Application of lactobacillus rhamnosus in preparation of product for improving and treating cognitive disorder

Abstract

The invention discloses application of lactobacillus rhamnosus to preparation of a product for improving and treating cognitive impairment. The invention discloses the new application of the rhamnosus cheese for the first time, and the rhamnosus cheese can improve or treat cognitive impairment related to aging by improving behavioral function change, brain inflammatory factors, oxidative stress activity and neurotransmitter level caused by aging of mice and regulating transcriptome, intestinal microbiome, metabolome and the like. The lactobacillus rhamnosus has a potential application prospect in the aspect of improving learning and memory ability impairment caused by senescence.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the use of Lactobacillus rhamnosus in the preparation of a product for improving and treating cognitive impairment. BACKGROUND

[0002] Aging is an irreversible biological process characterized by the gradual decline of physiological functions in the human body. Correspondingly, cognitive functions related to learning and memory often decline with age. With the change in population structure and the intensification of aging, neurodegenerative diseases related to aging, such as Alzheimer's disease (AD) and Parkinson's disease, pose a major challenge to the global aging population and affect their quality of life. In Alzheimer's disease, the aggregation of beta-amyloid protein exacerbates oxidative stress and neuroinflammation in the central nervous system (CNS), leading to irreversible decline in cognitive function, which is one of the core symptoms of Alzheimer's disease. In recent years, more and more studies have shown that the imbalance of gut microbiota is an important factor in the pathogenesis of Alzheimer's disease. With age, these pathological processes accelerate due to decreased DNA repair function, cell aging, mitochondrial dysfunction, and disruption of protein homeostasis. However, there is currently no drug that can completely prevent the development of Alzheimer's disease. Therefore, it is particularly important to find effective treatment methods to improve age-related neurodegenerative diseases.

[0003] The gut microbiota, composed of trillions of microorganisms, plays a key role in maintaining the physiological state of the host. The microbiota-gut-brain axis (MGBA) is a bidirectional communication network that connects the gut microbiota with the central nervous system through endocrine and immune pathways. A recent review suggests that disturbances in the MGBA have a significant impact on the development of Alzheimer's disease (AD). Lipopolysaccharide (LPS) and bacterial amyloid proteins produced by the gut microbiota can activate immune cells in the brain, triggering neuroinflammation and oxidative stress. Probiotics that prevent the progression of Alzheimer's disease (AD) show good therapeutic potential in the field of neurodegenerative diseases. Studies have shown that certain probiotics such as Clostridium butyricum, Bifidobacterium longum, and Bifidobacterium breve can enhance cognitive function in Alzheimer's disease models or patients by promoting the synthesis of short-chain fatty acids (SCFAs) in the gut. Research has found that the MGBA can improve depressive and anxious behaviors in animal models. In addition, exopolysaccharides derived from Lactobacillus rhamnosus (L. rhamnosus) can improve oxidative stress and regulate the gut microbiota in a mouse aging model. However, the specific mechanisms by which L. rhamnosus protects aging mice from the effects of the MGBA are not yet clear.

[0004] Therefore, exploring a probiotic fermentation product that positively affects age-related cognitive impairment and improves various indicators related to cognitive function under the condition of extremely low or no side effects through specific mechanisms is an urgent problem for those skilled in the art. SUMMARY

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide the use of Lactobacillus rhamnosus in the preparation of a product for improving and treating cognitive impairment.

[0006] To achieve the above-mentioned and other related purposes, the present application is obtained by the following technical scheme.

[0007] The first aspect of the present application protects the use of Lactobacillus rhamnosus or its culture in the preparation of a product for preventing, improving and / or treating cognitive impairment related to aging.

[0008] In some embodiments, the Lactobacillus rhamnosus is Lactobacillus rhamnosus KF7 strain, whose preservation number is CGMCC No. 6430.

[0009] In certain embodiments, the number of viable bacteria of the Lactobacillus rhamnosus is not less than 10 6 CFU / g, based on the total mass of the product.

[0010] In some embodiments, the culture of the Lactobacillus rhamnosus comprises fermentation broth and / or viable bacteria of the Lactobacillus rhamnosus.

[0011] In certain embodiments, the culture of the Lactobacillus rhamnosus is obtained by inoculating the Lactobacillus rhamnosus in a culture medium and culturing.

[0012] In some embodiments, the product has at least one of the following functions:

[0013] 1) inhibiting the level of inflammatory factors in the brain tissue of a mammal;

[0014] 2) increasing the level of anti-inflammatory factors in the brain tissue of a mammal;

[0015] 3) improving the level of oxidative stress in the brain tissue of a mammal;

[0016] 4) increasing the content of indole-3-lactic acid in the brain tissue of a mammal;

[0017] 5) increasing the content of indole-3-lactic acid in the feces of a mammal;

[0018] 6) inhibiting the content of monoamine oxidase in the brain tissue of a mammal;

[0019] 7) modulating the intestinal flora of a mammal.

[0020] In some embodiments, the inflammatory factor comprises interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α).

[0021] In some embodiments, the anti-inflammatory factor comprises interleukin 10 (IL-10).

[0022] In some embodiments, the improving the level of oxidative stress comprises increasing the level of superoxide dismutase 1 (Sod1), glutathione peroxidase (Gshpx), and decreasing the content of malondialdehyde (MDA).

[0023] In some embodiments, the modulating the intestinal flora comprises decreasing the relative abundance of Bacteroidota and increasing the relative abundance of Firmicutes.

[0024] In some embodiments, the mammal comprises a human.

[0025] In some embodiments, the product is selected from one or more of a drug, a functional food, a health product, a probiotic, and a pharmaceutical raw material.

[0026] In some specific embodiments, the functional food is a fermented food prepared by fermentation of Lactobacillus rhamnosus GG.

[0027] In some more specific embodiments, the raw milk is selected from one or both of raw milk and reduced milk. Preferably, the reduced milk is an aqueous solution of skimmed milk powder, and the concentration of the aqueous solution of skimmed milk powder is 6-20 wt%.

[0028] In some more specific embodiments, the fermentation is performed at a temperature of 30-40°C.

[0029] In some more specific embodiments, the fermentation is performed for a time of 12-30 h.

[0030] In some specific embodiments, the probiotic is in a dosage form selected from one or more of a lyophilized powder, a capsule, a tablet, and a granule.

[0031] In some more specific embodiments, the probiotic is a lyophilized powder obtained by drying a culture of Lactobacillus rhamnosus GG.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] The Lactobacillus rhamnosus provided by the application can significantly improve the cognitive and memory disorders of model mice, reduce the content of inflammatory factors, increase the content of anti-inflammatory factors, increase the nerve transmitter indole-3-lactic acid (ILA) in brain tissue, inhibit the expression of monoamine oxidase in brain tissue and reduce its activity, increase the content of indole-3-lactic acid (ILA) in feces, and can regulate intestinal flora and metabolites. The Lactobacillus rhamnosus provided by the application can be used for preparing a drug composition for improving cognitive impairment, and can also be applied to a drug, a fermented food or a health product as a microecological preparation, and has a very wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Effects of the Lactobacillus rhamnosus in Example 1 of the application on the behavioral indicators of the aging mouse model.

[0035] Figure 2 Effects of the Lactobacillus rhamnosus in Example 1 of the application on the neuroinflammation and oxidative stress of the aging mouse model.

[0036] Figure 3 Effects of the Lactobacillus rhamnosus in Example 1 of the application on the brain tissue neurotransmitters of the aging mouse model.

[0037] Figure 4 A graph showing the effects of the Lactobacillus rhamnosus on the phenotype improvement of the aging mouse model in the transcriptome analysis in Example 1 of the application.

[0038] Figure 5 A graph showing the effects of the Lactobacillus rhamnosus on the fecal non-targeted metabolome of the aging mouse model in Example 1 of the application.

