New application of lactobacillus delbrueckii subsp. Lactis strain

Abstract

The present invention relates to a composition for preventing, alleviating or treating cognitive disorders or Alzheimer's disease, comprising a Lactobacillus delbrueckii subsp. Lactis strain as an active ingredient. The present invention provides a composition for preventing, alleviating or treating cognitive disorders or Alzheimer's disease (AD), comprising a Lactobacillus delbrueckii subsp. Lactis strain as an active ingredient. The strain of the present invention has excellent effects of reducing amyloid beta protein (A beta) or tau protein (Tau) and improving cognitive function, and thus can be usefully used as a composition for preventing, alleviating or treating cognitive impairment or AD.

Description

Technical Field

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0144826, filed on November 2, 2022, and all contents disclosed in the corresponding Korean patent application document are incorporated herein as a part of this specification.

[0002] The present invention relates to a composition for preventing, improving or treating cognitive impairment or Alzheimer's disease. The composition comprises a Lactobacillus delbrueckii subsp. lactis strain as an active ingredient. Background Art

[0003] Alzheimer's disease (AD) is a degenerative brain disease characterized by a decline in cognitive function and is the most common form of dementia. AD gradually impairs various cognitive functions, including memory, language, judgment, and thinking, causing serious problems and inconvenience in daily life, but the exact cause and mechanism of the disease remain unknown. According to reports to date, abnormal accumulation of amyloid beta protein (Aβ) and tau protein (Tau) in brain tissue, decreased acetylcholine (ACh) due to the continued action of acetylcholinesterase (AChE), genetic factors, nerve cell damage caused by deterioration of blood-brain barrier function, and the resulting overactivation of immune cells in the brain are generally recognized as the main causes of cognitive decline. Therefore, most of the AD treatment drugs currently used are aimed at treating problems with signal transmission between brain nerve cells, and research on treatment development is also mainly limited to the brain.

[0004] Recent studies have found that intestinal microbes are related to the onset or treatment of brain diseases. Specifically, intestinal microbes play an important role in the two-way communication between the intestine and the brain through the endocrine, nervous and immune systems. That is, changes in the composition and metabolites of intestinal microbes not only directly affect appetite, sleep and mood regulation, but also affect brain functions such as memory and learning through the gut-brain axis mediated by systemic immunity, hormones and neurotransmitters. As the correlation between the intestine and the brain becomes clearer, research on the impact of intestinal microbes on brain diseases is being actively carried out.

[0005] However, even within the same species of lactic acid bacteria, which make up over 80% of the normal gut microbiome, microorganisms exhibit strain-specificity, exhibiting different biological activities depending on the strain. Therefore, the development of drugs for treating cognitive impairment or AD using these microorganisms remains challenging. Summary of the Invention

[0006]

Existing Technology Documents

[0007] (Patent Document 1) Korean Patent No. 10-2128098 (published on June 30, 2020)

[0008]

Technical Issues

[0009] By utilizing intestinal microorganisms to conduct various studies on the prevention, improvement or treatment of cognitive impairment or Alzheimer's disease (AD), the inventors have experimentally demonstrated that the Lactobacillus delbrueckii subsp. lactis strain can improve intestinal barrier permeability, reduce amyloid β protein (Aβ) and tau protein (Tau) (markers of brain degenerative diseases such as AD), inhibit the activity of reactive oxygen species (ROS) or acetylcholinesterase (AChE) to protect nerve cells, reduce inflammatory responses, and improve short-term memory ability, spatial perception ability or cognitive function in animal models, thereby completing the present invention.

[0010]

Technical solution

[0011] The present invention provides a pharmaceutical composition for preventing or treating cognitive impairment or Alzheimer's disease (AD). The pharmaceutical composition comprises Lactobacillus delbrueckii subsp. lactis deposited with the accession number KCTC14149BP as an active ingredient.

[0012] The term "Lactobacillus delbrueckii subsp. lactis strain" used in this specification refers to the strain deposited by the applicant with the Korea Institute of Biotechnology under the deposit number KCTC14149BP on March 3, 2020. The term "Lactobacillus delbrueckii subsp. lactis strain" in this specification can be used interchangeably with "Lactobacillus delbrueckii subsp. lactis," "Lactobacillus delbrueckii," "microbial cell," or "strain."

[0013] The term "strain" as used in this specification includes living cells, dead cells, cultures thereof, lysates, extracts, and cytoplasmic fractions. It also includes materials that have been post-processed or processed, such as by filtration, concentration, drying, extraction, or freezing. The term "culture" as used in this specification includes the strain itself, metabolites of the strain, the entire culture medium, and the culture fluid or culture supernatant after culturing the strain. There are no particular restrictions on the culture method, extraction method, separation method, concentration method, drying method, and dilution method of the strain.

[0014] The culture medium used for culturing microbial cells generally includes skim milk and other milk proteins, whey, casein, sugar, yeast extract, etc., and the culture method can adopt various commonly used aerobic or anaerobic methods.

[0015] For example, the culture temperature can be set to 35°C to 45°C. During the culture period, a neutralization culture method can be used, in which the pH of the culture medium is maintained at an acidic level, for example, a pH of 5 to 6, by using an alkali such as sodium hydroxide. In addition to this neutralization culture method, any appropriate culture method can be used, such as batch culture. After culture, the culture or its supernatant can be concentrated, dried, or diluted as needed.

[0016] Alternatively, the culture supernatant can be separated from the microbial cells by centrifugation or membrane separation, and the cell mass can be recovered in a concentrated state. Furthermore, the microbial cells can be subjected to ultrasonic treatment or enzyme treatment to extract components from the cells, and the culture fluid, its supernatant, microbial cells, or their extracts can be dried. These can be used as active ingredients in the compositions according to the present invention.

[0017] The physical properties of the cell mass of Lactobacillus delbrueckii subsp. lactis according to the present invention are as follows:

[0018] - Bacterial morphology: Gram-positive, rod-shaped or spherical;

[0019] - Physiological characteristics: The optimal growth temperature is 36°C to 38°C, the optimal pH is 5.5 to 5.8, and it is facultatively anaerobic. It is not motile and does not form spores; and

[0020] - Commonly isolated from fermented milk or cheese and considered safe for use in food (probiotics and dairy products).

[0021] The biochemical characteristics of Lactobacillus delbrueckii subsp. lactis are shown in Table 1. The 16S rRNA sequence of the strain is represented by SEQ ID NO: 1.

[0022] [Table 1]

[0023]

[0024]

[0025] In the present invention, Lactobacillus delbrueckii subsp. lactis can be used to prevent or treat cognitive impairment or AD.

[0026] The term "cognitive impairment" used in this specification refers to a disease that presents symptoms of decreased memory, attention, spatial perception ability, language ability, judgment, or a combination thereof.

[0027] In one embodiment of the present invention, cognitive impairment may be a disease caused by the accumulation of amyloid β protein (Aβ) or tau protein (Tau). Aβ may be soluble Aβ or insoluble Aβ, and specific examples include Aβ 40 , Aβ 42 or a combination thereof, but not limited thereto.

