Lactobacillus mucosus fermentation FM1 and use thereof

Abstract

The present application belongs to the field of microbial engineering, and provides a fermented Lactobacillus mui FM1 with life prolonging and anti-dementia probiotic functions and application thereof. The strain FM1 has good gastrointestinal environment tolerance. In vivo nematode model experiments show that the strain can prolong the life of wild type and dementia mutant nematodes, improve the reduced motor ability and cognitive memory ability caused by dementia tau protein, and significantly improve the down-regulation of tau protein phosphorylation and expression of related dementia related genes, and enhance the expression of immune defense and protein misfolding repair genes. The significant anti-aging and dementia activity of FM1 makes it have wide application value in various forms of probiotic food (such as probiotic solid beverage, etc.).

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering, specifically, it relates to a fermenting Lactobacillus mucinus FM1 with probiotic functions such as life extension and anti-dementia, and its application. Background Technology

[0002] Alzheimer's disease (AD) is currently the most widespread and prevalent dementia worldwide. Previous studies have shown that the gut microbiota of AD patients differs significantly from that of healthy individuals of the same age, and that the gut microbiota plays a crucial role in maintaining neurotransmitter secretion and brain activity. Research indicates that probiotics can regulate gut microbiota balance and prevent and alleviate various chronic diseases. Currently, probiotics mainly include two categories: Lactobacillus and Bifidobacterium, which are widely used in the food industry.

[0003] As a Gram-positive, non-spore-forming bacillus, lactobacillus is widely distributed in nature and can be isolated from the human gut, plant surfaces, dairy or fermented dairy products, meat products, and fermented dough.

[0004] Numerous studies have shown that Lactobacillus, as a probiotic, plays a vital role in maintaining intestinal microecological balance, enhancing the body's immune function, anti-oxidation, anti-inflammation, and preventing diarrhea. It is one of the essential physiological flora for maintaining bodily health and has been widely applied in the pharmaceutical, food, and feed industries. In the past decade, several different drugs have been reported for the treatment of Alzheimer's disease (AD); however, almost all compounds failed in clinical trials, with only a very few slowing disease progression. Furthermore, these drugs suffer from significant side effects, unstable efficacy, and limitations in the target population. In recent years, the gut-brain axis has become an important potential target for improving brain nutrition and metabolic activity. Therefore, expanding the isolation of Lactobacillus strains from the intestines of long-lived, healthy elderly individuals, selecting Lactobacillus strains with high viable counts, good safety profiles, and certain probiotic functions, and developing anti-aging and longevity-promoting probiotic foods will be of great significance to an aging society. Summary of the Invention

[0005] The purpose of this invention is to provide a fermentable Lactobacillus mucinus FM1 with probiotic functions such as life extension and anti-dementia, and its applications.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a strain FM1 isolated from the intestinal feces of healthy, long-lived elderly people in China, classified and named *Lactobacillus fermentum*. Limosilactobacillus fermentum The strain is now deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 35034, deposit date June 27, 2025.

[0007] Strain FM1 possesses the excellent characteristics of probiotics, including the ability to extend the lifespan of nematodes, reduce the level of the dementia protein Tau and its phosphorylation level, improve the decline in motor ability caused by Tau overexpression, and alter host cognitive and memory abilities. FM1's significant anti-aging and dementia activity makes it widely applicable in various forms of probiotic foods (such as probiotic solid beverages).

[0008] Secondly, the present invention provides a probiotic powder prepared from the fermented Lactobacillus FM1 after cultivation; wherein the viable count of the fermented Lactobacillus FM1 in the powder is ≥4.0 × 10⁻⁶. 11 CFU / g.

[0009] Thirdly, the present invention provides the use of the fermented Lactobacillus mucinus FM1 or the probiotic powder in the preparation of products for delaying aging, prolonging healthy lifespan, or preventing and / or improving dementia-related symptoms.

[0010] Furthermore, the dementia-related condition is Alzheimer's disease.

[0011] Furthermore, the improvement of dementia-related symptoms includes, but is not limited to: improving cognitive and memory abilities, improving motor abilities, reducing the levels of Tau protein and / or phosphorylated Tau protein (P-Tau), and / or upregulating the expression of the xbp-1 gene.

