JP2024069369A - Composition for improving neurite outgrowth - Google Patents

Abstract

The present invention aims to provide a composition that can be used for specific purposes, such as a composition for improving neurite outgrowth. The present invention relates to a composition that contains indole-3-lactic acid (ILA) or indole-3-lactic acid-producing bacteria as an active ingredient.

Description

The present invention relates to compositions that can be used for specific purposes, such as compositions for improving neurite outgrowth, promoting neuronal differentiation, or for obtaining effects based thereon.

Tryptophan is an essential amino acid with an indole ring that is derived from food proteins. Tryptophan is mainly digested and absorbed in the small intestine, but a significant amount of tryptophan remains in the large intestine, where it is metabolized by intestinal bacteria to produce various indole derivatives such as indole-3-lactic acid (ILA) and indole-3-propionic acid (IPA) (Non-Patent Document 1).

IPA has been found to have a strong neuroprotective effect against β-amyloid in Alzheimer's disease (Non-Patent Document 2) and against neuronal damage and oxidative stress in the ischemic hippocampus (Non-Patent Document 3). However, the effect of ILA on the health of neurons remains to be investigated in the future.

ILA and IPA may be produced by certain intestinal bacterial species. For example, strains of Bifidobacterium species commonly isolated from the intestines of human infants (e.g., Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, and Bifidobacterium bifidum) can produce high concentrations of ILA (Non-Patent Document 4).

Roager, H. M. & Licht, T. R. Microbial tryptophan catabolites in health and disease. Nat. Commun. 9, 1-10 (2018). Chyan, Y.-J. et al. Potent neuroprotective properties against the Alzheimer β-amyloid by an endogenous melatonin-related indole structure, indole-3-propionic acid. J. Biol. Chem. 274, 21937-21942 (1999). Hwang, I. K. et al. Indole-3-propionic acid attenuates neuronal damage and oxidative stress in the ischemic hippocampus. J. Neurosci. Res. 87, 2126-2137 (2009). Sakurai T. et al. Production of Indole-3-Lactic Acid by Bifidobacterium Strains Isolated from Human Infants. 2019 Sep 11;7(9).

The present invention aims to provide a composition that can be used for specific applications, such as a composition for improving neurite outgrowth, promoting neuronal differentiation, or obtaining effects based thereon. The present invention can also aim to provide means and methods for neuroprotection in vivo and in vitro.

The present inventors have conducted intensive research to solve the above problems. As a result, the inventors have discovered that indole-3-lactic acid (ILA) improves neurite outgrowth, and have completed the present invention.

That is, the present invention can be exemplified as follows.
[1]
A composition for promoting neuronal differentiation and/or improving neurite outgrowth, comprising the following component (A) and/or (B) as an active ingredient:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[2]
A composition for preventing, improving, and/or treating symptoms associated with neuropathy, comprising the following component (A) and/or (B) as an active ingredient:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[3]
The composition, comprising at least the component (B).
[4]
The composition, wherein the component (B) is a bacterium of the genus Bifidobacterium.
[5]
The composition, wherein the component (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.
[6]
The composition, which is a food or beverage composition.
[7]
The composition, wherein the composition is a pharmaceutical composition.
[8]
A method for promoting neuronal differentiation and/or improving neurite outgrowth, comprising administering to a subject the following components (A) and/or (B) as active ingredients:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[9]
A method for preventing, ameliorating, and/or treating symptoms associated with neuropathy, comprising administering to a subject the following ingredient (A) and/or (B) as an active ingredient:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[10]
The method, wherein at least said component (B) is administered to said subject.
[11]
The method as described above, wherein the component (B) is a bacterium belonging to the genus Bifidobacterium.
[12]
The method as described above, wherein the component (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.
[13]
The method, wherein the subject is a human.
[14]
Use of the following components (A) and/or (B) for promoting neuronal differentiation and/or improving neurite outgrowth:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[15]
Use of the following components (A) and/or (B) for preventing, improving and/or treating symptoms associated with neuropathy:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[16]
Use of the following components (A) and/or (B) in the preparation of said composition:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[17]
A composition for use in promoting neuronal differentiation and/or improving neurite outgrowth, comprising the following components (A) and/or (B) as active ingredients:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[18]
A composition for use in preventing, ameliorating, and/or treating symptoms associated with neuropathy, comprising the following components (A) and/or (B) as active ingredients:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.
[19]
The composition for use according to the above-mentioned composition for use, wherein the composition contains at least the component (B).
[20]
The composition for use according to the above-mentioned composition for use, wherein the component (B) is a bacterium of the genus Bifidobacterium.
[21]
The composition for use according to the above-mentioned composition for use, wherein the component (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.
[22]
The composition for use described in the composition for use, wherein the composition is a food or beverage composition.
[23]
The composition for use according to the above-mentioned composition for use, wherein the composition is a pharmaceutical composition.

Effect of 1 nM, 10 nM, 100 nM, and 1 μM tryptophan and its metabolites on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. PC12 cells were treated with NGF (25 ng/mL) and test substances (ILA, IPA, or Trp) for 5 consecutive days. (A) Percentage of neurite-bearing cells in PC12 cells. (B) Acetylcholinesterase (AchE) activity in PC12 cells. Data are the mean ± SD of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NGF-treated control. NTC: untreated control; PC: positive control; ILA: indole-3-lactic acid; IPA: indole-3-propionic acid; Trp: tryptophan. Effect of tryptophan and its metabolites on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. PC12 cells were treated with NGF (25 ng/mL) and ILA, IPA, or Trp (100 nM) for 5 consecutive days. (A) Percentage of cells with neurites in PC12 cells. (A) 100x magnification of immunostaining of βIII-tubulin (green) in PC12 cells. Scale bar, 100 μm. Nuclei were counterstained with DAPI (blue). (B) Percentage of cells with neurites in PC12 cells. (C) Acetylcholinesterase (AchE) activity in PC12 cells. Data are the mean ± SD of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NGF-treated control. NTC indicates a non-treated control, PC indicates a positive control, ILA indicates indole-3-lactic acid, IPA indicates indole-3-propionic acid, and Trp indicates tryptophan. Figure (photo) showing the effect of tryptophan and its metabolites on phosphorylation of TrkA, ERK1/2, and CREB in PC12 cells. Phosphorylation of TrkA, ERK1/2, and CREB in PC12 cells treated with NGF (25 ng/mL) and ILA, IPA, or Trp (100 nM) for 24 h was detected by Western blot analysis. Data are the mean ± SD of triplicate experiments. *P < 0.05, **P < 0.01 vs. NGF-treated control. NTC: untreated control, PC: positive control, ILA: indole-3-lactic acid, IPA: indole-3-propionic acid, Trp: tryptophan. Effect of tryptophan and its metabolites on the aryl hydrocarbon receptor (AhR) in PC12 cells. PC12 cells were pretreated with the AhR antagonist α-naphthoflavone (ANF; 1 μM) for 1 h and then continuously treated with NGF (25 ng/mL) and ILA, IPA, or Trp (100 nM) for 5 days. Non-pretreated PC12 cells served as null control. (A) AhR protein (95 kDa) in PC12 cells was detected by Western blot analysis using a monoclonal antibody specific for AhR. The corresponding blot of β-actin served as a loading control. (B) Acetylcholinesterase (AchE) activity in PC12 cells. Data are shown as the mean ± SD of triplicate experiments. *P < 0.05, **P < 0.01 vs. NGF-treated control. NTC indicates a non-treated control, PC indicates a positive control, ILA indicates indole-3-lactic acid, IPA indicates indole-3-propionic acid, and Trp indicates tryptophan. Effect of tryptophan and its metabolites on the aryl hydrocarbon receptor (AhR) in PC12 cells. PC12 cells were pretreated with the AhR antagonist CH223191 (1 μM) for 1 h and then continuously treated with NGF (25 ng/mL) and ILA, IPA, or Trp (100 nM) for 5 days. Non-pretreated PC12 cells served as null control. (A) AhR protein (95 kDa) in PC12 cells was detected by Western blot analysis using a monoclonal antibody specific for AhR. The corresponding blot of β-actin served as a loading control. (B) Acetylcholinesterase (AchE) activity in PC12 cells. Data are the mean ± SD of triplicate experiments. *P < 0.05, **P < 0.01 vs. NGF-treated control. NTC indicates a non-treated control, PC indicates a positive control, ILA indicates indole-3-lactic acid, IPA indicates indole-3-propionic acid, and Trp indicates tryptophan.