[0039] Figure 6 A graph showing the effects of the Lactobacillus rhamnosus on the fecal microorganisms of the aging mouse model in Example 1 of the application. DETAILED DESCRIPTION

[0040] D-galactose is a commonly used drug and is widely used to construct an animal aging model. Studies have found that excessive D-galactose metabolites accumulate in cells, leading to the generation of reactive oxygen species (ROS) and then triggering oxidative stress damage, which is highly similar to the mechanism of natural aging.

[0041] The present application applies Lactobacillus rhamnosus to a D-galactose-induced aging mouse model. The test results show that Lactobacillus rhamnosus exhibits significant antioxidant and anti-monoamine oxidase (MAO) activities. Further studies have found that it can regulate intestinal flora in vivo, increase the number of Lactobacillus murinus in the intestine, and thus increase the concentration of indole-3-lactic acid (ILA) in the intestine and brain. Further, indole-3-lactic acid (ILA) promotes the MAPK / ERK branch by activating the prolactin signaling pathway (the prolactin signaling pathway refers to the combination of prolactin Prl and its receptor Prlr), ultimately promotes the nuclear translocation of Nrf2, reduces neural inflammation and oxidative stress, and thus improves cognitive impairment. This finding reveals the potential application value of Lactobacillus rhamnosus in improving impaired learning and memory associated with aging.

[0042] The first aspect of the present application protects the use of Lactobacillus rhamnosus or a culture thereof in the preparation of a product for preventing, improving and / or treating cognitive impairment associated with aging.

[0043] In some embodiments, the Lactobacillus rhamnosus is Lactobacillus rhamnosus KF7 strain, whose accession number is CGMCC No. 6430.

[0044] In certain embodiments, the number of viable Lactobacillus rhamnosus is not less than 10 6 CFU / g, for example, 1×10 6 CFU / g, 1×10 7 CFU / g, 1×10 8 CFU / g, 1×10 9 CFU / g, 2×10 9 CFU / g, 5×10 9 CFU / g, 8×10 9 CFU / g, 1×10 10 CFU / g, 5×10 10 CFU / g, 1×10 11 CFU / g, etc., which can be selected by a person skilled in the art according to actual needs.

[0045] In some embodiments, the culture of Lactobacillus rhamnosus comprises a fermentation broth and / or viable cells of Lactobacillus rhamnosus. In certain embodiments, the culture of Lactobacillus rhamnosus is obtained by inoculating Lactobacillus rhamnosus in a culture medium. The culture medium can be MRS medium, which exemplary comprises the following ingredients: peptone, beef extract, glucose, sodium acetate, yeast extract, diammonium citrate, K2PO4·3H2O, MgSO4·7H2O, MnSO4, L-cysteine, Tween 80. The culture temperature is 30-40°C, for example, 30°C, 32°C, 35°C, 36°C, 37°C, 38°C, etc. The culture time is 12-30h, for example, 12h, 14h, 18h, 20h, 21h, 22h, 23h, 24h, 30h, etc. In a specific embodiment, Lactobacillus rhamnosus is inoculated in MRS medium and cultured at 37°C for 24h.

[0046] In some embodiments, the product has at least one of the following functions:

[0047] 1) inhibiting the level of inflammatory factors in the brain tissue of a mammal;

[0048] 2) increasing the level of anti-inflammatory factors in the brain tissue of a mammal;

[0049] 3) improving the level of oxidative stress in the brain tissue of a mammal;

[0050] 4) increasing the content of indole-3-lactic acid in the brain tissue of a mammal;

[0051] 5) increasing the content of indole-3-lactic acid in the feces of a mammal;

[0052] 6) inhibiting the content of monoamine oxidase in the brain tissue of a mammal;

[0053] 7) regulating the intestinal flora of a mammal.

[0054] In certain embodiments, the mammal comprises a human.

[0055] In certain embodiments, the inflammatory factors comprise interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α).

[0056] In certain embodiments, the anti-inflammatory factors comprise interleukin 10 (IL-10).

[0057] In certain embodiments, the improvement of the level of oxidative stress comprises increasing the levels of superoxide dismutase 1 (Sod1), glutathione peroxidase (Gshpx), and reducing the content of malondialdehyde (MDA).

[0058] In some embodiments, the gut microbiota is selected from Firmicutes, Bacteroidota. The modulating gut microbiota refers to reducing the relative abundance of Bacteroidota while increasing the relative abundance of Firmicutes. Further, the Firmicutes comprises Lactobacillus. Further, the Lactobacillus comprises one or more of L. murinus, L. johnsonii, and L. reuteri. The modulating gut microbiota in the present application refers to reducing the relative abundance of Bacteroidota while increasing the relative abundance of Firmicutes, specifically increasing the number of L. murinus, L. johnsonii, and L. reuteri, and the like, preferably increasing the relative abundance of Lactobacillus. murinus.

[0059] In some embodiments, the product is selected from one or more of a functional food, a probiotic, a medicine, a health product, and a pharmaceutical material.

[0060] In some embodiments, the functional food is a fermented food prepared by fermentation with R. rhamnosus. The fermented food can be a solid food, a liquid food, or a semi-solid food. The fermented food can be a dairy product, a legume product, or a fruit / vegetable product. The dairy product can be a fermented milk, a flavored fermented milk, a fermented milk beverage, a cream, a cheese, a milk-containing beverage, or a milk powder. The legume product can be a soy milk or a soy milk powder. The fruit / vegetable product can be a product prepared from at least one of Chinese cabbage, white radish, cucumber, sugar beet, yellow peach, or waxberry. In one embodiment, the functional food is a fermented milk prepared by inoculating R. rhamnosus into raw milk and fermenting the inoculated raw milk. The raw milk can be any raw milk suitable for preparing yogurt. For example, the raw milk can comprise a combination of one or more of raw milk and reduced milk. The raw milk can meet the requirements of GB 19301 Food Safety National Standard Raw Milk or the like. The reduced milk can meet the requirements of GB 19644 Food Safety National Standard Milk Powder or the like. The reduced milk is preferably full-fat milk powder. The raw milk can be derived from cow and / or sheep, or the like. Specifically, the seed liquid of R. rhamnosus is inoculated into the raw milk at a ratio of 5% (v / v) and the inoculated raw milk is fermented at 37°C for 20 h. The viable cell concentration of R. rhamnosus in the seed liquid is 7.5 x 10 9 CFU / mL.

[0061] In some embodiments, the dosage form of the bacterial agent comprises a lyophilized powder, a capsule, a tablet or a granule. The bacterial agent is a lyophilized powder obtained by drying a culture of Lactobacillus rhamnosus. The bacterial agent further comprises a protective agent and / or an auxiliary additive. The protective agent is selected from skimmed milk powder. The auxiliary additive comprises one or more of inulin, fructo-oligosaccharide, galacto-oligosaccharide, manno-oligosaccharide, trehalose, soybean oligosaccharide, resistant dextrin, spirulina, polydextrose, alpha-lactalbumin or lactoferrin. The Lactobacillus rhamnosus can be applied to the relevant product alone or in combination with other strains.

[0062] In some embodiments, the bacterial agent is prepared by mixing a culture of Lactobacillus rhamnosus with an aqueous solution of a protective agent and drying. Preferably, the drying is vacuum freeze-drying or other bacteria solution drying process. The concentration of the aqueous solution of the protective agent is 6-20 wt%, or 6 wt%, 8 wt%, 10 wt%, 10 wt%, 12 wt%, 14 wt%, 15 wt%, 17 wt%.

[0063] In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient is selected from one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants and antioxidants. In some embodiments, the dosage form of the medicament is selected from one or more of tablets, powders, granules, capsules, oral liquids, powders, pills, syrups, effervescent agents, emulsions, ointments, sprays, liniments, suppositories, lotions, foams and gels.

