Novel lactic acid bacteria and uses thereof

Abstract

The present invention relates to novel lactic acid bacteria, namely Lactobacillus plantarum P72 KCCM13445P, Bifidobacterium longum P77 KCCM13446P, or a mixture thereof, and uses thereof. The strains according to the present invention can be usefully employed for the prevention, treatment, or alleviation of neuropsychiatric disorders or inflammatory diseases.

Description

Novel lactic acid bacteria and their uses

[0001] The present invention relates to novel lactic acid bacteria, Lactobacillus plantarum P72 KCCM13445P, Bifidobacterium longum P77 KCCM13446P, or a mixture thereof, and uses thereof.

[0002] Neuropsychiatric disorders are conditions characterized by abnormal brain function, leading to difficulties in social and occupational adjustment, including interactions with others, and manifesting various behavioral and psychological abnormalities. The causes of mental illness are complex, encompassing genetic, psychological, biological, and environmental factors. However, many conditions remain unknown, and when the cause is identified, it typically occurs under severe stressful circumstances. These neuropsychiatric disorders are on the rise in modern society due to increasing social stress, and include depression, anxiety disorders, schizophrenia, and sleep disorders.

[0003] Sleep disorders are a common problem faced by modern people, affecting over 20% of the population at some point in their lives. These disorders are characterized by inability to achieve healthy sleep, difficulty maintaining daytime alertness despite adequate sleep, or difficulty falling asleep or staying awake due to disrupted sleep rhythms. Psychological issues can also play a significant role, resulting from excessive stress, anxiety, tension, and fear.

[0004] Pharmacological treatment accounts for the largest portion of treatment for neuropsychiatric disorders, including sleep disorders, and most of these are chemical agents. However, these treatments have drawbacks, such as rapid awakening due to the drug's half-life, hangover effects, and decreased daytime performance due to cumulative effects. Long-term use can also lead to serious side effects, including rebound insomnia, anxiety, withdrawal symptoms, tolerance, and dependence.

[0005] Accordingly, there is a continuous demand for the development of substances that can improve mental disorders such as cognitive impairment and sleep disorders while minimizing side effects.

[0006] The purpose of the present invention is to provide a Lactobacillus plantarum P72 strain deposited under the accession number KCCM13445P.

[0007] Another object of the present invention is to provide a Bifidobacterium longum P77 strain deposited under the accession number KCCM13446P.

[0008] Another object of the present invention is to provide a pharmaceutical composition comprising Lactococcus plantarum P72 strain, Bifidobacterium longum P77 strain, or a mixture thereof.

[0009] Another object of the present invention is to provide a food composition comprising Lactococcus plantarum P72 strain, Bifidobacterium longum P77 strain, or a mixture thereof.

[0010] 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 plantarum P72 strain, a Bifidobacterium longum P77 strain, or a mixture thereof.

[0011] One aspect of the present invention for achieving the above purpose relates to the Lactobacillus plantarum P72 strain deposited under accession number KCCM13445P.

[0012] The Lactobacillus plantarum P72 of the present invention is characterized as a novel lactic acid bacterium isolated and identified from the feces of a healthy human.

[0013] The 16S rDNA base sequence for identification and classification of Lactobacillus plantarum P72 of the present invention is as shown in SEQ ID NO: 1 attached to this specification. Therefore, Lactobacillus plantarum P72 of the present invention may include the 16S rDNA of SEQ ID NO: 1.

[0014] Analysis of the 16S rDNA base sequence of the above sequence number 1 showed 99% homology with known Lactobacillus plantarum strains, showing the highest molecular phylogenetic relationship with Lactobacillus plantarum. Therefore, the lactic acid bacterium was identified as Lactobacillus plantarum, named Lactobacillus plantarumP72, and deposited with the Korea Microbiological Conservation Center on December 22, 2023 (KCCM13445P).

[0015] The Lactobacillus plantarum P72 of the present invention is gram-positive. More specific physiological characteristics of Lactobacillus plantarum P72 can be analyzed according to a conventional method in the art, and specifically, Lactobacillus plantarum P72 can utilize L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, rhamnosus, mannitol, sorbitol, α-methyl-D-mannoside, N-acetyl-glucosamine, amygdalin, arbutin, salicin, cellobis, maltose, lactose, melibis, sucrose, trehalose, inulin, melizitose, raffinose, gentiobis, D-turanose, D-lyxose, and gluconate as a carbon source.

[0016] Another aspect of the present invention relates to Bifidobacterium longum strain P77 deposited under accession number KCCM13446P.

[0017] The Bifidobacterium longum P77 of the present invention is characterized as a novel lactic acid bacterium isolated and identified from the feces of a healthy human.

[0018] The 16S rDNA base sequence for identification and classification of Bifidobacterium longum P77 of the present invention is as shown in SEQ ID NO: 2 attached to this specification. Therefore, Bifidobacterium longum P77 of the present invention may include the 16S rDNA of SEQ ID NO: 2.

[0019] Analysis of the 16S rDNA base sequence of the above sequence number 2 showed 99% homology with known Bifidobacterium longum strains, demonstrating the highest molecular phylogenetic relationship with Bifidobacterium longum (Fig. 2). Therefore, the lactic acid bacterium was identified as Bifidobacterium longum, named Bifidobacterium longum P77, and deposited with the Korea Microbiological Conservation Center on December 22, 2023 (KCCM13446P).

[0020] The physiological characteristics of Bifidobacterium longum P77 of the present invention can be analyzed according to a conventional method in the art, and specifically, Bifidobacterium longum P77 can utilize urea, D-glucose, D-lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, D-raffinose, and D-trehalose as carbon sources.

[0021] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating neuropsychiatric or inflammatory diseases, comprising Lactococcus plantarum P72 strain, Bifidobacterium longum P77 strain, or a mixture thereof.

[0022] 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.

[0023] The above neuropsychiatric disorder may be one or more 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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 Associated with Another Medical Condition, Other Specified Depressive Disorder, and Unspecified Depressive Disorder.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Various sleep disorders occur when there is a problem with the switch or related neural circuit between the sleep center and the wake center.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] '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.

[0040] '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.

[0041] '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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In one embodiment of the present invention, when Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof was administered to an animal model in which stress-induced depressive disorder and / or sleep disorder was induced, it was confirmed that anxiety and depressive behaviors due to stress were significantly improved, the levels of brain-derived neurotrophic factor (BDNF), serotonin, and GABA, which were reduced in the stress-induced animal model, were restored, and blood stress indicators such as corticosterone, IL-6, and TNF-α were reduced. In particular, when Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof was administered, sleep latency time was reduced and sleep time was increased, and thus sleep disorders such as insomnia may be improved.

[0047] 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 colitis, arthritis, gout, hepatitis, obesity, keratitis, gastritis, enteritis, nephritis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis. More specifically, the inflammatory disease may be colitis, but is not limited thereto.

[0048] In one embodiment of the present invention, when Lactobacillus plantarum, Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof was administered to an animal model suffering from colitis due to stress, it was confirmed that the length of the colon, which is an indicator of colitis, was restored to a normal level, the expression of myeloperoxidase (MPO), TNF-α, IL-1β, and IL-6, which are indicators of colitis, was reduced, and the expression of IL-10 was increased. Through this, it was confirmed that a pharmaceutical composition comprising Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof can be usefully used for the prevention and treatment of inflammatory diseases, specifically, colitis.

[0049] The above Lactobacillus plantarum P72 or Bifidobacterium longum P77 strain may be a live cell, a dead cell, a culture, a lysate or an extract thereof, but may be applied without limitation in any form that can achieve the desired effect.

[0050] 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.

[0051] In the present invention, “extract” means a product obtained by extracting lactic acid bacteria with a known extraction solvent.

[0052] 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.

[0053] The mixture in the pharmaceutical composition may be a mixture of Lactobacillus plantarum P72 and Bifidobacterium longum P77 strains in a colony forming unit (CFU) ratio of 1:1 to 4:1, but is not limited thereto.

[0054] Additionally, specifically, the pharmaceutical composition may further comprise hempseed oil.

[0055] The above "hempseed oil" is an oil extracted from the kernel of hemp seeds from which the hallucinogenic component has been removed, and is high in unsaturated fatty acids and minerals. In one embodiment of the present invention, it was confirmed that when Lactobacillus plantarum P72 and hempseed oil were administered together, depressive disorder and sleep disorders were improved, and inflammatory indicators were improved. Therefore, the combination of hempseed oil and the lactic acid bacteria of the present invention can be utilized for the prevention, treatment, and / or improvement of neuropsychiatric disorders and inflammatory diseases.

[0056] 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.

[0057] Another aspect of the present invention relates to a food composition for preventing or improving neuropsychiatric or inflammatory diseases, comprising Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof.

[0058] The above “neuropsychiatric disease” and “inflammatory disease” are as described above.

[0059] Specifically, the neuropsychiatric disorder may be at least one selected from the group consisting of anxiety disorder, depression, stress-related disorder, mood disorder, insomnia, sleep disorder, memory disorder, cognitive disorder, and attention disorder, but is not limited thereto.

[0060] Also specifically, the inflammatory disease may be, but is not limited to, colitis.

[0061] There are no special 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 these, but may contain various flavorings or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates mentioned above 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.

[0062] 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 permitted as food.

[0063] When the Lactobacillus plantarum P72, Bifidobacterium longum P77 strain of the present invention, or a mixture thereof, 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 Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof may be appropriately determined depending on the purpose of use (prevention, health or improvement, therapeutic treatment).

[0064] Additionally, specifically, the food composition may further comprise hempseed oil. "Hempseed oil" is as described above.

[0065] 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 Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof. The composition may be a pharmaceutical composition.

[0066] The above “neuropsychiatric disease” and “inflammatory disease” are the same as those described above, and specifically, the inflammatory disease may be colitis, but is not limited thereto.

[0067] The subject refers to an animal, typically a mammal that can exhibit a beneficial effect from treatment using the lactic acid bacteria of the present invention. Examples of such subjects include primates such as humans.

[0068] Another aspect of the present invention relates to a method for improving a neuropsychiatric or inflammatory disease, comprising administering to a subject a composition comprising Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof. The composition may be, but is not limited to, a food composition.

[0069] The above “neuropsychiatric disease” and “inflammatory disease” are the same as those described above, and specifically, the inflammatory disease may be colitis, but is not limited thereto.

