Composition for promoting proliferation, differentiation and maturation of neural stem cells, promoting microglial activation and promoting astrocyte activation, and composition for maintaining and / or improving memory and / or learning ability

Abstract

[Problem] To provide a composition for promoting the proliferation, differentiation and maturation of neural stem cells, promoting microglial activation, and promoting astrocyte activation. [Solution] The present inventors found that a composition containing a lactic acid bacterium (in particular, Enterococcus faecium), a butyric acid bacterium (in particular, Clostridium butyricum) and a saccharifying bacterium (in particular, Bacillus subtilis) has effects of increasing the number of resting neural stem cells, promoting the differentiation of neural stem cells into neural cells and maturation thereof, promoting the proliferation of neural stem cells, promoting the maturation of immature neural cells, promoting microglial activation, promoting astrocyte activation, promoting adult neurogenesis, maintaining brain function homeostasis, and maintaining and / or improving memory and / or learning ability, to thereby complete the present disclosure.

Description

Composition for promoting proliferation, differentiation and maturation of neural stem cells, promoting activation of microglia and promoting activation of astrocytes, and composition for maintaining and / or improving memory and / or learning ability

[0001] The present disclosure relates to compositions for increasing the number of resting neural stem cells, compositions for promoting the differentiation and maturation of neural stem cells into neurons, compositions for promoting the proliferation of neural stem cells, compositions for promoting the maturation of immature neurons, compositions for promoting the activation of microglia, compositions for promoting the activation of astrocytes, and compositions for maintaining and / or improving memory and / or learning ability. This application claims priority to Japanese Patent Application No. 2023-74562, which is incorporated herein by reference.

[0002] Brain function is established by nerve cells (neurons) and glial cells (composed of microglia, astrocytes, and oligodendrocytes). Neurons are responsible for forming memory circuits, and most of them are generated during the fetal period. Astrocytes structurally and functionally maintain the brain's neural circuits and the blood-brain barrier, while microglia maintain central nervous system homeostasis as immune cells in the brain. Neural stem cells capable of differentiating into neurons and glial cells are present in the adult brain (particularly in the subventricular zone and hippocampal dentategyrus (DG)). The phenomenon of neurons being generated from adult neural stem cells, known as adult neurogenesis, has been reported (see Non-Patent Documents 1-3). Although the gut microbiota has been reported to contribute to adult neurogenesis in the hippocampus, many details remain unclear. Adult neurogenesis is known to involve several stages from neural stem cells to mature neurons, but it has not been clear at what stage of neurogenesis gut bacteria act, or to what extent they contribute to neuronal cell formation. Furthermore, the mechanism by which gut bacteria induce neurogenesis has not been elucidated at all (see Non-Patent Documents 4-7).

[0003] Patent Document 1 discloses a "composition for maintaining and / or improving memory and learning ability, containing cells of heterofermentative lactobacillus bacteria of the genus Lactobacillus." However, the composition disclosed herein has different components from the composition described in Patent Document 1.

[0004] Patent 6739603

[0005] Cell Stem Cell. 2019 May2;24(5):690-705.Aging (Albany NY). 2021 Jul 29;13(14):18689-18700.Nature. 2018 July; 559(7712):98-102.Curr Opin Neurobiol. 2015 Feb;30:51-8.Nat Med. 2019 Apr;25(4):554-560.Front Neurosci. 2022 Oct 28;16:1030694.Front Cell Dev Biol. 2020 May 29;8:407.PLoS One. 2010 Jan 29;5(1):e8809.Front Cell Neurosci. 2017 Aug 8;11:235.Trends Mol Med. 2019 Nov;25(11):967-979.Front Neurosci. 2022 Feb 16;16:824888.

[0006] The objective of this study is to clarify how a composition containing lactic acid bacteria, butyric acid bacteria, and / or saccharifying bacteria, which improves the intestinal flora and the barrier function of the large intestine, affects brain function, and to provide a composition that generates and promotes adult neurogenesis and is involved in maintaining homeostasis of brain function and maintaining and improving memory and learning abilities.

[0007] The present inventors conducted extensive research to solve the above-mentioned problems. They discovered that a composition containing lactic acid bacteria (particularly, Enterococcus faecium), butyric acid bacteria (particularly, Clostridium butyricum), and / or saccharifying bacteria (particularly, Bacillus subtilis) increases the number of resting neural stem cells, promotes neural stem cell proliferation, promotes the differentiation and maturation of neural stem cells into neurons, promotes the maturation of immature neurons, promotes microglial activation, promotes astrocyte activation, promotes adult neurogenesis, and maintains brain function homeostasis. Furthermore, they discovered that butyric acid bacteria maintain and / or improve memory and / or learning ability, and thus completed the present disclosure.

