Use of bacillus coagulans t-21 in reducing ulcerative colitis with cognitive dysfunction

Abstract

The application belongs to the field of biological medicine and microecological intervention technology, and relates to application of Bacillus coagulans T-21 in reducing ulcerative colitis combined with cognitive dysfunction. A dextran sodium sulfate (DSS) induced mouse ulcerative colitis combined with cognitive dysfunction model is established by taking a Bacillus coagulans T-21 strain as an active ingredient, and an intervention effect is evaluated. Results show that the Bacillus coagulans T-21 intervention can improve the cognitive related behavior performance and learning and memory ability of the model mice, thereby reducing ulcerative colitis related cognitive dysfunction. Meanwhile, the Bacillus coagulans T-21 intervention can increase the colon length, up-regulate the expression of tight junction proteins Occludin, Claudin-1 and Claudin-5 in colon tissue, improve the intestinal mucosal barrier damage, and down-regulate the expression levels of pro-inflammatory factors IL-1beta in colon tissue and IL-6 in hippocampus tissue, thereby reducing intestinal and central inflammatory reactions.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and microecological intervention technology, and more specifically relates to the application of Bacillus coagulans T-21 in alleviating ulcerative colitis complicated with cognitive dysfunction. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic intestinal diseases characterized by chronic inflammation of the intestines, with alternating periods of relapse and remission. It mainly includes ulcerative colitis (UC) and Crohn's disease (CD). In recent years, the global burden of IBD has continued to increase, and the affected population has expanded, making it one of the most significant diseases impacting patients' quality of life and public health burden. Ulcerative colitis, as one of the major types of IBD, has received widespread attention due to its prolonged course, high relapse rate, and accompanying multi-system abnormalities.

[0003] Patients with ulcerative colitis often experience clinical manifestations such as diarrhea, abdominal pain, and bloody, mucous stools. Persistent or recurrent intestinal inflammation can lead to damage to the structure and function of the intestinal mucosa. In addition to intestinal symptoms, increasing research indicates that patients with ulcerative colitis also frequently experience cognitive impairments, such as decreased attention, impaired learning and memory, and abnormal executive function, which are more pronounced during disease activity. Current treatments primarily focus on controlling intestinal inflammation and maintaining disease remission, but interventions for cognitive impairment associated with ulcerative colitis remain limited. Therefore, developing novel intervention strategies that can improve both intestinal inflammation and cognitive impairment has significant research and application value.

[0004] In recent years, the role of the gut-microbiota axis (MGB axis) in central nervous system dysfunction associated with inflammatory bowel disease has attracted attention. Gut microbiota imbalance, impaired intestinal barrier function, and abnormal activation of immune inflammation can affect central nervous system function through inflammatory mediators, the vagus nerve, neuroendocrine pathways, and microbial metabolites, further interfering with neural plasticity and cognitive regulatory networks in brain regions such as the hippocampus, thereby promoting the development of both gut inflammation and cognitive dysfunction. Therefore, gut-brain axis-targeted microecological interventions are considered a potential direction for simultaneously improving gut inflammation and cognitive dysfunction.

[0005] Bacillus coagulans is a spore-forming bacterium. In vitro studies have shown that some Bacillus coagulans strains have good survival capabilities in simulated gastrointestinal environments and exhibit certain adhesion and immunomodulatory activities, suggesting their potential as oral probiotic intervention strains. Animal studies further indicate that some Bacillus coagulans strains, or when used in combination with prebiotics, can improve the enteritis phenotype in experimental colitis models. This effect is related to changes in gut microbiota structure, regulation of short-chain fatty acid levels, downregulation of inflammatory factors, and improvement of intestinal mucosal barrier function. Meanwhile, existing research suggests that some Bacillus coagulans strains are associated with improvements in cognitive or emotional indicators in other disease or stress-related models, but the relevant evidence mainly comes from sleep deprivation models, D-galactose / AlCl3-induced cognitive impairment models, or irritable bowel syndrome combined with depression. On the other hand, clinical and neuroimaging studies suggest that patients with ulcerative colitis or inflammatory bowel disease may experience cognitive abnormalities in attention, memory, and executive functions. However, existing research mainly focuses on the intervention effects of Bacillus coagulans on intestinal inflammation, intestinal mucosal barrier damage, and intestinal flora imbalance, or its brain-gut axis-related effects in other models. Based on current published academic research, there is a lack of systematic studies on Bacillus coagulans, especially the specific strain Bacillus coagulans T-21, in alleviating cognitive impairment associated with ulcerative colitis. Therefore, it is necessary to further explore the application of Bacillus coagulans T-21 in alleviating cognitive impairment associated with ulcerative colitis to provide new microecological intervention strategies. Summary of the Invention

[0006] The Bacillus coagulans provided in this invention is Bacillus coagulans T-21 as described in application number 202311722148.1, which was deposited on October 16, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, with accession number GDMCC No: 63889.

[0007] The Bacillus coagulans T-21 provided by this invention can be used to prevent or improve ulcerative colitis models and cognitive impairment symptoms; its function is to improve intestinal mucosal barrier function, reduce inflammatory response, regulate intestinal flora imbalance and related metabolic pathways, and alleviate ulcerative colitis complicated with cognitive impairment.

[0008] This invention relates to the application of Bacillus coagulans T-21 in alleviating cognitive impairment associated with ulcerative colitis. Intervention with Bacillus coagulans T-21 can improve learning and memory abilities in model animals; it can also increase colon length, improve colonic tissue barrier damage, and upregulate the expression levels of tight junction proteins Occludin, Claudin-1, and Claudin-5; furthermore, it can downregulate the expression levels of the inflammatory cytokine IL-1β in colonic tissue and the inflammatory cytokine IL-6 in hippocampal tissue, thereby alleviating intestinal and central inflammatory responses.

[0009] Multi-omics analysis results showed that intervention with Bacillus coagulans T-21 could improve the gut microbiota imbalance and diversity abnormalities induced by DSS, and regulate related differentially expressed metabolites, metabolic pathways, and tissue protein profiles. Mechanistic analysis suggested that Bacillus coagulans T-21 may participate in the improvement of gut-brain axis-related pathological processes by regulating the expression of CXADR in colonic tissue and BDNF in hippocampal tissue. Therefore, this invention provides a new technical solution for microecological intervention in ulcerative colitis complicated with cognitive impairment. Attached Figure Description

[0010] Figure 1 The results of behavioral tests on mice in each group are shown.