[0070] The subject refers to an animal, typically a mammal that can exhibit a beneficial effect from treatment using the lactic acid bacteria of the present invention. Examples of such subjects include primates such as humans.

[0071] The Lactobacillus plantarum P72, Bifidobacterium longum P77 strain of the present invention, or a mixture thereof, is excellent in improving and / or treating neuropsychiatric disorders including cognitive impairment and sleep disorders. In addition, the Lactobacillus plantarum P72, Bifidobacterium longum P77 strain of the present invention, or a mixture thereof, has an effect of suppressing inflammatory responses and can be used in a composition for preventing or treating inflammatory diseases, and is particularly effective in preventing and treating colitis.

[0072] The effects of the present invention are not limited to the effects described above, 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.

[0073] Figure 1 shows the results of comparing the Lactobacillus plantarum P72 strain with similar strains using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method, along with the similarity between the two strains. Genomes were compared pairwise with the P72 strain to calculate gene-level similarity, and genes with homology were calculated by percentage and displayed in color. The closer to blue, the higher the similarity, and the closer to red, the lower the similarity.

[0074] Figure 2 shows the results of comparing the Bifidobacterium longum P77 strain with similar strains, as measured by UPGMA, and the full-length genome. Genomes were compared pairwise with the P77 strain to calculate gene-level similarity. Genes with homology were calculated by percentage and displayed by color. The closer to blue, the higher the similarity, and the closer to red, the lower the similarity.

[0075] Figure 3 shows the results of confirming the effect of Lactobacillus plantarum P72 strain and hemp seed oil on improving IS-induced sleep disorder and depression by immunofluorescence staining of GABA A1 receptors in the frontal lobe.

[0076] Figure 4 shows the results of confirming the effect of Lactobacillus plantarum P72 strain and hemp seed oil on improving IS-induced sleep disorder and depression by immunofluorescence staining of NF-κB positive cells in the frontal lobe.

[0077] Figure 5 shows the results of confirming the improvement effect on inflammation induced by IS of Lactobacillus plantarum P72 strain and hemp seed oil using immunofluorescence staining of NF-κB positive cells in the colon.

[0078] 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.

[0079] Example 1. Isolation and identification of lactic acid bacteria

[0080] 1-1. Isolation of lactic acid bacteria from human feces

[0081] Fresh feces (approximately 1 g) from healthy individuals were suspended in 9 mL of GAM broth (Nissui Pharmaceutical, Japan). The supernatant was then transferred to GAM agar medium (Nissui Pharmaceutical, Japan), MRS agar medium (BD, USA), or BL agar medium (Nissui Pharmaceutical, Japan), and anaerobically cultured at 37°C for approximately 48 h. The strains that formed colonies were isolated.

[0082] 1-2. Identification of isolated lactic acid bacteria

[0083] The physiological characteristics and 16S rDNA sequences of strains isolated from human feces were analyzed to identify the strains and assign strain names. The assigned strain names of the lactic acid bacteria are summarized in Tables 1 and 2 below.

[0084] There were one species of Lactobacillus casei (No. 1 in Table 1), one species of Lactobacillus acidophilus (No. 2 in Table 1), two species of Lactobacillus gasseri (No. 3 and 4 in Table 1), two species of Lactococcus lactis (No. 5 and 6 in Table 1), two species of Lactobacillus planantrum (No. 7 and 8 in Table 1), and two species of Lactobacillus pentosus (No. 9 and 10 in Table 1).

[0085] In addition, two species of Bifidobacterium longum (No. 11 and 12 in Table 1), one species of Bifidobacterium adolescentis (No. 13 in Table 1), one species of Bifidobacterium breve (No. 14 in Table 1), one species of Bifidobacterium bifidum (No. 15 in Table 1), one species of Bifidobacterium pseudocatenulatum (No. 16 in Table 1), one species of Bifidobacterium catenulatum (No. 17 in Table 1), and one species of Bifidobacterium infantis (No. 18 in Table 1) One species of Bifidobacterium animalis (No. 19 in Table 1) and one species of Bifidobacterium dentium (No. 20 in Table 1) were identified.

[0086] Management number Strain name Management number Strain name 1Lactobacillus caseiP6611Bifidobacterium longumP762Lactobacillus acidophilusP6712Bifidobacterium longumP773Lactobacillus gasseriP6813Bifidobacterium adolescentisP784Lactobacillus gasseriP6914Bifidobacterium breveP795Lactococcus lactisP7015Bifidobacterium bifidumP806Lactococcus lactisP7116Bifidobacterium pseudocatenulatumP817Lactobacillus planantrumP7217Bifidobacterium catenulatumP828Lactobacillus planantrumP7318Bifidobacterium infantisP839Lactobacillus pentosusP7419Bifidobacterium animalisP8410Lactobacillus pentosusP7520Bifidobacterium dentiumP85

[0087] 1-3. Measurement of activity of isolated lactic acid bacteria

[0088] 1-3-1. Measurement of inflammatory markers in macrophages

[0089] 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. After 5-10 minutes, the RPMI medium (macrophages) in the peritoneal cavity was removed, centrifuged at 1000 x g for 10 minutes, and washed twice with RPMI 1640 medium. The macrophages were seeded at 0.5 × 10 per well. 6 Dispense into 24-well plates and distribute the isolated lactic acid bacteria (final treatment concentration: 1x10 5CFU / mL) and heat-treated lipopolysaccharide (LPS) (100 ng / mL), an inflammatory response inducer, were added, cultured in a CO2 / air incubator for 24 hours, and the supernatant was obtained, and the expression levels of TNF-α and / or IL-6 were measured using an ELISA kit. The expression inhibitory activity of TNF-α and / or IL-6 was calculated according to Equation 1 below, and the results are shown in Tables 2 and 3.

[0090] [Formula 1]

[0091] Inhibition rate (%) = 100 x (IL-6 expression level of cells treated only with LPS - IL-6 expression level of cells treated with lactic acid bacteria and LPS) / (IL-6 expression level of cells treated only with LPS)

[0092] 1-3-2. Measurement of antioxidant activity (in vitro)

[0093] 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 (1 × 10 5 CFU / ㎖) 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 DPPH inhibitory activity was calculated according to Equation 2 below, and the results are shown in Tables 2 and 3.

[0094] [Formula 2]

[0095] Inhibition rate (%) = 100 x (absorbance of supernatant treated with vehicle only - absorbance of supernatant treated with lactic acid bacteria) / (absorbance of supernatant treated with vehicle only)

[0096] 1-3-3. Measurement of growth inhibition activity of Klebsiella oxytoca

[0097] The inhibitory activity against Klebsiella oxytoca, an intestinal bacterium that causes depression and anxiety, was measured. Lactic acid bacteria (1x10) were pre-incubated in 5 mL of BHI medium. 5 CFU / mL) were cultured, and Klebsiella oxytoca (1x10 5 Each of the 100 CFU / mL was transplanted and cultured under anaerobically at 37°C for 24 hours. These were transplanted onto MacConky agar medium and cultured aerobically for 24 hours. The grown Klebsiella oxytoca colonies were counted and the Klebsiella oxytoca growth inhibitory activity was calculated according to Equation 3 below. The results are shown in Tables 2 and 3.

[0098] [Formula 3]

[0099] Inhibition rate (%) = 100 x (Number of colonies on agar medium transplanted with medium cultured with only Klebsiella oxytoca - Number of colonies on agar medium transplanted with medium cultured with lactic acid bacteria and Klebsiella oxytoca) / (Number of colonies on agar medium transplanted with medium cultured with only Klebsiella oxytoca)

[0100] Serial number, strain name, inhibitory activity, IL-6DPPH, Klebsiella oxytoca, 1 Lactobacillus casei, P66+++-2, Lactobacillus acidophilus, P67++++, 3 Lactobacillus gasseri, P68++-+, 4 Lactobacillus gasseri, P69+++, 5 Lactococcus lactis, P70+++++, 6 Lactococcus lactis, P71+++++, 7 Lactobacillus planantrum, P72++++++++, 8 Lactobacillus planantrum, P73++-9, Lactobacillus pentosus, P74++-10, Lactobacilllus pentosus, P75+++*, Final concentration of lactic acid bacteria treated: 1×10 5 CFU / ml* +++, >80%; ++, >40 - 80%; +, >1 - 40%; -, <0

[0101] Serial number, strain name, inhibitory activity, TNF-α, IL-6, DPPH, Klebsiella oxytoca, 1, Bifidobacterium longum, P76++++++, 2, Bifidobacterium longum, P77++++++++++++, 3, Bifidobacterium adolescentis, P78+++++, 4, Bifidobacterium breve, P79++++, 5, Bifidobacterium bifidum, P80++++-, 6, Bifidobacterium pseudocatenulatum, P81++++++, 7, Bifidobacterium catenulatum, P82++++, 8, Bifidobacterium infantis, P83++++, 9, Bifidobacterium animalis, P84++++, 10, Bifidobacterium dentium, P85++++-*, Final concentration of lactic acid bacteria treated: 1×10 5 CFU / ml* +++, >80%; ++, >40 - 80%; +, >1 - 40%; -, <0

[0102] As shown in Table 2 above, the Lactobacillus plantarum P72 strain suppressed the expression of IL-6 and exhibited excellent antioxidant activity and Klebsiella oxytoca growth inhibition activity.

[0103] The above Lactobacillus plantarum P72 was patent deposited with the Korea Microbiological Conservation Center (Address: Yulim Building, 45 Hongje-nae 2-ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an authorized depository, and was assigned the accession number KCCM13445P. Lactobacillus plantarum P72 is Gram-positive, and as a result of comparing the 16S rDNA base sequence by BLAST search, no Lactobacillus plantarum strain with the same 16S rDNA base sequence was found, and it was confirmed to show 99% homology with the 16S rDNA sequence of a known Lactobacillus plantarum strain (SEQ ID NO: 1).