[0008] The present disclosure is as follows: 1. A composition comprising one or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria, and having any one or more of the following uses: (1) for increasing the number of resting neural stem cells (2) for promoting the proliferation of neural stem cells (3) for promoting the differentiation and maturation of neural stem cells into neurons (4) for promoting the maturation of immature neurons (5) for promoting adult neurogenesis (6) for maintaining and / or improving memory and / or learning ability (7) for promoting the activation of microglia (8) for promoting the activation of astrocytes (9) for maintaining brain functional homeostasis 2. The composition according to the preceding paragraph 1, comprising two or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. 3. The composition according to the preceding paragraph 1, comprising lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. 4. 4. The composition according to any one of preceding items 1 to 3, wherein the lactic acid bacteria are Enterococcus bacteria, the butyric acid bacteria are Clostridium bacteria, and the saccharifying bacteria are Bacillus bacteria. 5. The composition according to any one of preceding items 1 to 3, wherein the lactic acid bacteria are Enterococcus faecium or Enterococcus lactis species, the butyric acid bacteria are Clostridium butyricum species, and the saccharifying bacteria are Bacillus subtilis species. 6. The composition according to any one of preceding items 1 to 3, wherein the lactic acid bacteria are Enterococcus faecium T-110, the butyric acid bacteria are Clostridium butyricum TO-A, and the saccharifying bacteria are Bacillus subtilis TO-A. 7. 7. A composition for increasing the number of resting neural stem cells, comprising Enterococcus faecium or Enterococcus lactis, Clostridium butyricum, and Bacillus subtilis. 8. A composition for promoting the differentiation and maturation of neural stem cells into neurons, comprising Enterococcus faecium or Enterococcus lactis, Clostridium butyricum, and Bacillus subtilis.9. A composition for promoting the proliferation of neural stem cells, comprising Enterococcus faecium or Enterococcus lactis, Clostridium butyricum, and Bacillus subtilis. 10. A composition for promoting the maturation of immature nerve cells, comprising Enterococcus faecium or Enterococcus lactis, Clostridium butyricum, and Bacillus subtilis. 11. A composition for promoting the activation of microglia, comprising Enterococcus faecium or Enterococcus lactis, Clostridium butyricum, and Bacillus subtilis. 12. A composition for promoting the activation of astrocytes, comprising Enterococcus faecium or Enterococcus lactis, Clostridium butyricum, and Bacillus subtilis. 13. 1. A composition for promoting adult neurogenesis, comprising Enterococcus faecium or Enterococcus lactis, Clostridium butyricum, and Bacillus subtilis. 14. A composition for maintaining brain function homeostasis, comprising Enterococcus faecium or Enterococcus lactis, Clostridium butyricum, and Bacillus subtilis. 15. A composition for maintaining and / or improving memory and / or learning ability, comprising butyric acid bacteria. 16. The composition according to item 15, wherein the butyric acid bacteria are Clostridium bacteria. 17. The composition according to item 15, wherein the butyric acid bacteria are Clostridium butyricum TO-A. 18. A method for any one or more of the following purposes, comprising administering to a subject a composition comprising one or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria:(1) For increasing the number of resting neural stem cells (2) For promoting the proliferation of neural stem cells (3) For promoting the differentiation and maturation of neural stem cells into neurons (4) For promoting the maturation of immature neurons (5) For promoting adult neurogenesis (6) For maintaining and / or improving memory and / or learning ability (7) For promoting the activation of microglia (8) For promoting the activation of astrocytes (9) For maintaining brain function homeostasis 19. A method for maintaining and / or improving memory and / or learning ability, comprising administering a composition containing butyric acid bacteria to a subject. 20. Use of one or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria in the production of any one or more of the following compositions: (1) For increasing the number of resting neural stem cells (2) For promoting the proliferation of neural stem cells (3) For promoting the differentiation and maturation of neural stem cells into neurons (4) For promoting the maturation of immature neurons (5) For promoting adult neurogenesis (6) For maintaining and / or improving memory and / or learning ability (7) For promoting the activation of microglia (8) For promoting the activation of astrocytes (9) For maintaining homeostasis of brain function 21. Use of butyric acid bacteria in the manufacture of a composition for maintaining and / or improving memory and / or learning ability. 22. A food product for the following uses, comprising the composition according to any one of the preceding paragraphs 1 to 17. (1) For increasing the number of resting neural stem cells (2) For promoting the proliferation of neural stem cells (3) For promoting the differentiation and maturation of neural stem cells into neurons (4) For promoting the maturation of immature neurons (5) For promoting adult neurogenesis (6) For maintaining and / or improving memory and / or learning ability (7) For promoting the activation of microglia (8) For promoting the activation of astrocytes (9) For maintaining brain function homeostasis 23. The composition according to the preceding item 3, wherein the butyric acid bacteria are present in an amount of 100 to 2500 parts by mass and the saccharifying bacteria are present in an amount of 100 to 2500 parts by mass per 100 parts by mass of the lactic acid bacteria.

[0009] A composition comprising at least the lactic acid bacteria (particularly, Enterococcus faecium), butyric acid bacteria (particularly, Clostridium butyricum), and saccharifying bacteria (particularly, Bacillus subtilis) of the present disclosure has one or more of the following effects: (1) increasing the number of resting neural stem cells (2) promoting the differentiation and maturation of neural stem cells into neurons (3) promoting the proliferation of neural stem cells (4) promoting the maturation of immature neurons (5) promoting adult neurogenesis (6) promoting the activation of microglia (7) promoting the activation of astrocytes (8) maintaining brain function homeostasis In addition, a composition comprising at least the butyric acid bacteria (particularly, Clostridium butyricum) of the present disclosure has the effect of maintaining and / or improving memory and / or learning ability.

