Synbiotic composition and application thereof in improvement of Alzheimer's disease

Abstract

The invention is applicable to the technical field of microbial additives, and provides a synbiotic composition and application thereof in improvement of Alzheimer's Disease, the synbiotic composition comprises inulin and Lactobacillus seiulus AF91-01CMCA, and the preservation number of the Lactobacillus seiulus AF91-01CMCA is GDMCCNO: 64744. The combined use of the two components of the synbiotics composition can significantly improve the disease degree of an Alzheimer's disease model mouse, and the effect is superior to that of application of single probiotics or single prebiotics. The cognitive impairment of a tested object is obviously relieved; the brain A beta accumulation of a tested object is obviously reduced; the ILA level in a tested object is obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial additives, and in particular relates to a synbiotic composition and an application thereof in improving Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease, the onset of which is accompanied by cognitive impairment, memory loss, and language disorders. According to data released by the National Health Commission in 2022, there are more than 10 million AD patients in my country, and the treatment and prevention of AD has become a major public health issue. At present, the main therapeutic drugs for AD are memantine, donepezil, galantamine, etc. These drugs will bring some side effects while taking effect, and affect the quality of life of patients to varying degrees. Therefore, it is necessary to find a more moderate treatment strategy.

[0003] Recent studies have found that the intestinal flora of AD patients is significantly different from that of normal people, and this difference further leads to different intestinal metabolites. Indole lactic acid (ILA) is a metabolite produced by the decomposition of tryptophan by intestinal flora. Recent studies have found that the serum ILA level of AD patients is significantly lower than that of healthy people, and some intervention methods may improve AD by increasing the level of ILA in the body. In addition, studies have shown that ILA has anti-inflammatory effects, immune regulation, and the function of protecting nerve cells and promoting neural development. Therefore, increasing the level of ILA in the body through specific intervention methods may be a new strategy for treating AD.

[0004] It is known that some probiotics of the genus Lactobacillus have the ability to produce ILA and play an important role in the prevention and treatment of some inflammatory diseases. However, no studies have yet proven the role of these probiotics with the ability to produce ILA in alleviating Alzheimer's disease. Therefore, screening out a probiotic with high ILA production and constructing a synbiotic to promote its growth and metabolism, and functionally evaluating the effect of this synbiotic combination in alleviating Alzheimer's disease will help develop new synbiotic products and provide new solutions for the prevention and treatment of Alzheimer's disease. Summary of the invention

[0005] The purpose of the present 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] The present invention is achieved by: a Lactobacillus suifolius ( Lacticaseibacillus suilingensis )AF91-01CMCA, deposited in Guangdong Provincial Microbiological Culture Collection Center on June 13, 2024, and its deposit number is GDMCCNO:64744.

[0007] Another object of the present invention is to provide a synbiotic composition, the synbiotic composition comprising inulin and the Lactobacillus suifolius AF91-01CMCA described in claim 1, wherein the dosage ratio of the Lactobacillus suifolius AF91-01CMCA to the inulin is (1×10 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 the Lactobacillus suifolius AF91-01CMCA described in claim 1, and the dosage ratio of the Lactobacillus suifolius AF91-01CMCA to inulin is (1×10 10 ~1×10 12 ) CFU / mL:(20~30) g / L.

