surface of Bt@TA-Zn. After encapsulation with SA gel, it was found that it was densely attached to the bacterial cell surface in a spherical shape under the microscope. Figure 1 c). Confocal imaging further confirmed the successful modification of MPNs and SA gel. Figure 1 As shown in e, Rhodamine B-labeled TA exhibits red fluorescence, while FITC-labeled SA exhibits green fluorescence. The two fluorescencees are uniformly distributed on the Bt surface and can co-localize.

[0037] To ensure adequate and highly active oral bacterial delivery to the gut for their intended effect, it is crucial to enhance the aerobic tolerance and gastrointestinal environment of anaerobic bacteria. This was achieved by culturing Bt and Bt@TA-Zn in an aerobic / anaerobic, nutrient-free environment for 10 hours. Figure 3 As shown in a and b, there was no significant difference in activity between the two in anaerobic environments. However, in aerobic environments, the decrease in Bt survival rate was significantly greater than that of Bt@TA-Zn, and the colony density in Bt@TA-Zn plate counts was also higher, indicating that the TA-Zn network can enhance the oxygen tolerance of anaerobic bacteria. Bt, Bt@TA-Zn, and Bt@TA-Zn@SA were cultured in simulated gastric fluid at pH 2.0 for 2 h. The results showed that the acid tolerance of Bt@TA-Zn@SA was significantly improved. Figure 3 c). Subsequently, an in vitro gut microbiota ecosystem was constructed by extracting fecal bacteria from the feces of healthy wild-type C57BL / 6 mice, and an inflammatory gut microbiota ecosystem was constructed using 200 μM hydrogen peroxide to simulate a damaged gut. After culturing Bt, Bt@TA-Zn, and Bt@TA-Zn@SA in the inflammatory microbiota ecosystem for 3 days, the content of propionic acid (the main metabolite of Bt) in the supernatant of Bt@TA-Zn and Bt@TA-Zn@SA was significantly higher than that in Bt (…). Figure 3 d) indicates that the bilayer-modified probiotics exhibit better metabolic activity in the harsh intestinal microecological environment, producing more propionic acid with anti-inflammatory and immunomodulatory effects. The SA gel shell is stable and robust in acidic environments but gradually dissociates in alkaline environments, releasing Bt@TA-Zn. The phenolic hydroxyl groups in TA enhance its adhesion to intestinal epithelial cells. Co-incubation of Caco-2 cells with Bt and Bt@TA-Zn, respectively, followed by washing away unadhered airborne bacteria, and plate counting revealed a significant increase in the number of adherent bacteria using Bt@TA-Zn on the Caco-2 cell surface. Figure 3 e).

[0038] Then, small intestinal segments from wild-type C57BL / 6 mice were reversed and directly contacted with Cy7-labeled Bt, Bt@TA-Zn, and Bt@TA-Zn@SA bacterial solutions for the same duration. Analysis using a small animal in vivo imaging system revealed that the fluorescence retention rate of Bt@TA-Zn and Bt@TA-Zn@SA in the intestinal mucosa was significantly higher than that of Bt after washing. Figure 4a). The fluorescence retention rate of Bt was only about 9%, while the fluorescence retention rates of Bt@TA-Zn and Bt@TA-Zn@SA could reach 20% to 40%, indicating that the modified bacteria had a significantly improved ability to adhere to the intestine. Figure 4 b). Subsequently, mice were administered Cy7-labeled Bt and Bt@TA-Zn@SA via gavage. The fluorescence retention rates at different time points after gavage indicated that the retention rate of Bt@TA-Zn@SA in the mouse intestine was significantly higher than that of Bt, and the retention rate remained above 75% at 12 h. Figure 4 ce).

[0039] To investigate the in vivo properties of Bt@TA-Zn@SA, we first examined their safety in vitro and in vivo. Different concentrations of Bt, Bt@TA-Zn, and Bt@TA-Zn@SA were co-incubated with Caco-2 and RAW264.7 cells, and cell viability was assessed using CCK-8 assays. The results showed that high concentrations of bacterial solution maintained the viability of both cell types above 85%, indicating that the modified bacteria still possessed good cell compatibility. Figure 11 Then, the mice were administered 2 × 10⁻⁶ gavage continuously. 8 After 28 days of CFU Bt and Bt@TA-Zn@SA treatment, H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) in mice showed no significant pathological damage compared to the healthy control group, indicating that Bt@TA-Zn@SA has good biosafety and low toxicity. Figure 12 ).