[0010] A: Immunostaining of BrdU-positive cells (black) in the dentate gyrus of the hippocampus on day 1 after BrdU administration in each group of mice. Scale bar = 100 μm. B: The number of BrdU-positive cells in the granule cell layer of the dentate gyrus of the hippocampus per mouse in each group was counted and graphed. The number of BrdU-positive cells in the Bio3-administered mice group (Bio3) was statistically significantly higher than that in the SPF mice (SPF) and germ-free mice (GF) groups. *: p<0.05, **: p<0.01 (n=4 (SPF, Bio3), n=3 (GF)). A: Immunostaining of BrdU-positive cells (white) and expression of NeuN protein (black), which is specifically expressed in mature neurons, in the dentate gyrus of the hippocampus on day 21 after BrdU administration in each group of mice in each group of mice. Scale bar = 100 μm. B: The number of BrdU-positive cells in the granule cell layer of the hippocampal dentate gyrus was counted and graphed per mouse in each group. There was no statistically significant difference in the number of BrdU-positive cells between groups (n = 4). C: The number of BrdU- and NeuN-copositive cells in the granule cell layer of the hippocampal dentate gyrus was counted and graphed per mouse in each group. The group of mice administered Bio3 (Bio3) had a higher number of BrdU- and NeuN-copositive cells compared to the other groups, and this was statistically significantly higher compared to the GF group. *: p<0.05 (n = 4). D: The percentage of NeuN-expressing cells in BrdU-positive cells in each group is shown. The group of mice administered Bio3 (Bio3) had a statistically significantly higher percentage of NeuN-expressing cells compared to the GF group. **: p<0.01 (n=4). A: Immunostaining revealed the expression of calretinin protein (black), which is specifically expressed in mature neurons, in the hippocampal dentate gyrus region of mice from each group 21 days after BrdU administration. Scale bar = 100 μm. B: The number of calretinin-positive mature neurons in the granule cell layer of the hippocampal dentate gyrus per mouse from each group was counted and graphed. The Bio3-administered group had a significantly higher number of calretinin-positive mature neurons than the other groups.*: p<0.05, **: p<0.01 (n=4). A: Immunostaining revealed the expression of NeuroD1 protein (NeuroD1, black and white arrowheads), a transcription factor that promotes neuronal differentiation and maturation, in the hippocampal dentate gyrus of each group of mice. Scale bar = 50 μm. B and C: The number of NeuroD1-positive cells in the granule cell layer of the hippocampal dentate gyrus per mouse in each group was counted and graphed. (B) Brain tissue was collected from mice 1 day after BrdU administration (4 weeks after Bio3 or saline administration) and (C) 21 days after BrdU administration (7 weeks after Bio3 or saline administration). The number of NeuroD1-positive cells in the dentate gyrus was highest in the Bio3-treated group at both time points. *: p<0.05, **: p<0.01 (n=4). A: Resting neural stem cells (black arrowheads with white borders) co-expressing GFAP, nestin, and SOX2 proteins in the hippocampal dentate gyrus of each group of mice were shown. Scale bar = 100 μm. B: The number of resting neural stem cells in the granule cell layer of the hippocampal dentate gyrus was measured and graphed per mouse in each group. The number of resting neural stem cells in the GF group was significantly reduced compared to the SPF group. However, the number in the Bio3 group was similar to that in the SPF group. **: p<0.01 (n=4 (SPF), n=5 (GF, Bio3)). A: The upper panel shows microglia expressing IBA1 protein (white) in the cerebral cortex of each group of mice. The lower panel shows binarized fluorescence microscopic images of IBA1-positive microglia, which were used to measure the cell area in B. Scale bar = 20 μm. B: The area of ​​IBA1-positive microglia in the cerebral cortex of mice in each group was measured using ImageJ from the black and white images in A. The values ​​on the vertical axis are arbitrary units in ImageJ. The Bio3 group showed a significantly reduced area of ​​microglia compared to the SPF group.*: p<0.05 (n=4 (SPF, Bio3), n=3 (GF)). A: The upper panel shows astrocytes expressing GFAP protein (white) in the cerebral cortex of mice from each group. The lower panel shows fluorescent microscopic images of GFAP-positive astrocytes, binarized into black and white images, which were used to measure the cell area in B. Scale bar = 50 μm. B: From the black and white images in A, the average area of ​​GFAP-positive astrocytes per individual in the cerebral cortex of mice from each group was measured using ImageJ. The values ​​on the vertical axis are arbitrary units in ImageJ. The Bio3 group had the largest astrocyte area compared to the other groups, and was statistically significantly larger than the GF group in particular. *: p<0.05 (n=4). A: Study overview. B: Proliferation evaluation. A: Study overview. B: Neuronal differentiation evaluation. A-1: ​​Study overview - Evaluation agar medium. A-2: Test overview - Flow from starvation treatment of nematodes to behavioral observation. B: Results - Salt concentration chemotaxis index of nematodes.

[0011] The present disclosure relates to compositions for increasing the number of resting neural stem cells, compositions for promoting the differentiation and maturation of neural stem cells into neurons, compositions for promoting the proliferation of neural stem cells, compositions for promoting the maturation of immature neurons, compositions for promoting the activation of microglia, compositions for promoting the activation of astrocytes, compositions for promoting adult neurogenesis, compositions for maintaining brain function homeostasis, and compositions for maintaining and / or improving memory and / or learning ability. The present disclosure is described in detail below. The compositions of the present disclosure include, but are not limited to, pharmaceutical compositions, food compositions, feed compositions, etc.