[0012] Fourthly, the present invention provides the application of the fermented Lactobacillus mucin FM1 or the probiotic powder in the preparation of probiotic foods or health foods.

[0013] Furthermore, the probiotic food is a solid beverage, compressed candy, or fermented product.

[0014] Fifthly, the present invention provides a probiotic solid beverage comprising the probiotic powder and food-grade excipients. The excipients may be selected from one or more of the following: natural fruit powder, sweeteners, dietary fiber, and edible flavorings.

[0015] Furthermore, the sweetener is maltitol and / or erythritol.

[0016] Furthermore, the dietary fiber is resistant dextrin.

[0017] Furthermore, the viable count of *Lactobacillus mucinus* FM1 in the solid beverage is ≥2.0 × 10⁻⁶. 9 CFU / g.

[0018] Furthermore, the solid beverage is prepared from raw materials comprising the following mass percentages: 3%-6% probiotic powder, 22%-36% natural fruit powder, 30%-60% maltitol and / or erythritol, 5%-8% resistant dextrin, and 0.2%-0.4% edible flavoring.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: The *Lactobacillus FM1* fermentation strain provided by this invention exhibits good tolerance to the gastrointestinal environment. In vivo nematode model experiments show that this bacterium can prolong the lifespan of wild-type and dementia mutant nematodes, and improve the decline in motor ability and cognitive memory caused by dementia-related Tau protein. It can also significantly improve Tau protein phosphorylation, downregulate the expression of related dementia-related genes, and enhance the body's immune defense and the expression of protein misfolding repair genes.

[0020] The fermented Lactobacillus mucilaginosus FM1 of the present invention can obtain ultra-high cell concentration bacterial powder using the above-described fermentation culture method, with a colony forming unit ≥ 4.0 × 10⁻⁶. 11 CFU / g. The fermented Lactobacillus mucilaginosus powder provided by this invention can be used to prepare probiotic solid beverages with a good taste. Attached Figure Description

[0021] Figure 1 This is a colony morphology diagram of *Lactobacillus fermentans* FM1 on an MRS plate, representing a preferred embodiment of the present invention.

[0022] Figure 2 This is a Gram staining image of fermenting Lactobacillus FM1 in a preferred embodiment of the present invention.

[0023] Figure 3 This invention provides a preferred embodiment of the in vitro detection and analysis of artificial gastric juice resistance in fermenting Lactobacillus mucinus FM1.

[0024] Figure 4 This invention provides a preferred embodiment of the in vitro detection and analysis of artificial intestinal fluid resistance of fermenting Lactobacillus mucinus FM1.

[0025] Figure 5 This is a graph showing the change in lifespan of wild-type nematodes due to intervention with fermented Lactobacillus mucin FM1 in a preferred embodiment of the present invention.

[0026] Figure 6 The graph shows the change in lifespan of VH254 nematodes due to intervention with fermented Lactobacillus mucin FM1 in a preferred embodiment of the present invention.

[0027] Figure 7 In a preferred embodiment of the present invention, the use of fermented Lactobacillus mucin FM1 intervention improves the head-shaking rate of VH254 nematodes.

[0028] Figure 8In a preferred embodiment of the present invention, fermentation of Lactobacillus mucin FM1 was used to improve the short-term memory ability of VH254 nematodes.

[0029] Figure 9 In a preferred embodiment of the present invention, fermentation of Lactobacillus mucilaginosus FM1 intervenes to regulate the expression of phosphate transport-related genes.

[0030] Figure 10 In a preferred embodiment of the present invention, fermentation of Lactobacillus mucilaginosus FM1 intervenes in the regulation of chloride ion-related gene expression.

[0031] Figure 11 In a preferred embodiment of the present invention, fermented Lactobacillus mucin FM1 intervenes in the expression of AD-related genes.

[0032] Figure 12 In a preferred embodiment of the present invention, the fermentation of Lactobacillus mucin FM1 reduces the accumulation of Tau and P-Tau proteins in VH254 nematodes. Detailed Implementation

[0033] This invention provides the isolation, identification, activity detection, functional verification, preparation of mycelial powder, and application of *Lactobacillus mucinus* FM1, which has probiotic functions such as prolonging life and reducing dementia, in probiotic solid beverages, compressed candies, and fermented products.

