Compositions for promoting the proliferation, differentiation, and maturation of neural stem cells, promoting the activation of microglia, and promoting the activation of astrocytes; and compositions 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

[Technical Field]

[0001] This 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 nerve cells, compositions for promoting the proliferation of neural stem cells, compositions for promoting the maturation of immature nerve cells, 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 application No. 2023-74562, as incorporated herein by reference. [Background technology]

[0002] Brain function is constructed by nerve cells (neurons) and glial cells (consisting of microglia, astrocytes, and oligodendrocytes). Neurons are responsible for forming memory circuits, and the majority of them are formed during the embryonic stage. Astrocytes structurally and functionally maintain the brain's neural circuits and the blood-brain barrier, while microglia, as immune cells in the brain, maintain homeostasis in the central nervous system. Neural stem cells capable of differentiating into neurons and glial cells are also present in the adult brain (particularly in the subventricular zone and hippocampal dentate gyrus (DG)), and the phenomenon of new neurons being generated from adult neural stem cells, i.e., adult neurogenesis, has been reported (see Non-Patent Literature 1-3). While it has been reported that the gut microbiota contributes to adult neurogenesis in the hippocampus, many details remain unclear. Adult neurogenesis is known to involve several stages in the development of neural stem cells into mature nerve cells, but it has been largely unknown to date which stage of neurogenesis gut bacteria are involved, and to what extent they contribute to nerve cell formation. Furthermore, the mechanisms that induce neurogenesis have not been elucidated at all (see Non-Patent Literature 4-7).

[0003] Patent Document 1 discloses "a composition for maintaining and / or improving memory and learning ability, comprising the cells of a heterofermentative lactic acid bacillus of the genus Lactobacillus." However, the composition of this disclosure differs in composition from that described in Patent Document 1. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 6739603 [Non-patent literature]

[0005] [Non-Patent Document 1] Cell Stem Cell. 2019 May2;24(5):690-705. [Non-Patent Document 2] Aging (Albany NY). 2021 Jul 29;13(14):18689-18700. [Non-Patent Document 3] Nature. 2018 July; 559(7712):98-102. [Non-Patent Document 4] Curr Opin Neurobiol. 2015 Feb;30:51-8. [Non-Patent Document 5] Nat Med. 2019 Apr;25(4):554-560. [Non-Patent Document 6] Front Neurosci. 2022 Oct 28;16:1030694. [Non-Patent Document 7] Front Cell Dev Biol. 2020 May 29;8:407. [Non-Patent Document 8] PLoS One. 2010 Jan 29;5(1):e8809. [Non-Patent Document 9] Front Cell Neurosci. 2017 Aug 8;11:235.

Non-Patent Document 10

Non-Patent Document 11

Summary of the Invention

Problems to be Solved by the Invention

[0006] To elucidate what kind of influence a composition containing lactic acid bacteria, butyric acid bacteria, and / or saccharifying bacteria that improve the intestinal flora and the barrier function of the large intestine has on brain function, and to provide a composition that generates and promotes adult neurogenesis and is involved in maintaining the homeostasis of brain function and improving memory and learning ability is an object.

[0007] In order to solve the above problems, the present inventors conducted intensive research. Lactic acid bacteria (especially, Enterococcus faecium ), butyric acid bacteria (especially, Clostridium butyricum ), and / or saccharifying bacteria (especially, Bacillus subtilis ) were found to have the effects of increasing the number of quiescent neural stem cells, promoting the proliferation of neural stem cells, promoting the differentiation and maturation of neural stem cells into neurons, promoting the maturation of immature neurons, promoting the activation of microglia, promoting the activation of astrocytes, promoting adult neurogenesis, and maintaining brain function homeostasis, and the present disclosure was completed. Furthermore, it was found that butyric acid bacteria have the effect of maintaining and / or improving memory and / or learning ability, and the present disclosure was completed.