[0064] The present application finds that Lactobacillus rhamnosus can significantly improve cognitive impairment associated with aging, specifically in that: 1) significantly increasing the activity distance of the model mice in the open field test, the time spent in the center of the open field; significantly increasing the average escape latency of the model mice in the water maze test, the time spent in the platform quadrant, and the number of times crossing the platform quadrant; significantly prolonging the exploration time and recognition index of the model mice in the novel object recognition test; significantly prolonging the active avoidance latency of the model mice in the passive avoidance test and reducing the number of errors; significantly increasing the avoidance response rate of the model mice in the active avoidance test and reducing the number of errors; 2) reducing the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-a in the brain tissue of the model mice, and increasing the level of anti-inflammatory cytokine IL-10 in the brain tissue; 3) significantly increasing the levels of superoxide dismutase 1 (Sod1) and glutathione peroxidase (Gshpx) in the brain tissue of the model mice, and reducing the level of malondialdehyde (MDA); 4) inhibiting the activity of monoamine oxidase (MAO) in the brain tissue of the model mice; 5) increasing the content of neurotransmitter indole-3-lactic acid (Indolelactic acid, ILA) in the brain tissue and intestinal tract of the model mice; 6) regulating the intestinal flora of the model mice, reducing the relative abundance of Bacteroidota while increasing the relative abundance of Firmicutes, increasing the number of species of Firmicutes such as L. murinus, L. johnsonii and L. reuteri, and making the intestinal flora tend to be normal. In summary, the mechanism of action of Lactobacillus rhamnosus is that by increasing the number of L. murinus in the intestinal tract, it increases the concentration of its metabolite indole-3-lactic acid ILA in the intestine and brain. Further, indole-3-lactic acid ILA can activate the prolactin signaling pathway (which refers to the combination of prolactin Prl and its receptor Prlr) to promote the MAPK / ERK branch, ultimately promoting the nuclear translocation of Nrf2, reducing neural inflammation and oxidative stress, and ultimately improving cognitive impairment. Therefore, Lactobacillus rhamnosus can be used in the preparation of products for preventing, improving or treating the efficacy of aging-related cognitive impairment. As a probiotic, Lactobacillus rhamnosus strain has high safety and is not prone to drug resistance, and has a wide application prospect.

[0065] The specific embodiments of the present application are described below by way of specific examples. Those skilled in the art can easily understand other advantages and effects of the present application from the disclosure in the specification. The present application can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details in the specification without departing from the spirit of the present application.

[0066] Before particular embodiments of the present application are described, it is to be understood that the application is not limited to the particular specific embodiments described; it is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application. In this document, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0067] When a numerical range is given in the embodiments, it is understood that, unless otherwise stated by the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to those described in the embodiments of the present application can also be used to implement the present application according to the knowledge of the prior art possessed by those skilled in the art and the description of the present application.

[0068] In the following embodiments of the present application, Lactobacillus rhamnosus KF7 strain (same as strain KF7) is taken as an example, which is classified and named as Lactobacillus rhamnosus KF7, with the preservation number of CGMCC No.6430 and the preservation date of August 13, 2012. The preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No.3, Beichen West Road, Chaoyang District, Beijing.

[0069] Example 1 Preparation of fermented milk

[0070] 1.1 Preparation method of seed liquid

[0071] The strain KF7 was picked up with an inoculation loop to 10 mL of MRS liquid medium (purchased from Merck Co., Germany), and the single colony was uniformly dispersed in the liquid medium with a vortex shaker, and was cultured anaerobically at 37°C for 48 h.

[0072] Then, it was inoculated in the MRS liquid medium at an inoculation amount of 2% (v / v), and was cultured anaerobically at 37°C for 24 h. The culture was centrifuged at 15,000 rpm for 10 min, and the supernatant was discarded. The bacterial cells were washed twice with sterile distilled water, and were suspended with sterile distilled water at the original culture volume to obtain a seed liquid for fermentation, and the bacterial concentration of the seed liquid was 7.5 x 10 9 CFU / mL.

[0073] 1.2 Preparation method of skim milk

[0074] Skim milk powder was mixed with distilled water to obtain a solution with a mass percentage of 10 wt% of skim milk powder, then sterilized at 125°C for 5 min, cooled to room temperature to obtain skim milk.

[0075] 1.3, Preparation method of fermented milk

[0076] The seed solution obtained in step 1.1 was aseptically inoculated into the skim milk obtained in step 1.2 at an inoculation amount of 5% (v / v), and incubated at 37°C for 20 h to obtain fermented milk.

[0077] Further, to ensure the uniformity of the gavage samples in subsequent experiments on a mouse model of aging, the fermented milk was freeze-dried into a powder by a FreeZone 24 device at -50°C to obtain a freeze-dried powder with a viable bacterial count of 5-11 x 10 10 CFU / g, which was stored at -80°C for standby use.

[0078] Example 2 animal experiment

[0079] In this example 2, the freeze-dried powder obtained in example 1 was used to conduct experiments on a mouse model for studying aging and aging-related diseases, including behavioral tests, neuroinflammation and oxidative stress tests, neurotransmitter content tests, phenotypes, fecal non-targeted metabolomics, fecal microorganisms, etc. The behavioral tests included 5 tests, i.e. open-field test, novel object recognition test (NOR test), step-down test, active avoidance test (AA test) and morris water maze test (MWM test), to investigate their effects on aging and related behavioral and cognitive impairment. The tests included the following:

[0080] 2.1, Construction of a mouse model of aging

[0081] Forty-eight SPF 7-week-old male C57BL / 6 mice (Jiangsu Huachuang China Pharmaceutical Technology Co., Ltd.) were purchased and acclimated for 1 week. Except for the control group, the other groups received intraperitoneal injection of 240 mg / kg body weight of D-galactose (product number: D810319, Macklin, Shanghai, China) per day for a total of 8 weeks.

[0082] 2.2, Grouping and intervention

[0083] The 48 mice were randomly divided into four groups, with 12 mice in each group. The experimental procedure is shown in Figure 1 A. The specific intervention methods are as follows:

[0084] (1) Control group (Con): Intraperitoneal injection (Ip) of normal saline daily, and oral gavage (Oa) of 0.2 mL of 0.9% normal saline (Saline) daily, for 8 weeks of gavage.

[0085] (2) Model group (Mod): Intraperitoneal injection of 120 mg / kg body weight of D-galactose daily, and oral gavage of 0.2 mL of 0.9% normal saline (i.e. a dose of 20 ml / kg / d) daily, for 8 weeks of gavage.

[0086] (3) L. rhamnosus KF7 intervention group (KF7): Intraperitoneal injection of 120 mg / kg body weight of D-galactose daily, and oral gavage of 0.2 mL of L. rhamnosus KF7 solution (obtained by dissolving the freeze-dried powder of Example 1 in water) with a concentration of 5 x 10 9 CFU / mL daily, for 8 weeks of gavage.

[0087] (4) Donepezil (Don) group: Intraperitoneal injection of 120 mg / kg of D-galactose daily, and oral gavage of 1.0 mg / kg body weight of donepezil (Daiichi Sankyo Co., Ltd., Tokyo, Japan) solution daily (i.e. a dose of 1 mg / kg / d), for 8 weeks of gavage.

[0088] 2.3, Effect of L. rhamnosus on D-galactose-induced aging mouse model

[0089] 2.3.1, Behavioral test

[0090] 1) MWM water maze test

[0091] The MWM device includes a circular water tank with a diameter of 120 cm and a depth of 50 cm, filled with water and painted black, with a hidden platform with a diameter of 10 cm underneath. Four visual cues are placed in the north, south, east, and west directions, and a hidden platform is placed at the center of the southeast. In the training phase (days 1-4 after 8 weeks of gavage, 3 times a day), the mice are placed in the MWM device and allowed to swim freely for 60 seconds until they find the hidden platform. If the mice fail to find the platform within 60 seconds, they are carefully placed on the platform. Thirty seconds after the mice find the platform, they are returned to their respective cages. In the exploration phase, the mice swim freely for 60 seconds without a hidden platform. The VsiuTrack animal behavior analysis software (Shanghai Xinyan Information Technology Co., Ltd., China) is used to analyze the time and distance of the mice in the target quadrant (i.e. the platform quadrant), the number of target crossings, and the speed. Figure 1Figure B shows the trajectory, Figure C shows the escape latency, Figure D shows the time spent in the target quadrant, Figure E shows the number of times the mouse crossed the platform, and Figure F shows the movement of velocity. The terms "platform" and "target" can be interchanged.

