Novel lactic acid bacterium and use thereof

Abstract

The present invention relates to a novel lactic acid bacterium Lactococcus lactis P91 KCCM 13447P, Bifidobacterium animalis P97 KCCM 13448P, or a mixture thereof, and a use thereof. The strain and the mixture thereof, according to the present invention, can be effectively used for preventing, treating or alleviating neuropsychiatric diseases or inflammatory diseases.

Description

Novel lactic acid bacteria and their uses

[0001] The present invention relates to novel lactic acid bacteria, Lactococcus lactis P91 KCCM 13447P, Bifidobacterium animalis P97 KCCM 13448P or a mixture thereof, and uses thereof.

[0002] As we enter an aging society, the incidence of chronic degenerative diseases is rapidly increasing. Among these diseases, Parkinson's disease and cognitive decline are particularly common among the elderly, significantly reducing their quality of life and increasing their social and economic burden.

[0003] Parkinson's disease is a chronic, degenerative disease affecting the central nervous system. Its primary symptoms include motor decline, tremors, and rigidity. Non-motor prodromes that precede the onset of motor symptoms include neuropsychiatric and cognitive symptoms (depression, anxiety, cognitive decline, and attention deficits), sleep-related symptoms (REM sleep behavior disorder), and autonomic nervous system dysfunction (constipation, decreased or lost sense of smell). Furthermore, the disease progresses gradually and significantly interferes with the patient's daily life. It typically develops in the elderly and is accompanied by symptoms such as memory loss and impaired judgment, which reduce an individual's independence and increase the burden on caregivers. Parkinson's disease is believed to be caused by a combination of genetic and environmental factors, which lead to the deposition of alpha-synuclein in the midbrain, leading to the degeneration or significant reduction in the number of dopamine-producing cells in the substantia nigra.

[0004] Furthermore, it has been recently reported that enteritis and intestinal microbiota imbalance cause mental and neurological disorders including Parkinson's disease. For example, intestinal microbiota imbalance with overgrowth of Escherichia coli is reported to cause dementia and depression, and intestinal microbiota imbalance with overgrowth of Proteus mirabilis is reported to cause Parkinson's disease and cognitive impairment.

[0005] Therefore, there is an urgent need to develop new substances that can prevent, treat, and improve neuropsychiatric disorders by restoring the balance of intestinal microflora and reducing inflammation, thereby improving nervous system health.

[0006] The purpose of the present invention is to provide a Lactococcus lactis P91 strain deposited under the accession number KCCM 13447P.

[0007] Another object of the present invention is to provide a Bifidobacterium animalis P97 strain deposited under the accession number KCCM 13448P.

[0008] Another object of the present invention is to provide an antibacterial composition comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

[0010] Another object of the present invention is to provide a food composition comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

[0011] Another object of the present invention is to provide a method for treating or improving a neuropsychiatric disease or an inflammatory disease by administering to a subject a Lactococcus lactis P91 strain, a Bifidobacterium animalis P97 strain, or a mixture thereof.

[0012] One aspect of the present invention for achieving the above-mentioned purpose relates to the Lactococcus lactis P91 strain deposited under the accession number KCCM 13447P.

[0013] The Lactococcus lactis of the present invention may be a novel lactic acid bacterium isolated and identified from the feces of a healthy human.

[0014] The 16S rDNA base sequence for identification and classification of Lactococcus lactis P91 of the present invention is as shown in SEQ ID NO: 1 attached to this specification. Therefore, Lactococcus lactis P91 of the present invention may include the 16S rDNA of SEQ ID NO: 1.

[0015] Analysis of the 16S rDNA base sequence of the above sequence number 1 showed a high correlation with the known Lactococcus lactis, and whole genome analysis showed 97.4% homology with Lactococcus lactis ATCC19435 and 97.2% homology with Lactococcus lactis NBRC100931, confirming the highest molecular phylogenetic relationship with them (Figs. 1 and 2). Accordingly, the lactic acid bacterium was identified as Lactococcus lactis, named Lactococcus lactis P91, and deposited with the Korea Microbiological Conservation Center on December 22, 2023 (KCCM 13447P).

[0016] The Lactococcus lactis P91 of the present invention is a gram-positive coccus that does not produce spores. More specific physiological characteristics of Lactococcus lactis P91 can be analyzed according to conventional methods in the art, and specifically, L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, sucrose, trehalose, starch, and gentiobiose can be used as carbon sources. Can be.

[0017] Another aspect of the present invention relates to the Bifidobacterium animalis P97 strain deposited under accession number KCCM 13448P.

[0018] The above Bifidobacterium animalis P97 may be a novel lactic acid bacterium isolated and identified from the feces of a healthy human.

[0019] The 16S rDNA base sequence for identification and classification of Bifidobacterium animalis P97 of the present invention is as shown in SEQ ID NO: 2 attached to this specification. Therefore, Bifidobacterium animalis P97 of the present invention may include the 16S rDNA of SEQ ID NO: 2.

[0020] Analysis of the 16S rDNA base sequence of the above sequence number 2 showed a high correlation with the known Bifidobacterium animalis, and whole genome analysis showed 99.9% homology with Bifidobacterium animalis DSM10140 and 95.6% homology with Bifidobacterium animalis ATCC25527, confirming the highest molecular phylogenetic relationship with Bifidobacterium animalis (Figs. 3 and 4). Accordingly, the lactic acid bacterium was identified as Bifidobacterium animalis, named Bifidobacterium animalis P97, and deposited with the Korea Center for Microorganism Conservation on December 22, 2023 (KCCM 13448P).

[0021] Bifidobacterium animalis P97 of the present invention is a gram-positive rod. More specifically, the physiological characteristics of Bifidobacterium animalis P97 can be analyzed according to a conventional method in the art, and specifically, Bifidobacterium animalis P97 can decompose urea and utilize D-glucose, D-lactose, D-sucrose, D-maltose, salicin, D-xylose, L-arabinose, D-cellobiose, D-mannose, and D-raffinose as carbon sources.

[0022] Another aspect of the present invention relates to an antibacterial composition comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

[0023] In addition, another aspect of the present invention relates to a pharmaceutical composition for preventing or treating Proteus mirabilis infection, comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

[0024] Proteus mirabilis, described above, is a well-known species of the genus Proteus, accounting for 70% to 90% of infections caused by this genus. It causes intestinal inflammation and is a causative agent of chronic infections, cervicitis, cystitis, osteomyelitis, and rheumatoid arthritis, as well as alpha-synuclein accumulation.

[0025] In one embodiment of the present invention, it was confirmed that the Lactococcus lactis P91, Bifidobacterium animalis P97 or a mixture thereof inhibits the growth of Proteus mirabilis strains, and thus can be utilized for antibacterial purposes, and further, can be utilized for the prevention or treatment of Proteus mirabilis infection.

[0026] Another aspect of the present invention relates to a composition for inhibiting alpha-synuclein expression, comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

[0027] The above alpha-synuclein is a protein that helps in neurotransmission between brain cells. Alpha-synuclein protein should be removed normally through the protein degradation process by lysosomes, but if it is not removed normally and accumulates in cells, neurons die, causing neuropsychiatric diseases.

[0028] In one embodiment of the present invention, it was confirmed that Lactococcus lactis P91, Bifidobacterium animalis P97 or a mixture thereof inhibits alpha-synuclein expression, and thus can be used for the purpose of inhibiting alpha-synuclein expression and for the purpose of preventing, treating and improving alpha-synuclein-related diseases.

[0029] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating neuropsychiatric or inflammatory diseases, comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

[0030] In the present invention, "neuropsychiatric disorders" are caused by degeneration or damage to the brain or nerves, or by disruption of the brain's neurotransmitter systems (e.g., serotonin, dopamine, norepinephrine). Biological factors, such as complex interactions between brain neural circuits, neurotransmitter imbalances, and connectivity problems between brain regions, play a role, encompassing a variety of mental and behavioral disorders caused by structural or functional abnormalities in the brain.

[0031] Specifically, the above neuropsychiatric disease may be at least one selected from the group consisting of alpha-synucleinopathy, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.

[0032] The above "alpha-synucleinopathy" is a general term for all types of degenerative brain diseases in which alpha-synuclein proteins abnormally aggregate and accumulate within brain cells, damaging nerve cells. Normal alpha-synuclein proteins undergo structural deformation, become tangled with each other, and form insoluble aggregates. Depending on which type of brain cell (neuron or glial cell) and which part of the brain these aggregates primarily accumulate in, they manifest as diseases with different clinical symptoms, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.

[0033] Parkinson's disease (PD) is a representative alpha-synuclein disease and the second most common neurodegenerative disease after Alzheimer's disease. It is a chronic, progressive condition characterized by progressive motor function impairment. It primarily presents with motor symptoms such as resting tremor, muscle rigidity, bradykinesia, and postural instability. Non-motor symptoms such as depression, sleep disturbances, and cognitive decline are also common.

[0034] The main pathological characteristic is the selective destruction of dopamine-producing neurons distributed in the substantia nigra of the brain, and Lewy bodies, which are aggregates of alpha-synuclein, are found in the surviving neurons, and as dopamine secretion decreases due to the loss of dopamine neurons, movement abnormalities occur.

[0035] The above "Dementia with Lewy Bodies (DLB)" is a type of degenerative dementia that presents with cognitive decline and Parkinson's disease motor symptoms. The main symptoms include gradual cognitive decline (dementia), motor symptoms similar to Parkinson's disease (parkinsonism), recurrent visual hallucinations (seeing things as if they were hallucinations), severe cognitive function fluctuations, and REM sleep behavior disorder. As with Parkinson's disease, Lewy bodies are formed within nerve cells, but their distribution is not limited to the brainstem, such as the substantia nigra, but rather appears widely throughout the cerebral cortex, and extensive damage to the cerebral cortex is the direct cause of dementia symptoms such as memory and judgment decline.

[0036] The above "Multiple System Atrophy (MSA)" is a rare degenerative brain disease that presents with three symptoms: parkinsonism, cerebellar ataxia, and autonomic nervous system dysfunction. The disease progresses faster than Parkinson's disease. The main symptoms include motor symptoms similar to Parkinson's disease, cerebellar ataxia with difficulty maintaining balance and staggering, and autonomic nervous system dysfunction such as severe orthostatic hypotension or urinary dysfunction. In MSA, alpha-synuclein aggregates accumulate primarily in glial cells, especially oligodendrocytes, which support and assist nerve cells.

