Bifidobacterium breve and application thereof in relieving alzheimer's disease

Abstract

The application discloses Bifidobacterium breve and application thereof in relieving Alzheimer's disease and belongs to the technical field of probiotics. 18 Bifidobacterium strains are separated from the feces of children aged 0-3 years old, and are identified as Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium animalis through analysis and identification. A Bifidobacterium breve LE4 strain with potential ability of relieving Alzheimer's disease is screened out through in-vitro antioxidant and LPS clearance capacity screening. Through behavior experiment (nest building experiment, open field experiment, water maze experiment), histopathological analysis, molecular biology detection and other methods, the LE4 strain is comprehensively evaluated to have the effects of improving cognitive function, reducing Aβ deposition and tau protein phosphorylation, reducing neural inflammation, enhancing synaptic plasticity, repairing intestinal barrier, regulating intestinal flora and metabolites and other multi-target point intervention AD, and provides a new strategy for promoting AD microecological treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotics, more particularly to a Bifidobacterium breve and its application in relieving Alzheimer's disease. BACKGROUND

[0002] With the intensification of global population aging, Alzheimer's disease (AD) has become a major public health problem threatening human health. AD is mainly characterized by progressive cognitive impairment and memory loss. According to the 2024 report of the International Alzheimer's Disease Association (ADI), there are about 550 million dementia patients worldwide, and it is estimated that the number will rise to 1.39 billion by 2050, and the growth trend is particularly evident in low-income countries, which will cause a great burden on the society and the medical system. The drugs commonly used in clinical treatment of Alzheimer's disease, such as acetylcholinesterase inhibitors (such as donepezil, galantamine) and NMDA receptor antagonists (such as memantine), are mainly used to improve cognitive symptoms and delay disease progression, but their effects are limited to short-term intervention and are difficult to reverse neurodegenerative changes. Long-term use of some patients may also cause gastrointestinal discomfort, arrhythmia, dizziness, and depression, etc. Adverse reactions. Especially for elderly patients with multiple coexisting AD, drug dependence and safety issues are more prominent. Therefore, the development of alternative treatment methods with clear mechanisms of action and high safety has become the current research focus.

[0003] At present, the pathogenesis of AD is considered to be related to multiple factors, including β-amyloid (Aβ) deposition, tau protein abnormal phosphorylation, synaptic dysfunction, neuroinflammation, and metabolic imbalance, etc. Among them, intestinal flora imbalance is considered by more and more studies as one of the important external triggers of AD progression. Probiotics, especially Bifidobacterium, have been widely studied for AD prevention and intervention in recent years due to their multiple advantages in regulating intestinal microecology, reducing LPS levels, enhancing barrier function, and regulating immune inflammation. However, different strains have strain-specific functions, and there is currently a lack of systematic screening and verification of their mechanisms.

[0004] Although some probiotic products (including some published patent technologies such as CN118256400B, CN112972502B, etc.) have shown potential in improving cognitive function in animal models or preliminary clinical studies (such as observing improved performance in water maze tests, decreased inflammatory factor levels, etc.), the explanation of their mechanisms often stops at the relatively superficial level of phenomenon observation. This limitation is reflected in several key aspects:

[0005] Model selection and mechanism depth are limited: Many studies use models that are difficult to fully simulate the complex pathological process of AD (especially the chronic progression related to aging and genetics) (such as models focusing on single oxidative stress or rapid but with significant technical / biological defects of Aβ brain injection models). This limits the ability of researchers to explore the root cause of the disease and the long-term treatment effect. For example, Aβ injection models may introduce interference factors due to surgical trauma and metabolic differences, making it difficult to distinguish whether probiotics act on the core pathology or non-specific damage.

[0006] Evaluation indicators are relatively single: The evaluation of the intervention effect often focuses on limited behavioral tests (such as basic water maze spatial memory) and a few biochemical indicators (such as some inflammatory factors), lacking a comprehensive examination of the multidimensional pathophysiological changes of the disease (such as abnormal glucose and lipid metabolism, dynamic changes of key pathological protein networks, anti-inflammatory / pro-inflammatory balance, etc.).

[0007] "Intestinal-brain axis" mechanism exploration is superficial or in different directions: Although the relationship between intestinal microorganisms and brain health is increasingly valued, existing studies (such as CN115721017B) often only report the effects of probiotics on certain endpoints of the intestinal tract or immunity (such as activating specific anti-inflammatory indicators), failing to systematically and multi-dimensionally analyze how probiotics affect the central nervous system pathology through regulating the intestinal microecology and its metabolites (such as short-chain fatty acids), repairing intestinal barrier integrity, and inhibiting the production of key pro-inflammatory mediators (such as LPS) in the intestinal tract. The lack of depth in this mechanism exploration makes it difficult to reveal the fundamental path of probiotics' cognitive protection.

[0008] Given that AD pathological progression involves neuroinflammation, Aβ deposition, tau protein phosphorylation, metabolic disorders, blood-brain barrier damage, and intestinal barrier damage, developing a new complex intervention strategy with "strain specificity-mechanism clarity" as the core, screening intestinal probiotics that can be deeply analyzed at the mechanism level, have multi-target regulation ability, and have safety and transformation potential, is of great significance for promoting the scientific development and practical application of AD microecological therapy. SUMMARY

[0009] To solve the above problems, the present application provides a Bifidobacterium breve and its application in alleviating Alzheimer's disease.

[0010] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0011] Bifidobacterium breve LE4, which is preserved in Guangdong Microbial Culture Collection Center on March 6, 2025, has a preservation number of GDMCC No: 65984, and is located at No. 59, Building 5, 100, Martyrs' Avenue, Guangzhou.

[0012] Another object of the present application is to provide an application of the above-mentioned Bifidobacterium breve LE4 in preparing a product for relieving Alzheimer's disease, which is relieved by at least one of the following ways:

[0013] (a) inhibiting the TLR4-NF-κB / NLRP3 inflammatory pathway, and reducing the expression of TLR4, MYD88, p-p65, NLRP3 and IL-1β proteins in brain tissue;

[0014] (b) increasing the content of fecal short-chain fatty acids including acetate, propionate, butyrate, isobutyrate, iso-valerate and hexanoate;

[0015] (c) repairing the expression of intestinal tight junction proteins, and significantly increasing the expression of Occludin and ZO-1 proteins in the colon;

[0016] (d) reducing the deposition of Aβ plaques and the aggregation of phosphorylated tau proteins in brain tissue, inhibiting the activation of microglial cells and astrocytes, reducing the levels of inflammatory factors IL-1β and IL-18 in brain tissue, and increasing the level of anti-inflammatory factor IL-22;

[0017] (e) reducing the levels of IL-1β, IL-18 and LPS in serum, and increasing the level of IL-22;

[0018] (f) regulating the structure of intestinal flora, and restoring the ratio of Bacteroidetes / Firmicutes;

[0019] (g) up-regulating metabolites such as 24(S), 25-epoxycholesterol, and activating the linoleic acid metabolic pathway.

[0020] Another object of the present application is to provide a product for relieving Alzheimer's disease, which comprises one or more of the fermentation broth, live bacteria and freeze-dried powder of Bifidobacterium breve LE4.

[0021] Preferably, the number of live Bifidobacterium breve LE4 in the product for relieving Alzheimer's disease is ≥1×10 9 CFU / g.

[0022] Another object of the present application is to provide a medicament for alleviating Alzheimer's disease, which contains the above-mentioned Bifidobacterium breve LE4 and a pharmaceutically acceptable carrier.

[0023] Another object of the present application is to provide a preparation method of a medicament for alleviating Alzheimer's disease, which comprises culturing the Bifidobacterium breve LE4 strain.

[0024] Preferably, the culture method of the Bifidobacterium breve LE4 strain is as follows: the Bifidobacterium breve LE4 is inoculated into a modified MRS culture medium (liquid / solid) containing mupirocin and cysteine, and then subjected to anaerobic culture at 37 °C, followed by centrifugation, collection of the bacterial precipitate, washing with 0.01 M sterilized PBS buffer for 3 times, resuspension in the PBS buffer, and adjustment to a target concentration.

[0025] Preferably, the composition of the modified MRS culture medium (liquid / solid) containing mupirocin lithium salt and cysteine hydrochloride is as follows: 10.0 g / L proteose peptone, 5.0 g / L beef infusion powder, 4.0 g / L yeast infusion powder, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, 0.05 g / L mupirocin lithium salt, and 0.5 g / L cysteine hydrochloride; the solid culture medium additionally contains agar 15.0 g / L.