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

[0010] Another object of the present invention is to provide an application of a synbiotic composition in the preparation of a drug, wherein the drug is used to prevent and improve Alzheimer's disease, and the synbiotic composition comprises inulin and Lactobacillus suifolius AF91-01CMCA. The Lactobacillus suifolius AF91-01CMCA was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2024, and its deposit number is 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 of the subject; 2) reducing Aβ accumulation in the subject's brain; 3) increasing the level of ILA in the subject's body.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing and improving Alzheimer's disease, wherein the pharmaceutical composition comprises a synbiotic composition, wherein the synbiotic composition comprises inulin and Lactobacillus suifolius AF91-01CMCA, wherein the dosage ratio of Lactobacillus suifolius AF91-01CMCA to inulin is (1×10 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 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 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, wherein the food composition comprises a synbiotic composition, wherein the synbiotic composition is prepared by adding food-acceptable excipients and additives, wherein the synbiotic composition comprises inulin and Lactobacillus suifolius AF91-01CMCA, wherein the dosage ratio of Lactobacillus suifolius AF91-01CMCA to inulin is (1×10 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, wherein the synbiotic composition is used to prevent and improve Alzheimer's disease; the synbiotic composition is used to prepare food, special medical purpose formula food, 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 preparation method comprising the following steps: S1. Use whole genome sequencing technology to obtain the genome information of Lactobacillus suifolius AF91-01CMCA, use bioinformatics analysis technology to predict the functions of all gene sequences, screen out all carbohydrate utilization-related genes, and predict substrate and carbohydrate transporter information; S2. Construct corresponding metabolic pathways based on substrate and transporter information and their location on the genome, and then conduct functional genomic analysis of carbohydrate utilization to complete the prediction of strain-specific prebiotics; S3. Validate the predicted information in vitro, using the predicted prebiotic as the only carbon source and a semi-synthetic medium without a carbon source as a control, and use the BioScreen growth curve analyzer to measure the growth curve of the strain in the raw culture medium with the corresponding prebiotic as the substrate, thereby determining the effect of the prebiotic; S4, inoculate the candidate strains in ordinary culture medium and culture medium supplemented with prebiotics, centrifuge and prepare samples after 48 h of fermentation, and use Shimadzu LC-16 high performance liquid chromatography to determine the ILA production of the strains in ordinary culture medium and corresponding prebiotic culture medium; S5. Determine the composition and proportion of the synbiotics, and obtain a synbiotic composition according to the proportions.

[0017] The beneficial effects of the present invention are as follows: the synbiotic composition includes Lactobacillus suifolius AF91-01CMCA and inulin. The Lactobacillus suifolius AF91-01CMCA is a newly discovered strain isolated from fecal samples of healthy Chinese people; the synbiotic composition is a newly discovered synbiotic combination. Studies have shown that the combined use of the two can significantly improve the disease severity of Alzheimer's disease model mice, and the effect is better than the administration of probiotics or prebiotics alone. Specifically, it significantly alleviates the cognitive impairment of the subjects; significantly reduces the accumulation of Aβ in the brain of the subjects; and significantly increases the level of ILA in the subjects.

[0018] The synbiotic composition in this application has been screened and confirmed by in vitro experiments, and its ratio is reasonable. Compared with Lactobacillus suifolius AF91-01CMCA alone, the addition of inulin can promote the growth of Lactobacillus suifolius AF91-01CMCA and significantly increase its ability to produce ILA. In addition, the addition of inulin can promote the colonization and growth of Lactobacillus suifolius AF91-01CMCA in vivo and enhance its efficacy.

[0019] The method for taking the synbiotic composition in the present application has a reasonable dosage and significant efficacy, which effectively avoids the ineffectiveness of existing probiotic preparations due to unclear dosage or the possible adverse effects caused by excessive intake. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the growth curves of Lactobacillus suifolius AF91-01CMCA in culture media with different carbon source concentrations, including (A) 10 g / L, (B) 20 g / L, (C) 30 g / L, and (D) 40 g / L.

[0021] Figure 2 This is the growth curve of Lactobacillus suifolius AF91-01CMCA with different concentrations of inulin added.

[0022] Figure 3 The ILA production level of Lactobacillus suifolius AF91-01CMCA in the culture medium supplemented with 20 g / L inulin.

[0023] Figure 4 This is a test on the effect of synbiotics on alleviating cognitive impairment, including (A) exploration time of the target quadrant of the Barnes maze, and (B) discrimination index of the Y maze.

[0024] Figure 5 This is an experiment on reducing brain Aβ accumulation with synbiotics, including (A) Aβ fluorescence staining results, (B) Aβ quantification results in the CA1 region, (C) Aβ quantification results in the DG region, and (D) Aβ quantification results in the Cortex region.

