WO2025175626A1 - Probiotic formulation for preventing or treating alzheimer's disease and use thereof - Google Patents

Abstract

Provided are a probiotic formulation for preventing or treating Alzheimer's disease and use thereof. The microorganisms in the probiotic formulation consist of Akkermansia muciniphila and Bifidobacterium breve. Akkermansia muciniphila and Bifidobacterium breve can coordinate with each other after being compounded, so as to provide a synergistic effect on alleviating Alzheimer's disease and thereby a new strategy for preventing or treating Alzheimer's disease. Since Akkermania muciniphila and Bifidobacterium breve are both probiotics, a related functional product prepared from Akkermania muciniphila and Bifidobacterium breve may feature good safety and less likelihood of inducing resistance.

Description

A probiotic for preventing or treating Alzheimer's disease and its application Technical Field

The present application belongs to the technical field of probiotics and relates to a probiotic for preventing or treating Alzheimer's disease and its application.

Background Art

Alzheimer's disease (AD), also known as senile dementia, is a common neurodegenerative disease characterized by progressive cognitive decline. The main pathological feature of Alzheimer's disease is the accumulation of beta-amyloid protein plaques in the brain. The accumulation of these abnormal proteins is associated with neuronal death and neurotransmitter dysfunction, leading to significant cognitive decline. As my country enters a society with an aging population, the prevalence of Alzheimer's disease has increased significantly, becoming a key difficulty in clinical treatment. Current therapeutic drugs include cholinesterase inhibitors, memantine, and monoclonal antibodies, which are mainly used to relieve symptoms and slow down pathology in patients with Alzheimer's disease, but their efficacy is limited. They can only relieve disease symptoms or clear pathological proteins, and their effects are limited. Therefore, there is an urgent need to find a new treatment method.

Recent studies have suggested that the pathogenesis of AD may be linked to the gut microbiota. The gut microbiota can contribute to the development and progression of AD by promoting Aβ deposition, inducing neuroinflammation, disrupting blood-brain barrier permeability, and regulating neurotransmitters. The microbiota, an ecological community that coexists with the human body, plays a crucial regulatory role in human health and disease. Gut microbes constitute the vast majority of the human microbiome and are primarily composed of bacteria, most of which are anaerobic, but also fungi, viruses, protozoa, and monocytic organisms. The number and diversity of the gut microbiota maintain a dynamic balance to maintain homeostasis of the host immune system. However, factors such as poor diet, antimicrobial use, and lifestyle stress can disrupt this dynamic balance, impairing gut microbial activity and leading to gastrointestinal diseases. Furthermore, these diseases are not limited to the gastrointestinal tract; gut microbial imbalance is also associated with cardiovascular disease, type 2 diabetes, and renal failure. The gut microbiota produces essential vitamins and other important substances involved in central nervous system development and immune regulation. In addition to disrupting the stability of the intestinal environment, intestinal microbial imbalance can also affect behavior, learning, memory, and the occurrence of neurological diseases. The human intestinal microbiome is considered to be the second brain and may be one of the causes of AD and other neurodegenerative diseases.

However, the microbial preparations currently available for improving Alzheimer's disease are still very limited. Most microorganisms are aimed at maintaining the integrity of the intestinal structure and restoring the normal physiological function of the intestine. Research on Alzheimer's disease is limited, and the effectiveness of probiotics and their products in improving Alzheimer's disease remains to be improved. Therefore, providing a microbial preparation that can effectively improve Alzheimer's disease without causing adverse reactions in patients during treatment has become a pressing issue for those skilled in the art.

Summary of the Invention

The present application provides a probiotic agent for preventing or treating Alzheimer's disease and its application.

In a first aspect, the present application provides a probiotic for preventing or treating Alzheimer's disease, wherein the bacterial strains in the probiotic consist of Akkermansia muciniphila and Bifidobacterium breve.

This application has developed a new probiotic compounding method, compounding Akkermansia muciniphila and Bifidobacterium breve. It was found that the two have potential interactions and can cooperate with each other to synergize in improving the efficacy of Alzheimer's disease. When the bacterial dosage is consistent, the combination of the two bacteria significantly improves the above-mentioned efficacy compared with a single Akkermansia muciniphila or a single Bifidobacterium breve. Therefore, this probiotic agent provides a new strategy for preventing or treating Alzheimer's disease. Because Akkermansia muciniphila and Bifidobacterium breve are both probiotics, they are highly safe and less likely to develop resistance when used to prepare related efficacy products.

Preferably, the ratio of the viable counts of Akkermansia muciniphila to Bifidobacterium breve is 1:10-10:1, for example, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, etc. Other specific values within this numerical range can be selected and will not be described in detail here.