[0028] Tau can be soluble Tau or insoluble Tau, and can be phosphorylated Tau. Specific examples include phosphorylated Tau (Thr231), phosphorylated Tau (Ser202, Thr205), phosphorylated Tau (Thr181), phosphorylated Tau (Thr212, Ser214), phosphorylated Tau (Ser396), phosphorylated Tau (Ser422), etc., but are not limited thereto.

[0029] The disease caused by Aβ accumulation may be one or more selected from the group consisting of AD, Parkinson's disease dementia, Lewy body dementia, Huntington's disease dementia, preclinical Alzheimer's disease and Down syndrome, and the disease caused by Tau accumulation may be one or more selected from the group consisting of corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Pick's disease and frontotemporal dementia (FTD), but is not limited thereto.

[0030] The term "Alzheimer's disease (AD)" as used herein refers to the degenerative brain disease that represents the most common form of dementia. AD involves progressive memory loss. As the disease progresses, various cognitive functions, such as language, judgment, and reasoning, also decline, ultimately causing serious problems in daily life.

[0031] In the present invention, Lactobacillus delbrueckii subsp. lactis inhibits the production of Aβ or Tau in brain tissue.

[0032] In one embodiment of the present invention, the composition inhibits the activity of reactive oxygen species (ROS) or acetylcholinesterase (AChE) and protects nerve cells.

[0033] In one embodiment of the present invention, the composition inhibits inflammatory responses by reducing inflammatory cytokines such as nitric oxide (NO) and tumor necrosis factor-α (TNF-α).

[0034] In one embodiment of the present invention, the composition improves short-term memory ability, spatial perception ability or cognitive function.

[0035] In one embodiment of the present invention, the composition has the effect of reducing the permeability of the intestinal barrier.

[0036] In one embodiment of the present invention, the composition shows an increase in one or more intestinal microbiota selected from the group consisting of Erysipelotrichaceae, Erysipelotrichales, Turicibacter, Peptostreptococcales Tissierellales, Anaerovoracaceae and Eubacterium xylanophilum.

[0037] In one embodiment of the present invention, the composition shows a reduction in one or more intestinal microbiota selected from the group consisting of Peptococcales and Peptococaceae.

[0038] It is known that the gut microbiota may regulate neuroinflammation in various neurological diseases, including multiple sclerosis, Parkinson's disease, and AD. Changes in the gut microbiota can alter microbial-derived metabolites and peripheral immunity, which are associated with neurological diseases, and this may alter the immune response of the central nervous system (Sidhanth Chandra et al., Molecular Neurodegeneration 18(9), February 1, 2023).

[0039] The pharmaceutical composition of the present invention can be administered to a subject in need of preventing or treating cognitive impairment or AD by comprising an effective amount of the above-mentioned strain.

[0040] The term "administer" as used herein refers to the physical introduction of a composition into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Administration can be performed, for example, orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, spinally, or by other non-oral administration, such as by injection or infusion, but is not limited thereto. Administration can be, for example, a single administration, multiple administrations, or over one or more extended periods of time.

[0041] The pharmaceutical composition of the present invention can be formulated into various oral or non-oral administration forms as described below, but is not limited thereto.

[0042] Oral administration forms include, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsions, syrups, granules, and elixirs. In addition to the active ingredients described above, these formulations may also use one or more diluents or excipients, such as fillers, extenders, wetting agents, disintegrants, lubricants, binders, or surfactants. Disintegrants may include agar, starch, alginic acid or its sodium salt, anhydrous calcium dihydrogen phosphate, and the like; lubricants may include silicon dioxide, talc, stearic acid or its magnesium or calcium salt, polyethylene glycol, and the like; and binders may include magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, and low-substituted hydroxypropyl cellulose. Furthermore, lactose, glucose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like may be used as diluents, and, depending on the circumstances, conventionally known additive mixtures, absorbents, colorants, flavorings, sweeteners, and the like may also be used.

[0043] The composition may be sterile or may contain preservatives, stabilizers, hydrating agents or emulsifying promoters, salts for adjusting osmotic pressure, buffers and other therapeutic substances, and may be formulated according to conventional methods such as mixing, granulating or coating.

[0044] The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity.

[0045] According to the method that those skilled in the art can easily implement according to the present invention, the pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable carriers and / or excipients, and be prepared into unit dosage forms or be included in multidose containers. In this case, the preparation can be in the form of a solution, suspension, syrup or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and can additionally include a dispersant or stabilizer. The pharmaceutical composition of the present invention can only include the strain, or can also include the appropriate carrier, excipient or diluent conventionally used in the preparation of the pharmaceutical composition. Specifically, the carrier, excipient and diluent that can be included in the pharmaceutical composition can be, for example, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, but are not limited thereto. These can be used alone or in combination of two or more. In addition, if necessary, the pharmaceutical composition may further comprise other commonly used additives, such as antioxidants, wetting agents, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, buffers and / or antibacterial agents. It may further comprise dispersants, surfactants, binders, lubricants, etc., and may be formulated into powders, granules, tablets, liquids, capsules, suspensions, emulsions, syrups, creams, lotions, gels, ointments, aerosols, powder sprays, creams, suppositories, pills, etc.

[0046] Another aspect of the present invention provides a method for preventing or treating cognitive impairment or AD, comprising administering the Lactobacillus delbrueckii subsp. lactis strain deposited under the accession number KCTC14149BP.

[0047] In the prevention or treatment method according to the present invention, unless otherwise specified, each term has the same meaning as described in the pharmaceutical composition.

[0048] In the method for preventing or treating cognitive impairment or AD according to the present invention, the Lactobacillus delbrueckii subsp. lactis strain deposited under the accession number KCTC14149BP can be administered to the subject simultaneously, sequentially or separately with other therapeutic agents.

[0049] "Simultaneous" administration refers to the simultaneous administration of the Lactobacillus delbrueckii subsp. lactis strain of the present invention and the other therapeutic agent in a single formulation, or the simultaneous administration of the strain and the other therapeutic agent in separate formulations, in which case the routes of administration of the strain and the other therapeutic agent may be different. "Sequentially" administration refers to the relatively continuous administration of the strain and the other therapeutic agent, allowing only the minimum time required between administrations. "Separately" administration refers to the administration of the strain and the other therapeutic agent with a certain time interval between administrations. The method of administration of the strain and the other therapeutic agent can be appropriately selected by a physician or expert in the field based on the therapeutic effect, side effects, etc. on the subject.

[0050] Another aspect of the present invention provides a food composition for preventing or improving cognitive impairment or AD, wherein the food composition comprises the Lactobacillus delbrueckii subsp. lactis strain deposited under the accession number KCTC14149BP as an active ingredient.

[0051] In the food composition according to the present invention, unless otherwise specified, each term has the same meaning as described in the pharmaceutical composition.