[0104] Whole-genome analysis of Lactobacillus plantarum P72 was performed by first preparing a 20-kb sequencing library using the PacBio DNA Template Prep Kit 1.0, ligating a blunt-end SMARTbell adapter, and sequencing the library using PacBio P6C4 Chemistry on an 8-well SMART Cell v3 of the PacBio RSII. The PacBio sequencing data were assembled using the PacBio SMRT Analysis 2.3.0 program according to the HGAP2 Protocol (Pacific Biosciences, USA), and contigs were circularized using Circlator 1.4.0 (Sanger Institute). As a result, there were 6 contigs, the GC content was 44.4%, and the strain was most similar to Lactobacillus plantarum NCTC13644 (homology 99.1%), followed by Lactobacillus argentoratensis DSM16365 (homology 95.5%). P72 was confirmed to be a novel strain even in the full-length genome (Fig. 1).

[0105] In addition, as shown in Table 3 above, the Bifidobacterium longumP77 strain suppressed the expression levels of TNF-α and IL-6, and exhibited excellent antioxidant activity and Klebsiella oxytoca growth inhibition activity.

[0106] The above Bifidobacterium longum P77 was patent deposited with the Korea Microbiological Conservation Center (Address: Yulim Building, 45 Hongje-nae 2-ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an authorized depository, and was assigned the accession number KCCM13446P. Bifidobacterium longum P77 is Gram-positive, and when the 16S rDNA base sequence was compared by BLAST search, no Bifidobacterium longum strains with the same 16S rDNA base sequence were found, and it was confirmed that it showed 99% homology with the 16S rDNA sequence of known Bifidobacterium longum strains (SEQ ID NO: 2).

[0107] Whole-genome analysis of Bifidobacterium longum P77 was performed by first preparing a 20-kb sequencing library using the PacBio DNA Template Prep Kit 1.0, ligating a blunt-end SMARTbell adapter, and sequencing the library using PacBio P6C4 Chemistry on an 8-well SMART Cell v3 of the PacBio RSII. The PacBio sequencing data were assembled using the PacBio SMRT Analysis 2.3.0 program according to the HGAP2 Protocol (Pacific Biosciences, USA), and contigs were circularized using Circlator 1.4.0 (Sanger Institute). As a result, there was 1 contig, the GC content was 60.0%, and the strain was most similar to Bifidobacterium longumJCM1217 (homology 98.63%), followed by Bifidobacterium longumDSM20211 (homology 95.98%). P77 was confirmed to be a novel strain even in the full-length genome (Fig. 2).

[0108] 1-4. Physiological characteristics of isolated lactic acid bacteria

[0109] 1-4-1. Physiological characteristics of Lactobacillus plantarum P72

[0110] Lactobacillus plantarum P72 is a Gram-positive coccus. Among its physiological characteristics, carbon source utilization was analyzed through a sugar fermentation test using the API 50 CHL kit (BioMerieux, USA). The results are shown in Table 4 below. In Table 4, "+" indicates positive carbon source utilization, and "-" indicates negative carbon source utilization.

[0111] Carbon source P72Carbon source P72CONTROL-esculin-glycerol-salicylic acid+erythritol-cellobis+D-arabinose-maltose+L-arabinose+lactose+D-ribose+melibis+D-xylose+sucrose+L-xylose-trehalose+D-adonitol-inulin+methyl- β-D-xylopyranoside-melizitose+D-galactose+raffinose+D-glucose+starch-D-fructose+glycogen-D-mannose+xylitol-L-sorbose-gentiobis+rhamnosus+D-turanose+dulcitol-D-lyxose+inositol-D-tagatose-mannitol+D-fucose-sorbitol+L-fucose-α-methyl-D-mannoside+D-arabitol-α-methyl-D-glucoside-L-arabitol-N-acetyl-glucosamine+gluconate+amygdalin+2-keto-gluconate-arbutin+5-keto-gluconate-

[0112] 1-4-2. Physiological Characteristics of Bifidobacterium longum P77. Bifidobacterium longum P77 is a Gram-positive coccus. Among its physiological characteristics, carbon source utilization was analyzed through a sugar fermentation test using the API 20 A kit (BioMerieux, USA). The results are shown in Table 5 below. In Table 5, "+" indicates positive carbon source utilization, and "-" indicates negative carbon source utilization.

[0113] Carbon source P77Carbon source P77L-tryptophan-glycerol-urea+D-cellobis-D-glucose+D-mannose-D-mannitol-D-melezitose-D-lactose+D-raffinose+D-saccharose+D-sorbitol-D-maltose+L-rhamnose-salicylic acid-D-trehalose+D-xylose+catalase-L-arabinose+spores-gelatin-gram reaction+esculin ferric citrate+morphology-

[0114] Experimental Example 1. Confirmation of the novel strain's increased serotonin and BDNF production and TNF-α inhibitory effect.

[0115] 1-1. Cultivation of Lactobacillus plantarum P72 strain

[0116] Lactobacillus plantarum P72 was cultured in MRS medium and centrifuged at 5000 g at 4°C for 20 min. The collected cells were lyophilized, and the number of viable P72 strains was determined using plate counting on MRS agar. Heat-treated Lactobacillus plantarum P72 was prepared by heating at 60°C for 15 min, and live or heat-treated Lactobacillus plantarum P72 strains were suspended in sterile distilled water for in vitro and in vivo experiments.

[0117] 1-2. Cultivation of Bifidobacterium longum P77 strain

[0118] Bifidobacterium longum P77 was cultured in GAM or BL medium and centrifuged at 5000 x g, 4 °C for 20 min. The collected cells were lyophilized, and the number of viable P77 strains was determined using plate counting in GAM or BL medium. The lyophilized powder was heated at 75 °C for 15 min to prepare heat-treated Bifidobacterium longum P77, and live or heat-treated Bifidobacterium longum P77 strains were suspended in sterile distilled water for in vitro and in vivo experiments.

[0119] 1-3. Culture of SH-SY5Y cells and peritoneal macrophages

[0120] SH-SY5Y cells (purchased from the Korea Cell Line Bank, Seoul) were cultured in DMEM containing 5% fetal bovine serum and 1% antibiotic-antimycotic at 37°C in a mixed atmosphere of 5% CO2 and 95% air. SH-SY5Y cells (1Х10 6 cells / mL) with corticosterone alone or with lactic acid bacteria (1Х10 4 CFU / mL) and cultured for 24 h. Serotonin and BDNF levels were measured from the supernatant of the culture using respective measurement kits (DLD Diagnostika GmbH, Hamburg, Germany / R&D system, Minneapolis).

[0121] Peritoneal macrophages were isolated from mice. The isolated peritoneal macrophages were washed twice with RPMI 1640 medium, suspended in RPMI 1640 (RFA) containing 10% fetal bovine serum and 1% antibiotic-antimycotic, cultured at 37°C for 20 h, and washed three times with RFA. Attached macrophages (1 Х 10 6 cells / well) were cultured in the presence of LPS in the presence of lactic acid bacteria (Lactobacillus plantarum P72, 1 Х 10 4 CFU / mL) was treated for 24 hours. TNF-α levels were measured from the supernatant of the culture using a TNF-α measurement kit (R&D system, Minneapolis).

[0122] 1-4. Measurement of serotonin, brain-derived neurotrophic factor (BDNF), and tumor necrosis factor-α (TNF-α)

[0123] To determine whether the novel lactic acid bacteria can improve depression and sleep disorders, the amounts of serotonin, BDNF, and TNF-α isolated from cells cultured in 1-1 and 1-2 were measured after treatment with P72. The normal control group (NC) was treated with saline instead of the test substance.

[0124] As a result, as shown in Table 6, it was confirmed that serotonin and BDNF increased and TNF-α decreased by P72.

[0125] NC(Normal control) Corticosterone P72 (a) Serotonin (pg / mL) 16.1±2.6 10.9±0.4 23.0±3.2 (b) BDNF (pg / mL) 10.3±0.1 6.0±0.2 12.4±1.0 NCLPSP72 (c) TNF-α (pg / mL) 15.2±2.2 47.7±4.5 29.8±2.6

[0126] Additionally, as shown in Table 7, it was confirmed that serotonin and BDNF increased by P77.

[0127] NC(Normal control) Corticosterone P77(a) Serotonin (pg / mL) 16.5±2.2 10.2±0.3 22.2±2.1(b) BDNF (pg / mL) 10.8±0.2 6.2±0.1 12.2±1.1

[0128] Experimental Example 2. Confirmation of the improvement effect of restraint stress (IS) on sleep disorders and depression.

[0129] 2-1. Preparation of sleep disorder and depression model mice (Test substance: Lactobacillus plantarum P72)

[0130] C57BL / 6 mice (male, 18-21 g, 6 weeks old) were purchased from Coretech Co., Ltd. and were allowed to acclimate to the laboratory for 1 week before the experiment. Then, they were divided into 3 groups (NC, IS, P72) with 8 mice per group. After that, they were subjected to restraint stress (IS) once a day for 8 hours for 5 days, and 24 hours after the last restraint stress, they were administered the test substance (P72 group: 1X10 per mouse per day) once a day for 5 days. 9 CFU) was administered orally. The normal control (NC) and IS groups were administered saline instead of the test substance. In mice exposed to restraint stress, depressive and anxious behaviors were significantly increased.

[0131] 2-2. Preparation of sleep disorder and depression model mice (Test substance: Bifidobacterium longum P77)

[0132] C57BL / 6 mice (male, 18-21 g, 6 weeks old) were purchased from Coretech Co., Ltd. and were allowed to acclimate to the laboratory for 1 week before the experiment. Then, they were divided into 4 groups (NC, IS, P77, DP) with 8 mice per group. After that, they were subjected to restraint stress (IS) once a day for 8 hours for 5 days, and 24 hours after the last restraint stress, they were administered the test substance (P77 group: 1X10 per mouse per day) once a day for 5 days. 9 CFU) was administered orally. The normal control (NC) and IS groups were administered saline instead of the test substance. In mice exposed to restraint stress, depressive and anxious behaviors were significantly increased.

[0133] 2-3. Open field test (OFT)

[0134] Mice that had been subjected to restraint stress and administered test materials were placed one by one in an OFT box (40 cm wide, 40 cm long, 43 cm high) and measured in a laboratory equipped with a camera for 10 minutes. Using the EthoVision program, the total distance moved, the distance moved in the central area (20 cm wide, 20 cm long), and the time spent there were measured. The measurement results are shown in Table 8 or Table 9.

