Brain function improving agent

Abstract

The present invention addresses the problem of providing a bacterial preparation which increases the amount of BDNF in the brain, in particular, in the hippocampus and the like and is effective for a wide range of brain dysfunction such as dementia and mental diseases.　Provided is a brain function improving agent based on hippocampal BDNF containing lactic acid-producing bacteria, in particular, specific bifidobacteria and / or lactic acid bacteria.

Description

Brain function improving agent

[0001] The present invention relates to a brain function improving agent and a composition for improving brain function based on BDNF, and a method for improving brain function.

[0002] In recent years, the increase in the number of patients with brain function disorders has become a problem. Behind this increase are the diversification of mental diseases and the increase in their patients accompanying changes in lifestyle, and the increase in dementia patients accompanying the progress of aging. It is considered that the increase in these patients brings about an increase in medical costs, an increase in the burden of care, and further a decrease in the productivity of the whole society, and an early solution is desired.

[0003] However, for many of these diseases, there are few options for effective treatment methods. From this, intervening by continuously alleviating symptoms and continuously suppressing the onset risk has become a realistic treatment or prevention means. Here, in order to perform such an intervention, an intervention means with a small burden on the subject is required, but there have not been many means with a low burden and effective for brain function disorders provided so far.

[0004] In recent years, with the development of intestinal flora analysis using molecular biological techniques, many findings regarding the intestinal flora have been obtained. As a result, it has become clear that the intestinal bacteria contained in the intestinal flora not only affect the intestinal environment and the state of the digestive system, but also have various effects on the functions of the whole body. From this, bacterial preparations containing specific intestinal bacteria have attracted attention as means for preventive or therapeutic intervention for effective diseases.

[0005] Furthermore, such bacterial preparations are considered to be highly safe because the bacteria themselves are natural components, and side effects often remain mild, such as temporary abdominal distension and mild gas generation. From these facts, bacterial preparations may be suitable for long-term continuous intake with a small burden on patients due to continuous intake (Non-Patent Document 1).

[0006] US Agency for Healthcare Research and Quality: Safety of Probiotics to Reduce Risk and Prevent or Treat Disease: Evidence Reports / Technology Assessments, 2011 (200): 1-645

[0007] Brain-derived neurotrophic factor (BDNF) is a secreted protein that is primarily expressed in the central nervous system, mainly distributed in the hippocampus, amygdala, and cortex, and also expressed in peripheral systems such as the heart, adipose tissue, and skeletal muscle. As its name suggests, BDNF acts as a trophicing factor for nerve cells in the brain, contributing to the normal functioning of the brain. A decrease in BDNF is suggested to be involved in various brain dysfunctions, such as dementia and mental illnesses mentioned above.

[0008] The development of bacterial formulations has largely been conducted in the context of health promotion or the alleviation of broad-based diseases and symptoms. Therefore, little knowledge has been gained regarding the relationship between molecular-level changes and gut bacteria, particularly in organs other than the digestive system, such as their association with the expression of specific proteins. This is considered one of the reasons why the development of bacterial formulations has not led to the development of drugs effective for a wide range of diseases.

[0009] Therefore, the object of the present invention is to provide a bacterial preparation that enhances the amount of BDNF in the brain, particularly in the hippocampus, and is effective against a wide range of brain dysfunctions such as dementia and mental illness.

[0010] To solve the above problems, the present inventors conducted intensive research and found that among bacterial strains that are effective against intestinal cells, certain bacterial strains have the effect of enhancing BDNF levels in the hippocampus and other areas. The present invention is based on this new finding and provides the following: [1] A BDNF-based brain function improving agent comprising the bifidobacterium longum CECT 7894 strain. [2] The brain function improving agent according to [1], wherein the bifidobacterium comprises dead bacteria. [2-1] A BDNF-based brain function improving agent comprising the lactic acid bacterium Lactiprantibacillus plantarum CECT 7481 strain. [2-2] The brain function improving agent according to [2-1], wherein the lactic acid bacterium comprises dead bacteria. [2-3] A BDNF-based brain function improving agent comprising lactic acid-producing bacteria. [2-4] The brain function improving agent according to [2-3], wherein the lactic acid-producing bacteria is the bifidobacterium bifidobacterium longum CECT 7894 strain. [2-5] The brain function improving agent according to [2-3], wherein the lactic acid-producing bacteria is Lactiprantibacillus plantarum CECT 7481 strain, which is a lactic acid bacterium. [2-6] The brain function improving agent according to any one of [2-3] to [2-5], wherein the lactic acid-producing bacteria include dead bacteria. [3] The brain function improving agent according to any one of [1] to [2-6], wherein the BDNF is hippocampal BDNF. [4] A brain function improving composition based on BDNF, comprising Bifidobacterium longum CECT 7894 strain, which is a bifidobacterium. [4-1] A brain function improving composition based on BDNF, comprising Lactiprantibacillus plantarum CECT 7481 strain, which is a lactic acid bacterium. [4-2] A BDNF-based brain function improving composition comprising Bifidobacterium longum CECT 7894 strain, which is a bifidobacterium, and Lactiprantibacillus plantarum CECT 7481 strain, which is a lactic acid bacterium. [4-3] A BDNF-based brain function improving composition comprising lactic acid-producing bacteria. [4-4] The brain function improving composition according to [4-3], wherein the lactic acid-producing bacteria comprises Bifidobacterium longum CECT 7894 strain, which is a bifidobacterium, and / or Lactiprantibacillus plantarum CECT 7481 strain, which is a lactic acid bacterium. [5] The brain function improving composition according to any one of [4] to [4-4], wherein the bifidobacterium and / or lactic acid bacteria comprises dead bacteria.[5-1] A brain function improving composition according to either [4-3] or [4-4], wherein the lactic acid-producing bacteria include dead bacteria. [6] A brain function improving composition according to any one of [4] to [5-1], wherein the BDNF is hippocampal BDNF. [7] A brain function improving composition according to any one of [4] to [6], which is a food or beverage. [8] A brain function improving composition according to any one of [4] to [6], which is a pharmaceutical composition. [9] A brain function improving composition according to any one of [4] to [6], which is a composition for the prevention or treatment of brain dysfunction.

[10] A brain function improving composition according to [9], wherein the brain dysfunction is one or more diseases selected from the group consisting of dementia, neurodegenerative diseases and mental illnesses.

[11] A BDNF-based brain function improving method comprising administering Bifidobacterium longum CECT 7894 strain, which is a Bifidobacterium, to a subject. [11-1] A method for improving brain function based on BDNF, comprising administering the lactic acid bacterium Lactiprantibacillus plantarum CECT 7481 strain to a subject. [11-2] A method for improving brain function based on BDNF, comprising administering the bifidobacterium Bifidobacterium longum CECT 7894 strain and the lactic acid bacterium Lactiprantibacillus plantarum CECT 7481 strain to a subject. [11-3] The method for improving brain function according to

[11] , further comprising administering the lactic acid bacterium Lactiprantibacillus plantarum CECT 7481 strain to a subject. [11-4] The method for improving brain function according to [11-1], further comprising administering the lactic acid bacterium Bifidobacterium longum CECT 7894 strain to a subject. [11-5] A method for improving brain function based on BDNF, comprising administering lactic acid-producing bacteria to a subject. [11-6] The method for improving brain function according to [11-5], wherein the lactic acid-producing bacteria is Bifidobacterium longum CECT 7894 strain, which is a Bifidobacterium, and / or Lactiprantibacillus plantarum CECT 7481 strain, which is a Lactobacillus. This specification encompasses the disclosures of Japanese Patent Applications Nos. 2025-010439, 2025-010440, and 2025-174644, which form the basis of the priority of this application.

[0011] According to the brain function improving agent and brain function improving composition of the present invention, it is possible to improve brain function based on BDNF.