[0026] According to the above technical solution, compared with the prior art, the present application has the following beneficial effects:

[0027] 1) The present application isolates 18 strains of Bifidobacterium from the feces of children aged 0-3 years, which are identified to include Bifidobacterium longum, Bifidobacterium breve and animal Bifidobacterium, and a strain of Bifidobacterium breve LE4 with potential ability to alleviate Alzheimer's disease is screened out through in vitro antioxidant and LPS clearance capacity. Through behavioral experiments (nest building experiment, open field experiment, water maze experiment), histopathological analysis, molecular biology detection and other methods, the LE4 strain is comprehensively evaluated to have the effects of improving cognitive function, reducing Aβ deposition and tau protein phosphorylation, reducing neural inflammation, enhancing synaptic plasticity, repairing intestinal barrier, regulating intestinal flora and metabolites, and intervening AD at multiple targets, which provides a new strategy for promoting the microecological treatment of AD.

[0028] 2) The mouse model used in the present application is more similar to the actual progress of Alzheimer's disease in many aspects than the prior art, covering physiological level expression, progressive pathology and multi-dimensional cognitive impairment, improving the clinical relevance and reliability of the effect verification index. In contrast, some probiotic studies use single or rapid induction models (such as Aβ brain injection), which are difficult to fully simulate the complex pathology of AD, and the evaluation index is limited to simple behavior and some inflammatory factors, lacking a systematic analysis of metabolism, pathological protein network and inflammatory balance. In addition, existing research on the "gut-brain axis" mechanism stops at the level of immune activation, and does not reveal the complete pathway of how probiotics affect central nervous system pathology by regulating intestinal microecology, repairing barriers and inhibiting pro-inflammatory mediators, limiting the comprehensive understanding of the mechanism of cognitive protection.

[0029] 3) The mouse model of the present application highly simulates human AD in terms of expression level, physiological Aβ accumulation and cognitive impairment progression, and the verification index (Aβ42 / Aβ40 ratio, neuroinflammation, cognitive behavior, etc.) is more clinically relevant. In contrast, existing probiotic research relies on acute Aβ injection or single oxidative stress models, and evaluation is limited to basic water maze and a few inflammatory factors, making it difficult to distinguish between core pathology and non-specific damage; The "gut-brain axis" mechanism stops at the end-point effect report, lacks multi-level linkage analysis such as short-chain fatty acids, intestinal barrier repair and LPS inhibition, and is difficult to reveal the fundamental action path of probiotics. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 : 7 strains of Bifidobacterium with antioxidant activity inhibit the production of lipopolysaccharide (LPS) by intestinal microbiota, and the detection results are shown in the figure, * represents P<0.05, ** represents P<0.01;

[0031] Figure 2 : Phylogenetic tree of strain LE4 based on 16S rDNA sequence;

[0032] Figure 3 : Behavior analysis results of mice in each group in the nesting behavior experiment, wherein A: representative nesting shape of mice in each group, B: nesting quality score of mice in each group;

[0033] Figure 4 : Action trajectory analysis results of mice in each group in the open field experiment, wherein A: representative trajectory of mice in each group, B: standing frequency, movement distance, movement time and resting time statistical analysis of mice in each group;

[0034] Figure 5 : Action trajectory analysis results of mice in each group in the water maze experiment, wherein A: representative trajectory of mice in each group, B: latency change of mice in each group during positioning navigation training, C: number of times of crossing the platform during the spatial exploration period of mice in each group;

[0035] Figure 6 H&E staining results of liver of each group of mice;

[0036] Figure 7 Detection results of brain tissue Aβ pathological characteristics of each group of mice; wherein, A: Aβ immunohistochemical results, B: immunohistochemical relative quantification results, C: Western blot analysis detects the expression of Aβ in hippocampal tissue, D: WB quantification results of Aβ;

[0037] Figures 8A-8D Verification results of Bifidobacterium breve LE4 regulating Alzheimer's disease pathology of APP / PS1 mice, wherein, Figure 8A Expression results of p-tau, IBA-1 and GFAP using immunofluorescence, Figure 8B Immunofluorescence relative quantification results, Figure 8C Western blot analysis detects the expression of p-tau, IBA-1 and GFAP in hippocampal tissue, Figure 8D WB quantification results of p-tau, IBA-1 and GFAP;

[0038] Figure 9 Verification results of Bifidobacterium breve LE4 enhancing synaptic plasticity of APP / PS1 mice, wherein, A: Expression results of PSD95 and SYNAPSIN I using immunofluorescence, B. Immunofluorescence relative quantification results, C: Western blot analysis detects the expression of PSD95 and SYNAPSIN I in hippocampal tissue, D: WB quantification results of PSD95 and SYNAPSIN I;

[0039] Figure 10 Verification results of Bifidobacterium breve LE4 restoring intestinal barrier function of APP / PS1 mice, wherein, A: Expression results of Occludin and ZO-1 using immunofluorescence, B: Immunofluorescence relative quantification results, C: Western blot analysis detects the expression of Occludin and ZO-1 in colon tissue, D: WB quantification results of Occludin and ZO-1; * represents P<0.01;

[0040] Figure 11 Body weight and food intake change curve of each group of mice during 12 weeks of gavage, wherein, A: body weight, B: food intake;

[0041] Figure 12 Fasting blood glucose and 2h postprandial blood glucose detection results of each group of mice, wherein, A: fasting blood glucose, B: 2h postprandial blood glucose;

[0042] Figure 13 Detection results of TC, TG, INS and GLP-1 content in serum of each group of mice;

[0043] Figure 14 : The content of IL-1β, IL-18, IL-22, LPS, Aβ1-42 in the serum of each group of mice;

[0044] Figure 15 : The content of IL-1β, IL-18, IL-22, NLRP3, Aβ1-40, Aβ1-42 in the hippocampus of each group of mice;

[0045] Figure 16 : The content of LPS in the colon of each group of mice;

[0046] Figures 17A-17F : The results of diversity analysis of Bifidobacterium breve LE4 in restoring the intestinal microbiota of APP / PS1 mice, wherein, Figure 17A : The alpha diversity index of the fecal flora of mice, Figure 17B : The plot of mice fecal microorganisms, Figure 17C : The principal component analysis (PCA) of mice feces, Figure 17D : The relative abundance of changes in the phylum level of the fecal flora of mice, Figure 17E : The relative abundance of changes in the genus level of the fecal flora of mice, Figure 17F : LefSe analysis of mice fecal microorganisms;

[0047] Figures 18A-18C : Functional prediction of intestinal microbiota metabolic pathways, wherein, Figure 18A : Prediction of MetaCyc pathways in the intestinal microbiota of WT, AD and Bifidobacterium breve LE4 groups, Figure 18B : The abundance of KEGG 2-level functional categories of each group, Figure 18C : Significant differences in MetaCyc metabolic pathways between AD and Bifidobacterium breve LE4 groups;

[0048] Figures 19A-19D : The results of differential SCFA metabolism and related pathway analysis of Bifidobacterium breve LE4 intervened APP / PS1 mice, wherein, Figure 19A : The content of short-chain fatty acids (SCFAs) in the feces of each group of mice, Figure 19B : The heatmap of the content of short-chain fatty acids in the feces of each group of mice, Figure 19C : Comparison of metabolic pathways of WT and AD groups involving short-chain fatty acids, Figure 19D : Comparison of metabolic pathways of LE4 and AD groups involving short-chain fatty acids;

[0049] Figures 20A-20D : The results of fecal metabolomics and pathway analysis of Bifidobacterium breve LE4 intervened APP / PS1 mice, wherein, Figure 20A: Discriminant analysis of fecal metabolites of AD group and WT group mice by orthogonal partial least squares, Figure 20B : Discriminant analysis of fecal metabolites of AD group and LE4 group mice by orthogonal partial least squares, Figure 20C : Volcano plot of differential metabolites of LE4 group and AD group, Figure 20D : KEGG enrichment analysis of key differential metabolites;

[0050] Figure 21 : Verification results of Bifidobacterium breve LE4 inhibiting TLR4-NF-κB / NLRP3 molecular pathway, wherein, A, B: Western blot analysis detects the expression of TLR4-NF-κB / NLRP3 signaling pathway proteins in brain tissue, C, D: WB quantitative results of TLR4-NF-κB / NLRP3 signaling pathway; * represents P<0.05, ** represents P<0.01. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0052] The reagents involved in the embodiments of the present application are all purchased from the market channel, and the methods not mentioned are conventional experimental methods, which will not be described one by one here.