[0011] Figure 2 The results show the comparison of colon length among the mice in each group.

[0012] Figure 3 The results show the expression of colonic barrier-related proteins and inflammatory factors in mice of each group.

[0013] Figure 4 The results of metagenomic analysis of the gut microbiota of mice in each group are shown.

[0014] Figure 5 The results of the KEGG functional pathway analysis of the intestinal flora of mice in each group are shown.

[0015] Figure 6 The results of metabolomics analysis of intestinal contents of mice in each group are shown.

[0016] Figure 7 The results of proteomic analysis of colon tissue from each group of mice are shown.

[0017] Figure 8 The results of proteomics analysis of hippocampal tissue from each group of mice are shown.

[0018] Figure 9 The results show the detection levels of key proteins in the colon and hippocampus tissues of mice in each group. Detailed Implementation

[0019] The following is in conjunction with the appendix Figure 1-9The present application will be further described in detail with reference to the embodiments. It should be noted that: unless otherwise specified, the conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer, and the raw materials used in the following embodiments are all from commercially available sources unless otherwise specified.

[0020] The Bacillus coagulans T-21 used in the following embodiments of the present invention was produced by Kunming Aikete Biotechnology Co., Ltd.

[0021] The experimental materials and reagents involved in the following content are as follows: 1) Experimental animals: 40 male SPF grade C57BL / 6 mice, aged 6-8 weeks and weighing 23-26g, were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. and housed in an SPF grade animal room at a temperature of 22-25℃ and a humidity of 50-60%, with a circadian rhythm of 12h / 12h. They had free access to food and water and were acclimatized for 7 days before the experiment began.

[0022] 2) Main reagents: DSS (sodium dextran sulfate, molecular weight 36,000-50,000) was purchased from MP Biomedicals, USA; mesalazine enteric-coated tablets (Salfo) were purchased from Dr. Falk Pharma GmbH, Germany; tight junction protein (Occludin, Claudin-1, Claudin-5) antibodies were purchased from Abcam Biotechnology; inflammatory factor (IL-1β, IL-6) antibodies were purchased from CST Biotechnology; Anti-Rabbit IgG (H+L) and Anti-Mouse IgG (H+L) were purchased from VICMED Biotechnology; NC membrane was purchased from Cytiva (GE Life (Company Name); PVDF membranes were purchased from Millipore; BCA kits, PMSF protease inhibitors, RIPA lysis buffer, loading buffer, SDS-PAGE protein loading buffer (5×), and Ponceau S staining solution were purchased from Beyotime Biotechnology Co., Ltd.; SWE rapid high-resolution electrophoresis buffer (dry powder), ice-free rapid transfer buffer (dry powder), and TBS dry powder buffer were purchased from Sewell Biotechnology Co., Ltd. 3) Experimental equipment: Behavioral testing equipment (forced swimming test apparatus, tail suspension test apparatus) was purchased from Anhui Zhenghua Bio-instrument Equipment Co., Ltd.; other conventional experimental equipment were commonly used in the laboratory. Example 1

[0023] 1. Preparation of Bacillus coagulans T-21 bacterial suspension.

[0024] 1.1 Experimental Materials The strain used in this embodiment is Bacillus coagulans T-21, scientifically known as Bacillus coagulans T-21, with the preservation number GDMCC No. 63889. The strain used in the experiment was lyophilized powder of Bacillus coagulans T-21, with a viable cell count of 1×10¹¹ CFU / g.

[0025] 1.2 Experimental Methods Preparation of Bacillus coagulans T-21 bacterial suspension: Lyophilized Bacillus coagulans T-21 strain (GDMCC No. 63889) was taken, and the viable cell count of the lyophilized powder was 1×10¹¹ CFU / g. According to the experimental drug administration requirements, a bacterial suspension of the appropriate concentration was prepared with sterile water, thoroughly mixed by vortexing, and then set aside. The prepared bacterial suspension was used for subsequent animal intervention experiments.

[0026] 2. Construction of a mouse model of ulcerative colitis complicated with cognitive impairment and intervention with Bacillus coagulans T-21 2.1 Experimental Materials Experimental animals: SPF grade C57BL / 6 male mice, 6-8 weeks old, weighing 23-26g; after purchase, they were acclimatized under SPF conditions for 1 week before entering the experiment.

[0027] Main reagents: dextran sulfate sodium (DSS, molecular weight 36,000-50,000, MP Biomedicals); mesalazine enteric-coated tablets (Salfo, Dr. Falk Pharma GmbH, Germany).

[0028] 2.2 Experimental Methods 2.2.1 Model Construction and Validation Establishment of a model of ulcerative colitis complicated with cognitive impairment: Mice were given free access to 3% (w / v) DSS aqueous solution to induce acute colitis; from day 8 onwards, they were given free access to sterile water, and behavioral assessments were subsequently performed. After the behavioral assessments, relevant tissue samples were collected to detect intestinal barrier damage, changes in inflammatory factor expression, and their potential gut-brain axis mechanisms.

[0029] Model validation: After modeling, the degree of colitis was assessed by measuring colon length, and learning, memory and cognitive function were evaluated in combination with cognitive-related behavioral experiments to determine whether the mouse model of ulcerative colitis combined with cognitive dysfunction was successfully constructed.

[0030] 2.2.2 Grouping and Intervention Mice were randomly divided into four groups, approximately 15 mice per group: control group (CON group): free access to sterile water; model group (DSS group): free access to DSS solution; Bacillus coagulans T-21 intervention group (B.co intervention group): Bacillus coagulans was administered by gavage daily during the DSS treatment period at a dose of 2 × 10⁻⁶. 9 CFU / day / mouse; Positive control group (MES group): Mesalazine was administered by gavage daily at a dose of 200 mg / kg during DSS treatment. Intervention period: 7 consecutive days.

[0031] 2.3 Experimental Results Compared to the CON group, mice treated with DSS exhibited typical ulcerative colitis-related phenotypes, including shortened colon length, diarrhea, and positive fecal occult blood. Simultaneously, cognitive function tests showed that mice in the DSS group exhibited decreased learning and memory abilities and significant cognitive dysfunction. These results demonstrate that DSS successfully induced and established a mouse model of ulcerative colitis combined with cognitive impairment.

