Synbiotic compositions and use in improving alzheimer's disease

Abstract

The present application is suitable for the technical field of microbial additive, and provides a probiotic composition and application thereof in improving Alzheimer's disease, wherein the probiotic composition comprises inulin and Sui-ling lactobacillus AF91-01CMCA, the preservation number of the Sui-ling lactobacillus AF91-01CMCA is GDMCC NO: 64744. The two components of the probiotic composition are used in combination, which can significantly improve the disease degree of Alzheimer's disease model mice, and the effect is better than that of the administration of probiotics or prebiotics alone. Specifically, the cognitive impairment of the subject is significantly relieved, the brain A beta accumulation of the subject is significantly reduced, and the ILA level in the body of the subject is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial additive technology, and in particular relates to a synbiotic composition and its application in improving Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment, memory loss, and language difficulties. According to data released by the National Health Commission of China in 2022, there are over 10 million AD patients in my country, making its treatment and prevention a major public health issue. Currently, the main medications for AD treatment include methimazole, donepezil, and galantamine. While these drugs are effective, they also cause side effects and impact patients' quality of life to varying degrees. Therefore, a more gentle treatment strategy is needed.

[0003] Recent studies have revealed significant differences in the gut microbiota of Alzheimer's disease (AD) patients compared to healthy individuals, leading to variations in gut metabolites. Indole lactic acid (ILA), a metabolite produced by the breakdown of tryptophan by gut microbiota, has been found to have significantly lower serum ILA levels in AD patients than in healthy individuals. Some interventions may improve AD by increasing ILA levels. Furthermore, research has demonstrated that ILA possesses anti-inflammatory, immunomodulatory, and neuroprotective functions, promoting nerve cell development. Therefore, increasing ILA levels through specific interventions could be a novel strategy for treating AD.

[0004] Some probiotics of the Lactobacillus genus are known to produce ILA (intracellular inflammatory lesions) and play an important role in the prevention and treatment of some inflammatory diseases. However, no studies have yet demonstrated the role of these ILA-producing probiotics in alleviating Alzheimer's disease. Therefore, screening for a high-ILA-producing probiotic, constructing a synbiotic to promote its growth and metabolism, and conducting functional evaluation of the synbiotic combination's effect in alleviating Alzheimer's disease will contribute to the development of novel synbiotic products and provide new solutions for the prevention and treatment of Alzheimer's disease. Summary of the Invention

[0005] The purpose of this invention is to provide a synbiotic composition and its application in improving Alzheimer's disease, aiming to solve the technical problems existing in the prior art.

[0006] This invention is achieved as follows: a type of Lactobacillus suiliense (… Lacticaseibacillus suilingensis AF91-01CMCA was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 13, 2024, with accession number GDMCCNO:64744.

[0007] Another object of the present invention is to provide a synbiotic composition comprising inulin and *Lactobacillus suiliengensis* AF91-01CMCA as described in claim 1, wherein the dosage ratio of *Lactobacillus suiliengensis* AF91-01CMCA to inulin is (1 × 10⁻⁶). 9 ~1×10 13 CFU / mL: (10~40) g / L.

[0008] A further technical solution of the present invention is: the synbiotic composition comprises inulin and *Lactobacillus suiliengensis* AF91-01CMCA as described in claim 1, wherein the dosage ratio of *Lactobacillus suiliengensis* AF91-01CMCA to inulin is (1×10⁻⁶). 10 ~1×10 12 CFU / mL: (20~30) g / L.

[0009] A further technical solution of the present invention is: the concentration of the *Lactobacillus suilingensis* AF91-01CMCA is 1×10⁻⁶. 11 The concentration of inulin is 20 g / L, and the concentration is CFU / mL.

[0010] Another object of the present invention is to provide an application of a synbiotic composition in the preparation of a drug for the prevention and improvement of Alzheimer's disease. The synbiotic composition includes inulin and Lactobacillus suilingensis AF91-01CMCA, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 13, 2024, with the accession number GDMCC NO:64744.

