A combined bacterial agent for improving alzheimer's disease and application thereof

Abstract

This invention relates to the field of probiotic technology, specifically to a combination probiotic agent for improving Alzheimer's disease and its application. The combination probiotic agent includes *Lactobacillus gasseri* LFLG-245 and inactivated *Ackermania maughanii* AKKLF; *Lactobacillus gasseri* (… Lactobacillus gasseri LFLG-245 was deposited on March 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33952. (Ackermania pseudomallei) Akkermansia muciniphila AKKLF was deposited on March 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33955. The combined bacterial agent provided by this invention can improve Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotics, and particularly relates to a combined bacterial agent for improving Alzheimer's disease and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a common degenerative disease of the central nervous system, mainly characterized by memory loss, cognitive dysfunction, language and movement disorders, personality changes and other clinical features. According to the latest epidemiological statistics in 2025, there are more than 55 million dementia patients in the world, among which 60%-70% are AD, and it is expected to increase to 152.8 million in 2050. The prevention and treatment of Alzheimer's disease has become a medical, social and economic problem that needs to be solved in the current society.

[0003] The main pathological features of Alzheimer's disease include extracellular senile plaques (SPs), neurofibrillary tangles and neuronal loss, etc., but its pathogenesis is not fully understood. It is generally believed to be related to β-amyloid (β-amyloid, Aβ) toxicity, oxidative stress, abnormal phosphorylation of Tau protein, free radical damage, inflammatory response, neurotoxicity damage, etc., and brain ischemia and hypoxia can also cause cognitive dysfunction. At present, the drugs for treating Alzheimer's disease mainly include cholinesterase inhibitors (donepezil) and excitatory amino acid receptor antagonists (memantine), but these drugs can only improve symptoms, prevent further development of dementia, maintain residual brain function and reduce complications, and cannot cure the disease. For example, donepezil, as a commonly used drug for Alzheimer's disease, is widely used for symptom control, but still has gastrointestinal side effects (such as nausea, diarrhea, etc.). This not only leads to a decrease in long-term medication compliance, but also because its efficacy is only for symptom relief, it cannot intervene in the potential pathological mechanisms of the gut microbiota-brain axis and other Alzheimer's disease, and some patients have the risk of decreased efficacy over time.

[0004] In recent years, studies have shown that intestinal flora imbalance is related to the progression of Alzheimer's disease, but existing microbial preparations still face bottlenecks: single strain has limited effect, live bacterial preparations (such as probiotics) are easily destroyed by gastric acid and have low colonization rate, and inactivated bacterial preparations avoid the stability problem of live bacteria, but due to the lack of sustained metabolic activity of live bacteria, it is difficult to dynamically regulate the intestinal flora, and after the release of its components, it is easily metabolized and cleared, and long-term intervention cannot be achieved. SUMMARY

[0005] In view of the technical problems that the existing Alzheimer's disease treatment drugs (such as donepezil) have significant gastrointestinal side effects, low long-term medication compliance, and the existing microbial preparations have limited effect of single strain, the live bacteria are easily destroyed by gastric acid, the colonization rate is low, and the inactivated bacteria components are easily metabolized and cleared, thereby the effect sustainability is insufficient, the present application provides a combined bacterial agent for improving Alzheimer's disease and application thereof. The combined bacterial agent is prepared by synergistically matching Lactobacillus gasseri LFLG-245 and inactivated mucinophilic Akkermansia muciniphila AKKLF, which can not only improve the survival rate and colonization efficiency of live bacteria in the gastrointestinal tract, but also realize dynamic regulation and long-term intervention on intestinal flora through the synergistic effect of the active components of the two, thereby making up for the deficiencies of traditional drugs and single microbial preparations in terms of therapeutic effect sustainability and pathological intervention level.

[0006] In a first aspect, the present application provides a combined bacterial agent for improving Alzheimer's disease, comprising Lactobacillus gasseri (LFLG-245) and inactivated mucinophilic Akkermansia muciniphila (AKKLF). Lactobacillus gasseri )LFLG-245 and inactivated mucinophilic Akkermansia muciniphila (AKKLF). Akkermansia muciniphila )AKKLF;

[0007] Lactobacillus gasseri LFLG-245 was preserved in the China General Microbiological Culture Collection Center on March 24, 2025, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 33952, and the classification and naming is Lactobacillus gasseri. Lactobacillus gasseri ;

[0008] Mucinophilic Akkermansia muciniphila AKKLF was preserved in the China General Microbiological Culture Collection Center on March 24, 2025, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 33955, and the classification and naming is mucinophilic Akkermansia muciniphila. Akkermansia muciniphila .

[0009] Further, the dosage form of the combined bacterial agent is powder, pill, tablet, granule, capsule or liquid agent.

[0010] Further, the dosage form of the combined bacterial agent is liquid, and the preparation method of the combined bacterial agent comprises the following steps:

[0011] (1) After activation, the mucinophilic Akkermansia muciniphila AKKLF is inoculated into a brain heart infusion broth medium containing 0.5% porcine gastric mucin, and cultured under anaerobic conditions at 37°C for 48 h to obtain a bacterial liquid. The bacterial liquid is centrifuged, the bacterial precipitate is collected, and after resuspension, a mucinophilic Akkermansia muciniphila AKKLF live bacterial liquid is prepared, and after sterilization, a mucinophilic Akkermansia muciniphila AKKLF inactivated bacterial liquid is obtained;

[0012] (2) After activating Lactobacillus gasseri LFLG-245, it was inoculated onto MRS liquid medium and cultured at 37°C for 24 h to obtain bacterial suspension. The bacterial suspension was centrifuged, the bacterial precipitate was collected, and after resuspending, Lactobacillus gasseri LFLG-245 live bacterial suspension was prepared.

[0013] (3) Mix the inactivated Akkermansia muciniphila AKKLF bacterial solution and the live Lactobacillus gasseri LFLG-245 bacterial solution to obtain a combined bacterial agent.