[0032] 3. Detection of the ameliorative effect of Bacillus coagulans T-21 on neurobehavioral abnormalities in model mice. To evaluate the effect of Bacillus coagulans T-21 on improving neurobehavioral abnormalities in a DSS-induced ulcerative colitis model mouse, behavioral analyses were conducted on mice in each group using the open field test, spontaneous activity test, elevated cruciate maze test, tail suspension test, and forced swimming test (corresponding to...). Figure 1 (A-1E). The results showed that, compared with the DSS group, the spontaneous activity and exploratory behavior of mice in the B. coli intervention group were improved, the number of times they entered the elevated cruciate maze with open arms and the proportion of time spent with open arms increased, and the cumulative immobility time in the tail suspension test and forced swimming test was shortened, suggesting that Bacillus coagulans T-21 intervention can improve the neurobehavioral abnormalities in DSS-induced model mice. These results indicate that Bacillus coagulans T-21 has an ameliorative effect on DSS-induced ulcerative colitis-related neurobehavioral abnormalities, providing experimental evidence for its application in alleviating ulcerative colitis-related neurological function damage.

[0033] 3.1 Experimental Materials The instruments and equipment included: an open field test chamber (50cm×50cm×50cm), an autonomous activity monitoring system, an elevated cross maze device (arm length 30cm, arm width 5cm, maze height 15cm; open arm unobstructed, closed arm with a 15cm baffle), a suspended tail test device, and a forced swimming test device (cylindrical container with a diameter of 15cm and a height of 25cm), all purchased from Anhui Zhenghua Biological Instrument Equipment Co., Ltd.

[0034] 3.2 Experimental Methods Behavioral tests were conducted after the intervention period ended. After each animal test, the apparatus was cleaned and disinfected with 75% ethanol to reduce interference from odor cues on subsequent behavior. To minimize the interaction between different tests, behavioral assessments were conducted in the following order: open field test → spontaneous activity test → elevated cross maze test → tail suspension test → forced swimming test.

[0035] 3.2.1 Open Field Test: Mice were placed in the center of an open field chamber, and parameters such as total movement distance, number of crossings, time spent in the central area, and percentage of movement distance in the central area were recorded over 5 minutes using a video tracking system to assess spontaneous activity levels and exploratory behavior. Generally, a decrease in activity / exploration-related indicators suggests reduced exploration motivation and / or increased anxiety-like levels.

[0036] 3.2.2 Spontaneous Activity Experiment: Mice were placed in a spontaneous activity monitoring system, and the total movement distance, number of crossings, time spent in the central area, and proportion of movement distance in the central area were recorded within 5 minutes to further reflect changes in spontaneous activity ability and spatial exploration tendency.

[0037] 3.2.3 Elevated Cross Maze Test: Mice were placed in the center of the maze facing the open arms. The number of times mice entered the open arms and the time / percentage spent in the open arms were recorded within 5 minutes to evaluate the level of anxiety-like behavior. Reduced open-arm exploration usually indicates increased anxiety-like behavior.

[0038] 3.2.4 Tail Suspension Test: The mouse tail is fixed at approximately the proximal third and suspended upside down on the device. The cumulative immobility time over 6 minutes is recorded to assess depressive-like behavior. Prolonged cumulative immobility time usually indicates a worsening of the depressive-like phenotype.

[0039] 3.2.5 Forced swimming test: Add warm water (25±1℃) to a cylindrical container to a depth of about 15cm, place the mice in the water, and record the cumulative immobility time within 6 minutes to evaluate the depressive-like behavioral phenotype.

[0040] 3.3 Experimental Results like Figure 1 As shown in Figure A, compared with the CON group, the DSS group mice showed a significant decrease in total movement distance, number of crossings, time spent in the central area, and proportion of movement distance in the central area during the open field experiment, indicating that DSS treatment reduced the spontaneous activity ability and exploratory behavior of the mice. Compared with the DSS group, the B.co group showed a significant increase in the above indicators, indicating that Bacillus coagulans T-21 intervention can improve the abnormal activity ability and exploratory behavior of the model mice.

[0041] like Figure 1As shown in B, compared with the CON group, the DSS group mice showed significantly lower total movement distance, number of crossings, number of crossings, time spent in the central area, and proportion of movement distance in the central area during the spontaneous activity experiment; compared with the DSS group, the B.co group mice showed significantly higher values ​​for the above indicators, suggesting that Bacillus coagulans T-21 intervention can promote the recovery of spontaneous activity ability and exploratory behavior in mice.

[0042] like Figure 1 As shown in Figure C, compared with the CON group, the proportion of open arm entry and the proportion of open arm residence time in the DSS group were significantly decreased, indicating that the model mice exhibited obvious behavioral abnormalities. Compared with the DSS group, the proportion of open arm entry and the proportion of open arm residence time in the B.co group were significantly increased, indicating that Bacillus coagulans T-21 intervention could improve the above abnormal phenotypes.

[0043] Figure 1 As shown in Figure D, compared with the CON group, the cumulative immobility time in the tail suspension test was significantly prolonged in the DSS group; compared with the DSS group, the cumulative immobility time in the B.co group was significantly shortened, further indicating that Bacillus coagulans T-21 intervention can improve DSS-induced behavioral abnormalities in mice.

[0044] Figure 1 As shown in E, the cumulative immobility time in the forced swimming test did not differ significantly among the groups of mice. Combined with the results of the open field test, spontaneous activity test, elevated cruciate maze test, and tail suspension test, it can be concluded that Bacillus coagulans T-21 has a significant ameliorative effect on DSS-induced behavioral abnormalities in mice, providing experimental evidence for its application in alleviating neurological dysfunction associated with ulcerative colitis.

[0045] In conclusion, Bacillus coagulans T-21 can significantly improve DSS-induced spontaneous activity, exploratory behavior, and some emotion-related behavioral abnormalities in mice, suggesting its potential application value in alleviating neurological dysfunction associated with ulcerative colitis.

[0046] 4. Effects of Bacillus coagulans T-21 on colon morphology, intestinal barrier, and inflammatory factors in mice (corresponding) Figure 2 , Figure 3 ) The effect of Bacillus coagulans T-21 on colon length in a mouse model of DSS-induced enteritis combined with cognitive impairment (DSS) Figure 2 The expression changes of intestinal barrier-related proteins and inflammatory factors in colon tissue, as well as inflammatory factors in hippocampal tissue, were detected. Figure 3 ).