[0012] (Composition of the Present Disclosure) The composition of the present disclosure is a composition containing one or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. Lactic Acid Bacteria: The lactic acid bacteria are preferably of the genus Enterococcus, more preferably the species Enterococcus faecium or Enterococcus lactis, and even more preferably Enterococcus faecium T-110. Butyric Acid Bacteria: The butyric acid bacteria are preferably of the genus Clostridium, more preferably the species Clostridium butyricum, and even more preferably Clostridium butyricum TO-A. Saccharifying Bacteria: The saccharifying bacteria are preferably of the genus Bacillus, more preferably the species Bacillus subtilis, and even more preferably Bacillus subtilis TO-A. The above bacteria may be live or killed, but are preferably live. In addition, the composition of the present disclosure may be in the form of a powder, granules, orally disintegrating tablets, plain tablets, coated tablets, capsules, or liquid. The composition of the present disclosure is available from Toa Pharmaceutical Co., Ltd. (see http: / / www.toabio.co.jp / medicalitem). Enterococcus faecium T-110, Clostridium butyricum TO-A, and Bacillus subtilis TO-A are available from the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (NIET) as strains with unique accession numbers FERM BP-10867, FERM BP-10866, and FERM BP-07462, respectively. The paper "Int J Syst Evol Microbiol. 2021;71(8). DOI: 10.1099 / ijsem.0.004948" reports that a specific group of Enterococcus faecium strains (Clade B in the paper) belonged to the same cluster as Enterococcus lactis strains as a result of phylogenetic tree analysis based on core genome sequence comparison.The document deals with Enterococcus faecium T-110, which belongs to Clade B. The above-mentioned phylogenetic tree shows that T-110 diverges significantly from other Enterococcus faecium strains (Clades A1 and A2 in the document), and that it is in fact in the same cluster as Enterococcus lactis strains with a very high similarity to them (see Table 1 and Fig. 2 in the document). The contents of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria in the composition of the present disclosure are not particularly limited, but examples include the following: 100 to 2500 parts by mass, 100 to 1500 parts by mass, 100 to 1000 parts by mass, 100 to 500 parts by mass, 100 to 300 parts by mass, or 100 to 200 parts by mass of butyric acid bacteria per 100 parts by mass of lactic acid bacteria. The amount of saccharifying bacteria per 100 parts by mass of lactic acid bacteria is 100 to 2500 parts by mass, 100 to 1500 parts by mass, 100 to 1000 parts by mass, 100 to 500 parts by mass, 100 to 300 parts by mass, or 100 to 200 parts by mass. The content of butyric acid bacteria and the content of saccharifying bacteria can be selected and combined from the above ranges. For example, the amount of butyric acid bacteria can be 100 to 2500 parts by mass, and the amount of saccharifying bacteria can be 100 to 300 parts by mass.

[0013] (Subjects for Administration of the Composition of the Present Disclosure) The subjects for administration of the composition of the present disclosure are not particularly limited, but are organisms having nerve cells, preferably organisms having a central nervous system, more preferably vertebrates having a central nervous system, and even more preferably mammals including humans. In the case of humans, this includes both healthy individuals and patients. In particular, organisms in which adult neurogenesis can occur, i.e., adult humans, are targeted. However, given that compositions containing the present disclosure have been safely used as oral pharmaceuticals for approximately 60 years by people from infants to the elderly, it is possible to target humans of all ages.

[0014] (Method of Administration of the Composition of the Present Disclosure) The dosage, frequency of administration, and administration interval of the administration method of the present disclosure are not particularly limited and are appropriately selected depending on factors such as the purpose of prevention (particularly prevention of recurrence) and / or clinical treatment, the type of disease, the patient's weight, age, and severity of the disease. For example, the following dosage forms can be used, but are not particularly limited. Powder (daily dose): Lactic acid bacteria: 15-30 mg Butyric acid bacteria: 75-150 mg Saccharifying bacteria: 75-150 mg Orally disintegrating tablet or plain tablet (daily dose): Lactic acid bacteria: 6-12 mg Butyric acid bacteria: 30-60 mg Saccharifying bacteria: 30-60 mg These bacteria are preferably mixed and administered in the form of powder, granules, orally disintegrating tablets, plain tablets, coated tablets, capsules, or liquid, once or twice a day or in divided doses multiple times a day (morning, noon, evening). In addition, daily administration is preferred, but administration once every few days or several times every other week is also possible. In addition, the daily intake amount of the bacteria contained in the composition of the present disclosure is, for example, 10 ３ ~10 ２０ pcs, preferably 10 ５ ~10 １５ pcs, more preferably 10 ６ ~10 １３ There are individuals.

[0015] The composition (or use of the composition) of the present disclosure can be used as the following agents: An agent for promoting adult neurogenesis based on the effects of the composition on increasing the number of resting neural stem cells, promoting the differentiation and maturation of neural stem cells into neurons, promoting the proliferation of neural stem cells, and / or promoting the maturation of immature neurons; An agent for maintaining brain function homeostasis based on the effect of the composition on promoting the activation of microglia and / or astrocytes; An agent for maintaining and / or improving memory and / or learning ability based on the effect of the composition on maintaining and / or improving memory and / or learning ability. Furthermore, the composition of the present disclosure can be formulated into powders, tablets, granules, capsules, liquids, etc. by known methods by blending with additives such as carriers (e.g., starch, lactose, soy protein), excipients, binders, disintegrants, lubricants, stabilizers, suspending agents, etc.

[0016] The present disclosure will be described in detail below using specific examples, but the present disclosure is not limited to these examples. The following examples were carried out in accordance with the experimental animal handling guidelines of the University of Tokyo (approval number: P20-130) and the National Institute of Advanced Industrial Science and Technology (approval number: Animal 2022-0191).

[0017] (Preparation of Mice in Each Group) In each of the following Examples, three groups were prepared: a specific-pathogen-free mouse group (SPF group), a germ-free mouse group (GF group), and a group (Bio3 group) in which GF mice were colonized with three types of bacteria: lactic acid bacteria (Enterococcus faecium T-110: FERM BP-10867), butyric acid bacteria (Clostridium butyricum TO-A: FERM BP-10866), and saccharifying bacteria (Bacillus subtilis TO-A: FERM BP-07462).

[0018] (Rearing of GF group and Bio3 group) - Cage division 12-week-old GF mice (BALB / cA strain mice purchased from CLEA Japan and then bred in-house) kept in a germ-free environment were divided into two groups, and each group was aseptically moved from the vinyl isolator to a stainless steel isolator. One group was the GF group, which served as a control and was administered saline, and the other was the Bio3 group, which was administered a mixture of three bacteria: lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. Mice in each group were kept in the stainless steel isolator at the time of group division until the day of dissection. - Administration of the mixture of three bacteria to the Bio3 group and administration of saline to the GF group The Bio3 group received 5 x 10 each of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. 8The bacterial solution, adjusted to a CFU / ml concentration, was orally administered to each mouse at 200 μl per week (a total of five times). The GF group received the same oral administration, but with saline instead of the bacterial solution. Confirmation of colonization of the administered bacteria in the Bio3 group and sterility in the GF group: Feces were collected on the seventh day after oral administration at weeks 2 and 4, and homogenized on the same day. The homogenized suspension was then applied to selective media for each of the three bacteria, Lactobacillus, Butyric Acid Bacteria, and Saccharifying Bacteria, and cultured. In parallel, the suspension was prepared as a smear and subjected to morphological differentiation by Gram staining. Based on the Gram staining results and the detection of colonies on the selective media for each of the three bacteria, it was determined that the administered bacteria had colonized the intestines of the Bio3 mice. Furthermore, a sterility check was performed on the GF group on the day before necropsy to confirm sterility.