[0037] Although the above-mentioned Parkinson's disease, Lewy body dementia, and multiple system atrophy have some differences in symptoms and the types of cells primarily damaged, they have a common molecular pathology called abnormal accumulation of 'alpha-synuclein', and 'alpha-synuclein' can be set as a common treatment target.

[0038] In addition, the above neuropsychiatric disorder may be at least one selected from the group consisting of anxiety disorder, depression, stress-related disorder, mood disorder, sleep disorder, memory disorder, cognitive disorder, and attention disorder, but is not limited thereto.

[0039] The above "anxiety disorder" is a mental illness that causes disruption in daily life due to various forms of abnormal and pathological anxiety and fear. The amygdala is the area that detects threats and triggers a fear response, and when the activity of this amygdala increases abnormally, anxiety disorders occur. Furthermore, anxiety disorders occur when the function of the prefrontal cortex, which is responsible for rational judgment and emotional regulation, is impaired, making it difficult to effectively suppress excessive activity of the amygdala. Among neurotransmitters, dysfunction of the serotonin and GABA systems, which play an important role in emotional regulation, are also major causes of anxiety disorders.

[0040] For example, it may be one or more selected from the group including, but not limited to, generalized anxiety disorder, specific phobia, agoraphobia, social anxiety disorder, panic disorder, separation anxiety, and selective mutism.

[0041] The above-mentioned "depression" is a disease characterized by a decline in motivation and feelings of sadness, leading to various cognitive and psychosomatic symptoms and a decline in daily functioning. Depression can occur due to decreased brain function when the activity of the prefrontal cortex, which is responsible for emotional regulation, the hippocampus, which is crucial for memory and learning, and the nucleus accumbens, which is associated with motivation and reward, decreases or atrophies. Chronic stress, in particular, can suppress neurogenesis in the hippocampus, leading to depression and memory loss. Furthermore, imbalances in serotonin, norepinephrine, and dopamine can be a major cause of depression.

[0042] For example, it may be one or more selected from the group including, but not limited to, Major Depressive Disorder, Persistent Depressive Disorder, Dysthymia, Disruptive Mood Dysregulation Disorder, Premenstrual Dysphoric Disorder, Substance / Medication Induced Depressive Disorder, Depressive Disorder due to Another Medical Condition, Other Specified Depressive Disorder, and Unspecified Depressive Disorder.

[0043] The above "stress-related disorders" include post-traumatic stress disorder (PTSD), which occurs after experiencing an extreme stressful event, and refers to a condition in which an excessive stress response persists. Stress activates the hypothalamic-pituitary-adrenal axis (HPA axis) in the brain, causing the secretion of the stress hormone cortisol. Chronic stress or extreme trauma can disrupt the regulatory function of the HPA axis, resulting in abnormal cortisol secretion. This overactivates the amygdala, the center of emotional regulation, and suppresses the functions of the prefrontal cortex, which makes rational decisions, and the hippocampus, which is responsible for memory, causing fear responses even to minor stimuli.

[0044] The above "mood disorder" includes bipolar disorder, which is characterized by recurrent episodes of depression and mania (an abnormally excited and elevated mood state), and refers to a condition in which the range of mood changes is very large and unpredictable, causing difficulties in daily life and social relationships.

[0045] Mood disorders are primarily caused by instability in the brain's neural circuitry involved in emotional regulation. The connection between the amygdala, which processes emotions, and the prefrontal cortex, which controls rationality, weakens, disrupting the proper functioning of the emotional "switch." During periods of depression, overall brain activity is depressed, while during periods of mania, the dopamine system, associated with the reward circuitry, becomes overactive, leading to extreme brain changes.

[0046] The above "sleep disorder" includes insomnia, hypersomnia, narcolepsy, sleep-wake time disorders, and abnormal behavior during sleep. Sleep and wakefulness are processes in which various regions of the brain and neurotransmitters are precisely regulated, and are caused by dysfunction in the neural circuits and neurotransmitter systems that precisely coordinate sleep and wakefulness. If there is a problem with this system, the quantity and quality of sleep are impaired, such as difficulty falling asleep, difficulty maintaining sleep, or waking up too early, resulting in conditions such as fatigue and decreased concentration during the day. Symptoms include cataplexy, hypnagogic hallucinations, dreams, sleep, paralysis, and sleep attacks, and there is also periodic somnolence, which repeats light sleep periods for about a week. Sleep-wake time disorders include jet lag and sleep disorders of night workers, and abnormal behavior during sleep include sleepwalking, night terrors, and nocturnal enuresis.

[0047] As a sleep center, the ventrolateral preoptic nucleus (VLPO) located in the hypothalamus of the brain secretes the inhibitory neurotransmitter GABA to suppress the arousal system and induce sleep.

[0048] Additionally, as the arousal center, several monoaminergic nuclei located in the brainstem and posterior hypothalamus are responsible for arousal, and activate the cerebral cortex by secreting neurotransmitters that promote arousal, such as norepinephrine, serotonin, and histamine.

[0049] Various sleep disorders occur when there is a problem with the switch or related neural circuit between the sleep center and the wake center.

[0050] The above 'Insomnia' is a state of 'hyperarousal of the brain', where the sleep switch does not turn on properly or the wake switch does not turn off even at night.

[0051] Stress, anxiety, and worry stimulate the sympathetic nervous system and continuously stimulate the arousal center. This causes the brain to remain awake, even when lying down, without turning off. This makes falling asleep difficult and waking up easily from minor stimuli. Furthermore, if the inhibitory neurotransmitter GABA system is dysfunctional, the arousal system cannot be sufficiently suppressed, worsening the state of hyperarousal. Furthermore, if orexin (or hypocretin), a neuropeptide crucial for maintaining stable arousal, remains abnormally high even at night, it can lead to persistent arousal and contribute to insomnia.

[0052] The above insomnia includes sleep apnea, which is difficulty falling asleep; deep sleep disorder, which is difficulty sleeping even after falling asleep; and early morning awakening, which is waking up early in the morning.

[0053] Hypersomnia, also known as hypersomnia, is a sleep disorder characterized by extreme difficulty waking up from sleep. It can be caused by dysfunction in the brain's neural circuits that regulate the sleep-wake system. It includes narcolepsy, idiopathic hypersomnia, and recurrent hypersomnia.

[0054] 'Narcolepsy' is a disease in which unbearable drowsiness during the day occurs even after getting enough sleep at night. The main cause is the destruction or abnormality of the neurons in the hypothalamus that secrete orexin, which acts as a 'switch stabilizer' to maintain a stable state of wakefulness. Cataplexy, in which the body suddenly loses strength when experiencing strong emotions such as laughing, getting angry, or being surprised, is a characteristic symptom of narcolepsy. When orexin is deficient, the brainstem area that controls REM sleep is inappropriately activated by emotional stimulation, showing symptoms of sudden loss of muscle strength even though one is awake.

[0055] 'Idiopathic Hypersomnia' refers to cases where excessive sleepiness occurs for unknown reasons without narcolepsy, other sleep disorders, or internal medical diseases. Hypersensitivity of receptors for GABA, a representative neurotransmitter that suppresses brain activity and induces sleep, is also suggested.

[0056] 'Recurrent Hypersomnia' is a disease in which a state of extreme hypersomnia lasts for several days to several weeks, and then a pattern of returning to normal completely until the next episode repeats, and it includes Kleine-Levin Syndrome. When symptoms appear, the function of the hypothalamus, which is the center that controls appetite, sleep, and emotions, and the limbic system, which is responsible for emotions and memory, especially the thalamus and temporal lobe, may be temporarily and significantly reduced.

[0057] The above "memory impairment" refers to a condition in which one or more of the processes of memory acquisition (encoding), storage, and retrieval are partially or completely damaged, causing significant impairment in learning ability and daily life functions, and is caused by damage to specific neural mechanisms in the brain that create, store, and retrieve memories. One of the core mechanisms is the failure to 'storage' new memories. If the hippocampus is damaged due to stress or stroke, etc., anterograde amnesia occurs, in which new information cannot be converted to long-term memory, and if nerve cells are destroyed, comprehensive memory damage occurs, in which not only the storage of new memories but also existing stored memory files are deleted.

[0058] Furthermore, even when memories are fully stored in the brain, problems can arise due to problems with the "retrieval" process, which involves retrieving them. Furthermore, impaired prefrontal cortex function can lead to inability to recall specific information in a timely manner. Furthermore, impaired neuromodulators like acetylcholine can further deteriorate memory function, deteriorating the overall memory system and exacerbating memory impairment.

[0059] The aforementioned "cognitive impairment" refers to a condition in which impairments occur across the brain's higher-order mental functions, including memory, language, spatial perception, judgment, and problem-solving. When toxic proteins accumulate and destroy neurons, damage to the hippocampus can begin with initial memory loss. When concentrated in the frontal or temporal lobes, personality changes or language impairments can occur first. Furthermore, when neurons are damaged by impaired cerebral blood flow, executive function declines, such as reduced information processing speed and planning abilities, can become more pronounced.

[0060] The above "attention deficit" refers to a condition in which the ability to selectively focus on internal and external stimuli, sustained attention, and attentional shifting is lower than expected at the developmental stage, resulting in significant difficulty maintaining concentration or filtering out distracting stimuli during goal-directed behavior, learning, and problem-solving. It can be caused by developmental delays and functional decline in the prefrontal cortex-striatal neural circuit, and the dysfunction of this circuit can fundamentally be attributed to an imbalance between two neurotransmitters: dopamine, which controls motivation and reward, and norepinephrine, which regulates arousal and concentration.

[0061] In the present invention, "inflammatory disease" is a general term for diseases in which inflammation is the main lesion. The inflammatory disease of the present invention may be at least one selected from the group including arthritis, gout, hepatitis, obesity, keratitis, gastritis, enteritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis. More specifically, the inflammatory disease may be, but is not limited to, neuritis or enteritis.

[0062] The novel lactic acid bacteria of the present invention exhibit an effective inhibitory effect on the growth of Proteus mirabilis (P. mirabilis) strain and an effect of inhibiting TNF-α expression of macrophages, and are therefore effective in the prevention, treatment, and improvement of inflammatory diseases, specifically, neuritis and / or enteritis.