[0047] 4.1 Experimental Materials Reagents: Bacillus coagulans T-21 strain, manufactured by Kunming Aikete Biotechnology Co., Ltd.; DSS purchased from MPBiomedicals; BCA kit, PMSF protease inhibitor, RIPA lysis buffer, loading buffer, SDS-PAGE protein loading buffer (5×), and Ponceau S staining solution purchased from Beyotime Biotechnology Co., Ltd.; IL-1β antibody purchased from Cell Signaling Technology; Occludin antibody, Claudin-1 antibody, and Claudin-5 antibody purchased from Abcam; β-actin antibody purchased from Proteintech; 26616 marker and 26619 marker purchased from Thermo Fisher Scientific; NC membrane purchased from Cytiva (GELife); PVDF membrane purchased from Millipore; PAGE gel rapid preparation kit purchased from Yisheng Biotechnology Co., Ltd.; universal antibody dilution buffer, PBS phosphate buffer, qualitative filter paper, WB rapid blocking buffer, Tween-20, and Anti-Rabbit IgG (H+L). Anti-Mouse IgG (H+L) was purchased from VICMED Biotechnology Co., Ltd.; SWE rapid high-resolution electrophoresis buffer (dry powder), ice-free rapid transfer buffer (dry powder) and TBS dry powder buffer were purchased from Sewell Biotechnology Co., Ltd.; iodine disinfectant and 75% ethanol were purchased from Suzhou Zhongwang Health Technology Co., Ltd.; the remaining reagents were all common chemical reagents and were purchased from Shanghai Guoyao Group Chemical Reagent Co., Ltd.

[0048] 4.2 Experimental Methods 4.2.1 Colon length detection (corresponding to) Figure 2 After the intervention, the mice were euthanized by cervical dislocation, and the colon was quickly dissected and separated. The intestinal contents were gently removed with PBS to avoid traction damage. The length of the colon was measured with a ruler, photographed and recorded, and statistically analyzed.

[0049] 4.2.2 Detection of intestinal barrier proteins (corresponding) Figure 3Colon tissue was collected and lysed in pre-chilled RIPA lysis buffer (containing 1% PMSF protease inhibitor) for 30 min on ice. The mixture was then centrifuged at 14000 rpm for 20 min at 4°C. The supernatant was collected, and protein concentration was determined using the BCA method. 50 μg of protein sample was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blocked with a rapid blocking solution for 30 min. Primary antibodies (Occludin, Claudin-1, and Claudin-5 antibodies diluted 1:1000) were added and incubated overnight at 4°C. The membrane was washed three times with TBST (10 min each time), and then incubated with secondary antibody (diluted 1:10000) at room temperature for 2 h. After washing, the PVDF membrane was scanned using an Odyssey laser imaging system, and the band gray values ​​were analyzed using ImageJ software. The relative expression level was calculated using β-actin as an internal reference. Figure 3 A1 represents the Western blot bands for Occludin, Claudin-1, and Claudin-5 proteins. Figure 3 A2-3 is a statistical analysis of the relative expression levels of Occludin, Claudin-1, and Claudin-5.

[0050] 4.2.3 Detection of inflammatory factors in colon tissue (corresponding) Figure 3 B1~32): IL-1β protein expression in colon tissue was detected using Western blot. Figure 3 B1 is the IL-1β protein band detected by Western blot. Figure 3 B2 represents the statistical analysis of the relative expression levels of IL-1β.

[0051] 4.2.4 Detection of inflammatory factors in hippocampal tissue (corresponding) Figure 3 C1~C2): After the mice were sacrificed, the hippocampus tissue of the brain was quickly separated (operated on ice to avoid tissue degradation), and the expression of IL-1β protein was detected by Western blot. Figure 3 C1 represents the band detected by detecting IL-1β protein in hippocampal tissue. Figure 3 C2 represents the statistical analysis of the relative expression levels of IL-1β.

[0052] 4.3 Experimental Results like Figure 2 As shown, compared with the CON group, the colon length of mice in the DSS group was significantly shortened, suggesting that DSS induction can lead to colon tissue damage; compared with the DSS group, the colon length of mice in the B.co group was significantly increased, indicating that Bacillus coagulans T-21 can effectively alleviate DSS-induced colon shortening.

[0053] like Figure 3As shown in A1-A4, compared with the CON group, the relative expression levels of Occludin, Claudin-1, and Claudin-5 in the colon tissue of mice in the DSS group were significantly reduced, suggesting that DSS-induced enteritis can lead to damage to tight junction structures and decreased intestinal barrier function. Compared with the DSS group, the expression levels of the above tight junction proteins were significantly increased after intervention in the B.co group, indicating that Bacillus coagulans T-21 intervention can improve intestinal barrier integrity.

[0054] like Figure 3 As shown in B1-B2, compared with the CON group, the relative expression level of IL-1β protein in the colon tissue of the DSS group was significantly increased, suggesting that DSS induction can induce a significant local inflammatory response in the intestine. Compared with the DSS group, the relative expression level of IL-1β protein in the colon tissue of mice after B. coli intervention was significantly decreased, indicating that Bacillus coagulans T-21 intervention can downregulate the expression level of inflammatory factors in the colon tissue and improve the local inflammatory response.

[0055] like Figure 3 As shown in C1-C2, compared with the CON group, the relative expression level of IL-6 protein in the hippocampus of the DSS group was significantly increased, suggesting that DSS-induced intestinal inflammation may be accompanied by a central inflammatory response. Compared with the DSS group, the relative expression level of IL-6 protein in the hippocampus of mice after B. coli intervention was significantly decreased, indicating that Bacillus coagulans T-21 intervention can alleviate hippocampal inflammatory response. Combined with the results of intestinal length, intestinal mucosal barrier-related proteins, and inflammatory factors, it can be seen that Bacillus coagulans T-21 intervention has an ameliorative effect on DSS-induced colon-related pathological damage and inflammatory response, and has a certain regulatory effect on central inflammatory-related changes, suggesting its potential application value in alleviating ulcerative colitis complicated with cognitive dysfunction.

[0056] 5. The regulatory effect of Bacillus coagulans T-21 on the intestinal flora structure of mice (corresponding) Figure 4 , Figure 5 ) Metagenomic sequencing was used to analyze the gut microbiota of model mice to evaluate the effects of Bacillus coagulans T-21 intervention on the composition and diversity of gut microbiota. Combined with the results of microbiota functional enrichment analysis, the regulatory effect of Bacillus coagulans T-21 on DSS-induced gut microbiota dysfunction was detected.

[0057] 5.1 Experimental Materials Reagents: Bacillus coagulans strain T-21 was produced by Kunming Aikete Biotechnology Co., Ltd.; DSS was purchased from MPBiomedicals; iodine disinfectant and 75% ethanol were purchased from Suzhou Zhongwang Health Technology Co., Ltd.; PBS and qualitative filter paper were purchased from VICMED Biotechnology Co., Ltd.