[0012] This figure shows the results of the in vivo test in Example 1. In the figure, the standardized BDNF amount is the value obtained by standardizing the value of each group with the result of the control group set to 1. The experimental conditions for each group are also shown below each bar graph. For "Scopolamine," "-" indicates that scopolamine was not administered, and "+" indicates that scopolamine was administered. For "Additional Drugs," "-" indicates that no additional drugs were added to test the BDNF-enhancing effect, and for conditions where additional drugs were added to test the BDNF-enhancing effect, the name of the added drug is indicated. Error bars indicate the standard deviation, "ns" indicates that there was no significant difference, "*" indicates that the p-value was p<0.05, and "**" indicates that the p-value was p<0.01. This figure shows the results of the in vivo test using the CECT 7481 strain in Example 2. In the figure, the standardized BDNF amount is the value obtained by standardizing the value of each group with the result of the control group set to 1. The experimental conditions for each group are also shown below each bar graph. For "Scopolamine," "-" indicates that scopolamine was not administered, and "+" indicates that scopolamine was administered. For "Additional Drugs," "-" indicates that no additional drugs were added to test the BDNF-enhancing effect. For conditions where additional drugs were added to test the BDNF-enhancing effect, the name of the added drug is listed. Error bars indicate the standard deviation, and "*" indicates that the p-value was p<0.05. This figure shows the results of the in vivo test using the CECT 7746 strain in Example 2. In the figure, the standardized BDNF amount is the value obtained by standardizing the value of each group with the result of the control group set to 1. The experimental conditions for each group are also shown below each bar graph. For "Scopolamine," "-" indicates that scopolamine was not administered, and "+" indicates that scopolamine was administered. Regarding "additional drugs," "-" indicates that no additional drugs were added to test the BDNF-enhancing effect. Conditions where additional drugs were added to test the BDNF-enhancing effect are indicated by the name of the added drug. Error bars indicate the standard deviation; "ns" indicates no significant difference; and "*" indicates a p-value of p<0.05. This figure shows the results of the in vivo test in Example 3.Figure 4A shows the results for the control group, Figure 4B shows the results for the CECT 7746 strain administered group, Figure 4C shows the results for the CECT 7894 strain administered group, and Figure 4D shows the results for the CECT 7481 strain. In the figures, "Day 14" shows the results on day 14 of treatment before scopolamine administration, and "Day 16" shows the results on day 16 of treatment after scopolamine administration. The dashed lines in each figure show the outline of the mouse head viewed from above, and the color scale in the lower right of each figure indicates that the whiter the color, the stronger the luminescence intensity. In the figures, the scale bar represents 1 cm. This figure shows the results of the percentage change in the in vivo test in Example 3. In the figure, "Saline" shows the results for the control group, and the other series of bacterial strain names show the results for each bacterial strain administered group. Error bars show the standard error, "*" indicates that the p-value was p<0.05, and "#" indicates that the p-value was p<0.10. This figure shows the results of the frequency of freezing behavior in the in vivo test in Example 4. In the figure, "Saline-Saline" shows the results for the control group administered with saline solution without scopolamine, "Saline-Scopolamine" shows the results for the control group administered with scopolamine, and "CECT 7894-Scopolamine" and "CECT 7481-Scopolamine" show the results for the treatment groups of each bacterium administered with scopolamine, respectively. Error bars indicate the standard error, "†" indicates a p-value of p<0.00000001, "**" indicates a p-value of p<0.01, and "*" indicates a p-value of p<0.05. This figure shows the results of the frequency of freezing behavior in the in vivo test in Example 5. In the figure, "Saline-Saline" shows the results for the control group administered with saline solution without scopolamine, "Saline-Scopolamine" shows the results for the control group administered with scopolamine, and "CECT×2-Scopolamine" shows the results for the combined treatment groups of both bacteria administered with scopolamine. Error bars indicate the standard error, and "*" indicates that the p-value was p<0.05.

[0013] 1. Brain Function Improving Agent 1-1. Overview The first aspect of the present invention is a brain function improving agent. The brain function improving agent of the present invention contains specific lactic acid-producing bacteria, particularly Bifidobacterium or Lactobacillus, as an active ingredient, and improves brain function based on BDNF. The brain function improving agent of the present invention may be an active ingredient in the brain function improving composition described in the second aspect.

[0014] 1-2. Definitions The terms used herein are defined below.

[0015] "Lactic acid-producing bacteria" refers to bacteria capable of producing lactic acid, and in this specification, it specifically refers to bacteria known as beneficial intestinal bacteria in humans and animals. Examples include Bifidobacteria and Lactobacillus.

[0016] "Bifidobacteria" are Gram-positive, anaerobic rod-shaped bacteria belonging to the genus Bifidobacterium, and are known as beneficial gut bacteria in humans and animals. Bifidobacteria are lactic acid-producing bacteria that primarily produce lactic acid and acetic acid using sugars, and many of the bacteria included in the bifidobacteria genus, such as Bifidobacterium longum, are known to have effects such as improving the intestinal environment, enhancing immune function, and lowering cholesterol.

[0017] "CECT 7894 strain" refers to the bacterial strain of Bifidobacterium longum deposited with Coleccion Espanola de Cultivos Tipo (CECT) under accession number CECT 7894. In this specification, CECT 7894 strain also includes its derivative strains.

[0018] "Lactic acid bacteria" is a general term for bacteria (Gram-positive facultative anaerobic or anaerobic rods or cocci) that produce lactic acid by utilizing sugars such as glucose and lactose to obtain the energy necessary for growth. These bacteria are known as beneficial intestinal bacteria in humans and animals. Many of the bacteria included in lactic acid bacteria, such as the facultative anaerobic rod Lactiplantibacillus plantarum, are known to have effects such as improving the intestinal environment, enhancing immune function, and lowering cholesterol.

[0019] "CECT 7481 strain" refers to the bacterial strain of Lactiprantibacillus plantarum deposited with Coleccion Espanola de Cultivos Tipo (CECT) under accession number CECT 7481. In this specification, CECT 7481 strain also includes its derivative strains.

[0020] "BDNF (brain-derived neurotrophic factor)" refers to a secreted protein belonging to the neurotrophic factor family. BDNF binds to receptors such as TrkB and p75 and plays a role in supporting neuronal functions, including maintaining neuronal survival, inducing neuronal differentiation, regulating synaptic plasticity, and promoting neurotransmitter release. In this specification, BDNF specifically refers to BDNF found in the brain.

[0021] "Intracerebral region" refers to the parenchyma and / or cerebrospinal fluid of all or part of the brain. Unless otherwise specified, "intracerebral region" in this specification refers to the cerebrum and / or diencephalon, in addition to cerebrospinal fluid. The brain regions covered in this specification are not particularly limited, but are selected from one or more of the following groups: the cerebral cortex (frontal lobe, parietal lobe, temporal lobe, occipital lobe, etc.), the basal ganglia (caudate nucleus, putamen, globus pallidus, etc.), the limbic system (hippocampus, amygdala, cingulate gyrus, septum, entorhinal cortex, etc.), and the diencephalon (thalamus, hypothalamus, etc.). For example, the hippocampus is a suitable region.

[0022] The hippocampus is a brain region that is part of the limbic system and is involved in memory and spatial learning. It is particularly known for its function of storing short-term memories and sending some of them to the neocortex as long-term memories, and has been suggested to be associated with many brain dysfunctions such as dementia.

[0023] In this specification, "hippocampal BDNF" refers to BDNF present in the hippocampus. In this specification, "hippocampal BDNF" includes BDNF produced in the hippocampus, BDNF secreted in the hippocampus (including BDNF produced in other brain regions), and BDNF present in cells in the hippocampus (including BDNF present in cells whose cell bodies are located in other brain regions).

[0024] In this specification, "brain dysfunction" refers to a condition in which brain functions, such as cognitive function, are impaired due to brain damage, disease, stress, etc. While not specifically limited, examples of brain dysfunction include dementia, neurodegenerative diseases, and mental illnesses.

[0025] "Dementia" refers to a disease characterized by core symptoms such as memory impairment, disorientation, behavioral disorders, impaired comprehension and judgment, and executive function disorders, which appear as a result of damage to brain cells. For example, compositions or pharmaceutical compositions of the present invention that include synapse formation promoters, synapse maintenance agents, nerve cell function modifiers that cause changes (e.g., acceleration) in the electrophysiological properties of nerve cells (such as the properties of conduction and transmission of stimuli), or cytoskeletal modifiers that promote the maintenance and formation of the neuroskeleton, can be used in particular to improve or prevent the symptoms of dementia. Examples of dementia include Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia, depending on the underlying disease or disorder, but dementia in the present invention includes all of these, and the underlying disease or disorder is not particularly limited.

[0026] "Alzheimer's disease" refers to dementia characterized by atrophy of the medial temporal lobe (hippocampus) as seen in CT and MRI scans, and with event memory impairment as a core symptom of cognitive and memory impairment. It may also be accompanied by other cognitive impairments such as constructional atrophy, visuospatial cognitive impairment such as topographic disorientation, and recent memory impairment. It is a general term for dementia caused by Alzheimer's disease, which is characterized by amyloid-beta aggregation, and dementias with symptoms similar to Alzheimer's disease.

[0027] Vascular dementia refers to dementia that arises in association with cerebrovascular disease and is accompanied by impairments in complex attention, including information processing speed, and frontal lobe executive functions. Depending on the type of vascular disease, it is classified into polyinfarct dementia, small vessel disease dementia, hypoperfusion vascular dementia, hemorrhagic vascular dementia, etc. It frequently coexists with Alzheimer's disease.

[0028] Lewy body dementia is a type of dementia characterized primarily by the appearance of inclusion bodies (Lewy bodies) resulting from the abnormal accumulation of alpha-synuclein in nerve cells, accompanied by symptoms such as cognitive fluctuations, hallucinations, and / or idiopathic parkinsonism. It may also be accompanied by constructional disorders, severe hypersensitivity to antipsychotic drugs, and / or REM sleep behavior disorder. Lewy bodies may also be observed outside the central nervous system.

[0029] Frontotemporal dementia is a type of dementia characterized by brain atrophy in the frontal and / or temporal lobes, accompanied by progressive aphasia. It may also be accompanied by social cognitive impairments such as personality changes and executive function disorders, although memory impairment is often relatively mild. It is broadly classified into conditions involving tau accumulation and conditions involving TDP-43 accumulation, but conditions involving the accumulation of FUS (fused in sarcoma) are also known.

