Microbial agent based on synergistic effect of bifidobacterium pseudolongum and bifidobacterium animalis and application

Abstract

The invention discloses a microbial agent based on the synergistic effect of bifidobacterium pseudolongum and bifidobacterium animalis and application, and belongs to the technical field of biological medicine. The active ingredient of the microbial agent is a viable bacterium mixture of bifidobacterium pseudolongum and bifidobacterium animalis, or a metabolite co-cultured by the bifidobacterium pseudolongum and the bifidobacterium animalis. According to the microbial agent provided by the invention, an intestinal flora-butyric acid-peripheral / central immune axis is adjusted through a metabolic synergistic effect of the two strains, A beta deposition and neuroinflammation can be intervened from a pathologic source so as to intervene the progress of the Alzheimer's disease, and the safety is good. The synergistic effect of the bifidobacterium pseudolongum and the bifidobacterium animalis is applied to regulation of intestinal flora and intervention or auxiliary improvement of memory of the Alzheimer's disease, and a novel biological preparation and a technical path are provided for prevention and treatment of neurodegenerative diseases such as the Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to Bifidobacterium pseudolongum (… Bifidobacterium pseudolongum ) and Bifidobacterium animalis ( Bifidobacterium animalis It is used as an active ingredient in combination to prepare drugs that improve Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a latent, age-related neurodegenerative disease clinically characterized by progressive cognitive decline, accompanied by a variety of neuropsychiatric symptoms and behavioral disorders. As the most common type of dementia among the elderly, Alzheimer's disease not only severely reduces the quality of life of patients but also places a heavy burden on families and society.

[0003] The hallmark pathological features of Alzheimer's disease include β-amyloid (Aβ) plaque deposition and hyperphosphorylated tau protein tangles. Aβ oligomers can induce synaptic damage and tau protein hyperphosphorylation, leading to neuronal loss; simultaneously, Aβ aggregation triggers an inflammatory response, further exacerbating disease progression. Despite some progress in Alzheimer's disease research, its etiology remains unclear, and effective therapies to alter disease progression are lacking. Therefore, there is an urgent need to further explore its pathogenesis and develop innovative treatment strategies.

[0004] Currently, clinical management primarily focuses on symptom relief rather than addressing underlying pathological issues. In recent years, the preventative perspective has gained increasing attention. A Lancet committee report indicates that over one-third of dementia cases could be prevented by moderating modifiable lifestyle factors, particularly dietary habits. The Mediterranean diet, the Diet to Stop Hypertension (DASH), and the Mediterranean-DASH Delayed Neurodegenerative Intervention (MIND) have been shown to reduce the risk of Alzheimer's disease (Xu, J., et al Research progress and hotspots of the impact of the Mediterranean diet onaging from 2004 to 2023: A bibliometric analysis. Geriatr Nurs, 2025, 64, 103375. The core mechanism is closely related to the gut-brain axis's regulation of gut microbiota and brain function (Mafe, AN, et al. Could a Mediterranean Diet Modulate Alzheimer's Disease Progression?The Role of Gut Microbiota and Metabolite Signatures in Neurodegeneration.Foods, 2025, 14(9). For example, high-fat diet can induce gut dysbiosis, which leads to cognitive impairment in obese mice; both mouse models and human studies have shown that gut dysbiosis is associated with neuroinflammation and cognitive impairment (Bruce-Keller, A. J., et al . Obese-type gut microbiota induce neurobehavioral changes in the absence of obesity. Biol Psychiatry, 2015, 77(7), 607-615. Therefore, targeting gut microbiota through dietary intervention has become a potential strategy to delay the development of Alzheimer's disease.

[0005] Barley, as one of the oldest cultivated cereals, has been widely recognized for its health benefits. Studies have shown that early intervention with barley diet (BD) can significantly improve cognitive impairment in Alzheimer's disease mouse models, and this effect depends on the regulation of gut microbiota. Therefore, by analyzing the mechanism of gut microbiota regulation to improve Alzheimer's disease pathology, it is expected to provide new bacterial agents and application methods for the prevention and treatment of Alzheimer's disease. SUMMARY

[0006] The purpose of the present application is to provide a biological agent that can effectively improve Alzheimer's disease or assist in improving memory, and to provide a new strategy for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The present application determines that the relative abundance of Pseudobutyvibrio xylaniphilus and Bifidobacterium animalis in the intestinal contents of Alzheimer's disease (AD) model mice (5xFAD) fed with barley diet (BD) for a long time is significantly increased through 16S rRNA sequencing and abundance analysis. Bifidobacterium pseudolongum Bifidobacterium animalis Further, the Pseudobutyvibrio xylaniphilus and Bifidobacterium animalis are compounded at a ratio of 1:1 to prepare a bacterial suspension, and the AD model mice with antibiotic-treated gut microbiota (Antibiotics Treatment, ABX) are treated by gavage. The results show that the combination of the two bacteria can significantly improve the learning and memory ability of AD model mice.​

[0008] Therefore, the application provides use of B. pseudolongum and B. animalis in combination in preparation of a medicament for treating Alzheimer's disease.

[0009] Studies have shown that B. pseudolongum and B. animalis play a role in improving AD through synergistic effect (improving colonization efficiency and promoting mutual growth), and single bacteria cannot replace them.

[0010] As preferred, B. pseudolongum and B. animalis are mixed in equal amounts in the medicament.