[0014] Furthermore, in step (1), the viable count of the Akkermansia myxophilus AKKLF live bacterial solution was 1 × 10⁻⁶. 10 CFU / mL, sterilization method is pasteurization at 70℃ for 30 min; the viable count of Lactobacillus gasseri LFLG-245 in step (2) is 1×10 10 CFU / mL; In step (3), the volume ratio of Akkermansia muciniphila AKKLF inactivated bacterial solution to Lactobacillus gasseri LFLG-245 live bacterial solution is 1:9 to 9:1. For example, the volume ratio of Akkermansia muciniphila AKKLF inactivated bacterial solution to Lactobacillus gasseri LFLG-245 live bacterial solution can be selected from 1:9, 2:8, 3:7, 4:6, 1:1, 6:4, 7:3, 8:2 or 9:1.

[0015] Furthermore, the formulation of the combined microbial agent is a powder, and the combined microbial agent also includes pharmaceutically acceptable excipients; the excipients are selected from one or more of microcrystalline cellulose, lactose, magnesium stearate, trehalose, skim milk and sucrose.

[0016] Secondly, the present invention also provides the application of the above-mentioned combined bacterial agent in the preparation of a medicine for improving Alzheimer's disease.

[0017] Furthermore, the combination bacterial agent can be used alone or in combination with drugs for treating Alzheimer's disease to improve Alzheimer's disease; the drugs for treating Alzheimer's disease are selected from one or more of donepezil, galantamine, and rivastigmine.

[0018] Furthermore, improving Alzheimer's disease includes regulating the levels of inflammatory factors IL-6, hs-CRP, and / or TNF-α.

[0019] Furthermore, improvements in Alzheimer's disease include reducing Aβ plaque area, decreasing the p-Tau / Tau ratio, and / or reducing GSK-3β activity.

[0020] Furthermore, improving Alzheimer's disease includes increasing GABA synthesis efficiency; increasing GABA synthesis efficiency includes increasing intestinal GABA concentration, increasing serum GABA concentration, increasing GAD activity and / or gad gene expression.

[0021] The beneficial effects of this invention are as follows:

[0022] The present invention provides a combined bacterial agent for improving Alzheimer's disease, comprising *Lactobacillus gasseri* LFLG-245 and inactivated *Ackermania mutans* AKKLF. Experiments have demonstrated that this combined bacterial agent significantly reduces the levels of pro-inflammatory factors IL-6, hs-CRP, and TNF-α, alleviating neuroinflammatory responses. Simultaneously, it effectively reduces Aβ plaque deposition and regulates tau protein phosphorylation, intervening in the core pathological process of AD by lowering the p-Tau / Tau ratio and inhibiting GSK-3β activity. Furthermore, this combined bacterial agent significantly enhances GABA synthesis efficiency, specifically manifested as increased GABA concentrations in the gut and serum, enhanced GAD activity, and upregulated gad gene expression. Compared to single-strain or traditional drug treatments, the combination of live and inactivated bacteria used in this invention exhibits better synergistic effects in animal experiments, providing a safer and more effective microbial therapy for the treatment of Alzheimer's disease. In addition, experiments have confirmed that the combined bacterial agent provided by this invention, when used in combination with drugs for treating Alzheimer's disease, has a better effect. Replacing the dosage of existing Alzheimer's disease drugs with this combined bacterial agent during treatment can alleviate the gastrointestinal side effects caused by continuous use of existing Alzheimer's disease drugs to a certain extent and improve medication adherence. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a photograph of the colony morphology of Ackermann's AKKLF, a mycophile.

[0025] Figure 2 This is a Gram staining microscopic image (1000×) of the mucin Ackermann's AKKLF.

[0026] Figure 3 This is a photograph of the colony morphology of Lactobacillus gasseri LFLG-245.

[0027] Figure 4 This is a Gram staining microscopic image (1000×) of Lactobacillus freundii LFLG-245. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0029] It should be understood that the Akkermansia myxophilus AKKLF inactivated bacterial solution described in this invention includes dead cells, metabolites and / or cell lysates of Akkermansia myxophilus AKKLF; it may also be referred to as metabiotics.

[0030] Example 1: Isolation, screening and identification of bacterial strains

[0031] 1. Screening, purification and identification of Akkermansia myxophilus

[0032] (1) Sampling: Fresh feces from healthy children were collected in Beijing in September 2023.

[0033] (2) Strain isolation: Take 1 g of fresh feces from a healthy child and add it to 9 mL of PBS buffer and mix well to obtain a sample solution; dilute the sample solution with sterile PBS buffer to prepare dilutions of different concentration gradients, namely 10 mL and 10 mL of PBS buffer. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Then, using a spreader, 100 μL of the seven different concentration gradients were spread onto mucin plates containing vancomycin and incubated at 37°C under anaerobic conditions for 72 h.

[0034] The raw materials for the mucin agar plate medium include mucin (2 g / L), agar powder (15 g / L), KH₂PO₄ (0.4 g / L), Na₂HPO₄ (0.53 g / L), NH₄Cl (0.3 g / L), NaCl (0.3 g / L), MgCl₂ (0.1 g / L), CaCl₂ (0.11 g / L), NaHCO₃ (4 g / L), Na₂S·9H₂O (0.25 g / L), resazurite (0.5 mg / L), acidic trace element solution (1 mL / L), alkaline trace element solution (1 mL / L), vitamin solution (1 mL / L), and vancomycin (10 mg / L). The acidic trace element solution consists of FeCl₂ (0.95 g / L), H₃BO₃ (0.62 g / L), ZnCl₂ (0.068 g / L), CuCl₂·2H₂O (0.017 g / L), and MnCl₂ (0.063 g / L). The following ingredients were added: CoCl2 0.065 g / L, NiCl2 0.013 g / L, HCl 50 mmol / L; the alkaline trace element solution consisted of Na2SeO3 0.017 g / L, Na2WO4·2H2O 0.033 g / L, Na2MoO4·2H2O 0.024 g / L, NaOH 0.4 g / L; the vitamin solution consisted of thiamine 0.2 g / L, riboflavin 0.1 g / L, nicotinic acid 0.2 g / L, calcium pantothenate 0.1 g / L, pyridoxine 0.5 g / L, biotin 0.2 g / L, and para-aminobenzoic acid 0.1 g / L. After mixing the above ingredients thoroughly, the mixture was autoclaved at 121℃ for 15 min. Under aseptic conditions, the sterilized culture medium was poured into sterilized Petri dishes and allowed to cool before use.