[0058] 5.3 Experimental Results like Figure 4 As shown in Figure A, the richness and evenness of the gut microbiota in each group of mice were assessed using Alpha diversity analysis. The results showed differences in the Simpson, Shannon, and Chao indices among the groups. Compared to the CON group, the Shannon and Chao indices in the DSS group generally showed a decreasing trend, suggesting that the richness and diversity of the gut microbiota may be reduced after DSS treatment. Compared to the DSS group, the Shannon and Chao indices in the B. coli group showed an increasing trend, indicating that Bacillus coagulans T-21 intervention had a certain ameliorative effect on the changes in gut microbiota diversity induced by DSS. Furthermore, the Simpson index also showed differences among the groups, suggesting that DSS treatment may cause changes in the evenness of the microbiota and the distribution of dominant microbiota, and that these changes were somewhat improved after Bacillus coagulans T-21 intervention.

[0059] like Figure 4 As shown in Figure B, principal coordinate analysis (PCoA) based on gut microbiota community structure revealed different distribution trends in the CON, DSS, and B. co groups. Compared to the CON group, the DSS group showed an overall shift, suggesting that DSS treatment can alter the overall community structure of the gut microbiota. Compared to the DSS group, the B. co group showed some separation from the DSS group and a trend towards converging with the CON group, indicating that Bacillus coagulans T-21 intervention has a certain ameliorative effect on DSS-induced gut microbiota structure abnormalities.

[0060] like Figure 4 As shown in Figure C, metagenomic analysis revealed differences in the relative abundance of gut microbiota at the phylum level among the different groups of mice. In the CON group, Bacteroidetes, Verrucomicrobiota, and Bacillota accounted for a higher proportion. Compared to the CON group, the DSS group showed a significant decrease in Pseudomonadota and a decreasing trend in Verrucomicrobiota, suggesting that DSS treatment can alter the phylum-level composition of the gut microbiota. Compared to the DSS group, B. coli intervention resulted in a decrease in the relative abundance of Pseudomonadota, a rebound in the relative abundance of Bacteroidetes, and a further increase in Bacillota in some samples, indicating that Bacillus coagulans T-21 intervention can regulate the DSS-induced abnormalities in the phylum-level structure of the gut microbiota.

[0061] like Figure 4As shown in Figure D, metagenomic analysis revealed differences in the relative abundance of gut microbiota at the genus level among the different groups of mice. In the CON group, Akkermansia, Bacteroides, Clostridium, Muribaculum, and Duncanella accounted for a higher proportion. Compared to the CON group, the DSS group showed a significantly increased relative abundance of Escherichia, while the relative abundance of Akkermansia and Muribaculum decreased. Bacteroides and Clostridium showed some fluctuations among different samples within the group, suggesting a significant change in the genus-level composition of the gut microbiota after DSS treatment. Compared to the DSS group, the relative abundance of Escherichia decreased after B. coli intervention, while Bacteroides and Clostridium showed an increasing trend in some samples. The above results suggest that intervention with Bacillus coagulans T-21 can regulate the abnormal abundance of key bacterial genera induced by DSS and improve gut microbiota imbalance, which may be related to improved intestinal mucosal barrier function and reduced inflammatory response.

[0062] like Figure 4 As shown in Figure EF, a volcano plot was used to analyze the differentially expressed microbiota between groups. The horizontal axis represents log2 (fold change), indicating the fold change in microbiota abundance; the vertical axis represents -log10 (P value), indicating the statistical significance of the difference; the dashed line represents the screening threshold for differentially expressed microbiota. Points above the threshold line represent differentially expressed microbiota that meet the screening criteria, and different colors indicate their phylum-level classification. The results showed that compared with the CON group, the DSS group had more differentially expressed microbiota, suggesting that DSS treatment can lead to a wide range of changes in the composition of the gut microbiota. Compared with the DSS group, B. coli intervention still showed changes in various differentially expressed microbiota, suggesting that it has a regulatory effect on DSS-induced microbiota abnormalities. Combining the above phylum- and genus-level analysis results, it can be concluded that Bacillus coagulans T-21 intervention may improve DSS-induced gut microbiota imbalance, thereby being associated with improved intestinal mucosal barrier function, reduced inflammatory response, and relief of gut-brain axis-related abnormalities.

[0063] like Figure 5As shown, this study compared the changes in functional pathways of the gut microbiota in different groups of mice using KEGG Pathway Level 3 enrichment analysis. The results showed that, compared with the CON group, the DSS group had higher enrichment levels in pathways such as ABC transporters, Escherichia coli biofilm formation, phosphotransferase system (PTS), flagellar assembly, and bacterial chemotaxis. This suggests that DSS treatment altered the functional activities of the gut microbiota in transport and uptake, adhesion and colonization, motility and chemotaxis, as well as environmental responses; these changes may be related to enhanced intestinal mucosal stimulation and inflammation-related abnormalities.

[0064] Compared with the DSS group, the enrichment of pathways such as the bacterial secretion system and lipopolysaccharide biosynthesis decreased after B. co intervention, while the enrichment of pathways such as teichoic acid biosynthesis, peptidoglycan biosynthesis, biosynthesis of nucleotide sugars, biosynthesis of various nucleotide sugars, amino sugar and nucleotide sugar metabolism, and aminoacyl-tRNA biosynthesis increased.

[0065] The above results suggest that intervention with Bacillus coagulans T-21 can regulate DSS-induced abnormalities in the gut microbiota, reduce functional activities related to lipopolysaccharide-related stimulation and bacterial secretion, and enhance functional processes related to cell wall component synthesis and basal metabolism. This effect may be further related to improved intestinal mucosal barrier, reduced inflammatory response, and relief of gut-brain axis abnormalities.

[0066] 6. The regulatory effect of Bacillus coagulans T-21 on the intestinal metabolomics characteristics of mice (corresponding) Figure 6 ) 6.1 Experimental Materials Reagents: Bacillus coagulans strain T-21 was produced by Kunming Aikete Biotechnology Co., Ltd.; DSS was purchased from MPBiomedicals; iodine disinfectant and 75% ethanol were purchased from Suzhou Zhongwang Health Technology Co., Ltd.; PBS and qualitative filter paper were purchased from VICMED Biotechnology Co., Ltd.