[0030] In this specification, "neurodegenerative disease" refers to a disease in which the structure of nerve tissue degenerates over time. Specifically, examples include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, macular degeneration, multiple sclerosis, muscular dystrophy, Niemann-Pick disease, osteoporosis, and rheumatoid arthritis. Many of these are induced by the accumulation of specific proteins.

[0031] In this specification, "mental disorder" refers to a disorder characterized by mental and behavioral impairment. While not limited to specific conditions, mental disorders include, for example, mood disorders, developmental disorders, anxiety disorders, and other mental illnesses. Diagnosis can be made based on the World Health Organization's (WHO) International Classification of Diseases, 10th Revision (ICD-10) or the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, 5th Revision (DSM-5).

[0032] "Mood disorder" is a general term for disorders in which mood swings interfere with daily life. While not strictly limited, specific examples of mood disorders include depression and bipolar disorder.

[0033] "Depression" is a general term for disorders that impair daily and social life due to depressed mood, loss of interest, etc. While depression is classified into major depressive disorder (MDD) and dysthymic disorder depending on its severity, the term "depression" in this specification encompasses all of these subdivided types of depression. The causes of depression are not particularly limited; for example, endogenous, exogenous, and psychogenic factors are included, but the term "depression" in this specification encompasses all of these.

[0034] "Bipolar disorder" refers to a disorder characterized by extreme mood swings. In this specification, bipolar disorder includes all subtypes of bipolar disorder, such as bipolar I and bipolar II.

[0035] In this specification, "developmental disorder" refers to a disorder of brain function related to learning, language, attention, social interaction, or behavioral control. While not specifically limited, examples of developmental disorders include attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and learning disabilities.

[0036] Attention-deficit / hyperactivity disorder (ADHD) is a behavioral disorder characterized by inattention, impulsivity, and / or hyperactivity that is inappropriate for the age or developmental stage, and which impairs social activities and academic functioning. Typically, it is characterized by symptoms such as inattention, hyperactivity, and / or impulsivity. In this specification, ADHD includes all of the subdivided types of ADHD, such as inattentive type, hyperactive-impulsive type, and combined type.

[0037] Autism Spectrum Disorder (ASD) is a general term for developmental disorders characterized by persistent deficits in social communication and interpersonal interaction from early development, as well as behaviors, interests, or repetitive behaviors. Autism Spectrum Disorder includes classic autism, Asperger's syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), Rett syndrome, and childhood disintegrative disorder.

[0038] "Learning disability (LD)" refers to a condition in which there is no overall delay in intellectual development, but there are difficulties in acquiring and / or demonstrating one or more basic learning abilities such as listening and speaking, which impairs daily life and academic functioning.

[0039] In this specification, "anxiety disorder" is a general term for disorders that interfere with daily life due to excessive anxiety or fear. In this specification, anxiety disorders also include trauma and stressor-related disorders (such as acute stress disorder, adjustment disorder, and post-traumatic stress disorder) resulting from mental distress that occurs within a short period following a traumatic event. Specific examples of anxiety disorders include generalized anxiety disorder, specific phobias, and panic disorder.

[0040] Other mental illnesses include, for example, intellectual disability and schizophrenia.

[0041] "Intellectual disability" is a general term for disorders that involve impairments in cognitive and language-related intellectual functions that appear after the developmental stage, causing difficulties in daily life and social life.

[0042] "Schizophrenia" is a mental disorder characterized by hallucinations, delusions, negative symptoms, cognitive impairment, and integrative brain function. In this specification, schizophrenia encompasses all of its subtypes, including paranoid, disorganized, and catatonic schizophrenia.

[0043] In this specification, "significant" means statistically significant. Statistically significant means that there is a significant difference between the measured value and the control value of the test subject when the difference is statistically processed. For example, when the p-value (significance level) of the obtained value is small, specifically, less than 5% (p < 0.05), less than 1% (p < 0.01), or less than 0.1% (p < 0.001). The "p (value)" shown here indicates the probability that the test statistic accidentally becomes that value in the distribution based on the null hypothesis in a statistical test. Therefore, the smaller the "p", the lower the probability that the test statistic becomes that value, meaning that the null hypothesis is likely to be rejected. As the test method for statistical processing, a known test method capable of determining the presence or absence of significance can be appropriately used without particular limitation. For example, Student's t-test method, paired Student's t-test method, Welch's t-test method, Wilcoxon rank sum test, analysis of variance, Tukey's post hoc test, etc. can be used, but there is no particular limitation.

[0044] 1-3. The brain function improving agent constituting the present invention contains, as an essential constituent, lactic acid-producing bacteria, particularly Bifidobacterium longum CECT 7894 strain which is a Bifidobacterium or Lactiplantibacillus plantarum CECT 7481 strain which is a lactic acid bacterium. This will be specifically described below.

[0045] The Bifidobacterium longum CECT 7894 strain contained in the brain function improving agent of this aspect can include the cells or a part of the deposited strain deposited as CECT 7894 or its derivative strain.

[0046] The Lactiplantibacillus plantarum CECT 7481 strain contained in the brain function improving agent of this aspect can include the cells or a part of the deposited strain deposited as CECT 7481 or its derivative strain.

[0047] In this specification, "derived strain" refers to a new strain that has been obtained through genetic mutation from the original bacterial strain (in this invention, strain CECT 7894 and / or strain CECT 7481). The derived strains in this specification have an effect equivalent to or greater than that of strain CECT 7894 and / or strain CECT 7481 in terms of enhancing the amount of BDNF (hippocampal BDNF, etc.).

[0048] "Equivalent" means that the product is above a certain level and / or does not show a statistically significant difference compared to CECT 7894 strain and / or CECT 7481 strain. Specifically, the BDNF-enhancing effect should be, for example, 0.4 times, 0.45 times, 0.5 times, 0.6 times, 0.65 times, 0.7 times, 0.8 times, 0.9 times, or 1 time compared to CECT 7894 strain. More specifically, the BDNF-enhancing effect should be, for example, 0.7 times, 0.75 times, 0.8 times, 0.85 times, 0.9 times, 0.95 times, or 1 time compared to CECT 7481 strain or donepezil. Furthermore, there is no particular limit to the significance level in this case, but for example, the significance levels mentioned above in the definition section can be used.

[0049] The CECT 7894 strain and its derivatives can be prepared by culturing them under appropriate conditions using a culture medium commonly used for culturing Bifidobacteria. The culture medium used is not particularly limited; it may be a natural medium or a synthetic medium, as long as it contains a carbon source, nitrogen source, inorganic salts, etc., and is capable of efficiently culturing Bifidobacteria. Those skilled in the art can appropriately select a known culture medium suitable for the strain being used.

[0050] The CECT 7481 strain and its derivatives can be prepared by culturing them under appropriate conditions using a culture medium commonly used for culturing lactic acid bacteria. The culture medium used is not particularly limited; it may be a natural medium or a synthetic medium, as long as it contains a carbon source, nitrogen source, inorganic salts, etc., and is capable of efficiently culturing lactic acid bacteria. Those skilled in the art can appropriately select a known culture medium suitable for the strain being used.

[0051] Lactose, glucose, sucrose, fructose, galactose, and molasses can be used as carbon sources.

[0052] Organic nitrogen-containing substances such as hydrolyzed casein, hydrolyzed milk protein, and hydrolyzed soy protein can be used as nitrogen sources.

[0053] Inorganic salts such as phosphates, sodium, potassium, and magnesium can be used.

[0054] Suitable culture media for Bifidobacteria include, for example, MRS liquid medium (mMRS medium) supplemented with cysteine, an essential amino acid for Bifidobacteria, RCM medium, GAM medium, BL medium, Briggs Liver Broth, animal milk, skim milk, and milk whey. For example, sterile mMRS medium can be suitably used.

[0055] Suitable culture media for lactic acid bacteria include, for example, MRS liquid medium, GAM medium, BL medium, Briggs Liver Broth, animal milk, skim milk, and milk whey. For example, sterilized MRS medium can be suitably used.

[0056] While the culture conditions are not particularly limited, since CECT strain 7894 is an anaerobic bacterium, it is cultured under anaerobic conditions. Here, anaerobic conditions refer to a low-oxygen environment in which bacteria can grow, and methods for creating anaerobic conditions are widely known in the art. While not particularly limited, for example, an anaerobic chamber, anaerobic box, or oxygen absorber (Anelopack) can be used. (R) Anaerobic conditions can be created by using a sealed container or bag containing (etc.), or simply by sealing the culture vessel.

[0057] Furthermore, while there are no specific limitations on the culture conditions, since CECT strain 7481 is a facultative anaerobic bacterium, it should be cultured under either aerobic or anaerobic conditions. Here, aerobic conditions refer to conditions in which sufficient oxygen is present in the culture environment, and this can be achieved by performing the culture without using the anaerobic conditions described later.