[0052] The effective dose of the lactobacillus delbrueckii subspecies milk bacterial strain included in food compositions of the present invention can suitably be determined according to intended use (prevention, improvement or treatment).Usually, in food manufacturing process, the lactobacillus delbrueckii subspecies milk bacterial strain can be included in food compositions with the amount of 0.001 weight % to 20 weight %, 0.001 weight % to 15 weight % or 0.001 weight % to 10 weight %.For health beverage, with 100mL as benchmark, the content of bacterial strain can be 0.01g to 2g, be specially 0.02g to 2g, be more specifically 0.3g to 1g.But, when taking in for a long time in order to maintain health, hygiene or regulate health, can use the amount lower than above-mentioned scope.In the manufacture process of food compositions of the present invention, can suitably increase or reduce the content of the lactobacillus delbrueckii subspecies milk bacterial strain added in food compositions as required.

[0053] In addition, food composition can not only comprise lactobacillus, can also comprise the composition of conventional addition in food manufacturing process as active ingredient.Additional ingredients comprise for example protein, carbohydrate, fat, nutrient, flavoring and flavoring agent.Carbohydrate can comprise monosaccharide (for example glucose, fructose etc.), disaccharide (for example maltose, sucrose, oligosaccharide etc.) and polysaccharide (for example typical sugar such as dextrin, cyclodextrin and sugar alcohol such as xylitol, sorbitol, erythritol).As flavoring, natural flavoring (for example, thaumatin, stevia extract such as rebaudioside A, glycyrrhizin etc.) and synthetic flavoring (for example, saccharin, aspartame etc.) can be used.

[0054] For example, when the food composition of the present invention is prepared into a beverage, it may further comprise citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract or licorice extract in addition to the active ingredient strain of the present invention.

[0055] The food composition of the present invention includes all processed forms of natural materials, such as food, functional food, nutritional supplement, health food and food additives. Various types of food compositions can be manufactured into various forms according to conventional methods known in the art, and the conventional ingredients and materials added in this area can be added during the manufacturing process.

[0056] For example, as health food, milk-acid bacteria can be made into forms such as tea, fruit juice, beverage and drink, or also can be made into granular, capsule-like, powdered and take in.In addition, as food, milk-acid bacteria of the present invention can be added and manufactured into beverage, comprise alcoholic beverage, fruit and processed food thereof (for example, canned fruit, bottled fruit, jam, marmalade etc.), fish, meat and processed food thereof (for example, ham, sausage, corned beef etc.), bread and noodles (for example, udon, buckwheat noodles, hand-pulled noodles, spaghetti, macaroni etc.), fruit juice, various beverages, biscuits, toffee, milk-product (for example, sour milk, fermented milk, butter, cheese etc.), edible vegetable oil, margarine, vegetable protein, retort food, in frozen food and various flavorings (for example, soybean paste, soy sauce, sauce etc.).In addition, in order that milk-acid bacteria of the present invention is used with the form of food additives, it can be made into the form of pill, powder or concentrate.

[0057] In addition, unlike general medicines, food has the advantages of not having the side effects that may be caused by long-term drug administration and having excellent portability. Therefore, the food composition of the present invention can be used as an adjuvant to enhance or improve the preventive or therapeutic effects of cognitive impairment or Alzheimer's disease (AD), and can also be used simultaneously or sequentially with the pharmaceutical composition of the present invention and / or other compositions or therapies to maximize the above effects.

[0058] The present invention provides a composition for preventing, improving, or treating cognitive impairment or Alzheimer's disease (AD), comprising a Lactobacillus delbrueckii subsp. lactis strain as an active ingredient. The strain of the present invention has excellent efficacy in reducing amyloid β protein (Aβ) or tau protein (Tau) and improving cognitive function, and can therefore be effectively used as a composition for preventing, improving, or treating cognitive impairment or AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a branch diagram showing changes in the intestinal microbiota after administering the Lactobacillus delbrueckii subsp. lactis strain of the present invention to an Alzheimer's disease (AD) animal model. Green indicates an increase in bacteria, and red indicates a decrease in bacteria.

[0060] Figure 2 This figure shows changes in intestinal microbiota after administration of Lactobacillus delbrueckii subsp. lactis to AD animal models. NC refers to ADLP administered with PBS. APT mice, KCTC14149BP refers to ADLP administered with Lactobacillus delbrueckii subsp. APT mouse.

[0061] Figure 3 The images and graphs show the results of intestinal barrier permeability of experimental animals after Lactobacillus delbrueckii subsp. lactis was administered to AD animal models. WT refers to ADLP administered with PBS. WT Mice, NC refers to ADLP administered with PBS APT mice, KCTC14149BP refers to ADLP administered with Lactobacillus delbrueckii subsp. APT mouse.

[0062] Figure 4 The following are photos and graphs showing the results of immunostaining of amyloid β protein (Aβ) in brain tissue after administration of Lactobacillus delbrueckii subsp. lactis to AD animal models. In the immunostaining photos, blue indicates cell nuclei stained with DAPI, and green indicates Aβ plaques bound to the primary antibody. NC refers to ADLP administered with PBS. APT mice, KCTC14149BP refers to ADLP administered with Lactobacillus delbrueckii subsp. APT For mouse, 4G8 refers to the primary antibody biotin-labeled 4G8.

[0063] Figure 5 This figure shows the ELISA results of Aβ in brain tissue after Lactobacillus delbrueckii subsp. lactis was administered to AD animal models. NC refers to ADLP administered with PBS. APT mice, KCTC14149BP refers to ADLP administered with Lactobacillus delbrueckii subsp. APT Mouse, ** indicates P value < 0.01.

[0064] Figure 6 This is a graph showing the neuroprotective ability of this strain. NC refers to the control group, H2O2 refers to the H2O2-treated control group, MRS refers to the MRS-treated group, KCTC14149BP refers to the Lactobacillus delbrueckii subsp. lactis-treated group, and Soy-PS refers to the Soy-PS-treated group. *** indicates a P value < 0.001.

[0065] Figure 7This graph shows the antioxidant activity of Lactobacillus delbrueckii subsp. lactis, and shows the percentage (%) of glutathione content in nerve cells compared to the control group (NC). NC refers to the control group, H2O2 refers to the H2O2-treated control group, MRS refers to the MRS-treated group, KCTC14149BP refers to the Lactobacillus delbrueckii subsp. lactis-treated group, and Soy-PS refers to the Soy-PS-treated group. *** indicates a P value < 0.001.

[0066] Figure 8 This is a graph showing acetylcholinesterase (AChE) activity of Lactobacillus delbrueckii subsp. lactis. NC refers to the control group, H2O2 refers to the H2O2-treated control group, MRS refers to the MRS-treated group, KCTC14149BP refers to the Lactobacillus delbrueckii subsp. lactis-treated group, and Soy-PS refers to the Soy-PS-treated group. *** indicates a P value < 0.001.

[0067] Figure 9 The results of the anti-inflammatory activity of Lactobacillus delbrueckii subsp. lactis are shown, and the graph shows the amount of nitric oxide (NO) in microglia. NC refers to the control group, and Aβ refers to Aβ 1-42 Treatment control group, MRS refers to MRS treatment group, KCTC14149BP refers to Lactobacillus delbrueckii subsp. lactis treatment group, Soy-PS refers to Soy-PS treatment group, * indicates P value < 0.05, *** indicates P value < 0.001.