[0053] The following reagents are exemplary:

[0054] MRS medium (liquid / solid) containing mupirocin lithium salt and cysteine hydrochloride: 10.0 g / L proteose peptone, 5.0 g / L beef infusion powder, 4.0 g / L yeast extract powder, 20.0 g / L glucose, 2.0 g / L potassium phosphate dibasic, 2.0 g / L trisodium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, 0.05 g / L mupirocin lithium salt, 0.5 g / L cysteine hydrochloride; the solid medium additionally contains agar 15.0 g / L.

[0055] Triglyceride (TG) assay kit (single reagent GPO-PAP method, enzyme label meter and biochemical analyzer, item number: A110-1-1), total cholesterol (TC) assay kit (single reagent GPO-PAP method, enzyme label meter and biochemical analyzer, item number: A111-1-1) are purchased from Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd.

[0056] Mouse Interleukin 1 beta (IL-1 beta) ELISA Kit (Cat No: ml098416), Mouse Interleukin 18 (IL-18) ELISA Kit (Cat No: ml1066842), Mouse Interleukin 22 (IL-22) ELISA Kit (Cat No: ml063138), Mouse Amyloid beta 1-40 (A beta 1-40) ELISA Kit (Cat No: ml001859), Mouse Amyloid beta 1-42 (A beta 1-42) ELISA Kit (Cat No: ml002201), Mouse Lipopolysaccharide (LPS) ELISA Kit (Cat No: ml201801), Mouse Postsynaptic Density Protein 95 (PSD95) ELISA Kit (Cat No: ml106176), Mouse Brain-derived Neurotrophic Factor (BDNF) ELISA Kit (Cat No: ml002219), Mouse NLR Family Pyrin Domain Containing 3 (NLRP3) ELISA Kit (Cat No: ml037234), Mouse Insulin (INS) ELISA Kit (Cat No: ml001983), Mouse Glucagon-like Peptide 1 (GLP-1) ELISA Kit (Cat No: ml201801) were purchased from Shanghai Sunshine Biotechnology Co., Ltd.

[0057] Example 1 Screening of strains

[0058] The 18 strains of Bifidobacterium were isolated from the feces of children aged 0-3 years old by the Food Quality and Safety Innovation Team of the School of Food Science and Engineering, Jilin Agricultural University, and were sent to Shanghai Genechem Co., Ltd. for Sanger sequencing. After the sequencing results were compared with the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), it was determined that among the 18 strains, 5 were Bifidobacterium longum (No. BL3, BL4, BL5, BL11, BL22), 1 was Bifidobacterium breve (No. LE4), and 12 were animal Bifidobacterium (No. BD1, BD2, BD3...BD12).

[0059] The above 18 strains were inoculated into liquid modified MRS medium, and after anaerobic culture at 37 °C for 48 h, they were centrifuged at 4200 rpm for 20 min. The supernatant and bacterial pellet were collected and treated as follows:

[0060] The supernatant was filtered through a 0.22 μm sterile filter membrane to obtain cell-free supernatant, which was stored at 4 °C for later use;

[0061] The bacterial pellet was washed with 0.01 M sterilized PBS buffer for 3 times, and then divided into two groups and resuspended in PBS buffer, with the concentration adjusted to 1×10 9CFU / mL, one group as intact cells;

[0062] The other group of bacterial suspension was broken by ice bath ultrasonic wave (400 W, working for 5 s, interval for 5 s, for 60 cycles), then centrifuged at 4200 r / min for 10 min at 4 ℃, and the supernatant was collected to obtain cell-free extract, which was stored at 4 ℃ and used within 24 h.

[0063] 1. Primary screening of antioxidant capacity of strains in vitro

[0064] The intact cells, cell-free supernatant and cell-free extract of the 18 strains obtained above were subjected to DPPH, ABTS· + and ·OH radical scavenging capacity determination, specifically as follows:

[0065] (1) DPPH radical scavenging capacity determination

[0066] 1 mL of the sample to be tested was mixed with 1 mL of 0.2 mol / L DPPH anhydrous ethanol solution, incubated at 25 ℃ for 30 min in the dark, centrifuged at 10000 r / min for 1 min, and the absorbance (A1) of the supernatant was determined at 517 nm. Each group had 3 independent repeated experiments. The control group (A0) used an equal volume of distilled water instead of the sample (i.e. 1 mL distilled water + 1 mL DPPH anhydrous ethanol solution), and the blank group (A2) used an equal volume of anhydrous ethanol instead of DPPH (i.e. 1 mL sample to be tested + 1 mL anhydrous ethanol), and the rest of the operations were the same. The DPPH radical scavenging rate (%) was calculated according to the formula DPPH radical scavenging rate (%) = [1 - (A1-A2) / A0] x 100, and the results are shown in Table 1.

[0067]

[0068] As can be seen from Table 1, among the intact cells, Bifidobacterium longum BL3, BL4, BL5, BL11, BL22 and Bifidobacterium breve LE4 showed significant DPPH scavenging activity; the cell-free extract of Bifidobacterium breve LE4 showed the highest DPPH scavenging capacity (94.09 ± 0.03%), and the cell-free supernatant of Bifidobacterium animalis BD12 showed significant activity (78.08 ± 7.37%).

[0069] (2) ABTS· + radical scavenging capacity determination

[0070] ABTS radical cation (ABTS· +) was generated by mixing equal volume of 7 mmol / L ABTS aqueous solution and 2.45 mmol / L K2S2O8 solution at room temperature for 16 h in the dark. Before use, it was diluted with absolute ethanol and its absorbance at 734 nm (A0) was adjusted to 0.70 ± 0.02. 734

[0071] 2 mL of the sample to be tested was mixed with 4 mL of diluted ABTS• + solution, and the absorbance at 734 nm (A1) was measured after incubation at room temperature for 5 min in the dark. Three independent replicates were performed for each group. The control group (A0) used distilled water instead of the sample (i.e., 2 mL distilled water + 4 mL diluted ABTS• + solution), and the blank group (A2) used absolute ethanol instead of the diluted ABTS• + solution (i.e., 2 mL sample to be tested + 4 mL absolute ethanol), and the rest of the operations were the same. The ABTS• + radical scavenging rate (%) was calculated according to the formula: [1 - (A1 - A2) / A0] x 100. + The results are shown in Table 2.

[0072]

[0073] As shown in Table 2, in intact cells, Bifidobacterium animalis BD1 (99.02 ± 0.87%) and BD5 (96.79 ± 2.74%) showed the strongest ABTS• + scavenging activity (P < 0.05), and other strains also had significant efficacy; in cell-free supernatant, Bifidobacterium longum BL3, BL4, BL5, BL11, BL22 and Bifidobacterium breve LE4 had stronger ABTS• + scavenging activity; in cell-free extract, Bifidobacterium animalis BD2 (97.86 ± 1.26%) and BD12 (98.29 ± 0.15%) had significantly better ABTS• + scavenging ability than other strains (P < 0.05).

[0074] (3) Determination of •OH radical scavenging capacity

[0075] ​To 1 mL of the sample to be tested, 1 mL of 9 mmol / L salicylic acid solution, 1 mL of 0.05 mol / L FeSO4 solution (solvent is 0.1 mol / L HC1 solution, used to inhibit oxidation) and 1 mL of 8.8 mmol / L H2O2 solution were added in turn, mixed uniformly, and then placed in a 37 ℃ constant temperature water bath for 30 min in the dark. The absorbance (A1) was measured at 510 nm. Each group had 3 independent repeated experiments. The control group (A0) used an equal volume of distilled water instead of the sample to be tested, and the rest of the reagents were the same as the experimental group. The blank group (A2) used an equal volume of distilled water instead of the H2O2 solution, and the rest of the reagents were the same as the experimental group, which was used to deduct the background absorbance (such as Fe 2+ - non-specific absorption of salicylic acid complex). The ·OH radical scavenging rate of the sample was calculated according to the formula: ·OH radical scavenging rate (%) = [1- (A1-A0) / (A2-A0)] x 100. The results are shown in Table 3.