[0063] In one embodiment of the present invention, Lactococcus lactis P91, Bifidobacterium animalis P97 or a mixture thereof was administered to mice in which alpha-synuclein-related neuropsychiatric disorders (including Parkinson's disease) were induced by Proteus mirabilis or MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), and then a behavioral test was performed. As a result, it was confirmed that the treatment was effective in preventing, treating and improving such neuropsychiatric disorders and related memory and motor function decline.

[0064] In another embodiment of the present invention, biomarker analysis was performed on mice that underwent the above behavioral experiment, and it was confirmed that Lactococcus lactis P91, Bifidobacterium animalis P97, or a mixture thereof is effective in preventing, treating, and improving alpha-synuclein-related neuropsychiatric disorders and accompanying memory decline.

[0065] In another embodiment of the present invention, tissues of mice that performed the above behavioral experiment were stained and analyzed, and it was confirmed that Lactococcus lactis P91, Bifidobacterium animalis P97, or a mixture thereof is effective in preventing, treating, and improving alpha-synuclein-related neuropsychiatric diseases and related memory decline, and has the effect of inhibiting the expression and accumulation of alpha-synuclein, which is the core cause of the disease.

[0066] Specifically, in the pharmaceutical composition, the mixture may be a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 in a colony forming unit (CFU) ratio of 0.2:1 to 5:1, specifically 0.5:1 to 4:1, and more specifically 1:1 to 4:1, but is not limited thereto.

[0067] In addition, specifically, the Lactococcus lactis P91 or Bifidobacterium animalis P97 may be a live cell, a dead cell, a culture, a lysate or an extract thereof, respectively, but may be applied without limitation as long as it is in a form that can achieve the desired effect.

[0068] In the present invention, “live cells” means the novel lactic acid bacteria of the present invention themselves, “dead cells” means lactic acid bacteria sterilized by heating, pressurization, or drug treatment, and “disintegrated material” means lactic acid bacteria destroyed by enzyme treatment, homogenization, or ultrasonic treatment.

[0069] In the present invention, “extract” means a product obtained by extracting lactic acid bacteria with a known extraction solvent.

[0070] In the present invention, "culture" or "culture solution" means a product obtained by culturing lactic acid bacteria in a known medium, and the product may include a novel lactic acid bacteria. The medium may be selected from known liquid or solid media, and may be, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, or BL agar medium, but is not limited thereto.

[0071] The pharmaceutical composition according to the present invention can be formulated into a pharmaceutical formulation using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. In preparing the formulation, the pharmaceutical composition according to the present invention may additionally include a pharmaceutically acceptable carrier, provided that it does not inhibit the activity of the novel lactic acid bacteria.

[0072] Another aspect of the present invention relates to a food composition for preventing or improving neuropsychiatric or inflammatory diseases, comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

[0073] Specifically, the neuropsychiatric disorder may be at least one selected from the group consisting of alpha-synuclein disease, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In addition, the neuropsychiatric disorder may be at least one selected from the group consisting of anxiety disorder, depression, stress-related disorder, mood disorder, sleep disorder, memory disorder, cognitive disorder, and attention disorder, but is not limited thereto, and matters regarding the "neuropsychiatric disorder" are as described above.

[0074] The above “inflammatory disease” is the same as described above, and specifically, the inflammatory disease may be enteritis or neuritis.

[0075] Specifically, in the food composition, the mixture may be a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 strains in a colony forming unit (CFU) ratio of 0.2:1 to 5:1, specifically 0.5:1 to 4:1, and more specifically 1:1 to 4:1, but is not limited thereto.

[0076] There are no specific restrictions on the types of the above foods. Foods to which lactic acid bacteria can be added include dairy products including sausages, meat, bread, chocolate, snacks, candies, confectionery, ramen, pizza, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. When formulated as a beverage, the liquid ingredients added in addition to the novel lactic acid bacteria are not limited to this, but may contain various flavorings or natural carbohydrates as additional ingredients, as in conventional beverages. The above-mentioned natural carbohydrates may be monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), and polysaccharides (e.g., conventional sugars such as dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0077] In addition, specifically, the lactic acid bacteria included in the food composition of the present invention may be live cells, killed cells, cultures, lysates, or extracts thereof, but any form of lactic acid bacteria capable of achieving a preventive or therapeutic effect on inflammatory diseases or mental disorders may be used without limitation. The "live cells," "killed cells," "cultures," "lysates," and "extracts" are the same as those described above.

[0078] Specifically, the food may be a health functional food. The health functional food emphasizes the bioregulatory function of the food, and is a food that has been given added value by utilizing physical, biochemical, and biotechnological methods to function and manifest for a specific purpose. The ingredients of such health functional foods are designed and processed to sufficiently exert the body's regulatory functions related to biological defense, regulation of body rhythms, and prevention and recovery from disease. The food may contain food additives, sweeteners, or functional ingredients that are acceptable as food.

[0079] When the Lactococcus lactis P91, Bifidobacterium animalis P97 or mixture thereof of the present invention is used as a health functional food (or health functional beverage additive), the novel lactic acid bacteria may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The mixing amount of the Lactococcus lactis P91, Bifidobacterium animalis P97 or mixture thereof of the present invention may be appropriately determined depending on the purpose of use (prevention, health or improvement, therapeutic treatment).

[0080] Another aspect of the present invention relates to a method for preventing or treating a neuropsychiatric or inflammatory disease, comprising administering to a subject a composition comprising Lactococcus lactis P91, Bifidobacterium animalis P97 or a mixture thereof.

[0081] Specifically, the neuropsychiatric disorder may be at least one selected from the group consisting of alpha-synuclein disease, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In addition, the neuropsychiatric disorder may be at least one selected from the group consisting of anxiety disorder, depression, stress-related disorder, mood disorder, sleep disorder, memory disorder, cognitive disorder, and attention disorder, but is not limited thereto, and matters regarding the "neuropsychiatric disorder" are as described above.

[0082] The above “inflammatory disease” is the same as described above, and specifically, the inflammatory disease may be neuritis or enteritis.

[0083] The above-mentioned entity refers to an animal, and may be specifically a mammal that can exhibit beneficial effects from treatment using the lactic acid bacteria of the present invention. Specific examples of such entities include, but are not limited to, primates such as humans.

[0084] The Lactococcus lactis P91 KCCM 13447P, Bifidobacterium animalis P97 KCCM 13448P or a mixture thereof of the present invention is excellent in the prevention, treatment or improvement of neuropsychiatric diseases or inflammatory diseases.

[0085] The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0086] Figure 1 shows the phylogenetic characteristics of Lactococcus lactis P91 KCCM 13447P.

[0087] Figure 2 shows the results of comparing the whole genome of Lactococcus lactis P91 KCCM 13447P strain with similar strains.

[0088] Figure 3 shows the phylogenetic characteristics of Bifidobacterium animalis P97 KCCM13448P.

[0089] Figure 4 shows the results of comparing the whole genome of Bifidobacterium animalis P97 KCCM13448P strain with similar strains.

[0090] Figure 5 shows the results of confirming the induction of alpha-synuclein expression in a Proteus mirabilis strain in vitro.

[0091] Figure 6 shows the results of confirming the decline in motor function of the Proteus mirabilis strain in an animal model.

[0092] Figure 7 shows the results of comparing and observing pathological changes by immunofluorescence staining of brain tissues of a mouse model of neuropsychiatric disease induced by Proteus mirabilis Pm2 and a normal group (NC).

[0093] Figure 8 shows the results of ELISA and immunoblotting analysis of biochemical and molecular changes in the brain, blood, and intestinal tissues of a mouse model of neuropsychiatric disease induced by Proteus mirabilis Pm2 and a normal group (NC) (striatum: (a) to (d), substantia nigra: (e) to (h), hippocampus: (i) to (j), blood: (k) to (l), ileum: (m) to (o), colon: (p) to (r)).

[0094] Figure 9 shows the alpha-synuclein reducing effect when Lactococcus lactis P91 (PL), Bifidobacterium animalis P97 (PB) and / or a mixture thereof (PLB) is administered in a neuropsychiatric disease cell model (PM) induced by Proteus mirabilis Pm2.

[0095] Figure 10 shows the results of a behavioral experiment when Lactococcus lactis P91 (PL), Bifidobacterium animalis P97 (PB) and / or a mixture thereof (PLB) were administered to a mouse model of neuropsychiatric disease induced by Proteus mirabilis Pm2 (PM).

[0096] Figure 11 shows the results of confirming the histopathological effects in brain tissue when Lactococcus lactis P91 (PL), Bifidobacterium animalis P97 (PB) and / or a mixture thereof (PLB) are administered to a mouse model of neuropsychiatric disease induced by Proteus mirabilis Pm2 (PM).

[0097] Figure 12 shows the results of analyzing biochemical indicators in the striatum, substantia nigra, hippocampus, and blood when Lactococcus lactis P91 (PL), Bifidobacterium animalis P97 (PB), and / or a mixture thereof (PLB) were administered to a mouse model of neuropsychiatric disease induced by Proteus mirabilis Pm2 (PM) (striatum: (a) to (d), substantia nigra: (e) to (h), hippocampus: (i) to (l), blood: (m) to (o)).

[0098] Figure 13 shows the results of comparing the changes in alpha-synuclein and inflammatory cytokines in intestinal tissues when Lactococcus lactis P91 (PL), Bifidobacterium animalis P97 (PB) and / or a mixture thereof (PLB) were administered to a mouse model of neuropsychiatric disease induced by Proteus mirabilis Pm2 (PM) (ileum: (a) to (c), colon: (d) to (f)).

[0099] Figure 14 shows the Operational Taxonomic Unit (OUT) index according to administration of Proteus mirabilis NK2016 (PM), Lactococcus lactis P91 (PL), Bifidobacterium animalis P97 (PB) and / or a mixture thereof (PLB).

[0100] Figure 15 shows the results of confirming changes in microbial composition according to administration of Proteus mirabilis NK2016 (PM), Lactococcus lactis P91 (PL), Bifidobacterium animalis P97 (PB) and / or a mixture thereof (PLB).

[0101] Figure 16 shows the results of a behavioral experiment when a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 (MP) was administered to a Parkinson's disease mouse model (MT) induced by MPTP.

[0102] Figure 17 shows the results of confirming the histopathological effects in brain tissue when a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 (MP) is administered in a Parkinson's disease mouse model (MT) induced by MPTP.