[0034] The present invention adopts the following technical solution: This invention provides a self-screened strain of fermenting *Lactobacillus mucinus* FM1 (… Limosilactobacillus fermentum FM1 was isolated and screened from the intestinal feces of healthy, long-lived elderly people in China, and was classified and named accordingly. Limosilactobacillus fermentum It is deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 35034, deposited on June 27, 2025.

[0035] The *Lactobacillus fermentans* strain of this invention exhibits good anaerobic growth when a single colony is inoculated onto MRS solid medium at 37°C. The colonies are round, 1.5 mm-2.5 mm in diameter, relatively smooth, and milky white. They are Gram-positive, with straight rod-shaped cells, blunt ends, and can exist singly or in pairs, without spores. The FM1 strain demonstrates high resistance to gastric acid in simulated gastric acid experiments. Identification using the 16S rDNA sequence (SEQ ID NO:1) confirms that FM1 is *Lactobacillus fermentans*.

[0036] The fermented *Lactobacillus mucinus* strain of the present invention has good gastric acid tolerance and a high survival rate in a simulated gastric acid environment; the strain has a strong effect on resisting dementia and prolonging life in vivo.

[0037] The *Lactobacillus* strain of this invention significantly increases the lifespan, head-shaking rate, and memory and cognitive abilities of *E. spp.* VH254, a nematode overexpressing tau protein, while reducing the accumulation of tau and P-tau proteins, both associated with Alzheimer's disease. Furthermore, FM1 accelerates the degradation of tau protein accumulated in the endoplasmic reticulum (ER) due to misfolding by increasing the mRNA expression of the xbp-1 gene. It also possesses beneficial in vivo functions such as improving immune defense and fatty acid synthesis gene expression, enhancing phosphate transport, and reducing tau phosphorylation.

[0038] This invention also provides a fermented *Lactobacillus mucinus* FM1 bacterial powder with anti-aging, longevity-promoting, and anti-dementia properties. The bacterial powder is prepared by freeze-drying the fermentation product of *Lactobacillus mucinus* FM1 (vacuum degree 0.08 MPa, cold hydrazine temperature -60 ℃). The bacterial powder has an extremely high cell concentration, with colony-forming units ≥ 4.0 × 10⁻⁶. 11 CFU / g.

[0039] The above-mentioned application of *Lactobacillus fermentatus* FM1 to prepare *Lactobacillus fermentatus* powder involves the following steps: Under aseptic conditions, *Lactobacillus fermentatus* FM1 is streaked onto MRS plates for isolation, inoculated into culture medium, and anaerobically cultured at 37°C for 16 h to obtain a high-concentration cell culture medium. After centrifugation, the culture medium is mixed evenly with skim milk preservation solution (skim milk: 10%, w / w; sodium glutamate 0.1%, w / w) in a certain proportion, and then freeze-dried to obtain *Lactobacillus fermentatus* FM1 powder.

[0040] This invention provides a method for preparing a probiotic solid beverage, which involves using one or more of the following raw materials: fermented Lactobacillus FM1 mixed with natural fruit powder, erythritol, resistant dextrin, and edible flavoring, and then weighing, mixing, and packaging the ingredients.

[0041] The fermented Lactobacillus mucinus strain, the preparation method of fermented Lactobacillus mucinus powder, and the preparation method of probiotic solid beverage are all within the protection scope of this invention.

[0042] The fermented *Lactobacillus mucinus* FM1 of this invention possesses the excellent characteristics of probiotics, including the ability to prolong the lifespan of nematodes, reduce the level of the dementia protein Tau and its phosphorylation level, improve the decreased motility caused by Tau overexpression, and alter host cognitive and memory abilities. The fermented *Lactobacillus mucinus* FM1 of this invention can be used to obtain ultra-high cell concentration bacterial powder using the above-mentioned fermentation culture method, with a colony forming unit ≥ 4.0 × 10⁻⁶. 11 CFU / g. The fermented Lactobacillus mucilaginosus powder provided by this invention can be used to prepare probiotic solid beverages with a good taste.