[0092] Escape latency refers to the time (in seconds) required for a mouse to successfully locate the hidden platform (or remain there for 5 seconds) from its entry point into the water. A shorter average escape latency indicates better learning and memory. Platform quadrant dwell time refers to the percentage of time a mouse spends in the quadrant where it previously hid the platform. A shorter platform quadrant dwell time indicates better spatial memory, enabling the mouse to recognize and remember the target location. Platform quadrant crossing count refers to the number of times a mouse crosses the quadrant containing the platform within a set experimental time period. An increased number of crossings may indicate better platform location awareness or a more effective search strategy, allowing the mouse to return to the platform quadrant more frequently. Movement speed refers to the speed at which the mouse moves, i.e., the distance traveled per unit time (in mm / s).

[0093] from Figure 1 As shown in Figure B, compared with the Con (control group) group, the Mod (model group) mice exhibited trajectory disorder and took longer to find the platform. These symptoms were improved in the KF7 group and the Don (donepezil, positive drug) group.

[0094] from Figure 1 As shown in Figure C, the statistical results indicate that the average escape latency (in seconds) of the Mod group was significantly longer than that of the Con group (P<0.05), while the average escape latency of the Don and KF7 groups was significantly shorter than that of the Mod group (P<0.05). This suggests that the KF7 group mice learned and remembered the location of the platform more quickly, thus finding the hidden platform in a shorter time.

[0095] from Figure 1 The results from the middle DF showed that, compared with the Mod group, the Don group or KF7 group spent significantly longer time in the platform quadrant and crossed the platform quadrant more times at the same swimming speed (P<0.05). This indicates that the KF7 group mice had a stronger memory of the platform location and used spatial information more effectively to access the platform during the search process.

[0096] In summary, the behavior of KF7 group showed that the cognitive flexibility of mice was enhanced, i.e. they could better adapt to environmental changes and more effectively process and respond to spatial information.

[0097] 2) Novel object recognition (NOR) test

[0098] On day 66, the NOR test was performed, which consisted of three stages, namely, an adaptation period, a familiar object memory training period, and a novel object recognition period. 1) Adaptation period: Each mouse was placed in an empty rectangular box and allowed to freely explore for 3 min. After the exploration, the mouse was removed and the box was wiped with 75% ethanol to remove the odor. After waiting for 2 h, the mouse entered the next stage. 2) Familiar object memory training period: Two identical objects were placed in the rectangular box, and the mouse was allowed to freely explore for 5 min, after which the mouse was removed and waited for 1 h. 3) Novel object recognition period: One of the objects was replaced with a new object, and the mouse was again allowed to explore the device for 5 min. The time spent by the mouse exploring each object was recorded. Figure 1 G is a heat map of exploration behavior, where the box represents the old object. The circle represents the new object; H is a statistical chart of the time spent in novel object, and I is a statistical chart of the recognition index.

[0099] The heat map of exploration behavior represents the frequency of mouse activity in a particular area, with warmer colors (such as red, yellow) indicating more frequent activity and cooler colors (such as blue) indicating less activity. The time spent exploring novel objects is used to measure the time spent by mice exploring new objects, reflecting their cognitive ability and curiosity about new environments (unit: s). The longer the time spent exploring novel objects, the stronger the exploration behavior of the new object. The recognition index is calculated by calculating the ratio of the time spent by mice exploring new and old objects to assess their ability to recognize new objects. The higher the recognition index, the better the mouse can distinguish between new and old objects.

[0100] From Figure 1 G, it can be seen that in the representative trajectory, the mice in the Mod group tended to explore familiar objects more than new objects, while the Don and KF7 groups were the opposite.

[0101] From Figure 1 H-I, it can be seen that the time spent exploring novel objects and the recognition index of mice in the Mod group decreased, indicating that D-galactose caused impaired cognitive function; however, after administration of L. rhamnosus KF7 and donepezil, the exploration time and recognition index were prolonged, indicating that the impaired cognitive function was reversed (P<0.05).

[0102] 3) Open field test

[0103] Open field test is an experimental method widely used to study the behavior of rodents, which is used to assess the exploratory behavior, anxiety level and response to new environment of animals.

[0104] On day 65, mice were placed in an open field for 10 min of adaptation, then put in the center of a 50 cm long x 50 cm wide x 40 cm high experimental box, and freely explore for 5 min. Then record the distance of movement, average speed, resting time and distance of movement in the central area of the mice within 5 min. Figure 1 Fig. 8J-L shows the exploratory behavior heat map, distance of movement (Distance) statistics, and time in the center of open field (s) statistics.

[0105] The exploratory behavior heat map represents the frequency of mouse activity in the center of the open field, and the warmer the color (such as red, yellow), the more frequent the activity, and the cooler the color (such as blue), the less activity. The time in the center of the open field refers to the time (in seconds) that the mouse stays in the central area of the open field. The longer the time in the center of the open field, the more curious and less anxious the animal is. The distance of movement is used to record the total distance of movement (in cm) of the mouse during the open field test. The longer the distance of movement, the higher the movement ability and exploration willingness of the mouse.

[0106] From Figure 1 Fig. 8J-L shows the exploratory behavior heat map, distance of movement (Distance) statistics, and time in the center of open field (s) statistics.

[0107] 4) Passive avoidance test

[0108] Passive avoidance test is a behavioral experiment commonly used to assess the memory and learning ability of animals. This test is particularly suitable for measuring the long-term memory and fear conditioning of animals.

[0109] On day 67, passive avoidance test was performed. The experimental device included a plastic box with a stainless steel grid at the bottom and a foam platform. At the beginning of training, the mice were placed in the device for 5 min of adaptation. Then, an electric current was applied for 5 min. During this process, the mice would jump onto the platform to avoid the electric shock. The latency of each mouse to first step on the grid (i.e. the time from being placed in the device to the first step on the grid), the number of errors within 5 min (i.e. the number of times the mouse stepped on the grid and received an electric shock), and the number of electric shocks within 5 min were recorded. Figure 1M is the Active Avoidance Latency graph, and N is the Error times graph within 5 min.

[0110] Active avoidance latency represents the waiting time before the mouse first steps on the grid during the test phase. A longer latency indicates that the mouse has better memory ability to remember the association between the shock and the grid, thus avoiding stepping on the grid. Error times within 5 min refer to the number of times the mouse steps on the grid and receives an electric shock during the 5-minute test. Fewer error times indicate that the mouse has learned to avoid stepping on the grid, showing better learning and memory ability.

[0111] From Figure 1 M-N, compared with the Con group, the avoidance latency of the Mod group is shortened, and the error times are increased, while the trend of the Don group and the KF7 group is significantly opposite (P<0.05), indicating that the mice in the KF7 group have long-term memory and fear conditioning.

[0112] 5) Active avoidance test

[0113] Active avoidance test is a behavioral experiment used to assess the learning and memory ability of animals, especially their avoidance behavior to adverse stimuli such as electric shock.

