Probiotic preparation for enteritis treatment and diagnosis integration and preparation method thereof

Abstract

The invention discloses a preparation method of a probiotic preparation for enteritis treatment and diagnosis integration, and the preparation method comprises the following steps: 1) mixing metal ions and probiotic liquid, adding a ligand, reacting for 6-24 hours, centrifuging and washing to obtain an intermediate product LM; and 2) preparing the LM in the step 1) into a solution, adding polysaccharide, reacting for 2-8 hours, centrifuging and washing to obtain the probiotic preparation LMH. The probiotic preparation prepared by the preparation method can realize enteritis diagnosis based on excrement red fluorescence signal visualization and enteritis intervention treatment based on probiotics.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to a probiotic preparation for the integrated treatment and diagnosis of enteritis and its preparation method. Background Technology

[0002] Enteritis is characterized by chronic intestinal inflammation and damage to the epithelial barrier, and carries the potential risk of progressing to colorectal cancer. Early-stage enteritis can potentially be cured if accurately diagnosed and intervened. Therefore, constructing an integrated system that combines diagnostic and therapeutic functions is crucial for precision medicine and disease control in enteritis.

[0003] Currently, while endoscopy is considered the gold standard for diagnosing enteritis, its invasive nature is accompanied by cumbersome bowel preparation and poor patient compliance. Enteritis is caused by the immune system attacking digestive system tissues. During intestinal inflammation, macrophages polarize from the M2 phenotype to the M1 phenotype, leading to increased production of pro-inflammatory cytokines and decreased expression of anti-inflammatory cytokines. These changes trigger an increase in ROS production, thereby exacerbating oxidative stress, leading to apoptosis and further activating the inflammatory response. Therefore, ROS is a key signaling molecule reflecting the severity of inflammation. Currently, there are some strategies targeting the enteritis microenvironment (high levels of reactive oxygen species (ROS)) for enteritis diagnosis, but these methods only achieve simple enteritis diagnosis and have not yet achieved an organic integration of diagnosis and treatment. Therefore, developing a method that can achieve integrated diagnosis and treatment for the prevention and treatment of enteritis will have significant clinical implications.

[0004] Probiotics are an important component of the human gut microbiota, possessing excellent in vivo biocompatibility. They can exert therapeutic effects on enteritis by regulating colonic microbial diversity and reshaping gut microbiota homeostasis. Based on their excellent in vivo biocompatibility and natural gut compatibility, probiotics are widely used in daily life for gut microbiota regulation and intestinal disease prevention. However, delivering probiotics to the colon faces challenges not only in the harsh gastrointestinal environment of the administration route but also in achieving early diagnosis of enteritis during treatment. Currently, researchers have developed probiotic encapsulation strategies and improved probiotic delivery efficiency. However, existing strategies mainly focus on enhancing the probiotics' resistance to harsh gastrointestinal environments and their intestinal colonization ability, while probiotic encapsulation systems targeting enteritis diagnosis are still rarely reported. Meanwhile, in the pathogenesis of enteritis, various specific antigens and receptors (such as CD44, CD98, and mannose receptors) are overexpressed in lesions. Furthermore, compared to the normal anionic mucus layer, the colonic mucus in inflamed areas is cationic due to the high accumulation of surface cationic proteins (such as transferrin and eosinophil cationic proteins). High molecular weight hyaluronic acid (HA) is a negatively charged polymer that can specifically bind to the CD44 receptor and possesses anti-inflammatory properties. Therefore, promoting the targeted accumulation of probiotics in intestinal lesions through HA functionalization, combined with ROS-responsive properties, holds promise for achieving integrated diagnosis and treatment of enteritis, which would be a highly ideal strategy. Summary of the Invention

[0005] In view of this, the present invention provides a probiotic preparation for the integrated treatment and diagnosis of enteritis and its preparation method. Specifically, the present invention provides the following technical solution:

[0006] 1. A method for preparing a probiotic preparation integrating treatment and diagnosis of enteritis, comprising the following steps:

[0007] 1) Mix metal ions with probiotic solution, add ligand, react for 6 h ~ 24 h, centrifuge, wash, and obtain intermediate product LM;

[0008] 2) Prepare a solution of LM from step 1), add polysaccharide, react for 2 h ~ 8 h, centrifuge, wash, and obtain probiotic preparation LMH.