[0019] (Breeding of SPF mice) SPF mice (BALB / cA strain, Japan SLC) were bred according to standard methods, and the SPF, GF, and Bio3 groups were aged to the same age and subjected to intraperitoneal administration of bromodeoxyuridine (BrdU) and dissection as described below.

[0020] (Intraperitoneal administration of bromideoxyuridine (BrdU) to each mouse, followed by dissection and collection) - Intraperitoneal administration of bromideoxyuridine (BrdU). To monitor proliferating cells, all mice in this study were intraperitoneally administered BrdU. Specifically, after the fifth oral administration in the Bio3 and GF groups, and at the same time as the SPF group, 150 μl of 10 mg / ml BrdU suspended in saline and sterilized with a 0.22 μm PES filter was administered intraperitoneally using a 1 mL syringe with a 26 1 / 2 G needle (50 mg / kg body weight). Intraperitoneal administration of BrdU was performed for three consecutive days. Mice 1 day after the third administration and 21 days after administration were dissected and brain samples were collected using the following procedure, and then subjected to histological immunohistochemistry. Acclimation before dissection: Before dissection, mice were removed from their cages in the stainless steel isolator and left covered in the laboratory for 3 hours before dissection. Dissection order: Each group was dissected alternately to avoid any time differences between groups. Dissection: Each mouse was anesthetized with isoflurane, euthanized by exsanguination, and perfused with phosphate-buffered saline (PBS). Brain tissue was then perfused and fixed with 4% paraformaldehyde (PFA) and extracted. The extracted brain tissue was then incubated overnight in 4% PFA at 4°C for further fixation. To prevent tissue damage during freezing, the 4% PFA containing the brain tissue was replaced with 15% sucrose solution, incubated overnight at 4°C, and then replaced with 30% sucrose solution and further incubated overnight at 4°C. The brain tissue was then embedded in OCT compound (Sakura Fine Tech; 25608-930), frozen, and stored at -80°C.

[0021] Brain immunohistochemistry. Brain sections were prepared using a cryostat (Microm HM505e cryostat (Microm International GmbH, Walldorf, Germany)) at a thickness of 40 μm. After immersing the sections in PBS to remove the OCT compound, they were stored at -25°C in anti-freeze solution (Anti-freeze Cryoprotectant Solution for Fixed Sections, Bioenno Lifesciences; 006799-250) until staining. Immunohistochemistry was performed as follows: The tissue sections were washed with PBS and 0.1% Triton-X100 solution diluted in PBS (0.1% Triton-X100 in PBS). Afterwards, they were blocked with blocking solution (0.1% Triton-X100, 3% FBS in PBS) and left at room temperature for 1 hour. The primary antibody solution was then diluted with the blocking solution and incubated overnight at 4°C. After primary antibody incubation, sections were washed four times with 0.1% Triton-X100 solution. Secondary antibody dilutions were performed at room temperature for 3 hours in the dark using a blocking solution containing Hoechst 33258 (0.5 μg / ml, Dojindo Laboratories; 343-07961) for nuclear staining. After secondary antibody incubation, sections were washed four times with 0.1% Triton-X100 solution, dried, and mounted on APS-coated glass slides (MATSUNAMI; S8443). After washing once with PBS, sections were embedded in Fluorokeeper antifade buffer (Nacalai; 12593-64) and mounted with nail polish. The slides were then observed and photographed using a fluorescence microscope (THUNDERImager 3D Cell Culture system; Leica Microsystems). When staining with anti-BrdU antibody, the tissue sections were washed with PBS, then immersed in 2N HCl solution and treated at 37°C for 15 minutes. After washing with PBS, the sections were immersed in the above-mentioned blocking solution, and then tissue immunostaining was performed in the same manner as above.For staining with anti-NeuroD1 antibodies, tissue sections were attached to the slides described above, washed with PBS, and then immersed in Target Retrieval (Dako; S1699) and autoclaved at 105°C for 15 minutes for antigen retrieval. After washing with PBS and 0.1% Triton-X100 solution, the sections were blocked on the slides and then subjected to immunohistochemical staining in the same manner as described above. The primary antibodies used for tissue immunostaining were rat anti-BrdU (1:500, BIORAD; MCA6144), rabbit anti-NeuN (1:500, CellSignaling; 24307S), chicken anti-DCX (1:200, Abcam; ab153668), mouse calretinin (1:250, ProteinTech; 66496-1-IG), goat anti-NeuroD (NeuroD, SantaCruz; sc-1084), chicken anti-GFAP (1:500, Abcam; ab4674), rat anti-NESTIN (1:500, Wako; 012-26843), goat anti-SOX2 (1:500, Abcam; ab239218), and rabbit anti-IBA1 (1:500, Wako; 019-19741). The secondary antibodies used were CF568 anti-rat IgG antibody (500-fold dilution, Biotium; 20092), CF488 anti-mouse IgG antibody (500-fold dilution, Biotium; 20014), CF488 anti-rabbit IgG antibody (500-fold dilution, Biotium; 20015), CF568 anti-rabbit IgG antibody (500-fold dilution, Biotium; 20098), CF488 anti-chicken IgM antibody (500-fold dilution, Biotium; 20020), CF568 anti-goat IgG antibody (500-fold dilution, Biotium; 20106), and CF647 anti-goat IgG antibody (500-fold dilution, Biotium; 20048).