[0114] Active avoidance test uses a shuttle box connected by an archway (diameter 3 cm) with two compartments (each compartment size: 136 x 153 x 250 mm). The floor of the compartment is composed of 2 mm thick stainless steel rods with a 1 cm spacing. During the test, the mouse is first placed in one compartment, facing away from the door, and adapted for 5 min. After the adaptation period, the buzzer and light in the compartment will last for 5 seconds. If the mouse still stays in this compartment, it will be stimulated by 10-20 milliampere, 10-15 Hz electric foot current through the grid floor of the compartment until it enters the other compartment. The average interval of each test is 60 seconds. Each test contains 20 combinations of buzzer and light with electric foot current. Each mouse receives 20 tests per day for 2 consecutive days. On the last day, the final test results are recorded: (i) Active avoidance response latency; (ii) Escape response latency. Figure 1 O is the Total avoidance response rate graph, and N is the Avoidance latency graph.

[0115] The avoidance response rate is the ratio of the number of times an animal successfully performs an avoidance response (such as pressing a lever or jumping over an obstacle) to avoid or terminate an electric shock to the total number of tests during the test period. A higher avoidance response rate generally indicates that the animal has better learning ability and memory retention, i.e., the animal can learn and remember how to avoid an unpleasant stimulus through specific behaviors. The avoidance latency is the time from the start of the electric shock to the performance of the avoidance response by the animal. The avoidance latency is an indicator of the cognitive flexibility and response speed of the animal. A shorter avoidance latency indicates that the animal can learn and adapt to environmental changes more quickly and effectively perform avoidance behavior.

[0116] From Figure 2 It can be seen from Table 2 that, compared with the Con group, the avoidance response rate of the Mod group mice decreased, and the avoidance latency was prolonged (P<0.05), while the avoidance response rate of the Don group and the KF7 group was significantly improved, and the number of avoidance latency errors was reduced (P<0.05).

[0117] From the above five behavioral test results, it can be seen that D-galactose induced cognitive impairment and memory decline in aging mice, and the supplementation of L. rhamnosus KF7 can improve the performance of mice in the behavioral tests related to cognitive impairment.

[0118] 2.3.2, Effect on neuroinflammation in mice

[0119] Using the corresponding detection kit, the relative mRNA expression level, transcription level of interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) in brain tissue samples were detected.

[0120] The sample processing method is: first pretreat the brain tissue samples of each group of mice with 1× phosphate buffer. Then, the tissue is ground repeatedly in a glass tube tissue homogenizer to obtain a tissue homogenate, which is then centrifuged at 5 000×g at 4℃ for 10 min, and the supernatant is collected. The detection is carried out according to the method and conditions specified by the manufacturer.

[0121] Interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) are inflammatory factors associated with cognitive impairment. They can activate microglia and astrocytes in the central nervous system, leading to neuroinflammation, which can damage the integrity and function of neurons, leading to decreased cognitive ability. Inflammatory factors can also promote oxidative stress, leading to neuronal damage. In addition, inflammatory factors can disrupt the integrity of the blood-brain barrier, leading to vascular dysfunction and neuronal damage. Inflammatory factors can also affect the formation and function of synapses, affecting the learning and memory process.

[0122] Interleukin 10 (IL-10) is an anti-inflammatory cytokine that can inhibit the synthesis of various pro-inflammatory mediators, such as IL-1β and tumor necrosis factor alpha (TNFα), and limit inflammation. In neurodegenerative diseases, such as Alzheimer's disease (AD), the lack of IL-10 can accelerate neuroinflammation and disease progression.

[0123] From Figure 2 A-C, it can be seen that the mRNA levels of the genes encoding inflammatory cytokines IL-1β, IL-6 and TNF-a were significantly increased in the Mod group compared to the Con group (P < 0.05). After administration of donepezil or strain KF7, the mRNA levels of the aforementioned genes were significantly decreased (P < 0.05). From Figure 2 D, it can be seen that the transcription levels of the anti-inflammatory cytokine-encoding gene IL-10 were significantly increased in the brain tissue of the Don and KF7 groups compared to the Mod group (P < 0.05, Figure 2 D).

[0124] From Figure 2 E-H, it can be seen that the transcription levels of the inflammatory factors IL-1β, IL-6, TNF-a and IL-10 were consistent with the results of mRNA expression levels (P < 0.05).

[0125] 2.3.3, Effect on oxidative stress in mice

[0126] The content of malondialdehyde (MDA) in the brain tissue samples and the relative mRNA expression levels and transcription levels of superoxide dismutase 1 (Sod1) glutathione peroxidase (Gshpx) were detected using the corresponding detection kits.

[0127] MDA is the final product of lipid peroxidation reaction, and its increased level usually indicates enhanced oxidative stress and is associated with various cognitive impairments, which can impair the integrity and function of neurons, leading to decreased cognitive ability. Sod1 is an antioxidant enzyme present in the cytoplasm, mainly responsible for converting superoxide anion (O 2- ) into hydrogen peroxide (H2O2) and oxygen (O2), thereby reducing the production of reactive oxygen species (ROS) and protecting cells from oxidative damage and DNA damage. Gshpx is an enzyme that can scavenge hydrogen peroxide (H2O2), converting H2O2 into water (H2O) by using glutathione (Glutathione) as a reducing agent, thereby protecting cells from oxidative stress damage.

[0128] From Figure 2 I, it can be seen that the accumulation of MDA in the brain of D-galactose-induced mice (i.e., the Mod group) was significantly reversed after administration of donepezil or L. rhamnosus KF7 (P < 0.05).

[0129] From Figure 3 As shown in Fig. 4C, the mRNA expression levels of Sod1 and Gshpx in the Mod group were significantly decreased, while the mRNA levels of Sod1 and Gshpx were significantly increased in the L. rhamnosus KF7 group, and the effect of L. rhamnosus KF7 on the mRNA level of Gshpx was significantly better than that of donepezil (P<0.05).

[0130] From the above detection results, it can be seen that D-galactose can cause neuroinflammation and oxidative stress in the brain tissue of mice, and the supplementation of L. rhamnosus KF7 can regulate the transcription and translation of these inflammatory factors and antioxidant genes in the aging model mice.

[0131] 2.3.4, Effect on mouse monoamine oxidase

[0132] Using the corresponding detection kit, the activity of monoamine oxidase (MAO) in the brain tissue sample was detected, and the relative mRNA expression level and transcription level of monoamine oxidase A (Maoa) and monoamine oxidase B (Maob) were detected by real-time fluorescence detection method.

[0133] From Figure 3 As shown in Fig. 4C, the mRNA expression levels of Sod1 and Gshpx in the Mod group were significantly decreased, while the mRNA levels of Sod1 and Gshpx were significantly increased in the L. rhamnosus KF7 group, and the effect of L. rhamnosus KF7 on the mRNA level of Gshpx was significantly better than that of donepezil (P<0.05).

[0134] From Figure 3 As shown in Fig. 4C, the mRNA expression levels of Sod1 and Gshpx in the Mod group were significantly decreased, while the mRNA levels of Sod1 and Gshpx were significantly increased in the L. rhamnosus KF7 group, and the effect of L. rhamnosus KF7 on the mRNA level of Gshpx was significantly better than that of donepezil (P<0.05).

[0135] 2.3.5, Effect on mouse brain neurotransmitter

[0136] The quantitative analysis of neurotransmitters was performed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using an AB SCIEX 5500 QTRAP mass spectrometer in positive ion mode. 46 neurotransmitters were analyzed (the sensitivity of mass spectrometry can reach 46 kinds), and five categories include GABAergic system, dopaminergic system, serotonin, caninamine system and cholinergic system, etc.