The present invention is described in detail below.

<1> Active Ingredient In the present invention, the following components (A) and/or (B) are used as active ingredients: (A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.

That is, components (A) and/or (B) are also referred to as "active ingredients". For example, at least component (B) may be used as the active ingredient. The active ingredient can be used in vivo or in vitro. In particular, the active ingredient can be used in vivo, i.e., the active ingredient can be used by administering it to a subject.

By utilizing the active ingredient, specifically by administering the active ingredient to a subject, neurite outgrowth may be improved in the subject, i.e., an effect of improving neurite outgrowth may be obtained. This effect is also referred to as a "neurite outgrowth improving effect." Examples of neurite outgrowth include NGF-induced neurite outgrowth. NGF may be produced by the subject to which the active ingredient is administered. That is, the NGF produced by the subject may function together with the active ingredient. Thus, it is not necessary to administer NGF to the subject in addition to the active ingredient described herein. The neurite outgrowth improving effect can be confirmed, for example, by confirming an increase in the amount of neurite-bearing cells in a sample. In other words, the neurite outgrowth improving effect can be confirmed, for example, when the amount of neurite-bearing cells in a sample after administration of the active ingredient is greater than that before administration of the active ingredient. The "amount of neurite-bearing cells" may mean the absolute amount of neurite-bearing cells or the relative amount of neurite-bearing cells. The relative amount of neurite-bearing cells can be calculated, for example, as the ratio of the number of neurite-bearing cells to the total number of cells in a sample. The relative amount of cells with neurites can be calculated as the ratio of the number of cells with neurites after the use of the active ingredient to the total number of cells in the sample, and the effect of improving neurite outgrowth can be determined by comparing the relative amount of cells with neurites before and after the use of the active ingredient. In another embodiment, the absolute amount or relative amount of cells with neurites can be referenced using the absolute or relative number of cells with neurites from a reference subject (particularly a subject not using the active ingredient). The reference subject can be a group of subjects for which the average or median value is used as a reference. "Cells with neurites" can refer to cells that present a process that is at least 1.5 times the length of the cell body. "Cells with neurites" can refer to nerve cells (i.e., neurons) that present a process that is at least 1.5 times the length of the cell body. Processes include neurites, axons, and dendrites. The sample is not particularly limited as long as it contains nerve cells. The sample can be obtained, for example, by biopsy from a subject.

Improvement in neurite outgrowth may be an indicator of neuronal differentiation. Thus, by utilizing an active ingredient, specifically by administering the active ingredient to a subject, neuronal differentiation may be promoted, i.e., an effect of promoting neuronal differentiation may be obtained. This effect is also referred to as a "neuronal differentiation promoting effect." In other words, improvement in neurite outgrowth may be obtained by promoting neuronal differentiation. That is, the neurite outgrowth improving effect may be an example of a neuronal differentiation promoting effect. The neuronal differentiation promoting effect can be confirmed, for example, by confirming the neurite outgrowth improving effect.

The neuronal differentiation promoting effect can also be confirmed, for example, by confirming an increase in acetylcholinesterase (AchE) activity in the subject. In other words, the neuronal differentiation promoting effect can be confirmed, for example, when the AchE activity in a sample after administration of the active ingredient is greater than that before administration of the active ingredient. In this regard, the activity is preferably determined in samples obtained from the same subject before and after use of the active ingredient. However, the activity before use of the active ingredient can also be a reference activity obtained from a subject or group of subjects not using the active ingredient. The AchE activity can be measured, for example, using an Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA). The sample is not particularly limited as long as it contains nerve cells. The sample can be obtained, for example, by biopsy from the subject.

In addition, by utilizing the active ingredient, specifically by administering the active ingredient to a subject, an effect based on the promotion of neuronal differentiation and/or the improvement of neurite outgrowth may be provided. By the promotion of neuronal differentiation and/or the improvement of neurite outgrowth, for example, neuronal function may be protected and/or improved. That is, by the promotion of neuronal differentiation and/or the improvement of neurite outgrowth, for example, symptoms related to nerve disorder may be prevented, improved, and/or treated. That is, effects based on the promotion of neuronal differentiation and/or the improvement of neurite outgrowth include an effect of protecting nerve function, an effect of improving nerve function, and an effect of preventing, improving, and/or treating symptoms related to nerve disorder. Symptoms related to nerve disorder may or may not be a disease. In other words, symptoms related to nerve disorder may or may not be caused by a disease. Symptoms related to nerve disorder include neurodegenerative diseases, in other words, symptoms caused by neurodegenerative diseases. Neurodegenerative diseases include diseases accompanied by disorders of nerve cell differentiation or disorders of neurite outgrowth. Specific examples of neurodegenerative diseases include Alzheimer's disease (AD), dementia (dementia with Lewy bodies (DLB) and the like), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Huntington's disease, dystonia, transmissible spongiform encephalopathy (TSE), chorea-acanthocytosis (ChAc), adrenoleukodystrophy (ALD), multiple system atrophy (MSA), spinocerebellar degeneration (SCD), amyotrophic lateral sclerosis (AMSC), and cerebellar degeneration (CCD). These include amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal and bulbar muscular atrophy (SBMA), spinal muscular atrophy (SMA), Charcot-Marie-Tooth disease (CMT), and Batten disease.

It has been reported that the tryptophan metabolite kynurenine can cross the blood-brain barrier (Psychoneuroendocrinology. 2018;94:1-10. doi:10.1016/j.psyneuen.2018.04.019.). In the present invention, it is expected that the ILAs described herein can cross the blood-brain barrier.

In the above context and in light of the accompanying illustrative examples, the present invention also relates to the use of indole-3-lactic acid and/or indole-3-lactic acid producing bacteria in the prevention, amelioration and/or treatment of neurological disorders, such as neurodegenerative diseases. These diseases may include, among others: Alzheimer's disease (AD), dementia (such as dementia with Lewy bodies (DLB)), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Huntington's disease, dystonia, transmissible spongiform encephalopathy (TSE), chorea-acanthocytosis (ChAc), adrenoleukodystrophy (ALD), multiple system atrophy (MSA), spinocerebellar degeneration (BSA), and/or cerebellar degeneration (CBD). In a preferred embodiment of the present invention, the prevention, amelioration, and/or treatment of neurological disorders is associated with the promotion of neuronal differentiation, as surprisingly shown in the present specification and in the accompanying examples for indole-3-lactic acid (ILA). Thus, in the context of the present invention, it has surprisingly been found that indole-3-lactic acid (and/or indole-3-lactic acid-producing bacteria) exhibits a neuronal differentiation promoting effect, which is believed to ameliorate and/or improve the symptoms of neurological disorders such as neurodegenerative diseases. Such neuronal differentiation promoting effects are disclosed herein and illustrated in the accompanying technical examples by the surprising effects of indole-3-lactic acid (ILA) in neuronal differentiation and/or neurite outgrowth. Thus, in the context of the present invention, the present specification provides the use of indole-3-lactic acid (ILA) in the treatment and/or prevention of neurological disorders, such as neurodegenerative disorders.
Medical uses of lactic acid (ILA) and/or indole-3-lactic acid producing bacteria are disclosed. Indole-3-lactic acid (ILA) and/or indole-3-lactic acid producing bacteria can in this context be administered to a subject in need of prevention, amelioration and/or treatment of neurological disorders such as neurodegenerative diseases. Preferably, the subject is a mammal such as a primate such as a human, monkey, chimpanzee, or a mammal such as a horse, cow, sheep, goat, pig, dog, cat, etc. However, most preferably, the subject treated by the means and methods of the present invention is a human.