[0058] The culture method is not particularly limited, but for example, static culture, shaking culture, tank culture, or a combination thereof can be used.

[0059] The temperature conditions are not particularly limited as long as they are set appropriately, but for example, they can be 20°C to 50°C, 25°C to 42°C, 30°C to 42°C, 35°C to 42°C, or approximately 37°C. The temperature control means are not particularly limited, but for example, a constant temperature bath, mantle heater, jacket, etc. can be used.

[0060] Furthermore, while there are no specific limitations on the incubation time, it can be, for example, 3 to 100 hours or 10 to 50 hours. The pH of the culture medium can also be maintained, for example, between 3.5 and 8.0.

[0061] The bacterial cells may be included as live and / or dead cells. For example, at least some or all of the bacterial cells may be included as dead cells. For example, live bacterial cells can be obtained as a culture medium containing live bacterial cells after cultivation, or by separation techniques such as filtration and centrifugation from the culture medium. Furthermore, dead bacterial cells can be obtained by subjecting these live bacterial cells to known sterilization treatments such as autoclaving.

[0062] In this embodiment, the bacterial cells and / or a part thereof contained in the brain function improving agent can be bacterial cells that have undergone any treatment after culturing.

[0063] A microbial cell treatment product is obtained by subjecting microbial cells to physical, chemical, or biological treatment. While the specific treatment is not limited, examples include one or more treatments selected from the group consisting of sterilization, drying, destruction or crushing, dilution, concentration, and fermentation. Examples of microbial cell treatment products include dried microbial cells, destroyed microbial cells, crushed microbial cells, and fermented products. For example, dried microbial cells (such as freeze-dried microbial cells) can be suitably used.

[0064] Dried bacterial cells can be obtained by subjecting bacterial cells isolated from a culture medium, for example, bacterial cells that have been further sterilized, to a drying process. The specific drying process is not limited, but examples include drum drying, spray drying, vacuum drying, freeze-drying, or a combination thereof. For example, freeze-dried bacterial cells can be used. In this case, the bacterial cells can be in the form of a dried powder or granules.

[0065] Freeze-drying is typically carried out by freezing, degassing, and sublimation, but the specific method is not particularly limited. For example, it can be carried out using a commercially available freeze-dryer. The temperature conditions are not particularly limited, but for example, they can be below -20°C, below -30°C, below -50°C, below -60°C, below -70°C, or below -80°C.

[0066] Destroyed or lysed bacterial cells can be obtained by treating bacterial cells with methods such as crushing, grinding, enzymatic treatment, chemical treatment, or dissolution. Destroyed or lysed bacterial cells essentially consist of or include all of the solid and soluble components of the destroyed or crushed bacterial cells. Destroyed or lysed bacterial cells can be obtained, for example, by freeze-drying the destroyed or crushed material as is. The method of destruction or crushing is not particularly limited. For example, it can be carried out by known methods, physical crushing, enzymatic dissolution, chemical treatment, or a combination thereof.

[0067] For example, physical crushing can be carried out either wet or dry, by stirring using a homogenizer, ball mill, bead mill, dyno mill, planetary mill, etc., or by using a jet mill, French press, cell disruptor, etc. Enzymatic lysis can be carried out by destroying the cellular structure of the bacteria using an enzyme such as lysozyme. Chemical treatment can be carried out by destroying the cellular structure of the bacteria using a surfactant such as glycerol fatty acid ester or soybean phospholipid.

[0068] As part of the bacterial cells, for example, membrane components (cell membrane components, etc.) and / or cell wall components of bacterial cells from CECT 7894 strain and / or CECT 7481 strain can be used. Such parts of bacterial cells can be obtained by the drying treatment and / or crushing / disruption treatment described above. Therefore, processed bacterial cells can be used as part of the bacterial cells.

[0069] The bacterial cells can be prepared as a suspension or dilution by suspending or diluting them in a suitable solvent. The solvents that can be used are not particularly limited, but examples include water, physiological saline, phosphate-buffered saline (PBS), culture media, etc.

[0070] Furthermore, the treated bacterial cells may be subjected to further processing, as long as it has the effect of promoting BDNF expression in the target area (such as the hippocampus). Examples of such processing are described below.

[0071] Sterilization treatment can be used to prepare sterilized materials containing dead bacteria. The sterilization method is not particularly limited, but examples include filtration sterilization, radioactive sterilization, heat sterilization, pressurized sterilization, or a combination thereof.

[0072] The specific method of heat sterilization is not particularly limited. For example, it may be a low-temperature pasteurization method or a high-temperature pasteurization method. Low-temperature pasteurization methods include, for example, sterilization by placing the product in a relatively low temperature environment (around 60°C to 70°C, or 63°C to 67°C) for a certain period of time (20 minutes or more, 30 minutes or more, etc.), as well as sterilization methods such as low-temperature steam formaldehyde (LTSF) sterilization, low-temperature plasma sterilization, and ozone hydrogen peroxide mixed gas sterilization. High-temperature pasteurization methods include, for example, sterilization by placing the product in a high-temperature environment, as well as sterilization methods such as high-pressure steam sterilization (autoclave), high-frequency sterilization, and flame sterilization.

[0073] High-temperature sterilization can be performed, for example, by leaving the material at a certain temperature for a certain period of time. The temperature can be, for example, 72°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, or 90°C or higher. The duration is not particularly limited, but it can be, for example, 10 seconds or more, 15 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, or 30 minutes or more. Alternatively, ultra-high temperature flash sterilization, such as treating at a temperature of 120°C or higher for a few seconds (e.g., 1 to 3 seconds), may also be used.

[0074] A portion of the bacterial cells may be included as an extract. Typically, the extract can be obtained by extracting the bacterial cells using a suitable aqueous solvent or organic solvent. The extraction method is not particularly limited as long as it uses an aqueous solvent or organic solvent as the extraction solvent, but known methods include immersing, stirring, or refluxing the bacterial cells in aqueous or organic solvent (water, methanol, ethanol, etc.). Alternatively, the culture medium obtained when culturing the bacterial cells, or a component or fraction derived from the bacterial cells that has a BDNF-enhancing effect recovered therefrom, may be used as the extract. In this case, the method for recovering the target component or fraction is not particularly limited, but for example, it can be done using a known separation and purification method.

[0075] Such separation and purification methods are not particularly limited, but examples include methods utilizing solubility such as salt precipitation and organic solvent precipitation, methods utilizing molecular weight differences such as dialysis, ultrafiltration, and gel filtration, methods utilizing charge differences such as ion exchange chromatography, methods utilizing specific binding such as affinity chromatography, methods utilizing hydrophobicity such as hydrophobic chromatography and reversed-phase chromatography, or combinations thereof.

[0076] The above process may be performed once, multiple times, or in combination with other types of processes.

[0077] The brain function improving agent of this embodiment may consist of one type of bacterial cell and / or processed product, or it may consist of bacterial cells and / or processed products of multiple different bacteria. Furthermore, if it contains a processed product, it may be a processed product that has undergone one type of treatment, a processed product that has undergone multiple types of treatment, or a combination of multiple processed products that have undergone different treatments.

[0078] The brain function improving agent of this embodiment improves brain function based on BDNF (hippocampal BDNF, etc.).

[0079] BDNF-based brain function refers to brain function that changes based on the amount of BDNF, and broadly includes brain function that becomes abnormal due to a decrease in BDNF levels. Examples include brain function that is enhanced by an increase in BDNF levels, brain function that is reduced by an increase in BDNF levels, and brain function that is enhanced or reduced when the amount of BDNF is at a certain level. Examples of such brain functions include memory and learning function, cognitive function, and emotional function. The brain function improving agent in this embodiment can be a memory and learning function improving agent, a cognitive function improving agent, an emotional function improving agent, etc., depending on its purpose.

[0080] Improved brain function refers to the enhancement of desirable brain functions and / or the reduction of undesirable brain functions. For example, when applied to a person with brain dysfunction, it refers to abnormal brain functions becoming normal or near normal.

[0081] The brain function improving agent of this embodiment improves BDNF-based brain function by increasing the amount of BDNF. Therefore, whether or not the target brain function has improved can be determined by measuring that brain function, or by whether or not the amount of BDNF has increased.

[0082] The method for measuring BDNF levels in specific brain regions such as the hippocampus is not particularly limited, as long as it is a method capable of measuring protein levels in specific brain regions. Examples include methods based on PET or SPECT, or methods that use methods commonly used for measuring protein levels in tissue samples containing the target region (e.g., the hippocampus). The methods commonly used for measuring protein levels are not particularly limited, but examples include immunological detection methods, receptor-ligand binding analysis methods, aptamer analysis methods, gel filtration HPLC methods, mass spectrometry, or combinations thereof.