[0103] Figure 18 shows the results of analyzing biochemical indicators in the striatum, substantia nigra, hippocampus, and blood when a mixture (MP) of Lactococcus lactis P91 and Bifidobacterium animalis P97 was administered in a Parkinson's disease mouse model (MT) induced by MPTP (striatum: (a) to (d), substantia nigra: (e) to (h), hippocampus: (i) to (l), blood: (m) to (o)).

[0104] Figure 19 shows the results of comparing the changes in alpha-synuclein and inflammatory cytokines in intestinal tissues when a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 (MP) was administered in a Parkinson's disease mouse model (MT) induced by MPTP (ileum: (a) to (c), colon: (d) to (f)).

[0105] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited to the following examples.

[0106] Example 1. Isolation and identification of lactic acid bacteria

[0107] 1-1. Isolation of lactic acid bacteria

[0108] 1-1-1. Isolation of lactic acid bacteria from kimchi

[0109] Each cabbage kimchi was shredded, and the slurry was suspended in MRS broth (MRS Broth; Difco, USA). The supernatant was then transferred to MRS agar medium (MRS agar medium; Difco, USA) and anaerobically cultured at 37°C for approximately 48 hours. The strains that formed colonies were isolated.

[0110] 1-1-2. Isolation of lactic acid bacteria from human feces

[0111] Human feces were suspended in GAM broth (Nissui Pharmaceutical, Japan). The supernatant was then transferred to GAM or BL agar medium (Nissui Pharmaceutical, Japan) and anaerobically cultured at 37°C for approximately 48 hours. The strains that formed colonies were then isolated.

[0112] 1-2. Identification of isolated lactic acid bacteria

[0113] The physiological characteristics and 16S rDNA sequences of strains isolated from kimchi or human feces were analyzed to identify the strains and assign strain names. The assigned strain names are shown in Table 1 below. Control numbers 1 to 8 in Table 1 below correspond to lactic acid bacteria isolated from kimchi, and numbers 9 to 30 correspond to lactic acid bacteria isolated from healthy human feces.Specifically, two species of Lactobacillus sakei (No. 1 and 2 in Table 1), two species of Lactobacillus brevis (No. 3 and 4 in Table 1), three species of Lactobacillus plantarum (No. 5 to 7 in Table 1), one species of Lactobacillus curvatus (No. 8 in Table 1), two species of Lactobacillus fermentum (No. 9 and 10 in Table 1), one species of Lactobacillus gasseri (No. 11 in Table 1), one species of Lactobacillus reuteri (No. 12 in Table 1), Lactobacillus 1 species of Lactobacillus acidophilus (No. 13 in Table 1), 2 species of Lactobacillus casei (No. 14 and 15 in Table 1), 3 species of Lactococcus lactis (No. 16 to 18 in Table 1), 1 species of Bifidobacterium pseudocatenulatum (No. 19 in Table 1), 1 species of Bifidobacterium catenulatum (No. 20 in Table 1), 4 species of Bifidobacterium animalis (No. 21, 22, 29 and 30 in Table 1), 2 species of Bifidobacterium bifidum (No. 23 and 24), two species of Bifidobacterium longum (No. 25 and 26 in Table 1), and two species of Bifidobacterium adolescentis (No. 27 and 28 in Table 1).

[0114] Management numberGwantsmanymanagement numberGwantsmany1Lactobacillus sakeiP6216Lactococcus lactisP912Lactobacillus sakeiP6417Lactococcus lactisP923Lactobacillus brevisP6718Lactococcus lactisP934Lactobacillus brevisP6819Bifidobacterium pseudocatenulatumP945Lactobacillus plantarumP7120Bifidobacterium catenulatumP956Lactobacillus plantarumP7221Bifidobacterium animalisP967Lactobacillus plantarumP7322Bifidobacterium animalisP978Lactobacillus curvatusP7523Bifidobacterium bifidumP989Lactobacillus fermentumP7724Bifidobacterium bifidumP9910Lactobacillus fermentumP7825Bifidobacterium longumP10111Lactobacillus gasseriP7926Bifidobacterium longumP11312Lactobacillus reuteriP8127Bifidobacterium adolescentisP10613Lactobacillus acidophilusP8328Bifidobacterium adolescentisP10714Lactobacillus caseiP8629Bifidobacterium animalisP11115Lactobacillus caseiP8830Bifidobacterium animalisP113

[0115] 1-2-1. Physiological characteristics of Lactococcus lactis P91 (Lactococcus lactis P91) KCCM 13447P

[0116] Among the strains listed in Table 1 above, Lactococcus lactis P91 KCCM 13447P is a Gram-positive coccus that does not produce spores. The 16S rDNA sequence of Lactococcus lactis P91 had a GC content of 35.1% as a result of whole-genome analysis. No Lactococcus lactis strains with the same rDNA sequence were found. It showed 97.4% homology with the known Lactococcus lactis ATCC19435 and 97.2% homology with Lactococcus lactis NBRC100931, confirming that it has the highest molecular phylogenetic relationship with them.

[0117] Figure 1 shows the phylogenetic characteristics of Lactococcus lactis P91 KCCM 13447P, and Figure 2 shows the results of comparing the whole genome of Lactococcus lactis P91 KCCM 13447P strain with similar strains. Figure 2 calculates the gene-level similarity by comparing the genomes pairwise based on the Lactococcus lactis P91 strain, and calculates the percentage of genes with homology and displays them in color. The closer to blue, the higher the similarity, and the closer to red, the less similar.

[0118] Among the physiological characteristics of Lactococcus lactis P91, carbon source utilization was analyzed using the API 50CH Kit and API 20A Kit (manufacturer: BioMerieux's, USA). The results are shown in Table 2 below, where "+" indicates positive carbon source utilization, and "-" indicates negative carbon source utilization.

[0119] Carbon source P91 (API 50CH Kit)--glycerol-erythritol-D-arabinose-L-arabinose+D-ribose+D-xylose+L-xylose-D-adonitol-methyl-β-D-xylopyranoside-D-galactose+D-glucose+D-fructose+D-mannose+L-sorbose-L-rhamnose-dulcitol-inositol (Inositol) - Mannitol + Sorbitol - α-methyl-D-mannoside - α-methyl-D-glucoside - N-acetyl-glucosamine + Amygdalin + Arbutin + Esculin + Salicin + Cellobiose + Maltose + Lactose - Melibiose - Sucrose + Trehalose + Inulin - Melezitose - Raffinose - Starch + Glycogen - Xylitol (xylitol)-gentiobiose+D-turanose-D-lyxose-D-tagatose-D-fucose-L-fucose-D-arabitol-L-arabitol-gluconate-2-keto-gluconate-5-keto-gluconate-

[0120] The above novel strain Lactococcus lactis P91 was patented and deposited with the Korea Microbiological Conservation Center (Address: Yulim Building, 45 Hongje-nae 2-ga-gil, Seodaemun-gu, Seoul, Republic of Korea) and assigned the accession number KCCM13447P.

[0121] 1-2-2. Physiological characteristics of the novel lactic acid bacterium Bifidobacterium animalis P97

[0122] Among the strains listed in Table 1 above, Bifidobacterium animalis P97 is a Gram-positive rod that does not produce spores. The 16S rDNA sequence of Bifidobacterium animalis P97 had a GC content of 60.5% as a result of whole-genome analysis. No Bifidobacterium animalis strains with the same rDNA sequence were found. It showed 99.9% homology with the known Bifidobacterium animalis DSM10140 and 95.6% homology with Bifidobacterium animalis ATCC25527, confirming the highest molecular phylogenetic relationship with the Bifidobacterium animalis family.

[0123] Figure 3 shows the phylogenetic characteristics of Bifidobacterium animalis P97 KCCM13448P, and Figure 4 shows the results of comparing the whole genome of the Bifidobacterium animalis P97 KCCM13448P strain with similar strains. Figure 4 calculates the gene-level similarity by comparing the genomes pairwise based on the Bifidobacterium animalis P97 strain, and calculates the percentage of genes with homology and displays them in color. The closer to blue, the higher the similarity, and the closer to red, the lower the similarity.

[0124] Among the physiological characteristics of Bifidobacterium animalis P97, carbon source utilization was analyzed using the API 50CH Kit and API 20A Kit (manufacturer: BioMerieux's, USA). The results are shown in Table 3 below, where "+" indicates positive carbon source utilization, and "-" indicates negative carbon source utilization.

[0125] In addition, the biochemical characteristics (CAT, Spore, Gram, Cocci) of Bifidobacterium animalis P97 were analyzed using the API 20A Kit, and the results are shown in Table 3.

[0126] Carbon source P97 (API20A Kit) L-tryptophan - Urea + D-glucose + D-mannitol - D-lactose + D-sucrose + D-maltose + Salicin + D-xylose + L-arabinose + Gelatin - Esculin - Glycerol - D-cellobiose + D-mannose + D-melezitose - D-raffinose + D-sorbitol - D-rhamnose (D-rhamnose)-D-trehalose (D-trehalose)-CAT-Spore-Gram+Cocci-

[0127] The above novel strain Bifidobacterium animalis P97 was patented and deposited at the Korea Microbiological Conservation Center (Address: Yulim Building, 45 Hongje-nae 2-ga-gil, Seodaemun-gu, Seoul, Republic of Korea) and assigned the accession number KCCM 13448P.

[0128] Example 2. Isolation of intestinal bacteria that induce α-synuclein expression

[0129] The feces of patients with inflammatory bowel disease (IBD) and experimental rats (mice) with Parkinson's disease were suspended in sterilized TS medium, inoculated onto DHL agar medium, and cultured anaerobically at 37°C for 2 days. The cultured colonies were identified through Gram staining and 16S rRNA gene sequence analysis, and confirmed to be Proteus mirabilis strains. The isolated Proteus mirabilis strains were named NK1134 (Pm1) isolated from rats (mice), and NK2016 (Pm2), NK2013 (Pm3), and NK2015 (Pm4) isolated from the feces of patients with inflammatory bowel disease, respectively, and cultured anaerobically in TS medium at 37°C for 12 hours.

[0130] Experimental Example 1. Confirmation of induction of alpha-synuclein expression in Proteus mirabilis strains.

[0131] 1-1. In vitro

[0132] SH-SY5Y cells, a human neuroblastoma cell line, were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2.

[0133] For alpha-synuclein analysis, SH-SY5Y cells (1Х10 6 cells / mL) were added to Proteus mirabilis Pm1 to Pm4 (1Х10 4 After dispensing into 12-well plates in the presence or absence of 10 CFU / mL, the cells were cultured for 20 h. The alpha-synuclein expression level was then evaluated by enzyme-linked immunosorbent assay (ELISA).