[0011] Mechanism studies have shown that the medicament plays a role in improving Alzheimer's disease through the "gut flora-metabolite-central immune" axis, and the specific mechanism is as follows: a. Gut flora level: B. pseudolongum and B. animalis synergistically proliferate in the intestine (double bacteria co-oral administration significantly improves the colonization rate compared with single bacteria), and regulate the balance of intestinal flora; B. pseudolongum and B. animalis co-culture promotes the production of butyric acid.

[0012] b. Metabolite level: B. pseudolongum and B. animalis increase butyric acid production through metabolic synergy, and the produced butyric acid acts on the immune system through blood circulation, induces peripheral blood M2 macrophage polarization, improves M2 / M1 ratio and anti-inflammatory cytokine (IL-10, TGF-β) expression, reduces pro-inflammatory factor level, and realizes peripheral immune regulation.

[0013] c. Central immune and pathological level: peripheral immune regulation further reduces central nervous system inflammatory response, increases the number of hippocampal disease-associated microglia (DAM, such as CD11c + cells), enhances the co-localization of DAM cells and Aβ plaques and phagocytic activity; reduces hippocampal Aβ plaque deposition (reduces plaque area and diameter), and improves nerve damage.

[0014] d. Cognitive function level: the above pathological improvement ultimately improves the spatial learning and memory ability of the Alzheimer's disease model.

[0015] Based on the effect of B. pseudolongum and B. animalis in combination in improving the learning and memory ability of AD model, the application provides use of B. pseudolongum and B. animalis in combination in preparation of a functional food for assisting in improving memory.

[0016] Based on the effect of B. pseudolongum and B. animalis in combination in synergistically proliferating in the intestine and regulating the balance of intestinal flora, the application provides use of B. pseudolongum and B. animalis in combination in preparation of a functional food for regulating intestinal flora.

[0017] The present application also provides a bacterial agent for improving Alzheimer's disease or assisting in improving memory or regulating intestinal flora, wherein the active ingredient of the bacterial agent is a mixture of live bacteria of Bifidobacterium pseudolongum and Bifidobacterium animalis, or metabolites of co-cultured Bifidobacterium pseudolongum and Bifidobacterium animalis.

[0018] The present application can use active Bifidobacterium pseudolongum and Bifidobacterium animalis to prepare a live bacterial preparation. Preferably, the mixture of live bacteria is a mixture of Bifidobacterium pseudolongum and Bifidobacterium animalis in equal proportions.

[0019] The present application can also use metabolites of co-cultured Bifidobacterium pseudolongum and Bifidobacterium animalis to prepare a pharmaceutical preparation. The metabolites contain butyric acid. Butyric acid improves pathology by the following mechanisms: as a metabolite, butyric acid can act on the immune system through blood circulation, induce peripheral blood M2 macrophage polarization, increase the M2 / M1 ratio and anti-inflammatory cytokine (IL-10, TGF-β) expression, while reducing pro-inflammatory factor levels, achieving immune regulation; increasing the number of hippocampal disease-associated microglia (DAM), enhancing their colocalization with Aβ plaques and phagocytic activity.

[0020] The bacterial agent provided by the present application can be developed into various dosage forms to adapt to different application scenarios.

[0021] Preferably, the bacterial agent is a fecal microbiota transplantation (FMT) preparation containing Bifidobacterium pseudolongum and Bifidobacterium animalis. Studies have shown that the use of a fecal microbiota transplantation preparation with increased abundance of Bifidobacterium animalis and Bifidobacterium pseudolongum on AD models can significantly enhance the spatial learning and memory ability of AD models.

[0022] Preferably, the bacterial agent is an oral preparation. Through oral administration, Bifidobacterium pseudolongum and Bifidobacterium animalis proliferate synergistically in the intestinal tract, regulate intestinal flora balance, and promote the production of metabolite butyric acid.

[0023] The oral preparation is used to: increase peripheral blood butyric acid levels; induce macrophage polarization to M2 type, increase anti-inflammatory cytokine (IL-10, TGF-β) expression; promote the transformation of brain microglia to disease-associated microglia (DAM), and enhance the Aβ plaque clearance ability.

[0024] Preferably, the oral preparation contains sodium butyrate, which is used to enhance the immune regulation effect of the combined strains.

[0025] As a specific embodiment of the present application, the Bifidobacterium pseudolongum uses the strain with Korean Collection for Type Cultures (KCTC) number KCTC 3224; and the Bifidobacterium animalis uses the strain with Korean Collection for Type Cultures (KCTC) number KCTC 3219.

[0026] The synergistic bacterial agent provided by the application increases butyric acid production and regulates the immune system through the metabolic synergistic effect of the two bacteria, thereby reducing the deposition of beta-amyloid protein (Aβ) in the brain and significantly improving cognitive function, and belongs to the biological product category of using microorganisms to intervene in neurodegenerative diseases. The combined strains do not affect the weight gain of the AD model during the action process, and have good safety.

[0027] Another object of the application is to provide the use of the bacterial agent in the preparation of a medicament for treating neurodegenerative diseases. The neurodegenerative disease can be, but is not limited to, Alzheimer's disease.

[0028] The application has the following beneficial effects: (1) Synergistic effect: Bifidobacterium pseudolongum and animal bifidobacterium have a synergistic effect, which significantly improves the intestinal colonization rate and butyric acid production compared with single bacteria, and enhances the improvement effect on Alzheimer's disease.