[0011] A further technical solution of the present invention is that the drug has one or more of the following functions: 1) alleviating cognitive impairment in the test subjects; 2) reducing the accumulation of Aβ in the brain of the test subjects; 3) increasing the level of ILA in the body of the test subjects.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing and improving Alzheimer's disease, the pharmaceutical composition comprising a synbiotic composition, the synbiotic composition comprising inulin and Lactobacillus suiliaceus AF91-01CMCA, wherein the dosage ratio of Lactobacillus suiliaceus AF91-01CMCA and inulin is (1×10⁻⁶). 9 ~1×10 13 CFU / mL: (10~40) g / L or (1×10) 10 ~1×10 12 CFU / mL: (20~30) g / L.

[0013] A further technical solution of the present invention is: the pharmaceutical composition is prepared by adding a pharmaceutically acceptable non-toxic carrier to the synbiotic composition, and the dosage form of the pharmaceutical composition is one of powder, tablet, granule, capsule, solution, suspension, emulsion and lyophilized preparation.

[0014] Another object of the present invention is to provide a food composition for preventing and improving Alzheimer's disease, the food composition comprising a synbiotic composition, wherein the synbiotic composition is prepared by adding food-acceptable excipients and additives, the synbiotic composition comprising inulin and Lactobacillus suilieng AF91-01CMCA, wherein the dosage ratio of Lactobacillus suilieng AF91-01CMCA to inulin is (1×10⁻⁶). 9 ~1×10 13 CFU / mL: (10~40) g / L or (1×10) 10 ~1×10 12 CFU / mL: (20~30) g / L.

[0015] Another object of the present invention is to provide a use of a synbiotic composition for preventing and improving Alzheimer's disease; the synbiotic composition is used to make food, food for special medical purposes, nutritional supplement, functional food, health food and medicine.

[0016] Another object of the present invention is to provide a method for preparing a synbiotic composition, the method comprising the following steps:

[0017] S1. The genome information of Lactobacillus AF91-01CMCA in Suiling was obtained by whole-genome sequencing technology. The function of all gene sequences was predicted by bioinformatics analysis technology, all carbohydrate utilization-related genes were screened, and substrate and carbohydrate transporter information were predicted.

[0018] S2. Construct corresponding metabolic pathways based on substrate and transporter information and their location on the genome, and then perform functional genomic analysis of carbohydrate utilization to predict specific prebiotics of the strain.

[0019] S3. The predicted information was validated in vitro. The predicted prebiotic was used as the sole carbon source, and a carbon-free semi-synthetic medium was used as a control. The growth curve of the strain in the biological medium with the corresponding prebiotic as the substrate was measured using the BioScreen growth curve analyzer to determine the effect of the prebiotic.

[0020] S4. The candidate strains were inoculated into ordinary culture medium and culture medium with added prebiotics. After fermentation for 48 h, the samples were centrifuged and prepared. The ILA yield of the strains in ordinary and corresponding prebiotic culture media was determined by Shimadzu LC-16 high performance liquid chromatography.

[0021] S5. Determine the composition and proportion of the synbiotic, and obtain the synbiotic composition according to the proportion.

[0022] The beneficial effects of this invention are as follows: The synbiotic composition comprises *Lactobacillus suiliengensis* AF91-01CMCA and inulin. *Lactobacillus suiliengensis* AF91-01CMCA is a newly discovered strain, isolated from fecal samples of healthy Chinese individuals; the synbiotic composition is a newly discovered combination of synbiotics, and studies have shown that the combined use of both can significantly improve the disease severity in Alzheimer's disease model mice, with effects superior to the administration of probiotics or prebiotics alone. Specifically, it significantly alleviates cognitive impairment in subjects; significantly reduces Aβ accumulation in the brains of subjects; and significantly increases ILA levels in subjects.