[0067] 6.2 Experimental Methods 6.2.1 Collection and processing of intestinal contents: Immediately after euthanizing the mice, the abdominal cavity was dissected and the colon was separated. The contents of the colon were collected under sterile conditions, quickly aliquoted into sterile cryovials, and then stored at -80°C for long-term storage to minimize the risk of metabolite degradation and sample contamination.

[0068] 6.2.2 Metabolomics Detection: Metabolomics detection of intestinal contents samples was performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd., following their standardized procedures for sample pretreatment, instrument detection, and data acquisition and analysis.

[0069] 6.2.3 Metabolomics Analysis: The raw data were first filtered for features and missing values ​​were appropriately filled in. Then, normalization was performed to reduce systematic bias introduced by non-biological factors. Principal component analysis (PCA) was performed using the R package mixOmics to assess the clustering trend of the samples, the differences in metabolomics profiles between groups, and the degree of dispersion within groups. Differential metabolite screening was performed using the R package ropls (v1.42.0) to build a PLS-DA model and perform statistical tests. The screening thresholds were set as follows: fold change ≥ 1, model variable importance (VIP) ≥ 1, and p < 0.05. The cation and anion models were calculated separately and then summarized. The results were visualized using the R package ggplot2 to draw PLS-DA score plots, volcano plots, and heatmaps.

[0070] 6.3 Experimental Results like Figure 6 As shown, this study used partial least squares discriminant analysis (PLS-DA), differential metabolite volcano plots, Venn diagrams, and metabolite clustering heatmaps to analyze the intestinal metabolomics characteristics of mice in each group.

[0071] like Figure 6 A shows that the CON, DSS, and B. co group samples exhibit different distribution characteristics in the PLS-DA score map. The CON and DSS groups show a more pronounced separation, suggesting that DSS treatment alters the intestinal metabolic profile of mice. The B. co group also shows a certain degree of separation from the DSS group, suggesting that Bacillus subtilis T-21 intervention can, to some extent, regulate DSS-induced metabolic abnormalities. Figure 6As shown in B1-B2, the volcano plots further reveal a large number of differentially expressed metabolites in the DSS group, and a certain number of differentially expressed metabolite changes can still be observed after B.co intervention, suggesting that Bacillus coagulans T-21 may participate in alleviating ulcerative colitis complicated with cognitive dysfunction by regulating the intestinal metabolic disorders induced by DSS.

[0072] like Figure 6 C1-C2 Venn plot results showed that, through comparative analysis of differentially expressed metabolites, 25 metabolites that were upregulated in the DSS group compared to the CON group were downregulated after B. coli intervention; conversely, 69 metabolites that were downregulated in the DSS group compared to the CON group were upregulated after B. coli intervention. These results suggest that Bacillus coagulans T-21 intervention can reverse the abnormal metabolite changes induced by DSS, involving a total of 94 metabolites. Figure 6 The clustering heatmaps shown in D1-D2 further indicate that some metabolites with elevated overall expression in the DSS group showed a decreasing trend after B. coli intervention; conversely, some metabolites with decreased overall expression in the DSS group showed an increasing trend after B. coli intervention. These results suggest that Bacillus coagulans T-21 intervention can, to some extent, reverse DSS-induced intestinal metabolic abnormalities.

[0073] 7: Effects of Bacillus coagulans T-21 on the proteomic characteristics of mouse colon and hippocampus tissues (corresponding to...) Figure 7 , Figure 8 ) Proteomics methods were used to examine mouse colon and hippocampus tissues to analyze the changes in protein expression profiles in these two tissues after intervention with Bacillus coagulans T-21 (see colon). Figure 7 seahorse Figure 8 The study also screened and compared differentially expressed proteins to provide experimental evidence at the protein level for research on the mechanism of action of Bacillus coagulans T-21 in alleviating ulcerative colitis complicated with cognitive impairment. 7.1 Experimental Materials Reagents: Bacillus coagulans strain T-21 was produced by Kunming Aikete Biotechnology Co., Ltd.; DSS was purchased from MPBiomedicals; iodine disinfectant and 75% ethanol were purchased from Suzhou Zhongwang Health Technology Co., Ltd.; PBS and qualitative filter paper were purchased from VICMED Biotechnology Co., Ltd.

[0074] 7.2.1 Tissue protein extraction: 30 mg of colon tissue and 30 mg of hippocampal tissue were taken respectively, and Shanghai Meiji Biomedical Technology Co., Ltd. was commissioned to complete the protein extraction and proteomics detection.

[0075] 7.2.2 Proteomics Data Analysis: Preliminary quality control was performed on the protein expression matrix data provided by the company, and missing values ​​were imputed using the local similarity method, followed by quantile normalization. For sample correlation analysis, Pearson correlation coefficients were calculated using Hmisc v4.8-0 and visualized. Principal component analysis (PCA) was performed using FactoMineR v2.8 to reduce the dimensionality of the normalized data, and the first two principal components were extracted using factoextra v1.0.7 and a scatter plot was generated. Differentially expressed proteins were fitted and screened using a limma linear model, with a threshold set at |log2(FC)|≥0.5 and p<0.05; volcano plots were generated using ggplot2 v3.4.3, and heatmaps were generated using pheatmap v1.0.12.

[0076] 7.2 Experimental Results This study used high-throughput proteomics analysis to analyze the protein expression profiles of colon tissues from mice in each group, in order to evaluate the effect of Bacillus coagulans T-21 intervention on the proteomic characteristics of DSS-induced mouse colon tissues.

[0077] like Figure 7 As shown in Figure A, the box plot distributions of protein quantification indicators in each group of samples are generally similar. The dispersion among biological replicates in the CON group, DSS group, and B.co group is small, indicating that this proteomics detection has good intragroup consistency and comparability.

[0078] like Figure 7 As shown in Figure B, the correlation analysis of protein expression among samples revealed a high overall correlation between samples and good consistency in the correlation of replicates within each group, further indicating good experimental repeatability and reliable data. Meanwhile, certain differences in protein expression profiles were observed among different groups, suggesting that both DSS modeling and Bacillus coagulans T-21 intervention can have a holistic impact on the protein expression profile of mouse colon tissue.