[0068] Figure 10 The results of the anti-inflammatory activity of Lactobacillus delbrueckii subsp. lactis are shown, and the amount of tumor necrosis factor-α (TNF-α) in microglia is shown. NC refers to the control group, and Aβ refers to Aβ 1-42 Treatment control group, MRS refers to the MRS treatment group, KCTC14149BP refers to the Lactobacillus delbrueckii subsp. lactis treatment group, Soy-PS refers to the Soy-PS treatment group, * indicates P value < 0.05, *** indicates P value < 0.001.

[0069] Figure 11 This graph shows the effect of Lactobacillus delbrueckii subsp. lactis on improving short-term memory and spatial perception in experimental animals in a scopolamine-induced cognitive impairment animal model, as measured by a Y-maze test. G1 indicates the control group, G2 indicates the vehicle-treated group, G3 indicates the donepezil-treated group, G4 indicates the purpurogenol-treated group, G5 indicates the low-dose Lactobacillus delbrueckii subsp. lactis-treated group, G6 indicates the medium-dose Lactobacillus delbrueckii subsp. lactis-treated group, and G7 indicates the high-dose Lactobacillus delbrueckii subsp. lactis-treated group. ## indicates a P value < 0.01 compared to G1, and ** indicates a P value < 0.01 compared to G2.

[0070] Figure 12 This graph shows the effect of Lactobacillus delbrueckii subsp. lactis on improving cognitive ability in experimental animals in a scopolamine-induced cognitive impairment animal model, as measured by the passive avoidance test. G1 represents the control group, G2 the vehicle-treated group, G3 the donepezil-treated group, G4 the purpurogenol-treated group, G5 the low-dose Lactobacillus delbrueckii subsp. lactis-treated group, G6 the medium-dose Lactobacillus delbrueckii subsp. lactis-treated group, and G7 the high-dose Lactobacillus delbrueckii subsp. lactis-treated group. ## indicates a P value < 0.01 compared to G1, * indicates a P value < 0.05 compared to G2, and ** indicates a P value < 0.01 compared to G2. DETAILED DESCRIPTION

[0071] The present invention will be described in more detail below by way of examples. These examples are provided solely to more specifically describe the present invention, and it will be apparent to those skilled in the art that, according to the gist of the present invention, the scope of the present invention is not limited by these examples. In this specification, unless otherwise expressly stated, singular forms also include plural forms.

[0072] Throughout the specification, unless otherwise indicated, "%" used to express the concentration of a particular substance means (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid.

[0073] Preparation Example 1: Preparation of probiotics

[0074] The Lactobacillus delbrueckii subsp. lactis used in this experiment was isolated from raw milk fermentation products. The lactic acid bacteria starter was cultured in a flask containing MRS liquid medium at 37°C for 24 hours. The starter was inoculated into the optimized homemade culture medium. The culture was carried out at 37°C for 18 to 20 hours under the following conditions: pH was maintained at 5.5 to 6.0 and the stirring speed was 55RPM to 65RPM. Freeze-drying of 40X concentrated cells was performed according to the instructions. After freeze-drying, the colony forming units (CFU) per 1g of probiotic powder were determined by serial dilution method. The probiotics were suspended in 1×PBS and the density was adjusted to 5×10 9 CFU / 200μL.

[0075] Preparation Example 2: Preparation of Alzheimer's disease (AD) animal model

[0076] A transgenic ADLP (Alzheimer's disease-like pathology) animal model was prepared by crossing i) 5XFAD mice (Tg6977, Jackson Laboratory, Stock #006554) expressing the human amyloid precursor protein (APP) gene with the Swedish (K670N / M671L), Florida (I716V), and London (V717I) mutations and the human presenilin-1 (PSEN1) gene with the M146L and L286V mutations under the control of the Thy1 promoter with ii) JNPL3 mice (TauP301L-JNPL3, Taconic, Stock #2508 homozygous) under the control of the prion protein promoter.

[0077] ADLP due to co-expression of three human mutant genes APT Female mice show Alzheimer's disease pathology and cognitive impairment earlier than male mice, so only female mice were used in this experiment.

[0078] Example 1: Changes in the composition of the intestinal microbiota

[0079] To determine whether the strain of the present invention can prevent, alleviate or treat cognitive impairment or AD by changing the intestinal microbiota, changes in the intestinal microbiota composition of AD animal models after oral administration of the strain were analyzed.

[0080] Specifically, give ADLP from 2 to 2.5 months old APT The mice were administered with a 200 μL volume of PBS suspension of Lactobacillus delbrueckii subsp. lactis (strain treatment group) or PBS alone (negative control, NC). WT A normal control group (wild type, WT) of mice was administered 200 μL of PBS orally five times a week for five months.

[0081] After administration, fecal samples were collected before the experimental animals were sacrificed and stored at -80 ° C until analysis. Genomic DNA was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, 51304). The V3-V4 region of the 16S rRNA gene was amplified by PCR using primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′). The amplified PCR products were purified using HiAccuBead (AccuGene, ACN01.50). Metagenomic sequencing was performed using the Ion Torrent S5 sequencing system.

[0082] The generated sequences were analyzed using the QIIME2 pipeline (version 2022.2) to identify the overall genetic information of the intestinal microbiome. In order to confirm the differences in microbial genus species between the strain administration group and the NC group, the Linear Discriminant Analysis (LDA) effect size (LEfSe) algorithm was used at the genus and species level. Figure 1 and Figure 2 As shown, in the strain-administered group, the intestinal distribution of Erysipelothrixaceae, Erysipelothrixales, Zurichia, Peptostreptococcus-Lachnospirales, Anaerobacillaceae, and Eubacterium xylaniphilum increased, while the intestinal distribution of Peptococcales and Peptococcaceae decreased.

[0083] Example 2: Reduced intestinal permeability

[0084] Patients with cognitive impairment or AD exhibit "leaky gut" symptoms, that is, impaired intestinal barrier function and reduced intestinal permeability, which activates systemic inflammatory responses and leads to cognitive decline (Vanessa Stadlbauer et al., BMC Geriatrics 20(248), 20July 2020).

[0085] Therefore, to evaluate whether the strain of the present invention can prevent, alleviate or treat cognitive impairment or AD by improving impaired intestinal barrier function, changes in intestinal permeability were evaluated in an AD animal model after oral administration of the strain.

[0086] Specifically, the strain was administered to an AD animal model according to the method of Example 1. Following administration, the animals were fasted for 4 hours before intestinal permeability assessment. 0.6 mg / g of a 4 kDa fluorescent substance was then orally administered. Two hours after administration of the fluorescent substance, the substance present in the intestine was observed, and the residual amount was measured to assess intestinal permeability.