[0043] This invention also provides the application of the *Lactobacillus fermentum* FM1 bacterial powder in the preparation of probiotic solid beverages. The probiotic solid beverages comprise the following components by weight percentage: 3%-6% *Lactobacillus fermentum* FM1 freeze-dried bacterial powder, 22%-36% natural fruit powder, 30%-60% maltitol, 5%-8% resistant dextrin, and 0.2%-0.4% edible flavoring. The probiotic solid beverages are made from the above raw materials through weighing, mixing, and packaging processes. The viable probiotic count of the probiotic solid beverages is ≥2.0 × 10⁻⁶. 9 CFU / g.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0045] Example 1: Screening, Identification and Preservation of Strains 1. Sample collection: Freshly collected feces from healthy, long-lived elderly people (90 years old) in the Beijing area of ​​my country were immediately used for the next step of plate dilution and coating.

[0046] 2. Isolation and purification of the strain: 200 mg of the collected sample was added to 1.8 mL of sterile physiological saline and mixed thoroughly. 200 μL of the suspension was then added to 1.8 mL of sterile physiological saline, and the mixture was diluted tenfold. The resulting concentrations were then determined by repeated dilutions. -5 10 -6 and 10 -7 Spread 0.1 mL of each bacterial culture onto MRS plates, with three replicates for each dilution. Incubate anaerobically at 37°C for 20 h, and then streak a single colony for anaerobic incubation.

[0047] 3. Strain identification: Single colonies were selected and cultured in liquid for three generations. The strain DNA was extracted using a bacterial genomic DNA extraction kit, and 16S rDNA was amplified by PCR technology. Sequencing analysis was then performed to identify the species of the strain.

[0048] 4. Observation of strain morphology: After Gram staining, the selected strains are observed under a microscope. If the bacteria turn blue after staining, they are Gram-positive strains, and if the bacteria turn red, they are Gram-negative strains.

[0049] Experimental Results: Acid-resistant microbial strains were screened from the feces of healthy, long-lived elderly people in Beijing, my country. Through 16S rDNA sequencing analysis and morphological observation, strain FM1 was identified as *Lactobacillus fermentatus*, and its 16S rDNA sequence (SEQ ID NO: 1, n is a, t, g, or c) is similar to previously reported *Lactobacillus fermentatus* strains (…). Limosilactobacillus Fermentum strain CAU:237The sequence similarity was 99.9%. Single colonies of this bacterium, when inoculated onto MRS solid medium, grew well anaerobically at 37°C. The colonies were round, 0.5 mm–2.0 mm in diameter, relatively smooth, and milky white. Figure 1 Gram-positive, the bacteria are relatively straight or slightly curved, with blunt ends, and exist singly or in pairs, without spores. Figure 2 This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35034.

[0050] Example 2: Gastrointestinal Tolerance Test of Fermented Lactobacillus mucin FM1 Artificial gastrointestinal fluid tolerance test: FM1 bacteria were activated for 3 generations in 50mL centrifuge tubes. 800μL of FM1 bacterial culture was added to 40mL MRS broth medium, and the culture was incubated at 20℃ for 20h as one generation. The activated bacterial culture was then centrifuged at 4500r for 10min, and the bacterial sludge was collected. The sludge was then washed twice with 5mL PBS (8g / L NaCl, 0.2g / L KCl, 1.44g / L Na2HPO4, 0.24g / L KH2PO4, pH=7.4), vortexed, or mixed with a 5mL pipette tip. The supernatant was discarded, and the stock solution (bacterial content approximately 10) was obtained. 10 CFU / mL); 1 mL of the stock solution was inoculated into 4 mL of artificial gastric fluid (0.3% pepsin, 0.5% sodium chloride, pH=2.5), mixed thoroughly, and then incubated at 37℃. At 0h, 1h, 2h, and 3h, 100 μL of the bacterial solution was spread evenly on 90 mm MRS solid medium and incubated at 37℃ for 20h, after which the viable count was recorded. After 3h of incubation in artificial gastric fluid, 1 mL of the bacterial solution was inoculated into 4 mL of artificial intestinal fluid (0.1% trypsin, 1.8% bile salts, pH=8.0), mixed thoroughly, and then incubated at 37℃. At 0h, 3h, 6h, 12h, and 24h, 100 μL of the sample was spread on 90 mm MRS solid medium and incubated at 37℃ for 20h, after which the viable count was recorded. The survival rate was calculated using the following formula: C = (log N1) / (log N0) × 100% in C N1 represents the survival rate (%); N1 represents the number of viable bacteria at the corresponding time point (colony forming units CFU / mL); N0 represents the number of viable bacteria at 0h (CFU / mL).