[0137] The sample processing method is: after the brain of each group of 6 mice is weighed, homogenized in 100 μL of 1% formic acid. Then, 400 μL of acetonitrile containing 1% formic acid is added, vortexed for 60 seconds, and then low-temperature ultrasonic treatment is performed twice for 30 min. Then, after centrifugation at 14,000 x g for 20 min, the precipitate is discarded, the supernatant is taken and freeze-dried, and stored in an environment of -80°C. Neurotransmitters are detected according to the conventional method and condition, which can be referred to reference 1 (Gu, J.; Guo, M.; Yin, X.; Huang, C.; Qian, L.; Zhou, L.; Wang, Z.; Wang, L.; Shi, L.; Ji, G., A systematic comparison of neurotoxicity of bisphenol A and its derivatives in zebrafish. Sci Total Environ 2022, 805, 150210.). The expression patterns of five different neurotransmitters between the four groups are observed by cluster analysis, the differences in these neurotransmitter patterns are further observed by heat map analysis, and correlation analysis is performed. Figure 3Figure 1 shows the principal component analysis of 46 cognitive-related neurotransmitters in the blood of the Con, Mod and KF7 groups. Figure 2 shows the principal component analysis of 37 cognitive-related neurotransmitters in the blood of the Con, Mod and KF7 groups. Figure 3 shows the correlation analysis of the 37 cognitive-related neurotransmitters and the cognitive index of the mice. Figure 4 shows the linear fitting of the concentration of indole-3-lactic acid in the blood of the mice and the cognitive index of the mice. Figure 5 shows the linear fitting of the concentration of indole-3-lactic acid in the blood of the mice and the time spent in the new object recognition. In Figure 2, Cluster 1 is represented by indole-3-acetic acid, vasopressin and L-tryptophan; Cluster 2 is represented by β-alanine and propanoic acid; Cluster 3 is represented by dopamine and indolelactic acid; Cluster 4 is represented by glutamic acid and 3-indolepropionic acid; and Cluster 5 is represented by phenyethylamine and 2-aminophenol.

[0138] From Figure 3 Figures 1 and 2, it can be seen that by analyzing 46 cognitive-related neurotransmitters by LC-MS / MS, the principal component analysis of 37 detectable neurotransmitters shows that the spectral patterns of the Con, Mod and KF7 groups are similar.

[0139] From Figure 3 Figure 3, it can be seen that the correlation analysis shows that only two neurotransmitters, indolelactic acid (ILA) and kynurenine, are significantly correlated with the new object recognition index and the time spent in the target quadrant of the three groups of mice. The stars represent significance.

[0140] From Figure 4 Figures 4 and 5, it can be seen that the correlation coefficients of ILA and the recognition index and the time spent in the new object are 0.56 and 0.61, respectively, indicating that ILA is positively correlated with the cognitive level, and when the level of ILA is higher, the cognitive performance is likely to be better. In summary, the strain KF7 is significantly positively correlated with the cognitive level of the aging mice in the brain ILA level.

[0141] 2.3.6, Effect on the phenotype of mice

[0142] Total RNA of brain tissue (n=3) of each group of mice was purified using RNApure Fast Tissue and Cell Kit (Product No: CW0599S, CWBIO, Beijing, China), and the quality of the RNA was checked by ND-1000 (NanoDrop), Bioanalyzer 2100 (Agilent) and agarose gel electrophoresis. Polyadenylated mRNA was captured by magnetic beads (Product No: 88802, Thermo Fisher, Waltham, USA), and fragmenting treatment was performed using a magnesium ion RNA fragmentation module. Sequencing libraries with a fragment size of 300 ± 50 bp were constructed and sequenced using NovaSeqXPlus in PE150 sequencing mode.

[0143] The raw data was processed using Cutadapt-1.9 software to remove low-quality spliced sequences and repetitive sequences. The cleaned data was aligned to the genome by HISAT2-2.0.4 (https: / / daehwankimLab.github.io / hisat2 / ). The differential expression analysis between groups was performed using the negative binomial distribution of the DESeq2 R package (based on http: / / www.bioconductor.org / packages / release / bioc / vignettes / DESeq2 / inst / doc / DESeq2.htmL). Genes with log2|fold change|≥1 and P<0.05 were defined as differentially expressed genes (DEGs), and KOBAS (http: / / bioinfo.org / kobas) was used for KEGG enrichment analysis. Figure 4 Fig. A is a volcano plot of differentially expressed genes, Fig. B is a distribution plot of differentially expressed genes in cellular biological processes, Fig. C is a classification plot in BP biological processes, Fig. D is a KEGG pathway analysis plot of up-regulated differentially expressed genes, Fig. E is a KEGG pathway analysis plot of down-regulated differentially expressed genes, Fig. F is a gene enrichment analysis (GSEA) plot, Fig. G is an inflammatory signaling pathway plot, red color up-regulated, blue color down-regulated, Figs. I-K are mRNA expression level plots of Prl, Mapk15 and Nrf2 respectively, and Fig. H is the mechanism of action.

[0144] From Figure 4 As can be seen from Fig. A, the volcano plot of differentially expressed genes shows that there are significant differences in gene clustering between Mod mice and KF7 mice. Between Mod and KF7 groups, a total of 73 up-regulated differentially expressed genes (DEGs) and 125 down-regulated DEGs were identified.

[0145] From Figure 4As shown in Figure B, GO enrichment analysis of differentially expressed genes revealed significant enrichment in 190 BP (biological process) entries, 38 MF (metabolic function) entries, and 29 CC (cell partition) entries.

[0146] from Figure 4 As shown in C, in the significantly enriched BP term clusters, the largest module is related to "response", while the second largest module is mainly related to "regulation".

[0147] from Figure 4 As can be seen from D, KEGG pathway analysis showed that up-regulated differentially expressed genes (DEGs) are associated with multiple signaling pathways, including the prolactin (PRL) signaling pathway and the MAPK signaling pathway.

[0148] from Figure 4 As can be seen from the data, KEGG pathway analysis showed that down-regulated differentially expressed genes (DEGs) were more strongly associated with inflammatory signaling pathways, such as the T cell receptor signaling pathway, cytokine-cytokine receptor interaction, and Staphylococcus aureus infection.

[0149] from Figure 4 Further gene set enrichment analysis (GSEA) showed that KF7 suppressed the inflammatory response induced by D-galactose injection.

[0150] from Figure 4 As shown in the study, the PRL and MAPK signaling pathways identified by ORA were significantly upregulated in GSEA. After KF7 treatment, the expression levels of key genes in the PRL, MAPK, and NRF2 pathways (prolactin (Prl), prolactin receptor (Prlr), Mapk15, and Nrf2) were significantly increased in brain tissue.

[0151] from Figure 4 As can be seen from the results of RT-qPCR, the differential expression patterns of these genes were confirmed (P<0.05).

[0152] from Figure 5 Based on the above results, the molecular mechanism of cognitive impairment reduction mediated by L. rhamnosus KF7 was initially constructed.

[0153] 2.3.7 Effects on the non-targeted metabolome of mouse feces

[0154] Fecal metabolome analysis of mice was performed according to the conventional method and condition. It includes as follows:

[0155] Extraction step and condition: 1 mL of 70% volume ratio of pre-cooled to 4°C methanol was added to 2.0 mL centrifuge tube containing 100 mg of fecal sample, the sample was extracted at 4°C overnight, then centrifuged at 10,000 x g for 10 min, and the supernatant was immediately collected. CNWBOND Carbon-GCB SPE column (250 mg, 3 mL) was used and adsorbed by ANPEL (Shanghai, China), and the extract was then further analyzed by LC-ESI-MS / MS system. The identified metabolites of sample extract were annotated by HMDB (https: / / hmdb.ca / ) and KEGG database (http: / / www.kegg.jp / kegg / compound / ). The significantly differentially expressed metabolites between groups were determined by VIP value ≥ 1 and P value ≤ 0.05. For a given list of metabolites, significantly enriched pathways were identified by p value (<0.05). The results are shown in Figure 5 . Figure 5 Figure A, principal component analysis (PCA) of metabolites, abscissa: first principal component (maximum contribution to difference), ordinate: second principal component (second contribution to difference); Figure B, orthogonal partial least squares discriminant analysis (OPLS-DA), abscissa: first principal component, which is the principal component that removes all components orthogonal to Y, that is, the principal component that uses all variables related to the grouping factor for prediction, ordinate: orthogonal principal component, which is the variable information unrelated to Y, the feature is that there is always an abbreviation of Orthogonal on this coordinate, the abscissa represents the difference between groups, and the ordinate represents the difference between samples within the group, Figure C, Variable Importance in Projection (VIP), the greater the VIP of a metabolite, the more important it is for distinguishing different groups, and the more worthy of attention, generally VIP > 1 is considered as one of the criteria for screening differential metabolites, blue balls represent statistically significant differential metabolites, DEMs are differential expression metabolites; Figure D, distribution of differential metabolites (DEMs); Figure E, differential expression metabolites (DEMs) obtained by KEGG enrichment analysis; Figure F, heat map of the top 40 differential expression metabolites; Figure G, correlation of the top 40 differential expression metabolites with three cognitive behavior indicators; Figure H, concentration of ILA in brain tissue of 4 groups of different treated mice; Figure I, correlation of fecal ILA with ILA in brain tissue; Figure J, effect pathway of KF7 improved cognitive function after modification.