[0076]

[0077] As shown in Table 3, the intact cells and cell-free extracts of B. longum BL3 (66.39 ± 3.17%; 71.57 ± 0.72%), BL4 (67.70 ± 1.26%; 71.88 ± 0.78%), BL5 (69.85 ± 1.96%; 72.80 ± 0.31%), BL11 (67.74 ± 2.80%; 71.94 ± 0.95%), BL22 (69.56 ± 0.71%; 72.23 ± 0.46%) and B. breve LE4 (67.89 ± 0.63%; 71.55 ± 0.22%) all showed significant ·OH scavenging activity (scavenging rate > 65%). In the cell-free supernatant, B. longum BL4, BL11 and BL22 also showed significant ·OH scavenging activity (scavenging rate > 64%).

[0078] Based on the results of the above extracellular antioxidant experiment, at least two of the nine strains that showed statistically significant effects (P < 0.05) in the analysis of DPPH, ABTS· + and ·OH radical scavenging ability were selected for further study. Therefore, BL3, BL4, BL5, BL11, BL22, LE4, BD2, BD5 and BD12 were selected for further evaluation of their inhibitory effect on the production of lipopolysaccharide (LPS) in fecal microbial culture.

[0079] 2. Secondary screening of strains for inhibition of LPS production by fecal bacterial flora

[0080] Fresh feces from the natural defecation of 3-month-old male APP / PS1 mice (housed in an environment of 20-22°C, 55±5% humidity, and a 12-h / 12-h light-dark cycle) were collected and stored at -80°C for later use.

[0081] Dilute 1.0 g of fresh mouse feces with 15 mL of sterile GAM broth (Shandong Top Biotechnology Co., Ltd.), mix well, centrifuge at 1500 rpm for 5 min, and collect the supernatant to obtain GAM culture medium for later use. The bacterial concentration is 1 × 10⁻⁶. 9 Nine bacterial strains (BL3, BL4, BL5, BL11, BL22, LE4, BD2, BD5, and BD12) at CFU / mL were inoculated into the aforementioned GAM culture medium at a 5% inoculum. After anaerobic culture at 37 ℃ for 24 h, the bacteria were sonicated on ice (400 W, 5 s on, 5 s interval) for 1 h, centrifuged at 1400 rpm for 10 min, and the supernatant was filtered through 0.45 μm and 0.22 μm membranes sequentially. The filtrates were collected. The LPS content in the filtrates of the nine bacterial strains was detected using a mouse lipopolysaccharide (LPS) ELISA kit (catalog number: ml201801, Shanghai Enzyme-Link Biotechnology Co., Ltd.) according to the instructions. The filtrates of the same strains were sonicated on ice and centrifuged under the same conditions (5% PBS) and filtered through 0.45 μm and 0.22 μm membranes sequentially as the control group (CON). The results are shown in the figure. Figure 1 .

[0082] Depend on Figure 1 It was found that among the nine bacterial strains, compared with the control group (740.65±70.84 EU / L), BL4 (839.83±3.23 EU / L) and BL5 (861.07±32.95 EU / L) significantly increased the LPS level in the intestinal flora, with increases of 13.46% and 16.31%, respectively; BL11 and BL12 were similar to the control group; while BL3, LE4, BD2, BD5 and BD12 showed inhibitory effects. Among them, LE4 (476.41±14.67 EU / L), BL3 (546.01±45.34 EU / L) and BD12 (591.73±45.73 EU / L) showed particularly strong inhibitory effects, with inhibition rates of 35.62%, 26.21% and 20.04%, respectively, with LE4 showing the most significant inhibition (**P < 0.01). Therefore, strain LE4 was selected for subsequent in vivo evaluation.

[0083] Example 2: Molecular identification, preservation, and preparation of live bacteria for strain LE4

[0084] (1) Molecular identification and preservation of strain LE4

[0085] Strain LE4 was sequenced by ShangHai ShengFeng Bioengineering Co., Ltd. The 16S rDNA sequence information of strain LE4 was used to construct a phylogenetic tree on the NCBI website by BLAST, and the results are shown in Figure 2 The phylogenetic tree shows that strain LE4 is clustered with Bifidobacterium cebidarum strain CCUG 73785 with a support rate of 100%, confirming that the strain is Bifidobacterium breve. It is named Bifidobacterium breve LE4, the taxonomic name is Bifidobacterium breve, and it is preserved in Guangdong Microbial Culture Collection Center (GDMCC), with a preservation date of 2025.03.06, a preservation number of GDMCC No: 65984, and an address of 5th Floor, Building 59, 100 Middle Xianlie Road, Guangzhou.

[0086] (2) Preparation of live bacteria of Bifidobacterium breve LE4

[0087] Bifidobacterium breve LE4 was inoculated into modified MRS medium (liquid / solid) containing mupirocin lithium salt and cysteine hydrochloride, and cultured anaerobically at 37 °C for 48 h. After centrifugation at 4200 rpm for 20 min, the bacterial precipitate was collected and washed with 0.01 M sterilized PBS buffer for 3 times, then resuspended in PBS buffer, and the concentration was adjusted to 1×10 9 CFU / mL to obtain LE4 live bacteria.

[0088] Example 3 Improvement of behavioral defects of Bifidobacterium breve LE4 in APP / PS1 mice

[0089] All experimental protocols were approved by the Experimental Animal Ethics Committee of Jilin Agricultural University (Approval No: 20240415001, Approval Date: April 15, 2024). Thirteen 3-month-old male APP / PS1 double transgenic mice (APPswe / PS1dE9, C57BL / 6 × C3H background) and five littermate wild-type (WT) male mice were selected for the experiment and were raised in the Experimental Animal Center of Jilin Agricultural University. The feeding environment was at a temperature of 20-22°C, a humidity of 55±5%, a 12 h / 12 h light-dark cycle, 3-5 mice per cage, free access to irradiation sterilized experimental mouse maintenance feed (purchased from Beijing Huafukang Biotechnology Co., Ltd.) and free drinking water.

[0090] APP / PS1 mice were randomly divided into AD group (n=5) and B. breve LE4 intervention group (n=8), and WT mice were used as control group (n=5). The AD group and the WT group were gavaged with sterile PBS solution, and the LE4 group was gavaged with B. breve LE4 bacterial suspension (1x10 9 CFU / mL), and the dose was 0.1 mL / 10 g body weight. The gavage was performed once a day at a fixed time, and lasted for 12 weeks. The feces were collected one week before the behavior experiment, quickly frozen and stored at -80°C for standby.

[0091] After the last gavage for 24 h, the nest building behavior experiment, open field experiment and Morris water maze experiment were performed, as follows:

[0092] 1. Nest building behavior experiment

[0093] The nest building behavior experiment is a behavior experiment related to spatial learning and memory, which focuses on the cognitive function of mice, and pays more attention to the daily activities and executive function of mice. The three groups of mice were single-caged (temperature 20-22°C, humidity 55±5%, 12 h / 12 h light-dark cycle), and adapted for 48 h (free drinking and water). After the adaptation, 1 cm thick corn cob bedding was put into each cage, and 12 h later, sterile absorbent cotton (2.0 g / cage) was put into the cage. After 18 h, the nest building quality was scored according to the 4-point method, and the specific scoring criteria were as follows: 1 point, the absorbent cotton was scattered irregularly in the cage and was not bitten; 2 points, the absorbent cotton was loosely gathered on one side of the cage, but there was no shaped nest and no obvious bite marks; 3 points, the absorbent cotton was folded into a nest, but only a flat nest was formed, and there were no obvious bite marks; 4 points, the absorbent cotton was folded into a deep nest and was bitten into small pieces.

[0094] The nest building behavior reflects the daily life skills and motivation, and this function is impaired in AD mice, and the nest was destroyed at 48 h ( Figure 3 A). B. breve LE4 administration significantly improved the nest building behavior ( Figure 3 A), and a completely formed nest was produced within 48 h ( Figure 3 B).

[0095] 2. Open field experiment

[0096] The above 3 groups of mice were placed in an open (no cover) box (black, plastic material, 50 cm x 50 cm x 40 cm size) for testing, the bottom of the box was evenly divided into 5 x 5 squares by lines, the 9 squares in the middle were set as the central area, and the remaining squares were the peripheral area. In a quiet and dimly lit environment (the light intensity in the central area and the peripheral area was consistent), each mouse was placed in the center of the box and allowed to freely explore the environment for 5 minutes. During this period, the animal behavior acquisition system of Chengdu Tailian Software Co., Ltd. was used to automatically record and statistically analyze the standing frequency, central activity time, resting time, and total activity distance of each mouse. The system automatically analyzed the duration of each mouse in the central area and the exploration trajectory in the test box, and output the corresponding data. After each mouse was tested, the walls and bottom of the box were wiped with 75% alcohol to eliminate odors and affect subsequent experiments. After drying, the next mouse was placed in the box for further testing.