[0051] Experimental results are as follows Figures 3-4As shown. *Lactobacillus plantarum* P8 (reference 1, this strain was kindly provided by Professor Wang Jicheng of Inner Mongolia Agricultural University), which has good resistance to gastrointestinal fluid transport, was selected as a positive control. P8 and FM1 were cultured in artificially synthesized gastrointestinal fluid in vitro. Survival was observed, and compared with the positive control, the survival rate of FM1 strain in the artificial gastric fluid was 99% after 3 hours. Figure 3 This indicates that FM1 has good acid resistance. The survival rate dropped to a minimum of 89% after 24 hours in artificial intestinal fluid. Figure 4 This indicates that the vast majority of FM1 can survive in artificial intestinal fluid. It shows that Lactobacillus FM1 maintains a high survival rate in both strongly acidic and slightly alkaline intestinal fluids. As a probiotic, it can survive in large quantities not only in the stomach and the early small intestine, but also in the later part of the intestine, thus fully exerting its probiotic effects throughout the digestive tract. Figures 3-4 ).

[0052] Example 3: Functional Analysis of Fermented Lactobacillus FM1 in Extending the Lifespan of Nematodes Nematode strain: Wild-type nematode: Caenorhabditis elegans N2.

[0053] VH254 mutant nematode: Transgenic Caenorhabditis elegans VH254[F25B3.3::Tau352(PHP) +pha-1 (+)].

[0054] Reference 2 for N2 and VH254 of the nematode, from the Caenorhabditis Genetics Center (CGC) of the University of Minnesota.

[0055] Nematode culture: *C. elegans* is grown on a substrate coated with fresh... E. coli OP50 live bacteria are grown on NGM solid medium in a 20°C constant temperature incubator. When the bacterial solution is almost completely consumed, 5-8 L4 stage nematodes are picked up with a platinum wire and placed in a 60mm NGM culture dish coated with fresh bacterial solution.

[0056] Synchronization treatment of nematodes: First, nematodes are grown in 60mm petri dishes, ensuring approximately 200 nematodes per dish. Eggs within the nematodes should be clearly visible under a 4× microscope, and a high success rate is achieved when more than half of the nematodes are present during the oviposition period. Second, nematodes are collected by slowly adding 2mL of M9 buffer from the edge of the petri dish to the plate, then transferred to a 2mL sterile EP tube and centrifuged at 3500rpm for 1min, discarding the supernatant. Next, 1mL of M9 buffer is added to resuspend the nematodes, washing away any residual bacterial culture. The mixture is centrifuged at 3500rpm for 1min, and the M9 buffer is discarded. This process is repeated three times. Finally, 600μL of M9 buffer, 150μL of 5% w / v sodium hypochlorite, and 150μL of [other ingredients] are added. Lyse the nematodes with 5M sodium hydroxide, invert and mix for 10 min, centrifuge at 5000 r for 1 min, discard the supernatant and keep the precipitate (about 20 μL); finally, place the precipitate in a 60 mm petri dish coated with fresh OP50 live bacteria and incubate at 20℃ for 48 h. The hatched nematodes are the nematodes at the same developmental stage.

[0057] One hundred and twenty L4-stage VH254 mutant nematodes, after synchronization treatment, were transferred to NGM medium coated with fresh OP50 and FM1 live bacteria (bacterial content approximately 10). 9 CFU / mL), 20 nematodes per plate, 6 plates per group, OP50 as the control group, FM1 as the experimental group (Table 1). In the experiment, the first day of adulthood was defined as the first day of lifespan analysis. Experimental nematodes were transferred daily from plates at the peak of nematode oviposition to fresh NGM medium coated with the corresponding live bacteria and grown in a 20°C incubator. Nematodes that did not respond to mild stimuli were considered dead, and nematodes that were missing, sac-like, or had protruding vulvas were considered lost. The number of surviving nematodes, deaths, and losses were recorded daily. To ensure sufficient food and a good living environment for the nematodes, surviving nematodes were transferred to fresh medium every other day, and two independent lifespan measurements were performed.