The above effects (such as the effect of improving neurite outgrowth, the effect of promoting neuronal differentiation, and effects based on these) are collectively referred to as "neuro-promoting effects."

The present invention may provide for the use of an active ingredient to obtain a neuropromoting effect. That is, the present invention may provide for the use of an active ingredient for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, ameliorating, and/or treating symptoms associated with neurological disorders. The present invention may also provide for the use of an active ingredient for the manufacture of a composition for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, ameliorating, and/or treating symptoms associated with neurological disorders.

The present invention may provide an active ingredient used to obtain a neuropromoting effect. That is, the present invention may provide an active ingredient used to promote neuronal differentiation, improve neurite outgrowth, and/or prevent, improve, and/or treat symptoms associated with neurological disorders. The present invention may also provide an active ingredient used for the manufacture of a composition for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improve, and/or treat symptoms associated with neurological disorders.

The active ingredient may be used for therapeutic or non-therapeutic purposes. That is, the neurostimulatory effect may be obtained for therapeutic or non-therapeutic purposes, unless otherwise specified.

"Therapeutic purposes" may refer, for example, to a concept including medical procedures, and more specifically, to a concept including therapeutic treatment of the human body.

"Non-therapeutic purposes" may refer, for example, to a concept that does not include medical procedures, and more specifically, to a concept that does not include therapeutic treatment of the human body. Examples of non-therapeutic purposes include health promotion and cosmetic purposes.

"Prevention of a symptom or disease" may mean, for example, preventing and/or delaying the onset of a symptom or disease, or reducing the likelihood of onset of a symptom or disease. "Amelioration of a symptom or disease" or "treatment of a symptom or disease" may mean, for example, improvement of a symptom or disease, prevention or delay of a symptom or disease from worsening, or prevention or delay of a symptom or disease from progressing. "Amelioration of a symptom or disease" may mean, in particular, these events, and which are obtained for non-therapeutic purposes. "Treatment of a symptom or disease" may mean, in particular, these events, and which are obtained for therapeutic purposes.

In order to obtain a neuropromoting effect (neurite outgrowth improving effect, neuronal differentiation promoting effect, and effects based thereon, etc.), ILA may be significantly superior to indole-3-propionic acid (IPA). Similarly, in order to obtain a neuropromoting effect (neurite outgrowth improving effect, neuronal differentiation promoting effect, and effects based thereon, etc.), ILA-producing bacteria may be significantly superior to IPA (this is because ILA that can be produced by ILA-producing bacteria may be significantly superior to IPA in order to obtain a neuropromoting effect). Similarly, in order to obtain a neuropromoting effect (neurite outgrowth improving effect, neuronal differentiation promoting effect, and effects based thereon, etc.), both ILA and ILA-producing bacteria may be significantly superior to IPA-producing bacteria (this is because ILA may be significantly superior to IPA that can be produced by IPA-producing bacteria in order to obtain a neuropromoting effect). For example, the active ingredient may be superior to IPA or IPA-producing bacteria in promoting neurite outgrowth (particularly NGF-induced neurite outgrowth), promoting NGF-induced neurite outgrowth (particularly through promoting TrkA phosphorylation), promoting NGF-induced neurite outgrowth (particularly through promoting ERK signaling), or for obtaining effects based thereon. The superiority of such an active ingredient may be based, for example, on ILA and IPA as ligands of the aryl hydrocarbon receptor (AhR). That is, the active ingredient may exert such an advantage because the action of ILA and IPA appears to be exerted through binding to AhR, and ILA is a preferential AhR ligand over IPA.

ILA may be used in the form of a free form, a salt thereof, or a mixture thereof. In other words, unless otherwise specified, "indole-3-lactic acid (ILA)" may mean ILA in the form of a free form, a salt thereof, or a mixture thereof. The salt is not particularly limited as long as the neurostimulatory effect is achieved. Examples of the salt include ammonium salts, sodium salts, and potassium salts. As the salt, one type of salt may be used, or two or more types of salts may be used in combination.

As the ILA, a commercially available product may be used, or one obtained by appropriate production may be used. The method for producing ILA is not particularly limited, and for example, a known method may be used. ILA can be produced, for example, by chemical synthesis, enzyme reaction, extraction, or fermentation. Specifically, ILA can be produced, for example, by culturing ILA-producing bacteria. ILA may be purified to a desired degree, or it may not be. That is, as the ILA, a purified product may be used, or a material containing ILA may be used. Examples of materials containing ILA include a culture solution obtained by culturing ILA-producing bacteria, a culture supernatant separated from the culture solution, and/or processed products thereof (concentrates thereof (e.g., concentrates), dried concentrates thereof, fractions thereof, etc.). The ILA concentration in the material may be, for example, 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 30% by weight or more, 50% by weight or more, 70% by weight or more, or 90% by weight or more.

When a material containing ILA is used, the amount of ILA (e.g., the content in the composition of the present invention or the dosage in the method of the present invention) is calculated based on the amount of ILA itself contained in the material. When ILA is used in the form of a salt, the amount of ILA (e.g., the content in the composition of the present invention or the dosage in the method of the present invention) is calculated based on the mass of the salt converted to the equimolar mass of the free form.

"Indole-3-lactic acid producing bacteria (ILA producing bacteria)" refers to bacteria capable of producing ILA. The ILA producing bacteria may, for example, produce ILA from a carbon source and/or tryptophan. The ILA producing bacteria may specifically be bacteria capable of producing ILA in a subject when administered to the subject. More specifically, the ILA producing bacteria may be bacteria capable of producing ILA in the intestine of a subject when administered to the subject. The intestine may include the small intestine and large intestine.

Examples of ILA-producing bacteria include those of the genus Bifidobacterium. Bifidobacterium bacteria include Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum. thermophilum). Bifidobacterium bacteria include, in particular, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum. As the ILA-producing bacteria, one type of bacteria may be used, or two or more types of bacteria may be used in combination.

"Bifidobacterium longum" includes strains classified into any subspecies of Bifidobacterium longum, such as B. longum subsp. longum, B. longum subsp. infantis, and B. longum subsp. suis. "Bifidobacterium animalis" includes strains classified into any subspecies of Bifidobacterium animalis, such as B. animalis subsp. lactis. "Bifidobacterium pseudolongum" includes strains classified into any subspecies of Bifidobacterium pseudolongum, such as B. pseudolongum subsp. globosum and B. pseudolongum subsp. pseudolongum.

It has been reported that infant-type human-residential bifidobacteria (HRB) produce significantly higher concentrations of ILA than adult-type HRB and non-HRB (Non-patent document 4: Sakurai et al, Production of Indole-3-Lactic Acid by Bifidobacterium Strains Isolated from Human Infants. 2019 Sep 11;7(9).). Thus, an example of an ILA-producing bacterium used herein may be, in particular, infant-type HRB. Examples of infant-type HRB include Bifidobacterium longum (Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, etc.), Bifidobacterium breve, and Bifidobacterium bifidum.

Bifidobacterium longum, specifically, BB536 (NITE BP-02621), ATCC
Examples of Bifidobacterium longum include ATCC 15697, ATCC 15707, ATCC 25962, ATCC 15702, ATCC 27533, M-63 (NITE BP-02623), BG7, DSM 24736, SBT 2928, NCC 490 (CNCM I-2170), and NCC 2705 (CNCM I-2618). Examples of Bifidobacterium longum include BB536, ATCC 15697, ATCC 15707, and M-63. Examples of Bifidobacterium longum include BB536. Examples of Bifidobacterium longum include one type of strain, or two or more types of strains in combination.