[0134] As a result, as shown in Fig. 5, it was confirmed that administration of Proteus mirabilis Pm1 to Pm4 induced the expression of alpha-synuclein compared to the normal control group, the NC group. In particular, treatment with Pm2 showed higher alpha-synuclein expression than rat (mouse) isolated Pm1.

[0135] 1-2. Animal models and behavioral experiments

[0136] C57BL / 6 male mice (6 weeks old) were purchased from Daehan Biolink (Eumseong, Korea). They were housed in plastic cages at a temperature of 20–22°C, a humidity of 50±10%, and a 12-h light / dark cycle, with food and water provided ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyung Hee University (IACUC No. KHSASP-21306) and performed in accordance with the ethical policies and guidelines of the Committee, the Care and Use of Laboratory Animals, and the ARRIVE guidelines.

[0137] Mice were randomly divided into three groups of eight each (NC, Pm1 [NK1134], Pm2 [NK2016]), and Pm1 (Proteus mirabilis NK1134, 5 x 10 8 CFU / mouse / day) or Pm2 (Proteus mirabilis NK2016, 5 x 10 8 (CFU / mouse / day) was orally administered to mice daily for 14 days. The NC group, a normal control group, was orally administered saline.

[0138] Behavioral experiments including the rotarod test, grip strength test, and pole test were performed the day after the final administration of Proteus mirabilis.

[0139] As a result, as shown in Fig. 6, the normal control group showed normal motor ability on a rotating rod in the rotarod test, but when Proteus mirabilis Pm1 or Pm2 was administered, the time spent on the rotating rod was significantly reduced. This indicates that Proteus mirabilis Pm1 or Pm2 caused damage to the sense of balance and motor nerves, resulting in a decline in coordination ability (Fig. 6(a)).

[0140] In addition, in the grip strength test, muscle strength decreased when Proteus mirabilis Pm1 or Pm2 was administered compared to the muscle strength of the normal control group, indicating that Proteus mirabilis Pm1 or Pm2 affected neuromuscular function, resulting in muscle weakness (Figure 6(b)).

[0141] Additionally, the pole test assesses bradykinesia (a symptom of slowed movement), measuring the total time (TT) it takes for an animal to turn and descend from the top of a pole to the bottom. Compared to normal controls, Pm2 administration significantly increased TT. This suggests that the animals exhibited a typical symptom of alpha-synucleinopathy, characterized by severe impairment in the ability to initiate and execute movements, resulting in extremely slowed movement (Figure 6(c)).

[0142] 1-3. Brain tissue analysis

[0143] After the above behavioral experiments 1-2, brain tissue was immunofluorescently stained or cytokine and protein concentrations within the tissue were measured to confirm neuroinflammation and pathological protein accumulation.

[0144] Immunofluorescence staining

[0145] Sections of the substantia nigra (SN) and striatum (ST) were prepared and blocked with blocking solution (10% normal donkey serum and 0.3% Triton X-100 in PBS) for 1 hour. Then, to check for neuroinflammation and pathological changes, they were incubated overnight at 4°C with primary antibodies against GFAP (abcam, ab7260 dilution: 1 / 500), Iba1 (abcam, ab5076 dilution: 1 / 1000), p-Ser129 (abcam, ab51253 dilution: 1 / 500), NF-κB p65 (Cell signaling, 6956S dilution: 1 / 500), and TH (Invitrogen, PA1-4679 dilution: 1 / 500). After washing with PBS, the sections were incubated with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen, dilution: 1 / 500) for 2 h. Nuclei were visualized with 4',6-diamidino-2-phenylindole dilactate (DAPI, Sigma). All sections were observed using a confocal laser microscope.

[0146] As a result, as shown in Figure 7, dopamine neurons (TH: green) were well preserved in the normal group (NC). On the other hand, in the Pm2-administered group, green dopamine neurons were significantly reduced, indicating that dopamine neurons in the brain were reduced by Pm2.

[0147] In addition, microglia, another main player in neuroinflammation, Iba1 is indicated in red, and NF-kB, a key factor regulating intracellular inflammatory signal transduction, is indicated in green. In the normal group (NC), both Iba1 and NF-kB signals were very weak. On the other hand, in the Pm2-administered group, red microglia (Iba1) and green inflammatory signals (NF-kB) were significantly increased, showing that a strong inflammatory response was occurring in the brain.

[0148] In addition, pS129 (green) specifically stains only pathologically altered, toxic alpha-synuclein, and while no green pS129, a pathological protein, was observed in the normal group (NC), green pS129 aggregates were clearly accumulated throughout the brain tissue in the Pm2-administered group.

[0149] From this, it was confirmed that the pathological characteristics of dopaminergic neuron destruction, strong neuroinflammation, and alpha-synuclein accumulation were reproduced through Proteus mirabilis (Pm2) treatment (Fig. 7).

[0150] Measurement of cytokine and protein concentrations in tissues: ELISA and immunoblotting

[0151] Tissues from the striatum (ST), substantia nigra (SN), hippocampus, ileum, and colon were homogenized in RIPA lysis buffer containing 1% phosphatase inhibitor and protease inhibitor. The homogenized solution was centrifuged at 4°C and 10,000g for 20 min to prepare a supernatant containing proteins. For ELISA analysis, the prepared supernatant or blood was transferred to a 96-well plate, and the concentrations of alpha-synuclein (α-synuclein), inflammatory cytokines (TNF-α, IL-1β), and dopamine were measured using each ELISA kit. All proteins were quantified using a BCA assay kit.

[0152] Additionally, for immunoblotting analysis, the extracted proteins were applied to SDS-PAGE gels and electrophoresis was performed. Subsequently, the total expression level of alpha-synuclein in brain tissue was confirmed through alpha-synuclein (SNCA, Millipore, rabbit origin 1:200) antibody reaction.

[0153] As a result, as shown in Fig. 8, in the striatum (ST) and substantia nigra (SN), when Pm2 was administered, both alpha-synuclein and inflammatory substances such as TNF-α and IL-1β were significantly increased compared to the normal group (NC), while dopamine was significantly decreased (striatum: Figs. 8(a) to (d), substantia nigra: Figs. 8(e) to (h)). This shows that three characteristic pathological phenomena, namely alpha-synuclein accumulation, neuroinflammation, and dopaminergic neuron damage, were all clearly observed in the core areas of diseases related to alpha-synuclein pathology.

[0154] Furthermore, in the hippocampus, a region responsible for memory and learning, the inflammatory cytokines TNF-α and IL-1β significantly increased compared to the normal group (NC) when Pm2 was administered (Fig. 8(i), (j)). This indicates that neuroinflammation spread to the hippocampus region associated with cognitive function, inducing cognitive decline and memory loss, as seen in alpha-synuclein pathology.

[0155] In addition, blood is an indicator reflecting the systemic condition, and TNF-α and IL-1β were found to increase significantly in the Pm2-administered group compared to the normal group (NC) (Fig. 8(k), (l)). This indicates that the inflammatory response induced by Proteus mirabilis Pm2 was not limited to the brain, but caused systemic inflammation that spread throughout the body through the blood.

[0156] In addition, the inflammatory response in the intestine was analyzed, and it was confirmed that TNF-α and IL-1β were significantly increased in the ileum and colon compared to the normal group (NC) when Pm2 was administered (ileum: Fig. 8(n) and (o), colon: Fig. 8(q) and (r)).

[0157] Furthermore, the total expression level of alpha-synuclein protein in the intestinal tissue was confirmed by Western blot, and it was shown that alpha-synuclein protein was abnormally overexpressed when Pm2 was administered compared to the normal group (NC) (ileum: Fig. 8(m), colon: Fig. 8(p)).

[0158] Experimental Example 2. Inhibitory activity of Lactococcus lactis P91 and Bifidobacterium animalis P97 against inflammation, Proteus mirabilis strain, and alpha-synuclein.

[0159] 2-1. Antioxidant activity

[0160] DPPH (2,2-Diphenyl-1-picrylhydrazyl) was dissolved in ethanol to a concentration of 0.2 mM to prepare a DPPH solution. Lactic acid bacteria suspension (1x10) was added to 0.1 ml of the DPPH solution. 8 CFU / ㎖) or vitamin C solution (1 g / ㎖) was added and cultured at 37°C for 20 minutes. The culture solution was centrifuged at 3000 rpm for 5 minutes to obtain the supernatant. Afterwards, the absorbance of the supernatant was measured at 517 nm, and the antioxidant activity of the lactic acid bacteria was calculated. The results are shown in Table 4.

[0161] 2-2. Confirmation of TNF-α expression inhibition activity in macrophages

[0162] C57BL / 6 mice (male, 6 weeks old, 19-22 g) were administered 2 ml of sterile 4% thioglycolate into the peritoneal cavity, anesthetized 4 days later, and 8 ml of RPMI 1640 medium was administered into the peritoneal cavity of the mice. After 5-10 minutes, the RPMI medium (macrophages) in the peritoneal cavity was removed, centrifuged at 1000 x g for 10 minutes, washed twice with RPMI 1640 medium, and the macrophages were seeded at 0.5 Y10 per well. 6 were transplanted into 24-well plates with the number of isolated lactic acid bacteria (final treatment concentration: 1x10 4CFU / mL) and the inflammatory response-inducing strain Proteus mirabilis (1x10 4 The strain heat-treated at 90°C for 30 minutes (CFU / mL) was added, cultured in a CO2 / air incubator for 24 hours, and the supernatant was obtained, and the TNF-α expression level was measured using an ELISA kit (R&D system, USA). The measurement results are shown in Table 4.

[0163] 2-3. Confirmation of growth inhibition activity of Proteus mirabilis strain

[0164] Lactic acid bacteria (1x10) cultured in BHI medium in advance 5 Proteus mirabilis (1x10 CFU / mL) with 4 CFU / mL) were transplanted and cultured under anaerobically conditions at 37°C for 24 hours, then transplanted onto DHL agar medium and cultured aerobically for 24 hours, and the number of grown Proteus mirabilis colonies was measured. The measurement results are shown in Table 4.