[0079] like Figure 7As shown in Figure C, the principal component analysis (PCA) results based on the total protein quantification data showed that the variance explained by the first principal component (Dim1) was 41.0%, and that by the second principal component (Dim2) was 22.9%, collectively explaining 63.9% of the sample variation information, which can well reflect the overall protein expression differences among the samples. The results showed that the CON group, DSS group, and B.co group samples exhibited different distribution characteristics in the principal component space, with a clear separation trend among the groups. Specifically, the CON group and the DSS group were clearly separated, indicating that DSS induction can lead to a significant change in the protein expression profile of mouse colon tissue. The B.co group samples also showed a separation trend from the DSS group samples and exhibited different distribution characteristics than the DSS group, indicating that Bacillus coagulans T-21 intervention can regulate DSS-induced abnormal colon tissue protein expression.

[0080] like Figure 7 As shown in D, the differentially expressed protein volcano plot used |log2(fold change)|≥1 and P<0.05 as the differential screening threshold. Figure 7 As shown in D1, compared with the CON group, the DSS group had more differentially expressed proteins that reached the screening threshold, indicating that the pathological process of DSS-induced ulcerative colitis complicated with cognitive impairment is accompanied by extensive changes in the protein expression profile of colonic tissue; such as Figure 7 As shown in D2, compared with the DSS group, B.co intervention also showed a certain number of differentially expressed proteins that reached the screening threshold, indicating that Bacillus coagulans T-21 intervention can further adjust the abnormal protein expression profile induced by DSS.

[0081] like Figure 7 As shown in Figure E, Venn diagram analysis of differentially expressed proteins revealed that among the proteins upregulated in the DSS group compared to the CON group, 161 proteins showed a downregulation trend after B. coli intervention. Simultaneously, 240 proteins were specifically upregulated in the DSS group, while 62 proteins were specifically downregulated in the B. coli group. Conversely, among the proteins downregulated in the DSS group compared to the CON group, 143 proteins showed an upregulation trend after B. coli intervention. Simultaneously, 300 proteins were specifically downregulated in the DSS group, while 43 proteins were specifically upregulated in the B. coli group. These results indicate that DSS treatment can significantly alter the protein expression profile of colonic tissue, while B. coli intervention can inversely regulate some differentially expressed proteins. This suggests that Bacillus coagulans T-21 may participate in improving intestinal mucosal barrier damage and alleviating inflammatory responses by regulating abnormal colonic tissue protein expression profiles, and may further be associated with alleviating the pathological process of ulcerative colitis complicated by cognitive impairment.

[0082] Based on the differentially expressed proteins mentioned above, this study further performed GO functional annotation and KEGG pathway enrichment analysis on the differentially expressed proteins in colon tissue after Bacillus coagulans T-21 intervention.

[0083] like Figure 7 As shown in F1, GO biological process analysis revealed that differentially expressed proteins were mainly enriched in processes such as inflammatory response, neutrophil migration, neutrophil chemotaxis, and regulation of phagocytosis / positive regulation of phagocytosis. This indicates that the differentially expressed proteins related to Bacillus coagulans T-21 intervention are mainly involved in the local inflammatory immune response and inflammatory cell recruitment process in the colon.

[0084] like Figure 7 F2-F3 analysis and GO cell component and molecular function analysis showed that the differentially expressed proteins were mainly related to the NADPH oxidase complex, collagen-containing extracellular matrix, receptor ligand activity, endopeptidase regulator / inhibitor activity, and complement component C3b binding. This suggests that Bacillus coagulans T-21 intervention may be related to oxidative stress-related processes, extracellular matrix remodeling, inflammatory signaling regulation, and complement-related immune responses.

[0085] like Figure 7 F4 analysis showed that the differentially expressed proteins were mainly enriched in complement and coagulation cascades, neutrophil extracellular trap formation, leukocyte transendothelial migration, phagosomes, cell adhesion molecule interactions, and integrin signaling.

[0086] The above results suggest that Bacillus coagulans T-21 may be associated with improved intestinal mucosal barrier damage and reduced peripheral inflammatory response by alleviating local colonic inflammation, improving inflammatory cell recruitment and cell adhesion-related abnormalities, and further participate in the regulation of pathological processes related to ulcerative colitis and cognitive impairment.

[0087] High-throughput proteomics analysis was used to analyze the changes in protein expression profiles of hippocampal tissues in mice of different groups to evaluate the effect of Bacillus coagulans T-21 intervention on the proteomic characteristics of DSS-induced mouse hippocampal tissues.

[0088] like Figure 8 As shown in Figure A, the box plot distributions of protein quantification indicators in each group of samples are generally similar. The dispersion among biological replicates in the CON group, DSS group, and B.co intervention group is generally small, indicating that this proteomics test has good intragroup consistency and comparability.

[0089] like Figure 8 As shown in Figure B, the correlation analysis of protein expression among the samples in each group revealed a high overall correlation between the samples and good consistency in the correlation of repeated samples within each group, indicating good experimental repeatability and reliable data. Meanwhile, certain differences in protein expression profiles were observed among the samples from different groups, suggesting that both DSS treatment and Bacillus coagulans T-21 intervention can have a holistic impact on the protein expression profile of mouse hippocampal tissue.

[0090] like Figure 8 As shown in Figure C, the principal component analysis (PCA) results based on the total protein quantification data showed that the variance explained by the first principal component (Dim1) was 20.3%, the variance explained by the second principal component (Dim2) was 15.4%, and the cumulative variance explained reached 35.7%, which can fully reflect the differences in some overall protein expression among the samples in each group. The results showed different distribution characteristics in the principal component space, and there was a certain separation trend between the groups; among them, the distribution of the CON group and the DSS group was different, suggesting that DSS treatment can lead to changes in the protein expression profile of mouse hippocampal tissue; the B. coli intervention group and the DSS group samples also showed a separation trend and exhibited different distribution characteristics than the DSS group, suggesting that Bacillus coagulans T-21 intervention can regulate the abnormal protein expression in hippocampal tissue induced by DSS.

[0091] like Figure 8Figures D1-D2 show the volcano plots of differentially expressed proteins, with |log2(fold change)|≥1 and P<0.05 as the differential screening threshold. Compared with the CON group, the DSS group showed 20 significantly upregulated and 17 significantly downregulated differentially expressed proteins, indicating that DSS treatment is accompanied by significant changes in the protein expression profile of hippocampal tissue. Compared with the DSS group, the B. coli intervention group showed 35 significantly upregulated and 14 significantly downregulated differentially expressed proteins, suggesting that Bacillus coagulans T-21 intervention can further regulate the abnormal protein expression profile of hippocampal tissue induced by DSS.