[0156] From Figure 5From Table A-B, firstly, the non-targeted fecal metabolome of Mod and KF7 groups of mice was characterized. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) showed that there were significant differences in metabolite composition between KF7 and Mod groups.

[0157] From Table C, from the study of 135 differentially expressed metabolites, 57 significantly up-regulated metabolites and 78 significantly down-regulated metabolites were identified. Figure 5

[0158] From Table D, the 135 differentially expressed metabolites were mainly lipid and lipid-like molecules (27.41%), organic acids and their derivatives (20.74%), organic heterocyclic compounds (12.59%), etc. Figure 5

[0159] From Table E, by ORA (Over-Representation Analysis, study of the relevance between gene sets and biological functions, pathways or traits), the relevance of these differentially expressed metabolites (DEMs) to “Tryptophan catabolism” and “Tryptophan metabolism” was further confirmed. Figure 5 Tryptophan is a key essential amino acid for protein synthesis in the human body, and has become a key substance in the microbiota-gut-brain axis, and tryptophan metabolism imbalance can impair the cognitive function of the body.

[0160] From Table F, the heatmap analysis showed that there were L-tryptophan and indole-3-lactic acid (ILA) in the top 40 up-regulated differentially expressed molecules, and these molecules had been identified in the previous LC-MS analysis.

[0161] Figure 5 Subsequently, the correlation between these differentially expressed molecules and the behavioral indicators of mice was analyzed, including the recognition index, the new object residence time and the target quadrant residence time (G).

[0162] From Table G, PS (22:2 (13Z, 16Z) / 16:0), Xanthoxylin, L-Phenylalanine and Indole-3-lactic acid (ILA) all showed highly significant correlation with the three behavioral indicators (P<0.001). Figure 5

[0163] From Table H, the correlation between the differentially expressed molecules and the behavioral indicators of mice was analyzed, including the recognition index, the new object residence time and the target quadrant residence time (G). Figure 5

[0164] ​​​​​Given that indole-3-lactic acid (ILA) levels in the brain are associated with improved behavior in mice, an attempt was made to establish a correlation curve between brain indole-3-lactic acid (ILA) and fecal indole-3-lactic acid (ILA).

[0165] From Figure 5 it can be seen that the results show a significant positive correlation between the two.

[0166] From Figure 5 it can be seen that the ELISA results show that the brain indole-3-lactic acid (ILA) levels of mice in the Mod group were significantly lower than those in the Con group ( Figure 6 I). Supplementing L. rhamnosus KF7 significantly restored this trend (P < 0.05), while donepezil did not show a similar restorative effect (P > 0.05).

[0167] Based on the results of this part, the possible molecular mechanism by which L. rhamnosus KF7 exerts its effect was found ( Figure 6 J), which acts on indole-3-lactic acid (ILA) in the brain, then on the small molecule Prl located in the pituitary, and then on the molecule Prlr located in the hippocampus, affecting Mapk15, Nrf2.

[0168] 2.3.8. Effect on mouse fecal microbiota

[0169] 16S rRNA gene sequencing was performed according to the conventional method and condition. It includes the following:

[0170] DNA was extracted from mouse fecal samples using a bacterial genomic DNA kit (Product No. CW0552, CWBIO, Beijing, China). 16S rRNA sequencing was performed using the Illumina system (Illumina Inc., California, USA). Sequencing and bioinformatics analysis were completed by Majorbio Biopharm Technology Co., Ltd. (Shanghai, China). Operational taxonomic units (OTUs) were identified using USEARCH software (https: / / www.drive5.com / usearch). Microbial diversity was assessed by principal component analysis (PCA) and covariance analysis (PCoA) and microbial dysbiosis index (MDI) analysis. Differences in species abundance between groups were assessed by linear discriminant analysis effect size (LEfSe, http: / / galaxy.biobakery.org / ). PICRUSt2 (http: / / huttenhower.sph.harvard.edu / galaxy) was used to predict functional differences in different bacterial communities.

[0171] The effect of L. rhamnosus KF7 on the intestinal microbiota of mice treated with D-galactose was analyzed, and the key microorganisms that can promote the production of indole-3-lactic acid (ILA) were determined.

[0172] Figure 6 Figure 5A is a Chao1 curve of the 16S RNA sequencing results of the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group); Figure 6 Figure 5B is a PCA analysis of the composition of the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group); Figure 6 Figure 5C is an OPLS-DA analysis of the composition of the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group); Figure 6 Figure 5D is an MDI analysis of the 16S RNA sequencing results of the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group), and the higher the value, the more obvious the imbalance; Figure 6 Figure 5E is the relative abundance of the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group) at the phylum level; Figure 6 Figure 5F is the relative abundance of Bacteroidota in the feces of three groups of mice (Con group, Mod group, and KF-7 group); Figure 6 Figure 5G is the relative abundance of Firmicutes in the feces of three groups of mice (Con group, Mod group, and KF-7 group); Figure 6 Figure 5H is the F / B ratio of the relative abundance of Bacteroidota and the relative abundance of Firmicutes in the feces of three groups of mice (Con group, Mod group, and KF-7 group); Figure 6 Figure 5I is the relative abundance of the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group) at the genus level; Figure 6 Figure 5J is the relative abundance of Lactobacillus in the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group); Figure 6 Figure 5K is the relative abundance of Saccharomonas in the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group); Figure 6 Figure 5L is a species ternary analysis of the fecal microbiota of three groups of mice (Con group, Mod group, and KF-7 group) at the species level; Figure 6M, N and O are the relative abundance of Lactobacillus murinus, Lactobacillus johnsonii and Lactobacillus reuteri in the fecal microbiota of three groups of mice (Con group, Mod group and KF-7 group), respectively; Figure 6 P is the evolutionary branch diagram obtained by LEfSe analysis of the fecal microbiota of three groups of mice (Con group, Mod group and KF-7 group); Figure 6 Q is the main class of intestinal flora of mice in the KF-7 group; Figure 6 R is the correlation between the relative abundance of three Lactobacillus in the fecal microbiota of the KF-7 group, Lactobacillus murinus, Lactobacillus johnsonii and Lactobacillus reuteri, and the level of indole-3-lactic acid (ILA) in the brain (Brain) / feces (Fecal); Figure 6 S is a scatter plot showing the correlation between the relative abundance of Lactobacillus murinus in the fecal microbiota of the KF group and the level of ILA in the brain (Brain) / feces (Fecal); Figure 6 T is the pathway diagram of Rhamnosus KF7 slowing down the cognitive impairment caused by D-galactose.

[0173] From Figure 6 As can be seen from A, the Chao curve of 16S rRNA gene sequence rises rapidly and then remains almost unchanged, indicating that the depth of sequencing meets the requirements.