[0165] 2-4. Confirmation of alpha-synuclein inhibitory activity

[0166] SH-SY5Y cells obtained from the Korean Cell Line Bank were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics, and seeded at 2 x 10 per well in a 12-well plate. 6 Cells were divided into 10 wells. Afterwards, lactic acid bacteria (1 x 10 4 Proteus mirabilis (1x10 CFU / ml) heat-treated at 90°C for 30 min 4 CFU / mL) was added and cultured, and the expression level of alpha-synuclein was measured using an ELISA kit after 24 hours. The measurement results are shown in Table 4.

[0167] 김리균주명저해임(%)항사지이스트TNF-α발현얕제(대식세타)P. mirabilis성장양제알파-시너니다 이리이전(SH-SY5Y) 1Lactobacillus sakeiP62++--2Lactobacillus sakeiP64++++--3Lactobacillus brevisP67++++-+4Lactobacillus brevisP68++--5Lactobacillus plantarumP71+++++6Lactobacillus plantarumP72+++++7Lactobacillus plantarumP73+++++8Lactobacillus curvatusP75++-+9Lactobacillus fermentumP77++-+10Lactobacillus fermentumP78++-+11Lactobacillus gasseriP79++++++12Lactobacillus reuteriP81++++++13Lactobacillus acidophilusP83++-+14Lactobacillus caseiP86++++15Lactobacillus caseiP88++++16Lactococcus lactisP91++++++++17Lactococcus lactisP92++++++18Lactococcus lactisP93++++++19Bifidobacterium pseudocatenulatumP94++++20Bifidobacterium catenulatumP95+++-+21Bifidobacterium animalisP96+++-+22Bifidobacterium animalisP97++++++++++++23Bifidobacterium bifidumP98++++++24Bifidobacterium bifidumP99++++++25Bifidobacterium longumP101++-+26Bifidobacterium longumP113++++-+27Bifidobacterium adolescentisP106++++-+28BifidobacteriumadolescentisP107++--29Bifidobacterium animalisP111++++30Bifidobacterium animalisP113+++++Inhibition rate (%): -:<5% inhibition; +:5-25 % inhibition; ++:26-50% inhibition; +++:>50% inhibitionAntioxidant activity (%) = 100 x (Optical absorbance of supernatant treated with vehicle only - Absorbance of supernatant treated with lactic acid bacteria) / (Optical absorbance of supernatant treated with vehicle only)TNF-α inhibition rate (%) = 100 x (TNF-α expression level of cells treated with P. mirabilis only - TNF-α expression level of cells treated with lactic acid bacteria and P. mirabilis together) / (TNF-α expression level of cells treated with P. mirabilis only)P. Mirabilis growth inhibition (%) = 100 x (Number of colonies on agar medium transplanted with medium cultured with only P. mirabilis - Number of colonies on agar medium transplanted with medium cultured with both lactic acid bacteria and P. mirabilis) / (Number of colonies on agar medium transplanted with medium cultured with only P. mirabilis) Alpha-synuclein inhibition (%) = 100 x (Alpha-synuclein expression level of cells treated with only P. mirabilis - Alpha-synuclein expression level of cells treated with both lactic acid bacteria and P. mirabilis) / (Alpha-synuclein expression level of cells treated with only P. mirabilis)

[0168] As shown in Table 4 above, among the isolated lactic acid bacteria, Lactococcus lactis P91 and / or Bifidobacterium animalis P97 were found to have excellent antioxidant activity, which is the ability to remove active oxygen that causes cell damage and aging, and also exhibited a strong anti-inflammatory effect by having an excellent effect of suppressing the expression of TNF-α, a cytokine that causes inflammation.

[0169] In addition, it was confirmed that Lactococcus lactis P91, Bifidobacterium animalis P97 and / or a mixture thereof inhibited the inducing factor of alpha-synucleinopathy by inhibiting the Proteus mirabilis strain, and further effectively inhibited the expression of alpha-synuclein itself directly in a neuronal cell (SH-SY5Y) model (Fig. 9).

[0170] This suggests that the Lactococcus lactis P91, Bifidobacterium animalis P97 and / or the mixture thereof of the present invention exhibit excellent antioxidant and anti-inflammatory effects, thereby promoting the prevention, treatment and improvement of inflammatory diseases, and can also promote the prevention, treatment and improvement of neuropsychiatric diseases including alpha-synucleinopathy through the inhibition of the expression of Proteus mirabilis strain and alpha-synuclein.

[0171] Experimental Example 3. Confirmation of the neuropsychiatric improvement effects of Lactococcus lactis P91 and Bifidobacterium animalis P97 in mice with P. mirabilis-induced neuropsychiatric disorders.

[0172] 3-1. Creation of mice with neuropsychiatric disorders and administration of probiotics

[0173] Creation of P. mirabilis-induced Parkinson's disease mice and administration of probiotics

[0174] Mice (male C57BL / 6 mice, 6 weeks old, purchased from DBL and acclimated to the animal room for 7 days) were fed Proteus mirabilis (5x10 8 CFU) was administered orally once daily for 14 days (Pm2(PM)). The normal group (NC) was administered saline instead of Proteus mirabilis. The number of mice per experimental group was 6. Lactic acid bacteria were administered once daily for 2 weeks from the day after the final administration of Proteus mirabilis, Lactococcus lactis P91(PL) (1x10 9 CFU / mouse / day), Bifidobacterium animalis P97 (PB) (1x10 9CFU / mouse / day), mixture (PLB)) (Lactococcus lactis P91, Bifidobacterium animalis P97 [4:1] mix, 1x10 9 CFU / mouse / day) was administered orally.

[0175] 3-2. Behavioral experiment

[0176] The following behavioral experiments were performed once daily starting from the day after administration of Lactococcus lactis P91, Bifidobacterium animalis P97, and / or the mixture.

[0177] grip strength test

[0178] The grip strength of the mouse's forepaw was measured three times, five times each, using a grip strength meter, and the grip strength of the mouse model was expressed as the average of the three highest values. The measurement results are shown in Table 5 below.

[0179] pole test

[0180] A pole test was performed on a 55 cm high, 0.8 cm diameter bar. Mice were placed head-up on the bar, and the time it took for the mouse to turn 180° around the top and descend until all four legs touched the ground was measured. The measurement results are shown in Table 5 below.

[0181] Rotarod test

[0182] The Rotarod device is a rotatable cylindrical rod with a diameter of 7 cm, five 15 cm gaps, and a height of 60 cm. A mouse was placed on the rod rotating at 20 rpm, and the time it took for the mouse to fall (latency time; sec) was measured. The measurement results are shown in Table 5 below.

[0183] Y-maze test

[0184] The apparatus used in the Y-maze experiment consists of three branches, each measuring 40 cm in length, 3 cm in width, and 12 cm in height. Each branch forms a 120° angle and is made of black polyvinyl resin. Mice are carefully placed in one of the three Y-maze branches, labeled A, B, and C, and allowed to move freely for 8 minutes. The branch into which the mouse entered was recorded. Only when the tail completely entered was the experimental animal recorded as entering the branch, and re-entering the same branch was also recorded. One point (actual alternation) was awarded for entering three different branches in succession. The maximum alternation was determined by subtracting 2 from the total number of entries into the three branches.

[0185] The results of the behavioral experiment after administration of Lactococcus lactis P91 (PL), Bifidobacterium animalis P97 (PB) and / or a mixture (PLB) to Proteus mirabilis (Pm2)-induced neuropsychiatric mice are shown in Table 5 and Figure 10.

[0186] NCPm2PLPBPLB Grip strength test (g) 2.9 2.12.9 2.7 2.7 Pole test (s) 6.5 11.2 6.6 6.7 6.7 Rotarod test (s) 267 216 258 235 259 Y-maze test (SA %) 79.2 39.5 76.5 68.9 69.2 Spontaneous alternation (SA) % = 100 x [Actual alternation / Maximum alternation]

[0187] In the case of mice with Proteus mirabilis (Pm2)-induced neuropsychiatric disorders, compared to the normal group, decreased muscle strength (grip strength test), aggravated slowness of action speed (pole test), decreased motor coordination ability (rotarod test), and decreased memory ability (Y-maze test) were observed, confirming that motor and cognitive dysfunction similar to alpha-synuclein disease was induced. In all groups administered with Lactococcus lactis P91, Bifidobacterium animalis P97, and the mixture, the weakened muscle strength was significantly restored, the pole test time, which was greatly increased, was shortened to the normal level, indicating that action speed was restored, and the rotarod test time also recovered to the level of the normal group, confirming that motor coordination ability was restored. In addition, the Y-maze test scores were also significantly improved in all groups, confirming that memory was restored.

[0188] This suggests that Lactococcus lactis P91, Bifidobacterium animalis P97, and their mixture can all be very effective in improving, preventing, and treating neuropsychiatric disorders including alpha-synucleinopathy and their symptoms (motor and cognitive decline).

[0189] 3-3. Immunofluorescence analysis

[0190] Mice were perfused whole body with 4% paraformaldehyde, and brain tissues (striatum, substantia nigra, and hippocampus) were isolated, sectioned, and incubated overnight with primary antibodies against tyrosine hydroxylase (TH, Millipore, rabbit origin 1:2000), alpha-synuclein (SNCA, Millipore, rabbit origin 1:200), Iba1 (Cell Signaling, 1:200), or / and glial fibrillary acidic protein (GFAP, Cell signaling, 1:200), and then developed with antibodies conjugated with Alexa Fluor 594 (1:200, Invitrogen) or Alexa Fluor 488 (1:200).

[0191] The results of immunofluorescence analysis of Proteus mirabilis (Pm2)-induced neuropsychiatric disease mice are shown in Table 6 and Figure 11.

[0192] Tissue biomarker NCPm2PLPBPLB Substantia nigra pS129 + Iba1 + Cell count ( / mm 3 )1187231225182121798917885TH + GFAP + Cell count ( / mm 3 )3923124343345543325634891NF-κB + Iba1 + Cell intensity: 100295158182163TH + GFAP + Cell intensity: 10054828791

[0193] In mice with Proteus mirabilis (Pm2)-induced neuropsychiatric disorders, pS129 and inflammatory cell pS129 were found in the substantia nigra and striatum compared to the normal group. + Iba1+ The activity (number of cells and intensity) of the TH was significantly increased in the substantia nigra and striatum. + GFAP + The activity (cell number and intensity) was significantly reduced.

[0194] When PL, PB, and PLB of the present invention were administered, the death of dopaminergic neurons was suppressed, their function was restored, neuroinflammatory reactions were strongly suppressed, and a neuroprotective effect in the hippocampus was observed.