[0083] Here, an increase in BDNF levels refers to a relative increase compared to before or without the application of the brain function improving agent of this embodiment. The degree of increase is not particularly limited, but for example, it should be more than 1x, 1.1x or more, 1.15x or more, 1.17x or more, 1.18x or more, 1.2x or more, 1.25x or more, 1.3x or more, 1.4x or more, 1.5x or more, 1.6x or more, 1.65x or more, etc., compared to the comparison target. Furthermore, for example, it is sufficient if the increase is statistically significant compared to before or without the application of the brain function improving agent of this embodiment. The significance level in this case is not particularly limited, but for example, the significance levels exemplified in the definition section can be used. Furthermore, for example, BDNF levels can be judged as increased if they are equivalent to or greater than when donepezil is administered, or not statistically significantly lower than in that case. Equivalent to or greater than means the same as the above-mentioned content regarding the BDNF level enhancing effect.

[0084] The comparison group may be in a normal state, or in a state where BDNF levels are reduced in the entire brain or in a specific area (such as the hippocampus).

[0085] 2. Composition for Improving Brain Function 2-1. Overview A second aspect of the present invention is a composition for improving brain function. The composition of the present invention contains lactic acid-producing bacteria, particularly specific Bifidobacteria and / or lactic acid bacteria, as active ingredients as essential components. According to the composition of the present invention, it is possible to maintain brain function under conditions that result in improved brain function and / or decreased memory ability based on BDNF.

[0086] 2-2. Composition 2-2-1. Components The components of the composition of the present invention will now be described. In addition to the active ingredient, the composition of the present invention includes a solvent and / or a carrier as optionally selected components. Each component will be described in detail below.

[0087] (1) Active Ingredients The composition of the present invention contains, as an essential active ingredient, lactic acid-producing bacteria, particularly Bifidobacterium longum CECT 7894 strain and / or Lactiprantibacillus plantarum CECT 7481 strain, i.e., the brain function improving agent described in the first embodiment. It may also further contain, as necessary, one or more types of bacteria or agents having a brain function improving effect.

[0088] The composition of the brain function improving agent is described in detail in the first embodiment, so a specific explanation is omitted here. The composition of the present invention may contain one or more types of brain function improving agents.

[0089] The amount of active ingredient contained in the composition of the present invention is not particularly limited. Generally, the amount varies depending on the type of active ingredient, the dosage form, and the type of other components, such as the solvent and carrier, which will be described later. Therefore, it should be determined appropriately taking into account each of these conditions. It is sufficient that a single dose of this composition contains an effective amount of the active ingredient. However, if it is necessary to administer a large amount of this composition to the subject in order to obtain the pharmacological effect of the active ingredient, it may be administered in several divided doses to reduce the burden on the subject. In this case, the amount of active ingredient should be sufficient as long as the total amount contains an effective amount. "Effective amount" means the amount necessary for the active ingredient to exert its function and that does not cause little to no harmful side effects to the subject to which it is applied. This effective amount can vary depending on various conditions such as information about the subject, the route of application, and the number of applications. Therefore, when this composition is used as a medicine, the amount of active ingredient is ultimately determined by the judgment of a physician or pharmacist.

[0090] In this specification, "subject" refers to the target to which the brain function improving agent described in the first embodiment or the composition of this embodiment is applied. For example, this may be cells (including cultured cells), tissues, organs, or individuals. In the case of individuals, the animal species is not particularly limited, but is preferably a human individual. In the present invention, the subject may be healthy or suffering from some disease, and includes, for example, individuals with brain dysfunction (such as memory impairment), individuals at high risk of brain dysfunction (such as memory impairment), individuals with reduced BDNF levels, or individuals whose BDNF levels are expected to decrease.

[0091] Target animal species include mammals and birds, preferably primates such as humans, domesticated animals such as cattle, horses, sheep, goats, pigs, chickens, and turkeys, racing animals such as horses, and companion animals such as dogs and cats.

[0092] In this specification, "information about the subject" refers to various information about the characteristics and condition of the subject. For example, if the subject is a human individual, this may include age, weight, sex, overall health status, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.

[0093] The specific content of Bifidobacteria and / or Lactobacillus cells, which are the active ingredients of the composition in this embodiment, is not particularly limited, but the amount of Bifidobacteria and / or Lactobacillus can usually be in the range of 0.0001 to 99% by mass, 0.001 to 80% by mass, 0.001 to 75% by mass, etc. For example, it can be provided in a form that allows control of the daily dosage so that a desirable intake of the active ingredient can be obtained. Furthermore, the concentration of Bifidobacteria and / or Lactobacillus contained in the composition in this embodiment can be, for example, 10^5 cells / g to 10^12 cells / g, 10^7 cells / g to 10^12 cells / g, etc. When using a treated product, the number of cells is counted by converting it to the number of cells before treatment. There are no particular restrictions on the daily dosage, but for example, it can be 10^7 to 10^12 units / day, 10^7 to 10^11 units / day, 10^8 to 10^12 units / day, 10^8 to 10^11 units / day, 10^9 to 10^12 units / day, 10^9 to 10^11 units / day, 10^10 to 10^12 units / day, 10^10 to 10^11 units / day, etc.

[0094] The ratio of the Bifidobacterium and Lactobacillus of the present invention to each other when both are included is not particularly limited. For example, the amount of Lactobacillus of the present invention relative to Bifidobacterium can be 0.01 times or more, 0.05 times or more, 0.1 times or more, 0.2 times or more, 0.25 times or more, 0.3 times or more, 0.4 times or more, 0.5 times or more, 0.6 times or more, 0.7 times or more, 0.8 times or more, 0.9 times or more, 1 time or more, etc. Also, for example, the amount of Lactobacillus of the present invention relative to Bifidobacterium can be 100 times or less, 50 times or less, 25 times or less, 20 times or less, 10 times or less, 9 times or less, 8 times or less, 7 times or less, 6 times or less, 5 times or less, 4 times or less, 3 times or less, 2 times or less, 1.5 times or less, 1.4 times or less, 1.3 times or less, 1.2 times or less, 1.1 times or less, 1 time or less, etc. Furthermore, for example, the amount of lactic acid bacteria of the present invention relative to the bifidobacteria of the present invention can be 0.01 to 100 times, 0.05 to 50 times, 0.1 to 25 times, 0.1 to 20 times, 0.1 to 10 times, 0.2 to 9 times, 0.2 to 8 times, 0.2 to 7 times, 0.2 to 6 times, 0.2 to 5 times, 0.25 to 4 times, 0.3 to 3 times, 0.4 to 2 times, 0.5 to 1.5 times, 0.6 to 1.4 times, 0.7 to 1.3 times, 0.8 to 1.2 times, 0.9 to 1.1 times, 0.1 to 1 time, 0.5 to 1 time, etc.

[0095] (2) Solvents The compositions of the present invention may optionally contain pharmaceutically acceptable solvents. "pharmaceutically acceptable solvents" means solvents commonly used in the pharmaceutical technology field for the target animal species. Examples include water or aqueous solutions, or organic solvents. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, and sodium acetate buffers. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. An example of an organic solvent is ethanol.

[0096] (3) Carrier The composition of the present invention may optionally contain a pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" means an additive that is commonly used in the pharmaceutical technology field for the target animal species. Examples include excipients, binders, disintegrants, fillers, emulsifiers, flow additive modifiers, lubricants, and human serum albumin.

[0097] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, and Pluronic acid. (R) Examples include kaolin, silicic acid, or combinations thereof.

[0098] Examples of binders include starch paste made from plant starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof.

[0099] Examples of disintegrants include the aforementioned starch, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, or salts thereof.

[0100] Examples of fillers include petrolatum, the aforementioned sugars, and / or calcium phosphate.

[0101] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0102] Examples of fluid additive regulators and lubricants include silicates, talc, stearates, or polyethylene glycol.

[0103] In addition to the above, if necessary, the composition may also contain, as appropriate, solubilizers, suspending agents, diluents, dispersants, surfactants, analgesics, stabilizers, absorption enhancers, bulking agents, humectants, moisturizers, wetting agents, adsorbents, flavoring and deodorizing agents, disintegration inhibitors, coating agents, colorants, preservatives, antioxidants, fragrances, flavoring agents, sweeteners, buffering agents, isotonic agents, etc.

[0104] The carrier is used to avoid or inhibit the degradation of the active ingredient by enzymes, etc., within the target body, as well as to facilitate formulation and administration methods, and to maintain the dosage form and efficacy. It should be used as appropriate as needed.

[0105] If necessary, the material can be coated with a single or multiple layers of enteric-coated material to achieve enteric coating. The enteric-coated material should not dissolve in acidic pH ranges such as gastric juice, but should dissolve in neutral pH ranges, and such materials are widely known in the art. The specific enteric-coated material is not particularly limited, but examples include hypromellose phthalate, shellac, zein, lactoferrin, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl ethylcellulose, and cellulose acetate phthalate.

[0106] Furthermore, the composition of this embodiment may further contain one or more other drugs. The type of drug in this case is not particularly limited, but for example, it may include drugs that have an intestinal regulating effect, drugs that promote absorption from the intestines, etc.

[0107] (4) Drug Delivery System Particles (DDS Particles) The composition of this embodiment may optionally contain DDS particles. DDS particles are particles that contain an active ingredient or other carrier inside themselves, deliver the contents, especially the active ingredient, to the target site without degradation, and can control the drug distribution in the body both temporally and quantitatively. Since the active ingredient of the composition of this embodiment is a peptide or nucleic acid, the use of DDS particles is preferable to protect it from degradation by proteases and nucleases in the body after administration. The type of DDS particle is not limited. Examples include liposomes, polymer micelles, and viral particles.