[0087] The results are as follows Figure 3 As shown, it is confirmed that WT Compared with mice (WT), ADLP APT The amount of fluorescent substance remaining in the intestines of mice (NC) was significantly reduced. Compared to NC, the residual amount of fluorescent substance in the intestines of the strain-administered group increased. These results suggest that the Lactobacillus delbrueckii subsp. lactis strain of the present invention can prevent, alleviate, or treat cognitive impairment or AD by reducing increased intestinal permeability.

[0088] Example 3: Reduction of amyloid β protein (Aβ)

[0089] It is well known that cognitive impairment or AD is caused by the deposition of Aβ. Therefore, to evaluate whether the strain of the present invention can prevent, alleviate or treat cognitive impairment or AD, the changes in brain Aβ plaques in AD animal models after oral administration of the strain were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA).

[0090] 3-1. Immunohistochemistry

[0091] Specifically, the strain was administered to an AD animal model according to the method of Example 1. After administration, the experimental animals were anesthetized with a mixture of tiletamine-zolazepam and xylazine (1.2 mg / kg) and perfused with PBS. The brain tissue of the experimental animals was fixed with 4% paraformaldehyde for 24 hours, dehydrated in a 30% sucrose solution for about 72 hours, and then immunofluorescence staining was performed. The pretreated brain tissue was frozen at -80°C and then coronally sectioned using a microtome that can maintain -25°C. The brain tissue sections were washed with PBS, blocked and permeabilized, and then reacted with biotin-labeled primary antibodies for 4G8 (1:700, Covance, SIG-39240), GFAP (1:1,000, Invitrogen, 13-0300), and Iba1 (1:500, Wako, 019-19741). The brain tissue sections after reaction with the primary antibody were washed with PBS, then reacted with the fluorescently labeled secondary antibody, stained with DAPI, and observed under a confocal microscope. Figure 4 As shown, the area of Aβ plaques in the brain of the strain-administered group was significantly reduced by about 30% compared with the control group (NC).

[0092] These results indicate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention can prevent, alleviate or treat cognitive impairment or Alzheimer's disease by removing Aβ plaques accumulated in the brain.

[0093] 3-2.ELISA

[0094] Specifically, the strain was applied to the AD animal model according to the method of Example 1. After the administration was completed, the experimental animals were anesthetized with a mixture of Tiletamine-Zolazepam and Xylazine (1.2 mg / kg) and perfused with PBS. The brain tissue of the experimental animals was homogenized in RIPA buffer (50 mM Tris-HCL, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; 0.1% SDS; 0.5% sodium deoxycholate) for ELISA experiments, and the protein concentration in the supernatant of the tissue extract was quantified. 100 μg of protein was taken for ultracentrifugation to separate the RIPA soluble part and the RIPA insoluble part. Before the ELISA experiment, the RIPA insoluble part was resuspended in 70% formic acid and neutralized with 1 M Tris base solution. RIPA soluble Aβ was determined according to the protocol of the human Aβ-specific ELISA kit. 40 and Aβ 42 and RIPA-insoluble Aβ 40 and Aβ 42 concentration.

[0095] The results are as follows Figure 5 As shown, compared with the control group (NC), the RIPA-soluble Aβ in the brain of the strain administration group was 40 and Aβ 42 The levels of RIPA-insoluble Aβ were significantly reduced by approximately 27% and 19%, respectively. 40 and Aβ 42 levels were reduced by approximately 22% and 16%, respectively.

[0096] These results indicate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention can exert an effect of preventing, improving or treating cognitive impairment or Alzheimer's disease by reducing soluble or insoluble Aβ present in the brain.

[0097] Example 4: Neuroprotective Effect

[0098] In order to evaluate whether the strain of the present invention can prevent, improve or treat cognitive impairment or AD, the neuroprotective effect of the strain was evaluated, which was expressed as the reduction of reactive oxygen species (ROS).

[0099] Hydrogen peroxide (H2O2), a ROS, was added to neural cells and then subjected to an MTT assay. ROS, such as H2O2, are implicated in a variety of biological processes, particularly regulating cell differentiation, gene expression, and responses to cytokines. Therefore, maintaining ROS homeostasis is crucial for cell growth and survival (Sang Won Kang, Hanyang Med. Rev. 2013; 33:77-82).

[0100] Specifically, PC-12 neural cell line was cultured at 1.0×10 4 Cells were seeded at a concentration of 100 cells / well in a 96-well plate and cultured for 24 hours. H₂O₂, bacterial strains, and Soy-PS were selectively added to the corresponding wells, depending on whether they belonged to the control or experimental group. The control and experimental groups were categorized as follows. Soy phosphatidylserine (Soy-PS), a functional ingredient for brain health approved by the Korean Ministry of Food and Drug Safety, is the main phospholipid that forms brain cell membranes and is known to help prevent dementia and improve memory, cognitive function, learning ability, and attention deficit disorder (ADHD). Therefore, it was used as a positive control.

[0101] (1) Control group (NC): No H2O2, strains or Soy-PS added

[0102] (2) H2O2-treated control group (H2O2): 800 μM H2O2 was added

[0103] (3) MRS medium treatment group (MRS): 0.05 mL, 0.1 mL, 0.5 mL, and 1.0 mL of MRS medium and 800 μM H2O2 were added

[0104] (4) Strain treatment group (KCTC14149BP): 0.05%, 0.1%, 0.5% and 1.0% of the strain and 800 μM H2O2 were added

[0105] (5) Soy-PS treatment group (Soy-PS): Soy-PS at concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL and 800 μM H2O2 were added

[0106] After adding the substances and reacting for 24 hours as described above, MTT solution was added to each well and allowed to react for 4 hours. Subsequently, 100 μL of DMSO was added to each well to dissolve the purple formazan. Absorbance was measured at 570 nm using a spectrophotometer to determine cell viability (%). Cell viability was expressed as a percentage compared to the control group (NC). The experiment was repeated three times and statistical analysis was performed (n = 3 for each experimental group).

[0107] like Figure 6As shown, in PC-12 cells, the strain-treated group (KCTC14149BP) exhibited significant cytoprotective effects compared to the H2O2-treated control group, with an approximately 1.3-fold increase at 0.05% and an approximately 1.2-fold increase at 0.1%. Compared to the H2O2-treated control group, the Soy-PS-treated group (Soy-PS) increased cell viability by approximately 1.2-fold at concentrations of 200 μg / mL and 400 μg / mL, which is comparable to the results observed in the strain-treated group (KCTC 14149BP) at concentrations of 0.05% and 0.1%.

[0108] These results indicate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention increases neuronal cell viability and exhibits neuroprotective effects. Because this effect is similar to that of the previously used brain health functional ingredient Soy-PS, it suggests that this strain may have a preventive, ameliorative, or therapeutic effect on cognitive impairment or Alzheimer's disease.