[0035] (3) Purification: Select plump white single colonies and streak them onto mucin agar plates for purification. After culturing at 37°C under anaerobic conditions for 72 h, select single colonies again (colony morphology as shown in the image). Figure 1 As shown), after Gram staining, it was determined to be a Gram-negative bacterium (Gram staining microscopic image as shown). Figure 2 As shown in the figure, the mucin was cultured in liquid mucin medium until turbid, then stored in glycerol tubes at -80°C. Compared to mucin plate medium, the liquid mucin medium does not contain agar powder, but the preparation method is the same.

[0036] All clones were sent to a sequencing institution (Shanghai Sangon Biotech) to detect 16S rDNA. The strain identified as Akkermansia myxophilus was named Akkermansia myxophilus AKKLF.

[0037] The specific primer sequences used for identification include:

[0038] Forward primer (Seq_1): 5'-CAGCACGTGAAGGTGGGGAC-3';

[0039] Reverse primer (Seq_2): 5'-CCTTGCGGTTGGCTTCAGAT-3'.

[0040] The 16S rDNA gene sequence (Seq_3) of the myxotrophic Akkermansia AKKLF is as follows:

[0041]

[0042] The myxotrophic Akkermansia AKKLF was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 24, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33955, and classified as *Ackermansia myxotroph*. Akkermansia muciniphila .

[0043] 2. Screening, purification, and identification of Lactobacillus gasseri

[0044] (1) Sampling: Fresh feces from healthy children were collected in Shenzhen, Guangdong Province in August 2024.

[0045] (2) Strain isolation: Take 1 g of fresh feces from a healthy child and add it to 9 mL of PBS buffer and mix well to obtain a sample solution; dilute the sample solution with sterile PBS buffer to prepare dilutions of different concentration gradients, namely 10 mL and 10 mL of PBS buffer. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Then, using a spreader, 100 μL of each of the seven different concentration gradients was spread onto MRS plates and incubated at 37°C for 48 h.

[0046] The preparation method of MRS plate culture medium is as follows:

[0047] Take 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium hydrogen citrate, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.02 g of manganese sulfate, 1 mL of Tween-80, and 18 g of agar powder. Dissolve them in deionized water and bring the volume to 1 L. Autoclave at 121℃ for 15 min. Then, under aseptic conditions, pour the sterilized culture medium into sterilized petri dishes and let it cool before use.

[0048] (3) Purification: Single colonies were picked and streaked onto MRS agar plates for purification. After incubation at 37°C for 48 h, single colonies were picked again (colon morphology as shown in the image). Figure 3 As shown), after Gram staining, it was determined to be a Gram-positive bacterium (Gram staining microscopic image as shown). Figure 4 As shown), it was incubated in MRS liquid medium for 24 h until the medium became turbid, and then stored in glycerol tubes at -80°C.

[0049] Compared to MRS plate culture medium, MRS liquid culture medium does not contain agar powder in its raw materials, but the preparation method is the same.

[0050] All clones were sent to a sequencing institution (Shanghai Sangon Biotech) to detect 16S rDNA, and the strain identified as Lactobacillus gasseri was named Lactobacillus gasseri LFLG-245.

[0051] The specific primer sequences used for identification include:

[0052] Forward primer (Seq_4): 5'-GGATCATGTGGTAAAGGTGCAGTA-3';

[0053] Reverse primer (Seq_5): 5'-TCCACTAGCAGTTTGTAGAACCAAT-3'.

[0054] The 16S rDNA gene sequence (Seq_6) of this Lactobacillus gasseri LFLG-245 is as follows:

[0055]

[0056] Lactobacillus gasseri LFLG-245 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 24, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33952, and classified as Lactobacillus gasseri. Lactobacillus gasseri .

[0057] Example 2: Preparation of a combination bacterial agent to improve Alzheimer's disease

[0058] (1) Akkermansia myxophilus AKKLF, frozen at -80℃, was activated and inoculated at a rate of 1% (v / v) onto brain and heart broth containing 0.5% porcine gastric mucin (Qingdao Haibo, BHI medium, pH 6.5). The culture was incubated at 37℃ for 48 h under anaerobic conditions (80% N2, 10% CO2, 10% H2) to obtain the bacterial suspension. After incubation, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in PBS buffer to prepare a viable Akkermansia myxophilus AKKLF suspension, and the viable bacterial concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL. The live Akkermansia myxophilus AKKLF bacterial solution was pasteurized at 70℃ for 30 min to obtain the inactivated Akkermansia myxophilus AKKLF bacterial solution.

[0059] (2) Lactobacillus gasseri LFLG-245, frozen at -80℃, was activated and inoculated onto MRS liquid medium (preparation method see Example 1) at an inoculation rate of 1% (v / v). The medium was incubated at 37℃ for 24 h to obtain the bacterial suspension. After incubation, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in PBS buffer to prepare a viable Lactobacillus gasseri LFLG-245 suspension, and the viable bacterial concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL.

[0060] (3) Mix the inactivated Akkermansia muciniphila AKKLF bacterial solution and the live Lactobacillus gasseri LFLG-245 bacterial solution at a volume ratio of 1:1 to obtain a combined bacterial agent, which is a liquid preparation.