[0058] Table 1. Grouping of the nematode experiment

[0059] Gut microbiota release various metabolites from dietary carbohydrates, such as short-chain fatty acids (SCFAs), butyrate, and H2, which play a positive role in the host's immune function, metabolism, and homeostasis. Long-term consumption of yogurt containing *Lactobacillus delbrueckii* increases butyrate production, indicating that probiotics can delay aging by regulating gut microbiota and metabolites. *Lactobacillus fermentum*, a species of *Lactobacillus*, significantly extends the lifespan of wild-type *C. elegans* N2 and mutant *VH254* compared to *Escherichia coli* OP50 with long-term consumption. Figure 5 and Figure 6, P <0.001, n=120).

[0060] Example 5: Effects of fermentation of Lactobacillus mucin FM1 on motility activity of *Nematodea dementia* and analysis of expression function of related dementia genes. Experiment to determine the head-swaying rate of nematodes: Head swaying behavior was defined as a change in direction with an angle greater than 90°. Ten L4-stage VH254 mutant nematodes, after synchronization treatment, were transferred to 60 mm NGM culture dishes coated with fresh OP50 and FM1 live bacteria. The head-swaying rate was measured on days 3 and 6 after incubation at 20°C until adulthood. The nematodes were then transferred to NGM plates without bacterial suspension, and the number of head swaying events within 30 seconds was observed and recorded using a Leica ICC50W microscope. Images were acquired and saved using Leica LAS EZ software. Three independent measurement experiments were conducted.

[0061] Experimental analysis of short-term memory in VH254 nematodes: (1) The L4 stage nematodes after synchronization treatment were picked and placed on 60mm NGM culture dishes coated with fresh OP50 and FM1 live bacteria, 400 nematodes per group, 200 nematodes per plate, for a total of 2 plates, and cultured at 20℃ for 4 days. (2) 200 nematodes were taken from each of the experimental and control groups to perform a blank chemotaxis test. The specific operation steps are as follows: ① Draw a pair of cross lines on a 90mm NGM plate. Add 1μL of 10mM imidazole anesthetic symmetrically to the left and right of the horizontal midline, 20mm from the edge. Note that the anesthetic should be added within 15 minutes before the start of the experiment. ② After the anesthetic has dried, add 1 μL of isoamyl alcohol (1:100 diluted in water) to one side of the same position, and add M9 buffer to the other side. Since isoamyl alcohol has a volatile odor, the petri dish lid should only be opened when necessary. ③ Place 200 nematodes at the starting position of the lower half of the vertical center line in a petri dish, at the same distance from isoamyl alcohol and M9 buffer to ensure the accuracy of the experiment. Incubate at room temperature for 1 hour and record the number of nematodes anesthetized by imidazole within 1 cm of the spots at isoamyl alcohol and M9 buffer.

[0062] (3) 200 nematodes were taken from each of the experimental and control groups for isoamyl alcohol training. The specific operation steps are as follows: ① First, collect 200 nematodes in a 1.5 mL EP tube using M9 buffer and starve them at room temperature for 1 hour; ② Transfer the nematodes to a 60mm NGM culture dish containing fresh OP50 and FM1 live bacteria. At the same time, add 5μL of undiluted isoamyl alcohol to the lid of the dish, immediately cover the dish, and incubate at room temperature for 1 hour. ③ Collect nematodes in 1.5 mL EP tubes using M9 buffer, and then transfer the nematodes to 90 mm NGM plates for chemotaxis testing.

[0063] (4) Calculate the chemotaxis index of nematodes based on the experimental results, using the following method: CI = (N1-N0) / N Where CI is the chemotactic index (%); N1 is the number of nematodes anesthetized at isoamyl alcohol; N0 is the number of nematodes anesthetized at M9 buffer. N is the total number of nematodes in the petri dish.

[0064] Differential gene expression analysis in nematodes: Total RNA was extracted from 600 nematodes in each group using TRIzol reagent, and the transcriptional levels of relevant genes were detected using the TBGreen Premix Ex Taq Real-Time PCR Kit (TaKaRa). 2 -ΔΔCt The method calculates the relative quantification of mRNA expression and normalizes it to the expression of the internal reference genes tba-1 or snb-1.