[0023] The synbiotic composition in this application has been screened and confirmed through in vitro experiments to have a reasonable formulation. Compared with Lactobacillus suilingensis AF91-01CMCA alone, the addition of inulin can promote the growth of Lactobacillus suilingensis AF91-01CMCA and significantly increase its ability to produce ILA. In addition, the addition of inulin can promote the in vivo colonization and growth of Lactobacillus suilingensis AF91-01CMCA, enhancing its efficacy.

[0024] The method of administration of the synbiotic composition in this application is reasonable and the dosage is significant, effectively avoiding the adverse effects that may result from unclear dosage in existing probiotic preparations, such as no effect after administration or excessive administration. Attached Figure Description

[0025] Figure 1 The growth curves of *Lactobacillus suilingensis* AF91-01CMCA in different carbon source media are shown, where (A) 10 g / L, (B) 20 g / L, (C) 30 g / L, and (D) 40 g / L.

[0026] Figure 2 Growth curves of *Lactobacillus subsuilensis* AF91-01CMCA added to inulin at different concentrations.

[0027] Figure 3 The ILA production level of Lactobacillus suiliensis AF91-01CMCA in a culture medium supplemented with 20 g / L inulin.

[0028] Figure 4 The study aimed to improve cognitive impairment using biosimilars, including (A) Barnes maze target quadrant exploration time and (B) Y maze discrimination index.

[0029] Figure 5The results of the assay to reduce Aβ accumulation in the brain by synbiotic are shown in (A) Aβ fluorescence staining, (B) Aβ quantitative results in the CA1 region, (C) Aβ quantitative results in the DG region, and (D) Aβ quantitative results in the Cortex region.

[0030] Figure 6 The present invention provides an experiment to increase the level of ILA in the body by synbiotic, wherein (A) fecal ILA content and (B) serum ILA content. Detailed Implementation

[0031] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further described below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0032] In some specific implementation examples, the synbiotic composition includes probiotics and prebiotics. The probiotic is *Lactobacillus suis* AF91-01CMCA, and the prebiotic is inulin. The synbiotic composition contains 2 × 10⁻⁶ probiotics. 10 The product contains CFU / d of probiotics and inulin at a dose of 0.02 g / mL.

[0033] The technical solution of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, the selected embodiments are only for illustrating the present invention and do not limit the scope of the present invention.

[0034] Example 1:

[0035] Isolation and identification of Lactobacillus suiliensis AF91-01CMCA

[0036] 1. Sample collection

[0037] The isolated sample was obtained from the feces of a healthy person. The feces were collected into a sterile sample tube and brought back to the laboratory for sorting within 1 hour.

[0038] 2. Separation and purification

[0039] Freshly collected samples were immediately transferred to an anaerobic chamber. 0.2g of sample was added to 1ml of sterile PBS (phosphate-buffered saline), thoroughly vortexed, and then serially diluted and spread. A modified PYG medium was used as the culture medium, with the following formula (1L): 8g tryptone, 2g soybean peptone, 1g polyprotein, 1g casein, 10g yeast extract, 5g beef extract, 5g glucose, 2g dipotassium hydrogen phosphate, 0.5g maltose, 0.5g cellobiose, 0.5g soluble starch, 0.25g sodium sulfide, 0.5mL Tween 80, and Cysteine-HCl•H2O. 0.5g, 0.5mL glycerol, 5g sodium acetate, 10ml heme solution (each liter of heme solution contains 500mg heme, 10ml sodium hydroxide solution (1M)), 10ml vitamin solution (each liter of vitamin solution contains 2mg biotin, 10mg pyridoxine hydrochloride, 5mg thiamine hydrochloride dihydrate, 5mg riboflavin, 5mg niacin, 5mg calcium pantothenate, 0.1mg vitamin B12, 5mg p-aminobenzoic acid, 5mg lipoic acid), 40mL inorganic salt solution (each liter of inorganic salt solution contains 0.25g calcium chloride dihydrate, 0.5g magnesium sulfate heptahydrate, phosphate) 1g of dipotassium hydrogen phosphate, 1g of potassium dihydrogen phosphate, 10g of sodium bicarbonate, and 2g of sodium chloride; 10ml of trace elements (each liter of trace elements contains 1.5g of nitric acid, 3g of magnesium sulfate heptahydrate, 1g of sodium chloride, 0.1g of ferric sulfate heptahydrate, 0.18g of cobalt sulfate heptahydrate, 0.1g of calcium chloride dihydrate, 0.18g of zinc sulfate heptahydrate, 0.01g of copper sulfate pentahydrate, 0.02g of potassium aluminum sulfate dodecahydrate, 0.01g of boric acid, 0.01g of sodium molybdate, 0.03g of nickel chloride hexahydrate, and 0.3mg of sodium selenite pentahydrate); 1ml of resazurin; and distilled water to a final volume of 1L. Adjust the pH to 6.8-7.0. The plate was placed in anaerobically at 37℃ with an anaerobic gas composition of N2:CO2:H2 = 90:5:5. After 3 days of incubation, single colonies were picked and streaked to obtain pure cultures of each bacterial strain.