[0108] 2-2-2. Dosage Form and Application Method The dosage form of the composition of the present invention is not particularly limited. It is acceptable as long as it is a form that can be delivered to the target site in the target body without inactivating the active ingredient.

[0109] The specific dosage form will vary depending on the method of administration, which will be described later. Since the methods of administration can be broadly divided into parenteral administration and oral administration, the dosage form should be appropriate for each method of administration. Oral administration is the preferred method of administration.

[0110] If the method of administration is oral, preferred dosage forms include solid preparations (including tablets, capsules, drops, and lozenges), granules, powders, and liquid preparations (including oral aqueous preparations, emulsions, and syrups). If a solid preparation is used, it may be a dosage form with a coating known in the art, such as a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multi-layer tablet, as needed.

[0111] For example, if the method of administration is parenteral, the dosage form can be local or systemic via the circulatory system. Local administration includes, for example, intramuscular, subcutaneous, tissue, and organ administration, while systemic parenteral administration includes enteral, suppository, and intracardiac administration, such as intravenous, intra-arterial, and intra-lymphatic administration. If it is a liquid preparation, for example, it can be an injectable preparation. Injectable preparations can be formulated by mixing a solvent with the aforementioned excipients, emulsifiers, suspensions, surfactants, stabilizers, pH adjusters, etc., in a unit dose form generally accepted for pharmaceutical production. Furthermore, if it is administered by enteral or suppository administration, it can be formulated in the same way as the dosage form for oral administration.

[0112] The specific shapes and sizes of each of the above dosage forms are not particularly limited, as long as they fall within the range of dosage forms known in the relevant art. The composition of the present invention may be manufactured according to conventional methods in the relevant art.

[0113] The composition according to this embodiment may also be provided as a fermentation product prepared by fermenting raw milk, skim milk powder, or soy milk using bacterial cells. For example, it can be prepared by inoculating Bifidobacteria and / or Lactobacillus into raw milk, skim milk powder, or soy milk, etc., and carrying out fermentation under fermentation conditions known in the art. The fermentation conditions are the same as those described above as culture conditions. The resulting fermentation product may be used as is, or it may be subjected to other treatments such as filtration, sterilization, dilution, or concentration.

[0114] The composition of this embodiment can be used in combination with other drugs. The type of other drugs in this case is not particularly limited. For example, it may be a drug that has a brain function improving effect (for example, a composition containing the brain function improving agent described in the first embodiment, or a composition containing other bacteria, etc.), or it may be a drug that has other effects (a drug that has an intestinal regulating effect, a drug that promotes absorption from the intestines, etc.).

[0115] 2-3. Applicable Diseases The applicable diseases of the composition of this embodiment are not limited to any brain dysfunction based on BDNF. For example, one or more diseases selected from the group consisting of dementia, neurodegenerative diseases and mental illnesses, as exemplified in the section on the definition of the first embodiment.

[0116] The compositions of this embodiment can be provided, for example, as compositions for the prevention or treatment of dementia (such as Alzheimer's disease), compositions for the prevention or treatment of neurodegenerative diseases (such as Parkinson's disease), or compositions for the prevention or treatment of mental illnesses (such as developmental disorders or intellectual disabilities).

[0117] Furthermore, the composition of this embodiment can be used to improve brain function based on BDNF (e.g., hippocampal BDNF) in healthy individuals. Brain function based on BDNF and its improvement are described in accordance with the description in the first embodiment.

[0118] 2-4. Other Embodiments The compositions of this embodiment can be provided as food and beverages (functional food and beverages, etc.), animal feed, pharmaceutical compositions, or compositions for the prevention or treatment of brain dysfunction.

[0119] The present invention also relates to the use of lactic acid-producing bacteria, particularly Bifidobacterium longum CECT 7894 strain and / or Lactiprantibacillus plantarum CECT 7481 strain, in the production of brain function-improving compositions based on BDNF (e.g., hippocampal BDNF).

[0120] The present invention also relates to the use of lactic acid-producing bacteria, particularly Bifidobacterium longum CECT 7894 strain and / or Lactiprantibacillus plantarum CECT 7481 strain, in the manufacture of compositions for the prevention or treatment of brain dysfunction based on BDNF (e.g., hippocampal BDNF).

[0121] Furthermore, the present invention also relates to a BDNF-based brain function improving agent comprising Bifidobacterium longum CECT 7894 strain, a bifidobacterium, for use in combination with Lactiplantibacillus plantarum CECT 7481 strain, a lactic acid bacterium.

[0122] The present invention also relates to a BDNF (e.g., hippocampal BDNF) quantity enhancer comprising lactic acid-producing bacteria, particularly Bifidobacterium longum CECT 7894 strain or Lactiprantibacillus plantarum CECT 7481 strain, and to a BDNF (e.g., hippocampal BDNF) quantity enhancer comprising lactic acid-producing bacteria, particularly Bifidobacterium longum CECT 7894 strain and / or Lactiprantibacillus plantarum CECT 7481 strain.

[0123] The lactic acid-producing bacteria, Bifidobacterium longum CECT 7894 strain, Lactiprantibacillus plantarum CECT 7481 strain, composition components, target subjects, target diseases, etc., in these inventions shall be in accordance with the description in this embodiment.

[0124] The present invention also relates to a method for improving brain function based on BDNF (e.g., hippocampal BDNF), which includes administering lactic acid-producing bacteria, particularly Bifidobacterium longum CECT 7894 strain and / or Lactiprantibacillus plantarum CECT 7481 strain (e.g., the brain function improving agent described in the first embodiment or the brain function improving composition described in this embodiment) to a subject.

[0125] The present invention also relates to a method for preventing or treating BDNF-based brain dysfunction (e.g., hippocampal BDNF) by administering lactic acid-producing bacteria, particularly Bifidobacterium longum CECT 7894 strain and / or Lactiprantibacillus plantarum CECT 7481 strain (e.g., the brain function improving agent described in the first embodiment or the brain function improving composition described in this embodiment) to a target.

[0126] The lactic acid-producing bacteria, Bifidobacterium longum CECT 7894 strain, Lactiprantibacillus plantarum CECT 7481 strain, target population, and administration method in these preventive or therapeutic methods and methods for improving brain function shall be in accordance with the description in this embodiment. When administering both bacteria together, they may be administered simultaneously or separately. When administered simultaneously, they may be administered in the form of a composition containing Bifidobacterium longum CECT 7894 strain and Lactiprantibacillus plantarum CECT 7481 strain, or as separate compositions. When administering the two bacteria separately, the order is not particularly limited. For example, one may be administered first and the other later, or one may be administered prior to and / or after simultaneous administration. The quantity ratio of the two bacteria when administered together is not particularly limited, but for example, it may be the quantity ratio exemplified in the second embodiment.

[0127] <Example 1. Comparison of the efficiency of enhancing BDNF levels in the hippocampus by bacterial cells> (Objective) To compare the effects on intestinal cells depending on the type of bacteria from which the bacterial cells are derived, and to investigate the effect on BDNF levels in the hippocampus.

[0128] (Methods) 1. Preparation of bacterial cells The CECT 7894 strain was obtained from AB-Biotics, SA. First, 100 μL of the Bifidobacterium strain to be used was taken from the stock and inoculated into 5 mL of mMRS medium and cultured statically under anaerobic conditions at 37°C for 24 hours (G1 culture). 800 μL of the culture solution after G1 culture was inoculated into 40 mL of fresh mMRS medium and cultured under the same conditions as G1 culture for 24 hours (G2 culture). mMRS medium was prepared by adding L-cysteine ​​hydrochloride (Kishida Chemical) to MRS medium (Becton Dickinson) at a final concentration of 0.05 (w / v)%, and anaerobic culture was performed using an aneropack. (R) The procedure was performed in a container containing (Mitsubishi Gas Chemical Company).

[0129] The culture medium after G2 incubation was centrifuged at 18,800 × g for 10 minutes to obtain the bacterial cells as precipitate. After removing the supernatant, sterile water was added to wash the bacterial cells, and the bacterial cells were collected again as precipitate by centrifugation.

[0130] The washed bacterial cells were suspended in 3 mL of sterile water and heat-sterilized in a 90°C water bath for 30 minutes. The bacterial suspension after heat sterilization was frozen at -80°C and freeze-dried using a freeze-dryer (Tokyo Rikagaku Kiki Co., Ltd.) to prepare a dead bacterial sample. The freeze-dried dead bacterial sample was filtered through a wire mesh to obtain a finely powdered bacterial sample, which was stored at -80°C.

[0131] For comparison, Bifidobacterium longum strain JCM 1217 was obtained from the RIKEN BioResource Research Center (RIKEN BRC), and for comparison, Streptococcus dentisani strain CECT 7746 was obtained from AB-Biotics, SA. Strain JCM 1217 was prepared using the same method as strain CECT 7894. Strain CECT 7746 was cultured using MRS medium under aerobic conditions in a static culture, except that the preparation method for strain CECT 7894 was followed.