[0008] The present disclosure is as follows. 1. A composition containing a bacterium selected from one or more of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria, for any one or more of the following uses. (1) For increasing the number of quiescent 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 2. The composition according to item 1 above, comprising bacteria selected from 2 or more of lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria 3. The composition according to item 1 above, comprising lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria 4. The lactic acid bacteria are Enterococcus bacteria of the genus, the butyric acid bacteria are Clostridium bacteria of the genus, and the saccharifying bacteria are Bacillus bacteria of the genus, the composition according to any one of items 1 to 3 above 5. The lactic acid bacteria are Enterococcus faecium species or Enterococcus lactis species, the butyric acid bacteria are Clostridium butyricum species, and the saccharifying bacteria are Bacillus subtilis species, the composition according to any one of items 1 to 3 above 6. The lactic acid bacteria are Enterococcus faecium T-110, the butyric acid bacteria are<00001​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​A composition containing seeds for promoting the differentiation and maturation of neural stem cells into nerve cells. 9. Enterococcus faecium species or Enterococcus lactis seed, Clostridium butyricum Seeds and Bacillus A composition containing seeds for promoting the proliferation of neural stem cells. 10. subtilis species or Enterococcus seed, faecium Seeds and Enterococcus A composition containing seeds for promoting the maturation of immature nerve cells. 11. lactis species or Clostridium seed, butyricum Seeds and Bacillus A composition containing seeds for promoting the activation of microglia. 12. subtilis species or Enterococcus seed, faecium Seeds and Enterococcus A composition containing seeds for promoting the activation of astrocytes. 13. lactis species or Clostridium seed, butyricum Seeds and Bacillus A composition containing seeds for promoting adult neurogenesis. 14. subtilis species or Enterococcus seed, faecium Seeds and Enterococcus A composition containing seeds for maintaining brain function homeostasis. 15. A composition containing butyrate-producing bacteria for maintaining and / or improving memory and / or learning ability. 16. The butyrate-producing bacteria The composition described in item 15 above, which is a bacterium of the genus. 17. The butyrate-producing bacteria lactis The composition described in item 15 above, which is TO-A. 18. A method of use for one or more of the following purposes, comprising administering a composition containing one or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria to a target. (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 nerve cells (4) For promoting the maturation of immature nerve cells (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 of use for maintaining and / or improving memory and / or learning ability, comprising administering a composition containing butyrate-producing bacteria to a target subject. 20. Use of one or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria in the manufacture 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 nerve cells (4) For promoting the maturation of immature nerve cells (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 21. Use of butyrate-producing bacteria in the manufacture of compositions for maintaining and / or improving memory and / or learning ability. 22. Foods for the following uses, comprising any one of the compositions described in item 1 to 17 above. (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 nerve cells (4) For promoting the maturation of immature nerve cells (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 item 3 above, wherein the amount of butyric acid bacteria is 100 to 2500 parts by mass and the amount of saccharifying bacteria is 100 to 2500 parts by mass per 100 parts by mass of lactic acid bacteria. [Effects of the Invention]

[0009] The lactic acid bacteria disclosed herein (especially, Clostridium ), butyrate-producing bacteria (especially, butyricum ) and saccharifying bacteria (especially, Bacillus A composition containing at least ) has one or more of the following effects: (1) Increase in the number of resting neural stem cells (2) Promotion of differentiation and maturation of neural stem cells into nerve cells (3) Promoting the proliferation of neural stem cells (4) Promoting the maturation of immature nerve cells (5) Promotion of adult neurogenesis (6) Promoting the activation of microglia (7) Promoting the activation of astrocytes (8) Maintaining brain function homeostasis In addition, the butyrate-producing bacteria of this disclosure (especially, subtilis A composition containing at least ) has the effect of maintaining and / or improving memory and / or learning ability. [Brief explanation of the drawing]