[0109] Example 5: Antioxidant Activity

[0110] To evaluate whether the strains of the present invention can prevent, improve, or treat cognitive impairment or AD, their antioxidant activity was assessed. Specifically, the activity of glutathione peroxidase (GSH-Px) in neurons treated with H2O2 as a reactive oxygen species (ROS) was measured by quantifying the amount of glutathione (GSH). GSH, a component of the antioxidant enzyme GSH-Px, can reduce H2O2 to water (H2O), thereby preventing DNA damage and inducing apoptosis in abnormal cells, demonstrating antioxidant activity.

[0111] Specifically, PC-12 neural cell line was cultured at 1.0×10 4 Cells were seeded at a concentration of 100 cells / well in a 96-well plate and cultured for 24 hours. H₂O₂, bacterial strains, and Soy-PS were selectively added to the corresponding wells, depending on whether they were in the control or experimental group. The control and experimental groups were categorized as follows.

[0112] (1) Control group (NC): No H2O2, strains or Soy-PS added

[0113] (2) H2O2-treated control group (H2O2): 800 μM H2O2 was added

[0114] (3) MRS medium treatment group (MRS): 0.1 mL, 0.5 mL, and 1.0 mL of MRS medium and 800 μM H2O2 were added

[0115] (4) Strain treatment group (KCTC14149BP): 0.1%, 0.5%, and 1.0% of the strain were added, as well as 800 μM H2O2

[0116] (5) Soy-PS treatment group (Soy-PS): Soy-PS at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL and 800 μM H2O2 were added

[0117] After adding the substances and reacting for 24 hours as described above, the control and experimental groups were washed twice with PBS (pH 7.4) and then homogenized in RIPA buffer. RIPA buffer consists of 150 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), 25 mM NaF, 20 mM EGTA, 1 mM DTT, 1 mM Na3VO4, and a protease inhibitor cocktail. After homogenization, the mixture was centrifuged at 1,890 x g for 10 minutes at 4°C to remove the cell supernatant. After adding 400 μL of glutathione assay buffer, the solution was centrifuged at 12,000 x g for 30 minutes at 36°C to obtain the cell supernatant as a sample.

[0118] For glutathione analysis, a total of 160 μL of glutathione reaction buffer, glutathione reductase, and generation mixture (BioVision) were added to a 96-well plate and incubated at room temperature for 10 minutes. Subsequently, 20 μL of standard solution or sample (cell supernatant) was added to each well and reacted again for 10 minutes at room temperature. 20 μL of substrate solution was then added to induce color development, and the absorbance was measured at 405 nm using a spectrophotometer. The GSH concentration in the sample was calculated from the standard calibration curve and expressed as a percentage (%) relative to the control group (NC). The experiment was repeated three times and statistical analysis was performed (n = 3 per experimental group).

[0119] The results are as follows Figure 7 As shown, compared to the H2O2-treated control group, the strain-treated group (KCTC14149BP) showed a significant increase of approximately 1.9-fold in GSH levels in PC-12 cells at both 0.5% and 1.0% H2O2 concentrations. Specifically, the strain-treated group (KCTC14149BP) exhibited approximately 84% of the GSH level in the Soy-PS-treated group (Soy-PS), indicating that the strain of the present invention and Soy-PS exhibit similar antioxidant activity.

[0120] These results demonstrate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention has enhanced antioxidant activity and exhibits neuroprotective effects. Because these effects are similar to those of the previously used brain health ingredient Soy-PS, this suggests that the strain may have preventive, ameliorative, or therapeutic effects on cognitive impairment or Alzheimer's disease.

[0121] Example 6: Inhibition of acetylcholinesterase (AChE)

[0122] In order to evaluate whether the strain of the present invention can prevent, improve or treat cognitive impairment or AD, the effect of the strain on the inhibition of acetylcholinesterase (AChE) activity was evaluated.

[0123] Specifically, AChE activity was induced with hydrogen peroxide (H2O2), and the AChE activity levels of each control and experimental group were measured. 150 μL of PBS was added to each well of a 96-well plate. H2O2, bacterial strains, and Soy-PS were optionally added to the control and experimental groups. The control and experimental groups were categorized as follows:

[0124] (1) Control group (NC): No H2O2, strain or Soy-PS was added, but 0.2 U / mL AChE was added.

[0125] (2) H2O2-treated control group (H2O2): 800 μM H2O2 and 0.2 U / mL AChE were added.

[0126] (3) MRS medium treatment group (MRS): Different amounts of MRS medium (0.1 mL, 0.5 mL, 1.0 mL) as well as 800 μM H2O2 and 0.2 U / mL AChE were added.

[0127] (4) Strain treatment group (KCTC14149BP): strains were added at concentrations of 0.1%, 0.5%, and 1.0%, as well as 800 μM H2O2 and 0.2 U / mL AChE.

[0128] (5) Soy-PS treatment group (Soy-PS): Soy-PS at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, as well as 800 μM H2O2 and 0.2 U / mL AChE were added.

[0129] After adding the above substances, the samples were kept at 37°C for 5 minutes. Then, 30 μL of 10 mM DTNB and 20 μL of 15 mM ATCI were added and incubated at 37°C for 30 minutes. Absorbance was measured at 415 nm using a fluorescence spectrophotometer. AChE inhibitory activity was expressed as nM tacrine equivalents. The experiment was repeated three times and statistical analysis was performed (n = 3 experimental groups).

[0130] The results are as follows Figure 8As shown, compared with the H2O2-treated control group, the AChE activity of the strain-treated group (KCTC14149BP) was significantly reduced, with a reduction of about 70% at concentrations of 0.1% and 1.0%, and a reduction of about 60% at a concentration of 0.5%. Specifically, compared with the H2O2-treated control group, the Soy-PS-treated group (Soy-PS) was reduced by about 70% at 50 μg / mL, about 90% at 100 μg / mL, and about 80% at 200 μg / mL, which was similar to the results observed in the strain-treated group (KCTC14149BP), confirming that the strain of the present invention and Soy-PS exhibit similar effects in inhibiting AChE activity.

[0131] These results indicate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention inhibits AChE activity and exhibits a neuroprotective effect. Since these effects are similar to those of the previously used brain health functional ingredient Soy-PS, this suggests that the strain may have a preventive, ameliorative, or therapeutic effect on cognitive impairment or Alzheimer's disease.

[0132] Example 7: Anti-inflammatory activity

[0133] In order to evaluate whether the strain of the present invention can prevent, improve or treat cognitive impairment or AD, the effect of the strain on anti-inflammatory activity was evaluated. The deposition of Aβ in the brain is associated with an inflammatory response, which is mediated by the activation of microglia, which are immune cells surrounding Aβ plaques. Microglia are activated to remove Aβ by phagocytosis (an immune response), and they play a key role in brain inflammation by rapidly responding to signals from the extracellular environment. Aβ-activated microglia produce inflammatory mediators such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and increase the synthesis and secretion of inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α), so their activation needs to be regulated. Previous studies have shown that long-term use of anti-inflammatory agents to regulate this inflammatory process can inhibit and improve the progression of AD (Hui-Jin Mun et al., JKSCI, Vol.25No.6,165-170).