[0195] Specifically, the number of dopaminergic neurons and the density of nerve terminals (cell intensity), which had been rapidly reduced in the Pm2 group, were significantly recovered in all of the PL, PB, and PLB groups, indicating that the lactic acid bacteria of the present invention have a strong neuroprotective effect that prevents the death of dopaminergic neurons, which is one of the main characteristics of alpha-synuclein disease.

[0196] In addition, the activity of inflammatory cells, which had rapidly increased in the Pm2 group, was significantly reduced in all of the PL, PB, and PLB groups, indicating that the lactic acid bacteria of the present invention can effectively control neuroinflammation by calming excessive immune responses in brain tissue.

[0197] In addition, factors related to hippocampal neuronal growth and survival, which had decreased in the Pm2 group, increased again in all of the PL, PB, and PLB groups, showing that the therapeutic effect of the lactic acid bacteria of the present invention can affect the hippocampus, which is responsible for memory, and thus recovery of cognitive function can occur.

[0198] These results suggest that Lactococcus lactis P91, Bifidobacterium animalis P97, and their mixture can effectively prevent, treat, and / or improve alpha-synucleinopathy through neuroprotection, anti-inflammation, and increasing neurotrophic factors.

[0199] 3-4. Measurement of brain and intestinal tissue biomarkers (ELISA)

[0200] After completing the behavioral test, mice were sacrificed, and brain tissues (striatum, substantia nigra, hippocampus) and intestinal tissues (gut and colon) were isolated. The tissues were homogenized with RIPA lysis buffer, and the amounts of BDNF, TNF-α, IL-10, IL-1β, and dopamine in the centrifuged supernatant were measured using ELISA kits. The ELISA test method was performed according to the manufacturer's instructions.

[0201] The biomarker measurements of brain and intestinal tissues of Proteus mirabilis (PM)-induced neuropsychiatric disease mice are shown in Table 7 and Figure 12.

[0202] NCPMPLPBPLBSubstantia nigraTNF-α (pg / mg)172239183185183IL-1β (pg / mg)6179635861IL-10 (pg / mg)268231251239241Dopamine (pg / mg)0.0150.0130.0140.0150.015StriatumTNF-α (pg / mg)149228175165162IL-1β (pg / mg)5672636162IL-10 (pg / mg)232183185208194HippocampusTNF-α (pg / mg)157233159183173IL-1β (pg / mg)76119849185IL-10 (pg / mg)601354415485467BDNF (pg / mg)14511045130213981499Gut / colonTNF-α (pg / mg)215265225228220IL-1β (pg / mg)157181159161167IL-10 (pg / mg)621586595601605SNCA (pg / mg)0.230.390.280.290.28Gut / IleumTNF-α (pg / mg)189247218215209IL-1β (pg / mg)132179153157149IL-10 (pg / mg)325277302309311SNCA (pg / mg)0.270.530.370.340.32

[0203] In mice with Proteus mirabilis (PM)-induced neuropsychiatric disorders, compared to the normal group, TNF-α and IL-1β increased in the substantia nigra (SN), striatum (ST), hippocampus, intestine / colon, intestine / ileum, and blood, indicating increased inflammation, while IL-10 decreased, indicating a decreased anti-inflammatory function. In addition, the level of dopamine, an important neurotransmitter, was significantly reduced in the substantia nigra, and brain-derived neurotrophic factor (BDNF), which helps the survival and growth of neurons, was significantly reduced in the hippocampus. In particular, it was confirmed that the level of alpha-synuclein (SNCA) was significantly increased in the intestinal tissue.

[0204] When PL, PB, and PLB of the present invention were administered, it was confirmed that inflammation was suppressed in the substantia nigra, striatum, and hippocampus, which are key areas of brain tissue, intestinal inflammation and alpha-synuclein were normalized, and neuroprotection and functional recovery were achieved, and that there was a systemic anti-inflammatory effect that suppressed the inflammatory state of the whole body through the blood.

[0205] Specifically, we confirmed that TNF-α and IL-1β, which had been significantly increased in the PM group, were significantly reduced in the substantia nigra, striatum, hippocampus, intestine / colon, intestine / ileum, and blood, and that IL-10, an anti-inflammatory cytokine, was restored, suppressing brain tissue, intestinal tissue, and systemic inflammation. In addition, we confirmed that BDNF and dopamine levels were restored in the substantia nigra, striatum, and hippocampus, thereby protecting neurons.

[0206] In addition, in the intestinal tissues of the large intestine / colon and intestine / ileum, alpha-synuclein was significantly increased from the intestine in the PM group, and when PL, PB, and PLB of the present invention were administered, alpha-synuclein in the intestinal tissue was significantly reduced. This indicates that the present invention can exhibit a therapeutic effect on alpha-synucleinopathy by improving the intestinal environment through Lactococcus lactis P91, Bifidobacterium animalis P97, and a mixture thereof.

[0207] 3-5. Analysis of intestinal pathological changes

[0208] Changes in the expression of alpha-synuclein and inflammatory cytokines in the ileum and colon were measured. Alpha-synuclein was observed using immunoblot, and inflammatory cytokines were measured using ELISA. For the measurement of cytokines and proteins in tissues, immunoblot analysis for alpha-synuclein and cytokine ELISA measurements were performed in the same manner as in Experimental Examples 1-3 above.

[0209] As a result, as shown in Fig. 13, in the case of Proteus mirabilis (Pm)-induced neuropsychiatric disease mice, alpha-synuclein and inflammatory cytokines TNF-α and IL-1β were both increased in both the ileum and the large intestine.

[0210] On the other hand, it was confirmed that when PL, PB, and PLB of the present invention were administered, the overexpression of alpha-synuclein and the level of inflammation in the ileum tissue were significantly suppressed and showed a level similar to that of the normal group, and it was confirmed that the overexpression of alpha-synuclein and the inflammatory response in the colon tissue were also significantly suppressed and restored to a normal level.

[0211] Proteus mirabilis induced alpha-synuclein overexpression and inflammatory responses in the intestines (ileum and colon), the starting point of the gut-brain axis, while Lactococcus lactis P91, Bifidobacterium animalis P97, and a mixture thereof showed effects in controlling these intestinal pathological changes. This suggests that improving the intestinal environment through the lactic acid bacteria of the present invention may enable the prevention, treatment, and / or improvement of neuropsychiatric disorders, including alpha-synuclein disease.

[0212] Experimental Example 4. Intestinal Microbiota Analysis

[0213] The composition of the intestinal microbial community was analyzed to determine whether dysbiosis was induced by Proteus mirabilis NK2016 (Pm2, PM) and whether it was restored by the lactic acid bacteria of the present invention. Specifically, genes were amplified using barcoded primers targeting the V4 region of the bacterial 16S rRNA gene using DNA extracted from fecal samples as a template. Each amplified product (amplicon) was sequenced using an Illumina iSeq 100 (Illumina iSeq 100, San Diego, CA) instrument. The final analyzed sequence data (reads) were deposited with the National Center for Biotechnology Information (NCBI) in the United States, and the accession number is PRJNA1229807.

[0214] As a result, the OTU index of Fig. 14 is a unit that statistically represents microbial diversity, and as shown in Fig. 14, there is no statistically significant difference in the OTU index value between the normal group, PM, PL, PB, and PLB groups.

[0215] In addition, Figure 15 shows the results of confirming whether there was a change in the microbial composition even though there was no significant difference in the complexity of the intestinal microorganisms, and shows that intestinal imbalance was induced in the PM group and recovered in the PL, PB, and PLB groups.

[0216] Specifically, Fig. 15(a) shows an analysis at the Phylum level. In the normal group (NC), the normal intestinal environment is dominated by two large groups, Bacteroidetes and Firmicutes, and maintains a stable balance. In the PM group, the thickness of Firmicutes decreased and Proteobacteria increased compared to the normal group. Proteobacteria are associated with inflammation, and an increase in this ratio suggests that the intestinal environment has changed to an inflammatory one. In the PL, PB, and PLB groups, the ratio of Proteobacteria, which had increased in the PM group, decreased again, showing that the balance was restored.

[0217] In addition, Fig. 15(b) shows an analysis at the family level, in which the healthy intestinal environment of the normal group is formed by the largest proportion of Lachnospiraceae, which produces butyric acid, and Muribaculaceae, which plays a role in decomposing polysaccharides, forming a stable community, and various microbial groups coexist.

[0218] The PM group showed a decrease in the proportion of beneficial bacteria, Lactobacillaceae, while the proportion of harmful bacteria, such as Desulfovibrionaceae, increased, causing an imbalance in the intestines. In the PL, PB, and PLB groups, the proportion of Lactobacillus, which had decreased in the PM group, increased significantly again, indicating that the important constituent microorganisms of a healthy intestinal environment were restored, and the harmful bacteria that had increased in the PM group showed a suppression pattern.

[0219] These results suggest that the lactic acid bacteria of the present invention can suppress systemic inflammation by restoring intestinal balance, and can exhibit an improvement and treatment effect on neuropsychiatric disorders by suppressing inflammation in brain tissue and protecting nerve cells.

[0220] Experimental Example 5. Confirmation of the Parkinson's disease and cognitive improvement effects of Lactococcus lactis P91 and Bifidobacterium animalis P97 in MPTP-induced Parkinson's disease mice.

[0221] 5-1. Creation of Parkinson's disease mice and administration of probiotics

[0222] Mice (male C57BL / 6 mice, 6 weeks old, purchased from DBL and acclimated to the animal room for 7 days) were administered MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Sigma, Hercules, CA, USA) intraperitoneally at 30 mg / kg / day for 5 days (MT). The normal group (NC) was administered saline instead of MPTP. The number of mice per experimental group was 8. Lactic acid bacteria were administered once daily for 14 days from the day after the final MPTP administration, using the lactic acid bacteria Lactococcus lactis P91 (1x10 9 CFU / mouse / day), Bifidobacterium animalis P97 (1x10 9 CFU / mouse / day), mixture (Lactococcus lactis P91, Bifidobacterium animalis P97 [4:1] mix, 1x10 9 CFU / mouse / day) was administered orally (MP).

[0223] 5-2. Behavioral experiment

[0224] The grip strength test, pole test, Rotarod test, and Y-maze test were performed the day after administration of the mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97, and the performance method was the same as the behavioral experiment in 3-2. The results of the behavioral experiment are shown in Table 8 and Figure 16. In Figure 16, Mt is the MPTP administration group, and MM is the group treated with the mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 after MPTP administration.