[0009] Furthermore, the probiotics are one or a mixture of several of the following: Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bacillus subtilis, Bacillus coagulans, Bacillus licheniformis, Akkermansia myxotroph, Clostridium butyricum, and Saccharomyces boulardii.

[0010] Furthermore, the concentration of the probiotics in the reaction system in step 1) is 107 to 109 CFU / mL.

[0011] Further, the metal ion mentioned in step 1) is one or a mixture of several of manganese ions, iron ions, magnesium ions, chromium ions, zinc ions, cobalt ions and copper ions, and the concentration of the metal ion in the reaction system is 0.1 ~ 4 mg / mL.

[0012] Furthermore, the manganese ion is Mn(OAc)3·2H2O or MnSO4, the iron ion is FeCl3 or FeSO4, the magnesium ion is MgCl2, the chromium ion is CrCl3, the cobalt ion is CoCl2·6H2O, the zinc ion is ZnSO4, and the copper ion is CuSO4.

[0013] Further, the ligand in step 1) is one or a mixture of several of the following: 4, 4′, 4′′′, 4′″-(porphyrin-5,10,15,20-tetra(benzoic acid), 1,2,4,5-tetracarboxybenzene, 2-methylimidazolium, 2-ethylimidazolium, 2,6-pyridinedicarboxylic acid, pyromellitic acid, oxalic acid, o-phenylrheline, and tetraazacyclododecane, and the concentration of the ligand in the reaction system is 0.05 to 0.15 mg / mL.

[0014] Furthermore, the mass ratio of the probiotics, metal ions, and ligands in step 1) is 100:(20~50):(1~10).

[0015] Further, the polysaccharide mentioned in step 2) is one or a mixture of several of the following: chitin, hyaluronic acid, dextran, chitosan, sodium alginate, carrageenan, alginate, chondroitin sulfate, green algae polysaccharide, and snail glycosaminoglycan, and the concentration of the polysaccharide in the reaction system is 0.1~10 mg / mL.

[0016] Further, in step 2), the mass ratio of LM to polysaccharide is 100:1 to 100:10.

[0017] 2. The probiotic preparation prepared by the above-mentioned method for the integrated treatment and diagnosis of enteritis is characterized in that the probiotic preparation can be used for the diagnosis of enteritis based on the visualization of fecal red fluorescence signals and for the intervention and treatment of enteritis based on probiotics.

[0018] The beneficial effects of this invention are as follows: This invention constructs a hybrid formulation of probiotics (Lactobacillus reuteri, LR) coated with ROS-responsive metal-organic framework (MOF) and polysaccharide hyaluronic acid (HA) for the integrated diagnosis and treatment of enteritis. The intermediate product, probiotic-metallic LR@MOF (LM), is obtained by coating the surface of LR with MOF that responds to the high ROS content and fluorescence at the enteritis site. Subsequently, through electrostatic interactions and carboxyl-manganese ion coordination, high molecular weight HA is further coated to form the hybrid LR@MOF@HA (LMH). This formulation can be used for the treatment and diagnosis of enteritis, effectively protecting probiotics against harsh environments and significantly improving the storage stability and ease of use of probiotics. After oral administration, LMH effectively resists the erosion of strong gastrointestinal acid and digestive enzymes, maintaining probiotic activity. At the enteritis lesion site, with the help of the highly expressed CD44 receptor and abnormally elevated ROS levels in the colon, LMH can achieve: 1) targeted enrichment at the enteritis site; 2) ROS-responsive coating degradation. MOF and HA degradation not only release probiotics to exert anti-inflammatory and gut microbiota-regulating effects, but also generate synergistic anti-inflammatory effects through ROS consumption and release red fluorescence signals. By collecting mouse feces, illuminating them with light-emitting diode (LED) light sources, and analyzing changes in the intensity of red fluorescence in the feces, we successfully achieved: 1) enteritis diagnosis based on visualized red fluorescence signals in feces; 2) probiotic-based therapeutic intervention, ultimately realizing integrated diagnosis and treatment of enteritis. Attached Figure Description

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0020] Figure 1 These are transmission electron micrographs of LM, an intermediate product of H2TCPP ligands at different concentrations.