[0022] (Proliferation-promoting effect of a three-type mixed bacteria on neural stem cell proliferation) The results of immunostaining BrdU-positive cells in the dentate gyrus region of the hippocampus of mice in each group, one day after administration of BrdU for three consecutive days, are shown in Figure 1. The results in Figure 1 confirmed that a three-type mixed bacteria of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria promotes the proliferation of neural stem cells (particularly neural stem cells present in the dentate gyrus of the hippocampus and capable of proliferation).

[0023] (Effect of a three-type bacterial mixture on promoting differentiation and maturation of neural stem cells into neurons) The results of immunostaining of BrdU-positive cells and the expression of NeuN protein, which is specifically expressed in mature neurons, in the hippocampal dentate gyrus region of mice in each group that received three consecutive BrdU administrations on day 21 are shown in Figure 2. The results in Figure 2 confirmed that a three-type bacterial mixture of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria promotes the differentiation and maturation of neural stem cells (particularly those in the dentate gyrus of the hippocampus) into neurons.

[0024] (Effect of the three-type mixed bacteria on promoting the maturation of immature neurons) The results of immunostaining for calretinin protein, which is specifically expressed in mature neurons, in the hippocampal dentate gyrus region of mice from each group 21 days after BrdU administration are shown in Figure 3. The results in Figure 3 confirmed that the three-type mixed bacteria (lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria) promote the maturation of immature neurons (particularly immature neurons in the hippocampal dentate gyrus).

[0025] (The effect of a three-type bacterial mixture on promoting the differentiation and maturation of neural stem cells into neurons through increased expression of NeuroD1 in neural stem cells) The results of immunostaining for the expression of NeuroD1 protein, a transcription factor that promotes neuronal differentiation and maturation, in the hippocampal dentate gyrus region of mice in each group are shown in Figure 4. The results in Figure 4 confirmed that a three-type bacterial mixture of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria promoted the differentiation and maturation of neural stem cells into neurons through the expression of NeuroD1 in neural stem cells (particularly neural stem cells in the dentate gyrus of the hippocampus).

[0026] (Effect of a three-type mixed bacteria on restoring the decrease in the number of resting neural stem cells following sterilization) The results of immunostaining of resting neural stem cells that co-express GFAP protein, nestin protein, and SOX2 protein in the hippocampal dentate gyrus region of mice in each group are shown in Figure 5. The results in Figure 5 confirmed that a three-type mixed bacteria of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria restored the decrease in the number of resting neural stem cells following sterilization (increased the number of resting neural stem cells).

[0027] (Microglial activation promoting effect of a three-type bacterial mixture) Figure 6 shows the results of immunostaining microglia expressing IBA1 protein in the cerebral cortex of mice in each group. It is known that microglia cell volume decreases when activated. The results in Figure 6 confirmed that a three-type bacterial mixture of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria promotes microglial activation.

[0028] (Effect of a three-type mixed bacteria on promoting astrocyte activation) Figure 7 shows the results of immunostaining of astrocytes expressing GFAP protein in the cerebral cortex of mice in each group. It is known that astrocytes increase in cell volume when activated. The results in Figure 7 confirmed that a three-type mixed bacteria of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria promotes astrocyte activation.

[0029] (Preparation of Human Neural Stem Cells and Culture Supernatants of Each Bacterium to Confirm the Effect of Each Bacterium) The results shown in Examples 2 to 8 are the results of observations of brain cells in experiments in which mice were orally administered a mixture of the three bacteria described in Example 1: lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. Using human neural stem cells, we confirmed that each of these three bacteria alone is effective on neural stem cells, and furthermore, that they also exhibit effects on human cells. Human neural stem cells were obtained from a cell line purchased from Phoenix Songs Biologicals, Inc., derived from the human cerebral cortex of a 14-week-old male fetus with informed consent. Test cells were prepared according to the method described in Patent No. 6976600 or International Publication No. WO2019 / 009205. To prepare the culture supernatants of each bacteria to be added to human neural stem cells, all strains were grown in the human neural stem cell medium described in Patent No. 6976600 or International Publication No. WO2019 / 009205. For the culture of each strain, Escherichia coli ATCC8739 (control strain) and saccharifying bacteria (Bacillus subtilis TO-A: FERM BP-07462, hereafter referred to as BS) were inoculated into L-shaped tubes and cultured at 37°C under aerobic conditions with linear shaking at 100-110 rpm. Lactic acid bacteria (Enterococcus faecium T-110: FERM BP-10867, hereafter referred to as EF) and butyric acid bacteria (Clostridium butyricum TO-A: FERM BP-10866, hereafter referred to as CB) were cultured statically at 37°C under anaerobic conditions. For both strains, culture was terminated when the turbidity of the culture medium reached or just before a steady state. After centrifugation, the supernatant was sterilized using a 0.2 μm pore size filter. Each test solution of this supernatant was diluted 10-fold and added to human neural stem cells, and the effects on neural stem cell proliferation as shown in Example 10 and on differentiation into neurons as shown in Example 11 were evaluated.