[0135] 2-4. Elevated Plus Maze Task (EPMT) Test

[0136] The elevated plus maze experimental apparatus was made of black Plexiglas and consisted of two open arms (30 x 7 cm) 50 cm above the floor and two enclosed arms (30 x 7 cm) with walls 20 cm high, each extending 7 cm (7 x 7 cm) from a central platform. The movements of mice placed in the elevated plus maze were recorded in a room with a brightness of 20 lux and a video camera installed above.

[0137] Mice were placed facing the open passage with their heads in the center of the elevated plus maze (EPMT), and the time and number of times they spent in the open and enclosed passages for 5 minutes were measured. Arm entry was defined as entering the passage with all four paws. The time spent in the open arms among the total experimental time was calculated for each mouse as (Time spent in open arm) / (Time spent in open arm + Time spent in enclosed arm) x 100). After each behavioral experiment, any remaining odor was removed with 70% ethanol. The measurement results are shown in Table 8 or Table 9.

[0138] 2-5. Tail Suspension Test (TST)

[0139] The mouse was suspended 1 cm from the tip of its tail in a 35 cm diameter, 50 cm high chamber using a fixture. The mouse's immobility was measured for a total of 6 minutes. Immobility refers to a state in which the mouse does not move and only engages in minimal activity. The measurement results are shown in Table 8 or 9.

[0140] 2-6. Isoflurane sleep test (test substance: Lactobacillus plantarum P72)

[0141] From the day after performing the depression-like behavior test, isoflurane (2%) was exposed in a transparent chamber (15 x 15 x 15 cm), and the time to loss of the righting reflex and the time to recovery of the righting reflex were measured. The time to loss of the righting reflex was defined as the sleep induction time, and the time from the loss of the righting reflex to the recovery of the righting reflex was calculated as the sleep time. The measurement results are shown in Table 8.

[0142] 2-7. Pentobarbital sleep test (test substance: Bifidobacterium longum P77)

[0143] The test substance was administered orally at 10:00 AM the day after the depression-like behavior test. In addition, the positive control drug (DP, 20 mg / kg diphenhydramine) was administered orally 30 minutes before the injection of pentobarbital (40 mg / kg, pentobarbital sodium, Hanlim Pharmaceutical, dissolved in saline). The normal control (NC) and IS groups were administered saline instead of the test substance. The behavioral test was measured from 2:00 PM.

[0144] Pentobarbital was administered intraperitoneally to mice, and the time to loss of the righting reflex and the time to recovery of the righting reflex were measured for 60 minutes starting 30 minutes later. The time to loss of the righting reflex was defined as the sleep induction time, and the time from the loss of the righting reflex to its recovery was calculated as the sleep time. The measurement results are shown in Table 9.

[0145] As shown in Table 8, the results of the above experiment showed that the total moving distance (TD), the central region moving distance (DC), and the central region residence time (TC) were all reduced by restraint stress (IS) compared to the normal control group, and both the residence time in the open arm and the number of entries into the open arm were reduced. However, it was confirmed that the total moving distance (TD), the central region moving distance (DC), and the central region residence time (TC) reduced by IS were all significantly increased by P72 administration.

[0146] In addition, it was confirmed that the residence time (OT) and number of entries (OE) in the open arm, which were significantly reduced by IS, were significantly recovered through the administration of P72, and it was confirmed that the immobility time, which was significantly increased by IS, was significantly reduced through the administration of P72.

[0147] In addition, in a sleep test induced by isoflurane, it was confirmed that sleep induction time increased and sleep time decreased due to restraint stress, but sleep induction time was reduced and sleep time increased through P72 administration, thereby improving sleep disorders through P72.

[0148] NCISP72(a) Total distance traveled (TD) (m) 33.1±2.0 28.2±0.7 35.9±3.9(b) Central distance traveled (DC) (m) 6.6±1.6 3.7±0.7 6.6±0.9(c) Central dwell time (TC) (s) 103.0±26.4 42.3±7.0 95.2±14.8(d) Time spent in open arms (OT) (%) 14.2±2.6 6.5±2.7 17.0±5.6(e) Number of entries in open arms (OE) (%) 35.2±6.1 14.6±5.6 38.0±5.8(f) Immobility time (IT) (s)144.2±10.4205.8±15.8162.2±6.4(g) Sleep latency time (s)28.2±3.135.5±2.627.2±2.2(h) Sleep duration (s)39.2±3.432.5±2.439.7±8.2

[0149] In addition, as shown in Table 9, the total moving distance (TD), the moving distance in the central region (DC), and the dwell time in the central region (TC) were all reduced by restraint stress (IS) compared to the normal control group, and both the dwell time in the open arm and the number of entries into the open arm were also reduced. However, it was confirmed that the total moving distance (TD), the moving distance in the central region (DC), and the dwell time in the central region (TC) reduced by IS were all significantly increased by administration of P77. It was confirmed that the dwell time (OT) and the number of entries (OE) in the open arm, which were significantly reduced by IS, were significantly recovered by administration of P77, and it was confirmed that the immobility time, which was significantly increased by IS, was significantly reduced by administration of P77.

[0150] In addition, in a sleep test induced by pentobarbital, it was confirmed that sleep induction time increased and sleep time decreased due to restraint stress, but sleep induction time was reduced and sleep time increased through P77 administration, thereby improving sleep disorders through P77.

[0151] NCISP77DP (a) Total distance traveled (TD) (m) 35.1±2.1 29.4±2.3 37.2±3.9 30.8±1.2 (b) Central distance traveled (DC) (m) 6.4±1.4 4.4±0.7 6.2±1.2 4.5±0.5 (c) Central dwell time (TC) (s) 89.1±23.0 41.3±2.6 86.2±14.2 45.8±5.1 (d) Time spent in open arms (OT) (%) 11.4±3.3 4.2±1.0 14.5±2.6 4.6±1.2 (e) Number of entries in open arms (OE) (%)49.5±5.124.5±5.041.2±3.224.2±4.1(f) Immobility Time (IT) (s)121.5±5.5181.5±12.6133.2±12.5172.5±15.2(g) Sleep Latency Time (s)149.2±10.3212.3±10.5166.7±5.2194.2±8.5(h) Sleep Duration (min)42.1±6.217.4±2.434.3±4.646,2±3.2

[0152] 2-8. Protein and gene expression analysis

[0153] 2-8-1. ELISA

[0154] Brain (frontal lobe) and colon tissues collected from mice were homogenized and lysed in RIPA buffer (150 mM sodium chloride, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, 2 mM EDTA, pH 7.5) containing a phosphatase inhibitor cocktail (Roche), and centrifuged at 10,000 g at 4 °C for 20 minutes.

[0155] After collecting the supernatant, TNF-α, IL-1β, IL-6, IL-10, and myeloperoxidase were measured using ELISA kits (R&D system, Minneapolis, MN), corticosterone was measured using a kit from eBioscience (TX), GABA was measured using a kit from Mybiosource (San Diego, CA), and serotonin was measured using a kit from DLD Diagnositika GmbH (Hamburg, Germany). The results are shown in Table 11 or Table 12.

[0156] 2-8-2. qPCR

[0157] mRNA (2 μg) was isolated from the frontal lobe and thalamus using the RNeasy Mini kit, and cDNA was synthesized using a cDNA synthesis kit (TaKaRa). Real-time qPCR for serotonin 1A receptor (5-HT1AR), serotonin 1B receptor (5-HT1BR), serotonin 2A receptor (5-HT2AR), serotonin 7 receptor (5-HT7R), γ-aminobutyric acid type A receptor subunit alpha 1 (GABRA1), γ-aminobutyric acid type A receptor subunit alpha 2 (GABRA2), melatonin receptor type 1 (MT1R), melatonin receptor type 2 (MT2R), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed using SYBER premix Ex Taq II (TaKaRa). PCR was performed under the following conditions: initial denaturation at 95°C for 30 seconds, denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, for 40 cycles. Gene expression levels were calculated based on GAPDH. The primer sequences used for PCR are as shown in Table 10, and the results are shown in Table 11 or Table 12.

[0158] Sequence number Gene type Direction Base sequence (5'-3') 3 GABRA1 Forward (F) 5′- GAGTCGTCCAATCCAGCAC-3′ 4 Reverse (R) 5′- AGCCAGAAGGAAACCTGTGA-3′ 5 GABRA2 Forward (F) 5′- TTACAGTCCAAGCCGAATGTCCC-3′ 6 Reverse (R) 5′- ACTTCTGAGGTTGTGTAAGCGTAGC-3′ 7 MT1R Forward (F) 5′- TGTCAGCGAGCTGCTCAATG-3′ 8 Reverse (R) 5′- GGTACACAGACAGGATGACCA-3′ 9 MT2R Forward (F) 5′- GAACAGCTCAATCCCTAACTGC-3′ 10 Reverse (R) 5′- ACGACTACTGTAGATAGCATGGG-3′ 11 5-HT1AR Forward (F) 5′- CCGTGAGAGGAAGACAGTCTAAGA-3′12Reverse (R) 5′- GGTTGAGCAGGGAGTTGGAGTAG-3′135-HT1BRForward (F) 5′- CCAGCGGTCCATCCACAGAG-3′14Reverse (R) 5′- CCAGCGGTCCATCCACAGAG-3′15GAPDHForward (F) 5′-TGCAGTGGCAAAGTGGAGAT-3′16Reverse (R) 5′-TTTGCCGTGAGTGGAGTCATA-3′

[0159] As shown in Table 11, the results of the above experiment showed that IS increased brain corticosterone, decreased GABA levels and GABA receptor expression in the prefrontal cortex, and decreased serotonin (5-HT) levels and 5-HT and melatonin receptor expression in the prefrontal cortex. In addition, IS decreased serotonin (5-HT) levels, decreased 5-HT and melatonin receptor expression, increased corticosterone and TNF-α expression in the prefrontal cortex, and decreased IL-10 expression.

[0160] However, administration of P72 increased the GABA and serotonin levels, GABA receptors (GABRA1 and GABRA2), melatonin receptors (MT1R), and 5-HT receptors (5-HT1AR, 5-HT1BR) expression, which were reduced by IS. In addition, it was confirmed that when P72 was administered, the expression of corticosterone and TNF-α induced by IS was reduced, whereas the expression of IL-10, which was suppressed by IS, was increased.