[0021] Figure 2 These are transmission electron micrographs and surface scanning analysis of the probiotic preparation LMH.

[0022] Figure 3 This is the fluorescence spectrum of LMH at different hydrogen peroxide concentrations.

[0023] Figure 4 These are the growth curves of LR, LM, and LMH in LB medium.

[0024] Figure 5 The bacterial activities of LR, LM, and LMH at different time points in simulated gastric juice, bile salts, and simulated intestinal juice are measured.

[0025] Figure 6 The diagnosis of inflammatory bowel disease (IBD) using MOF and LMH at different severities of IBD.

[0026] Figure 7This is a graph showing the effects of LMH treatment for inflammatory bowel disease.

[0027] Figure 8 It refers to the storage activity of probiotic preparations at different temperatures. Detailed Implementation

[0028] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0029] Example 1: Preparation of intermediate product LM

[0030] Mix 1 mL of Mn(OAc)3·2H2O solution (2.8 mg / mL) with 1 mL of Lactobacillus reuteri (LR) suspension (1.0 × 10⁻⁶). 9 The mixture was mixed with 200 μL of 4, 4′, 4′′′, 4′″-(porphyrin-5,10,15,20-tetra(benzoic acid)) (H2TCPP) solution (5 mg / mL). The mass ratio of probiotics, metal ions and ligands was 100:28:10. After reacting for 12 h, the mixture was centrifuged and washed to obtain the intermediate product LM.

[0031] Example 2 Preparation of probiotic preparation LMH

[0032] The LM material prepared using 4, 4′, 4′′′, 4′″-(porphyrin-5,10,15,20-tetra(benzoic acid)) with a final concentration of 0.1 mg / mL, selected from Example 1, was subjected to a 1.0 × 10 9 After adding 2 mL of hyaluronic acid (HA) solution to CFU LM, the concentration of HA solution was 2.0 mg / mL, and the mass ratio of LM to HA was 48:1. The reaction was carried out at room temperature for 6 h, and LMH was obtained after centrifugation and washing.

[0033] Comparative Example 1: Preparation of LM-1

[0034] Mix 1 mL of Mn(OAc)3·2H2O solution (2.8 mg / mL) with 1 mL of Lactobacillus reuteri (LR) suspension (1.0 × 10⁻⁶). 9 The mixture was mixed with 10 μL of 4, 4′, 4′′′, 4′″-(porphyrin-5,10,15,20-tetra(benzoic acid)) (H2TCPP) solution (5 mg / mL). The mass ratio of probiotics, metal ions and ligands was 200:56:1. After reacting for 12 h, the mixture was centrifuged and washed to obtain the intermediate product LM-1.

[0035] Comparative Example 2: Preparation of LM-2

[0036] Mix 1 mL of Mn(OAc)3·2H2O solution (2.8 mg / mL) with 1 mL of Lactobacillus reuteri (LR) suspension (1.0 × 10⁻⁶). 9 The mixture was mixed with 400 μL of 4, 4′, 4′′′, 4′″-(porphyrin-5,10,15,20-tetra(benzoic acid)) (H2TCPP) solution (5 mg / mL). The mass ratio of probiotics, metal ions and ligands was 100:28:20. After reacting for 12 h, the mixture was centrifuged and washed to obtain the intermediate product LM-2.

[0037] Preparation of Comparative Example 3 MOF

[0038] 1 mL of Mn(OAc)3·2H2O solution was mixed with 200 μL of 4, 4′, 4′′′, 4′″-(porphyrin-5,10,15,20-tetratetra(benzoic acid)) (H2TCPP) solution (5 mg / mL), with a metal ion to ligand mass ratio of 28:10. After reacting for 12 h, the product MOF was obtained by centrifugation and washing.

[0039] Test Example 1: Material Morphology Characterization

[0040] The morphology of the hybrid material was characterized using a transmission electron microscope (HT-7800); mapping analysis of the hybrid material was performed using a FEI Tecnai G2F30 microscope.