[0097] The action trajectory analysis results of the mice in each group shown in Figure 4 indicate that after B. breve LE4 treatment, the mice showed increased standing frequency and more central area activity, indicating enhanced exploration ability and reduced anxiety.

[0098] 3. Morris water maze (MWM) experiment

[0099] The water maze experiment forces the experimental mice to swim to learn to find the hidden platform in the water, and then tests the learning and memory ability of the experimental animals to space location and direction (spatial orientation). It is the preferred classical experiment for behavioral research, especially for learning and memory spatial memory research.

[0100] The water maze circular pool (diameter 120 cm, water depth 16 cm) was divided into four quadrants, and the water temperature was maintained at 21-22°C. After the above 3 groups of mice were gavaged for 24h in the room with the water maze, they were subjected to 5 days of positioning navigation training. During the training period, each mouse was subjected to 4 pseudo-random attempts per day to locate the hidden platform under the water 0.5 cm from the water surface (diameter 65 mm, height 15 cm, transparent acrylic material) in four different quadrants. Each attempt lasted 60 s, and if it was not found within 60 s, the mouse was guided to the platform and stayed for 15 s. On the 6th day, the platform was removed and the mice were subjected to a spatial exploration test: the mice were placed in the water from quadrant I for 60 s, and the number of times the mice crossed the platform and the original residence time and swimming distance were recorded. The trajectories of each mouse were recorded using the WMT-100 Morris water maze experiment system software (Chengdu Tailian Software Co., Ltd.) during the navigation training and spatial exploration test, and the representative trajectories of the 3 groups of mice were analyzed. The latency period during positioning navigation training, the number of times the platform was crossed during spatial exploration testing, and the results are shown in Figure 5 .

[0101] As shown in Figure 5 Figure B, from the first day, the mice in the Bifidobacterium breve LE4 intervention group (LE4) showed significantly shorter escape latency than the other 2 groups (**P < 0.01). As shown in Figure 5 Figures A and 5C, the mice in the Bifidobacterium breve LE4 intervention group crossed the former platform site more frequently than the wild type WT group in the place navigation training, indicating that the memory retention of the mice in the Bifidobacterium breve LE4 intervention group was improved.

[0102] In summary, the Bifidobacterium breve LE4 intervention improved the memory ability of the APP / PS1 mice, alleviated the changes in anxiety behavior and daily life ability, and had potential for application in the auxiliary treatment of Alzheimer's disease products.

[0103] Example 4 Effect of Bifidobacterium breve LE4 on the histopathology of the brain, colon and liver of APP / PS1 mice

[0104] After the end of the behavioral experiment in Example 3, all mice were deeply anesthetized with sodium pentobarbital (anesthetic dose 150 mg / kg), blood, brain tissue, colon, liver tissue, and stored at -80°C.

[0105] The brain tissue, hippocampal tissue, colon, and liver tissue described above were paraffin-embedded and 5 μm sections were prepared according to the method of the technical literature (“Lactobacillus regulate muscle fiber type conversion in Chinese native pigs via tryptophan metabolism”, Song B, Azad MAK, Zhu Q., et al, NPJ biofilms and microbiomes, Vol. 11, No. 1). The following experiments were performed, respectively:

[0106] (1) Histopathological analysis of liver

[0107] The liver sections were stained with hematoxylin and eosin (H&E) according to the method shown in the technical literature (“Study on the relationship between the weight loss effect of Astragalus polysaccharides on obese mice and the regulation of intestinal flora”, He Xuyun, et al, World Chinese Medicine, Vol. 11, No. 11, pp. 2379-2384, publication date: November 30, 2016), and the microscopic examination results are shown in Figure 6 . As shown in Figure 6It can be seen that compared with wild-type mice (WT group), the structure of APP / PS1 mice in the AD group showed abnormalities, characterized by focal macrophage aggregation, inflammatory infiltration of hepatocytes and increased intracellular lipid droplets; B. breve LE4 administration reduced liver inflammation and restored lipid droplet morphology.

[0108] (2) Brain tissue immunohistochemical analysis

[0109] Place the 5 μm brain tissue section in the following order: environmental deparaffinizing solution I for 10 min → environmental deparaffinizing solution II for 10 min → environmental deparaffinizing solution III for 10 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → anhydrous ethanol III for 5 min → distilled water rinse (prevent drying throughout the process), cool naturally, add PBS (pH 7.4) to decolorize and shake bed wash 3 times (5 min / time), add 3% H2O2 solution, room temperature, light-proof for 25 min; PBS (pH 7.4) decolorization shake bed wash 3x5 min, after drying, the tissue is circled with a group of histological pens, and 3% BSA is added and blocked for 30 min; drop the prepared Aβ primary antibody (rabbit anti, dilution ratio 1:1000, manufacturer Servicebio, item number GB115755), wet box at 4 °C overnight, PBS (pH 7.4) decolorization shake bed wash 3x5 min; after the section is slightly dried, drop the rabbit anti secondary antibody (HRP labeled) to cover the tissue in the circle, incubate at room temperature for 50 min; place the slide in PBS (PH 7.4) for 3x5 min; after the section is slightly dried, drop the freshly prepared DAB color developing solution in the circle, rinse the section with tap water to stop color development; and use hematoxylin for restaining. Examine the section under an optical microscope, and use ImageJ software to quantify the Aβ positive signal.

[0110] (3) Immunofluorescence analysis of brain tissue and colon

[0111] The 5 μm brain tissue sections, colon tissue sections were placed in turn into environmental protection dewaxing solution I for 10 min→ environmental protection dewaxing solution II for 10 min→ environmental protection dewaxing solution III for 10 min→ anhydrous ethanol I for 5 min→ anhydrous ethanol II for 5 min→ anhydrous ethanol III for 5 min→ distilled water rinsing (prevent dry piece throughout the process), natural cooling, add PBS (pH 7.4) decoloring shaking table washing 3 times (5 min / time), spin dry after group organization pen around the tissue draw a circle, drop 3% BSA, block 30 min; drop prepared one antibody (see table 4 for specific information), 4 °C in wet box overnight, PBS (pH 7.4) decoloring shaking table wash 3x5 min; add corresponding fluorescein isothiocyanate (FITC) fluorescent second antibody (fluorescent second antibody information: FITC labeled goat anti-rabbit IgG; article number: GB22303; excitation wavelength is 492 nm, emission wavelength 520 nm; dilution ratio: 1:100), light-proof room temperature 50 min; PBS (pH 7.4) decoloring shaking table wash 3x5 min; slightly spin dry after spontaneous fluorescence quencher 5 min, water flushing 10 min, carry out mounting processing. Finally, use 2 μg / mL DAPI (Servicebio; article number: G1012-100ML) to recolor the cell nucleus. Use inverted fluorescence microscope (resolution 2048x2048 pixels, 0.325 μm / pixel) to capture image, and use ImageJ software to quantify fluorescence intensity.

[0112]

[0113] (4) Brain and colon immunoblot analysis

[0114] Take the brain tissue and colon tissue of each group of mice stored in example 4, respectively, place in ice-cold lysis buffer (formula: RIPA lysis buffer (strong, article number: The samples were homogenized at low temperature using a tissue homogenizer. The total protein concentration of the homogenized samples was determined using an enhanced BCA protein assay kit (Beyotime, China) to determine the total protein concentration of the homogenized samples. The homogenized samples were denatured at 95 °C for 5 min and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the following conditions: initial voltage of 80 V for 30 min, followed by 120 V for 70 min. The transfer buffer consisted of Tris 5.8 g, glycine 2.9 g, SDS 0.37 g, 200 mL methanol, and distilled water to a final volume of 1 L. Proteins were transferred to a PVDF membrane at a constant current of 300 mA for 90 min. The membrane was then blocked with 5% skim milk-TBST solution at room temperature for 90 min. Primary antibodies (as shown in Table 5) were added at 4 °C (each primary antibody was incubated separately; all antibodies were diluted according to the manufacturer's protocol) and incubated overnight at 4 °C. After primary antibody incubation, the membrane was washed three times with TBST (10 min / wash) to remove unbound primary antibody. Horseradish peroxidase-conjugated secondary antibody (1:20000 dilution, Albisi) was added at room temperature and incubated for 1 h to allow specific binding between the secondary and primary antibodies. After secondary antibody incubation, the membrane was washed three times with TBST (10 min / wash) to remove free secondary antibody. The protein bands after the above treatment were visualized using an enhanced ECL detection kit (Seven Innovations (Beijing) Biotechnology Co., Ltd.) (Severex SCG-W3000), and quantification was performed using ImageJ software. β-actin (dilution ratio 1:20000, manufacturer Proteintech) was used as an internal control for data normalization.