Specific examples of Bifidobacterium breve include M-16V (NITE BP-02622), MCC1274 (FERM BP-11175), ATCC 15700, B632 (DSM 24706), Bb99 (DSM 13692), ATCC 15698, DSM 24732, UCC2003, YIT4010, YIT4064, BBG-001, BR-03, C50, and R0070. Specific examples of Bifidobacterium breve include M-16V, MCC1274, and ATCC 15700. Specific examples of Bifidobacterium breve include M-16V. Specific examples of Bifidobacterium breve include one type of strain, or two or more types of strains in combination.

Bifidobacterium bifidum specifically includes ATCC 29521, NITE BP-02429, NITE BP-02431, OLB6378, and BF-1. Bifidobacterium bifidum specifically includes ATCC 29521, NITE BP-02429, and NITE BP-02431. Bifidobacterium adolescentis specifically includes ATCC 15703. Bifidobacterium dentium specifically includes DSM 20436. Bifidobacterium animalis specifically includes DSM 10140, Bb-12, DN-173 010, GCL2505, and CNCM I-3446. Specific examples of Bifidobacterium pseudolongum include JCM 5820 and ATCC 25526. Specific examples of Bifidobacterium thermophilum include ATCC 25525.

Bifidobacterium longum subsp. longum BB536 (NITE BP-02621) was internationally deposited on January 26, 2018 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD; postal code: 292-0818, address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) based on the Budapest Treaty, and has been assigned the deposit number NITE BP-02621.

Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623) was internationally deposited on January 26, 2018 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD; postal code: 292-0818, address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) based on the Budapest Treaty and has been assigned the deposit number NITE BP-02623.

Bifidobacterium breve M-16V (NITE BP-02622) was internationally deposited on January 26, 2018 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD; postal code: 292-0818, address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) based on the Budapest Treaty and assigned the deposit number NITE BP-02622.

Bifidobacterium breve MCC1274 (FERM BP-11175) was internationally deposited on August 25, 2009 at the National Institute of Advanced Industrial Science and Technology (currently the National Institute of Technology and Evaluation Patent Organism Depositary (IPOD), Postal Code: 292-0818, Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) based on the Budapest Treaty and has been assigned the deposit number FERM BP-11175.

Bifidobacterium bifidum NITE BP-02429 was internationally deposited on February 21, 2017 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD; postal code: 292-0818, address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) based on the Budapest Treaty and assigned the deposit number NITE BP-02429.

Bifidobacterium bifidum NITE BP-02431 was internationally deposited on February 21, 2017 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD; postal code: 292-0818, address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) based on the Budapest Treaty and assigned the deposit number NITE BP-02431.

These strains are available from, for example, the American Type Culture Collection (ATCC, Address: 10801
Microorganisms can be obtained from the Belgian Coordinated Collections of Microorganisms (BCCM, Address: Rue de la Science 8, 1000 Brussels, Belgium), or from the depository institutions where the respective strains have been deposited.

The strains specified by the above-mentioned examples of strain names are not limited to the strains deposited or registered with a specified institution under the strain names (hereinafter, for convenience of explanation, also referred to as "deposited strains"), but also include strains substantially equivalent to the deposited strains (hereinafter, also referred to as "derived strains"). That is, for example, "Bifidobacterium longum BB536" is not limited to the strain deposited with the above-mentioned depository institution under the deposit number NITE BP-02621, but also includes strains substantially equivalent to the deposited strain. "Strains substantially equivalent to the deposited strain" refers to strains that belong to the same species as the deposited strain, can produce ILA, and have an identity of preferably 99.86% or more, more preferably 99.93% or more, and even more preferably 100% to the nucleotide sequence of the 16SrRNA gene of the deposited strain, and preferably have the same biological properties as the deposited strain. A strain substantially equivalent to the deposited strain may be, for example, a derivative strain obtained using the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains naturally occurring from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include irradiation with X-rays, irradiation with ultraviolet rays, and treatment with mutagens (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), etc.). Strains naturally occurring from the deposited strain include strains naturally occurring during use of the deposited strain. Use of the deposited strain includes culturing (e.g., subculture) of the deposited strain. A derivative strain may be constructed by one type of modification, or by two or more types of modifications.

As the ILA-producing bacteria, commercially available products may be used, or those obtained by appropriate production may be used. Commercially available products include Bifidobacterium longum subsp. longum BB536, Bifidobacterium breve M-16V, and Bifidobacterium longum subsp. infantis M-63, all manufactured by Morinaga Milk Industry Co., Ltd.

The term "indole-3-lactic acid producing bacteria (ILA producing bacteria)" as an active ingredient may specifically mean the bacterial cells of the ILA producing bacteria. The bacterial cells of the ILA producing bacteria can be easily obtained by culturing the ILA producing bacteria. The culturing method is not particularly limited as long as the ILA producing bacteria can grow. As the culturing method, for example, a method normally used for culturing Bifidobacterium genus bacteria can be used as is or with appropriate modifications. The culturing temperature may be, for example, 25 to 50°C, preferably 35 to 42°C. The culturing may be preferably performed under anaerobic conditions, for example, while aerating with anaerobic gas such as carbon dioxide gas. Alternatively, the culturing may be performed under microaerobic conditions such as liquid static culture. The culturing may be performed, for example, until the ILA producing bacteria grow to a desired extent.

The medium used for the culture is not particularly limited as long as the ILA-producing bacteria can produce ILA, and preferably as long as the ILA-producing bacteria can produce ILA and grow. As the medium, for example, a medium normally used for culturing Bifidobacterium bacteria can be used as is or after appropriate modification. The medium may contain, for example, a carbon source, a nitrogen source, an inorganic salt, an organic component, a milk component, or a combination thereof. Examples of carbon sources include sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysates, and blackstrap molasses, and any of these sugars can be used depending on the assimilation ability of the ILA-producing bacteria. Examples of nitrogen sources include ammonia, ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium nitrate, or nitrates. Examples of inorganic salts include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Examples of organic components include peptone, soy flour, defatted soybean meal, meat extract, and yeast extract. Examples of milk components include milk proteins. Examples of milk proteins include casein, whey, and their hydrolyzates. These components may be used alone or in appropriate combinations. Specific examples of media that can be used to culture ILA-producing bacteria include Reinforced Clostridial medium, de Man, Rogosa, and Sharpe medium (MRS medium), modified MRS medium (mMRS medium), TOS propionate medium (TOSP medium), and TOS propionate mupirocin medium (TOSP Mup medium).

As the ILA-producing bacteria, the ILA-producing bacteria itself or a fraction containing the ILA-producing bacteria can be used. That is, as the ILA-producing bacteria, for example, the culture solution of the ILA-producing bacteria may be used as it is, or the bacteria collected from the culture solution may be used. In addition, the bacteria or a fraction containing the bacteria may be subjected to a treatment before use. The treatment is not particularly limited as long as it does not impair the neuropromoting effect. The treatment may preferably be one that does not significantly reduce the survival rate of the bacteria. Examples of the treatment include dilution, concentration, freezing, and drying. That is, examples of the ILA-producing bacteria (specifically, the bacteria of the ILA-producing bacteria) include the culture solution of the ILA-producing bacteria, the bacteria collected from the culture solution, and processed products thereof (dilutions, concentrates, frozen products, dried products, etc.). Examples of the treatment include freezing of the culture solution, spray drying, freeze drying, and the oil drop method. The bacteria contain live bacteria so that ILA is produced after administration. The bacteria may or may not contain dead bacteria.

As an active ingredient, for example, a fraction containing both ILA and ILA-producing bacteria may be used. For example, a culture medium of ILA-producing bacteria or a processed product thereof may contain both ILA and ILA-producing bacteria.

<2> Composition of the Present Invention The composition of the present invention is a composition containing an active ingredient.

That is, the composition of the present invention is a composition containing the following components (A) and/or (B):
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.

The composition of the present invention can be administered to a subject. Specifically, the composition of the present invention can be administered to a subject as described in the method of the present invention. The composition of the present invention can be used, for example, to obtain a neurostimulatory effect.