[0061] Example 3: Preparation of a combination bacterial agent to improve Alzheimer's disease

[0062] (1) Akkermansia myxophilus AKKLF, frozen at -80℃, was activated and inoculated at a rate of 1% (v / v) onto brain and heart broth containing 0.5% porcine gastric mucin (Qingdao Haibo, BHI medium, pH 6.5). The culture was incubated at 37℃ for 48 h under anaerobic conditions (80% N2, 10% CO2, 10% H2) to obtain the bacterial suspension. After incubation, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in PBS buffer to prepare a viable Akkermansia myxophilus AKKLF suspension, and the viable bacterial concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL. The live Akkermansia myxophilus AKKLF bacterial solution was pasteurized at 70℃ for 30 min to obtain the inactivated Akkermansia myxophilus AKKLF bacterial solution.

[0063] (2) Lactobacillus gasseri LFLG-245, frozen at -80℃, was activated and inoculated onto MRS liquid medium (preparation method see Example 1) at an inoculation rate of 1% (v / v). The medium was incubated at 37℃ for 24 h to obtain the bacterial suspension. After incubation, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in PBS buffer to prepare a viable Lactobacillus gasseri LFLG-245 suspension, and the viable bacterial concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL.

[0064] (3) Mix the inactivated bacterial solution of Akkermansia muciniphila AKKLF and the live bacterial solution of Lactobacillus gasseri LFLG-245 at a volume ratio of 1:1, concentrate and freeze dry, then mix and grind with microcrystalline cellulose to obtain a combined bacterial agent in powder form.

[0065] Depending on the actual needs, Akkermansia muciniphila AKKLF inactivated bacterial solution and Lactobacillus gasseri LFLG-245 live bacterial solution can be mixed and pharmaceutically acceptable excipients, excipients and other adjuvants and / or carriers can be added to prepare different dosage forms, such as pills, tablets, granules or capsules.

[0066] Comparative Example 1: Preparation of live Akkermansia myxophilus AKKLF culture

[0067] Akkermansia myxophilus AKKLF, frozen at -80℃, was activated and inoculated at a rate of 1% (v / v) onto brain and heart broth containing 0.5% porcine gastric mucin (Qingdao Haibo, BHI medium, pH 6.5). The culture was incubated anaerobically (80% N2, 10% CO2, 10% H2) at 37℃ for 48 h to obtain the bacterial suspension. After incubation, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in PBS buffer to prepare a viable Akkermansia myxophilus AKKLF suspension, and the viable cell concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL.

[0068] Comparative Example 2: Preparation of inactivated Akkermansia myxophilus AKKLF bacterial solution

[0069] Akkermansia myxophilus AKKLF, frozen at -80℃, was activated and inoculated at a rate of 1% (v / v) onto brain and heart broth containing 0.5% porcine gastric mucin (Qingdao Haibo, BHI medium, pH 6.5). The culture was incubated anaerobically (80% N2, 10% CO2, 10% H2) at 37℃ for 48 h to obtain the bacterial suspension. After incubation, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in PBS buffer to prepare a viable Akkermansia myxophilus AKKLF suspension, and the viable cell concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL. The live Akkermansia myxophilus AKKLF bacterial solution was pasteurized at 70℃ for 30 min to obtain the inactivated Akkermansia myxophilus AKKLF bacterial solution.

[0070] Comparative Example 3: Preparation of live Lactobacillus gasseri LFLG-245 culture

[0071] Lactobacillus gasseri LFLG-245, frozen at -80℃, was activated and inoculated onto MRS liquid medium (preparation method as shown in Example 1) at a 1% (v / v) inoculation rate. The medium was incubated at 37℃ for 24 h to obtain the bacterial suspension. After incubation, the suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The pellet was resuspended in PBS buffer to prepare a viable Lactobacillus gasseri LFLG-245 suspension, and the viable cell concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL.

[0072] Comparative Example 4: Preparation of Combined Microbial Agents

[0073] (1) Akkermansia myxophilus AKKLF, frozen at -80℃, was activated and inoculated at a rate of 1% (v / v) onto brain and heart broth containing 0.5% porcine gastric mucin (BHI medium, pH 6.5). The culture was then incubated at 37℃ for 48 h under anaerobic conditions (80% N2, 10% CO2, 10% H2) to obtain the bacterial suspension. After incubation, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in PBS buffer to prepare a viable Akkermansia myxophilus AKKLF suspension, and the viable cell concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL.

[0074] (2) Lactobacillus gasseri LFLG-245, frozen at -80℃, was activated and inoculated onto MRS liquid medium (preparation method see Example 1) at an inoculation rate of 1% (v / v). The medium was incubated at 37℃ for 24 h to obtain the bacterial suspension. After incubation, the bacterial suspension was centrifuged at 8000 rpm for 10 min, and the bacterial pellet was collected. The bacterial pellet was resuspended in PBS buffer to prepare a viable Lactobacillus gasseri LFLG-245 suspension, and the viable bacterial concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL.

[0075] (3) The viable bacterial suspensions of Akkermansia muciniphila AKKLF and Lactobacillus gasseri LFLG-245 were mixed at a volume ratio of 1:1 and the viable bacterial concentration was verified to be 1×10⁻⁶ using plate counting. 10 CFU / mL was used to obtain a combined bacterial agent.

[0076] Experimental Example 1: Effects of Combined Bacterial Agents on Alzheimer's Disease

[0077] 1. Animal model preparation

[0078] One hundred female SD rats, aged 7-9 months and weighing 220-238 g, were selected and acclimatized for one week. Ninety rats were randomly selected to establish an Alzheimer's disease model: 45 mg / kg sodium nitrite and 120 mg / kg D-galactose were injected subcutaneously into the neck and back of the rats once daily for four weeks until dementia symptoms appeared (such as increased spontaneous activities like scratching the ears and cheeks). The Morris water maze test was then used to verify the model's success. The results showed that compared to healthy rats without the model, the escape latency of the 90 rats was significantly prolonged, and the percentage of swimming distance in the target quadrant during the spatial exploration test was significantly reduced, indicating successful model establishment.