[0041] To characterize the distribution of the MOF coating on probiotics, the hybrid material was characterized using transmission electron microscopy. The MOF coating content on the probiotic surface varied significantly with different amounts of H2TCPP added. Figure 1 These are transmission electron microscopy (TEM) images of LR, Mn(OAc)3·2H2O, and H2TCPP at different mass ratios. The LM (LM sample of Example 1) was obtained with 200 μL of H2TCPP added. Figure 1 a) It can be seen that MOF can completely coat LR to form a uniform probiotic coating. When the amount of H2TCPP added is 10 μL ( Figure 1 b), LM-1 of Comparative Example 1 was obtained, but the MOF coating did not completely cover LR. When the amount of H2TCPP added was 400 μL ( Figure 1 c) LM-2 of Comparative Example 2 was obtained, with MOF coating over-coating LR. A small amount of H2TCPP cannot completely achieve uniform MOF coating on LR; as the H2TCPP content increases, MOF coating on LR can be achieved. When the H2TCPP content is too high, there will be a large amount of free MOF.

[0042] Transmission electron microscopy (TEM) images of the LMH in Example 2 show that the LR surface has a clearly uniform MOF coating thickness ( Figure 2 a). Elemental distribution shows that LMH contains a high and uniform amount of Mn, proving that the MOF coating was successfully applied to the surface of Lactobacillus reuteri. Figure 2 b).

[0043] Test Example 2: Reactive Oxygen Species (ROS) Response Fluorescence Recovery Test

[0044] To verify the ROS-responsive fluorescence performance of the probiotic reagent, the fluorescence of solutions with different concentrations of ROS (H₂O₂) was tested. The LMH from Example 2 was dispersed in 1 mL of H₂O₂ solutions of different concentrations (0 μM, 50 μM, 100 μM, 150 μM, 200 μM) and incubated at 37 °C for 2 h. After 2 h of incubation, the fluorescence intensity of the solution was measured. Figure 3 As can be seen, the fluorescence intensity in the LMH of Example 2 increases with the increase of H2O2 concentration, indicating that the LMH of Example 2 of the present invention can be applied to the early diagnosis of IBD by responding to ROS in the microenvironment of inflammatory bowel disease (IBD).

[0045] Test Example 3: Resuscitation Test of Bacteria Responding to Reactive Oxygen Species (ROS)

[0046] To characterize the therapeutic performance of probiotic preparations in the high ROS-content microenvironment of IBD, the raw probiotics LR from Example 1, LM from Example 1, and LMH from Example 2 were added to LB medium with or without H2O2 (100 μmol), respectively. The suspensions of LR, LM, and LMH were diluted to 500 μL of fresh LB medium and cultured with gentle shaking at 37 °C. At predetermined time points, the OD600 values ​​of the culture medium were measured using a NanoDrop One spectrophotometer to quantitatively analyze bacterial growth.

[0047] from Figure 4As can be seen, the OD600 values ​​of the probiotics in the LR and LR+H2O2 groups in Example 1 gradually increased over time, exhibiting normal proliferation behavior. This indicates that H2O2 has virtually no impact on the probiotic proliferation activity. The LM group in Example 1 and the LMH group in Example 2 showed no significant proliferation behavior in an environment without H2O2, which may be because the coating limited the proliferation of probiotics in terms of both physical space and material exchange. This indicates that the LM group in Example 1 and the LMH group in Example 2 will not have probiotic proliferation under normal physiological conditions, further improving safety. The LM+H2O2 and LMH+H2O2 groups began to show normal proliferation activity after 4 hours, because the MOF coating limited the normal proliferation of probiotics; during the 0-4 hour period, the H2O2 caused responsive degradation of the coating, releasing the probiotics and enabling subsequent normal proliferation behavior. This indicates that the LM group in Example 1 and the LMH group in Example 2, due to the MOF coating's responsiveness to ROS degradation, can respond to ROS at the IBD site, achieving specific proliferation and improving efficacy.