[0134] Example 7-1: Reduction of Nitric Oxide (NO)

[0135] Specifically, the amount of nitric oxide (NO) in microglia during Aβ-induced inflammation was measured.

[0136] BV-2 microglial cells were cultured at a rate of 1.0 × 10 4 Cells were seeded into 96-well plates at a concentration of 100 cells / well and cultured for 24 hours. 1-42, strains, and Soy-PS were selectively added to the control and experimental groups in the wells. The control and experimental groups were classified as follows:

[0137] (1) Control group (NC): no Aβ added 1-42 , strains or Soy-PS

[0138] (2) Aβ 1-42 Treatment control group (Aβ): add 20 μM Aβ 1-42

[0139] (3) MRS medium treatment group (MRS): Different amounts of MRS medium (0.1 mL, 0.5 mL, 1.0 mL) and 20 μM Aβ were added 1-42

[0140] (4) Strain treatment group (KCTC14149BP): 0.1%, 0.5%, and 1.0% of the strain and 20 μM Aβ were added. 1-42

[0141] (5) Soy-PS treatment group (Soy-PS): Soy-PS at concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL and 20 μM Aβ were added 1-42

[0142] After adding the substance to the control and experimental groups and reacting for 24 hours, the cell cultures were harvested and the amount of NO was determined using the Griess reagent system. The NO content was converted based on the sodium nitrite standard curve. The experiment was repeated three times and statistically analyzed (n=3 for each experimental group).

[0143] The results are as follows Figure 9 As shown, with Aβ 1-42 Compared with the control group (Aβ), the amount of NO in BV-2 cells in the strain-treated group (KCTC14149BP) decreased by about 20% at concentrations of 0.1%, 0.5%, and 1.0%. 1-42 Compared with the control group (Aβ), the Soy-PS-treated group (Soy-PS) decreased by approximately 10% at 50 μg / mL, by approximately 30% at 100 μg / mL and 200 μg / mL, and by approximately 40% at 400 μg / mL, showing a similar degree of reduction observed in the strain-treated group (KCTC14149BP).

[0144] These results indicate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention reduces NO levels and exhibits anti-inflammatory activity. Because this effect is similar to that of the previously used brain health functional ingredient Soy-PS, it suggests that this strain may have a preventive, ameliorative, or therapeutic effect on cognitive impairment or Alzheimer's disease.

[0145] Example 7-2: Reduction of inflammatory cytokines

[0146] Specifically, the amount of inflammatory cytokine TNF-α in microglia during Aβ-induced inflammation was measured.

[0147] BV-2 microglial cells were cultured at a rate of 1.0 × 10 4 Cells were seeded into 96-well plates at a concentration of 100 cells / well and cultured for 24 hours. 1-42 , strains, and Soy-PS were selectively added to the control and experimental groups in the wells. The control and experimental groups were classified as follows:

[0148] (1) Control group (NC): no Aβ added 1-42 , strains or Soy-PS

[0149] (2) Aβ 1-42 Treatment control group (Aβ): add 20 μM Aβ 1-42

[0150] (3) MRS medium treatment group (MRS): Different amounts of MRS medium (0.1 mL, 0.5 mL, 1.0 mL) and 20 μM Aβ were added 1-42

[0151] (4) Strain treatment group (KCTC14149BP): 0.1%, 0.5%, and 1.0% of the strain and 20 μM Aβ were added. 1-42

[0152] (5) Soy-PS treatment group (Soy-PS): Soy-PS at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL and 20 μM Aβ were added 1-42

[0153] After the substance was added to the control group and the experimental group and allowed to react for 24 hours, the cell culture was collected and the amount of TNF-α was determined. The amount of TNF-α in the sample (cell culture) was determined using a standard curve. The experiment was repeated three times and statistically analyzed (n=3 for each experimental group).

[0154] The results are as follows Figure 10 As shown, with Aβ 1-42 Compared with the control group (Aβ), the amount of TNF-α in BV-2 cells in the strain-treated group (KCTC14149BP) decreased by about 20% at a concentration of 0.1%, and by about 10% at concentrations of 0.5% and 1.0%. 1-42Compared with the treated control group (Aβ), the Soy-PS treated group (Soy-PS) decreased by about 20% at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, which was similar to the TNF-α levels observed in the strain treated group (KCTC 14149BP), confirming that the strain of the present invention and Soy-PS exhibit comparable effects in reducing TNF-α.

[0155] These results indicate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention reduces TNF-α levels, demonstrating anti-inflammatory activity. Because this effect is similar to that of the previously used brain health ingredient Soy-PS, this suggests that the strain may have a preventive, ameliorative, or therapeutic effect on cognitive impairment or Alzheimer's disease.

[0156] Example 8: Y-maze test

[0157] In order to evaluate whether the strain of the present invention can prevent, improve or treat cognitive impairment or AD, the effects of the strain on short-term memory and instantaneous spatial perception ability were evaluated using a Y-maze test.

[0158] The Y-maze test assesses short-term memory and spatial perception by evaluating the animal's ability to remember previously explored arms. A lower tendency for the animal to re-enter the same arm indicates better memory performance.

[0159] Specifically, scopolamine, which induces memory impairment, was administered together with saline (vehicle), a known therapeutic drug for cognitive impairment or AD (donepezil or purpurogenol), or the strain of the present invention, and then a Y-maze test was performed.

[0160] Scopolamine is a muscarinic receptor antagonist that blocks muscarinic receptors located on postsynaptic cholinergic neurons. It prevents acetylcholine (a neurotransmitter released from presynaptic neurons) from binding to muscarinic receptors, and this inhibition of neurotransmission impairs learning and memory. Therefore, scopolamine is widely used in studies evaluating improvements in cognitive functions such as learning and memory (Dae-eok Kim et al., Journal of Oriental Neuropsychiatry, 2018; 29(3): 121–134).

[0161] Donepezil is an acetylcholinesterase inhibitor approved by the FDA for the treatment of AD. Acetylcholine (ACh) is a neurotransmitter in the autonomic nervous system that contributes to learning and memory. In AD patients, ACh levels are reduced. Therefore, ACh inhibitors such as donepezil are used to inhibit ACh, which breaks down ACh into acetate and choline.

[0162] Purpurogenol is a functional ingredient that has been certified to improve cognitive function. It inhibits acetylcholinesterase (AChE) in the hippocampus, which is closely related to memory and learning, thereby enhancing memory (Ki Yong Lee et al., Korean Journal of Pharmacognosy, 2008; 39(2): 86–90).

[0163] The Y-shaped maze consisted of three arms with dimensions of 40 cm in length, 3 cm in width, and 15 cm in height, and the angle between the arms was set at 120°.

[0164] Specifically, the experimental animals were divided into 7 groups, namely a control group (G1) and experimental groups (G2 to G7), and the dosage of each substance was 200 μL.