[0040] 3. Preservation of microbial strains

[0041] The obtained pure culture strain was cultured until the concentration was approximately 10. 9 CFU / mL, take 400 μL of bacterial culture and add 400 μL of 40% glycerol to make the glycerol concentration reach 20%, and then store at -80℃.

[0042] Prepare the lyophilized bacterial strain powder according to the following steps: Autoclave the ampoules and cryoprotectant. Streak the overnight cultured bacterial solution and incubate at 37°C for 24 hours. After confirming no contamination, proceed with the following steps: Collect the bacterial solution by centrifugation and wash with sterile physiological saline. Add 2–3 mL of skim milk cryoprotectant to resuspend the solution and prepare a colony count of 102. 8 ~1010 The bacterial suspension was prepared at a concentration of 100 cells / mL and dispensed into sterile ampoules. The ampoules were pre-frozen at -80 °C for 1–2 hours. Following the standard operating procedure for freeze dryers, the ampoules were freeze-dried for 8–20 hours until completely lyophilized. After freeze-drying, the sample ampoules were removed, and the area below the cotton plug at the neck of the ampoule was sealed using a strong flame, following the standard operating procedure for vacuum ampoule sealing machines.

[0043] 4. 16S rDNA identification

[0044] After culturing the obtained isolated strains in liquid PYG medium for 24 h, PCR amplification was performed using universal 16S rDNA primers. The amplification system consisted of: 2.5 μL 10× PCR buffer, 2 μL dNTP (2.5 mM), 0.5 μL 27F (5'-AGAGTTTGATCATGGCTCAG-3'), 0.5 μL 1492R (5'-TAGGGTTACCTTGTTACGACTT-3'), 0.3 μL Taq enzyme, 1 μL template, and 18.2 μL ddH2O. The PCR amplification conditions were: 94℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 40 s, and 72℃ extension for 1 min 30 s, for 10 cycles; 95℃ denaturation for 30 s, 56℃ annealing for 40 s, and 72℃ extension for 1 min 30 s, for 25 cycles; 72℃ extension for 10 min, and storage at 4℃. The obtained 16S rDNA amplification product was subjected to electrophoresis detection, purification, and 3730 sequencing to obtain a 1400bp 16S rDNA sequence.Sequencing results were viewed using SeqScanner v1.0. Sequences with acceptable sequencing quality were selected and aligned online using EZBioCloud (https: / / www.ezbiocloud.net / identify). The sequences are shown below. The identification results show that the bacteria is most closely related to *Lacticaseibacillus baoqingensis*, with a similarity of 98.7%, indicating that this bacterium is a new species of the genus *Ligilactobacillus*.