[0010] EnterococcusA: Immunostaining of BrdU-positive cells (black) in the hippocampal dentate gyrus region on day 1 post-administration in mice from each group that received BrdU for 3 consecutive days is shown. Scale bar = 100 μm. B: The number of BrdU-positive cells in the granule cell layer of the hippocampal dentate gyrus per mouse in each group was measured and graphed. The group of mice administered Bio3 (Bio3) had a statistically significantly higher number of BrdU-positive cells compared to 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 shows the expression of BrdU-positive cells (white) and NeuN protein (black), which is specifically expressed in mature neurons, in the hippocampal dentate gyrus region of mice in each group that were administered BrdU for three consecutive days, 21 days after administration. Scale bar = 100 μm. B: The number of BrdU-positive cells in the granule cell layer of the hippocampal dentate gyrus per mouse in each group was measured and graphed. There was no statistically significant difference in the number of BrdU-positive cells between the groups. (n=4) C: The number of BrdU and NeuN co-positive cells in the granule cell layer of the hippocampal dentate gyrus per mouse in each group was measured and graphed. The group of mice administered Bio3 (Bio3) had a higher number of BrdU and NeuN co-positive cells compared to the other groups, and was statistically significantly higher compared to the GF group. *: p<0.05 (n=4). D: The proportion of NeuN expression in BrdU-positive cells in each group is shown. The group of mice treated with Bio3 (Bio3) had a statistically significantly higher proportion of cells expressing NeuN compared to the group of GF mice. **: p<0.01 (n=4). faeciumA: Immunostaining shows 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 in each group was measured and graphed. The Bio3 administration group had a significantly higher number of Calretinin-positive mature neurons compared to the other groups. *: p<0.05, **: p<0.01 (n=4). A: Immunostaining shows the expression of the transcription factor NeuroD1 protein (NeuroD1, black and white arrowheads), which promotes neuronal differentiation and maturation, in the hippocampal dentate gyrus region of mice from each group. 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 from each group was measured and graphed. B is a graph of brain tissue collected from mice 1 day after BrdU administration (4 weeks after Bio3 or saline administration), and C is a graph of brain tissue collected 21 days after BrdU administration (7 weeks after Bio3 or saline administration), stained, and then the number of NeuroD1-positive cells was measured. In both time periods, the number of NeuroD1-positive cells in the hippocampal dentate gyrus was highest in the Bio3 administration group. *: p<0.05, **: p<0.01 (n=4). Enterococcus A: Psitoneal neural stem cells (white border, black arrowhead) co-expressing GFAP protein, nestin protein, and SOX2 protein are shown in the hippocampal dentate gyrus region of mice from each group. Scale bar = 100 μm. B: The number of quiescent neural stem cells in the granule cell layer of the hippocampal dentate gyrus per mouse from each group was measured and graphed. The GF group had a significantly lower number of quiescent neural stem cells compared to the SPF group. However, the Bio3 group was similar to the SPF group. **: p<0.01 (n=4 (SPF), n=5 (GF, Bio3)). A: The upper panel shows microglia expressing the IBA1 protein (white) in the cerebral cortex of mice from each group. The lower panel is a binarized grayscale image of fluorescence microscopy images of IBA1-positive microglia, which was 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 from each group was measured using ImageJ from the grayscale image in A. The values ​​on the vertical axis are arbitrary units in ImageJ. The Bio3 group showed a significantly reduced microglial area compared to the SPF group. *: p<0.05 (n=4 (SPF, Bio3), n=3 (GF)). lactis A: The upper panel shows astrocytes expressing GFAP protein (white) in the cerebral cortex of mice from each group. The lower panel is a black and white image obtained by binarizing fluorescence microscopy images of GFAP-positive astrocytes, and this image was used to measure the cell area in B. Scale bar = 50 μm. B: From the black and white image in A, the average area per individual of GFAP-positive astrocytes 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, especially compared to the GF group. *: p<0.05 (n=4). A: Overview of the experiment. B: Proliferation evaluation. Clostridium A: Overview of the study. B: Evaluation of neuronal differentiation. A-1: Test Overview - Agar medium for evaluation. A-2: Test Overview - Flowchart from starvation treatment of nematodes to behavioral observation. B: Results - Salt concentration chemotaxis index of nematodes. [Modes for carrying out the invention]

[0011] This 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 nerve cells, compositions for promoting the proliferation of neural stem cells, compositions for promoting the maturation of immature nerve cells, 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 this disclosure are not particularly limited, but include pharmaceutical compositions, food compositions, feed compositions, and the like.

[0012] (Compositions of this Disclosure) The composition disclosed herein is a composition comprising one or more bacteria selected from lactic acid bacteria, butyric acid bacteria, and saccharifying bacteria. Lactic acid bacteria Lactic acid bacteria are preferably butyricum It is a genus, and Bacillus species or subtilis Enterococcus faecium Enterococcus lactis Clostridium butyricum Bacillus subtilis Clostridium Clostridium butyricum Enterococcus faecium Clostridium butyricum Bacillus subtilis Clostridium butyricum

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[0013] (Target subjects for administration of the compositions disclosed herein) The subjects to whom the compositions of this disclosure can be administered 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, while the target organism is one in which adult neurogenesis can occur, i.e., adults in humans, the composition containing this strain has been safely used as an orally administered medicine for approximately 60 years in people of all ages, from infants to the elderly.