[0174] From Figure 6 As can be seen from B-C, through principal component analysis (PCA) and covariance analysis (PCoA), according to the β-diversity analysis, the changes of OTUs between the Con group, Mod group and KF7 group can be seen through principal component analysis and principal coordinate analysis, and the intestinal flora of the three groups of mice has significant differences, and after supplementing the strain KF7, the intestinal flora of mice tends to be normal.

[0175] Microbial dysbiosis index (MDI) is used to quantify the severity of microbial imbalance, and the higher the value, the more obvious the imbalance.

[0176] From Figure 6 As can be seen from D, through microbial dysbiosis index (MDI) analysis, it is found that D-galactose significantly increases the MDI index, and after administration of strain KF7, the MDI is significantly reduced (P<0.05).

[0177] from Figure 6 As shown in the study, analysis of the relative abundance of fecal microbiota at the phylum level in three groups of mice (Con group, Mod group, and KF-7 group) using LEfSe revealed that Firmicutes, Bacteroidetes, Verrucomicrobiota, and Patescibacteria constituted the main components of the fecal microbiota.

[0178] from Figure 6 According to the results from FG, further analysis using LEfSe on the relative abundance of Firmicutes and Bacteroides in the fecal microbiota of the three groups of mice (Con group, Mod group, and KF-7 group) revealed that compared with the Con group, the relative abundance of Bacteroides increased in the Mod group, while the relative abundance of Firmicutes decreased. Furthermore, the administration of strain KF7 reduced the relative abundance of Bacteroides while increasing the relative abundance of Firmicutes, indicating that regulating Firmicutes and Bacteroides can improve cognitive impairment.

[0179] from Figure 6 As shown in Figure H, further calculation of the ratio of Firmicutes to Bacteroidetes revealed a significant decrease in the F / B ratio in the Mod group (P<0.05), while the F / B ratio in the KF7 group increased. Figure 6 An imbalance in the gut microbiota usually leads to a decrease in the fat-to-bacteria ratio.

[0180] from Figure 6 As shown in Figure I, analysis of the relative abundance of fecal microbiota at the genus level in three groups of mice (Con, Mod, and KF-7) using LEfSe revealed that Lactobacillus and Akkermansia constituted the main components of the microbial community. In Figure I, norank_f_Muribaculaceae refers to various rodentarian bacilli that have not been classified into ranks.

[0181] from Figure 6From Table K and J, by analyzing the relative abundance of the fecal microbiota of the three groups of mice (Con group, Mod group and KF-7 group) through LEfSe, it was found that the use of KF7 reversed the decrease in the number of Lactobacillus and Saccharomonas (C) caused by D-galactose, and the effect on Lactobacillus was more significant (P < 0.05).

[0182] From Figure 6 Table L, by three-dimensional analysis at the species level, it was found that at the species level, the number of Lactobacillus murinus, Lactobacillus johnsonii and Lactobacillus reuteri in the Con group and the KF7 group increased significantly.

[0183] From Figure 6 Table M-O, by analyzing the relative abundance of Lactobacillus murinus, Lactobacillus johnsonii and Lactobacillus reuteri in the fecal microbiota of the three groups of mice (Con group, Mod group and KF-7 group) through LEfSe, it was found that the use of KF7 reversed the decrease in the number of L.murinus, L.johnsonii and L.reuteri species induced by D-galactose, and the effect on L.murinus was more significant (P < 0.05).

[0184] From Figure 6 Table P, further by analyzing the evolutionary branch diagram of the fecal microbiota of the three groups of mice (Con group, Mod group and KF-7 group) through LEfSe, to evaluate the distribution of dominant bacteria in the KF7 group.

[0185] From Figure 6 Table Q, by analyzing the LDA value of the intestinal flora of mice in the KF7 group through LEfSe, among the top 10 LDA score classifications, the Lactobacillaceae family, Lactobacillales order, Lactobacillus genus and Lactobacillus murinus species played a role in the KF7 group, which was different from other groups.

[0186] From Figure 6The results showed that by analyzing the correlation between the relative abundance of lactobacilli in the KF7 group and the fecal / brain ILA level, a significant positive correlation was found between Lactobacillus murinus and Lactobacillus reuteri and the fecal / brain indole-3-lactic acid (ILA) level (P<0.05).

[0187] from Figure 6 As shown in the analysis, by analyzing the correlation between the relative abundance of Lactobacillus murinus and the fecal / brain ILA level, the correlation coefficients between Lactobacillus murinus and fecal / brain levels were 0.83 and 0.54, respectively.

[0188] from Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 Figure 6 The study revealed that strain KF7 increased the number of *Lactobacillus* species (L. murinus) in the gut of D-galactose-induced aging mice, thereby increasing the concentration of its metabolite indole-3-lactic acid (ILA) in the gut and brain. Furthermore, indole-3-lactic acid (ILA) may promote MAPK / ERK branching by activating the prolactin signaling pathway (the prolactin signaling pathway refers to the binding of prolactin Prl to its receptor Prlr), ultimately promoting Nrf2 nuclear translocation and alleviating neuroinflammation and oxidative stress.

[0189] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Use of Lactobacillus rhamnosus or a culture thereof in the preparation of a product for preventing, improving and / or treating cognitive impairment associated with aging.

2. Use according to claim 1, characterized in that, The Lactobacillus rhamnosus is Lactobacillus rhamnosus KF7 strain, whose accession number is CGMCC No. 6430.

3. Use according to claim 2, characterized in that, The culture of the Lactobacillus rhamnosus comprises a fermentation broth and / or viable bacteria of the Lactobacillus rhamnosus; and / or, the viable cell number of the Lactobacillus rhamnosus is not less than 10 6 CFU / g, based on the total mass of the product.

4. Use according to claim 3, characterized in that, The culture of the Lactobacillus rhamnosus is obtained by inoculating the Lactobacillus rhamnosus into a culture medium and culturing.

5. The use according to claim 1, characterized in that, The product has at least one of the following functions: 1) inhibiting the level of inflammatory factors in the brain tissue of a mammal; 2) increasing the level of anti-inflammatory factors in the brain tissue of a mammal; 3) improving the level of oxidative stress in the brain tissue of a mammal; 4) increasing the content of indole-3-lactic acid in the brain tissue of a mammal; 5) increasing the content of indole-3-lactic acid in the feces of a mammal; 6) inhibiting the activity of monoamine oxidase in the brain tissue of a mammal; 7) regulating the intestinal flora of a mammal.

6. Use according to claim 5, characterized in that, The mammal includes a human; And / or, the inflammatory factors include IL-1β, IL-6 and TNF-α; And / or, the anti-inflammatory factors include IL-10; And / or, the improvement of the level of oxidative stress includes increasing the levels of Sod1 and Gshpx and reducing the content of MDA; And / or, the regulation of the intestinal flora includes reducing the relative abundance of Bacteroidota and increasing the relative abundance of Firmicutes; And / or, the product is selected from one or more of functional food, bacterial agent, medicine, health product and medical raw material.

7. Use according to claim 6, characterized in that, The functional food is a fermented food prepared by fermentation of Lactobacillus rhamnosus; And / or, the dosage form of the bacterial agent is selected from one or more of lyophilized powder, capsule, tablet and granule.

8. Use according to claim 7, characterized in that, The preparation method of the functional food is that Lactobacillus rhamnosus is fermented in raw milk to obtain; And / or, the bacterial agent is a lyophilized powder obtained by drying the culture of Lactobacillus rhamnosus.

9. Use according to claim 8, characterized in that, The raw milk is selected from one or both of raw milk and reduced milk; And / or, the fermentation temperature is 30-40℃; And / or, the fermentation time is 12-30h.

10. Use according to claim 9, characterized in that, The reduced milk is an aqueous solution of skimmed milk powder, and the concentration of the aqueous solution of skimmed milk powder is 6-20wt%.

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