[0025] Figure 6This is an experiment provided by an embodiment of the present invention for improving the ILA level in vivo with synbiotics, wherein (A) the fecal ILA content and (B) the serum ILA content. DETAILED DESCRIPTION

[0026] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0027] In some specific implementation cases, the synbiotic composition includes probiotics and prebiotics, the probiotics are Lactobacillus suifolius AF91-01CMCA, the prebiotics are inulin, and the synbiotic composition contains a dosage of 2×10 10 CFU / d of probiotics and containing inulin at a dose of 0.02 g / mL.

[0028] The technical solution of the present invention is further described in detail below by embodiments and in conjunction with the accompanying drawings. However, the selected embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention.

[0029] Embodiment 1: Isolation and Identification of Lactobacillus suifolius AF91-01CMCA 1. Sample collection The separation sample comes from the feces of a healthy person. The feces is collected into a sterile sample tube and brought back to the laboratory for sorting within 1 hour.

[0030] 2. Separation and purification The collected fresh samples were immediately transferred to an anaerobic operating box. 0.2 g of the sample was taken into 1 ml of sterile PBS (phosphate buffer), shaken thoroughly to mix, and then gradient dilution was applied. The culture medium used a modified version of PYG culture medium. The specific formula was (1 L): 8 g of tryptone, 2 g of soy peptone, 1 g of polyprotein, 1 g of casein, 10 g of yeast powder, 5 g of beef extract, 5 g of glucose, 2 g of dipotassium hydrogen phosphate, 0.5 g of maltose, 0.5 g of cellobiose, 0.5 g of soluble starch, 0.25 g of sodium sulfide, 800.5 mL of Tween, and 100 g of Cysteine-HCl•H2O. 0.5g, glycerol 0.5mL, sodium acetate 5g, heme solution 10ml (each liter of heme solution contains 500mg of heme, sodium hydroxide solution (1M) 10ml), vitamin solution 10ml (each liter of vitamin solution contains 2mg of biotin, 10mg of pyridoxine hydrochloride, 5mg of thiamine hydrochloride dihydrate, 5mg of riboflavin, 5mg of niacin, 5mg of dextro-calcium pantothenate, 0.1mg of vitamin B12, 5mg of p-aminobenzoic acid, 5mg of lipoic acid), inorganic salt solution 40mL (each liter of inorganic salt solution contains 0.25g of calcium chloride dihydrate, 0.5g of magnesium sulfate heptahydrate, phosphoric acid 1g potassium dihydrogen, 1g potassium dihydrogen phosphate, 10g sodium bicarbonate, 2g sodium chloride), 10ml trace elements (each liter of trace elements contains 1.5g nitrilotriacetic acid, 3g magnesium sulfate heptahydrate, 1g sodium chloride, 0.1g iron sulfate heptahydrate, 0.18g cobalt sulfate heptahydrate, 0.1g calcium chloride dihydrate, 0.18g zinc sulfate heptahydrate, 0.01g copper sulfate pentahydrate, 0.02g potassium aluminum sulfate dodecahydrate, 0.01g boric acid, 0.01g sodium molybdate, 0.03g nickel chloride hexahydrate, 0.3mg sodium selenite pentahydrate), 1ml resazurin, add distilled water to 1L, and adjust the pH to 6.8~7.0. The coated plate was placed at 37℃ for anaerobic culture, and the anaerobic gas composition was N2:CO2:H2=90:5:5. After 3 days of culture, single colonies were picked for streaking separation to obtain pure culture strains of each single bacteria.

[0031] 3. Strain preservation The obtained pure culture strain was cultured to a concentration of about 10 9 cfu / mL, take 400μL of bacterial solution and add 400μL of 40% glycerol to make the glycerol concentration reach 20%, and then store it at -80℃.