[0115]

[0116] A comprehensive analysis of the above experimental results is conducted:

[0117] Depend on Figure 7 The results of Aβ pathological features analysis of brain tissue from each group of mice showed that Aβ plaque deposition in the brains of APP / PS1 (AD) mice was significantly higher than that in the WT control group (P < 0.05) and significantly decreased after treatment with Bifidobacterium breve LE4 (P < 0.05). Western blot analysis confirmed these findings, showing elevated hippocampal Aβ levels in AD mice, which were significantly reduced by Bifidobacterium breve LE4 intervention (P < 0.05).

[0118] Validation results of B. breve LE4 modulating Alzheimer's disease pathology in APP / PS1 mice showed that compared with WT, the accumulation of phosphorylated tau (p-tau) in AD brain was increased, along with up-regulation of microglial marker IBA-1 and astrocyte marker GFAP expression, all of which were significantly reduced after administration of B. breve LE4. Western blotting confirmed the down-regulation of p-tau, IBA-1 and GFAP in the hippocampus of B. breve LE4-treated mice (P < 0.05). In summary, these results indicated that B. breve LE4 reduced p-tau accumulation and inhibited glial cell activation, thereby providing neuroprotection and alleviating cognitive deficits in APP / PS1 mice.

[0119] Validation results of B. breve LE4 modulating Alzheimer's disease pathology in APP / PS1 mice showed that compared with WT, the accumulation of phosphorylated tau (p-tau) in AD brain was increased, along with up-regulation of microglial marker IBA-1 and astrocyte marker GFAP expression, all of which were significantly reduced after administration of B. breve LE4. Western blotting confirmed the down-regulation of p-tau, IBA-1 and GFAP in the hippocampus of B. breve LE4-treated mice (P < 0.05). In summary, these results indicated that B. breve LE4 reduced p-tau accumulation and inhibited glial cell activation, thereby providing neuroprotection and alleviating cognitive deficits in APP / PS1 mice. Figure 9 Validation results of B. breve LE4 enhancing synaptic plasticity in APP / PS1 mice showed that compared with the WT group, APP / PS1 (AD) mice exhibited significantly elevated hippocampal expression of presynaptic protein Synapsin I and postsynaptic density protein PSD-95, which might reflect a compensatory response to early synaptic pathology Figure 9 A,B). Upon B. breve LE4 treatment, Synapsin I and PSD-95 levels were further increased. These observations were confirmed by Western blotting Figure 9 C,D), which demonstrated significant up-regulation of PSD-95 in the B. breve LE4-treated group (P < 0.05). In summary, these data indicated that B. breve LE4 enhanced presynaptic and postsynaptic membrane function, thereby alleviating synaptic dysfunction in APP / PS1 mice.

[0120] Immunofluorescence analysis of colon tissue showed that the fluorescence intensity of ZO-1 and claudin was significantly reduced in APP / PS1 (AD) mice compared with the WT control group (P < 0.05 Figure 10 A,B). B. breve LE4 treatment significantly restored the expression of ZO-1 and claudin, resulting in a clear, continuous and well-organized staining pattern. Western blotting Figure 10 C,D) confirmed the up-regulation of these tight junction proteins upon administration of B. breve LE4 (P < 0.05). In summary, these results indicated that B. breve LE4 enhanced intestinal barrier integrity in APP / PS1 mice by restoring tight junction protein expression.

[0121] In summary, B. breve LE4 intervention improved the inflammatory response in the liver of AD mice, significantly reduced the pathological features in the brain tissue of AD mice, restored the disrupted intestinal barrier, and has the potential to be applied in the auxiliary treatment of AD products.

[0122] Example 5 Regulation of B. breve LE4 in metabolism and inflammation

[0123] 1. Body weight and glucose metabolism detection

[0124] To evaluate the effects of B. breve LE4 on cognitive function and whole body physiology, body weight and food intake were recorded weekly during the 12-week period of B. breve LE4 gavage in each group of mice, and the results are shown in Figure 11 Although there was no significant difference in food intake between groups, the B. breve LE4 treatment group showed significantly reduced body weight compared to the AD group during the 3rd to 10th week (P < 0.05).

[0125] Given the increase in water intake observed in AD mice, the fasting blood glucose and 2-hour postprandial blood glucose levels of each group of mice were measured the day before the collection of feces, and the results are shown in Figure 12 Although the fasting blood glucose of the AD group remained within the normal range (4.5-8.1 mmol / L), both fasting and 2-hour postprandial blood glucose levels were elevated compared to the WT group. B. breve LE4 intervention effectively restored glucose levels to those observed in WT mice, indicating that B. breve LE4 can regulate glucose metabolism.

[0126] 2. Analysis of gut-brain axis molecular markers

[0127] To further evaluate serum glucose and lipid metabolism markers, the total cholesterol (TC) and triglyceride (TG) contents in the serum of each group of mice were determined using commercially available biochemical kits from Nanjing Jiancheng Biological Engineering Research Institute according to the manufacturer's instructions; mouse brain tissue and colon tissue were mixed with 4 °C pre-cooled PBS at a solid-liquid ratio of 1:9, then homogenized at 4 °C until no tissue was visible to the naked eye, and the supernatant was collected by centrifugation at 12000 rpm for 10 min at 4 °C. The contents of IL-1β, IL-18, IL-22, Aβ1-40, Aβ1-42, LPS, PSD95, BDNF, NLRP3, INS, and GLP-1 in the serum and tissue extract supernatant of each group of mice were determined using ELISA kits from Shanghai Enzyme-Linked Bioengineering Co., Ltd. (Shanghai, China) according to the instructions. The results are as follows:

[0128] The total cholesterol (TC) (see Figure 13 A) and triglyceride (TG) (see Figure 13 B) levels in the AD group were significantly elevated, but were significantly reduced after treatment with B. breve LE4. In addition, the serum insulin (INS) (see Figure 13 C) and glucagon-like peptide-1 (GLP-1) (see Figure 13D) were all decreased, but significantly increased after B. breve LE4 administration, which is consistent with the observed improvement in body weight and glycemic control.

[0129] AD mice also exhibited significant neuroinflammation, reflected by elevated serum levels of proinflammatory cytokines IL-1 b (see Figure 14 A) and IL-18 (see Figure 14 B), both of which were significantly decreased in the B. breve LE4 group. IL-22 was significantly decreased in AD mice (see Figure 14 C) and restored after B. breve LE4 treatment. Notably, serum levels of lipopolysaccharide (LPS) were significantly elevated in the AD group (see Figure 14 D), but significantly decreased after B. breve LE4 intervention (P < 0.05). In addition, serum Ab1-42 concentration was increased in AD mice, but decreased after treatment with B. breve LE4, showing a significant decrease (see Figure 14 E).

[0130] Neuroinflammatory markers in brain tissue were also assessed. Expression of IL-1 b (see Figure 15 A), IL-18 (see Figure 15 B), and NLRP3 (see Figure 15 D), a component of the inflammasome, were significantly elevated in the AD group and significantly inhibited in mice treated with B. breve LE4 (P < 0.05), while IL-22 expression was significantly upregulated after B. breve LE4 intervention (see Figure 15 C). These changes suggest that B. breve LE4 alleviates AD-associated neuroinflammation by reducing proinflammatory cytokines and enhancing anti-inflammatory responses. In addition, Ab1-40 (see Figure 15 E) and Ab1-42 (see Figure 15 F) content in the hippocampus was significantly decreased after LE4 intervention, consistent with the results of pathological section and WB shown in Example 4.

[0131] Given the elevated serum LPS levels in AD mice, colon expression of LPS was examined (see Figure 16 ), and it was assessed that B. breve LE4 treatment significantly decreased LPS levels (P < 0.05) and IL-1 b expression in the colon. These findings, combined with the immunofluorescence data showing restoration of tight junction protein expression, support the hypothesis that LPS increase in AD can lead to gut barrier disruption, which can be improved by B. breve LE4 intervention.

[0132] In summary, B. breve LE4 intervention significantly decreased serum, hippocampal, and colon inflammatory factor levels in AD mouse models, significantly increased anti-inflammatory factor IL-22, reduced Ab1-40 and Ab1-42 content in the hippocampus, and has the potential to be applied to the production of adjuvant therapy for AD.