Based on the potential interaction between Akkermansia muciniphila and Bifidobacterium breve, the present application also found that when the two strains are combined at the above-mentioned specific ratio of viable bacteria count, their efficacy in improving Alzheimer's disease is more significant.

Preferably, in the probiotic agent, the total viable bacteria count is not less than 1×10 ⁸ CFU/mL or 1×10 ⁸ CFU/g, for example, 1×10 ⁸ CFU/mL (CFU/g), 2×10 ⁸ CFU/mL (CFU/g), 5×10 ⁸ CFU/mL (CFU/g), 8×10 ⁸ CFU/mL (CFU/g), 1×10 ⁹ CFU/mL (CFU/g), 5×10 ⁹ CFU/mL (CFU/g), 1×10 ¹⁰ CFU/mL (CFU/g), or 1×10 ¹¹ CFU/mL (CFU/g), etc. Other specific values within this numerical range can be selected and will not be repeated here.

Preferably, the dosage form of the probiotic includes lyophilized powder, capsule, tablet or granule.

The dosage form of the probiotics involved in this application is not limited, and the dosage form includes the most commonly used freeze-dried powder The freeze-dried powder can be prepared by the following method:

Akkermansia muciniphila and Bifidobacterium breve are inoculated into culture medium respectively for cultivation to obtain culture fluid; the culture fluid is centrifuged to obtain bacterial cells; the bacterial cells are resuspended with a freeze-drying protective agent to obtain a resuspension; the resuspension is freeze-dried to obtain the product, and then the two are compounded according to a proportion.

Preferably, the probiotic further comprises a freeze-drying protectant.

Preferably, the lyoprotectant comprises any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

Preferably, the probiotic further comprises auxiliary additives.

Preferably, the auxiliary additives include any one or a combination of at least two of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharide, stachyose, polydextrose, α-lactoprotein or lactoferrin.

In a second aspect, the present application provides use of the probiotic for preventing or treating Alzheimer's disease according to the first aspect in the preparation of a medicament for preventing, alleviating or treating Alzheimer's disease.

Preferably, the drug further includes excipients, which include any one or a combination of at least two of excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

Compared with the prior art, this application has the following beneficial effects:

This application has developed a new probiotic compounding method, compounding Akkermansia muciniphila and Bifidobacterium breve. It was found that the two have potential interactions and can cooperate with each other to synergize in improving the efficacy of Alzheimer's disease. When the bacterial dosage is consistent, the combination of the two bacteria significantly improves the above-mentioned efficacy compared with a single Akkermansia muciniphila or a single Bifidobacterium breve. Therefore, this probiotic agent provides a new strategy for preventing or treating Alzheimer's disease. Because Akkermansia muciniphila and Bifidobacterium breve are both probiotics, they are highly safe and less likely to develop resistance when used to prepare related efficacy products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the animal intervention method according to an embodiment.

FIG2 is a statistical graph showing the latency of each group of mice to find the target hole during the training phase.

FIG3 is a graph showing the statistical results of the latency of each group of mice to find the target hole during the detection phase.

FIG4 is a graph showing the statistical results of the percentage of time each group of mice spent in the target hole area during the detection phase.

FIG5 is a graph showing the statistical results of the average speed of each group of mice during the detection phase.

FIG6 shows immunofluorescence staining of telencephalon sections of mice in each group.

FIG7 is a graph showing the statistical results of the hippocampal β-amyloid protein levels in each group of mice.

DETAILED DESCRIPTION

The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

The Akkermansia muciniphila (AKK) involved below is the commercially available strain ATCC BAA-835, purchased from Beijing Biobo Biotechnology Co., Ltd.; the Bifidobacterium breve (B. breve) involved below is the commercially available strain BNCC 186529, purchased from Beijing Biobo Biotechnology Co., Ltd.

The culture medium formulas involved in the following examples are as follows:

MRS medium (g/L): peptone 10 g/L, beef extract 10 g/L, glucose 20 g/L, sodium acetate 2 g/L, yeast powder 5 g/L, diammonium hydrogen citrate 2 g/L, K ₂ PO ₄ ·3H ₂ O 2.6 g/L, MgSO ₄ ·7H ₂ O 0.1 g/L, MnSO ₄ 0.05 g/L, polysorbate 80 1 mL/L, cysteine hydrochloride 0.5 g/L.

The following method for preparing a bacterial suspension comprises inoculating Akkermansia muciniphila or Bifidobacterium breve into MRS liquid culture medium, culturing the culture in an anaerobic environment (75% _N2 , 20% _CO2 , 5% _H2 ) at 37°C for 18 hours for activation, and performing two activation cycles to obtain an activated solution; inoculating the activated solution into MRS liquid culture medium at an inoculum rate of 2% (v/v), culturing the culture in an anaerobic environment (75% _N2 , 20% _CO2 , 5% _H2 ) at 37°C for 24 hours to obtain a bacterial suspension; centrifuging the bacterial suspension at 5000 rpm at 4°C for 10 minutes, filtering to obtain bacterial cells, and resuspending the bacterial cells in phosphate buffered saline.