[0079] 2. Grouping and Dosing

[0080] Ten healthy rats that were not modeled were used as the normal control group and were given 1 mL of physiological saline by gavage daily.

[0081] Ninety rats that successfully developed the model were randomly divided into nine groups of ten each. The gavage medications and dosages for each group were as follows:

[0082] Model group: 1 mL of normal saline was administered by gavage daily;

[0083] Positive control group: 1 mL of physiological saline containing donepezil was administered by gavage daily. The daily dose of donepezil was calculated as 0.88 mg / kg of rat body weight.

[0084] AKKLF live bacterial solution group: 1 mL of Akkermansia myxophilus AKKLF live bacterial solution prepared by comparative example 1 was administered by gavage daily;

[0085] AKKLF inactivated bacterial solution group: 1 mL of Akkermansia myxophilus AKKLF inactivated bacterial solution prepared by comparative example 2 was administered by gavage daily;

[0086] LFLG-245 live bacteria solution group: 1 mL of Lactobacillus gasseri LFLG-245 live bacteria solution prepared in Comparative Example 3 was administered by gavage daily;

[0087] Group A of combined bacterial agents: 1 mL of the combined bacterial agent prepared in Example 2 was administered by gavage daily;

[0088] Group B of combined microbial agents: 1 mL of the combined microbial agent prepared in Comparative Example 4 was administered by gavage daily;

[0089] Group A: 0.5 mL of the combined bacterial agent prepared in Example 2 and 0.5 mL of physiological saline containing donepezil were administered by gavage daily. The daily gavage dose of donepezil was calculated as 0.44 mg / kg of rat body weight.

[0090] Group B: 0.5 mL of the combined bacterial agent prepared in Comparative Example 4 and 0.5 mL of physiological saline containing donepezil were administered by gavage daily. The daily gavage volume of donepezil was calculated as 0.44 mg / kg of rat body weight.

[0091] Each group was administered the above-mentioned dosage via gavage once daily for four consecutive weeks. The experimental procedure and research adhered to the principles and requirements established by the Animal Ethics Committee of the Chinese Association for Laboratory Animal Science.

[0092] 3. Behavioral tests

[0093] The Morris water maze experiment is an experiment in which laboratory animals are forced to swim and learn to find platforms hidden in the water. It is mainly used to test the learning and memory abilities of laboratory animals in terms of spatial location and orientation (spatial orientation), and its application is very common in Alzheimer's disease (AD) research.

[0094] (1) Experimental equipment

[0095] The Morris water maze detection system consists of two parts: an ABS powder-coated cylindrical water tank and the VisuTrack animal behavior analysis system (Shanghai Xinruan Information Technology Co., Ltd.). The water tank has a diameter of 120 cm and a height of 50 cm. The platform has a diameter of 6 cm and a height of 14 cm. The water tank is divided into four quadrants (NE, SE, SW, NW) along the four cardinal directions. The midpoint of the arc on the quadrant wall is the optional entry point for the animal. The platform can be placed in the center of any quadrant. The VisuTrack image acquisition and analysis system records the animal's swimming trajectory data for indicator extraction and analysis.

[0096] (2) Experiment content

[0097] Adaptation training: Before the experiment, rats were placed in a water maze (without a platform) and allowed to swim freely for 5 minutes to familiarize them with the maze environment and eliminate their fear of the environment.

[0098] Orientation and navigation experiment: Four times a day for days 1-4. Rat were placed in the water from the midpoint of the edges of four different quadrants, with their heads facing the pool wall. The time it took for the rat to find the hidden platform (escape latency) was recorded. If the rat did not find the platform within 90 seconds, it was guided to the platform and remained there for 10 seconds.

[0099] Space exploration experiment: On day 5, the platform was removed, and the rats were placed in the water from the quadrant opposite to the platform. The swimming distance to the target quadrant and the number of times the rats crossed the original platform position within 90 seconds were recorded.

[0100] The experiment lasted for 5 days, with each day starting at a fixed time and each time slot consisting of 4 training sessions.

[0101] (3) Results and Analysis

[0102] Statistical analysis was performed using SPSS 20.0 software. Results are expressed as mean ± standard deviation. The t-test was used for comparisons between groups, and p < 0.05 was considered statistically significant. The results are shown in Tables 1 and 2 below.

[0103] Table 1. Time taken for rats to reach the platform during the navigation experiment (unit: seconds)

[0104]

[0105] Note: Compared with the normal control group, ▲▲ indicates a highly significant difference (p<0.01), and ▲ indicates a significant difference (p<0.05); compared with the model group, ** indicates a highly significant difference (p<0.01), and * indicates a significant difference (p<0.05); compared with the AKKLF inactivated bacterial solution group, ## indicates a highly significant difference (p<0.01), and # indicates a significant difference (p<0.05).

[0106] In Table 1, the shorter the time it takes for a rat to reach the platform, the better its learning and memory abilities.

[0107] Table 2 Results of Space Exploration Experiments

[0108]

[0109] Note: Compared with the normal control group, ▲▲ indicates a highly significant difference (p<0.01), and ▲ indicates a significant difference (p<0.05); compared with the model group, ** indicates a highly significant difference (p<0.01), and * indicates a significant difference (p<0.05); compared with the AKKLF inactivated bacterial solution group, ## indicates a highly significant difference (p<0.01), and # indicates a significant difference (p<0.05).

[0110] In Table 2, the more times a rat crosses the platform, the better its learning and memory ability; the higher the percentage of swimming distance in the target quadrant, the better its learning and memory ability.