[0225] NCMTMP (MPTP + mixture) Grip strength test (g) 2.8 2.0 2.6 Pole test (s) 5.9 10.9 6.5 Rotarod test (s) 258 204 248 Y-maze test (SA %) 75.4 5 1.3 6 9.1

[0226] Mice induced with Parkinson's disease by MPTP showed decreased muscle strength (grip strength test), increased slowness of movement (pole test), decreased motor coordination (rotarod test), and decreased memory (Y-maze test) compared to the normal group, indicating that representative motor and cognitive dysfunctions of Parkinson's disease were induced.

[0227] In all groups administered the mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97, weakened muscle strength significantly recovered, the pole test time, which had been greatly extended, was shortened to a normal level, indicating a recovery in behavioral speed, and the rotarod test time also recovered to the normal group level, confirming the recovery of motor coordination ability. In addition, Y-maze test scores also significantly improved in all groups, confirming the recovery of memory.

[0228] This suggests that a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 is effective in preventing, treating, and improving Parkinson's disease.

[0229] 5-3. Immunofluorescence analysis

[0230] The method was performed in the same manner as the method 3-3 above, and the results of immunofluorescence analysis in MPTP-induced Parkinson's disease mice are as shown in Table 9 and Figure 17. In Figure 17, MT is the MPTP administration group, and MP is the group treated with a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 after MPTP administration.

[0231] Tissue biomarker NCMTMP Substantia nigra pS129 + Iba1 + Cell count ( / mm 3 )101222732817724TH + GFAP + Cell count ( / mm3 )421262578336539StriatumSNCA + Iba1 + Cell intensity 100305183TH + GFAP + Cell intensity: 1005688

[0232] Mice with Parkinson's disease induced by MPTP had higher levels of inflammatory cells pS129 in the substantia nigra and striatum compared to the normal group. + Iba1 + The activity (cell number and intensity) of TH was significantly increased. + GFAP + The activity (cell number and intensity) was significantly reduced.

[0233] In the group administered a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97, dopaminergic neuron death was suppressed, function was restored, neuroinflammatory responses were strongly suppressed, and a neuroprotective effect in the hippocampus was observed.

[0234] Specifically, the number of dopaminergic neurons and the density of nerve terminals (cell intensity), which had been rapidly reduced upon MPTP administration, were significantly restored in the mixture administration group, indicating that the lactic acid bacteria of the present invention have a strong neuroprotective effect that prevents the death of dopaminergic neurons, which is one of the main characteristics of Parkinson's disease.

[0235] In addition, the activity of inflammatory cells, which had significantly increased upon MPTP administration, was significantly reduced in the mixture administration group, indicating that the lactic acid bacteria of the present invention can effectively control neuroinflammation by calming excessive immune responses in brain tissue.

[0236] These results suggest that Lactococcus lactis P91, Bifidobacterium animalis P97, and their mixture may effectively prevent, treat, and / or improve Parkinson's disease through neuroprotection, anti-inflammation, and increased neurotrophic factors.

[0237] 5-4. Biomarker Measurements of Brain and Intestinal Tissues

[0238] The method for measuring biomarkers using ELISA in brain and intestinal tissues is as follows: 3-4. The results are as follows: Table 10 and Figure 18. In Figure 18, MT is the MPTP administration group, and MP is the group treated with a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 after MPTP administration.

[0239] NCMTMPSubstantia nigraTNF-α (pg / mg)243341249Dopamine (pg / mg)0.0210.0130.020StriatumTNF-α (pg / mg)125202147Dopamine (pg / mg)0.0210.0120.021HippocampusTNF-α (pg / mg)164266219BDNF (pg / mg)10986891043Gut / colonTNF-α (pg / mg)232302182SNCA (pg / mg)0.190.270.22Gut / IleumTNF-α (pg / mg)119302182SNCA (pg / mg)0.220.430.27

[0240] In MPTP-induced neuropsychiatric disease mice, compared to the normal group, TNF-α and IL-1β increased in the substantia nigra (SN), striatum (ST), hippocampus, intestine / colon, intestine / ileum, and blood, indicating increased inflammation. In addition, IL-10 decreased in the blood, indicating a decreased anti-inflammatory function. In addition, the level of dopamine, an important neurotransmitter, was also significantly reduced in the substantia nigra, and in the hippocampus, brain-derived neurotrophic factor (BDNF), which promotes the survival and growth of neurons, was significantly reduced. In particular, it was confirmed that the level of alpha-synuclein (SNCA) was significantly increased in the intestinal tissue.

[0241] When the mixture of P91 and P97 of the present invention was administered, it was confirmed that inflammation was suppressed in the substantia nigra, striatum, and hippocampus, which are key areas of brain tissue, intestinal inflammation and alpha-synuclein were normalized, and neuroprotection and functional recovery were achieved, and that there was a systemic anti-inflammatory effect that suppressed the inflammatory state of the whole body through the blood.

[0242] Specifically, TNF-α and IL-1β, which had been significantly increased upon MPTP administration, were significantly reduced in the substantia nigra, striatum, hippocampus, intestine / colon, intestine / ileum, and blood, and IL-10, an anti-inflammatory cytokine, was restored in the blood. In addition, BDNF and dopamine levels were also restored in the substantia nigra, striatum, and hippocampus, confirming the protective effect on neurons.

[0243] In addition, in the intestinal tissues of the large intestine and ileum, alpha-synuclein was significantly increased from the intestine when MPTP was administered, and alpha-synuclein in the intestinal tissue was significantly reduced when the mixture of P91 and P97 of the present invention was administered. This indicates that the mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 of the present invention can exhibit a therapeutic effect on Parkinson's disease by improving the intestinal environment.

[0244] 5-5. Analysis of intestinal pathological changes

[0245] Changes in the expression of alpha-synuclein and inflammatory cytokines in the ileum and colon were measured, and the experimental method was as in 3-5.

[0246] As a result, as shown in Fig. 19, when MPTP was administered, both alpha-synuclein and inflammatory cytokines TNF-α and IL-1β increased in both the ileum and the large intestine (in Fig. 19, MT is the MPTP administration group, and MP is the group treated with a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 after MPTP administration).

[0247] On the other hand, when a mixture of Lactococcus lactis P91 and Bifidobacterium animalis P97 was administered, the overexpression of alpha-synuclein and the level of inflammation in the ileum tissue were significantly suppressed, and the overexpression of alpha-synuclein and the level of inflammation in the colon tissue were also significantly suppressed and restored to normal levels.

[0248] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0249] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0250]

[0251] [Accession number]

[0252] Name of depositor: Korea Microbiological Conservation Center (overseas)

[0253] Accession number: KCCM13447P

[0254] Date of acceptance: 20231222

[0255]

[0256] Name of depositor: Korea Microbiological Conservation Center (overseas)

[0257] Accession number: KCCM13448P

[0258] Date of acceptance: 20231222

[0259]

[0260]

[0261]

[0262]

[0263]

Claims

1. Lactococcus lactis P91 strain deposited under accession number KCCM 13447P.

2. In paragraph 1, The above strain is a Lactococcus lactis P91 strain comprising a 16S rDNA base sequence of sequence number 1.

3. Bifidobacterium animalis P97 strain deposited under accession number KCCM 13448P.

4. In paragraph 3, The above strain is a Bifidobacterium animalis P97 strain comprising the 16S rDNA base sequence of sequence number 2.

5. An antibacterial composition comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

6. A pharmaceutical composition for preventing or treating Proteus mirabilis infection, comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

7. A composition for inhibiting alpha-synuclein expression, comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

8. A pharmaceutical composition for preventing or treating neuropsychiatric or inflammatory diseases, comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

9. In paragraph 8, A pharmaceutical composition, wherein the above neuropsychiatric disease is at least one selected from the group consisting of alpha-synuclein disease, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.

10. In paragraph 8, A pharmaceutical composition, wherein the above neuropsychiatric disorder is at least one selected from the group consisting of anxiety disorder, depression, stress-related disorder, mood disorder, sleep disorder, memory disorder, cognitive disorder, and attention disorder.

11. In paragraph 8, A pharmaceutical composition wherein the inflammatory disease is neuritis or enteritis.

12. In paragraph 8, A pharmaceutical composition wherein the mixture comprises Lactococcus lactis P91 strain and Bifidobacterium animalis P97 strain mixed in a colony forming unit (CFU) ratio of 0.2:1 to 5:

1.

13. In paragraph 8, A pharmaceutical composition wherein the Lactococcus lactis P91 strain or Bifidobacterium animalis P97 strain is a live cell thereof, a dead cell thereof, a culture thereof, a lysate thereof, or an extract thereof.

14. A food composition for preventing or improving neuropsychiatric or inflammatory diseases, comprising Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof.

15. In the above 14th paragraph, A food composition wherein the above neuropsychiatric disease is at least one selected from the group consisting of alpha-synuclein disease, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.

16. In the above paragraph 14, A food composition wherein the above neuropsychiatric disorder is at least one selected from the group consisting of anxiety disorder, depression, stress-related disorder, mood disorder, sleep disorder, memory disorder, cognitive disorder, and attention disorder.

17. In the above 14th paragraph, The above inflammatory disease is neuritis or enteritis, food composition.

18. In Article 14 A food composition wherein the mixture is a mixture of Lactococcus lactis P91 strain and Bifidobacterium animalis P97 strain in a colony forming unit (CFU) ratio of 0.2:1 to 5:

1.

19. In Article 14 A food composition wherein the Lactococcus lactis P91 strain or the Bifidobacterium animalis P97 strain is a live cell thereof, a dead cell thereof, a culture thereof, a lysate thereof, or an extract thereof.

20. A method for treating a neuropsychiatric disease or an inflammatory disease, comprising administering to a subject a Lactococcus lactis P91 strain, a Bifidobacterium animalis P97 strain, or a mixture thereof.

21. A method for improving a neuropsychiatric disease or an inflammatory disease, comprising administering to a subject a Lactococcus lactis P91 strain, a Bifidobacterium animalis P97 strain, or a mixture thereof.

22. Use of Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof for the treatment of neuropsychiatric or inflammatory diseases.

23. Use of Lactococcus lactis P91 strain, Bifidobacterium animalis P97 strain or a mixture thereof for improving neuropsychiatric or inflammatory diseases.

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