[0030] (Proliferation effect of bacteria alone on human neural stem cells) The influence of the culture supernatant of each bacteria alone on the proliferation of human neural stem cells was evaluated using the Click-iT EdU Cell Proliferation Kit (Thermo Fisher Scientific) according to the recommended procedure. -2 ~10 -8 After incubation for 1 day, 2.5 μM 5-ethynyl-2'-deoxyuridine (EdU) was added and the cells were incubated for 2 hours (see Figure 8A). The cells were then fixed with 4% PFA, reacted with Click-iT, and the cell nuclei were stained with Hoechst 33342. The stained cells were visualized under a fluorescent microscope, and the cell proliferation rate was calculated by dividing the number of EdU-positive cells by the number of Hoechst-positive cells. As shown in Figure 8B, it was confirmed that EF culture supernatant significantly improved the proliferation of human neural stem cells. Specifically, EF 10 -5 , 10 -6 and 10 -8 The dilution significantly improved the proliferation of human neural stem cells. It was confirmed that the culture supernatant of the control strain, E. coli, was unable to significantly improve the proliferation of human neural stem cells.

[0031] (Effect of each bacterium alone on differentiation of human neural stem cells into neurons) Whether or not the culture supernatant of each bacterium alone induces differentiation of human neural stem cells into neurons was evaluated by indirect immunostaining of β III-tubulin protein, i.e., Tuj-1, which is specifically expressed in differentiated neurons. Specifically, after 3 days of culture of human neural stem cells, 10 -2 ~10 -8After 7 days of incubation with the culture supernatant of each strain diluted to 1:1, the cells were fixed with 4% PFA and stained with an antibody against Tuj-1 (R&D Systems; MAB1195) as the primary antibody and a CF568 anti-mouse IgG antibody (1:500 dilution, Biotium, 20105) as the secondary antibody. Cell nuclei were also stained with Hoechst 33342 (see Figure 9A). The stained cells were visualized under a fluorescent microscope, and the percentage of differentiated neurons was calculated by dividing the number of Tuj-1-positive cells by the number of Hoechst-positive cells. As a secondary evaluation of the culture supernatant of the selected strains, human neural stem cells (0.6 x 10) cultured in a 24-well plate were used. 5 All samples were evaluated simultaneously on a single plate using 100 cells / well of BS. The results of the primary and secondary evaluations are shown in Figure 9. As shown in the results in Figure 9B, it was confirmed that the culture supernatants of all the single bacteria other than E. coli, BS, EF, and CB, significantly enhanced the induction of differentiation of human neural stem cells into neurons. Specifically, 10 cells of BS －７ Dilution of EF 10 －６ Dilutions of 10 and 10 of CB －８ The dilution significantly enhanced the induction of differentiation of human neural stem cells into neurons. It was confirmed that the culture supernatant of the control strain, E. coli, was unable to significantly improve the proliferation of human neural stem cells.

[0032] (Memory and Learning Effects of CB Strains) The nematode Caenorhabditis elegans is known to be able to memorize and learn the salt concentration of its environment. For example, starved nematodes memorize and learn the salt concentration of their rearing environment, exhibiting behaviors that avoid the salt concentration they experienced under starvation. Taking advantage of this characteristic, we divided two groups of nematodes into two groups: one group fed only the standard diet (Escherichia coli OP50 strain) for four days (from day 3 to day 7 after the start of rearing) (standard diet group), and the other group fed CB along with E. coli OP50 (CB test group). We then observed changes in salt-avoidance behavior during starvation. The test and evaluation methods were based on those described in the literature (Neuron. 2006; 51: 613-25. DOI: 10.1016 / j.neuron.2006.07.024). The specific evaluation method is as follows. To create a salt gradient for evaluation of C. elegans, a cylindrical agar plate (5 mm diameter) containing 100 mM NaCl was placed on one end of the plate and allowed to stand for 24 hours, as shown in Figure 10A-1. To allow the nematodes to memorize and learn about starvation and the presence or absence of salt in their rearing environment, two treatments were performed, as shown in Figure 10A-2. Nematodes were treated with either naive or NaCl treatment, respectively. The behaviors associated with memory and learning were observed using the evaluation agar plate and the nematodes that underwent each treatment. Specifically, nematodes were placed in the center of the evaluation agar plate and allowed to stand at 25°C for 1 hour. The number of nematodes present in each of the high-salt concentration regions (A), intermediate-salt concentration regions (C), and low-salt concentration regions (B) was counted, as shown in FIG. 10A-1, and the salt concentration chemotaxis index of the nematodes was calculated using the formula (AB) / (total number of nematodes - C). Specifically, a chemotaxis index closer to 1 indicates that the nematodes are attracted to salt, while a chemotaxis index closer to -1 indicates that the nematodes are repelled by salt. Since NGM medium, commonly used for conventional nematode rearing, including this example, contains 50 mM NaCl, it is known that naive-treated nematodes are attracted to salt.Furthermore, the salt chemotaxis index of NaCl-treated nematodes starved in the presence of salt was lower than that of naive-treated nematodes, which is a reasonable response. As shown in Figure 10B, the salt chemotaxis index of the naive-treated nematodes in this example was similar to that of the CB test group and the normal diet group. However, the salt chemotaxis index of the NaCl-treated nematodes in the CB test group was lower than that of the normal diet group. In other words, the CB test group tended to exhibit stronger salt aversion behavior when starved than the normal diet group. These results demonstrate that CB intake maintains and / or improves memory and learning ability.