By utilizing the composition of the present invention, specifically by administering the composition of the present invention to a subject, neurite outgrowth may be improved in the subject, i.e., a neurite outgrowth improving effect may be obtained. That is, the composition of the present invention may be, for example, a composition for improving neurite outgrowth.

By using the composition of the present invention, specifically by administering the composition of the present invention to a subject, neuronal differentiation may be promoted, i.e., a neuronal differentiation promoting effect may be obtained. That is, the composition of the present invention may be, for example, a composition for promoting neuronal differentiation. A composition for improving neurite outgrowth may be an example of a composition for promoting neuronal differentiation.

By using the composition of the present invention, specifically by administering the composition of the present invention to a subject, an effect based on the promotion of neuronal differentiation and/or the improvement of neurite outgrowth may be obtained. That is, the composition of the present invention may be, for example, a composition for obtaining an effect based on the promotion of neuronal differentiation and/or the improvement of neurite outgrowth. Specifically, the composition of the present invention may be, for example, a composition for preventing, improving, and/or treating symptoms associated with neurological disorders. A composition for obtaining an effect based on the promotion of neuronal differentiation and/or the improvement of neurite outgrowth (such as a composition for preventing, improving, and/or treating symptoms associated with neurological disorders) may be an example of a composition for promoting neuronal differentiation and/or a composition for improving neurite outgrowth.

The same description of the subject to which the active ingredient in the method of the present invention is administered can be applied mutatis mutandis to the subject to which the composition of the present invention is administered.

The composition of the present invention may be, for example, a food and drink composition, a pharmaceutical composition, or a feed composition. The composition of the present invention may be, in particular, a food and drink composition or a pharmaceutical composition. That is, the present invention may provide, for example, a food and drink composition for obtaining a neuropromoting effect (for example, for promoting neuronal differentiation, improving neurite outgrowth, and/or for preventing, improving, and/or treating symptoms associated with neurological disorders). The present invention may also provide, for example, a pharmaceutical composition for obtaining a neuropromoting effect (for example, for promoting neuronal differentiation, improving neurite outgrowth, and/or for preventing, improving, and/or treating symptoms associated with neurological disorders). The present invention may also provide, for example, a feed composition for obtaining a neuropromoting effect (for example, for promoting neuronal differentiation, improving neurite outgrowth, and/or for preventing, improving, and/or treating symptoms associated with neurological disorders). The composition of the present invention that is a food and drink composition, a pharmaceutical composition, or a feed composition is also referred to as the "food and drink composition of the present invention", the "pharmaceutical composition of the present invention", or the "feed composition of the present invention", respectively.

The composition of the present invention may consist of an active ingredient, or may contain additional ingredients in addition to the active ingredient.

The additional component is not particularly limited as long as it does not impair the neurostimulatory effect. Any additional component that is acceptable depending on the mode of use of the composition of the present invention can be used. Examples of the additional component include components that can be used by being blended into food and beverage products, pharmaceutical products, or feed. Specific examples of the additional component include the components exemplified for the food and beverage compositions, pharmaceutical compositions, or feed compositions described below. As the additional component, one type of component may be used, or two or more types of components may be used in combination.

The content and ratio of the components (i.e., the active ingredient and optionally additional ingredients) in the composition of the present invention are not particularly limited as long as the neurostimulatory effect is achieved. The content and ratio of the components in the composition of the present invention can be appropriately set depending on various conditions such as the type of active ingredient, the type of additional ingredient, the type, dosage form, and method of use of the composition of the present invention, the type, age, and health condition of the subject to be administered.

The content of the active ingredient in the composition of the present invention may be, for example, 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 30% by weight or more, 50% by weight or more, 70% by weight or more, or 90% by weight or more, or 99.99% by weight or less, 99% by weight or less, 90% by weight or less, 70% by weight or less, 50% by weight or less, 30% by weight or less, 10% by weight or less, 5% by weight or less, or 1% by weight or less, or any compatible combination thereof.

The content of the active ingredient in the composition of the present invention, converted into the amount of ILA-producing bacteria cells, may be, for example, ^1x104 cells/g or more, ^1x105 cells/g or more, ^1x106 cells/g or more, ^1x107 cells/g or more, or ^1x108 cells/g or more, or ^1x1013 cells/g or less, ^1x1012 cells/g or less, or ^1x1011 cells/g or less, or any combination of these ranges. The content of the active ingredient in the composition of the present invention may be, for example, ^1x104 cells/mL or more, 1x105 cells/mL or more, ^1x106 cells/mL or more, ^1x107 cells/mL or more, or ^1x108 cells/mL or more, ^{or 1x1013} cells/mL or less, ^1x1012 cells/mL or less, or ^1x1011 cells/mL or less, or a combination thereof, when converted into the amount of ILA-producing bacteria. The content of the active ingredient in the composition of the present invention may be, for example, ^1x104 to ^1x1013 cells/g, ^1x105 to ^1x1013 cells/g, ^1x106 to ^1x1012 cells/g, preferably ^1x107 to ^1x1011 cells/g, and more preferably ^1x108 to ^1x1010 cells/g, when converted into the amount of ILA-producing bacteria. Specifically, the content of the active ingredient in the composition of the present invention may be, for example, ^1x104 to ^1x1013 cells/mL, 1x105 to ^1x1013 cells/mL, ^1x106 to ^1x1012 cells/mL, preferably ^1x107 to ^1x1011 cells/mL, and more preferably ^1x108 to ^1x1010 cells/mL, calculated as the amount of ILA ^- producing bacteria. "Cells" may be read as "cfu.""Cfu" stands for colony forming unit.

The content of the active ingredient in the composition of the present invention may be set, for example, so as to achieve the dosage of the active ingredient mentioned in the method of the present invention.

The shape of the composition of the present invention is not particularly limited. Any shape that is acceptable depending on the mode of use of the composition of the present invention can be adopted as the shape of the composition of the present invention. Specific examples of the shape of the composition of the present invention include the shapes exemplified for the food and drink compositions, pharmaceutical compositions, and feed compositions described below.

In one embodiment, the present invention relates to a composition for medical use comprising the following components (A) and/or (B) as active ingredients:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.

In one aspect, the present invention relates to a composition for medical use, comprising as active ingredients the following components (A) and/or (B) used in the presence of nerve growth factor (NGF):
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.

In a particular embodiment, the present invention relates to a composition comprising indole-3-lactic acid or indole-3-lactic acid producing bacteria for use in the prevention, amelioration, and/or treatment of symptoms associated with neurological disorders. In a particular embodiment, the present invention relates to a composition comprising indole-3-lactic acid or indole-3-lactic acid producing bacteria for use in achieving a neuropromoting effect in a subject. In a particular embodiment, the present invention relates to a composition comprising indole-3-lactic acid or indole-3-lactic acid producing bacteria for use in promoting neuronal differentiation or improving neurite outgrowth in a subject. In a particular embodiment, the present invention relates to a composition comprising indole-3-lactic acid or indole-3-lactic acid producing bacteria for use in the prevention, amelioration, and/or treatment of symptoms associated with diseases involving impaired neuronal differentiation or impaired neurite outgrowth. In some embodiments, the composition for use according to the present invention is a pharmaceutical composition or a food and drink composition. In some embodiments, the composition for use according to the present invention is a pharmaceutical composition.

<Food and drink composition>
The food and drink composition of the present invention is not particularly limited as long as it contains an active ingredient. The food and drink composition may be provided in any form, such as a liquid, a paste, a solid, or a powder.