[0032] Prepare the strain vacuum freeze-dried powder according to the following steps. Sterilize the ampoule tube and protective agent by high pressure and set aside for use. Streak the overnight cultured bacterial solution and culture it at 37℃ for 24 hours. After observing that no bacteria contamination is found, proceed with the following operations. Collect the bacterial solution by centrifugation and wash it with sterile saline. Add 2-3mL skim milk protective agent and suspend it to make a colony count of 10 8 ~10 10pcs / mL bacterial suspension, and dispense into sterile ampoule tubes, and pre-freeze in a -80 ℃ refrigerator for 1 to 2 hours. According to the "Standard Operating Procedures for Freeze Dryers", freeze-dry in a freeze dryer for 8 to 20 hours until freeze-dried. After freeze-drying, take out the sample ampoule tube, and according to the "Standard Operating Procedures for Vacuum Ampoule Sealing Machines", use a strong flame to pull the part below the cotton plug on the neck of the ampoule tube to seal it.

[0033] 4. 16S rDNA identification After the isolated strain was cultured in liquid PYG medium for 24 hours, PCR amplification was performed using 16S rDNA universal primers. The amplification system was: 10×PCR buffer 2.5μL, dNTP (2.5mM) 2μL, 27F (5'-AGAGTTTGATCATGGCTCAG-3') 0.5μL, 1492R (5'-TAGGGTTACCTTGTTACGACTT-3') 0.5μL, Taq enzyme 0.3μL, template 1μL, ddH2O 18.2μL. PCR amplification conditions were: 94℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 60℃ annealing for 40s, 72℃ extension for 1min30s, 10 cycles; 95℃ denaturation for 30s, 56℃ annealing for 40s, 72℃ extension for 1min30s, 25 cycles; 72℃ extension for 10min, and storage at 4℃. The obtained 16S rDNA amplification product was subjected to electrophoresis detection, purification, and 3730 sequencing to obtain a 16S rDNA sequence with a length of 1400 bp.The sequencing results were checked using SeqScanner v1.0, and sequences with qualified sequencing quality were selected for online sequence alignment in EZBioCloud (https: / / www.ezbiocloud.net / identify). The sequences are as follows: The identification results showed that the strain was most closely related to Lacticaseibacillus baoqingensis, with a similarity of 98.7%, indicating that the strain was a new species of the genus Ligilactobacillus.

[0034] Example 2 Screening of Carbon Sources for Lactobacillus suifolius AF91-01CMCA 1. Materials and Methods 1.1 Experimental Materials Bacterial strain: Lactobacillus suifolius AF91-01CMCA. Main reagents: GAM medium, inulin, ILA standard, chromatographic methanol, sodium dihydrogen phosphate, glacial acetic acid.

[0035] 1.2 Experimental methods The whole genome sequencing of Lactobacillus suifolius AF91-01CMCA was performed, and the sequencing results were compared and annotated using the CAZy carbohydrate enzyme database to obtain the gene information related to the carbon source metabolism of Lactobacillus suifolius AF91-01CMCA, and the substrate and carbohydrate transporter information were further predicted. The corresponding metabolic pathways were constructed based on the substrate and transporter information and their location on the genome, and then functional genomic analysis of carbohydrate utilization was performed to complete the prediction of specific prebiotics of Lactobacillus suifolius AF91-01CMCA.

[0036] Secondly, the prediction results were verified in vitro. Using a semi-synthetic medium (GAM) without a carbon source as a control, Lactobacillus suifolius AF91-01CMCA was inoculated into a medium containing different prebiotics and cultured for 48 h. The OD of the culture medium was measured every 1 h using a BioScreen growth curve analyzer. 600 nm absorbance value, and a growth curve was drawn according to the measured absorbance value to determine the effect of prebiotics on the growth of Lactobacillus suifolius AF91-01CMCA. The amount of prebiotics added to the culture medium was set at different concentrations, namely 10 g / L, 20 g / L, 30 g / L, and 40 g / L.

[0037] Finally, Lactobacillus suifolius AF91-01CMCA was inoculated in GAM medium and GAM+20 g / L inulin medium. After fermentation for 48 h, the samples were centrifuged and the ILA production of the strain in ordinary and corresponding prebiotic medium was determined using Shimadzu LC-16 high performance liquid chromatography analyzer.