[0111] As shown in Tables 1 and 2, the escape latency of rats in the normal control group gradually shortened, indicating that the rats' familiarity with the environment gradually increased. The escape latency of rats in the model group was significantly longer than that of the normal control group, the number of times they crossed the platform was less than that of the normal control group, and the percentage of swimming distance in the target quadrant was significantly lower than that of the normal control group, indicating that the model was successfully established.

[0112] Compared to the model group, the escape latency of rats in the positive control group, AKKLF inactivated bacterial solution group, AKKLF live bacterial solution group, LFLG-245 live bacterial solution group, combination bacterial agent A group, combination bacterial agent B group, combined A group, and combined B group were significantly reduced, the number of times they crossed the platform was increased, and the percentage of swimming distance in the target quadrant was significantly increased, indicating that they all had the effect of improving cognitive impairment in Alzheimer's disease model rats. Among them, compared with the AKKLF inactivated bacterial solution group, the combination bacterial agent A group and the combined A group had a more significant effect on improving cognitive impairment in Alzheimer's disease model rats. This indicates that the combination of Akkermansia myxophilus AKKLF inactivated bacterial solution and Lactobacillus gasseri LFLG-245 live bacterial solution is the optimal strategy for improving cognitive impairment in Alzheimer's disease model rats, with an effect close to the normal level (p>0.05) and superior to the positive control group (p<0.05).

[0113] 4. Serum inflammatory factor level test

[0114] After the behavioral test was completed, blood was collected from the heart of the rats by puncture. The blood was centrifuged at 3000 r / min for 2 min at 4℃ to separate the serum. The serum was divided into two parts for use. One part was used to detect the level of inflammation in the serum by enzyme-linked immunosorbent assay.

[0115] (1) Experimental materials

[0116] The detection of IL-6, hs-CRP, and TNF-α by ELISA was performed according to the instructions of the ELISA kit (Wuhan Yilairuit Biotechnology), and the ELISA reader used was a TECAN (Switzerland) multi-functional ELISA reader.

[0117] (2) Experiment content

[0118] ① Plotting the standard curve

[0119] Dilute IL-6, hs-CRP, and TNF-α standards to different concentration gradients (e.g., 0 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL) using diluent. Add 100 μL of each concentration of standard to the corresponding well of an ELISA plate, repeating three times per well. Add 100 μL of specific enzyme-labeled antibody to each well, mix well, and incubate at 37°C for 2 h. Wash the ELISA plate three times with washing buffer, soaking for 30 seconds each time, and finally pat dry on absorbent paper. Add 100 μL of chromogenic reagent to each well, mix gently, and incubate at room temperature in the dark for 15 min. Add 50 μL of stop solution to each well and mix gently. Measure the absorbance of each well at 450 nm using a microplate reader. Plot a standard curve with standard concentration on the x-axis and absorbance on the y-axis.

[0120] ② Sample testing

[0121] Rat serum samples were diluted 1:10 with diluent. 100 μL of the diluted serum sample was added to the corresponding well of an ELISA plate, with three replicates per well. 100 μL of specific enzyme-labeled antibody was added to each well, mixed, and incubated at 37°C for 2 h. The ELISA plate was washed three times with washing buffer, immersing for 30 seconds each time, and then patted dry on absorbent paper. 100 μL of chromogenic reagent was added to each well, gently mixed, and incubated at room temperature in the dark for 15 min. 50 μL of stop solution was added to each well, and gently mixed. The absorbance of each well was measured at 450 nm using a microplate reader, and the concentrations of IL-6 (interleukin-6), hs-CRP (C-reactive protein), and TNF-α (tumor necrosis factor-α) in the sample were calculated based on the standard curve.

[0122] (3) Results and Analysis

[0123] The experimental results are shown in Table 3.

[0124] Table 3. Levels of inflammatory factors in each group

[0125]

[0126] Note: Compared with the normal control group, ▲▲ indicates a highly significant difference (p<0.01), and ▲ indicates a significant difference (p<0.05); compared with the model group, ** indicates a highly significant difference (p<0.01), and * indicates a significant difference (p<0.05); compared with the AKKLF inactivated bacterial solution group, ## indicates a highly significant difference (p<0.01), and # indicates a significant difference (p<0.05).

[0127] As shown in Table 3, the levels of inflammatory factors IL-6, hs-CRP, and TNF-α in the model group rats were significantly higher than those in the normal control group (p<0.01), indicating that the model was successfully established. Compared with the model group, the levels of inflammatory factors in the positive control group, AKKLF live bacteria group, AKKLF inactivated bacteria group, LFLG-245 live bacteria group, combined bacterial agent A group, combined bacterial agent B group, combined A group, and combined B group were significantly lower (p<0.05) or extremely significantly lower (p<0.01), indicating that these groups can inhibit the inflammatory response in Alzheimer's disease rats by regulating the three inflammatory factors IL-6, hs-CRP, and TNF-α. Moreover, compared with the AKKLF inactivated bacterial solution group, the level of inflammatory factors in the combined bacterial agent A group was significantly reduced (p<0.05), and the level of inflammatory factors in the combined A group and the combined B group was extremely significantly reduced (p<0.01), indicating that the inflammatory suppression effect of the combined bacterial agent A group was better than that of the AKKLF inactivated bacterial solution group, and the combined A group showed a better effect, which has clinical translational significance.

[0128] 5. AD pathological marker level test

[0129] After cardiac blood collection, the rats were euthanized by decapitation, and the entire brain was rapidly dissected. The left hippocampus and cortical tissue were excised along the sagittal plane and fixed in 4% paraformaldehyde for 24 h, followed by dehydration in 30% sucrose solution for 48 h. The remaining right brain tissue was immediately flash-frozen in liquid nitrogen and stored at -80°C for protein extraction.