[0048] In summary, this demonstrates that the LMH of Example 2 of the present invention can achieve specific proliferation in response to ROS in the microenvironment of IBD, thereby improving the therapeutic effect.

[0049] Test Example 4: Performance Test Against Harsh Gastrointestinal Environment

[0050] To evaluate the protective effect of LMH on probiotics in a simulated gastrointestinal tract (GIT) environment in Example 2, the following simulation conditions were used: Simulated gastric juice (SGF): pH 1.5, containing 0.2 g NaCl and 0.32 g pepsin per 100 mL ddH2O, with pH adjusted to 1.5 using HCl; Simulated intestinal juice containing H2O2 (SIF): pH 6.8, containing 0.68 g KH2PO4 and 1 g trypsin per 100 mL ddH2O, with pH adjusted to 6.8 using NaOH; containing 200 μM H2O2; bile salt solution concentration was 0.3 mg / mL.

[0051] The LR from Example 1, the LM from Example 1, and the LMH from Example 2 (1 × 10) 8 CFU / mL) were cultured with SGF, SIF, and bile salts at 37 °C and 200 rpm with shaking. Samples were collected at predetermined time points, washed with PBS, and resuspended to a bacterial concentration of 1.0 × 10⁻⁶. 8CFU / mL. 100 μL of suspension was added to a 96-well plate, and bacterial viability was determined using the CCK-8 assay. The viability of LR (Example 1), LM (Example 1), and LMH (Example 2) in simulated gastric juice (SGF) was investigated first. LR (Example 1), LM (Example 1), and LMH (Example 2) were incubated together with SGF containing pepsin. Figure 5 The bacterial activities of LR, LM, and LMH at different time points in simulated gastric juice, bile salts, and simulated intestinal juice are measured.

[0052] The activity changes of probiotics over time in simulated gastric fluid in Example 1 (LR), Example 1 (LM), and Example 2 (LMH) are as follows: Figure 5 As shown in Figure a. Due to the dual protection of MOF and HA, the LMH in Example 2 exhibited excellent resistance to strong acid and pepsin damage, maintaining over 72% bacterial activity even after 4 hours of incubation in SGF, significantly higher than LR (24%) and LM (48%). Overall, the coating demonstrates high potential in enhancing the resistance of probiotics to gastric acid.

[0053] Based on the above results, the LMH in Example 2 improved the survival rate of probiotics in the harsh intestinal microenvironment of IBD. To confirm this, the survival rates of probiotics in bile salts and SIF containing H2O2 were further evaluated using LR from Example 1, LM from Example 1, and LMH from Example 2. The changes in probiotic LR activity over time in bile salts are shown below. Figure 5 As shown in b, after 4 h of incubation in bile salts, the bacterial activity of LR was 20%; the bacterial activity of the LM group was 33.7%; while the bacterial activity of the LMH group, protected by MOF and HA coatings, remained at 74%. The changes in the activity of probiotic LR over time in simulated intestinal fluid (SIF) are shown in Figure b. Figure 5 As shown in b, due to the protective effect of the coating, the probiotics in LMH maintained a high activity (80%) after 4 h of SIF, which was significantly higher than that of LR (46%) and LM (61%).

[0054] In summary, the LMH of the present invention can maintain the activity of probiotics in a harsh gastrointestinal environment.

[0055] Test Example 5: IBD Diagnostic Effectiveness Assessment

[0056] To evaluate the diagnostic efficacy of LMH in Example 2 for inflammatory bowel disease, an IBD model was constructed for animal experiments to detect enteritis.

[0057] The experiment used 6-8 week old female C57BL / 6 mice and established IBD models of different severities by feeding them with 3% DSS (sodium dextran sulfate, molecular weight 36,000-50,000 kDa) aqueous solution for different number of days. The experiment was divided into two groups: MOF and LMH. The mice were orally administered LMH (Example 2) and MOF (Comparative Example 3), respectively. Fecal samples were collected within 24 hours, and the feces were irradiated with an LED light and photographed to analyze the red fluorescence signal. Simultaneously, colonic tissue was extracted and photographed. The content of pro-inflammatory factors in the colonic tissue was detected using ELISA. Equal volumes of mouse feces were dissolved in ethanol, centrifuged, and the supernatant was measured using a microplate reader.