[0014] (Method of administering the composition of this disclosure) The dosage, frequency and interval of administration of the administration method described herein are not particularly limited and will be appropriately selected depending on the purpose of prevention (especially prevention of recurrence) and / or clinical treatment, the type of disease, the patient's weight, age, the severity of the disease, and other factors. For example, the following dosage forms can be used, but are not limited to them. Powder (daily dose) Lactic acid bacteria: 15~30 mg Butyric acid bacteria: 75~150 mg Saccharifying bacteria: 75~150 mg Orally disintegrating tablets or uncoated tablets (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, uncoated tablets, coated tablets, capsules, or liquid, once or twice a day, or multiple times a day (morning, noon, and evening). In addition, daily administration is preferred, but administration every few days or several times every other week is also possible. Furthermore, as a guideline for the daily intake of bacteria contained in the composition disclosed herein, for example, in the case of a human, it is 10 3 ~10 20 pieces, preferably 10 5 ~10 15 10 comfortable 6 ~10 13 It is an individual.

[0015] The compositions (or uses of the compositions) of this disclosure are 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 nerve cells, promoting the proliferation of neural stem cells, and / or promoting the maturation of immature nerve cells. A homeostatic agent for brain function, based on the effect of the composition in 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 compositions disclosed herein can be formulated into powders, tablets, granules, capsules, liquids, etc., by incorporating known carriers such as starch, lactose, and soy protein, as well as excipients, binders, disintegrants, lubricants, stabilizers, suspending agents, and other additives, using well-known methods.

[0016] The disclosure will be explained in detail below with specific examples, but this disclosure is not limited to these examples. The following examples were conducted in accordance with the guidelines for handling laboratory animals established by the University of Tokyo (Approval Number: P20-130) and the National Institute of Advanced Industrial Science and Technology (Approval Number: 2022-0191). [Examples]

[0017] (Preparation of mice for each group) In each of the following examples, there are three groups: a specific-pathogen-free mouse group (SPF group), a germ-free mouse group (GF group), and a lactic acid bacteria group. Enterococcus feces T-110:FERM BP-10867), butyric acid bacteria ( Clostridium buttermilk TO-A:FERM BP-10866) and saccharifying bacteria ( Bacillus subtle We prepared a group (Bio3 group) in which three types of bacteria (TO-A:FERM BP-07462) were colonized in GF mice.

[0018] (Rearing of GF group and Bio3 group) • Cage separation Twelve-week-old GF mice (BALB / cA strain mice purchased from CLEA Japan and then bred in-house) raised in a sterile environment were divided into two groups, and each group was aseptically transferred from a vinyl isolator to a stainless steel isolator. One group was designated as the control group (GF group) and administered Saline, while the other group was designated as the Bio3 group and administered a mixture of three bacteria: lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria. The mice in each group were raised in the stainless steel isolators used at the time of division until the day of dissection. • Administration of a three-bacterial mixture to Bio3 group and administration of Saline to GF group Bio3 contains lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria, each in a quantity of 5 × 10⁻⁶. 8 A bacterial suspension, adjusted to a CFU / ml concentration, was orally administered to each mouse at a dose of 200 µl once a week (a total of 5 times). For the GF group, physiological saline (Saline) was used instead of the bacterial suspension and administered orally in the same manner. • Confirmation of bacterial colonization in Bio3 group and confirmation of sterility in GF group Fecal samples were collected on the 7th day after oral administration in weeks 2 and 4, and the homogenized suspensions were immediately spread on selective media for each of the three bacterial strains (lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria) and cultured. Simultaneously, smears of the suspensions were prepared and morphological identification was performed by Gram staining. Based on the Gram staining results and the detection of colonies in the selective media for each of the three bacteria, it was determined that the administered bacteria had colonized the intestines of the Bio3 group mice. Furthermore, the GF group underwent a sterility check the day before dissection to confirm its sterility.

[0019] (Raising SPF mice) SPF mice (BALB / cA strain, Japan SLC) were reared according to standard procedures, and the SPF, GF, and Bio3 groups were subjected to intraperitoneal administration of Bromodeoxyuridine (BrdU) and dissection as described below, after being brought to the same age.