[0045] Example 2

[0046] Carbon source screening of Lactobacillus suiliensis AF91-01CMCA

[0047] 1. Materials and Methods

[0048] 1.1 Experimental Materials

[0049] Strain: *Lactobacillus suilingensis* AF91-01CMCA. Main reagents: GAM medium, inulin, ILA standard, chromatographic methanol, sodium dihydrogen phosphate, glacial acetic acid.

[0050] 1.2 Experimental Methods

[0051] Whole-genome sequencing analysis was performed on Lactobacillus suilingensis AF91-01CMCA. The sequencing results were compared and annotated using the CAZy carbohydrate enzyme database to obtain information on carbon source metabolism-related genes of Lactobacillus suilingensis AF91-01CMCA. Substrate and carbohydrate transporter information were further predicted. Based on the substrate and transporter information and their location on the genome, corresponding metabolic pathways were constructed, and then functional genomic analysis of carbohydrate utilization was performed to complete the prediction of specific prebiotics of Lactobacillus suilingensis AF91-01CMCA.

[0052] Next, the predicted results were validated in vitro. Using carbon-free semi-synthetic medium (GAM) as a control, *Lactobacillus suilingensis* AF91-01CMCA was inoculated into mediums containing different prebiotics and cultured for 48 hours. The OD of the medium was measured every 1 hour using a BioScreen growth curve analyzer. 600 The absorbance values ​​were measured at nm, and growth curves were plotted based on these values ​​to determine the effect of prebiotics on the growth of *Lactobacillus suiliengensis* AF91-01CMCA. Different concentrations of prebiotics were added to the culture medium: 10 g / L, 20 g / L, 30 g / L, and 40 g / L.

[0053] Finally, *Lactobacillus suilingensis* AF91-01CMCA was inoculated into GAM medium and GAM+20 g / L inulin medium. After fermentation for 48 h, the samples were centrifuged and prepared. The amount of ILA produced by the strain in ordinary and corresponding prebiotic media was determined using a Shimadzu LC-16 high performance liquid chromatograph.

[0054] The composition of the synbiotic was determined based on the growth curve results and the results of ILA production during fermentation.

[0055] Test results

[0056] 1. Carbon source prediction for Lactobacillus suilingensis AF91-01CMCA

[0057] The comparison results with the CAZy carbohydrate enzyme database are shown in Table 1. The analysis results indicate that *Lactobacillus suilingensis* AF91-01CMCA possesses inulinase, β-mannosidase, β-xylanase, and α-arabinosidase. These enzymes are related to the hydrolysis of inulin, xylooligosaccharides, and mannose, thus suggesting that it has the ability to utilize these carbon sources.

[0058] Table 1. Carbon source metabolic enzymes possessed by Lactobacillus suiliengensis AF91-01CMCA

[0059]

[0060] 2. Growth curve analysis results

[0061] Depend on Figure 1 It can be seen that among the three carbon source-supplemented culture media, inulin had the best growth-promoting effect on *Lactobacillus suilingensis* AF91-01CMCA, and the growth-promoting effect of inulin was the best at different carbon source supplementation concentrations. Figure 2 It can be seen that the growth of Lactobacillus suilingensis AF91-01CMCA is better with the increase of inulin concentration, but the growth-promoting effect of inulin concentrations of 20 g / L, 30 g / L and 40 g / L is similar.

[0062] 3. ILA production level during fermentation

[0063] Depend on Figure 3 It can be seen that, compared with GAM medium, the ILA production of Lactobacillus AF91-01CMCA in GAM medium supplemented with 20 g / L inulin was significantly increased, reaching about 25 μg / mL.

[0064] In summary, based on the growth curves and liquid phase results, the synbiotic combination was determined to be Lactobacillus suilingensis AF91-01CMCA and inulin.

[0065] Example 3

[0066] Synbiotics alleviate cognitive impairment in Alzheimer's disease

[0067] This invention uses animal experiments to evaluate the efficacy of the synbiotic composition of this invention, in order to demonstrate the effectiveness of the synbiotic composition.