[0130] (1) Experiment content

[0131] ① Quantitative analysis of fixed Aβ plaques

[0132] Thioflavin S staining method: After OCT embedding of brain tissue, 10 μm thick coronal frozen sections were prepared. After rinsing with PBS, 0.1% thioflavin S staining solution (dissolved in 50% ethanol) was added, and the sections were incubated in the dark for 8 min. After staining, the sections were separated twice with 70% ethanol for 10 seconds each time, rinsed with pure water, and mounted with glycerol. Images of the hippocampal CA1 region and cortex were taken using a fluorescence microscope (excitation wavelength 450 nm, emission wavelength 510 nm). The area percentage of Aβ plaques was calculated using ImageJ software (plaque area / total field of view × 100%). Three sections were analyzed for each rat, and the mean value was taken.

[0133] ② Detection of phosphorylated Tau protein (p-Tau)

[0134] Protein blotting: Frozen brain tissue (hippocampus) was added to RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors), homogenized, and centrifuged at 4°C (1200 rpm, 15 min). The supernatant was collected to determine protein concentration. 30 μg of protein was loaded onto a PVDF membrane, separated by 10% SDS-PAGE electrophoresis, and transferred to the membrane. After membrane blocking, the membrane was sequentially incubated with primary antibody (p-TauAT8 antibody, 1:1000; total Tau antibody, 1:2000) and HRP-labeled secondary antibody (1:5000). After ECL development, the band grayscale values ​​were analyzed using ImageLab software, and the ratio of p-Tau to total Tau was calculated.

[0135] ③ GSK-3β activity assay

[0136] Collect the supernatant from the brain tissue homogenate and follow the instructions for the GSK-3β activity assay kit (Abcam, ab131368). Mix the sample with the substrate (GSK-3β specific peptide) and add the reaction buffer (containing ATP and Mg). 2+ Incubate at 37℃ for 30 min. After adding the stop solution, measure the absorbance at 450 nm and calculate the GSK-3β activity based on the standard curve.

[0137] (2) Results and Analysis

[0138] The experimental results are shown in Table 4.

[0139] Table 4 Results of AD pathological marker tests in each group

[0140]

[0141] Note: Compared with the normal control group, ▲▲ indicates a highly significant difference (p<0.01), and ▲ indicates a significant difference (p<0.05); compared with the model group, ** indicates a highly significant difference (p<0.01), and * indicates a significant difference (p<0.05); compared with the AKKLF inactivated bacterial solution group, ## indicates a highly significant difference (p<0.01), and # indicates a significant difference (p<0.05).

[0142] Table 4 shows that for the three AD pathological markers—Aβ plaque area, p-Tau / Tau ratio, and GSK-3β activity—the model group rats showed extremely significant increases in the test results compared to the normal control group (p<0.01), indicating that the AD model was successfully established and exhibited typical pathological features of Aβ deposition, Tau protein hyperphosphorylation, and GSK-3β overactivation. Compared to the model group, the positive control group, AKKLF live bacteria group, AKKLF inactivated bacteria group, LFLG-245 live bacteria group, combined bacterial agent A group, combined bacterial agent B group, combined A group, and combined B group showed significantly decreased (p<0.05) or extremely significant (p<0.01) test results, indicating that these groups can exert neuroprotective effects and improve Alzheimer's disease by regulating Aβ metabolism, Tau phosphorylation, or the GSK-3β signaling pathway. Moreover, compared to the AKKLF inactivated bacterial solution group, the pathological marker test results of the combined bacterial agent group A, the combination group A, and the combination group B showed extremely significant reductions (p<0.01). In addition, the combination group A showed superior effects, indicating that the combination of Akkermansia myxophilus AKKLF inactivated bacterial solution and Lactobacillus gasseri LFLG-245 live bacterial solution, when used in combination with drugs, can produce an additive effect, possibly through the synergistic regulation of AD progression via multiple targets including gut microbiota-metabolism-neuroinflammation.

[0143] 6. Gamma-aminobutyric acid (GABA) metabolism capacity test

[0144] (1) Sample collection

[0145] After rats were euthanized by decapitation, the following samples were collected under aseptic conditions:

[0146] ①Intestinal contents: Take the contents from the terminal ileum to the colon, weigh them, add 0.1 M phosphate buffer (pH 7.4) and dilute at a ratio of 1:9 (w / v), vortex for 5 min, centrifuge at 12000×g for 15 min at 4℃, take the supernatant and filter it through a 0.22 μm filter membrane, aliquot and freeze at -80℃ for GABA detection.

[0147] ② Serum: Another sample of serum preserved for serum inflammatory factor testing.

[0148] (2) Experimental methods

[0149] ① Detection of intestinal GABA by high performance liquid chromatography

[0150] An Agilent 1260 Infinity II HPLC system was used, with a ZORBAX Eclipse Plus C18 column (4.6 × 250 mm, 5 μm). The mobile phase was 0.1 M phosphate buffer (pH 3.0), the flow rate was 1.0 mL / min, the column temperature was 30℃, the detection wavelength was 210 nm, and the injection volume was 20 μL. GABA was separated using a gradient elution program: isocratic elution for 0–5 min, followed by linear increase of the acetonitrile ratio to 20% for 5–10 min. The GABA concentration was calculated using the external standard method, with a standard curve ranging from 0.1–50 μg / mL (R0). 2 >0.99).

[0151] ② Detection of serum GABA by enzyme-linked immunosorbent assay (ELISA)

[0152] The rat GABA ELISA kit (catalog number: MBS726793) was used, strictly following the instructions: Samples were co-incubated with horseradish peroxidase-labeled GABA antibody for 1 h, washed, and then TMB chromogenic solution was added. The absorbance was measured at 450 nm, and the concentration was calculated using a standard curve. Simultaneously, glutamate decarboxylase (GAD65 / 67) activity was detected: tissue homogenate was reacted with substrate L-glutamate (final concentration 10 mM) at 37℃ for 30 min, and the amount of GABA generated was determined colorimetrically.

[0153] (3) Results and Analysis

[0154] The experimental results are shown in Table 5.