[0038] The composition of the synbiotic was determined based on the growth curve results and the ILA production results of the fermentation.

[0039] Test results 1. Prediction of carbon source of Lactobacillus suifolius AF91-01CMCA The comparison results of CAZy carbohydrate enzyme database are shown in Table 1. The analysis results showed that Lactobacillus suifolius AF91-01CMCA has inulinase, β-mannosidase, β-xylanase, and α-arabinosidase, which are related to the hydrolysis of inulin, oligoxylose, and oligomannose, so it is speculated that it has the ability to utilize these carbon sources.

[0040] Table 1 Carbon source metabolizing enzymes possessed by Lactobacillus suifolius AF91-01CMCA 2. Growth curve analysis results Depend on Figure 1It can be seen that among the three carbon source supplemented culture media, inulin has the best growth-promoting effect on Lactobacillus suifolius AF91-01CMCA, and at different carbon source supplementation concentrations, inulin has the best growth-promoting effect. Figure 2 It can be seen that with the increase of inulin concentration, the growth of Lactobacillus suifolius AF91-01CMCA became better, but the inulin concentrations of 20 g / L, 30 g / L, and 40 g / L had similar growth-promoting effects on the strain.

[0041] 3. ILA level produced by fermentation Depend on Figure 3 It can be seen that compared with GAM medium, the ILA production of Lactobacillus suifolius AF91-01CMCA in GAM medium supplemented with 20 g / L inulin was significantly increased, reaching about 25 μg / mL.

[0042] In summary, based on the growth curve and liquid phase results, the synbiotic combination was determined to be Lactobacillus suifolius AF91-01CMCA and inulin.

[0043] Example 3 Biobiotics alleviate cognitive impairment in Alzheimer's disease The present invention uses animal experiments to evaluate the efficacy of the synbiotic composition of the present invention to illustrate the effectiveness of the synbiotic composition.

[0044] 1. Experimental Animal Grouping The experiment selected 35 7-month-old 5×FAD mice (AD model mice) and 10 C57 mice (Con normal control mice) and divided them into 5 groups, control group (Con), Alzheimer's disease model group (AD), model supplemented with prebiotics group (AI), model supplemented with probiotics group (AL), model supplemented with synbiotics group (AS), with 8-10 mice in each group. The mice were intervened with control, probiotics, prebiotics, and synbiotics for 4 weeks.

[0045] 2. Processing methods for each group Control treatment: normal drinking water, and 100 μL of PBS supplemented by gavage every day.

[0046] Prebiotic treatment: Dissolve inulin in sterile water, adjust to a concentration of 20g / L, and supplement with drinking water.

[0047] Probiotic treatment: The cultured Lactobacillus suifolius AF91-01CMCA was suspended in an appropriate amount of PBS until the viable count reached 1×10 11 CFU / mL, freshly prepared and used. 200μL of bacterial suspension was supplemented by intragastric administration every day.

[0048] Synbiotic treatment: daily intragastric administration of bacterial suspension and drinking water supplementation of prebiotics.

[0049] 3. Animal behavior testing methods After 4 weeks of feeding, behavioral analysis was performed to evaluate the cognitive ability of the mice.