The food and drink composition may be, for example, a food or drink itself, or may be a material used in the manufacture of a food or drink. Examples of such materials include seasonings, food additives, and other food ingredients. Specific examples of the food and drink composition include flour products, instant foods, processed agricultural products, processed marine products, processed livestock products, dairy products (fermented milk, cheese, infant formula, etc.), oils and fats, basic seasonings, complex seasonings, frozen foods, confectionery, beverages, and other commercially available foods and drinks. Specific examples of the food and drink composition include health foods, functional foods, enteral nutritional foods, special purpose foods, health functional foods (specified health foods, nutrient functional foods, functional foods, etc.), nutritional supplements, and quasi-drugs. The food and drink composition may be, for example, a supplement such as a tablet-shaped supplement.

The food and drink composition of the present invention can be produced, for example, by combining an active ingredient with an additional ingredient. The operation of combining an active ingredient with an additional ingredient is also referred to as "addition of an active ingredient". The method for producing the food and drink composition of the present invention is not particularly limited. The food and drink composition of the present invention can be produced, for example, by the same method as that of a normal food and drink using the same raw materials as that of a normal food and drink, except for the addition of the active ingredient. The same applies when the food and drink composition of the present invention is produced as a material used in the production of a food and drink. The addition of the active ingredient may be carried out at any stage of the production process of the food and drink composition. The addition of the active ingredient may be carried out, for example, during or after the production of the food and drink composition. That is, for example, the food and drink composition of the present invention may be produced by adding the active ingredient to a food and drink that has been prepared in advance. In addition, the food and drink composition of the present invention may be produced through a fermentation process by a fermentation process by an active ingredient (specifically, a fermentation process by an ILA-producing bacterium). Examples of food and drink compositions produced through a fermentation process include fermented products such as fermented milk and probiotic drinks. That is, the active ingredient (specifically, an ILA-producing bacterium) may be used, for example, as a starter for the production of a fermented product. Of course, the active ingredient can also be added to a pre-prepared fermentation product.

The food and beverage composition of the present invention can also be used to produce another food and beverage composition. That is, for example, when the food and beverage composition of the present invention is provided as a material (seasoning, food additive, other food and beverage ingredients, etc.) used in the production of food and beverages, another food and beverage composition may be produced by adding the food and beverage composition of the present invention. Such another food and beverage composition is also an example of the food and beverage composition of the present invention. With regard to the addition of the food and beverage composition of the present invention in the production of a food and beverage composition, the description regarding the addition of an active ingredient in the production of a food and beverage composition can be applied mutatis mutandis.

The food and drink composition of the present invention may be provided and sold as a food and drink labeled with the intended use (including health uses), such as for promoting neuronal differentiation, improving neurite outgrowth, and/or for preventing, improving, and/or treating symptoms associated with neurological disorders. The food and drink composition of the present invention may be provided and sold as a food and drink labeled with the intended target.

"Display" includes all acts aimed at informing consumers of the above-mentioned uses, and any expression that can recall or infer the above-mentioned uses falls under "display" regardless of the purpose, content, object, or medium of the display. Display may be implemented in particular using expressions that enable consumers to directly recognize the above-mentioned uses.

Specific examples of labeling include the act of transferring, delivering, displaying for transfer or delivery, or importing a product related to the food and beverage composition of the present invention or a product packaging on which the above-mentioned uses are written, and the act of displaying or distributing the product and writing the above-mentioned uses in advertisements, price lists, or transaction documents related to the product, or writing the above-mentioned uses in information containing these contents and providing them by electromagnetic means (such as the Internet). Examples of labeling include, in particular, labeling on packaging, containers, catalogs, pamphlets, promotional materials (POP, etc.) at the point of sale, or other documents.

Examples of the labeling include labeling as health foods, functional foods, enteral nutritional foods, special purpose foods, health functional foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.), dietary supplements, and quasi-drugs. The labeling may preferably be labeling approved by the government or the like (for example, labeling approved based on various systems established by the government and made in a manner based on such approval). Labeling approved by the government or the like includes labeling approved by the Consumer Affairs Agency. Labeling approved by the Consumer Affairs Agency includes labeling approved under the system of health functional foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.) and similar systems. Specific examples of labeling approved by the Consumer Affairs Agency include labeling as foods for specified health uses, labeling as foods for conditional specified health uses, labeling that affects the structure and function of the body, labeling that reduces the risk of disease, and labeling of functionality based on scientific evidence. More specifically, the labeling approved by the Consumer Affairs Agency includes labeling as a food for specified health uses (particularly labeling of health uses) and similar labeling as stipulated in the Cabinet Office Ordinance on Permission for Labeling of Special Uses Provided in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009).

The content of the active ingredient in the food and beverage composition of the present invention may be, for example, within the ranges described above. In particular, the content of the active ingredient in the food and beverage composition of the present invention may be, for example, ^1x104 to ^1x1013 cells/g, ^1x105 to ^1x1013 cells/g, ^1x106 to ^1x1012 cells/g, preferably ^1x107 to ^1x1011 cells/g, and more preferably ^1x108 to ^1x1010 cells/g, calculated as the amount of ILA-producing bacteria. Furthermore, the content of the active ingredient in the food and beverage composition of the present invention, particularly when converted into the amount of ILA-producing bacteria cells, may be, for example, ^1x104 to ^1x1013 cells/mL, ^1x105 to ^1x1013 cells/mL, ^1x106 to ^1x1012 cells/mL, preferably ^1x107 to ^1x1011 cells/mL, and more preferably ^1x108 to ^1x1010 cells/mL.

<Pharmaceutical Composition>
The pharmaceutical composition of the present invention is not particularly limited as long as it contains an active ingredient.

The pharmaceutical composition of the present invention may be appropriately formulated into a desired dosage form. The dosage form of the pharmaceutical composition of the present invention is not particularly limited. The dosage form of the pharmaceutical composition of the present invention may be appropriately selected depending on various conditions such as the administration method. The pharmaceutical composition of the present invention may be for oral administration or for parenteral administration. The pharmaceutical composition of the present invention may be particularly for oral administration. In the case of oral administration, the dosage form may be a solid preparation such as a powder, granules, tablets, or capsule, or a liquid preparation such as a solution, syrup, suspension, or emulsion. In the case of parenteral administration, the dosage form may be a suppository or ointment.

The method of formulation is not particularly limited. Formulation can be carried out, for example, by a known method according to the dosage form. Physiologically acceptable additives can be used in formulation. Examples of additives include various organic and inorganic components. Specific examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, pH adjusters, colorants, diluents, surfactants, and solvents. These additives can be appropriately selected depending on various conditions such as the dosage form.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and calcium carboxymethyl cellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

In addition to the above excipients, binders include gelatin, polyvinylpyrrolidone, macrogol, etc.

Disintegrants include the above excipients as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone.

Lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as beechm and glomerulus; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and starch derivatives.

Stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.

Flavoring agents include sweeteners, acidulants, and fragrances.

The content of the active ingredient in the pharmaceutical composition of the present invention may be, for example, within the ranges described above. In particular, the content of the active ingredient in the pharmaceutical composition of the present invention may be, for example, ^1x104 to ^1x1013 cells/g, ^1x105 to ^1x1013 cells/g, ^{1x106 to 1x1012 cells/g, preferably 1x107 to 1x1011 cells} /g, and more preferably ^1x108 to ^1x1010 cells/g, calculated as the amount of ILA-producing bacteria. Furthermore, the content of the active ingredient in the pharmaceutical composition of the present invention, particularly when converted into the amount of ILA-producing bacteria cells, may be, for example, ^1x10 to ^1x10 cells/mL, ^1x10 to ^1x10 cells/mL, ^1x10 to ^1x10 cells/mL, preferably ^1x10 to ^1x10 cells/mL, and more preferably ^1x10 to ^1x10 cells/mL.

<Feed composition>
The feed composition of the present invention is not particularly limited as long as it contains an active ingredient. The feed composition may be a pet food or livestock feed. The feed composition may be provided in any form such as a powder, granule, crumble, pellet, cube, paste, liquid, etc.