[0029] (2) Clear mechanism: The bacterial agent provided by the application plays a role through the "gut flora-butyric acid-peripheral / central immune" axis, and the mechanism is clear. It can intervene in Aβ deposition and neuroinflammation from the pathological root to intervene in the progression of Alzheimer's disease, rather than just relieving symptoms.

[0030] (3) Multi-dimensional improvement: The bacterial agent provided by the application can regulate butyric acid levels and immune cell function, while reducing pathological characteristics (Aβ plaques) and improving cognitive function (NOR / NPR / MWM test significantly improved), providing a new and efficient biological agent for the prevention and treatment of AD.

[0031] (4) Biological safety: The bacterial agent provided by the application does not affect the weight gain of the AD model, and has good safety. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Long-term consumption of barley can improve the learning and memory ability of 5xFAD mice. Wherein A is the discrimination index (DI) of NOR (left) or NPR (right) test of WT and 5xFAD mice fed with CD at 2 months. B is the immunofluorescence staining of Aβ (12F4, red; DAPI, blue) on the coronal section of WT and 5xFAD mice at 6 months old. C-D are bacterial data of 16S rDNA sequence of feces of WT and 5xFAD mice at 2 months. E-F are bacterial data of WT and 5xFAD mice at 6 months. H is a schematic diagram of the experimental design. I is the trajectory heat map of 5xFAD mice during the entire NPR test. J is the discrimination index (DI) of NOR (left) and NPR (right) test of WT and 5xFAD mice fed with CD or BD. K-L are mouse data in the water maze test.

[0033] Figure 2B. ani and B. pse synergistically improve Alzheimer's disease. A, Schematic of experimental design. B, DI values of bacterial-treated 5xFAD mice fed with BD in NPR test. C, DI values of bacterial-treated 5xFAD mice fed with CD in NPR test. D, Relative abundance of B. pseudolongum and B. animatus detected during gavage of ABX-treated mice. E-F, Absolute abundance of B. animatus when B. pseudolongum was added in CD or BD medium. G, Schematic of bacterial cross-feeding experimental design. H, Absolute abundance of B. animatus after incubation in M9 minimal medium.

[0034] Figure 3 B. ani and B. pse synergistically improve Alzheimer's disease. A, Schematic of experimental design. B, DI values of bacterial-treated 5xFAD mice fed with BD in NPR test. C, DI values of bacterial-treated 5xFAD mice fed with CD in NPR test. D, Relative abundance of B. pseudolongum and B. animatus detected during gavage of ABX-treated mice. E-F, Absolute abundance of B. animatus when B. pseudolongum was added in CD or BD medium. G, Schematic of bacterial cross-feeding experimental design. H, Absolute abundance of B. animatus after incubation in M9 minimal medium.

[0035] Figure 4 B. ani and B. pse synergistically improve Alzheimer's disease. A, Schematic of experimental design. B, DI values of bacterial-treated 5xFAD mice fed with BD in NPR test. C, DI values of bacterial-treated 5xFAD mice fed with CD in NPR test. D, Relative abundance of B. pseudolongum and B. animatus detected during gavage of ABX-treated mice. E-F, Absolute abundance of B. animatus when B. pseudolongum was added in CD or BD medium. G, Schematic of bacterial cross-feeding experimental design. H, Absolute abundance of B. animatus after incubation in M9 minimal medium.

[0036] Figure 5A is representative HE staining of WT and 5xFAD mice colon. B-C is the log abundance of SCFA and butyrate in serum of WT and 5xFAD mice fed CD or BD at 6 months. D is the UMAP of blood immune cells. E is the percentage of monocytes, dendritic cells and macrophages in mouse blood. F is the macrophage content. G is the UMAP of macrophages. H-I is the proportion of M2-like macrophages in mouse blood. J is the mRNA level of inflammatory cytokines in blood of WT and 5xFAD mice fed CD or BD.

[0037] Figure 6 BD-AniPse-butyrate pathway improves hippocampal pathology and induces microglial response in Alzheimer's disease mice. A is Aβ (D54D2, red; DAPI, blue) immunofluorescence staining of coronal brain sections of 6 mice. B is the average number of Aβ positive plaques and the percentage of area covered by Aβ in hippocampus. C is Aβ plaques labeled with 12F4. D is the quantification of Aβ plaque diameter in hippocampus. E is the mRNA level of inflammatory cytokines (IL-6, IL-1β, IL-10 and TGF-β) in hippocampus. F is Iba1 (green) staining to assess microglial cell count. G is the average quantification of microglial cell count in dentate gyrus of 6 matched brain sections per mouse. H is Iba1 (green) staining to show microglial cell morphology. I is Sholl analysis quantification of 6 matched brain sections per mouse.

[0038] Figure 7 Barley diet modulates microglial polarization to DAMs and promotes microglial phagocytosis of Aβ. A is the UMAP of brain immune cells. B is the UMAP of microglial cells. C is the percentage of microglial cells. D is the DAM and activated microglial cell phenotypes in mouse hippocampus. E is the percentage of DAM, activated and steady-state microglial cells in the total microglial cell population in mouse hippocampus. F is the proportion of DAM / activated microglial cells in mouse hippocampus. G is representative confocal images of ThioS (pink), Iba-1 (green) and CD11c (red) staining in hippocampus. H is the percentage of CD11c+ microglial cells in hippocampus. I-J is microglial cell imaging to assess plaque coverage of microglial cells. DETAILED DESCRIPTION

[0039] The application will be further described in conjunction with specific examples. The following examples are only used to illustrate the present application, and are not used to limit the scope of the application. Any modification or replacement of the method, step or condition of the present application, without departing from the spirit and essence of the present application, shall fall within the scope of the present application.