[0068] 1. Grouping of laboratory animals

[0069] Thirty-five 7-month-old 5×FAD mice (AD model mice) and ten C57 mice (Con normal control mice) were selected and divided into five groups: control group (Con), Alzheimer's disease model group (AD), model supplemented with prebiotics group (AI), model supplemented with probiotics group (AL), and model supplemented with synbiotics group (AS), with 8-10 mice in each group. The mice underwent 4 weeks of intervention with control, probiotics, prebiotics, and synbiotics.

[0070] 2. Processing methods for each group

[0071] Control group: Normal drinking water, supplemented with 100μL PBS by gavage daily.

[0072] Prebiotic treatment: Dissolve inulin in sterile water to a concentration of 20g / L and supplement with drinking water.

[0073] Probiotic treatment: The cultured Lactobacillus AF91-01CMCA from Suiling was suspended in an appropriate amount of PBS until the viable count reached 1×10⁻⁶. 11CFU / mL, prepare fresh before use. Administer 200 μL of bacterial suspension daily via gavage.

[0074] Synbiotic treatment: Daily gavage supplementation of bacterial suspension and drinking water supplementation of prebiotics.

[0075] 3. Animal behavioral testing methods

[0076] Behavioral analysis was performed on mice after 4 weeks of feeding to evaluate their cognitive abilities.

[0077] 3.1 New Object Recognition Test

[0078] A novel object recognition test was used to evaluate the short-term memory ability of mice. The test included an adaptation day, a training day, and a test day. On the adaptation day, mice were placed in an open cube (length × width × height; 40 cm × 40 cm × 40 cm) and allowed to explore freely for 5 minutes. On the training day, two identical objects were placed in the upper left and lower right corners of the cube, and the mouse was placed in the center and allowed to explore freely for 5 minutes. On the test day, one of the objects from the training day was replaced with a new object (the new object had a different shape and color than the old object), and the mouse was allowed to explore freely for 5 minutes. During this period, the mouse's activity trajectory and the time it spent sniffing the new and old objects were recorded using software. The discrimination index was calculated as: (new object exploration time - old object exploration time) / (new object exploration time + old object exploration time) × 100%.

[0079] 3.2 Barnes Maze Test

[0080] The day before the experiment, animals were individually placed in the target box from the target hole for 4 minutes to acclimatize. The animals were then placed in a 20 cm x 27 cm plastic cylinder in the center of the maze and their movement was restricted for 5 seconds. The cylinder was removed, and the software was activated. An escape attempt was counted when all four limbs of the animal entered the target box, and the animal remained in the box for 30 seconds. Each animal was observed for a maximum of 4 minutes at a time. If the animal still could not find the target box during this period, it was removed from the maze and placed in the target box for 30 seconds. This interval was used to clean the maze. Animals were trained once a day for four consecutive days. From the second training session onwards, the maze was rotated by one or more holes before each training session, but the target box remained fixed in the same direction. This was to prevent the animals from relying on scent and instead using memory to locate the target box. The latency to reach the target box and the number of errors for each animal were recorded. On the fifth day, the target box was removed, and the mice were allowed to explore freely for 120 seconds. The number of times any hole was explored, the time spent near the target box, and the time it took to first find the target box were recorded.

[0081] Test results

[0082] Depend on Figure 4It was found that, compared with WT mice, AD mice exhibited significantly reduced cognitive abilities, manifested as decreased target quadrant exploration time and a lower discrimination index. However, after 4 weeks of synbiotic supplementation, AD mice showed a significant increase in target quadrant exploration time and a significant improvement in the discrimination index during the new object test, with effects superior to probiotic or prebiotic supplementation alone. These results indicate that synbiotics can alleviate cognitive impairment in Alzheimer's mice.

[0083] Example 4

[0084] Synbiotics reduce Aβ accumulation in the brain

[0085] 1. The experimental grouping and treatment are the same as in Example 2.