[0058] Figure 6 The tests used in Example 2 (LMH) and Comparative Example 3 (MOF) were to diagnose inflammatory bowel disease of different severities. Figure 6 a shows the fecal fluorescence statistics of IBD model mice after oral administration of MOF or LMH at different severities. The more severe the IBD, the stronger the red color of the collected feces, indicating that the Red value can reflect the disease activity index (i.e., the severity of inflammatory bowel disease). No obvious red signal was observed in the feces on day 0 of DSS induction, indicating that LMH in Example 2 can be used for the diagnosis of inflammatory bowel disease; as the DSS induction time increased from 1 day to 7 days, the red signal in the feces gradually increased significantly. Figure 6 a) This demonstrates that the LMH in Example 2 can diagnose inflammatory bowel disease by observing the intensity of red fluorescent signals in feces. It also shows that the LMH in Example 2 not only has the ability to treat inflammatory bowel disease, but also has the ability to diagnose inflammatory bowel disease like MOF.

[0059] To better illustrate the different degrees of IBD severity, colonic tissue was extracted from mice after fecal collection, photographed, and colon length was measured. To further analyze the fecal red signal for diagnosing IBD, mice were orally administered LMH (Example 2) and MOF (Comparative Example 3), and the correlation between the Disease Activity Index (DAI), fecal extract fluorescence, colonic inflammatory factor levels, and red signal was investigated. Figure 6 b~e represent the fit between the disease activity index, colon length, inflammatory factor levels, and Red value. The fitted curves for the disease activity index and Red value are shown below. Figure 6 As shown in b, the LMH group reflects a good linear relationship between the disease activity index and the Red value. The fitted curves for colon length and Red value are shown below. Figure 6As shown in Figure c, the LMH group reflects a good linear relationship between colon length and Red value. This indicates that oral LMH monitoring of fecal Red value can further reflect the colon length, i.e., the severity of inflammatory bowel disease. Therefore, the LMH in Example 2 can be applied to the early diagnosis of enteritis in vivo. The fitting curve of IL-6 and Red value is shown in Figure c. Figure 6 As shown in Figure d, the LMH group reflects a good linear relationship between IL-6 and Red values, indicating that oral LMH monitoring of fecal Red values ​​can further reflect the IL-6 status, i.e., the severity of inflammatory bowel disease. Therefore, the LMH in Example 2 can be applied to the early diagnosis of enteritis in vivo. The fitting curves of TNF-α and Red values ​​are shown in Figure d. Figure 6 As shown in e, the LMH group can reflect a good linear relationship between TNF-α and Red value, indicating that the fecal Red value can be monitored by oral LMH to further reflect the status of TNF-α, i.e. the severity of enteritis. Therefore, the LMH in Example 2 can be applied to the early diagnosis of inflammatory bowel disease in vivo.

[0060] In summary, this demonstrates that the LMH of Example 2 of the present invention can be used to diagnose inflammatory bowel disease via oral administration.

[0061] Test Case 6: Evaluation of IBD Treatment Efficacy

[0062] To evaluate the therapeutic effect of probiotic preparations on IBD, 6-8 week old female C57BL / 6 mice were randomly divided into the following groups 7 days (-7 days) before the start of the experiment: healthy group, PBS group, probiotic LR treatment group, LM treatment group of Example 1, LMH group of Example 2, and control group 5-ASA treatment group. Except for the healthy group, all other groups of mice were fed 3% DSS (sodium dextran sulfate, molecular weight 36000-50000 kDa) aqueous solution for 7 days to induce the IBD model. Subsequently, each group of mice was orally administered 200 μL containing 1 × 10⁻⁶ probiotics. 8 Different formulations of CFU were used for intervention: PBS group plus PBS, LR treatment group plus LR, LM treatment group plus LM, LMH treatment group plus LMH formulation, and 5-ASA treatment group plus 5-ASA formulation. During the experiment, daily changes in mouse body weight were recorded, and fecal consistency, fecal occult blood, and degree of weight loss were observed to calculate the Disease Activity Index (DAI) score. On day 5, mice were sacrificed, colon tissue was harvested and photographed, and colon length was measured to assess the severity of colitis. Figure 7 This is a graph showing the effects of LMH treatment for inflammatory bowel disease.