[0020] (Intraperitoneal administration of Bromodeoxyuridine (BrdU) to each mouse, followed by dissection and tissue collection) • Intraperitoneal administration of Bromodeoxyuridine (BrdU) To monitor the proliferation of cells, BrdU was administered intraperitoneally to all mice in this study. Specifically, in the Bio3 and GF groups, 10 mg / ml of BrdU, suspended in Saline and sterilized through a 0.22 μm PES filter, was administered intraperitoneally (150 uls, assuming 50 mg / kg body weight) using a 1 mL syringe with a 26 1 / 2 G needle, at the same timing as the aforementioned two groups. Intraperitoneal administration of BrdU was performed for three consecutive days. The brains of mice dissected one day after the third administration and 21 days after administration were subjected to histoimmunostaining using the procedure described below. • Habituation before dissection Before dissection, the mice were removed from their cages in stainless steel isolators and left covered in the laboratory for three hours before the dissection began. • Order of dissection To avoid creating temporal differences between the groups due to the order of dissection, each group was dissected alternately. ·Dissection Each mouse was anesthetized with isoflurane and euthanized by bleeding, followed by perfusion with phosphate-buffered saline (PBS). Then, perfusion fixation was performed with 4% paraformaldehyde solution (PFA), and brain tissue was extracted. The extracted brain tissue was incubated overnight in 4% PFA at 4°C for further tissue fixation. Subsequently, to prevent tissue damage during freezing, the 4% PFA containing the brain tissue was replaced with a 15% sucrose solution and incubated overnight at 4°C. After that, it was replaced with a 30% sucrose solution and incubated again overnight at 4°C. Subsequently, the brain tissue was embedded using OCT compound (Sakura FineTech; 25608-930), frozen at -80°C, and stored.

[0021] (Histological immunohistochemistry of the brain) Brain sections were prepared using a cryostat (Microm HM505e cryostat (Microm International GmbH, Walldorf, Germany)) with a thickness of 40 μm. The tissue sections were immersed in PBS solution to remove the OCT compound, and then stored at -25°C in an anti-freeze cryoprotectant solution for fixed sections (Bioenno Lifesciences: 006799-250) until staining. The following immunohistochemical staining was performed. Tissue sections were washed with PBS and a 0.1% triton-X100 solution diluted in PBS (0.1% triton-X100 in PBS), then a blocking solution (0.1% triton-X100, 3% FBS in PBS) was added, and the sections were allowed to stand at room temperature for 1 hour to block. Next, the primary antibody solution was replaced with a solution diluted with the blocking solution, and the antibody reaction was carried out overnight at 4 °C. After the primary antibody reaction, the sections were washed four times with a 0.1% triton-X100 solution, and the secondary antibody dilution solution, diluted with a blocking solution containing Hoechst 33258 (0.5 μg / ml, Dojin Chemical Research Institute; 343-07961) for nuclear staining, was used to carry out the antibody reaction for 3 hours at room temperature in the dark. After the secondary antibody reaction, the sections were washed four times with a 0.1% triton-X100 solution, dried, and mounted on APS-coated glass slides (MATSUNAMI; S8443). After washing once with PBS, the slides were embedded in FluoroKeeper anti-fading agent (Nacalai: 12593-64) and secured with nail polish. The prepared slides were observed and photographed using a fluorescence microscope (THUNDERImager 3D Cell Culture system; Leica Microsystems). When staining with anti-BrdU antibody, tissue sections were washed with PBS, immersed in 2N HCl solution and treated at 37°C for 15 minutes, washed with PBS, immersed in the blocking solution described above, and then subjected to tissue immunostaining in the same manner as described above. When staining with anti-NeuroD1 antibody, tissue sections were mounted on the aforementioned slides, washed with PBS, and then immersed in Target Retrieval (Dako; S1699). Antigen retrieval was performed by autoclaving (105 °C for 15 minutes). After washing with PBS and 0.1% triton-X100 solution, blocking was performed on the slide, and then immunohistochemical staining was carried out in the same manner as described above. The primary antibodies used in histoimmunostaining were rat anti-BrdU antibody (500x, BIORAD; MCA6144), rabbit anti-NeuN antibody (500x, CellSignaling; 24307S), chicken anti-DCX antibody (200x, abcam; ab153668), mouse Calretinin antibody (250x, ProteinTech; 66496-1-IG), goat anti-NeuroD antibody (NeuroD, SantaCruz; sc-1084), chicken anti-GFAP antibody (500x, abcam; ab4674), rat anti-NESTIN antibody (500x, Wako; 012-26843), goat anti-SOX2 antibody (500x, abcam; ab239218), and rabbit anti-IBA1 antibody (500x, 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). [Examples]

[0022] (Effect of a mixture of three types of bacteria on promoting the proliferation of neural stem cells) Figure 1 shows the results of immunostaining of BrdU-positive cells in the hippocampal dentate gyrus region of mice in each group that received BrdU for three consecutive days, on day 1 after administration. The results shown in Figure 1 confirm that a mixture of three types of bacteria—lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria—promotes the proliferation of neural stem cells (particularly neural stem cells present in the hippocampal dentate gyrus that have proliferative capacity). [Examples]