[0040] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0041] Example 1: Long-term intake of barley can improve the learning and memory ability of Alzheimer's disease mice, and its effect is related to the regulation of intestinal flora 1. Experimental design First, evaluate 2-month-old Alzheimer's disease (AD) model mice (5xFAD). This stage is the initial stage of AD, and then 2-month-old AD mice are given barley diet (BD) or control diet (CD) respectively, and the behavior test is performed until 6 months of age to explore the effect of barley diet on AD and its correlation with intestinal flora.

[0042] 2. Experimental animals and treatment 8-week-old SPF 5xFAD and C57BL / 6 wild-type (WT) mice were selected, and a 12-hour light / dark cycle was followed (all experiments were performed during the light cycle).

[0043] The mice were fed with breeding feed (Lab Diets 5K52), and the control group was fed with standard feed (Lab Diet 5010) during the experimental stage, and the barley diet group (BD) was fed with granular barley (Minrinmai 2009001).

[0044] 3. Immunofluorescence staining and pathological detection Brain tissue sample processing: After the mice were anesthetized with isoflurane, the heart was perfused with 1xPBS, and the brain was separated into left and right hemispheres: the left hemisphere was fixed with 4% paraformaldehyde, and 50μm thick sections were prepared.

[0045] Immunofluorescence staining and quantitative analysis: After the sections were blocked with 3% goat serum, the primary antibody was incubated: anti-Aβ (D54D2, 1:1500; 12F4, 1:100), anti-Iba1 (1:500), and anti-GFAP (1:1000), 4°C overnight; the next day, Alexa Fluor 488 / 594 secondary antibody (1:500) was incubated, DAPI was used to stain the nucleus, and Leica DMI8 confocal microscope imaging was used, and Fiji software was used to quantitatively analyze the Aβ plaque area and microglial cell and astrocyte activation status.

[0046] 4. Behavioral test The experimenter used blind operation, and the mice were adapted to the behavior test environment 1 day in advance.

[0047] A. New object recognition (NOR) test Training phase: the mice explored two identical objects for 10 minutes in an open field box; Test phase (1 hour later): Replace one object with a new object, record the exploration time within 10 minutes, calculate the discrimination index (DI), formula as follows: DI = [Exploration time of new object (seconds) - Exploration time of familiar object (seconds)] / [Exploration time of new object (seconds) + Exploration time of familiar object (seconds)] x 100%.

[0048] B, New position recognition (NPR) test The process is the same as NOR. In the test phase, one object is moved to a new position, and DI is calculated.

[0049] C, Morris water maze (MWM) test Training phase: 4 times a day for 5 consecutive days, record the latency of mice finding the hidden platform; Probe test: Remove the platform on the 6th day, record the number of times crossing the original platform location and the target quadrant residence time within 60 seconds.

[0050] 5, Results analysis Initial characteristics of 2-month-old AD mice: behavioral phenotype: there was no significant difference in the behavioral phenotype of 2-month-old 5xFAD mice and wild-type (WT) mice (A of Figure 1 ); pathological features: no obvious Aβ deposition was observed in the hippocampus (B of Figure 1 ); intestinal flora: compared with WT, the composition of intestinal flora in 2-month-old AD mice had already been significantly different (such as reduced abundance of Bifidobacterium), and this flora imbalance persisted until 6 months old (C-G of Figure 1 ); This indicates that AD may be improved by adjusting the composition of intestinal flora, providing a theoretical basis for subsequent barley diet intervention.

[0051] Behavioral evaluation results of 6-month-old AD mice: behavioral defects of control diet group: 6-month-old 5xFAD-CD mice showed learning and memory deficits in the discrimination index (DI) of the new position recognition (NPR) task; improved behavior of barley diet group: after 4 months of BD feeding, 5xFAD-BD mice showed enhanced exploration behavior in the NPR task (I, J of Figure 1 ).

[0052] Morris water maze (MWM) verification results: learning ability: 5xFAD-BD mice significantly improved their learning ability in the training phase, with significantly shortened latency to reach the target platform (K of Figure 1 ); memory retention: in the probe test, 5xFAD-BD mice increased the number of times entering the target area and spent more time in the target quadrant, with better memory retention (L of Figure 1 ); Conclusion: Long-term intake of barley can significantly improve the cognitive impairment of AD model mice, and this effect may be related to the regulation of intestinal flora, providing direct evidence for the subsequent screening of functional strains (such as Pseudobifidobacterium and animal bifidobacterium) from the intestinal flora of barley-fed mice.

[0053] Example 2: Barley diet alleviates Alzheimer's disease by regulating intestinal microbiota, in which Pseudobifidobacterium and animal bifidobacterium are core functional bacteria 1. Experimental design: 16S rRNA sequencing was used to analyze the effect of BD on the intestinal flora of 6-month-old AD mice. Antibiotic (ABX) clearance of flora and fecal microbiota transplantation (FMT) experiments were combined to verify the core role of intestinal flora in BD improving AD and identify key functional strains.