[0161] NCISP72(a) GABA (pg / mg)15.6±1.910.8±1.815.2±2.8(b) GABRA1 (fold change)1.0±0.10.8±0.10.9±0.1(c) GABRA2 (fold change)1.0±0.20.7±0.00.9±0.2(d) MT1R (fold change)1.0±0.10.8±0.21.4±0.2(e) Serotonin (pg / mg)17.8±2.49.1±2.420.1±4.5(f) 5-HT1AR (fold change)1.0±0.20.7±0.11.0±0.1(g) 5-HT1BR (fold change)1.0±0.10.7±0.21.2±0.1(h) Corticosterone (ng / mg) 1.5±0.2 2.0±0.1 1.6±0.1(i) TNF-α (pg / mg) 49.4±2.4 60.0±4.8 51.7±3.9(j) IL-10 (ng / mg) 0.12±0.01 0.10±0.01 0.12±0.01

[0162] Furthermore, administration of P77 increased GABA and serotonin levels, GABA receptors (GABRA1 and GABRA2), melatonin receptors (MT1R), and 5-HT receptor (5-HT1AR) expression, which were reduced by IS. In addition, it was confirmed that when P77 was administered, the expression of corticosterone and TNF-α induced by IS was reduced, whereas the expression of IL-10, which was suppressed by IS, was increased.

[0163] NCISP77DP (a) GABA (pg / mg) 15.4±1.5 10.2±1.6 14.8±2.3 115.1±1.2 (b) GABRA1 (fold change) 1.0±0.1 0.7±0.1 0.9±0.1 0.9±0.0 (c) GABRA2 (fold change) 1.0±0.1 0.7±0.1 0.9±0.1 0.8±0.1 (d) MT1R (fold change) 1.0±0.1 0.8±0.1 1.2±0.1 0.9±0.1 (e) Serotonin (pg / mg) 16.7±1.4 9.6±2.1 17.5±3.4 17.2±3.2 (f) 5-HT1AR (fold change) change)1.0±0.10.7±0.11.0±0.10.8±0.1(g) Corticosterone (ng / mg)1.6±0.22.2±0.11.7±0.12.0±0.2(h) TNF-α (pg / mg)45.2±2.162.2±5.149.2±4.959.2±5.2(i) IL-10 (ng / mg)0.11±0.010.09±0.010.11±0.010.12±0.01

[0164] Experimental Example 3. Confirmation of the anti-inflammatory effect induced by restraint stress (IS).

[0165] Colitis was induced by the IS stress described above, and as shown in Table 13 and / or Table 14, the length of the colon was shortened, the expression of myeloperoxidase (MPO), TNF-α, IL-1β, and IL-6 was increased, and the expression of IL-10 was decreased.

[0166] However, it was confirmed that the length of the colon was restored by administration of P72 or P77, the expression of MPO, TNF-α, IL-1β, and IL-6 was reduced, and the expression of IL-10, which was suppressed by IS, was increased.

[0167] Through the above results, it was confirmed that the novel lactic acid bacteria of the present invention can be applied to the improvement or treatment of inflammatory diseases by improving inflammation indicators.

[0168] NCISP72(a) Colon length (cm)6.3±0.05.2±0.26.3±0.2(b) MPO (ng / mg)0.12±0.010.16±0.040.11±0.02(c) TNF-α (ng / mg)0.13 ±0.010.15±0.010.13±0.01(d) IL-1β (ng / mg)0.07±0.000.10±0.020.07±0.01(e) IL-6 (pg / mg)67.6±6.580.6±9.760.1±6.5(f) IL-10 (ng / mg)0.13±0.010.09±0.010.12±0.01

[0169] NCISP77DP(a) Colon length (cm)6.4±0.15.2±0.36.2±0.25.4±0.2(b) MPO (ng / mg)0.11±0.020.15±0.020.12±0.020.14±0.02(c) TNF-α (ng / mg)0.12±0.020.16±0.020.13±0.030.15±0.02(d) IL-1β (ng / mg)0.06±0.010.11±0.020.08±0.020.10±0.02(e) IL-6 (pg / mg)63.2±5.483.5±7.665.2±5.679.3±8.2(f) IL-10 (ng / mg)0.12±0.010.08±0.020.11±0.010.11±0.02

[0170] Experimental Example 4. Confirmation of the improvement effect of sleep disorders and depression induced by cFM transplantation of Lactobacillus plantarum P72 strain.

[0171] To prepare mice with depression and inflammatory bowel disease induced by cultured fecal microbiota transplantation (cFM), cultured fecal microbiota from patients with inflammatory bowel disease were transplanted into mice. Mice were divided into three groups (NC, cFM, LP) of six each, and cultured fecal microbiota (2X10 8 (CFU / mouse, suspended in 0.1 mL of saline) was administered orally to mice in the cFM and LP groups once a day for 5 days. Saline was administered to the normal control group (NC).

[0172] LP group was administered test substance P72 (1Х10) 24 hours after the last cFM treatment. 9 CFU / mouse / day was administered orally once daily for 7 days. The cFM group was administered saline instead of the test substance.

[0173] 4-1. Behavioral experiment

[0174] The OFT, EPMT, and TST behavioral experiment methods are the same as Experimental Examples 2-3 to 2-5 above.

[0175] 4-2. Pentobarbital sodium sleep test

[0176] The day after the depression-like behavior test, pentobarbital (40 mg / kg, pentobarbital sodium, Hanlim Pharmaceutical, dissolved in saline) was administered intraperitoneally to the mice, and the time to loss of the righting reflex and the time to recovery of the righting reflex were measured for 60 minutes. The time to loss of the righting reflex was defined as the sleep induction time, and the time from the loss of the righting reflex to the recovery of the righting reflex was calculated as the sleep time. The measurement results are shown in Table 15.

[0177] Cultured fecal microbiota transplantation (cFM) reduced TD, DC, and TC in OFT compared to the normal control group, reduced OT and OE in EPMT, and increased immobility time in TST.

[0178] However, it was confirmed that the TD, CD, TC, OT, and OE reduced by cFM were significantly restored through administration of P72, and the immobility time was reduced.

[0179] In addition, it was confirmed that sleep latency increased and sleep time decreased by cFM, but sleep induction time decreased and sleep time increased through administration of P72, thereby improving sleep disorders through P72.

[0180] NCcFMP72(a) TD (m)35.1±1.030.3±0.634.1±3.2(b) DC (m)7.2±0.64.6±0.46.5±0.9(c) TC (s)111.4±14.654.5±9.478.8±10.1(d) OT (%)32.2±8.117.5±5.738.6±9.9(e) OE (%)46.0±5.423.6±7.149.1±3.9(f) Immobility time (s)121.3±6.1151.2±11.292.6±17.2(g) Sleep induction time (s)154.2±5.7184.2±13.5162.7±10.1(h) Sleep time (min)47.8±3.124.3±2.735.0±6.1

[0181] 4-3. Protein and gene expression analysis

[0182] Protein and gene expression analysis was performed in the same manner as in Experimental Example 2-8 above.

[0183] As a result, as shown in Table 16, cFM decreased GABA levels and the expression of GABA receptors (GABRA1 and GABRA2) and melatonin receptors (MT1R) in the prefrontal cortex. In addition, cFM decreased serotonin (5-HT) levels, decreased the expression of serotonin receptors 5-HT1AR and 5-HT1BR, increased the expression of TNF-α, and decreased the expression of IL-10.

[0184] However, administration of P72 increased the GABA and serotonin levels, GABA receptors (GABRA1 and GABRA2), melatonin receptors (MT1R), and 5-HT receptors (5-HT1AR, 5-HT1BR) expression, which were reduced by cFM. In addition, it was confirmed that when P72 was administered, the expression of TNF-α and IL-6 induced by cFM was reduced, and the expression of IL-10, which was suppressed by cFM, was increased.

[0185] NCcFMP72(a) GABA (pg / mg)16.5±2.89.9±1.915.2±2.9(b) GABRA1 (fold change)1.0±0.00.8±0.11.0±0.1(c) GABRA2 (fold change)1.0±0.10.7±0.11.0±0.1(d) MT1R (fold change)1.0±0.10.8±0.11.1±0.1(e) Serotonin (pg / mg)14.3±2.011.2±2.014.8±2.2(f) 5-HT1AR (fold change)1.0±0.00.8±0.11.0±0.1(g) 5-HT1BR (fold change)1.0±0.20.6±0.11.2±0.2(h) Corticosterone (ng / mg)1.5±0.31.9±0.21.5±0.1(i) TNF-α (pg / mg)51.8±3.961.9±2.456.8±3.2(j) IL-10 (ng / mg)0.12±0.010.09±0.000.10±0.00

[0186] Experimental Example 5. Confirmation of the anti-inflammatory effect of cFM transplantation.

[0187] Colitis was induced by cultured fecal microbiota transplantation (cFM). As shown in Table 17, the colon was shortened, the expression of myeloperoxidase (MPO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 was increased, and the expression of IL-10 was decreased.

[0188] However, it was confirmed that the length of the colon was restored by administration of P72 or P77, the expression of MPO, TNF-a, IL-1β, and IL-6 was reduced, and the expression of IL-10, which was suppressed by cFM, was increased.

[0189] NCcFMP72(a) Colon length (cm)6.1±0.25.5±0.26.3±0.2(b) MPO (ng / mg)0.17±0.010.20±0.020.15±0.02(c) TNF-α (ng / mg)0.07±0.010.09±0.010.07±0.01(d) IL-1β (pg / mg)33.6±2.245.3±5.633.8±4.0(e) IL-6 (ng / mg)0.08±0.010.10±0.020.08±0.01(f) IL-10 (ng / mg)0.14±0.010.12±0.010.15±0.02

[0190] NCcFMP77DP(a) Colon length (cm)6.4±0.25.4±0.26.2±0.36.3±0.3(b) MPO (ng / mg)0.15±0.020.22±0.040.17±0.030.16±0.04(c) TNF-α (ng / mg)0.11±0.10.16±0.20.12±0.10.12±0.1(d) IL-6 (ng / mg)0.08±0.010.10±0.020.08±0.010.08±0.01(e) IL-10 (ng / mg)0.15±0.020.12±0.020.14±0.020.15±0.03

[0191] Experimental Example 6. Confirmation of the improvement effect of cFM transplantation of P7277, a composite composition containing Bifidobacterium longum P77 strain and Lactobacillus plantarum P72, on sleep disorders and depression.