[0165] (1) Control group (G1): No scopolamine, donepezil, purpurogenol or bacterial strains were administered

[0166] (2) Vehicle-treated group (G2): scopolamine and vehicle were administered

[0167] (3) Donepezil-treated group (G3): Administer scopolamine and 5 mg / kg donepezil

[0168] (4) Purpurogenol treatment group (G4): scopolamine and 5 mg / kg purpurogenol were administered

[0169] (5) Low-dose strain treatment group (G5): scopolamine and 1×10 8 CFU strain / unit

[0170] (6) Medium-dose strain treatment group (G6): scopolamine and 5×10 8 CFU strain / unit

[0171] (7) High-dose strain treatment group (G7): scopolamine and 1×10 9 CFU strain / unit

[0172] The control and experimental groups were placed in the center of a Y-shaped maze, where they were allowed to explore the maze for 8 minutes. The movements of each individual were analyzed using an EthoVision XT16 (Noldus, USA). The experimental results were expressed as spontaneous alternation behavior (%) and calculated according to the following formula:

[0173] [Formula 1]

[0174] Spontaneous alternation (%) = (number of spontaneous alternations / (total number of entries into an arm - 2)) × 100

[0175] The number of spontaneous alternations refers to the number of times an animal enters three different arms in succession, and the total number of arm entries refers to the total number of times an animal enters any arm. Statistical analysis was performed using 10 animals per group (n = 10 for each experimental group).

[0176] The results are as follows Figure 11 As shown, short-term memory ability and instantaneous spatial perception ability were evaluated, and the low-dose strain-treated group (G5), the medium-dose strain-treated group (G6), and the high-dose strain-treated group (G7) showed a concentration-dependent increase. In addition, compared with the vehicle-treated group (G2), the medium-dose strain-treated group (G6) increased by about 1.3 times, and the high-dose strain-treated group (G7) increased by about 1.4 times. Specifically, compared with the vehicle-treated group (G2), the donepezil-treated group (G3) and the purpurogenol-treated group (G4) increased by about 1.3 times, which was similar to the results in the medium-dose (G6) and high-dose (G7) strain-treated groups.

[0177] These results indicate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention can improve short-term memory and transient spatial perception, and these effects are similar to those of donepezil or purpurogenol, which are therapeutic agents for cognitive impairment or Alzheimer's disease. This means that the strain can have a preventive, ameliorative, or therapeutic effect on cognitive impairment or Alzheimer's disease.

[0178] Example 9. Passive avoidance test

[0179] In order to evaluate whether the strain of the present invention can prevent, improve or treat cognitive impairment or AD, the effect of the strain on cognitive ability was evaluated by a passive avoidance test.

[0180] The passive avoidance test is an experiment to assess cognitive ability by taking advantage of rodents' preference for staying in dark places.

[0181] Specifically, saline (as a vehicle), donepezil (an agent used to treat cognitive impairment or AD), purpurogenol (a functional ingredient believed to improve cognitive function), or the strain of the present invention was co-administered with scopolamine, which induces memory impairment, and then a passive avoidance test was performed.

[0182] The experimental animals were divided into 7 groups, namely a control group (G1) and experimental groups (G2 to G7), and the dose of each substance was 200 μL.

[0183] (1) Control group (G1): No scopolamine, donepezil, purpurogenol or bacterial strains were administered

[0184] (2) Vehicle-treated group (G2): Administered with scopolamine and vehicle (saline)

[0185] (3) Donepezil-treated group (G3): Administer scopolamine and 5 mg / kg donepezil

[0186] (4) Purpurogenol treatment group (G4): scopolamine and 5 mg / kg purpurogenol were administered

[0187] (5) Low-dose strain treatment group (G5): scopolamine and 1×10 8 CFU strain / unit

[0188] (6) Medium-dose strain treatment group (G6): scopolamine and 5×10 8 CFU strain / unit

[0189] (7) High-dose strain treatment group (G7): scopolamine and 1×10 9 CFU strain / unit

[0190] The control and experimental groups were placed in a chamber consisting of a light chamber and a dark chamber of equal size. When the animals entered the dark chamber from the light chamber, a 0.5 mA current was applied for 3 seconds. Twenty-four hours later, the animals were placed back in the light chamber, and the time it took to enter the dark chamber was measured (maximum 300 seconds) to assess memory.

[0191] The results are as follows Figure 12 As shown, cognitive ability was evaluated, and the low-dose strain-treated group (G5), the medium-dose strain-treated group (G6), and the high-dose strain-treated group (G7) showed a concentration-dependent increase. In addition, compared with the vehicle-treated group (G2), the high-dose strain-treated group (G7) significantly increased by about 2.2 times. Specifically, the donepezil-treated group (G3) showed about 74% memory recovery, while the purpurogenol-treated group (G4) showed about 87% memory recovery, demonstrating that the strain of the present invention, donepezil, or purpurogenol showed similar results in terms of cognitive function recovery.

[0192] These results indicate that the Lactobacillus delbrueckii subsp. lactis strain of the present invention improves cognitive ability. Since this effect is similar to that of the AD therapeutic agents donepezil or purpurogenol, it is shown that the strain can prevent, improve or treat cognitive impairment or Alzheimer's disease.

[0193] [Accession number]

[0194] Depository: Korea Type Culture Collection (KCTC), Bioresource Center of Korea Institute of Biotechnology

[0195] Accession number: KCTC 14149BP

[0196] Deposit date: 20200303

[0197]

Claims

1. A pharmaceutical composition for treating or preventing cognitive impairment or Alzheimer's disease, comprising a Lactobacillus delbrueckii subsp. lactis strain deposited under the accession number KCTC14149BP as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein The strain is one or more selected from the group consisting of living cells, dead cells, and cultures, lysates, extracts, and cytoplasmic fractions thereof.

3. The pharmaceutical composition according to claim 1, wherein The cognitive impairment is manifested as symptoms of decreased memory, attention, spatial perception, language ability or a combination thereof.

4. The pharmaceutical composition according to claim 1, wherein The cognitive disorder is amyloid β protein accumulation disorder or tau protein accumulation disorder.

5. The pharmaceutical composition according to claim 4, wherein The amyloid β protein is a soluble amyloid β protein or an insoluble amyloid β protein.

6. The pharmaceutical composition according to claim 1, wherein The composition exhibits an increase in one or more intestinal microbiota selected from the group consisting of Erysipelotrichaceae, Erysipelotrichales, Turicibacter, Peptostreptococcales Tissierellales, Anaerovoracaceae, and Eubacterium xylanophilum.

7. The pharmaceutical composition according to claim 1, wherein The composition exhibits a reduction in one or more intestinal microbiota selected from the group consisting of Peptococcales and Peptococaceae.

8. A food composition for alleviating or preventing cognitive impairment or Alzheimer's disease, the food composition comprising a Lactobacillus delbrueckii subsp. lactis strain deposited with the accession number KCTC14149BP as an active ingredient.

9. The food composition according to claim 8, wherein The strain is one or more selected from the group consisting of living cells, dead cells, and cultures, lysates, extracts, and cytoplasmic fractions thereof.

10. The food composition according to claim 8, wherein The cognitive impairment is manifested as symptoms of decreased memory, attention, spatial perception, language ability or a combination thereof.

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