[0040] Furthermore, dextran sulfate sodium (DSS) was used to induce intestinal injury in mice to evaluate the repair effect of Bt@TA-Zn@SA on damaged intestines. The results showed that PBS, Bt, Bt@TA-Zn, and TA-Zn had limited repair effects. H&E staining revealed that DSS caused severe histological damage to the mouse intestinal tract, including epithelial defects, crypt disappearance, and goblet cell depletion. Treatment with 2 × 10⁻⁶ ppm of DSS significantly reduced the repair effect. 7 When CFU was administered via gavage, Bt@TA-Zn@SA showed less histological damage ( Figure 5 a). Impaired intestinal barrier function allows disordered symbiotic microorganisms to translocate into the intestinal wall. Subsequently, innate immune cells take up and respond to these microorganisms and their mediators, triggering immune cell activation and the production of cytokines and chemokines. This recruits more immune cells into the intestinal wall and activates adaptive immunity, initiating an inflammatory cascade. Further evaluation of the intestinal barrier repair effect of Bt@TA-Zn@SA revealed that Alsin blue-periodic acid Schiff (AB-PAS) staining showed limited repair effects of PBS, Bt, Bt@TA-Zn, and TA-Zn on intestinal mucus and the cell barrier, while Bt@TA-Zn@SA treatment significantly improved the number and function of goblet cells. Figure 5 b), the expression of mucin 2 (MUC2) was also significantly increased. Figure 6 a). Gene and protein level analysis of intestinal barrier tight junction proteins also showed that the expression levels of ZO-1 and Occludin were significantly increased after Bt@TA-Zn@SA treatment. Figure 6 (b, c) Immunofluorescence results showed that the continuity and expression intensity of tight junction proteins were significantly restored. Figure 6 e). Simultaneously, the expression of intestinal cannabinoid type 1 receptor (CB1R), which positively regulates intestinal epithelial barrier function, was also significantly upregulated. Figure 6 d) This suggests that Bt@TA-Zn@SA can effectively repair the intestinal barrier and help reduce excessive immune activation. This effect is attributed to the adequate delivery of highly active probiotics to the damaged intestine, where they work synergistically to achieve anti-inflammatory effects and promote goblet cell differentiation and barrier repair through their anti-inflammatory metabolite propionic acid and slowly released zinc ions with pro-repair properties.

[0041] To further evaluate the regulatory effect of Bt@TA-Zn@SA on intestinal mucosal immunity, flow cytometry analysis of mouse Pareto's nodes revealed pro-inflammatory Th17 cells (RORγt) within Pareto's nodes. + The proportion of Th17 cells was significantly increased, while the proportion of anti-inflammatory Treg cells (CD25⁺Foxp3⁺) decreased. After intervention with Bt, TA-Zn, or Bt@TA-Zn, the proportion of Th17 cells only decreased slightly, and the proportion of Treg cells only slightly increased. However, the Bt@TA-Zn@SA group significantly downregulated the proportion of Th17 cells. Figure 7 a, c), while significantly increasing the proportion of Treg cells ( Figure 7 (b, d) The corresponding Th17 / Treg cell ratio also decreased. This result indicates that Bt@TA-Zn@SA can effectively correct the mucosal immune imbalance in the intestinal tract caused by inflammation, regulate the balance between Th17 and Treg cells, and remodel the immune microenvironment towards anti-inflammatory effects.

[0042] Furthermore, analysis of mouse feces using 16S rDNA high-throughput sequencing revealed that Bt@TA-Zn@SA significantly increased the number of observable species in the mouse gut microbiota. Figure 8 (a) indicates that it can effectively reverse the loss of microbial species in the damaged gut. α-diversity analysis showed that the Chao1 index of the mouse gut microbiota was significantly decreased, while the Chao1 index of the Bt@TA-Zn@SA group was significantly increased (a). Figure 8(b) The above results all confirm that Bt@TA-Zn@SA can effectively improve the loss of diversity and abundance of gut microbiota in inflamed gut. Venn diagram analysis further showed that the number of unique species in the gut microbiota of the Bt@TA-Zn@SA group was significantly higher than that of the model group, and the number of shared species with the control group was also greater than that of the model group, which intuitively reflects the improvement of gut microbiota species diversity by Bt@TA-Zn@SA. Figure 8 c).

[0043] Given that Bt@TA-Zn@SA exhibits excellent repair effects on severely damaged intestines and modulatory effects on intestinal mucosal immunity under inflammatory conditions, a collagen-induced RA mouse model was further constructed. Each treatment group was administered Bt and Bt@TA-Zn@SA suspensions (2×10⁻⁶) by gavage every two days. 8 CFU / animal), after 28 days of administration, the arthritis score curve of Bt@TA-Zn@SA was found to be significantly flatter than that of the RA group and the Bt group (CFU / animal). Figure 9 a). Simultaneously, observation of mouse paw morphology revealed significant swelling and redness in the paws of RA mice. After Bt@TA-Zn@SA treatment, the swelling and redness significantly subsided, approaching the levels of the healthy control group, demonstrating a significantly better effect than Bt intervention. Micro-CT scans of mouse paws also showed that Bt@TA-Zn@SA significantly improved bone damage associated with arthritis compared to Bt. Figure 9 b), H&E staining of mouse paws also showed a significant reduction in joint and bone damage caused by Bt@TA-Zn@SA. Figure 9 c); Simultaneously, safranin-fast green staining of mouse joints showed that Bt@TA-Zn@SA had a significantly better protective effect against cartilage destruction than Bt, and TRAP staining also showed a significant reduction in osteoclasts in the Bt@TA-Zn@SA group ( Figure 9 c). Furthermore, the expression level of the pro-inflammatory factor TNF-α in Bt@TA-Zn@SA joint tissue was also significantly reduced ( Figure 9 (d, e); The above results all indicate that Bt@TA-Zn@SA can effectively alleviate immune-mediated arthritis in mice and reduce the destruction of bone and articular cartilage.