[0092] like Figure 8 As shown in E, the Venn intersection analysis of differentially expressed proteins revealed that among the 20 proteins upregulated in the DSS group compared to the CON group, one protein was significantly downregulated after B. coli intervention; and among the 17 proteins significantly downregulated in the DSS group compared to the CON group, 17 proteins were significantly upregulated after B. coli intervention. These results indicate that Bacillus coagulans T-21 intervention can have a reverse regulatory effect on some differentially expressed proteins induced by DSS, and may be related to the improvement of abnormal protein expression in hippocampal tissue and the relief of central inflammation-related abnormalities.

[0093] like Figure 8 As shown in F, based on the differentially expressed proteins mentioned above, GO functional annotation and KEGG pathway enrichment analysis were further performed on the differentially expressed proteins in the hippocampus after intervention with Bacillus coagulans T-21.

[0094] like Figure 8 As shown in F1, GO bioprocess analysis revealed that the differentially expressed proteins were mainly enriched in processes such as protein activation cascade, negative regulation of coagulation, negative regulation of fibrinolysis, and S-adenosylmethionine metabolic process. This suggests that the differentially expressed proteins related to Bacillus coagulans T-21 intervention are mainly involved in inflammation-related cascade, coagulation / fibrinolysis balance, and methyl donor metabolism.

[0095] like Figure 8As shown in F2, GO molecular functional analysis revealed that the differentially expressed proteins were mainly located in structures such as the G protein-coupled receptor dimeric complex, the Golgi transcisterna, and the Golgi medial cisterna, indicating that intervention with Bacillus coagulans T-21 may be related to membrane receptor signal transduction and protein processing and transport.

[0096] As shown in the figure Figure 8 As shown in F3, GO molecular functional analysis revealed that the differentially expressed proteins were mainly enriched in S-methyltransferase activity, small GTPase binding, and actin binding.

[0097] like Figure 8 As shown in F4, KEGG pathway enrichment analysis revealed that differentially expressed proteins were mainly enriched in pathways related to complement and coagulation cascades, cysteine ​​and methionine metabolism, glycine, serine, and threonine metabolism, and folate transport and metabolism. These results suggest that Bacillus coagulans T-21 intervention may participate in the regulation of pathological processes associated with ulcerative colitis and cognitive impairment by modulating central inflammation-related processes.

[0098] 8. Validation of core targets and elucidation of mechanisms (corresponding to) Figure 9 ) To further verify the molecular changes associated with Bacillus coagulans T-21 in improving DSS-induced ulcerative colitis complicated with cognitive impairment, this study used Western blotting to detect changes in the protein expression of Coxsackievirus and Adenovirus Receptor (CXADR) in colonic tissue and Brain-Derived Neurotrophic Factor (BDNF) in brain tissue after intervention.

[0099] Changes in the key colonic protein CXADR and its possible mechanisms of action: like Figure 9 A1-A2 show the detection results of the key colonic protein CXADR. The results showed that compared with the CON group, the expression level of CXADR protein in the colonic tissue of mice in the DSS group was decreased; compared with the DSS group, the expression level of CXADR protein increased after B. coli intervention. Possible mechanism of action of CXADR: CXADR is a molecule related to epithelial cell junctions and barrier stability, and its expression changes are closely related to the state of the intestinal barrier. DSS-induced colonic mucosal damage was accompanied by downregulation of CXADR expression, suggesting that the local intestinal barrier was disrupted; while after B. coli intervention, CXADR expression was upregulated compared with the DSS group. These results suggest that Bacillus coagulans T-21 may be related to the improvement of intestinal mucosal barrier damage and the reduction of local colonic inflammation, thereby participating in the regulation of the pathological processes related to ulcerative colitis and cognitive impairment.

[0100] The possible mechanisms underlying changes in the key brain protein BDNF: like Figure 9 B1-B2 represent the detection results of the key brain tissue protein BDNF. The results showed that, compared with the CON group, the expression level of BDNF protein in the brain tissue of mice in the DSS group was decreased; after B. coli intervention, the expression level of BDNF protein increased. Possible mechanism of action of BDNF: BDNF is an important neurotrophic molecule related to the maintenance of neuronal function and synaptic plasticity, and its expression changes may be related to hippocampal tissue functional status and cognitive abnormalities. The decrease in BDNF level in hippocampal tissue after DSS treatment suggests that it may be accompanied by abnormal hippocampal tissue functional status; while the increase in BDNF expression after B. coli intervention suggests that Bacillus coagulans T-21 may improve the abnormal state of hippocampal tissue by regulating the expression of neurotrophic molecules in hippocampal tissue, and may be related to the alleviation of pathological processes related to ulcerative colitis and cognitive impairment.

[0101] In summary, intervention with Bacillus coagulans T-21 may participate in the regulation of the pathological process associated with ulcerative colitis and cognitive impairment by improving local intestinal barrier-related abnormalities, reducing inflammatory responses, and further related to improved expression of neurotrophic molecules in the hippocampus. These results provide molecular-level support for the role of Bacillus coagulans T-21 in improving ulcerative colitis and cognitive impairment, from both the perspectives of intestinal barrier-related molecules and hippocampal neurotrophic molecules.

[0102] The applicant declares that while the technical solutions described above are illustrative of this invention, the invention is not limited to the specific embodiments described, and therefore does not imply that the invention must be implemented in the manner described above. Those skilled in the art should understand that any improvements, equivalent substitutions of materials, and selections of methods for this invention fall within the scope of protection and disclosure of this invention.

[0103] Furthermore, the various technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid repetition, the present invention will not describe various combination methods separately.

Claims

1. A Bacillus coagulans T-21, characterized in that: The application of Bacillus coagulans T-21 in the prevention or improvement of ulcerative colitis.

2. The Bacillus coagulans T-21 as described in claim 1, characterized in that: The application of Bacillus coagulans T-21 in alleviating cognitive impairment associated with ulcerative colitis.

3. A Bacillus coagulans T-21, characterized in that: The application of Bacillus coagulans T-21 in improving gut microbiota.

4. The Bacillus coagulans T-21 according to claim 3, characterized in that: The application of Bacillus coagulans T-21 in improving the Bacteroidetes phylum in the intestine.

5. The Bacillus coagulans T-21 according to claim 3, characterized in that: The application of Bacillus coagulans T-21 in inhibiting Escherichia coli in the intestine.

6. The Bacillus coagulans T-21 according to any one of claims 1-5, characterized in that: The product used in the application uses Bacillus coagulans T-21 as the active ingredient, and the dosage form is powder. The viable count of Bacillus coagulans T-21 is not less than 1×10¹¹ CFU / g.

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