[0133] Example 6: Effects of Bifidobacterium breve LE4 on intestinal flora structure

[0134] PCR amplification of *Bifidobacterium breve* LE4 was performed using universal primers 341F and 806R, published in the technical literature ("Nutrient supply controls the linkage between species abundance and ecological interactions in marine bacterial communities," Dai T, Wen D, Bates CT, et al., *Nature Communications*, Vol. 13, 2022). The PCR reaction system (50 μL) consisted of: 0.25 μL of 5× high-fidelity DNA polymerase, 5 μL of 5× Reaction Buffer, 5 μL of 5× High GC Buffer, 2 μL of 10 μM dNTPs, 2 μL of template DNA, 1 μL each of 10 μM forward and reverse primers, and 8.75 μL of water. The PCR program was: 98 °C pre-denaturation for 5 min; 98 °C denaturation for 30 s, 52 °C annealing for 30 s, and 72 °C extension for 45 s. The amplification process was performed at 25 cycles; a final extension at 72 °C for 5 min was completed, followed by storage at 12 °C. The amplification results were subjected to 2% agarose gel electrophoresis, and the target fragment was excised and recovered using an Axygen gel extraction kit. The PCR amplicon was then subjected to paired-end sequencing (2 × 250 bp) on the Illumina MiSeq platform. The raw sequencing reads were processed into amplicon sequence variants (ASVs) using the DADA2 pipeline. β-diversity analysis was performed using principal coordinate analysis (PCoA) based on the Jaccard distance metric, implemented in the q2-diversity module of QIIME2, and visualized in Emperor.

[0135] like Figure 17A The results of assessing microbial diversity using alpha diversity indices (including Chao1, Shannon, Simpson, and Observed_species) showed that all four indices were significantly reduced in AD mice, indicating a loss of microbial diversity, which was partially recovered after treatment with Bifidobacterium breve LE4.

[0136] Venn diagram analysis of mouse fecal microbiota ( Figure 17B) showed that WT, AD and B. breve LE4 groups had 2099, 508 and 1646 unique OUT / ASVs, respectively, with 372 OUT / ASVs common to all groups. This finding indicates that B. breve LE4 increased microbial richness in APP / PS1 mice.

[0137] Principal Coordinate Analysis (PCoA) of each group of mice fecal samples as shown in Figure 17C indicated that AD group samples were far from the clear clustering of WT group, while B. breve LE4 treated samples partially overlapped with WT group, indicating that B. breve LE4 partially restored the microbial community structure.

[0138] Classification bar plots at phylum and genus levels showed significant compositional differences between the three groups. At phylum level (see Figure 17D ), in AD mice, Clostridia abundance increased from 53.6% in WT to 68.4%, while Bacteroidia decreased, leading to an elevated Bacteroidia / Clostridia ratio (from 1.86 to 3.98). B. breve LE4 treatment decreased Clostridia (to 62.03%) and increased Bacteroides (to 21.95%), adjusting the ratio to 2.83. Genus level analysis (see Figure 17E ) showed elevated levels of Lactobacillus, Bifidobacterium and Prevotella in AD mice, while Allobaculum, Alicyclobacillus, Desulfovibrio, Lactococcus, Bacteroides and Faecalibacterium levels decreased. Administration of B. breve LE4 significantly decreased Lactobacillus and partially restored WT levels of other genera.

[0139] Differential taxa were identified by LEfSe (LDA SCORE > 2), highlighting AD-enriched microbiota such as Lactobacillus, Bacillus and Globicatella, which can serve as potential microbial biomarkers Figure 17F ). Conversely, B. breve LE4 treatment depleted these taxa and significantly enriched Ruminococcus and Dorea.

[0140] In summary, B. breve LE4 modulated the composition of the intestinal microbiota in APP / PS1 mice and can help restore the disrupted microbial homeostasis in Alzheimer’s disease pathology, with potential for application in an adjunct therapy product for AD.

[0141] Example 7 Prediction of microbiota function and metabolic pathway enrichment analysis

[0142] To investigate the functional potential of the gut microbiota, the PICRUSt2 computer tool (which can perform phylogenetic studies on communities by reconstructing unobserved states) was used in conjunction with the MetaCyc database (https: / / metacyc.org / ) and the KEGG database (https: / / www.kegg.jp / ) to predict and analyze the gut microbiota function of each group of mice.

[0143] As shown in Figure 18, the gut microbiota gene profiles of each group of mice predicted based on the MetaCyc and KEGG databases indicate that the MetaCyc pathway analysis ( Figure 18A The results indicate that the enzyme is enriched in categories such as biosynthesis, degradation / utilization / assimilation, detoxification, precursor metabolite and energy production, glycan pathway, macromolecular modification and metal cluster-related metabolism.

[0144] KEGG analysis ( Figure 18B The study revealed a rich array of metabolic pathways involved in cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and biological systems, with metabolism-related genes accounting for approximately 60% of the total. Notably, the number of genes involved in lipid metabolism and glycan biosynthesis was comparable. Comparative analysis of different metabolic pathways among the groups revealed significant functional shifts. Figure 18C Notably, several pathways, including the ASYN-PWY (ornithine degradation pathway), were significantly altered in the *Bifidobacterium breve* LE4 group compared to the AD group. This suggests that *Bifidobacterium breve* LE4 intervention remodels the metabolic potential of the gut microbiota, potentially contributing to its beneficial effects on host physiology and Alzheimer's disease pathology, and demonstrating its potential application in adjunctive therapy products for AD.

[0145] Example 8: Validation of Bifidobacterium breve LE4 enhancing SCFA levels

[0146] The feces collected in Example 3 were quantitatively monitored for short-chain fatty acids (SCFAs) by GC-MS. The specific steps are as follows:

[0147] (1) Determination of standard products and standard curves

[0148] The acetic acid, propionic acid, butyric acid, isobutyric acid, pentanoic acid, isopentanoic acid pure standard were measured, and 100 mg / mL stock solution was prepared by mixing with water, and the stock solution was diluted with water to obtain 0.06, 0.3, 1.5, 6, 30, 75, 150, 300, 750, 1500 μg / mL series of working standard solutions; the hexanoic acid pure standard was measured, and 100 mg / mL stock solution was prepared by mixing with ether, and the stock solution was diluted with ether to obtain 0.06, 0.3, 1.5, 6, 30, 75, 150, 300, 750, 1500 μg / mL series of working standard solutions. The internal standard is 4-methylvaleric acid internal standard solution (375 μg / mL, solvent is ether). For each concentration point, take a separate test tube and add 200 μL of 6 kinds of acid series working standard solution (corresponding concentration), 100 μL of 15% phosphoric acid, 20 μL of hexanoic acid series working standard solution, 20 μL of internal standard and 260 μL of ether to prepare 10 independent mixed standard solutions (total volume is 600 μL), and the concentration is from low to high, that is, 0.02 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 2 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL ten standard curve points. The stock solution is stored at -20°C, and each working solution is prepared and used immediately.

[0149] (2) Sample extraction

[0150] From the feces of each group of mice collected in Example 3, 40 mg was added to 500 μL water, 100 mg glass beads were homogenized for 60 s, and centrifuged at 12000 rpm at 4°C for 10 min. 200 μL supernatant was taken, 100 μL 15% phosphoric acid, 20 μL 375 μg / mL 4-methylvaleric acid ether solution, and 280 μL ether were added in turn, homogenized for 1 min, and centrifuged at 4°C at 12000 rpm for 10 min. The supernatant was taken, and the sample for testing was obtained.

[0151] (3) Machine testing

[0152] Chromatographic conditions: Thermo Trace 1300 gas chromatograph (Thermo Fisher Scientific, USA) was used for analysis, equipped with Agilent HP-INNOWAX capillary column (30 m x 0.25 mm ID x 0.25 μm), injection volume was 1 μL, split ratio was 10:1, carrier gas was helium, flow rate was 1 mL / min, injection port temperature was 250℃, column temperature program: initial 90℃, increased to 120℃ at a rate of 10℃ / min, increased to 150℃ at a rate of 5℃ / min, increased to 250℃ at a rate of 25℃ / min, finally 250℃ for 2 min.

[0153] Mass spectrometric conditions: Thermo ISQ 7000 single quadrupole mass spectrometer (Thermo Fisher Scientific, USA) was used, ionization mode was electron impact (EI) source, electron energy was 70 eV, scan mode was SIM (selected ion monitoring), ion source temperature was 300℃, transmission line temperature was 250℃.