[0132] 2. STC-1 cells (obtained from the American Type Culture Collection), an in vitro test mouse enteroendocrine cell line, were cultured in cell culture medium at 37°C under a 5% CO2 atmosphere. The cell culture medium used was DMEM medium supplemented with 10% FBS (Thermo Fisher SCIENTIFIC) and 1% Antibiotic-Antimycotic (Thermo Fisher SCIENTIFIC). When the cells had grown to approximately 70% confluence, they were harvested by trypsin treatment. The harvested cells were seeded at a cell density of 2 × 10^5 cells / well into a 24-well plate and cultured for 48 hours in cell culture medium of the same composition.

[0133] After removing the culture medium from each well, the cells were washed with PBS, and 500 μL of the reaction solution was added. The cells were treated at 37°C under a 5% CO2 atmosphere for 6 hours to induce GLP-1 production. The reaction solution used was DMEM(-) medium (Thermo Fisher SCIENTIFIC) supplemented with 1 mM glucose, to which powdered bacterial samples were added to achieve final concentrations of 10 μg / mL, 30 μg / mL, and 50 μg / mL. A solution without powdered bacterial samples was used as a negative control. As a positive control, a solution with glutamine added to achieve a final concentration of 4 mM was used instead of powdered bacterial samples.

[0134] The culture supernatant after treatment was collected and frozen at -80°C. Quantitative determination of GLP-1 in the culture supernatant after treatment was performed using the GLP-1 ELISA kit (Fujifilm Wako Pure Chemical Industries, Ltd., 299-75501) according to the manufacturer's specified protocol.

[0135] The experiment was repeated three times. Statistical analysis was performed using Student's t-test.

[0136] 3. In vivo study 3-1. Administration to mice Male C57BL / 6 mice (CIEA Japan) approximately 8 weeks old were acclimatized for several days before being used in the experiment. Powdered bacterial cell sample suspended in physiological saline was administered orally once daily by force-feeding at a dose of 1 × 10^9 cells / day. Control mice were administered physiological saline orally in the same manner.

[0137] 0.15 mg of scopolamine (Tocris Bioscience), dissolved in physiological saline, was administered intraperitoneally to mice one hour after the 12th day of administration to suppress BDNF expression in the brain. Some control mice were not administered scopolamine, and some control mice were administered donepezil (Tokyo Chemical Industries) intraperitoneally 30 minutes before scopolamine administration. Donepezil was used as a positive control because it has been reported to suppress the decrease in brain BDNF expression caused by scopolamine administration.

[0138] The entire brain was removed from mice that were euthanized three hours after administration of scopolamine, and the hippocampus was recovered from both hemispheres. The recovered hippocampal tissue was frozen in liquid nitrogen and stored at -80°C.

[0139] Experiments were conducted with 5 individuals under each condition.

[0140] 3-2. Measurement of Protein and BDNF Content First, 15 mg to 20 mg of hippocampal tissue was lysed under ice-cold conditions, and total protein was extracted using the Minute Total Protein Extraction kit (Invent, SN-002) according to the manufacturer's specified protocol.

[0141] The relative BDNF amount (pg / mg) was calculated by dividing the BDNF concentration by the total protein concentration. The total protein concentration was quantified using the BCA protein assay kit (Thermo Fisher SCIENTIFIC) according to the manufacturer's specified protocol. The BDNF concentration was quantified using the Mature BDNF ELISA kit (Fujifilm Wako Pure Chemical Industries) according to the manufacturer's specified protocol.

[0142] Statistical analysis was performed using Student's t-test.

[0143] (Results) The results are shown in Table 1 and Figure 1. As shown in Table 1, the in vitro tests revealed that the amount of GLP-1 produced by enteroendocrine cells increased when using the three bacterial strains tested compared to the negative control without bacterial cell addition. In particular, when using CECT 7746 and CECT 7894 strains, the increase was found to be 1.3 to 1.4 times that of the negative control. This increase was induced by the addition of trace amounts of 10 μg / mL and 30 μg / mL.

[0144]

[0145] This confirmed that ingesting any of the bacterial strains produced an effect on intestinal cells. Therefore, it was confirmed that all of the bacterial strains produced a certain effect in the body upon ingestion. In particular, the effect was found to be higher when using CECT strain 7746 and CECT strain 7894.

[0146] Furthermore, in vivo studies were conducted to clarify the effects of these bacterial strains on the brain. The results are shown in Figure 1.

[0147] Compared to the control group, scopolamine administration significantly reduced BDNF levels in the hippocampus, and administration of donepezil further restored levels to near their original levels. When CECT strain 7746 was administered instead of donepezil, there was no significant increase in the reduced BDNF levels. On the other hand, when CECT strain 7894 was administered, BDNF levels recovered significantly, and unexpectedly, increased more than when donepezil was administered.

[0148] This indicates that the CECT 7894 strain according to the present invention is particularly useful as a factor that enhances BDNF levels in the hippocampus and other areas.

[0149] <Example 2. Comparison of the efficiency of enhancing BDNF levels in the hippocampus by bacterial cells> (Objective) To compare the effects on intestinal cells depending on the type of bacteria from which the bacterial cells are derived, and to investigate the effect on BDNF levels in the hippocampus. This example corresponds to Example 1 of Japanese Patent Application No. 2025-010440.

[0150] (Methods) 1. Preparation of bacterial cells CECT 7481 strain was obtained from AB-Biotics, SA. First, 100 μL of the lactic acid bacteria strain to be used was taken from the stock and inoculated into 5 mL of MRS medium (Becton Dickinson) and cultured statically at 37°C for 24 hours under aerobic conditions (G1 culture). 800 μL of the culture solution after G1 culture was inoculated into 40 mL of fresh MRS medium and cultured for 24 hours under the same conditions as G1 culture (G2 culture). The acquisition of bacterial cells, preparation and storage of dead bacterial samples, in vitro tests and in vivo tests were performed in the same manner as in Example 1.

[0151] For comparison, Lactiplantibacillus plantarum NBRC 15891 strain was obtained from the National Institute of Technology and Evaluation (NITE), and Streptococcus dentisani CECT 7746 strain was obtained from AB-Biotics, SA. The culture of NBRC 15891 strain and CECT 7746 strain was carried out following the same method as the preparation of the bacterial cells for CECT 7481 strain.

[0152] (Results) The results are shown in Table 2 and Figures 2 and 3. As shown in Table 2, the in vitro tests revealed that the amount of GLP-1 produced by enteroendocrine cells increased when using the three bacterial strains tested compared to the negative control without bacterial cell addition. In particular, when using CECT 7746 and CECT 7481 strains, the increase was found to be approximately 1.1 to 1.3 times that of the negative control. This increase was induced by the addition of a small amount of 10 μg / mL.

[0153]

[0154] This confirmed that ingesting any of the bacterial strains produced an effect on intestinal cells. Therefore, it was confirmed that all of the bacterial strains produced a certain effect in the body upon ingestion. In particular, the effect was found to be higher when using CECT strain 7746 and CECT strain 7481.

[0155] Furthermore, in vivo studies were conducted to clarify the effects of these bacterial strains on the brain. The results are shown in Figures 2 and 3.

[0156] Compared to the control group, scopolamine administration significantly reduced BDNF levels in the hippocampus, and administration of donepezil further restored them to near their original levels. When the CECT 7481 strain was administered instead of donepezil, the reduced BDNF levels increased by 14 points, unexpectedly restoring them to a level comparable to that achieved with donepezil (Figure 2). On the other hand, when the CECT 7746 strain was administered (Figure 3), there was no significant increase in BDNF levels reduced by scopolamine administration, only a 1-point increase.

[0157] This indicates that the CECT 7481 strain according to the present invention is particularly useful as a factor that enhances BDNF levels in the hippocampus and other areas.

[0158] <Example 3. Evaluation of Brain BDNF Gene Expression Induction Activity Using Genetically Modified Mice> (Objective) Using genetically modified mice (BDNF-Luc mice) in which changes in BDNF gene expression in the brain can be measured by luminescence intensity, the brain BDNF gene expression induction activity by administration of lactic acid bacteria and bifidobacteria was evaluated.

[0159] (Methods) Approximately 8-week-old male BDNF-Luc mice (C57BL / 6 strain) capable of expressing the same amount of luciferase as BDNF under the control of the BDNF promoter were orally administered a powdered bacterial cell sample suspended in physiological saline at a dose of 1 × 10^9 cells / day for 16 days via force-feeding once daily. Control mice were similarly orally administered physiological saline without the powdered bacterial cell sample. CECT strains 7746, 7894, and 7481 were used individually as the powdered bacterial cell samples.