The feed composition of the present invention can be produced, for example, by combining an active ingredient with an additional ingredient. The operation of combining an active ingredient with an additional ingredient is also referred to as "addition of an active ingredient". The method for producing the feed composition of the present invention is not particularly limited. The feed composition of the present invention can be produced, for example, by using the same raw materials as in normal feed and by the same method as in normal feed, except for the addition of the active ingredient. The active ingredient may be added at any stage of the production process of the feed composition. The active ingredient may be added, for example, during or after the production of the feed composition. That is, for example, the feed composition of the present invention may be produced by adding the active ingredient to a previously prepared feed. The feed composition of the present invention may also be produced through a fermentation process using the active ingredient (specifically, a fermentation process using an ILA-producing bacterium). An example of a feed composition produced through a fermentation process is silage.

<3> Method of the Present Invention The method of the present invention is a method comprising administering an active ingredient to a subject. This step is also referred to as an "administration step." The subject to which the active ingredient is administered is also referred to as an "administration subject."

That is, the method of the present invention is a method comprising administering to a subject the following components (A) and/or (B):
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.

The method of the present invention can be carried out, for example, to obtain a neurostimulatory effect.

By utilizing the method of the present invention, specifically by administering an active ingredient to a subject, neurite outgrowth may be improved in the subject, i.e., a neurite outgrowth improving effect may be obtained. In other words, the method of the present invention may be, for example, a method for improving neurite outgrowth.

By utilizing the method of the present invention, specifically by administering an active ingredient to a subject, neuronal differentiation may be promoted, i.e., a neuronal differentiation promoting effect may be obtained. That is, the method of the present invention may be, for example, a method for promoting neuronal differentiation. A method for improving neurite outgrowth may be an example of a method for promoting neuronal differentiation.

By utilizing the method of the present invention, specifically by administering an active ingredient to a subject, an effect based on the promotion of neuronal differentiation and/or the improvement of neurite outgrowth may be obtained. That is, the method of the present invention may be, for example, a method for obtaining an effect based on the promotion of neuronal differentiation and/or the improvement of neurite outgrowth. Specifically, the method of the present invention may be, for example, a method for preventing, ameliorating, and/or treating symptoms associated with a neurological disorder. A method for obtaining an effect based on the promotion of neuronal differentiation and/or the improvement of neurite outgrowth (such as a method for preventing, ameliorating, and/or treating symptoms associated with a neurological disorder) may be an example of a method for promoting neuronal differentiation or a method for improving neurite outgrowth.

Note that "administering an active ingredient to a subject" can be used interchangeably or equivalently to "allowing a subject to ingest the active ingredient." Ingestion may be voluntary (i.e., voluntary intake) or forced (i.e., forced intake). That is, the administration step may be, for example, a step of supplying the active ingredient (which may be blended in food, drink, or feed) to a subject, thereby allowing the subject to ingest the active ingredient freely. Administration may be oral or parenteral. Administration may typically be oral. Parenteral administration includes tube administration, rectal administration, and intranasal administration.

The conditions for administering the active ingredient (e.g., the subject, administration period, number of administrations, dosage, and other conditions related to administration) are not particularly limited as long as the neurostimulatory effect is achieved. The conditions for administering the active ingredient can be set appropriately depending on various conditions such as the type of active ingredient, the type of subject, age, and health condition.

The subject of administration is not particularly limited as long as the neuropromoting effect is achieved. The subject of administration may be a mammal. Examples of mammals include primates such as humans, monkeys, and chimpanzees, rodents such as mice, rats, hamsters, and guinea pigs, and various other mammals such as rabbits, horses, cows, sheep, goats, pigs, dogs, and cats. Examples of mammals include humans in particular. The subject of administration (e.g., a mammal) may be, for example, a pet animal, livestock, or a laboratory animal. The subject of administration may be a male subject or a female subject. The subject of administration may be, for example, a subject of any age, such as an infant, a child, an adult, a middle-aged person, or an elderly person. The subject of administration may be, for example, a healthy subject or an unhealthy subject. An example of an unhealthy subject is a subject exhibiting symptoms related to a neurological disorder.

The dosage of the active ingredient may be, for example, in terms of the dosage of ILA, 0.001 mg/kg body weight/day or more, 0.01 mg/kg body weight/day or more, 0.1 mg/kg body weight/day or more, 1 mg/kg body weight/day or more, 10 mg/kg body weight/day or more, 100 mg/kg body weight/day or more, or 1000 mg/kg body weight/day or more, or 10,000 mg/kg body weight/day or less, 1000 mg/kg body weight/day or less, 100 mg/kg body weight/day or less, 10 mg/kg body weight/day or less, 1 mg/kg body weight/day or less, 0.1 mg/kg body weight/day or less, or 0.01 mg/kg body weight/day or less, or any combination thereof that is not contradictory. Specifically, the dosage of the active ingredient may be, for example, 0.001 to 10,000 mg/kg body weight/day, preferably 0.1 to 1,000 mg/kg body weight/day, more preferably 1 to 100 mg/kg body weight/day, and even more preferably 10 to 100 mg/kg body weight/day.

The dosage of the active ingredient is, for example, 1x10 ⁶ cells/kg, calculated as the dosage of ILA-producing bacteria.
The dosage may be 1x10 6 to 1x10 ¹² cells/kg body weight/day, 1x10 7 to 1x10 ¹¹ cells/kg body weight/day, or 1x10 ⁸ cells/kg body weight/day or more, or 1x10 ¹² cells/kg body weight/day or less, 1x10 ¹¹ cells/kg body weight/day or less, or any compatible combination thereof. The dosage of the active ingredient may be, specifically, for example, 1x10 ⁶ to 1x10 ¹² cells/kg body weight/day, preferably 1x10 ⁷ to 1x10 ¹¹ cells/kg body weight/day, and more preferably 1x10 ⁸ to 1x10 ¹⁰ cells/kg body weight/day, calculated in terms of the dosage of ILA-producing bacteria. "Cells" may be read as "cfu".

The administration period of the active ingredient may be, for example, 1 day or more, 3 days or more, 1 week or more, 2 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 6 months or more, 9 months or more, or 12 months or more, or 10 years or less, 5 years or less, 1 year or less, or 6 months or less, or any non-consistent combination thereof. The active ingredient may be administered, for example, throughout the subject's life or for a portion of the subject's life. The active ingredient may be administered, for example, at least until a neurostimulatory effect is achieved. The active ingredient may be administered, for example, daily or once every few days. The active ingredient may be administered, in particular, daily. The dosage of the active ingredient at each administration may be constant or not.

For example, the active ingredient may be administered to a subject as it is, or may be prepared as a composition, such as a food or drink composition, a pharmaceutical composition, or a feed composition, containing the active ingredient, and then administered to a subject. For compositions containing an active ingredient, the description of the composition of the present invention can be applied mutatis mutandis. The active ingredient may be administered alone or in combination with additional ingredients. Examples of additional ingredients include food or drink, pharmaceuticals, feed, and ingredients contained therein. Furthermore, for additional ingredients used in the method of the present invention, the description of additional ingredients contained in the composition of the present invention can be applied mutatis mutandis.

The active ingredient can be administered to a subject, for example, by using the composition of the present invention (specifically, by administering the composition of the present invention to a subject). That is, one aspect of the method of the present invention may be a method including administering the composition of the present invention to a subject. That is, "administration of an active ingredient" also includes administration of the composition of the present invention. The administration conditions of the composition of the present invention (e.g., the subject to be administered, the administration period, the number of administrations, the dosage, and other conditions related to administration) are not particularly limited as long as the neuropromoting effect is achieved. The administration conditions of the composition of the present invention can be appropriately set according to various conditions such as the type and content of the active ingredient, the type and content of the additional ingredient, the type and dosage form of the composition, the type of the subject to be administered, the age, and the health condition. The administration conditions of the composition of the present invention can be applied mutatis mutandis to the description of the administration conditions of the active ingredient. That is, the composition of the present invention may be administered to a subject such as those exemplified above. In addition, the dosage of the composition of the present invention can be set so that, for example, the dosage of the active ingredient as exemplified above is obtained. In addition, the composition of the present invention may be administered alone or in combination with an additional ingredient.