[0192] To prepare mice with depression and inflammatory bowel disease induced by cultured fecal microbiota transplantation (cFM), cultured fecal microbiota from patients with inflammatory bowel disease were transplanted into mice. Mice were divided into four groups of six each (NC, cFM, P77, P7277) and cultured fecal microbiota (2X10 8 (CFU / mouse, suspended in 0.1 mL of saline) was administered orally once a day for 5 days to mice in the cFM, P77, and P7277 groups. Saline was administered to the normal control group (NC).

[0193] Twenty-four hours after the last cFM treatment, the BL group was administered the test substance P77 (1Х10 9 CFU / mouse / day, and the P7277 group was administered a test substance (1Х10) mixed [4:1] of P72 and P77. 9 CFU / mouse / day was administered orally once daily for 7 days. The cFM group was administered saline instead of the test substance.

[0194] 6-1. Behavioral experiment

[0195] The OFT, EPMT, and TST behavioral experiment methods are the same as Experimental Examples 2-3 to 2-5, and the sleep disorder test method is the same as Experimental Example 2-6. The measurement results are as shown in Table 19.

[0196] Cultured fecal microbiota transplantation (cFM) reduced TD, DC, and TC in OFT compared to the normal control group, reduced OT and OE in EPMT, and increased immobility time in TST.

[0197] However, it was confirmed that TD, CD and TC, OT and OE reduced by cFM were significantly restored through administration of P77 or P7277, and immobility time was reduced.

[0198] In addition, it was confirmed that sleep latency time increased and sleep time decreased by cFM, but sleep induction time decreased and sleep time increased through administration of P77 or P7277, thereby improving sleep disorders.

[0199] NCcFMP77P7277(a) TD (m)33.5±1.228.9±0.833.2±3.634.5±2.7(b) DC (m)8.3±0.74.2±0.57.1±0.77.8±0.6(c) TC (s)113.1±12.155.6±6.979.3±9.488.8±10.2(d) OT (%)31.2±6.516.3±5.133.7±6.438.2±9.1(e) OE (%)45.2±4.724.2±6.244.2±4.849.2±3.5(f) Dead time (s)122.2±5.3162.2±10.1125.3±11.2102.6±11.2(g) Sleep induction time (s)145.7±9.6209.2±12.4182.5±7.3176.6±9.2(h) Sleep time (min)42.5±5.818.2±4.335.9±5.237.2±5.3

[0200] 6-2. Protein and gene expression analysis

[0201] Protein and gene expression analysis was performed in the same manner as in Experimental Example 2-8 above.

[0202] As a result, as shown in Table 20, cFM decreased GABA levels and the expression of GABA receptors (GABRA1 and GABRA2) and melatonin receptors (MT1R) in the prefrontal cortex. In addition, cFM decreased serotonin (5-HT) levels, decreased the expression of the serotonin receptor 5-HT1AR, increased the expression of TNF-α, and decreased the expression of IL-10.

[0203] However, administration of P77 increased GABA and serotonin levels, GABA receptors (GABRA1 and GABRA2), melatonin receptors (MT1R), and 5-HT receptor (5-HT1AR) expression, which were reduced by cFM. In addition, it was confirmed that when P77 was administered, TNF-α expression induced by cFM was reduced, and IL-10 expression suppressed by cFM was increased.

[0204] NCISP77DP (a) GABA (pg / mg) 14.2±2.1 8.2±1.4 13.4±1.8 14.5±1.6 (b) GABRA1 (fold change) 1.0±0.1 0.8±0.1 1.0±0.1 1.1±0.1 (c) GABRA2 (fold change) 1.0±0.1 0.7±0.1 1.0±0.1 1.1±0.1 (d) MT1R (fold change) 1.0±0.1 0.7±0.1 1.0±0.1 1.1±0.1 (e) Serotonin (pg / mg) 14.5±1.7 10.4±1.5 14.1±1.9 14.6±2.1 (f) 5-HT1AR (fold change)1.0±0.10.8±0.11.0±0.11.0±0.1(g) Corticosterone (ng / mg)1.3±0.21.8±0.11.6±0.21.5±0.1(h) TNF-α (pg / mg)52.5±4.668.4±2.256.7±3.354.2±2.8(i) IL-10 (ng / mg)0.14±0.020.09±0.020.11±0.020.12±0.02

[0205] Experimental Example 7. Confirmation of the effect of P7277, a composite composition containing Bifidobacterium longum P77 strain and Lactobacillus plantarum P72, on improving sleep disorders and depression induced by restraint stress (IS).

[0206] Mice with sleep disturbance and depression induced by IS were used, and the effects of the P77 strain and LB (a mixed composition of P77 and P72) were confirmed.

[0207] Mice were divided into four groups (NC, IS, P77, P7277) and each group consisted of eight mice. Restraint stress (IS) was applied to the mice in the seven groups once a day for five days, and the test substance (1Х10) was applied once a day for seven days starting 24 hours after the last restraint stress. 9 P77 of CFU / mouse / day; 0.8Х10 9 P72 and 0.2Х10 in CFU / mouse / day 9A mixture of P77 (P7277) (CFU / mouse / day) was administered orally at 10:00 AM. The normal control (NC) and IS groups were administered saline instead of the test substance. Behavioral tests were performed from 2:00 PM.

[0208] 7-1. Behavioral experiment

[0209] The OFT, EPMT, and TST behavioral experiment methods are the same as Experimental Examples 2-3 to 2-5, and the sleep disorder test method is the same as Experimental Example 2-6. The measurement results are as shown in Table 21.

[0210] In OFT, TD, DC, and TC were all reduced compared to the normal control group by IS, in EPMT, OT and OE were reduced, and in TST, immobility time was increased.

[0211] However, administration of P77 or P7277 improved TD, CD, and TC suppressed by IS. In addition, administration of P77 or P7277 significantly improved OT induced by IS in EPMT, and significantly reduced immobility time in TST.

[0212] In addition, it was confirmed that sleep latency was increased and sleep time was decreased by IS, but sleep latency was decreased and sleep time was increased through administration of P77 or P7277, and sleep disorders were improved through P77 or P7277.

[0213] Overall, P7277 was found to be most effective against stress-induced sleep disturbances and depression.

[0214] Mean ± SDNCISP77P7277(a) TD (m)35.2±3.131.4±2.534.7±3.236.2±4.1(b) DC (m)7.2±1.14.2±0.76.1±0.36.9±1.2(c) TC (s)78.4±12.342.3±2.175.3±9.377.5±11.2(d) OT (%)12.3±3.24.1±1.012.1±2.213.2±1.6(e) OE (%)42.4±4.322.7±2.241.3±4.142.9±3.1(f) Dead time (s)106.3±6.5171.3±11.6136.4±9.4129.2±9.8(g) Sleep induction time (s)27.1±2.634.5±1.528.5±2.227.9±1.5(h) Sleep time (s)37.8±3.232.4±1.137.9±1.438.3±1.2

[0215] 7-2. Protein and gene expression analysis

[0216] Protein and gene expression analysis was performed in the same manner as in Experimental Example 2-8 above.

[0217] As a result, as shown in Table 22, IS reduced GABA levels and the expression of GABA receptors (GABRA1 and GABRA2) and melatonin receptors (MT1R, MT2R) in the prefrontal cortex. In addition, IS reduced serotonin (5-HT) levels and the expression of serotonin receptors 5-HT1AR and 5-HT1BR.

[0218] However, administration of P77 or P7277 increased the GABA and serotonin levels, which were reduced by IS, and the expression of GABA receptors (GABRA1 and GABRA2), melatonin receptors (MT1R, MT2R), and 5-HT receptors (5-HT1AR). In particular, it was confirmed that the amount of serotonin was significantly increased when P77 or P7277 was administered.

[0219] Mean ± SD NCISP77P7277(a) GABA (pg / mg)22.4±2.513.2±2.519.4±3.220.4±2.1(b) GABRA1(fold change)1.0±0.10.8±0.11.0±0.11.1±0.1(c) GABRA2(fold change)1.0±0.10.8±0.10.9±0.11.0±0.1(d) MT1R(fold change)1.0±0.10.8±0.11.0±0.11.1±0.1(e) MT2R(fold change)1.0±0.10.7±0.11.0±0.11.1±0.1(f) Serotonin (ng / mg) 25.5±2.4 16.2±1.6 23.4±1.4 24.8±2.2 (g) 5-HT1AR (fold change) 1.0±0.1 0.8±0.1 1.0±0.1 1.1±0.1

[0220] In addition, as shown in Table 23, IS increased brain corticosterone, increased the expression of TNF-α and IL-6, and decreased the expression of IL-10. However, it was confirmed that administration of P77 or P7277 decreased the expression of corticosterone, TNF-α, and IL-6 induced by IS, while increased the expression of IL-10 suppressed by IS.

[0221] Mean ± SD NCISP77P7277 (a) Corticosterone (ng / mg) 1.6 ± 0.1 2.1 ± 0.1 1.7 ± 0.1 1.6 ± 0.1 (b) TNF-α (ng / mg) 0.12 ± 0.01 0.17 ± 0.02 0.14 ± 0.02 0.13 ± 0.01 (c) IL-6 (pg / mg) 56.2 ± 2.1 7 1.2 ± 4.5 59.4 ± 4.2 57.3 ± 3.4 (d) IL-10 (ng / mg) 0.15 ± 0.01 0.11 ± 0.01 0.13 ± 0.01 0.14 ± 0.00

[0222] Experimental Example 8. Confirmation of the anti-inflammatory effect of P7277, a composite composition containing Bifidobacterium longum P77 strain and Lactobacillus plantarum P72, on restraint stress (IS)-induced inflammation.

[0223] Colitis was induced by the IS stress described above, and as shown in Table 24, the length of the colon was shortened, the expression of myeloperoxidase (MPO), TNF-a, IL-1β, and IL-6 increased, and the expression of IL-10 decreased.

[0224] However, it was confirmed that the length of the colon was restored by administration of P77 or P7277, the expression of MPO, TNF-α, IL-1β, and IL-6 was reduced, and the expression of IL-10 suppressed by IS was increased. Through the above results, it was confirmed that P77 or P7277 of the present invention can be applied to the improvement or treatment of inflammatory diseases by improving inflammatory indicators.