[0154] The results of fecal short-chain fatty acid (SCFA) level detection of mice in each group are shown in Table 2. Figure 19A It can be seen from Table 2 that the administration of B. breve LE4 significantly increased the fecal SCFA level of APP / PS1 mice, and the levels of acetic acid, propionic acid and butyric acid increased by 2.06 times, 2.55 times and 4.15 times (P<0.001, P<0.05 and P<0.01) respectively. Other SCFAs also showed an upward trend, although these changes were not statistically significant. These results indicate that the supplementation of B. breve LE4 can alleviate the SCFA imbalance in APP / PS1 mice. Figure 19A KEGG pathway analysis (FIG. 11A, D) showed that WT mice contained more metabolites related to propionate metabolism, as well as the digestion and absorption of carbohydrates and proteins, compared with AD group. It can be seen that AD mice showed decreased levels of metabolites related to these pathways compared with B. breve LE4 group, indicating that B. breve LE4 may alleviate AD pathology by regulating propionate, carbohydrate and protein metabolism, and has potential for application in the product of adjuvant therapy for AD.

[0155] Figure 19C Example 9 Verification of B. breve LE4 regulating intestinal metabolites

[0156] Example 9 Verification of B. breve LE4 regulating intestinal metabolites

[0157] ​In order to clarify the regulatory effect of B. breve LE4 on intestinal metabolites, non-targeted metabolomics analysis was performed on the fecal samples of each group of mice collected in Example 3 using UPLC-Q Exactive-MS liquid chromatography-mass spectrometry technology, and the operation process was as follows:

[0158] The fecal samples of each group of mice were thawed at 4 ℃ and vortexed for 1 min, 50 μL of each was added to a 96-well protein precipitation plate containing 300 μL of 2-chlorophenylalanine solution (4 ppm, solvent: 80% methanol solution), shaken for 5 min, then transferred to an A200 positive pressure nitrogen blowing module, and run at low pressure for 10 min; transferred to the test area and sealed, and the detection sample was obtained.

[0159] The UPLC-Q Exactive-MS analysis conditions were as follows: an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm; Waters) was used, the chromatographic conditions were a flow rate of 0.3 mL / min, a sample volume of 2 μL, and a column temperature of 40 ℃; for LC-ESI(+)-MS analysis, the mobile phase consisted of 0.1% formic acid aqueous solution (A2) and acetonitrile (B2), while LC-ESI(-)-MS used 5 mM ammonium formate (A3) and acetonitrile (B3), and both analyses used the same gradient program: 8%–98% B in 8 minutes, 98% B for 2 minutes, and then column re-equilibration. The Q Exactive instrument was operated in full MS-ddMS2 mode for mass spectrometry detection. Full scan MS1 spectra were acquired in the m / z 100–1000 range with a resolution of 70000, while MS / MS spectra were collected with a resolution of 17500, and the normalized collision energy was 30 eV. The spray voltage was set to 3.50 kV (ESI+) and -2.50 kV (ESI-), and the capillary temperature was maintained at 325 ℃. Metabolite identification was performed by matching the experimental mass number with the HMDB, METLIN, MassBank, LipidMaps, mzCloud and PANOMIX metabolomics databases, with a mass error tolerance of 30 ppm.

[0160] As Figure 20A and 20BThe shown partial least squares discriminant analysis (PLS-DA) showed different clustering patterns between the WT group, the AD group and the LE4 group, showing the impact of B. breve LE4 administration on the fecal metabolome of APP / PS1 mice. Significant metabolic differences were observed between the AD model and the B. breve LE4 intervention group. After excluding the unannotated metabolites in the HMDB and KEGG databases, 14 metabolites were identified that showed significant changes between these groups (as shown in Table 6). The volcano plot of the differential metabolites between the LE4 group and the AD group Figure 20C ) revealed significant upregulation of 24(S), 25-epoxycholesterol, a-hydroxy-N-desmethyl tamoxifen and N-acetylglucosamine-N-acetylgalactosamine, and significant downregulation of N-acetyl leucine and arginine. KEGG pathway enrichment analysis Figure 20D ) revealed significant changes in metabolic pathways, including amino acid biosynthesis (arginine, proline, phenylalanine, tyrosine and tryptophan), steroid hormone biosynthesis, mTOR signaling pathway, clavulanic acid biosynthesis and linoleic acid metabolism.

[0161]

[0162] Example 10 Validation of B. breve LE4 inhibition of the TLR4-NF-κB / NLRP3 molecular pathway

[0163] The serum LPS and IL-1 β levels of APP / PS1 mice were significantly reduced by B. breve LE4 intervention, which can be related to the TLR4 / NF-κB signaling pathway. Western blot analysis of the TLR4-NF-κB / NLRP3 signaling pathway in brain tissue (Figure 21 ) showed that after B. breve LE4 treatment, the hippocampal expression of TLR4 (**P < 0.01), MYD88 (**P < 0.01), IKKα (**P < 0.01) and phosphorylated p65 (p-p65) (**P < 0.01) was significantly downregulated, indicating that the canonical NF-κB pathway was inhibited Figure 21 A, C). In addition to NF-κB activation, APP / PS1 mice showed elevated NLRP3 expression in the hippocampus.

[0164] To further assess the effect of B. breve LE4 on NLRP3-mediated neuroinflammation, Western blotting of hippocampal tissue was performed for NLRP3, casepase-1 p20, ASC and IL-1 β (experimental method as in Example 4). The results are shown in Figure Figure 21As shown in B and D, treatment with Bifidobacterium breve LE4 significantly reduced the levels of hippocampal casepase-1 p20 (**P < 0.01), NLRP3 (**P < 0.01), ASC (**P < 0.01), and IL-1β (*P < 0.05), consistent with the ELISA results of Example 5.

[0165] Therefore, it can be seen that Bifidobacterium breve LE4 exerts its neuroprotective effect by inhibiting the activation of the NLRP3 inflammasome and weakening neuroinflammatory signaling in the TLR4–NF–κB / NLRP3 pathway, and has the potential to be used in adjunctive therapy products for AD.

[0166] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0167] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A short bifidobacterium, characterized in that, The Bifidobacterium breve is Bifidobacterium breve ( Bifidobacterium breve LE4, deposited at Guangdong Provincial Center for Microbial Culture Collection, deposited on March 6, 2025, with accession number GDMCCNo: 65984, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of Bifidobacterium breve LE4 according to claim 1 in the preparation of drugs to alleviate Alzheimer's disease, characterized in that, Relieve it through at least one of the following methods: (a) Inhibition of the TLR4-NF-κB / NLRP3 inflammatory pathway downregulates the expression levels of TLR4, MYD88, p-p65, NLRP3 and IL-1β proteins in brain tissue; (b) Increase the content of short-chain fatty acids in feces, including acetic acid, propionate, butyric acid, isobutyric acid, isovaleric acid and hexanoic acid; (c) Repairing the expression of intestinal tight junction proteins significantly increases the expression levels of Occludin and ZO-1 proteins in the colon; (d) Reduces Aβ plaque deposition and phosphorylated tau protein aggregation in brain tissue, inhibits the activation of microglia and astrocytes, reduces the levels of inflammatory factors IL-1β and IL-18 in brain tissue, and increases the level of anti-inflammatory factor IL-22. (e) Decreases serum IL-1β, IL-18 and LPS levels, while increasing IL-22; (f) Regulate the gut microbiota structure and restore the Bacteroidetes / Firmwallis ratio; (g) Upregulates metabolites such as 24(S) and 25-epoxycholesterol and activates the linoleic acid metabolic pathway.

3. A drug for alleviating Alzheimer's disease, characterized in that, The drug comprises one or more of the fermentation broth, live cells, and lyophilized powder of Bifidobacterium breve LE4 as described in claim 1.

4. The drug for alleviating Alzheimer's disease according to claim 3, characterized in that, The number of viable Bifidobacterium breve LE4 bacteria in the drug is ≥1×10⁻⁶. 9 CFU / g.

5. A drug for alleviating Alzheimer's disease, characterized in that, The drug contains the Bifidobacterium breve LE4 as described in claim 1 and a pharmaceutically acceptable carrier.

6. The method for preparing the drug for alleviating Alzheimer's disease according to claim 5, characterized in that, The preparation method includes culturing Bifidobacterium breve LE4 strain.

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