The experimental animals involved in the following are 5×FAD strain mice (Alzheimer's disease model mice), purchased from THE JACKSON LABORATORY.

The data of the following experiments were statistically processed using SPSS 20.0 software. The results are expressed as (x±s). The t-test was used for comparison between the groups. P < 0.05 was considered statistically significant (* represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001).

Example

This example verifies the ability of probiotics to improve Alzheimer's disease:

(1) Animal grouping and intervention methods: 24 5×FAD strain mice were randomly divided into model group (n=10), AKK intervention group (n=10), B. breve intervention group (n=10), and AKK + B. breve combined intervention group (n=10, viable bacterial count ratio 1:1). Wild-type mice served as the control group (n=10). 5×FAD mice were treated with an antibiotic mixture (ABX, purchased from Sigma-Aldrich) daily for one week starting one week before 7 months of age. Subsequently, at 7 months of age, they were given saline, AKK suspension, B. breve suspension, or a mixed suspension of AKK and B. breve (each group received 200 μL of the suspension once daily, with a total concentration of 5×10 ⁷ CFU/mL in each group) daily for 3 months. The procedure is shown in Figure 1. The control group received saline.

(2) Specimen collection and testing:

(2.1) After the intervention, mice in each group were subjected to the Barnes maze test. The Barnes maze test is used to study the spatial learning and memory abilities of mice, as well as their ability to adapt to new environments. The specific procedures are as follows:

The Barnes maze apparatus (Chengdu Techman Instrument Co., Ltd., ST-120) consists of a rotatable white acrylic disc (0.75 m diameter) and 0.58 m height. It features 18 holes (5 cm diameter) evenly spaced along its rim. During the experiment, the apparatus was placed under bright light (600 lux). One of the holes was selected as the target hole, and an escape box was placed below it. Mice first explored the platform freely for 5 min on two consecutive days and then acclimated to the escape box for 2 min.

During the training phase, each mouse performed two trials daily. In one trial, each mouse was placed in the center of the maze covered with an opaque cylinder. After a 15-second habituation period, the cylinder was gently removed, and the mouse was allowed to freely explore the maze for 180 seconds until it found the target hole. If the mouse did not find the target hole, the latency was considered 180 seconds. After the first trial, the maze and escape box were wiped with 70% ethanol. The mouse was considered to have found the target hole when its body touched the hole. If the entire body was on the platform, the mouse was considered to have entered the target hole. The initial latency of the mouse to find the target hole was recorded for each trial. Trials were conducted twice daily for seven consecutive days. Memory retrieval was assessed on the fifth day after the last training trial, with the escape box removed for 90 seconds. If the animal did not find the target hole within 90 seconds, the latency was considered 90 seconds. The location of the target hole remained the same as during the training phase. Anymaze software was used to analyze the latency to find the target hole and the time spent in the quadrant where the target hole was located.

The latency to find the target hole during the training and test phases for each group was statistically analyzed, as shown in Figures 2 and 3, respectively. The percentage of time the animals spent in the target area during the test phase was also statistically analyzed, as shown in Figure 4. The average speed of mice in each group is statistically analyzed in Figure 5.

As shown in Figure 2, during the Barnes maze training phase, compared with the model group (AD+saline group), The latency of mice in the AKK intervention group (AD+AKK group) and the B.breve intervention group (AD+B.breve group) to find the target hole was significantly reduced, suggesting that AKK or B.breve can improve the spatial learning ability of AD mice; compared with the AD+AKK group or AD+B.breve group, the latency of mice in the AKK+B.breve combined intervention group (AD+AKK+B.breve group) to find the target hole was significantly reduced, suggesting that the combined use of AKK and B.breve has a better effect on improving the spatial learning ability of AD mice (*p<0.05, **p<0.01, ***p<0.001, ns, no significant difference).

As shown in Figures 3 and 4, in the Barnes maze test stage, compared with the model group (AD+saline group), the latency of mice in the AKK intervention group (AD+AKK group) and the B.breve intervention group (AD+B.breve group) to find the target hole was significantly reduced, and the percentage of time in the target hole area was significantly increased, suggesting that AKK or B.breve can improve the memory retrieval ability of AD mice; compared with the AD+AKK group or AD+B.breve group, the latency of mice in the AKK+B.breve combined intervention group (AD+AKK+B.breve group) to find the target hole was significantly reduced, and the percentage of time in the target hole area was significantly increased, suggesting that the combined use of AKK and B.breve can better improve the memory retrieval ability of AD mice (*p<0.05, **p<0.01, ***p<0.001, n.s., no significant difference).