[0054] 2. Intestinal flora detection: 16S rRNA amplicon sequencing: Mouse fecal DNA was extracted, and the 16S rRNA V4 region was amplified. The flora composition was analyzed by Qiime2, and the relative abundance of Pseudobifidobacterium and animal bifidobacterium was calculated.

[0055] 3. Antibiotic treatment (ABX): Some mice were given water containing vancomycin (500 μg / ml), ampicillin (1 mg / mL), and neomycin (1 mg / mL) for 2 weeks to clear the intestinal flora, which was used to verify the flora dependence.

[0056] 4. Results analysis 4.1 Effect of BD on intestinal flora composition Flora isolation: 16S rRNA sequencing showed that the intestinal flora composition of 6-month-old 5xFAD and CD groups was significantly separated (principal component analysis could clearly distinguish the two group clusters); genus changes: LEfSe analysis showed that after 4 months of BD feeding, the relative abundance of Bifidobacterium and Allobaculum in the intestines of AD mice increased significantly; the abundance of Muribaculaceae and Sutterella also increased, while the abundance of Clostridiales was higher in the CD group (Figures 1 Figure 2 A-C). The results showed that BD can significantly change the intestinal flora composition of 5xFAD mice.

[0057] 4.2 Verification of the necessity of intestinal flora in BD improving AD ABX clearance experiment: After ABX treatment (clearing intestinal flora) in AD mice, even if given BD, the spatial learning and memory ability defects were not improved (NOR and NPR test DI values did not change significantly) (Figures 2 Figure 2E). This result directly demonstrated that gut microbiota is an indispensable factor for BD-mediated cognitive improvement.

[0058] 4.3 Functional verification of BD-enriched microbiota (FMT experiment) ABX-treated AD mice were transplanted with fecal microbiota from BD-fed or CD-fed mice, and then all were fed with CD for 4 months to evaluate cognitive function; it was found that the DI values of NOR and NPR tests of mice receiving BD microbiota were significantly higher than those of mice receiving CD microbiota; even under CD feeding conditions, BD microbiota can significantly enhance the spatial learning and memory ability of mice Figure 2 F). The results showed that BD-enriched gut microbiota itself can play an anti-AD role, independent of the continued intake of barley diet.

[0059] 4.4 Identification of key functional strains Strain deletion and recovery: compared with WT, there was a significant deletion of Bifidobacterium in the intestines of 2-month-old 5xFAD mice, and after 4 months of BD feeding, the abundance of this genus in the intestines of 6-month-old AD mice was significantly recovered Figure 2 G, H of FIG. 4).

[0060] Species level verification: At the species level, BD significantly increased the abundance of B. animalis and B. pseudolongum in animals Figure 2 I of FIG. 4). The results showed that B. animalis and B. pseudolongum are the main functional strains that mediate the improvement of AD by BD.

[0061] This part of the experiment proves the causal chain of “barley diet → enrichment of B. pseudolongum and B. animalis → improvement of AD” through microbiota sequencing, ABX clearance and FMT transplantation, providing direct experimental basis for the subsequent development of synergistic bacterial agents based on these two bacteria.

[0062] Example 3: B. pseudolongum and B. animalis regulate Alzheimer's disease through synergistic action, and single bacteria cannot replace its function 1. Experimental design: To clarify the direct effect of B. pseudolongum and B. animalis on AD, this part uses antibiotic (ABX) treatment to clear the gut microbiota of mice, and through the separate gavage of single bacteria or 1:1 mixed bacteria, combined with in vitro co-culture experiments, to explore the interaction of the two bacteria and the synergistic regulation mechanism of AD.

[0063] 2. Strains and culture Bifidobacterium pseudolongum was purchased from the Korean Collection for Type Cultures (KCTC), strain number KCTC 3224; and Bifidobacterium animalis was purchased from the same collection (KCTC), strain number KCTC 3219. The strains were cultured in lactose-containing BHI broth at 37°C under anaerobic conditions for 26 hours until OD (Organic Degradation) was reached. 600 The bacterial cells were collected after centrifugation at 5000g for 10 minutes until the concentration reached 0.5.

[0064] 3. Microbial intervention and health monitoring: *Bifidobacterium pseudolongum* and *Bifidobacterium animalis* were mixed in a 1:1 ratio to prepare 5×10... 9 The bacterial suspension of CFU / mL was administered to ABX-treated 5xFAD mice by gavage at a rate of 200 μL daily for 4 weeks (until 6 months of age); the control group was administered an equal volume of sterile PBS by gavage. Body weight was monitored weekly during the gavage period to ensure no significant abnormalities were observed.

[0065] 4. Results Analysis 4.1 Validation of synergistic effects in vivo Colonization efficiency: 28 days after ABX treatment and gavage, the relative abundance of both bacteria was significantly higher in 5xFAD mice treated with mixed bacteria compared to single-bacterial gavage, indicating that the synergistic effect of the two bacteria can improve intestinal colonization efficiency. Figure 3 (D).

[0066] Cognitive function recovery: Gavage with mixed bacteria significantly restored the learning and memory abilities of ABX-treated 5xFAD mice; while gavage with either Bifidobacterium pseudolongum or Bifidobacterium animalis alone failed to improve their location recognition ability. Figure 3 B).

[0067] Diet independence verification: After SPF-grade CD mice were gavaged with both bacteria for 28 days, it was also observed that the mixed bacteria improved cognitive function, confirming that this synergistic effect was independent of the continuous intake of barley diet. Figure 3 (C).