[0050] 3.1 New Object Recognition Test The 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, the mice were placed in an open cubic box (length × width × height; 40 cm × 40 cm × 40 cm), and the mice were allowed to explore freely for 5 minutes. On the training day, two identical objects were placed at the upper left and lower right of the box, and the mice were placed in the center of the box and allowed to explore freely for 5 minutes. On the test day, one of the objects on the training day was replaced with a new object (the new object was different in shape and color from the old object), and the mice were allowed to explore freely for 5 minutes. During this period, the software was used to record the activity trajectory of the mice and their sniffing time for the new and old objects. Discrimination index calculation: (new object exploration time - old object exploration time) / (new object exploration time + old object exploration time) × 100% 3.2 Barnes maze test One day before the experiment, the animals were placed in the target box from the target hole for 4 min. The animals were placed in a plastic cylinder (20 cm*27 cm) in the center of the maze to restrict their activities for 5 s. The cylinder was removed and the software was started. If all four limbs of the animal entered the target box, it was counted as an escape, and the animal was allowed to stay in the box for 30 s. Each animal was observed for a maximum of 4 min at a time. If the animal still could not find the target box during this period, the animal was removed from the maze and placed in the target box for 30 s. The maze was cleaned during this interval. The animals were trained once a day for 4 consecutive days. Starting from the second training, the maze was rotated one to several holes before each training, but the target box was always fixed in the same direction. The purpose of this was to prevent the animals from relying on smell and memory to determine the location of 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 s. The number of times any hole was explored, the time spent near the target box, and the time when the target box was first found were recorded.

[0051] Test results Depend on Figure 4 It can be seen that compared with WT mice, the cognitive ability of AD mice was significantly reduced, as shown by the reduction of exploration time of the target quadrant and the reduction of the discrimination index. After 4 weeks of synbiotic supplementation, the exploration time of AD mice in the target quadrant increased significantly, and the discrimination index in the new object test was also significantly improved, and the effect was better than that of probiotic or prebiotic supplementation alone. The above results show that synbiotics can alleviate cognitive impairment in Alzheimer's mice.

[0052] Example 4 Synbiotics reduce Aβ accumulation in the brain 1. The experimental grouping and treatment were the same as in Example 2.

[0053] 2. Experimental Methods The mice were killed after 4 weeks of intervention, and their brain tissues were collected and fixed in 4% paraformaldehyde. They were then embedded, sectioned, dewaxed, permeabilized, antigen repaired, inactivated endogenous enzyme activity, blocked, incubated with primary antibodies, incubated with secondary antibodies, and mounted with DAPI, and photographed under a fluorescence microscope to observe the accumulation of Aβ in the brain.

[0054] Test results Excessive accumulation of Aβ is one of the pathological features of Alzheimer's disease. Figure 5 It can be seen that there is a large amount of Aβ accumulation in the CA1, DG, and Cortex areas of the brain of AD mice, and 4 weeks of synbiotic intervention can significantly reduce brain Aβ deposition, and the effect is better than that of probiotic or prebiotic supplementation alone.

[0055] Example 5 Synbiotics increases ILA levels in the body 1. The experimental grouping and treatment were the same as in Example 2.

[0056] 2. Experimental Methods After 4 weeks of intervention, the mice were fed feces and then killed to collect serum. The ILA levels in feces and serum were detected by high performance liquid chromatography. The specific method is as follows: Fecal sample preparation: Weigh 0.10 g of mouse feces, add 1 mL of methanol, vortex mix, and water bath at 40°C for 20 minutes, mixing once every 5 minutes. After the water bath, place it in a -20°C refrigerator for 20 minutes, take it out and centrifuge it at 13000r / min at 4°C for 10 minutes. Take the supernatant after centrifugation and filter it through a 0.22 µm microporous filter membrane (organic phase), add it to a brown vial, and place it in a 4°C refrigerator as the sample to be tested.

[0057] Serum sample preparation: 30 μL of serum was diluted to 300 μL with methanol, and then placed at -20°C for 20 min. The sample was then centrifuged at 4°C, 13,000 r / min for 10 min. The supernatant was filtered through a 0.22 μm organic filter membrane, added to a brown bottle, and placed in a 4°C refrigerator for testing.

[0058] Liquid phase detection: ZORBAX XDB C18 chromatographic 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°C, detection wavelength fluorescence detector (excitation wavelength / emission wavelength): 282 / 352 nm, injection volume: 10 μL.

[0059] Test results Depend on Figure 6 It can be seen that after 4 weeks of intervention, synbiotic supplementation significantly increased the ILA levels in the feces and serum of mice compared with the AD group.

[0060] Data and statistical analysis All experimental data were analyzed using the Duncan method of one-way ANOVA in SPSS 26.0 software (P < 0.05), and graphed using Graphpad 8.0. The experimental results were expressed as mean ± standard deviation, and each experiment was repeated three times.