[0086] 2. Experimental Methods

[0087] Mice were euthanized after a 4-week intervention, and their brain tissue was collected, fixed in 4% paraformaldehyde, and then subjected to steps such as embedding, sectioning, dewaxing, permeabilization, antigen retrieval, inactivation of endogenous enzyme activity, blocking, primary antibody incubation, secondary antibody incubation, and DAPI mounting before being photographed under a fluorescence microscope to observe the accumulation of Aβ in the brain.

[0088] Test results

[0089] Excessive accumulation of Aβ is one of the pathological features of Alzheimer's disease, caused by... Figure 5 It is known that AD mice have a large amount of Aβ accumulation in the CA1, DG, and Cortex regions of their brains. Four weeks of synbiotic intervention can significantly reduce Aβ deposition in the brain, and the effect is better than probiotic or prebiotic supplementation alone.

[0090] Example 5

[0091] Synbiotics increase ILA levels in the body

[0092] 1. The experimental grouping and treatment are the same as in Example 2.

[0093] 2. Experimental Methods

[0094] Mice were treated for 4 weeks, and their feces were collected. They were then sacrificed, and their serum was collected. High-performance liquid chromatography (HPLC) was used to detect ILA levels in the feces and serum. The specific methods are as follows:

[0095] Fecal sample preparation: Weigh 0.10 g of mouse feces, add 1 mL of methanol, vortex mix, and incubate in a water bath at 40℃ for 20 min, mixing once every 5 min. After the water bath, place in a -20℃ refrigerator for 20 min, then remove and centrifuge at 13000 r / min for 10 min at 4℃. Filter the supernatant after centrifugation through a 0.22 µm microporous membrane (organic phase), add to a brown vial, and store as the test sample in a 4℃ refrigerator.

[0096] Serum sample preparation: Take 30 μL of serum and dilute it with methanol to 300 μL. Then, place it at -20℃ for 20 min and centrifuge it at 4℃ and 13000 r / min for 10 min. Filter the supernatant through a 0.22 μm organic filter membrane, add it to a brown bottle, and place it in a 4℃ refrigerator for testing.

[0097] Liquid chromatography: A ZORBAX XDB C18 column (4.6×250 μm, 5 μm) was used as the analytical column. Mobile phase A: 15 mmol / L sodium dihydrogen phosphate solution (pH 2.8), mobile phase B: chromatographic methanol, flow rate: 1.0 ml / min, gradient elution program: 0-12 min, A:B=42:58, 12-28 min, A:B=50:50, 28-35 min, A:B=85:15, column temperature: 30℃, detection wavelength fluorescence detector (excitation wavelength / emission wavelength): 282 / 352 nm, injection volume: 10 μL.

[0098] Test results

[0099] Depend on Figure 6 It can be seen that after 4 weeks of intervention, compared with the AD group, the addition of synbiotic significantly increased the levels of ILA in the feces and serum of mice.

[0100] Data and Statistical Analysis

[0101] All experimental data were analyzed using the Duncan method of one-way ANOVA in SPSS 26.0 software (P < 0.05), and plotted using Graphpad 8.0. All results are expressed as mean ± standard deviation, and each experiment was performed in triplicate.

[0102] Example 6

[0103] Preparation of food compositions containing Lactobacillus suiliensis AF91-01CMCA

[0104] Application Example 1: Preparation of Synbiotic Microcapsules Containing Lactobacillus suiliensis AF91-01CMCA and Inulin

[0105] S1: Preparation of synbiotic suspension. *Lactobacillus suilingensis* AF91-01CMCA was inoculated at a 5% inoculum in 20 mL of sterile MRS medium for activation culture at 37℃ for 24 h. Subsequently, the activated *Lactobacillus suilingensis* AF91-01CMCA was inoculated again at a 5% inoculum in 20 mL of sterile MRS medium and cultured at 37℃ for 48 h. The cultured bacterial solution was centrifuged at 4000 r / min for 10 min at 4℃, the supernatant was discarded, and the bacterial sludge precipitate was washed three times with sterile PBS. The sludge was then dissolved in 2 mL of sterile 50 g / L inulin solution to obtain the synbiotic suspension.