[0068] 4.2 In vitro interaction mechanism Growth-promoting effect: In vitro co-culture experiments showed that *Bifidobacterium pseudolongum* significantly promoted the growth of *Bifidobacterium animalis* (the bacterial count increased twofold within 4 hours), while *Bifidobacterium animalis* had no significant effect on the growth of *Bifidobacterium pseudolongum*. Figure 3 (E, F).

[0069] Cross-feeding experiment: B. pseudolongum was inoculated in agar containing CD or BD components, and animal B. longum was inoculated in the upper M9 minimal medium. The results showed that the growth of animal B. longum was significantly enhanced in the BD agar group (Fig. 2B), suggesting that B. pseudolongum could degrade specific components in the barley diet to provide nutritional support for animal B. longum. Figure 3

[0070] The results showed that B. pseudolongum and animal B. longum could improve AD by synergistic action (enhancing colonization efficiency and promoting mutual growth), and single bacteria could not replace them. The synergistic mechanism of the two bacteria provided direct experimental evidence for the subsequent development of mixed bacterial agents, and this effect was independent of the continuous intake of barley diet, which had independent application potential.

[0071] Example 4: Butyric acid produced by B. pseudolongum and animal B. longum can reduce inflammation and Alzheimer's disease pathology, and is the key metabolite mediating its effect 1. Experimental design: Metabolomics analysis of the colon contents of WT and 5xFAD mice (fed with CD / BD) was performed to screen for bacteria-dependent differential metabolites. Combined with in vitro bacterial metabolic experiments and exogenous butyric acid intervention, the key role of butyric acid (BA) in the improvement of AD by the two bacteria was determined.

[0072] 2. Metabolomics analysis: The colon contents were collected, extracted with 80% methanol, and the content of short-chain fatty acids (especially butyric acid) was detected by UHPLC-MS / MS. TraceFinder3.2.0 software was used for quantification.

[0073] 3. Butyric acid intervention: 40mM sodium butyrate (Sigma, ARK2161-250G) was added to the drinking water of part of the mice for 8 weeks to verify the effect of the metabolite by applying exogenous butyric acid.

[0074] 4. Results analysis 4.1 Screening of differential metabolites by metabolomics Overall difference: Metabolomics analysis of the colon contents of 5xFAD mice showed that a total of 1009 metabolites were detected, and partial least squares discriminant analysis (PLS-DA) showed that the metabolic profiles of the BD group and the CD group were significantly separated (Fig. 3A). Figure 4

[0075] Key categories: The differential metabolites were mainly enriched in fatty acid secondary metabolites; further analysis showed that the levels of short-chain fatty acids (SCFAs) and butyric acid (BA) in the BD group were significantly increased (Fig. 3B-E). Figure 4

[0076] 4.2 Ability of the two bacteria to produce butyric acid synergistically ​​​In vitro metabolic experiments showed that both animal bifidobacterium and pseudolong bifidobacterium could utilize barley-derived substrates to produce BA, and the BA yield in co-culture of the two bacteria was significantly higher than that in single-bacterium culture, confirming the synergistic metabolic effect of the two bacteria. Figure 4 F).

[0077] 4.3 Exogenous butyric acid improves AD Intervention effect: Administration of sodium butyrate (BA precursor) through drinking water can significantly increase the concentration of BA in the feces and serum of 5xFAD mice ( Figure 4 H); after 8 weeks of administration of exogenous sodium butyrate, NPR test showed that cognitive function was significantly improved ( Figure 4 I). The results showed that BA needs long-term action to alleviate AD cognitive impairment, which confirms that the two bacteria mediate AD improvement by continuously producing BA.

[0078] 4.4 Downstream regulation pathway of butyric acid Pathway enrichment analysis showed that differential metabolites were mainly involved in lipid metabolism and immune system pathways ( Figure 4 J); physiological and biochemical tests confirmed that both BD and BA treatment can significantly up-regulate the expression of anti-inflammatory cytokines (such as IL-10, TGF-β) in the colon ( Figure 4 K). The results showed that BA can reduce inflammation by regulating the immune pathway, laying a foundation for improving AD pathology.

[0079] The results showed that pseudolong bifidobacterium and animal bifidobacterium increased butyric acid production through synergistic action, and butyric acid, as a key metabolite, can improve AD cognitive impairment and regulate immune inflammation through long-term action, which is the core molecular bridge connecting the two bacteria and AD improvement.

[0080] Example 5: Butyric acid promotes M2 macrophage polarization through peripheral immune regulation and improves systemic inflammatory state 1. Experimental design: This part aims to explore the specific mechanism of butyric acid (BA) through peripheral immune regulation. Sodium butyrate is used to intervene in 5xFAD mice, combined with colon inflammation score, serum metabolite detection, and mass cytometry (CyTOF) analysis of peripheral blood immune cell phenotype, focusing on the changes of macrophage subtypes and cytokine levels.

[0081] 2. Immune cell phenotype analysis Preparation of single cell suspension: Take peripheral blood, digest with RPMI containing collagenase IV (2 mg / mL) and DNase (20 μg / mL) for 1 hour, filter and remove red blood cells with ACK lysis buffer to prepare single cell suspension; CyTOF detection: After the cells were labeled with 250 nM cisplatin to mark dead cells, the metal-labeled surface antibodies (CD4, CD8, CD11b, etc.) and intracellular antibodies (IL-10, TGF-β, etc.) were incubated, and the Helios mass spectrometry flow cytometer was used for detection. The proportion of M2 type macrophages (CD206 + ) and the levels of anti-inflammatory cytokines (IL-10, TGF-β) were analyzed by FlowJo and R software.