[0063] Figure 7A is a flowchart of the IBD model construction and treatment process. The specific procedures are as follows: Mice were fed drinking water containing 3% DSS for 7 days to induce an inflammatory bowel disease (IBD) model, followed by daily treatment with different methods for 5 days. During the experiment, daily changes in mouse body weight were recorded. Figure 7 (b) The body weight of mice in the LMH group was closer to that of mice in the Health group after 5 days of treatment compared to PBS and other treatment groups, indicating that the LMH group had a better therapeutic effect. This demonstrates that the LMH preparation can restore the body weight of mice with IBD.

[0064] Meanwhile, during the treatment process, the mice's fecal viscosity, blood loss, and weight loss were also monitored to assess their DAI. Figure 7 c represents the curve of disease activity index (DAI) over time during the entire model induction and treatment period. It can be seen that the DAI index rose rapidly during DSS induction, while the DAI in the LMH group was significantly lower than in any other group after treatment. This indicates that LMH in Example 2 can restore the disease activity index in mice.

[0065] After treatment, the colon tissue of mice in each group was removed, photographed, and the colon length was measured. Figure 7 d and Figure 7 f is a statistical chart of colon length. It can be seen that the DSS-induced PBS group had the shortest colon length (4.7 cm). LMH, compared to 5-ASA, had a longer colon length, achieving the best therapeutic effect. Furthermore, the colon length in the LMH group was essentially the same as that in the healthy group, indicating that the LMH of Example 2 could restore the colon length to a healthy state after treating IBD and had excellent therapeutic effects. This demonstrates that the LMH preparation of Example 2 can achieve IBD treatment by restoring the intestinal barrier through antioxidant activity and regulation of the intestinal flora.

[0066] Figure 7 H&E staining analysis showed that the intestinal morphology of LMH in Example 2 was better than that of PBS and other treatment groups, with intact colonic epithelium. This confirms that LMH in Example 2 can effectively treat IBD. This indicates that the LMH preparation in Example 2 can effectively reduce DSS-induced intestinal damage and achieve excellent IBD treatment results.

[0067] In summary, the LMH of Embodiment 2 of the present invention can effectively treat inflammatory bowel disease.

[0068] Test Example 7: Probiotic Storage Stability Test

[0069] Maintaining good bacterial activity during storage is an important performance indicator for probiotics in treating intestinal diseases, and it also ensures that probiotics can be used immediately. To evaluate the effect of LMH from Example 2 on bacterial activity under different in vitro storage conditions, LR from Example 1, LM from Example 1, and LMH from Example 2 (1.0 × 10⁻⁶) were compared. 8 CFU were stored at room temperature, 4 °C, -20 °C, and -80 °C, respectively. Samples were taken out at preset time points, and bacterial activity was determined using the CCK-8 assay. The bacterial activities of LR in Example 1, LM in Example 1, and LMH in Example 2 after storage under different conditions for different times were compared.

[0070] Figure 8 This refers to the bacterial activity of probiotic preparations after storage at different temperatures. The bacterial activities of LR in Example 1, LM in Example 1, and LMH in Example 2 after 4 weeks of storage under different temperature conditions are shown below. Figure 8 As shown in Figure a, after 4 weeks of storage at room temperature, 4°C, -20°C, and -80°C, the bacterial activity of the LR group decreased significantly, especially at -80°C where the bacterial activity decreased to ~12%. The LM group, possibly due to the protective effect of the MOF surface coating, showed less reduction in probiotic activity, with a decrease to ~40% at -80°C. In contrast, the LMH group showed the least reduction in bacterial activity under different environments, and the probiotics maintained high bacterial activity, remaining above 70% even at -80°C.