[0065] The results showed that, when the head-shaking rate of nematodes at 3 days and 6 days was analyzed, it was found that on day 6, the head-shaking rate of nematodes in group FM1 was significantly higher than that in the control group. Figure 7 (P<0.01, n=40), indicating that FM1 can indeed improve the motility of P-Tau nematodes. The head-shaking rate of nematodes also showed a significant decreasing trend with increasing age.

[0066] Probiotic FM1 was started during the L4 stage of nematodes. Short-term memory ability was then studied in 4-day-old VH254 nematodes. Chemotaxis analysis revealed that the clinical inertia (CI) of nematodes was significantly higher than that of the control group before isoamyl alcohol training, and this difference became more pronounced after training. Figure 8 (P<0.001, n=200). Although feeding OP50 CI also increased, it was not as significant as FM1, indicating that probiotic FM1 can significantly improve the short-term memory of VH254 nematodes.

[0067] In addition, the expression of genes related to transcriptional activity and AD in nematodes was analyzed to further explore the molecular mechanism by which FM1 improves dementia-like behavior in nematodes. Figure 9(n=600). The study found that the expression of genes related to transcriptional activity, sma-9 (Gene ID: 181271), hsp-60 (Gene ID: 175316), and cyp-23A1 (Gene ID: 173787), was significantly upregulated (P<0.01), while the expression of genes mut-14 (Gene ID: 178952) and hsp-1 (Gene ID: 178507) showed no difference (P>0.05). In addition, the transcriptional level of the xbp-1 (Gene ID: 175541) gene, which is used to degrade the accumulation of Tau protein caused by misfolding in the endoplasmic reticulum (ER), was significantly increased. We also examined several genes related to defense and immunity in nematodes and found that the expression levels of clc-3 (Gene ID: 180625), hlh-30 (Gene ID: 177157), atf-7 (Gene ID: 175587), and daf-16 (Gene ID: 172981) were significantly increased. Figure 10 (P<0.01), while there was no difference in the expression of the clc-1 (Gene ID: 181293) gene. P >0.05). The expression level of fat-7 (Gene ID: 179100), a gene associated with fatty acid biosynthesis, was significantly upregulated. Figure 11 , P <0.01. However, there were no significant differences in the expression of che-3 (Gene ID: 172593), che-11 (Gene ID: 179666), ift-74 (Gene ID: 174102), and ift-81 (Gene ID: 185183) genes related to chemotaxis. Figure 11 , P >0.05).

[0068] Common signaling pathways include insulin / IGF-1 and jnk / MAPK, which have significant impacts on host lifespan. daf-16 is a downstream forkhead transcription factor in the insulin / IGF-1 signaling pathway and is also involved in nematode defense responses against other organisms, neuronal development, reproduction, and more. atf-7 is involved in multiple life activities, including defense responses against bacteria, regulation of innate immune responses, and transcriptional regulation by RNA polymerase II. It is a human homolog in nematodes associated with Alzheimer's disease.

[0069] Western blotting experiments on proteins in 8-day-old nematodes revealed that FM1 intervention significantly reduced the levels of Tau and P-Tau proteins in the nematodes. Figure 12 , P <0.001, n=700).

[0070] Example 6: Preparation of fermented Lactobacillus mucilaginosus FM1 bacterial powder 1. Activation of bacterial strain: After thawing the Lactobacillus fermentans FM1 stored in cryovials, the strains were streaked on MRS plates under aseptic conditions and anaerobically cultured at 37°C for 20 h. Single colonies were picked from the MRS plates and streaked on slant plates and anaerobically cultured at 37°C for 20 h for storage.

[0071] 2. Primary culture: Take one loopful of bacteria from each vigorous growth slant and scrape it into 100 mL of primary culture medium using a sterilized inoculation loop. Incubate anaerobically at 37°C for 16 h to obtain the primary culture medium of fermenting Lactobacillus mucilaginosus FM1.

[0072] 3. Secondary culture: The primary culture of fermenting Lactobacillus FM1 was inoculated into the secondary culture medium in a 5 L fermenter at an inoculation rate of 1.5%-2% and a liquid volume of 3 L-4 L. The fermentation temperature was 37℃ and the rotation speed was 100 rpm-120 rpm. The culture was carried out for 15-20 h to obtain the secondary seed culture.