[0106] S2: Preparation of Synbiotic microcapsules. Synbiotic suspension was mixed with 4% (w / v) pectin solution at a bacterial-to-pectin ratio of 1:4. After thorough mixing, the mixture was slowly dripped into 400 mmol / L sterile CaCl2 solution using an extrusion method to obtain synbiotic microcapsules.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Lactobacillus suiliensis ( Lacticaseibacillus suilingensis )AF91-01CMCA, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 13, 2024, with accession number GDMCC NO:64744.

2. A synbiotic composition, characterized in that, The synbiotic composition comprises inulin and *Lactobacillus suilienae* AF91-01CMCA as described in claim 1, wherein the dosage ratio of *Lactobacillus suilienae* AF91-01CMCA to inulin is (1 × 10⁻⁶). 9 ~1×10 13 CFU / mL: (10~40) g / L.

3. The synbiotic composition according to claim 2, characterized in that, The synbiotic composition comprises inulin and *Lactobacillus suilienae* AF91-01CMCA as described in claim 1, wherein the dosage ratio of *Lactobacillus suilienae* AF91-01CMCA to inulin is (1 × 10⁻⁶). 10 ~1×10 12 CFU / mL: (20~30) g / L.

4. The synbiotic composition according to claim 3, characterized in that, The concentration of the *Lactobacillus suiliensis* AF91-01CMCA was 1×10⁻⁶. 11 The concentration of inulin is 20 g / L, and the concentration is CFU / mL.

5. The application of synbiotic compositions in the preparation of pharmaceuticals, characterized in that, The drug is used to prevent and improve Alzheimer's disease. The synbiotic composition includes inulin and Lactobacillus suilingensis AF91-01CMCA. Lactobacillus suilingensis AF91-01CMCA was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 13, 2024, with the accession number GDMCC NO:64744.

6. The application according to claim 5, characterized in that, The drug has one or more of the following functions: 1) alleviating cognitive impairment in the subjects; 2) reducing the accumulation of brain alpha beta in the subjects; 3) increasing the level of intracellular alpha (ILA) in the subjects.

7. A pharmaceutical composition for preventing and improving Alzheimer's disease, characterized in that, The pharmaceutical composition includes a synbiotic composition comprising inulin and Lactobacillus suiliaceus AF91-01CMCA, wherein the dosage ratio of Lactobacillus suiliaceus AF91-01CMCA to inulin is (1×10⁻⁶). 9 ~1×10 13 CFU / mL: (10~40) g / L.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage ratio of *Lactobacillus suiliensis* AF91-01CMCA and inulin was (1×10⁻⁶). 10 ~1×10 12 CFU / mL: (20~30) g / L.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that, The pharmaceutical composition is prepared by adding a pharmaceutically acceptable, non-toxic carrier to the synbiotic composition, and the dosage form of the pharmaceutical composition is one of the following: powder, tablet, granule, capsule, solution, suspension, emulsion, and lyophilized preparation.

10. A synbiotic food composition, characterized in that, The synbiotic food composition is made by adding food-acceptable excipients and additives. The synbiotic food composition includes inulin and *Lactobacillus suiliaceus* AF91-01CMCA, with the dosage ratio of *Lactobacillus suiliaceus* AF91-01CMCA to inulin being (1×10⁻⁶). 9 ~1×10 13 CFU / mL: (10~40) g / L.

11. The synbiotic food composition according to claim 10, characterized in that, The dosage ratio of *Lactobacillus suiliensis* AF91-01CMCA and inulin was (1×10⁻⁶). 10 ~1×10 12 CFU / mL: (20~30) g / L.

12. The use of the synbiotic composition of claim 2 in the preparation of food, characterized in that, The food products mentioned are nutritional supplements, functional foods, or health foods.

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