[0023] (Effect of a mixture of three types of bacteria on promoting the differentiation and maturation of neural stem cells into nerve cells) Figure 2 shows the results of immunostaining of BrdU-positive cells in the hippocampal dentate gyrus region and the expression of NeuN protein, which is specifically expressed in mature neurons, in mice from each group that received BrdU for three consecutive days, 21 days after administration. The results shown in Figure 2 confirm that a mixture of three types of bacteria—lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria—promotes the differentiation and maturation of neural stem cells (particularly neural stem cells present in the hippocampal dentate gyrus) into nerve cells. [Examples]

[0024] (Effect of a mixture of three types of bacteria on promoting the maturation of immature nerve cells) Figure 3 shows 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. The results shown in Figure 3 confirm that a mixture of three types of bacteria—lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria—promotes the maturation of immature nerve cells (particularly immature nerve cells located in the hippocampal dentate gyrus). [Examples]

[0025] (Effect of a mixture of three types of bacteria on promoting the differentiation and maturation of neural stem cells into nerve cells through increased expression of NeuroD1 in neural stem cells) Figure 4 shows 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 from each group. The results shown in Figure 4 confirm that a mixture of three types of bacteria—lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria—promotes the differentiation and maturation of neural stem cells (particularly neural stem cells in the hippocampal dentate gyrus) into neurons through the expression of NeuroD1 in these cells. [Examples]

[0026] (Effect of restoring the number of resting neural stem cells, which is reduced by sterilization using a mixture of three types of bacteria) Figure 5 shows the results of immunostaining of quiescent neural stem cells co-expressing GFAP protein, nestin protein, and SOX2 protein in the hippocampal dentate gyrus region of mice from each group. The results shown in Figure 5 confirm that a mixture of three types of bacteria—lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria—recovers the decrease in the number of quiescent neural stem cells associated with sterilization (increases the number of quiescent neural stem cells). [Examples]

[0027] (Effect of promoting microglial activation by a mixture of three types of bacteria) Figure 6 shows the results of immunostaining of microglia expressing the IBA1 protein in the cerebral cortex of mice from each group. It is known that when microglia are activated, their cell volume decreases. The results shown in Figure 6 confirm that a mixture of three types of bacteria—lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria—promotes the activation of microglia. [Examples]

[0028] (The effect of a mixture of three types of bacteria on promoting astrocyte activation) Figure 7 shows the results of immunostaining of astrocytes expressing GFAP protein in the cerebral cortex of mice from each group. It is known that astrocytes increase in volume when activated. The results shown in Figure 7 confirm that a mixture of three types of bacteria—lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria—promotes the activation of astrocytes. [Examples]

[0029] (Preparation of human neural stem cells and culture supernatants of each bacterium to confirm the effects of each individual bacterium) The results shown in Examples 2-8 are observations of brain cells in mice after orally administering a mixture of the three types of bacteria described in Example 1: lactic acid bacteria, butyrate-producing bacteria, and saccharifying bacteria. It was confirmed using human neural stem cells that each of these three types of bacteria is effective on neural stem cells on its own, and furthermore, that they also show effects on human cells. Human neural stem cells were purchased from Phoenix Songs Biologicals, derived from the human cerebral cortex of a 14-week-old male fetus from whom informed consent was obtained, and the preparation of the test cells was carried out according to the method described in Patent No. 6976600 or International Publication No. WO2019 / 009205. For the preparation of the culture supernatant of each bacterium to be added to the human neural stem cells, each strain was grown in the medium for human neural stem cells described in Patent No. 6976600 or International Publication No. WO2019 / 009205. As the culture method for each strain, Escherichia to be cultivated ATCC8739 (control strain) and saccharifying bacteria ( Bacillus subtle TO-A:FERM BP-07462. Hereinafter BS) were inoculated into L-shaped tubes and cultured at 37 °C under aerobic conditions with linear shaking at 100 - 110 rpm, and lactic acid bacteria ( Enterococcus feces T-110:FERM BP-10867. Hereinafter EF) and butyric acid bacteria ( Clostridium buttermilk TO-A:FERM BP-10866. Hereinafter CB) were statically cultured under anaerobic conditions at 37 °C. For each strain, the culture was terminated when the turbidity of the culture solution reached a steady value or just before that, and the supernatant after centrifugation was sterilized with a 0.2 μm pore size filter. Each test solution in a 10-fold dilution series of this supernatant was added to human neural stem cells, and the effects on the proliferation of neural stem cells shown in Example 10 and the effects on the differentiation into neurons shown in Example 11 were evaluated respectively.