[0071] To investigate the long-term storage stability of probiotics, the storage time of LR (Example 1), LM (Example 1), and LMH (Example 2) was extended by nearly two months. The bacterial activity of LR (Example 1), LM (Example 1), and LMH (Example 2) under different temperature storage conditions for 8 weeks was as follows: Figure 8 As shown in b. LR bacteria showed almost 0% bacterial activity at any temperature, indicating no bacterial activity; LM bacteria showed approximately 11% bacterial activity at -80°C, indicating decreased bacterial activity; while most bacteria in the LMH group maintained good bacterial activity, with bacterial activity still exceeding 49% at -80°C.

[0072] In summary, the LMH of Embodiment 2 of the present invention exhibits good storage stability under different environments.

[0073] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a probiotic preparation integrating treatment and diagnosis of enteritis, characterized in that, The preparation steps are as follows: 1) Mix metal ions with probiotic solution, add ligand, react for 6 h ~ 24 h, centrifuge, wash, and obtain intermediate product LM; 2) Prepare a solution of LM from step 1), add polysaccharide, react for 2 h ~ 8 h, centrifuge, wash, and obtain probiotic preparation LMH.

2. The method for preparing a probiotic preparation for integrated treatment and diagnosis of enteritis according to claim 1, characterized in that, The probiotics mentioned are one or a mixture of several of the following: Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bacillus subtilis, Bacillus coagulans, Bacillus licheniformis, Akkermansia myxophilus, Clostridium butyricum, and Saccharomyces boulardii.

3. The method for preparing a probiotic preparation for integrated treatment and diagnosis of enteritis according to claim 1, characterized in that, The concentration of probiotics in the reaction system in step 1) is 10. 7 Up to 10 9 CFU / mL.

4. The method for preparing a probiotic preparation for integrated treatment and diagnosis of enteritis according to claim 1, characterized in that, The metal ion mentioned in step 1) is one or a mixture of several of manganese ions, iron ions, magnesium ions, chromium ions, zinc ions, cobalt ions and copper ions, and the concentration of the metal ion in the reaction system is 0.1 ~ 4 mg / mL.

5. The method for preparing a probiotic preparation for integrated treatment and diagnosis of enteritis according to claim 1, characterized in that, The manganese ion is Mn(OAc)3·2H2O or MnSO4, the iron ion is FeCl3 or FeSO4, the magnesium ion is MgCl2, the chromium ion is CrCl3, the cobalt ion is CoCl2·6H2O, the zinc ion is ZnSO4, and the copper ion is CuSO4.

6. The method for preparing a probiotic preparation for integrated treatment and diagnosis of enteritis according to claim 1, characterized in that, The ligand in step 1) is one or a mixture of several of the following: 4, 4′, 4′′′, 4′″-(porphyrin-5,10,15,20-tetra(benzoic acid), 1,2,4,5-tetracarboxybenzene, 2-methylimidazolium, 2-ethylimidazolium, 2,6-pyridinedicarboxylic acid, pyromellitic acid, oxalic acid, o-phenylrheline, and tetraazacyclododecane. The concentration of the ligand in the reaction system is 0.05 to 0.15 mg / mL.

7. The method for preparing a probiotic preparation for integrated treatment and diagnosis of enteritis according to claim 1, characterized in that, The mass ratio of the probiotics, metal ions, and ligands mentioned in step 1) is 100:(20~50):(1~10).

8. A method for preparing a probiotic preparation for integrated treatment and diagnosis of enteritis according to claim 1, characterized in that, Step 2) The polysaccharide is one or a mixture of several of the following: chitin, hyaluronic acid, dextran, chitosan, sodium alginate, carrageenan, alginate, chondroitin sulfate, green algae polysaccharide, and snail glycosaminoglycan. The concentration of the polysaccharide in the reaction system is 0.1~10 mg / mL.

9. A method for preparing a probiotic preparation for integrated treatment and diagnosis of enteritis according to claim 1, characterized in that, In step 2), the mass ratio of LM to polysaccharide is 100:1 to 100:

10.

10. The probiotic preparation prepared according to any one of claims 1-9 for the integrated treatment and diagnosis of enteritis, characterized in that, Probiotic preparations can be used for the diagnosis of enteritis based on the visualization of red fluorescent signals in feces, and for the intervention and treatment of enteritis based on probiotics.

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