[0082] 3. Results analysis 3.1 Butyric acid improves the intestinal inflammatory state After butyric acid treatment, the colon inflammation symptoms of 5xFAD mice were significantly reduced, and the intestinal tissue inflammation score was reduced by 40% (Fig. 1A), indicating that butyric acid can improve local intestinal inflammation; compared with the WT-CD group, the concentration of short-chain fatty acids (SCFAs) and butyric acid in the serum of 5xFAD mice treated with butyric acid was significantly increased (Fig. 1B, C), confirming that butyric acid from the intestine can enter the peripheral blood circulation and play a systemic role. Figure 5 Figure 5

[0083] 3.2 Changes in peripheral blood immune cell subsets (CyTOF analysis) Immune cell clustering: 42 heavy metal isotope-labeled antibody combinations were used to label the peripheral blood mononuclear cells (PBMCs) of WT-CD, 5xFAD-CD and 5xFAD-BD groups of mice, and UMAP analysis showed that there were significant differences in immune cell clustering among the groups (Fig. 2A). Figure 5

[0084] Specific cell enrichment: The number of monocytes, dendritic cells (DCs) and macrophages in the 5xFAD-BD group was significantly increased compared with the 5xFAD-CD group (Fig. 2D, E). Figure 5

[0085] M2 type macrophage polarization: Macrophage subtype analysis showed that the proportion of M2 type macrophages (CD206 + ) in the 5xFAD-BD group was significantly increased (Fig. 2F-H), and the M2 / M1 ratio was increased by 2.3 times compared with the CD group (Fig. 2I). Figure 5 Figure 5

[0086] 3.3 Changes in cytokine levels The levels of anti-inflammatory cytokines (IL-10, TGF-β) in peripheral blood were significantly increased, and the levels of pro-inflammatory cytokines (TNF-α, IL-6) were decreased (Fig. 3J), which was consistent with the trend of cytokine changes in the colon tissue, indicating that butyric acid can systemically regulate immune balance. Figure 5

[0087] ​​​​​​​Results showed that butyric acid, as a key metabolite of Pseudobutyrate bifidobacterium and animal bifidobacterium, can improve intestinal inflammation, enter peripheral blood circulation, specifically promote M2 macrophage polarization and up-regulate anti-inflammatory cytokine expression, optimize the peripheral immune microenvironment, and lay the foundation for subsequent reduction of central nervous system inflammation.

[0088] Example 6: Barley diet-bacteria-butyric acid axis (BD-AniPse-BA axis) reduces Alzheimer's disease pathological damage and regulates microglial cell activation state 1. Experimental design: Based on the regulatory effect of butyric acid on peripheral immunity, this part focuses on the central nervous system to explore the effects of BD, bacteria (AniPse: animal bifidobacterium + Pseudobutyrate bifidobacterium) and butyric acid (BA) on the hippocampus of AD model mice, and focuses on evaluating Aβ plaque deposition and microglial cell activation to clarify the correlation between "peripheral immunity-central pathology".

[0089] 2. Immunofluorescence staining and pathological detection Brain tissue sample processing: After isoflurane anesthesia, the mouse was heart perfused with 1×PBS, and the left and right hemispheres were taken: the left hemisphere was fixed with 4% paraformaldehyde to make 50 μm thick sections; the right hemisphere was frozen for protein extraction.

[0090] Immunofluorescence staining and quantitative analysis: After blocking the sections with 3% goat serum, incubate the first antibody: anti-Aβ (D54D2, 1:1500; 12F4, 1:100), anti-Iba1 (1:500), 4°C overnight; the next day, incubate Alexa Fluor 488 / 594 secondary antibody (1:500), DAPI stain the nucleus, use Leica DMI8 confocal microscope for imaging, and use Fiji software for quantitative analysis of Aβ plaque area and microglial cell branch number.

[0091] 3. Results analysis 3.1 Aβ plaque deposition is reduced Immunofluorescence staining showed that Aβ plaques (marked by D54D2) accumulated in the hippocampus of 6-month-old 5xFAD-CD mice; the number of Aβ plaques in the 5xFAD-BD group was significantly reduced, and the diameter of the plaques marked by 12F4 was reduced by 35%; both AniPse and butyric acid intervention can reproduce the effect of BD, and can significantly reduce the burden of Aβ plaques (A-D); Figure 6 After the activation of the BD-AniPse-BA axis, the expression of anti-inflammatory cytokines (such as IL-10) in the hippocampus was significantly up-regulated (E), suggesting that the relief of central inflammation may be indirectly regulated through peripheral immunity. Figure 6

[0092] 3.2 The activation state of microglial cells is improved ​Compared with the WT-CD group, the 5xFAD-CD group had fewer microglia (Iba1) + The number of microglia increased, and intervention with BD, double bacteria, or butyrate further regulated the increase in the number of microglia. Figure 6 (F, G); Microglial morphology analysis showed that microglial branches were reduced and processes were simplified in the BD group, exhibiting activated state characteristics (F, G); Figure 6 The presence of H and I indicates a shift in its functional state toward phagocytosis of Aβ.