[0044] Subsequently, measurements of colon length in arthritis-affected mice revealed shortened colons, suggesting potential intestinal damage. Bt@TA-Zn@SA, however, restored intestinal length. Figure 10 a); Simultaneously, similar results were found in the detection of intestinal barrier-related tight junction proteins. In RA mice, the levels of MUC2, Claudin-1, Claudin-2, and CB1R were all decreased, while the levels of these indicators were significantly upregulated after Bt@TA-Zn@SA treatment, showing better efficacy than Bt ( Figure 10 This indicates that Bt@TA-Zn@SA can effectively repair the damaged intestinal barrier in RA mice.

[0045] Furthermore, assessments of spleen weight and the proportion of immune cells in arthritic mice revealed ( Figure 10 (fh) In RA mice, spleen weight was significantly increased, the proportion of Th17 cells among CD4⁺ T cells was significantly increased, and the proportion of Treg cells was decreased. Bt@TA-Zn@SA can significantly reduce spleen weight in arthritic mice, while downregulating the proportion of Th17 cells and upregulating the proportion of Treg cells, thereby regulating the systemic immune balance and alleviating arthritis.

[0046] In summary, a layer-by-layer encapsulation technique using polyphenols and SA gel has been employed to modify the surface of Bacteroides polymorpha with zinc ions layer by layer. The efficacy of probiotics modified with a double-layer metal crosslinking material in treating rheumatoid arthritis (RA) has been investigated. Using conventional methods, MPNs loaded with nutrient metal ions are modified onto the bacterial surface to enhance their oxygen resistance and adhesion and colonization ability in the damaged intestine. Then, a pH-responsive SA gel loaded with immunomodulatory zinc ions is encapsulated with sodium alginate, endowing the bacteria with resistance to the gastrointestinal environment and a sustained-release effect of zinc ions in the intestine. This achieves both nutritional supplementation and targeted delivery of orally administered probiotics to the damaged intestine in a highly active state. Furthermore, the anti-inflammatory metabolites synergistically work with zinc ions to achieve anti-inflammatory effects, repair the intestinal barrier, regulate intestinal mucosal immunity, and thus regulate systemic immune balance, alleviating the effects of extraintestinal IMIDs.

[0047] The above are merely specific embodiments of the present invention and experimental examples used to verify the inventive concept of the present invention, but are not intended to limit the present invention. All modifications that can be directly derived or conceived by those skilled in the art from the content disclosed in the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. An anaerobic probiotic modified with nutrient metal ions, characterized in that, It is obtained by sequentially depositing nutrient metal ions on the surface of anaerobic probiotics with polyphenols and sodium alginate.

2. The anaerobic probiotic according to claim 1, characterized in that, The anaerobic probiotics are selected from one of the following families: Bifidobacterium, Lactobacillus, Bacteroidetes, Clostridium, Verrucocephala, Veillonella, and Eubacteriales.

3. The anaerobic probiotic according to claim 1, characterized in that, The polyphenolic substance is tannic acid.

4. The anaerobic probiotic according to claim 1, characterized in that, The nutrient metal ions include one of zinc ions, copper ions, selenium ions, magnesium ions, manganese ions, and chromium ions.

5. A method for preparing anaerobic probiotics modified with nutrient metal ions, characterized in that, Includes the following steps: (1) Add polyphenolic substance solution and nutrient metal ion solution to anaerobic probiotic liquid. Polyphenolic substance and nutrient metal ion form metal polyphenol network (MPNs) through coordination and encapsulate the surface of anaerobic probiotics. (2) Add nutrient metal ion solution and sodium alginate solution to the anaerobic probiotic solution modified with MPNs to obtain nutrient metal ion modified anaerobic probiotics.

6. The use of the anaerobic probiotic modified with nutrient metal ions as described in claim 1 in the preparation of a biological agent for treating parenteral immune-mediated inflammatory diseases.

7. The application according to claim 6, characterized in that, The parenteral immune-mediated inflammatory diseases include one of the following: rheumatoid arthritis, primary sclerosing cholangitis, multiple sclerosis, psoriasis, atopic dermatitis, connective tissue diseases, and spondyloarthritis.

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