[0160] Six hours after bacterial administration on day 14, BDNF-Luc mice were anesthetized by 2% isoflurane inhalation anesthesia. Under anesthesia, the luciferase substrate TokeOni (Kurogane Kasei Co., Ltd.) was administered intraperitoneally at a dose equivalent to 100 mg / kg body weight. Luminescence measurements were performed in brain regions, including the hippocampus, using IVIS LuminaII (PerkinElmer), and the luminescence intensity at a wavelength of 680 nm was obtained as the expression level of the BDNF gene. The procedure for luminescence measurement was roughly as follows: Bioluminescence imaging (BLI) was performed 4 or 6 minutes after substrate administration with an exposure time of 2 minutes. A pseudo-color bioluminescence image showing the spatial distribution of emitted photons was superimposed on an image of the mouse head taken in the chamber. A specific region containing the mouse brain region was selected from the image data, and the average radiance of that region was calculated as the luminescence intensity.

[0161] On days 15 and 16, 0.03 mg of scopolamine (Fujifilm Wako Pure Chemical Industries) dissolved in physiological saline was administered intraperitoneally to mice 30 minutes after each administration of lactic acid bacteria. Six hours after the scopolamine administration on day 16, the luciferase substrate was administered intraperitoneally using the same method as before, and luminescence measurements were performed.

[0162] For each individual, the luminescence intensity obtained from the luminescence measurement on day 16 was calculated as the expression level of the BDNF gene after scopolamine administration. Based on a comparison with the luminescence intensity obtained on day 14, the percentage change was calculated using the following formula: (Percentage change (%)) = 100 × {(Luminescence intensity on day 16) - (Luminescence intensity on day 14)} / (Luminescence intensity on day 14)

[0163] Statistical analysis of the results for each group administered with powdered bacterial cells was performed using Welch's t-test against the results for the control group. Experiments were conducted with 3 to 5 individuals for each condition.

[0164] (Results) The results are shown in Figures 4 and 5. As shown in Figures 4 and 5, in control mice, administration of scopolamine twice on days 15 and 16 reduced the amount of luminescence in the brain measurement site, i.e., the expression level of the brain BDNF gene, confirming that the decrease in brain BDNF levels due to scopolamine administration, as seen in Examples 1 and 2, can be detected in this experimental system (in Figures 4A and 5, labeled "Saline"). In mice administered with the CECT 7746 strain, the expression level of the brain BDNF gene, which had decreased due to scopolamine administration, recovered somewhat, but did not reach the activity level before scopolamine administration (Figure 4B), and the recovery was not significant (in Figure 5, labeled "CECT 7746").

[0165] On the other hand, in mice administered with CECT 7894 or CECT 7481 strains, the expression level of the brain BDNF gene, which had been reduced by scopolamine, recovered to levels higher than those before scopolamine administration. Unexpectedly, brain BDNF gene expression generally increased compared to before scopolamine administration (Figures 4C and 4D), and this recovery showed a significant difference or trend (in Figure 5, "CECT 7894" and "CECT 7481").

[0166] This indicates that the CECT 7894 and CECT 7481 strains according to the present invention are particularly useful for enhancing BDNF levels in brain regions including the hippocampus, and that this enhancement is possible even under conditions where BDNF levels are reduced.

[0167] <Example 4. Evaluation of the effect of bacterial administration on improving brain function> (Objective) To evaluate the effect of enhancing BDNF in the brain by administering lactic acid bacteria and bifidobacteria on brain function.

[0168] (Methods) Male C57BL / 6N mice approximately 8 weeks old were orally administered a powdered bacterial cell sample suspended in physiological saline at a dose of 1 × 10^9 cells / day for 16 days via force-feeding once daily. Control mice were similarly administered physiological saline orally. Contextual fear conditioning tests were performed on mice that had been administered either the powdered bacterial cell sample or physiological saline for 16 days, according to the following procedure.

[0169] On day 16, 30 minutes after administration, 0.06 mg of scopolamine dissolved in physiological saline was administered intraperitoneally to mice. 30 minutes after scopolamine administration, each mouse was placed in a chamber equipped with an electric grid and observed for 180 seconds. Fear conditioning was performed by presenting a 0.4 mA electric shock for 2 seconds (148-150 seconds), while freezing behavior was measured. The frequency of freezing behavior was measured as the percentage of freezing time to the total measurement time.

[0170] Twenty-four hours after fear conditioning, the mice were placed back into the same chamber, one by one, and observed for 180 seconds to measure their freezing behavior.

[0171] Furthermore, the control group, which received oral administration of physiological saline for 16 days, was divided into two groups. In one of these groups, a contextual fear conditioning test was performed, with scopolamine-free physiological saline being administered instead of scopolamine. Experiments were conducted with 8 to 10 individuals for each condition.

[0172] (Results) The results are shown in Figure 6. As shown in Figure 6, control individuals who underwent fear conditioning without being administered scopolamine retained the fear memory caused by the electric shock and exhibited freezing behavior when placed in the same chamber 24 hours later, at a high frequency of 50-60% (in Figure 6, "Saline-Saline"). On the other hand, in control individuals who underwent fear conditioning after being administered scopolamine, the frequency of freezing behavior 24 hours later was significantly reduced to about 10%, and the retention of fear memory was significantly suppressed (in Figure 6, "Saline-Scopolamine").

[0173] On the other hand, in mice administered with CECT 7894 or CECT 7481 strains, the frequency of freezing behavior, which had decreased with scopolamine administration, significantly increased, and the retention of fear memories, which had been suppressed by scopolamine administration, was restored (Figure 7, "CECT 7894-Scopolamine" and "CECT 7481-Scopolamine").

[0174] This demonstrates that the CECT 7894 and CECT 7481 strains according to the present invention actually improve brain function under conditions that cause a decline in memory ability by enhancing BDNF levels in brain regions including the hippocampus.

[0175] <Example 5. Evaluation of the effect of co-administration of bacteria on improving brain function> (Objective) To evaluate the effect of co-administration of lactic acid bacteria and bifidobacteria of the present invention on brain function.

[0176] (Method) As a powdered bacterial sample, a mixture of CECT strain 7894 and CECT strain 7481 was used in a ratio of 1:1, and the administration was carried out in the same manner as in Example 4, except that the dose was 2 × 10^9 cells / day (1 × 10^9 cells / day for each strain). Statistical analysis was performed by Tukey's multiple comparison test.

[0177] (Results) The results are shown in Figure 7. As shown in Figure 7, fear conditioning of control individuals, similar to Example 4, showed a significant decrease in the frequency of freezing, which was about 50% in the group not administered scopolamine, to about 10% with scopolamine administration (in Figure 7, "Saline-Saline" and "Saline-Scopolamine").

[0178] On the other hand, in mice administered with both CECT strain 7894 and CECT strain 7481, the frequency of freezing behavior, which had decreased with scopolamine administration, recovered to the same level as the group not administered scopolamine (approximately 40%), and there was no significant difference between the two groups (Figure 7, "CECT×2-Scopolamine"). This recovery far exceeded the additive effect of recovery achieved by administering each bacterium individually.

[0179] This demonstrates that by using the CECT 7894 and CECT 7481 strains according to the present invention in combination, brain function can be improved by enhancing BDNF levels in brain regions including the hippocampus, and robust brain function can be maintained even under conditions that cause a decline in memory ability. All publications, patents and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A brain function improving agent based on BDNF, derived from lactic acid-producing bacteria.

2. The brain function improving agent according to claim 1, wherein the lactic acid-producing bacteria is Bifidobacterium longum CECT 7894 strain, which is a type of Bifidobacterium.

3. The brain function improving agent according to claim 1, wherein the lactic acid-producing bacterium is Lactiprantibacillus plantarum CECT 7481 strain, which is a lactic acid bacterium.

4. The brain function improving agent according to claim 1, wherein the lactic acid-producing bacteria include dead bacteria.

5. The brain function improving agent according to any one of claims 1 to 4, wherein the BDNF is hippocampal BDNF.

6. A BDNF-based composition for improving brain function, containing lactic acid-producing bacteria.

7. The brain function improving composition according to claim 6, wherein the lactic acid-producing bacteria include Bifidobacterium longum CECT 7894 strain, which is a Bifidobacterium, and / or Lactiprantibacillus plantarum CECT 7481 strain, which is a Lactobacillus.

8. The brain function improving composition according to claim 6, wherein the lactic acid-producing bacteria include dead bacteria.

9. The brain function improving composition according to claim 6, wherein the BDNF is hippocampal BDNF.

10. A composition for improving brain function according to any one of claims 6 to 9, which is a food or beverage.

11. A pharmaceutical composition, the brain function improving composition according to any one of claims 6 to 9.

12. A brain function improving composition according to any one of claims 6 to 9, which is a composition for the prevention or treatment of brain dysfunction.

13. The brain function improving composition according to claim 12, wherein the brain dysfunction is one or more diseases selected from the group consisting of dementia, neurodegenerative diseases, and mental disorders.

14. A method for improving brain function based on BDNF, including administering lactic acid-producing bacteria to the subject.

15. The method for improving brain function according to claim 14, wherein the lactic acid-producing bacteria is Bifidobacterium longum CECT 7894 strain, which is a Bifidobacterium, and / or Lactiprantibacillus plantarum CECT 7481 strain, which is a Lactobacillus.

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