[0093] The results showed that the BD-AniPse-BA axis significantly improved central nervous system pathological damage in AD model mice by reducing Aβ deposition and regulating microglia activation. Its effect may be achieved by enhancing systemic immunity and regulating microglia activity, and it is a key link connecting peripheral immunity and central nervous system pathology.

[0094] Example 7: Barley diet regulates microglia state by upregulating anti-inflammatory cytokines, promoting their differentiation into disease-associated microglia (DAM). 1. Experimental Design: To clarify the regulatory mechanism of barley diet (BD) on central microglia function, this section uses flow cytometry (CyTOF) to analyze the hippocampal immune cell subsets of 5xFAD mice. Figure 7 Combined with immunofluorescence staining, this study focuses on exploring the changes in microglial subtypes (especially disease-associated microglia, DAM) and their interaction with Aβ plaques, as well as their impact on peripheral immune cells.

[0095] 2. Immune cell phenotype analysis Preparation of single-cell suspension: Brain tissue was taken, digested with RPMI containing collagenase IV (2 mg / ml) and DNase (20 μg / ml) for 1 hour, filtered, and erythrocytes were removed with ACK lysis buffer to prepare single-cell suspension; CyTOF assay: After cells were labeled with 250 nM cisplatin and then incubated with metal-labeled surface antibodies (CD4, CD8, CD11b, etc.) and intracellular antibodies (IL-10, TGF-β, etc.), the results were analyzed using Helios mass cytometry. DAM (CD11c) was analyzed using FlowJo and R software. + )quantity.

[0096] 3. Results Analysis 3.1 Hippocampal immune cell subset analysis (CyTOF) Microglia subtype classification: Microglia were classified into activated and homeostatic subtypes based on surface markers. Compared with the 5xFAD-CD group, the 5xFAD-BD group showed an increase in activated microglia and a decrease in homeostatic microglia. Figure 7 AC).

[0097] DAM ratio increased: Further analysis of the activated microglia subtypes showed that the proportion of disease-associated microglia (DAM) in 5xFAD-BD group increased by 30% (p < 0.05, Fig. 2D-F), suggesting that BD promoted the differentiation of microglia into DAM. Figure 7

[0098] 3.2 Immunofluorescence verification of DAM function CD11c + Microglia increased: Immunofluorescence staining of DAM marker CD11c showed that the number of CD11c + microglia in 5xFAD-BD group increased significantly, and the co-localization rate with Aβ plaques increased by 45% (p < 0.05, Fig. 2G-H). Figure 7

[0099] Aβ aggregation enhanced: The aggregation ability of microglia around Aβ plaques in the hippocampus of BD group 5xFAD mice was significantly enhanced, suggesting that the phagocytic activity of Aβ was improved (p < 0.05, Fig. 2I-J). Figure 7

[0100] The results showed that barley diet regulated the microglial state (increased activation, decreased homeostasis) by up-regulating anti-inflammatory cytokines, promoted the differentiation of microglia into DAM, enhanced the interaction with Aβ plaques and phagocytic ability; this process was regulated by peripheral immunity (especially M2 macrophages), and was a key terminal link in the improvement of AD pathology by the "gut microbiota-metabolites-peripheral immunity-central immunity" axis.

[0101] The above merely describes specific embodiments of the present application, which aims to enable those skilled in the art to understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any equivalent transformation or replacement according to the essence of the present application should be covered within the protection scope of the present application.​​​

Claims

1. Bifidobacterium pseudolongum ( Bifidobacterium pseudolongum ) and Bifidobacterium animalis ( Bifidobacterium animalis Its application in the preparation of drugs for treating Alzheimer's disease or in the preparation of functional foods that help improve memory or regulate gut microbiota.

2. The application as described in claim 1, characterized in that, The drug contains an equal amount of Bifidobacterium pseudolongum and Bifidobacterium animalis.

3. The application as described in claim 1, characterized in that, Bifidobacterium pseudolongum and Bifidobacterium animalis synergistically proliferate in the gut, regulating the balance of gut microbiota; through metabolic synergy, they increase butyrate production, regulate peripheral and central immunity, thereby reducing β-amyloid protein deposition in the brain and improving cognitive function.

4. A bacterial agent for improving Alzheimer's disease, assisting in improving memory, or regulating gut microbiota, characterized in that, The active ingredient of the bacterial agent is a mixture of live bacteria of Bifidobacterium pseudolongum and Bifidobacterium animalis, or a metabolite of co-cultured Bifidobacterium pseudolongum and Bifidobacterium animalis.

5. The microbial agent as described in claim 4, characterized in that, The live bacteria mixture is a mixture of Bifidobacterium pseudolongum and Bifidobacterium animalis in equal proportions.

6. The microbial agent as described in claim 4, characterized in that, The metabolites include butyric acid.

7. The microbial agent as described in claim 4, characterized in that, The microbial agent is a fecal microbiota transplantation preparation containing Bifidobacterium pseudolongum and Bifidobacterium animalis.

8. The microbial agent as described in claim 4, characterized in that, The bacterial agent is an oral preparation.

9. The microbial agent according to any one of claims 4-8, characterized in that, The *Bifidobacterium pseudolongum* strain used is strain KCTC 3224 from the Korean Center for Type Culture Collection; the *Bifidobacterium animalis* strain used is strain KCTC 3219 from the Korean Center for Type Culture Collection.

10. The use of the bacterial agent according to any one of claims 4-9 in the preparation of a medicament for treating neurodegenerative diseases.

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