[0061] Example 6 Preparation of food composition containing Lactobacillus suis AF91-01CMCA Application Example 1: Preparation of synbiotic microcapsules containing Lactobacillus suifolius AF91-01CMCA and inulin S1: Preparation of synbiotic suspension. Lactobacillus suifolius AF91-01CMCA was inoculated with 5% inoculum into 20 mL sterilized MRS medium for activation culture and cultured at 37°C for 24 h. Subsequently, the activated Lactobacillus suifolius AF91-01CMCA was inoculated with 5% inoculum into 20 mL sterilized MRS medium and cultured at 37°C for 48 h. The cultured bacterial solution was centrifuged at 4000 r / min for 10 min at 4°C, the supernatant was discarded, and the bacterial sludge precipitate was rinsed three times with sterile PBS, and then the bacterial sludge was dissolved in 2 mL of sterile 50 g / L inulin solution to obtain a synbiotic suspension.

[0062] S2: Preparation of synbiotic microcapsules. The synbiotic suspension was mixed with 4% (w / v) pectin solution at a ratio of 1:4. After thorough mixing, the mixture was slowly dripped into 400 mmol / L sterile CaCl2 solution by extrusion to obtain synbiotic microcapsules.

[0063] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Lactobacillus suifolius AF91-01CMCA, characterized in that It was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 13, 2024, and its deposit number is GDMCC NO:64744.

2. A synbiotic composition, characterized in that: The synbiotic composition comprises inulin and the Lactobacillus suifolius AF91-01CMCA described in claim 1, wherein the dosage ratio of the Lactobacillus suifolius AF91-01CMCA to inulin is (1×10 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 the Lactobacillus suifolius AF91-01CMCA described in claim 1, wherein the dosage ratio of the Lactobacillus suifolius AF91-01CMCA to inulin is (1×10 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 suifolius AF91-01CMCA is 1×10 11 CFU / mL, and the concentration of inulin is 20g / L.

5. The use of the synbiotic composition in the preparation of a medicine, characterized in that: The drug is used to prevent and improve Alzheimer's disease. The synbiotic composition includes inulin and Lactobacillus suifolius AF91-01CMCA. The Lactobacillus suifolius AF91-01CMCA was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2024, and its preservation number is GDMCC NO: 64744.

6. The use according to claim 5, characterized in that: The drug has one or more of the following functions: 1) alleviating cognitive impairment of the subject; 2) reducing Aβ accumulation in the subject's brain; 3) increasing the level of ILA in the subject's body.

7. A pharmaceutical composition for preventing and improving Alzheimer's disease, characterized in that: The pharmaceutical composition comprises a synbiotic composition, wherein the synbiotic composition comprises inulin and Lactobacillus suifolius AF91-01CMCA, and the dosage ratio of the synbiotic composition to inulin is (1×10 9 ~1×10 13 ) CFU / mL:(10~40) g / L or (1×10 10 ~1×10 12 ) CFU / mL:(20~30) g / L.

8. The pharmaceutical composition according to claim 7, 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 powder, tablet, granule, capsule, solution, suspension, emulsion and lyophilized preparation.

9. A food composition for preventing and improving Alzheimer's disease, characterized in that: The food composition comprises a synbiotic composition, wherein the synbiotic composition is prepared by adding food-acceptable auxiliary materials and additives, wherein the synbiotic composition comprises inulin and Lactobacillus suifolius AF91-01CMCA, wherein the dosage ratio of Lactobacillus suifolius AF91-01CMCA to inulin is (1×10 9 ~1×10 13 ) CFU / mL:(10~40) g / L or (1×10 10 ~1×10 12 ) CFU / mL:(20~30) g / L.

10. Use of a synbiotic composition, characterized in that: The synbiotic composition is used for preventing and improving Alzheimer's disease; the synbiotic composition is used for preparing food, special medical purpose formula food, nutritional supplement, functional food, health food and medicine.

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