[0225] Mean ± SD NCISP77P7277(a) Colon length (cm)6.4±0.25.4±0.36.3±0.26.3±0.2(b) MPO (ng / mg)0.15±0.010.22±0.020.18±0.020.17±0.01(c) TNF-α (ng / mg)0.11±0.010.16±0.020.13±0.030.12±0.02(d) IL-1β (pg / mg)62.4±4.184.8±7.468.2±5.164.4±4.7(e) IL-6 (pg / mg)61.5±2.988.2±4.166.9±2.565.2±5.3(f) IL-10 (ng / mg)0.15±0.010.12±0.010.13±0.020.14±0.01

[0226] Experimental Example 9. Confirmation of the effect of P7277, a complex composition containing the inactivated Bifidobacterium longum P77 strain and Lactobacillus plantarum P72, on improving sleep disorders and depression induced by restraint stress (IS).

[0227] After acclimating to the laboratory for one week prior to the experiment, C57BL / 6 mice (male, 18-21 g, 6 weeks old) were divided into four groups (NC, IS, P77, P7277) with eight mice per group. After that, restraint stress (IS) was applied once a day for 8 hours for 5 days, and the test substance (2X10) was administered once a day for 5 days starting 24 hours after the last restraint stress. 9 Heat-treated killed cells P77 (CFU / mouse / day); 1.6Х10 9 Heat-treated killed cells P72 and 0.4Х10 CFU / mouse / day 9 A mixture of heat-treated P77 cells (CFU / mouse / day) was orally administered. The normal control (NC) and IS groups were administered saline instead of the test substance. In mice exposed to restraint stress, depressive and anxious behaviors were significantly increased.

[0228] 9-1. Behavioral experiment

[0229] The OFT, EPMT, and TST behavioral experiment methods are the same as Experimental Examples 2-3 to 2-5, and the sleep disorder test method is the same as Experimental Example 2-6. The measurement results are as shown in Table 25.

[0230] In the OFT, TD, DC, and TC were all reduced compared to the normal control group due to restraint stress, in the EPMT, OT and OE were reduced, and in the TST, immobility time was increased.

[0231] However, it was confirmed that the TD, CD and TC, OT and OE reduced by restraint stress were significantly recovered through administration of heat-treated P77 or P7277, and the immobility time was reduced.

[0232] In addition, it was confirmed that sleep induction time increased and sleep time decreased due to restraint stress, but sleep induction time decreased and sleep time increased through administration of heat-treated P77 or P7277, thereby improving sleep disorders through heat-treated P77 or P7277.

[0233] NCISP77 (heat treatment) P7277 (heat treatment) (a) TD (m) 33.6±1.2 29.2±0.9 32.9±2.1 34.2±2.7 (b) DC (m) 6.9±0.8 3.9±0.6 6.8±0.5 7.2±0.6 (c) TC (s) 115.5±11.3 59.2±3.9 82.7±7.3 92.2±10.8 (d) OT (%) 34.2±4.6 14.2±3.1 32.3±2.9 34.1±5.6 (e) OE (%) 47.5±4.9 25.5±5.3 46.1±4.2 48.4±3.9 (f) Dead time (s)103.2±7.454.6±12.196.7±13.5108.5±17.2(g) Sleep induction time (s)27.5±2.836.1±1.729.2±2.228.4±1.9(h) Sleep time (s)38.1±2.334.5±1.738.1±1.239.4±1.8

[0234] 9-2. Protein and gene expression analysis

[0235] Protein and gene expression analysis was performed in the same manner as in Experimental Example 2-8 above.

[0236] As a result, as shown in Table 26, GABA levels in the prefrontal cortex were reduced by restraint stress. In addition, serotonin (5-HT) levels were also reduced by restraint stress, but TNF-α expression was increased.

[0237] However, administration of heat-treated P77 or P7277 increased the levels of GABA, GABRA1, GABRA2, serotonin, 5-HT1AR, and MT1R, which were reduced by restraint stress. In addition, administration of heat-treated P77 or P7277 confirmed that TNF-α expression induced by restraint stress was reduced.

[0238] NCISP77 (heat treatment) P7277 (heat treatment) (a) GABA (pg / mg) 16.4±1.9 10.2±1.5 15.3±2.1 16.2±1.8 (b) Serotonin (pg / mg) 16.3±1.3 10.1±1.3 14.4±1.7 14.8±2.2 (c) Corticosterone (ng / mg) 1.3±0.1 1.9±0.1 1.5±0.2 1.4±0.1 (d) TNF-α (pg / mg) 62.2±4.1 84.3±5.3 65.6±3.9 64.3±3.2 (e) GABRA1 (fold change) 1.0±0.0 0.8±0.1 1.0±0.1 1.1±0.1 (f) GABRA2 (fold change)1.0±0.10.7±0.11.0±0.11.0±0.1(g) MT1R(fold change)1.0±0.10.7±0.11.0±0.11.1±0.1(h) 5-HT1AR(fold change)1.0±0.00.8±0.11.0±0.11.0±0.1

[0239] Experimental Example 10. Confirmation of the effect of Lactobacillus plantarum P72 strain and hempseed oil on improving sleep disorders and depression induced by restraint stress (IS) [1]

[0240] After acclimating to the laboratory for one week prior to the experiment, C57BL / 6 mice (male, 18-21 g, 6 weeks old) were divided into four groups (NC, IS, hLP, hLH) with eight mice per group. After that, restraint stress (IS) was applied once a day for 8 hours for 5 days, and the test substance (hLP group: 1Х10 9 Heat-treated killed cells P72 (CFU / mouse / day); LH, 0.4Х10 9 P72 (CFU / mouse / day) and 0.12 mg / kg HO (hemp seed oil) were administered orally. The normal control (NC) and IS groups were administered saline instead of the test substance. Mice exposed to restraint stress showed a significant increase in depressive and anxious behaviors.

[0241] 10-1. Behavioral experiment

[0242] The OFT, EPMT, and TST behavioral experiment methods are the same as Experimental Examples 2-3 to 2-5, and the sleep disorder test method is the same as Experimental Example 4-2. The measurement results are as shown in Table 27.

[0243] In the OFT, TD, DC, and TC were all reduced compared to the normal control group due to restraint stress, in the EPMT, OT and OE were reduced, and in the TST, immobility time was increased.

[0244] However, it was confirmed that the TD, CD, TC, OT, and OE decreased by restraint stress were significantly restored through the administration of P72, and the immobility time was reduced.

[0245] In addition, it was confirmed that sleep induction time increased and sleep time decreased due to restraint stress, but sleep induction time decreased and sleep time increased through administration of P72, thereby improving sleep disorders through P72.

[0246] NCIShLPhLH(a) TD (m)36.5±1.131.2±0.835.2±3.135.5±3.2(b) DC (m)7.8±0.74.5±0.87.1±0.67.4±0.9(c) TC (s)122.3±12.557.4±4.979.9±7.389.7±10.1(d) OT (%)38.3±6.516.5±3.835.±3.837.9±9.9(e) OE (%)47.5±4.925.5±5.346.1±4.248.4±3.9(f) Dead time (s)103.2±7.454.6±12.196.7±13.5108.5±17.2(g) Sleep latency (s)115.7±5.9154.3±15.6129.1±9.8126.1±10.1(h) Sleep time (min)46.2±4.225.5±2.537.3±5.238.6±6.1

[0247] 10-2. Protein and Gene Expression Analysis

[0248] Protein and gene expression analysis was performed in the same manner as in Experimental Example 2-8 above.

[0249] As a result, as shown in Table 28, GABA levels in the prefrontal cortex were reduced by restraint stress. In addition, serotonin (5-HT) levels were also reduced by restraint stress, but TNF-α expression was increased.

[0250] However, administration of P72 increased GABA and serotonin levels, which were reduced by restraint stress. Furthermore, it was confirmed that P72 administration reduced TNF-α expression induced by restraint stress.

[0251] NCIShLPhLH(a) GABA (pg / mg)17.2±2.29.5±1.215.5±2.316.5±2.2(b) Serotonin (pg / mg)15.2±1.710.5±2.114.7±2.114.9±2.5(c) Corticosterone (ng / mg)1.4±0.22.0±0.11.5±0.31.5±0.2(d) TNF-α (pg / mg)58.3±3.272.5±5.855.6±3.856.4±2.9

[0252] Experimental Example 11. Confirmation of the effect of Lactobacillus plantarum P72 strain and hempseed oil on improving IS-induced sleep disorders and depression [2]

[0253] As in Experimental Example 2 above, mice with sleep disorders and depression induced by IS were used, and the combined effect of the P72 strain and hemp seed oil was confirmed.

[0254] The mouse is divided into 7 groups (NC, IS, LP) L , LP H , HO H , LH and DP) and each group consisted of eight mice. Restraint stress (IS) was applied to the mice in the above seven groups once a day for 5 days, and the test substance (LP) was applied once a day for 7 days starting 24 hours after the last restraint stress.L , 0.4Х10 9 P72; LP in CFU / mouse / day H , 1Х10 9 P72; HO in CFU / mouse / day H , 0.24 g / kg of HO (hemp seed oil); LH, 0.4Х10 9 P72 (CFU / mouse / day) and 0.12 mg / kg HO) were administered orally at 10:00 AM. The positive control drug (DP, 20 mg / kg diphenhydramine) was administered orally 30 minutes before pentobarbital injection. The normal control (NC) and IS groups were administered saline instead of the test substance. Behavioral tests were performed from 2:00 PM.

[0255] 11-1. Behavioral experiment

[0256] The OFT, EPMT, and TST behavioral experiment methods are the same as Experimental Examples 2-3 to 2-5, and the sleep disorder test method is the same as Experimental Example 4-2. The measurement results are as shown in Table 29.

[0257] In OFT, TD, DC, and TC were all reduced compared to the normal control group, in EPMT, OT and OE were reduced, and in TST, immobility time was increased.

[0258] However, when P72 and / or hemp seed oil were administered, TD, CD, and TC suppressed by IS were improved, while hemp seed oil alone (HO) was administered H ) compared to LH, TD and DC recovered more significantly when P72 and hemp seed oil were administered together. In addition, IS-induced OT in EPMT was significantly improved and immobility time in TST was significantly reduced when P72 and / or hemp seed oil were administered together.