Example

[0030] (Proliferation effect of bacteria alone on human neural stem cells) The presence or absence of the effect of the culture supernatant of each bacterium 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 of the kit. Specifically, 10 -2 ~10 -8After adding the culture supernatant of each bacterial strain diluted in 545, the cells were cultured for 1 day, and then 2.5 μM 5-ethynyl-2′-deoxyuridine (EdU) was added and cultured for 2 hours (see Figure 8A). Subsequently, the cells were fixed with 4% PFA, and after the Click-iT reaction, the cell nuclei were stained with Hoechst 33342. The stained cells were visualized using a fluorescence 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 the culture supernatant of EF significantly improved the proliferative capacity of human neural stem cells. Specifically, EF 10 -5 , 10 -6 and 10 -8 The diluted solution significantly improved the proliferative capacity of human neural stem cells. The control strain E.coli It was confirmed that the culture supernatant could not significantly improve the proliferative capacity of human neural stem cells. [Examples]

[0031] (Effects of each bacterium on the differentiation of human neural stem cells into neurons) The ability of individual bacterial culture supernatants to induce differentiation of human neural stem cells into neurons was evaluated using indirect immunohistochemistry for βIII-tubulin protein, or Tuj-1, which is specifically expressed in differentiated neurons. Specifically, human neural stem cells were cultured for 3 days and then tested for 10 -2 ~10 -8 After adding the culture supernatant of each bacterial strain diluted to 400ml and culturing for 7 days, the cells were fixed with 4% PFA and stained using an antibody against Tuj-1 (R&D Systems; MAB1195) as the primary antibody and CF568 anti-mouse IgG antibody (500-fold dilution, Biotium; 20105) as the secondary antibody, and the cell nuclei were also stained with Hoechst 33342 (see Figure 9A). The stained cells were visualized using a fluorescence microscope, and the percentage of cells differentiated into 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 bacterial strains, human neural stem cells (0.6 x 10⁶) cultured in 24-well plates were used. 5All samples were evaluated simultaneously in a single plate using a sample size (individual samples / well). The results of the primary and secondary evaluations are shown in Figure 9. As shown in the results in Figure 9B, E. coli It was confirmed that the culture supernatants of all other monocellular strains of BS, EF, and CB significantly enhanced the differentiation induction of human neural stem cells into neurons. Specifically, BS 10 -7 Diluted solution of EF 10 -6 Diluted solution of and 10 of CB -8 The diluted solution significantly enhanced the induction of differentiation of human neural stem cells into neurons. The control strain E.coli It was confirmed that the culture supernatant could not significantly improve the proliferative capacity of human neural stem cells. [Examples]

[0032] (Memory and learning effect of CB strains) Nematodes Caenorhabditis elegant It is known that nematodes can remember and learn the salt concentration in their environment. For example, nematodes that have been starved will remember and learn the salt concentration in their rearing environment at that time, and will avoid environments with the salt concentration they have experienced while starved. Taking advantage of this characteristic, for four days from the third to the seventh day of rearing, the standard food is Escherichia to be cultivated Nematode groups fed only the OP50 strain (normal diet group), CB E. coli A group of nematodes (CB test group) was established that ingested the OP50 strain together, and changes in their behavior to avoid salt concentrations when they were in a state of starvation were observed. The test method and evaluation method were based on the method 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 concentration gradient for agar plates used for nematode evaluation, a cylindrical (5 mm in diameter) 100 mM NaCl-containing agar plate was cut as shown in Figure 10A-1, placed on one end of the plate, and left to stand for 24 hours. To enable nematodes to remember and learn about starvation and the presence or absence of salt in their rearing environment at that time, two types of treatments shown in Figure 10A-2 were performed, resulting in nematodes that were not subjected to starvation (Naive treatment) and nematodes that were subjected to starvation in the presence of salt (NaCl treatment). The behaviors associated with memory and learning were observed using the evaluation agar plates and nematodes that had undergone each treatment. Specifically, nematodes were placed in the center of the evaluation agar plate and left to stand for 1 hour at 25°C. Then, as shown in Figure 10A-1, the number of nematodes in each area of ​​high salt concentration (A), intermediate concentration (C), and low salt concentration (B) was counted, 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 were attracted to the salt, while a value closer to -1 indicates that the nematodes avoided the salt. Furthermore, since NGM medium, commonly used for the normal rearing of nematodes, including in this embodiment, contains 50 mM NaCl, it is known that naive-treated nematodes are attracted to salt. In addition, as a salt concentration chemotactic index, it is a reasonable response that NaCl-treated nematodes that have experienced starvation in the presence of salt will have a lower value than naive-treated nematodes. As shown in Figure 10B, the results of the salt concentration chemotaxis index in this example showed no difference between the CB test group and the normal feed group in naive-treated nematodes. However, in NaCl-treated nematodes, the CB test group showed a lower value than the normal feed group, meaning that the CB test group tended to exhibit stronger avoidance behavior towards salt concentration when starved than the normal feed group. This result indicates that CB intake maintains and / or improves memory and learning ability.