[0073] 4. Preparation of Lactobacillus fermentans FM1 bacterial powder: The secondary seed liquid was centrifuged (5000 g, 10 min) to obtain active Lactobacillus fermentans sludge, which was then mixed with skim milk (20%, w / v) at a ratio of 1:1.5 (by weight) for 30 min to obtain a high-concentration Lactobacillus fermentans FM1 bacterial suspension, which was then freeze-dried to produce Lactobacillus fermentans FM1 bacterial powder.

[0074] Experimental results: Plate count showed that the viable count (colony-forming units) of fermenting *Lactobacillus mucinus* FM1 was ≥4.0 × 10⁻⁶. 11 CFU / g.

[0075] Example 7: Preparation of a probiotic solid beverage made from fermented Lactobacillus mucin FM1 In this embodiment, the probiotic solid beverage containing Lactobacillus fermentum FM1 is prepared from the following raw materials in the indicated mass percentages: 3%-6% freeze-dried Lactobacillus fermentum FM1 bacterial powder, 22%-36% natural fruit powder, 30%-60% erythritol, 5%-8% resistant dextrin, and 0.2%-0.4% edible flavoring.

[0076] In this embodiment, the specific steps for preparing a probiotic solid beverage containing Lactobacillus fermentum FM1 are as follows: the raw materials are crushed and sieved (80 mesh), then each raw material is weighed according to the proportion, thoroughly mixed, and the viable bacteria count of the solid beverage is tested.

[0077] Experimental Results: Three batches of probiotic solid beverage were tested for indicators, and the results are shown in Table 2. The experiment demonstrates that the probiotic solid beverage of this invention has a live probiotic count ≥2.0 × 10⁻⁶. 9 CFU / g.

[0078] Table 2 Detection of Indicators for Probiotic Solid Beverages

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0080] References: [1] Bao Y, Zhang Y, Li H, et al. In vitro screen of Lactobacillusplantarum as probiotic bacteria and their fermented characteristics insoymilk[J]. Annals of Microbiology, 2012, 62(3): 1311-1320. [2] Brandt R, Gergou A, Wacker I, et al. A Caenorhabditis elegansmodel of tau hyperphosphorylation: induction of developmental defects by transgenic overexpression of Alzheimer's disease-like modified tau[J]. Neurobiology of aging, 2009, 30(1): 22-33.

Claims

1. Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum FM1, with accession number CGMCC No.35034.

2. Probiotic powder, characterized in that, The product is prepared by culturing the *Lactobacillus FM1 fermentation* as described in claim 1; wherein the viable count of *Lactobacillus FM1 fermentation* in the bacterial powder is ≥4.0 × 10⁻⁶. 11 CFU / g.

3. The use of the fermented Lactobacillus mucin FM1 of claim 1 or the probiotic powder of claim 2 in the preparation of products for delaying aging, prolonging healthy lifespan, or preventing and / or improving dementia-related symptoms.

4. The application according to claim 3, characterized in that, The dementia-related condition mentioned is Alzheimer's disease.

5. The application according to claim 3 or 4, characterized in that, The improvements in dementia-related symptoms include: improving cognitive and memory abilities, improving motor abilities, reducing the levels of Tau protein and / or phosphorylated Tau protein, and / or upregulating the expression of the xbp-1 gene.

6. The use of the fermented Lactobacillus mucin FM1 of claim 1 or the probiotic powder of claim 2 in the preparation of probiotic foods or health foods.

7. The application according to claim 6, characterized in that, The probiotic food is a solid beverage, compressed candy, or fermented product.

8. A probiotic solid beverage, characterized in that, It comprises the probiotic powder of claim 2, and food-grade excipients; The excipients are selected from one or more of natural fruit powder, sweeteners, dietary fiber, and edible flavorings.

9. The probiotic solid beverage according to claim 8, characterized in that, The sweetener is maltitol and / or erythritol; The dietary fiber is resistant dextrin; The viable count of *Lactobacillus mucinus* FM1 in the solid beverage is ≥2.0 × 10⁻⁶. 9 CFU / g.

10. The probiotic solid beverage according to claim 9, characterized in that, It is prepared from raw materials containing the following mass percentages: 3%-6% probiotic powder, 22%-36% natural fruit powder, 30%-60% maltitol and / or erythritol, 5%-8% resistant dextrin, and 0.2%-0.4% edible flavoring.

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