[0259] In addition, it was confirmed that sleep latency was increased and sleep time was decreased by IS, but sleep latency was decreased and sleep time was increased through administration of P72, and sleep disorders were improved through P72 and / or hemp seed oil.

[0260] In general, for stress-induced sleep disturbances and depression, LPH (1Х10 9 The efficacy of P72 (CFU / mouse dose) was found to be the greatest, and it was confirmed that the combination of P72 and hemp seed oil could also significantly improve sleep disorders and depression.

[0261] Mean ± SDNCISLP L LP H HO H LHDP(a) TD (m)34.8±2.230.9±2.133.7±1.038.8±4.033.0±2.038.8±4.030.8±1.3(b) DC (m)6.5±1.54.5±0.86.1±0.98.0±0.54.9±0.96.3±1.44.6±0.3(c) TC (s)91.0±24.241.5±2.982.8±16.188.1±19.651.9±10.087.7±14.545.7±5.0(d) OT (%)11.9±4.54.4±1.113.6±2.716.6±3.611.3±2.316.6±3.64.7±1.4(e) OE (%)49.7±5.324.8±5.239.1±4.444.5±6.236.9±6.040.9±3.323.3±4.0(f) Immobility time (s)126.0±6.1180.2±14.3133.2±6.4134.3±8.4167.3±9.9139.1±13.7179.6±16.9(g) Sleep induction time (s)156.3±9.8205.3±11.2182.4±4.5168.3±4.1193.6±11.2168.3±4.1195.6±9.3(h) Sleep time (min)41.2±7.518.3±1.631.0±5.339.2±1.626.3±2.433.8±5.747.2±2.8

[0262] 11-2. Protein and gene expression analysis

[0263] Protein and gene expression analysis was performed in the same manner as in Experimental Example 2-8 above.

[0264] As a result, as shown in Table 30 and Fig. 3, IS reduced GABA levels and the expression of GABA receptors (GABRA1 and GABRA2) and melatonin receptors (MT1R, MT2R) in the prefrontal cortex. In addition, IS reduced serotonin (5-HT) levels and the expression of serotonin receptors 5-HT1AR and 5-HT1BR.

[0265] However, administration of P72 alone or in combination with hemp seed oil increased GABA and serotonin levels, which were reduced by IS, and the expression of GABA receptors (GABRA1 and GABRA2), melatonin receptors (MT1R, MT2R), and 5-HT receptors (5-HT1AR, 5-HT1BR). In particular, it was confirmed that the amount of serotonin was significantly increased when P72 and hemp seed oil were administered in combination.

[0266] In addition, as shown in Table 31, IS increased brain corticosterone, increased the expression of TNF-α and IL-6, and decreased the expression of IL-10. However, it was confirmed that administration of P72 alone or a combination of P72 and hemp seed oil decreased the expression of corticosterone, TNF-α, and IL-6 induced by IS, whereas increased the expression of IL-10, which was suppressed by IS.

[0267] 11-3. Immunofluorescence staining

[0268] Brain (frontal lobe and thalamus) and colon tissues collected from mice were fixed with 4% paraformaldehyde, cell-protected in 30% sucrose solution, frozen, and sectioned using a cryostat.

[0269] Sections were incubated with primary antibodies against GABRA1, NF-κB, Iba1, and / or CD11c for 16 h at 4°C, washed twice with saline, and incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:1,000, Invitrogen) or Alexa Fluor 594 (1:500, Invitrogen). Nuclei were stained with DAPI, and immunostained tissue sections were observed using a confocal laser microscope.

[0270] As a result, as shown in Fig. 3, it was confirmed through immunofluorescence staining that the expression of GABRA1 was reduced by IS exposure and then restored by administration of P72 alone or a combination of P72 and hemp seed oil.

[0271] Additionally, as shown in Fig. 4, NF-κB was detected through immunofluorescence staining. + Iba1 + We confirmed that the number of cells increased by IS exposure and decreased by administration of P72 alone or a combination of P72 and hemp seed oil. This suggests that P72 alone or a combination of P72 and hemp seed oil can alleviate sleep disorders and depression by regulating the serotonin and GABA systems through inhibition of NF-κB activation.

[0272] Mean ± SDNCISLP L LP H HO HLHDP(a) GABA (pg / mg)20.7±3.311.3±2.117.6±2.218.6±3.014.6±2.617.8±1.916.8±1.0(b) GABRA1(fold change)1.0±0.10.8±0.10.9±0.11.0±0.10.8±0.10.9±0.10.8±0.0(c) GABRA2(fold change)1.0±0.00.8±0.10.9±0.00.9±0.00.9±0.01.0±0.00.9±0.1(d) MT1R(fold change) change)1.0±0.10.8±0.10.9±0.11.1±0.10.8±0.01.0±0.10.9±0.0(e) MT2R(fold change)1.0±0.10.7±0.10.9±0.11.0±0.10.8±0.01.1±0.00.8±0.1(f) 세로토니(ng / mg)28.7±4.015.1±2.621.4±2.328.9±3.418.4±3.028.9±3.418.4±4.0(g)5-HT1AR(fold change)1.0±0.10.8±0.01.0±0.11.1±0.10.9±0.01.0±0.10.8±0.0(h)5-HT1BR(fold change)1.0±0.10.6±0.10.9±0.10.9±0.10.7±0.00.9±0.20.6±0.1

[0273] Mean ± SDNCISLP L LP H HO H LHDP(a) 1.5±0.11.9±0.11.6±0.11.6±0.11.8±0.11.5±0.11.9±0.2(ng / mg) TNF-α(ng / mg)0.14±0.000.16±0.010.14±0.000.13±0.010.15±0.000.13±0.010.14±0.01(c) IL-6(cg / mg)51.8±2.065.1±5.356.5±7.554.4±3.462.6±2.455.1±3.260.3±6.8(d) IL-10(ng / mg)0.14±0.010.10±0.000.11±0.010.12±0.010.11±0.000.12±0.000.11±0.01

[0274] Experimental Example 12. Confirmation of the anti-inflammatory effect of Lactobacillus plantarum P72 strain and hemp seed oil through IS.

[0275] Colitis was induced by the IS stress described above, and as shown in Table 32 and Figure 5, the length of the colon was shortened, the expression of myeloperoxidase (MPO), TNF-a, IL-1β, and IL-6 increased, and the expression of IL-10 decreased.

[0276] However, administration of P72 alone or a combination of P72 and hemp seed oil restored colon length, and the expression of MPO, TNF-α, IL-1β and IL-6 and NF-κB + CD11c + It was confirmed that cells were reduced and IL-10 expression, which was suppressed by IS, increased. Through the above results, it was confirmed that the P72 of the present invention or the combination of P72 and hemp seed oil can be applied to the improvement or treatment of inflammatory diseases by improving inflammatory indicators.

[0277] Mean ± SDNCISLP L LP H HO HLHDP (a) Large intestine length (cm)6.1±0.25.2±0.15.8±0.26.2±0.25.5±0.26.1±0.25.2±0.2(b) MPO (ng / mg)0.16±0.020.21±0.010.19±0.010.17±0.010.20±0.020.18±0.010.22±0.02(c) TNF-α (ng / mg)0.11±0.010.14±0.010.11±0.020.11±0.020.12±0.010.10±0.020.14±0.12(d) IL-1β (pg / mg)59.6±4.580.2±9.561.8±4.858.3±5.369.2±8.854.9±6.573.1±9.9(e) IL-6 (pg / mg)68.8±2.178.8±4.771.4±1.466.8±2.373.9±4.661.8±6.174.5±3.8(f) IL-10(ng / mg)0.14±0.010.11±0.000.13±0.010.14±0.010.12±0.010.13±0.010.11±0.01

[0278] 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.

[0279] 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.

Claims

Lactobacillus plantarum P72 strain deposited under accession number KCCM13445P. In the first paragraph, The above strain is Lactobacillus plantarum P72 strain, which contains the 16S rDNA base sequence of sequence number 1. Bifidobacterium longum P77 strain deposited under accession number KCCM13446P. In the first paragraph, The above strain is a Bifidobacterium longum P77 strain comprising the 16S rDNA base sequence of sequence number 2. A pharmaceutical composition for preventing or treating a neuropsychiatric or inflammatory disease, comprising Lactococcus plantarum P72 strain, Bifidobacterium longum P77 strain, or a mixture thereof. In paragraph 5, 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. In paragraph 5, A pharmaceutical composition wherein the above inflammatory disease is colitis. In paragraph 5, The above Lactobacillus plantarum P72 or Bifidobacterium longum P77 strain, A pharmaceutical composition comprising a live cell, a dead cell, a culture thereof, a lysate thereof or an extract thereof. In paragraph 5, The above mixture is a pharmaceutical composition in which the Lactococcus plantarum P72 strain and the Bifidobacterium longum P77 strain are mixed in a colony forming unit (CFU) ratio of 1:1 to 4:

1. In paragraph 5, A pharmaceutical composition, wherein the composition further comprises hempseed oil. A food composition for preventing or improving neuropsychiatric or inflammatory diseases, comprising Lactococcus plantarum P72 strain, Bifidobacterium longum P77 strain, or a mixture thereof. In Article 11, 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. A food composition according to claim 11, wherein the inflammatory disease is colitis. In Article 11, The above Lactobacillus plantarum P72 or Bifidobacterium longum P77 strain, A food composition comprising a live cell, a dead cell, a culture thereof, a lysate thereof or an extract thereof. In claim 11, the mixture is a food composition in which the Lactococcus plantarum P72 strain and the Bifidobacterium longum P77 strain are mixed in a colony forming unit (CFU) ratio of 1:1 to 4:

1. A food composition according to claim 11, wherein the composition further comprises hemp seed oil. A method for treating a neuropsychiatric or inflammatory disease, comprising administering to a subject a composition comprising Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof. A method for improving a neuropsychiatric or inflammatory disease, comprising administering to a subject a composition comprising Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof. Use of a composition comprising Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof, for treating neuropsychiatric or inflammatory diseases. Use of a composition comprising Lactobacillus plantarum P72, Bifidobacterium longum P77 strain, or a mixture thereof, for improving neuropsychiatric or inflammatory diseases.

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