[0033] Based on the results of Examples 2 to 11 described above, the composition of this disclosure has an effect of promoting adult neurogenesis. More specifically, adult neurogenesis refers to the phenomenon in which differentiation from neural stem cells into mature neurons occurs even in adults, after the fetal period in which the majority of neurons are formed. In this invention, from the perspective of adult neurogenesis, we investigated the factors expressed in each stage of neurogenesis 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 of differentiation, was confirmed by the amount of BrdU uptake. In Example 3, the number of cells expressing NeuN, which is characteristic of mature neurons, at the final stage of differentiation, increased significantly. Thus, the composition of this disclosure was confirmed to promote adult neurogenesis. To elaborate further, we confirmed in Examples 5 and 4, respectively, an increase in the expression of NeuroD, which is expressed during the differentiation process into nerve cells, and the subsequent increase in cells expressing Calretinin. Furthermore, in Example 6, it was confirmed that the composition of this disclosure promotes adult neurogenesis beyond the conventional perspective, specifically increasing the number of resting neural stem cells that form the basis of adult neurogenesis. On the other hand, in Examples 7 and 8, it was confirmed that the composition of this disclosure not only promotes differentiation into neurons as described above, but also promotes the activation of glial cells, namely microglia and astrocytes. The effects of each bacterium individually are shown in Examples 9 to 11. Example 10 confirmed that lactic acid bacteria alone promote the proliferation of human neural stem cells. In addition, Example 11 confirmed that any of the saccharifying bacteria, lactic acid bacteria, and butyrate-producing bacteria individually promote the differentiation of human neural stem cells into neurons.

[0034] Based on the results of Examples 2 to 12 described above, the compositions of this disclosure have an effect on maintaining brain function homeostasis. For more details, Non-Patent Document 9 classifies the morphological changes of microglia in detail, and Non-Patent Document 10 describes the functions of microglia in maintaining homeostasis. Specifically, microglia control axonal binding, programmed cell death, neurite formation, synaptic homeostasis ("pruning"), and synapse formation, playing an important role in maintaining the normal function of nerve cells in the brain (brain function homeostasis maintenance effect). Furthermore, microglia control the proliferation and differentiation of neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs), promote myelin formation, 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 the health of the central nervous system, and are responsible for the circulation of neurotransmitters, support for nerve cell metabolism, and maintenance of the blood-brain barrier. Their interactions with microglia are described in detail in Non-Patent Document 11. In detail, microscopic observations (changes in cell area) of Examples 7 and 8 using the composition of this invention revealed activation of microglia and astrocytes, respectively, confirming its potential for maintaining homeostasis of brain function. Furthermore, the results from Example 12 confirmed that at least the intake of butyrate-producing bacteria maintains and / or improves memory and learning ability. [Industrial applicability]

[0035] This disclosure provides 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

1. A composition for promoting adult neurogenesis, comprising Enterococcus bacteria (a lactic acid bacterium), Clostridium bacteria (a butyrate-producing bacterium), and Bacillus bacteria (a saccharifying bacterium).

2. The composition according to claim 1, wherein the lactic acid bacteria is of the species Enterococcus faecium or Enterococcus lactis, the butyric acid bacteria is of the species Clostridium butyricum, and the saccharifying bacteria is of the species Bacillus subtilis.

3. The composition according to claim 1 or 2, wherein the lactic acid bacteria is Enterococcus faecium T-110, the butyric acid bacteria is Clostridium butyricum TO-A, and the saccharifying bacteria is Bacillus subtilis TO-A.

4. The composition according to claim 1 or 2, wherein one or more of the aforementioned bacteria are viable bacteria.

5. The composition according to claim 1 or 2, wherein one or more of the aforementioned bacteria is a bacterial culture solution.

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