[0155] Table 5. Results of GABA metabolism-related indicators in each group

[0156]

[0157] Note: Compared with the normal control group, ▲▲ indicates a highly significant difference (p<0.01), and ▲ indicates a significant difference (p<0.05); compared with the model group, ** indicates a highly significant difference (p<0.01), and * indicates a significant difference (p<0.05); compared with the AKKLF inactivated bacterial solution group, ## indicates a highly significant difference (p<0.01), and # indicates a significant difference (p<0.05).

[0158] As shown in Table 5, for the four GABA metabolism-related indicators—intestinal GABA, serum GABA, GAD activity, and gad gene expression—the GABA metabolism-related indicators in the model group rats were significantly lower than those in the normal control group (p<0.01), indicating that the model was successfully established and the GABAergic system was impaired. Compared with the model group, the GABA metabolism-related indicators in the positive control group, AKKLF live bacteria group, AKKLF inactivated bacteria group, LFLG-245 live bacteria group, combined bacterial agent A group, combined bacterial agent B group, combined A group, and combined B group showed significantly higher (p<0.05) or extremely significantly higher (p<0.01) GABA metabolism-related indicators, indicating that these groups can improve GABA synthesis efficiency and alleviate Alzheimer's disease. Moreover, compared to the AKKLF inactivated bacterial solution group, the levels of inflammatory factors in the combined bacterial agent B group were significantly reduced (p<0.05), while the GABA synthesis efficiency of the combined A and B groups was significantly increased (p<0.01), indicating that the GABA synthesis efficiency of the combined bacterial agent B group was better than that of the AKKLF inactivated bacterial solution group. The reason for this is that the combined bacterial agent can upregulate gad gene expression by releasing stable metabolites (such as γ-aminobutyric acid precursors). Furthermore, the combined A group showed a better effect, which may be because the combination of Akkermansia myxophilus AKKLF inactivated bacterial solution and Lactobacillus gasseri LFLG-245 live bacterial solution, used in combination with donepezil, can directly promote GABA production through the dual pathway of "upregulating gad expression - activating GAD enzyme".

[0159] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A combination bacterial agent for improving Alzheimer's disease, characterized in that, Including Lactobacillus gasseri ( Lactobacillus gasseri LFLG-245 and inactivated Akkermansia myxophilus ( Akkermansia muciniphila AKKLF; Lactobacillus gasseri LFLG-245 was deposited on March 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33952, and classified as Lactobacillus gasseri. Lactobacillus gasseri ; Akkermansia myxophilus AKKLF was deposited on March 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33955, and classified as Akkermansia myxophilus. Akkermansia muciniphila .

2. The combined microbial agent as described in claim 1, characterized in that, The dosage forms of combined microbial agents are powder, pills, tablets, granules, capsules, or liquids.

3. The combined microbial agent as described in claim 2, characterized in that, The combined microbial agent is in liquid form, and its preparation method includes the following steps: (1) After activating Akkermansia mucin AKKLF, it was inoculated onto a broth medium containing 0.5% porcine gastric mucin and cultured at 37°C for 48 h under anaerobic conditions to obtain a bacterial suspension. The bacterial suspension was centrifuged, the bacterial precipitate was collected, and after resuspending, a live Akkermansia mucin AKKLF bacterial suspension was prepared. After sterilization, an inactivated Akkermansia mucin AKKLF bacterial suspension was obtained. (2) After activating Lactobacillus gasseri LFLG-245, it was inoculated onto MRS liquid medium and cultured at 37°C for 24 h to obtain bacterial suspension. The bacterial suspension was centrifuged, the bacterial precipitate was collected, and after resuspending, Lactobacillus gasseri LFLG-245 live bacterial suspension was prepared. (3) Mix the inactivated Akkermansia muciniphila AKKLF bacterial solution and the live Lactobacillus gasseri LFLG-245 bacterial solution to obtain a combined bacterial agent.

4. The combined microbial agent as described in claim 3, characterized in that, In step (1), the viable count of the Akkermansia myxophilus AKKLF live bacterial solution was 1×10⁻⁶. 10 CFU / mL, sterilization method is pasteurization at 70℃ for 30 min; the viable count of Lactobacillus gasseri LFLG-245 in step (2) is 1×10 10 CFU / mL; In step (3), the volume ratio of Akkermansia myxophila AKKLF inactivated bacterial solution and Lactobacillus gasseri LFLG-245 live bacterial solution is 1:9, 2:8, 3:7, 4:6, 1:1, 6:4, 7:3, 8:2 or 9:

1.

5. The combined microbial agent as described in claim 2, characterized in that, The combined microbial agent is in powder form and also includes pharmaceutically acceptable excipients; the excipients are selected from one or more of microcrystalline cellulose, lactose, magnesium stearate, trehalose, skim milk and sucrose.

6. The use of the combined bacterial agent as described in any one of claims 1-5 in the preparation of a medicament for improving Alzheimer's disease.

7. The application as described in claim 6, characterized in that, Combination bacterial agents can be used alone or in combination with drugs for treating Alzheimer's disease to improve Alzheimer's disease; the drugs for treating Alzheimer's disease are selected from one or more of donepezil, galantamine, and rivastigmine.

8. The application as described in claim 6 or 7, characterized in that, Improving Alzheimer's disease involves regulating the levels of inflammatory factors IL-6, hs-CRP, and / or TNF-α.

9. The application as described in claim 6 or 7, characterized in that, Improving Alzheimer's disease includes reducing Aβ plaque area, decreasing the p-Tau / Tau ratio, and / or reducing GSK-3β activity.

10. The application as described in claim 6 or 7, characterized in that, Improving Alzheimer's disease includes increasing GABA synthesis efficiency; increasing GABA synthesis efficiency includes increasing intestinal GABA concentration, increasing serum GABA concentration, increasing GAD activity and / or gad gene expression.

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