As shown in Figure 5, there was no significant difference in the average speed of mice in each group, indicating that bacterial solution intervention did not affect the motor ability of mice (*p<0.05, **p<0.01, ***p<0.001, n.s., no significant difference).

(2.2) β-amyloid protein expression level detection test, the specific operation is as follows:

After transcardial perfusion with PBS and 4% paraformaldehyde, the telencephalon was harvested. The telencephalon was placed in 4% paraformaldehyde at 4°C overnight and then dehydrated in 15% and 30% sucrose until it sank to the bottom of the tube. The telencephalon was sectioned at 40 μm thickness using a cryostat and then subjected to immunofluorescence staining.

Brain slices were first washed three times with PBS and then blocked with 10% goat serum containing 0.3% Triton X-100 for 2 hours at room temperature. Subsequently, sections were incubated in primary antibodies diluted in blocking buffer (5% normal goat serum containing 0.3% Triton X-100) at 4°C for 24 hours. Sections were washed three times with phosphate-buffered saline and then incubated in secondary antibodies diluted in blocking buffer (5% normal goat serum containing 0.3% Triton X-100) at room temperature for 2 hours. Cell nuclei were stained with 6-diamidino-2-phenylindole (DAPI) for 10 minutes. Sections were then washed three times with PBS and mounted on slides. Fluorescent images were captured using an Olympus VS120 slide scanner. The primary antibodies used were mouse anti-Aβ (1:500, Covance, HY-001193, clone 4G8) and the secondary antibody was Alexa Fluor Cy5 goat anti-mouse (1:500, Invitrogen).

The fluorescence images of each group are shown in Figure 6, and the statistical results of the hippocampal β-amyloid protein levels of mice are shown in Figure 7. As shown in the figure, compared with the model group (AD+saline group), the AKK intervention group (AD+AKK group) The level of β-amyloid protein in the hippocampus of mice in the AD+AKK and B.breve intervention groups (AD+B.breve group) was significantly decreased, suggesting that AKK or B.breve alleviated Aβ pathology in AD mice; compared with the AD+AKK group or AD+B.breve group, the level of β-amyloid protein in the hippocampus of mice in the AKK+B.breve combined intervention group (AD+AKK+B.breve group) was significantly decreased, suggesting that the combined use of AKK and B.breve can more effectively improve Aβ pathology in AD mice (*p<0.05, **p<0.01, ***p<0.001, ns, no significant difference).

The applicant declares that while the above-mentioned embodiments are used to illustrate the technical solutions of this application, this application is not limited to these embodiments, and does not imply that this application must rely on these embodiments in order to be implemented. Persons skilled in the art should understand that any improvements to this application, equivalent replacements for the raw materials of the products of this application, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

The preferred embodiments of the present application are described in detail above. However, the present application is not limited to the specific details of the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the scope of protection of the present application.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner unless there is any contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

Claims (10)

A probiotic for preventing or treating Alzheimer's disease, wherein the bacterial strains consist of Akkermansia muciniphila and Bifidobacterium breve. The probiotic for preventing or treating Alzheimer's disease according to claim 1, wherein the ratio of the viable counts of Akkermansia muciniphila to Bifidobacterium breve is 1:10-10:1. The probiotic for preventing or treating Alzheimer's disease according to claim 1, wherein the total viable bacteria count in the probiotic is not less than 1×10 ⁸ CFU/mL or 1×10 ⁸ CFU/g. The probiotic for preventing or treating Alzheimer's disease according to claim 1, wherein the dosage form of the probiotic comprises a lyophilized powder, a capsule, a tablet or a granule. The probiotic for preventing or treating Alzheimer's disease according to claim 1, wherein the probiotic further comprises a lyoprotectant. The probiotic for preventing or treating Alzheimer's disease according to claim 5, wherein the lyoprotectant comprises any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol. The probiotic for preventing or treating Alzheimer's disease according to claim 1, wherein the probiotic further comprises an auxiliary additive. The probiotic for preventing or treating Alzheimer's disease according to claim 7, wherein the auxiliary additive comprises any one or a combination of at least two of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soy oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharide, stachyose, polydextrose, α-lactoprotein or lactoferrin. Use of the probiotic for preventing or treating Alzheimer's disease according to any one of claims 1 to 8 in the preparation of a medicament for preventing, alleviating or treating Alzheimer's disease. The use according to claim 9, wherein the drug further comprises excipients, and the excipients include any one or a combination of at least two of excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.