[0033] The results of Examples 2 to 11 above demonstrate that the composition of the present disclosure has the effect of promoting adult neurogenesis. Specifically, adult neurogenesis refers to the phenomenon in which neural stem cells differentiate into mature neurons, even in adults after the fetal stage, when the majority of neurons are formed. From the perspective of adult neurogenesis, the present invention examined factors expressed at each stage of neurogenesis as described in Non-Patent Document 8 (Figure 10). Specifically, in Example 2, a significant increase in the division and proliferation of neural stem cells, which are the starting point for differentiation, was confirmed by measuring BrdU incorporation, and in Example 3, a significant increase in the number of cells expressing NeuN, a protein characteristic of mature neurons, at the final stage of differentiation, confirming that the composition of the present disclosure promotes adult neurogenesis. More specifically, in Examples 5 and 4, an increase in NeuroD, which is expressed during differentiation into neurons, and a subsequent increase in cells expressing Calretinin were confirmed, respectively. Furthermore, in Example 6, it was confirmed that the composition of the present disclosure promotes adult neurogenesis beyond the conventional perspective, i.e., it even increases the number of resting neural stem cells, which are the basis for adult neurogenesis. On the other hand, it was confirmed in Examples 7 and 8 that the composition of the present disclosure not only promoted the above-mentioned differentiation into neurons, but also promoted the activation of glial cells, microglia and astrocytes. The effects of each bacterium alone are shown in Examples 9 to 11. It was confirmed in Example 10 that lactic acid bacteria alone promoted the proliferation of human neural stem cells. In addition, it was confirmed in Example 11 that any of saccharifying bacteria, lactic acid bacteria, and butyric acid bacteria alone promoted the differentiation of human neural stem cells into neurons.

[0034] The results of Examples 2 to 12 demonstrate that the composition of the present disclosure has the effect of maintaining brain function homeostasis. Specifically, Non-Patent Document 9 provides a detailed classification of microglial morphological changes, and Non-Patent Document 10 describes the function of microglia in maintaining homeostasis. Specifically, microglia control axonal fasciculation, programmed cell death, neurite formation, synaptic homeostasis ("pruning"), synapse formation, and other processes, playing an important role in maintaining normal neuronal function in the brain (brain function homeostasis). Furthermore, microglia control the proliferation and differentiation of neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs), promote myelination, and increase astrocyte activation and proliferation. Microglia also play a role in eliminating unnecessary cells and foreign substances through phagocytosis. Astrocytes have various functions, including maintaining a healthy central nervous system, and are responsible for circulating neurotransmitters, supporting neuronal metabolism, and maintaining the blood-brain barrier. Their interactions with microglia are also described in detail in Non-Patent Document 11. Specifically, the results of microscopic observation (changes in cell area) in Examples 7 and 8 using the composition of the present invention revealed activation of microglia and astrocytes, respectively, confirming the suggestion of maintaining homeostasis of brain function. Furthermore, the results of Example 12 confirmed that at least the intake of butyric acid bacteria maintains and / or improves memory and learning ability.

[0035] The present disclosure can provide compositions for increasing the number of resting neural stem cells, compositions for promoting the proliferation of neural stem cells, compositions for promoting the differentiation and maturation of neural stem cells into neurons, compositions for promoting the maturation of immature neurons, compositions for promoting the activation of microglia, compositions for promoting the activation of astrocytes, compositions for promoting adult neurogenesis, compositions for maintaining brain function homeostasis, and compositions for maintaining and / or improving memory and / or learning ability.

Claims

**Claim 1** A composition for any one or more of the following uses, comprising a bacterium selected from one or more of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. (1) For promoting adult neurogenesis (2) For increasing the number of quiescent neural stem cells (3) For promoting the proliferation of neural stem cells (4) For promoting the differentiation and maturation of neural stem cells into neurons (5) For promoting the maturation of immature neurons (6) For promoting the activation of astrocytes (7) For promoting the activation of microglia **Claim 2** The composition according to claim 1, comprising two or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. **Claim 3** The composition according to claim 1, comprising lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. **Claim 4** The composition according to any one of claims 1 to 3, wherein the lactic acid bacteria are bacteria of the genus Enterococcus, the butyric acid bacteria are bacteria of the genus Clostridium, and the saccharifying bacteria are bacteria of the genus Bacillus. **Claim 5** The composition according to any one of claims 1 to 3, wherein the lactic acid bacteria are of the species Enterococcus faecium or Enterococcus lactis, the butyric acid bacteria are of the species Clostridium butyricum, and the saccharifying bacteria are of the species Bacillus subtilis. **Claim 6** The composition according to any one of claims 1 to 3, wherein the lactic acid bacteria are Enterococcus faecium T-110, the butyric acid bacteria are Clostridium butyricum TO-A, and the saccharifying bacteria are Bacillus subtilis TO-A. **Claim 7** A composition for promoting adult neurogenesis, comprising the species Enterococcus faecium or Enterococcus lactis, the species Clostridium butyricum, and the species Bacillus subtilis. **Claim 8** A composition for increasing the number of quiescent neural stem cells, comprising the species Enterococcus faecium or Enterococcus lactis, the species Clostridium butyricum, and the species Bacillus subtilis. **Claim 9** A composition for promoting the differentiation and maturation of neural stem cells into neurons, comprising the species Enterococcus faecium or Enterococcus lactis, the species Clostridium butyricum, and the species Bacillus subtilis.

10. A composition for promoting the proliferation of neural stem cells, comprising Enterococcus faecium species or Enterococcus lactis species, Clostridium butyricum species, and Bacillus subtilis species.

11. A composition for promoting the maturation of immature neural cells, comprising Enterococcus faecium species or Enterococcus lactis species, Clostridium butyricum species, and Bacillus subtilis species.

12. A composition for promoting the activation of microglia, comprising Enterococcus faecium species or Enterococcus lactis species, Clostridium butyricum species, and Bacillus subtilis species.

13. A composition for promoting the activation of astrocytes, comprising Enterococcus faecium species or Enterococcus lactis species, Clostridium butyricum species, and Bacillus subtilis species.

14. A composition for maintaining brain function homeostasis, comprising Enterococcus faecium species or Enterococcus lactis species, Clostridium butyricum species, and Bacillus subtilis species.

15.

15. A composition for maintaining and / or improving memory and / or learning ability, comprising Clostridium butyricum.

16.

16. The composition according to claim 15, wherein the Clostridium butyricum is a Clostridium bacterium.

17.

17. The composition according to claim 15, wherein the Clostridium butyricum is Clostridium butyricum TO-A.