The present invention will now be described in more detail with reference to the following non-limiting examples.

1. Materials and Methods Test Compounds and Reagents Indole-3-lactic acid (ILA) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Indole-3-propionic acid (IPA) was purchased from Merck (Tokyo, Japan). Tryptophan, papaverine hydrochloride, α-naphthoflavone (AFN), CH223191, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Dimethyl sulfoxide (DMSO) and methanol were obtained from Wako (Osaka, Japan). ANF and CH223191 were dissolved in DMSO at a concentration of 10 mM. These stock solutions were serially diluted with sterile MilliQ water to prepare analytical samples. Nerve growth factor (NGF; 2.5S) was purchased from Alomone Labs (Jerusalem, Israel).

<1-1> Cell culture
PC12 cells (rat adrenal pheochromocytoma cell line) were obtained from the European Collection of Authenticated
Cell Cultures (ECACC 88022401; Salisbury, UK). Suspension cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI; Gibco Life Technologies, Grand Island, NY, USA) supplemented with ₁₀ % (v/v) heat-inactivated horse serum (HS; Gibco Life Technologies), 5% (v/v) fetal bovine serum (FBS; Gibco Life Technologies), and 0.1% (v/v) penicillin/streptomycin (Gibco Life Technologies) at 37°C in a 5% CO2 atmosphere. Medium was changed every 3 days.

<1-2> Dose-response of tryptophan and its metabolites
PC12 cells (passage number <13) were seeded at a density of 10,000 cells/mL per well on type IV collagen-coated 24-well culture plates (Iwaki, Shizuoka, Japan) in complete growth medium (RPMI supplemented with 10% (v/v) HS, 5% (v/v) FBS, and 1% (v/v) penicillin/streptomycin) and cultured for 24 h. The cells were then replaced with low-serum medium (1% v/v HS) for 24 h. The cells were then treated with NGF (25 ng/mL) and a wide range of final concentrations (1 μM, 100 nM, 10 nM, and 1 nM) of test compounds (ILA, IPA, or tryptophan) for 5 consecutive days. On the third day, the low-serum medium was replaced with the test compounds. Papaverine hydrochloride added under the same conditions was used as a positive control. Untreated controls (cells without NGF) and NGF controls (cells treated with NGF only) were also cultured under the same conditions. Then, the cells were subjected to immunofluorescence staining and acetylcholinesterase assay for quantification of neurite outgrowth. Then, the maximum effective concentration of the test compound was selected for further analysis.

<1-3> Immunofluorescence staining
After 5 days of treatment, cells in clear collagen IV-coated plates were washed twice with room temperature phosphate-buffered saline (PBS) and fixed with absolute methanol (pre-cooled to −20°C) for 5 min. Cells were washed three times with ice-cold PBS and incubated for 1 h in blocking buffer PBST (1% (w/v) bovine serum albumin, 10% (v/v) normal goat serum, and 0.3 M glycine in PBS containing 0.1% (v/v) Tween-20). Cell bodies and processes were then labeled overnight at 4°C with anti-βIII-tubulin mouse primary antibody (1 μg/mL; Abcam, Cambridge, UK) diluted in PBST containing 1% (w/v) bovine serum albumin, followed by Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (2 μg/mL; Abcam) for 1 h at room temperature in the dark. For nuclear staining, cells were counterstained with Cellstain® 4',6'-Diamidino-2-phenylindole (DAPI; Dojindo Molecular Technologies, Kumamoto, Japan) for 10 min at 37°C, after which the plates were subjected to image acquisition and analysis of neurite outgrowth.

<1-4> Quantification of neurite outgrowth Plates containing fluorescently labeled cells were observed at 100x magnification under an inverted fluorescence microscope (DP73; Olympus, Tokyo, Japan) using a multi-bandpass emission filter and corresponding excitation filters for the blue channel (nuclei) and green channel (cell body and processes). Four images were taken per well. Cells presenting processes at least 1.5 times longer than the length of the cell body were considered positive and counted as cells with neurites. Counting was performed in a blinded manner. The percentage of cells with neurites was calculated as the percentage of the number of neurites divided by the total number of cells. Each data point corresponds to counts obtained from three independent wells.

<1-5> Analysis of acetylcholinesterase (AchE) activity
To investigate the effect of tryptophan and its metabolites on AchE activity, a biochemical marker of neuronal differentiation in PC12 cells, thiocholine generated from hydrolysis of acetylthiocholine by endogenous AchE enzyme in each sample was quantified by a fluorescent colorimetric method. Briefly, PC12 cells were grown and treated for 5 consecutive days as described above. Then, cells were lysed with ice-cold NP-40 cell lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 2 mM EDTA (pH 8.0), and 1% (v/v) NP-40. AchE activity in cell lysates was determined using the Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) as per the manufacturer's instructions. The assay signal was read at Ex/Em = 490/520 nm using a fluorescence absorbance microplate reader. AchE activity was determined from a standard curve and normalized to the protein concentration in each sample. Protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Invitrogen, Paisley, UK) with bovine serum albumin as the standard.

＜1-6＞Analysis of phospho-TrkA, ERK, phospho-ERK, CREB, and phospho-CREB proteins by Western blot
PC12 cells (passage number <13) were seeded at a density of 50,000 cells/mL per well in type IV collagen-coated 24-well culture plates in complete growth medium and cultured for 24 h, then transferred to low serum medium (1% v/v HS) for 24 h and exposed to NGF (25 ng/mL) and test compounds (positive control; ILA, IPA, or tryptophan at a final concentration of 100 nM) for 24 h. Untreated controls (cells without NGF) and NGF controls (cells treated with NGF only) were also cultured under the same conditions. Following treatment, cells were washed with ice-cold PBS, scraped in ice-cold NP-40 cell lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 2 mM EDTA (pH 8.0), and 1% (v/v) NP-40, and incubated on ice for 15 min. The cell lysate was collected by centrifugation (8,000 g, 15 min) at 4°C, and the protein concentration was determined using a BCA kit with bovine serum albumin as the standard substance.

After boiling for 5 min, cell lysates (20 μg) were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane using the iBlot dry blotting system (Invitrogen, Paisley, UK). Nonspecific reactions were blocked with Bullet Blocking One solution (Nacalai Tesque, Kyoto, Japan) by shaking at room temperature for 5 min. Blots were incubated overnight at 4°C with the appropriate antibodies: anti-phospho-TrkA (Tyr490) (1:2000), anti-phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000) (Cell Signalling Technology, Danvers, MA, USA), anti-β-actin (1:5000), anti-CREB (1:1000), anti-phospho-CREB (Ser133) (1:1000) (Abcam, Cambridge, UK), and anti-ERK (pan-ERK) (1:5000) (BD, Franklin Lakes, NJ, USA). After washing three times with Tris-buffered saline containing 0.1% Tween-20 (TBST), blots were incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000) (Abcam). Blots were washed with TBST and proteins were detected using Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare, Chicago, Illinois, USA) according to the manufacturer's instructions, and chemiluminescent signals were visualized using a ChemiDoc Imager and quantified using Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).

<1-7> Western blot analysis of AhR receptor
PC12 cells (50,000 cells/mL/well) were cultured in collagen IV-coated 24-well culture plates as described above. The cells were then exposed to AhR receptor antagonists (ANF and CH223191) at a final concentration of 1 μM for 1 h, followed by continuous treatment with NGF (25 ng/mL) and test compounds (positive control; ILA, IPA, or tryptophan at a final concentration of 100 nM) for 5 days. Control cells without ANF and CH223191 were also grown and treated under the same conditions. After 3 days, the medium and test compounds were replaced. Following treatment, cell lysates containing 20 μg of total protein were harvested, separated by 8% SDS-PAGE, and transferred to PVDF membranes using the iBlot dry blotting system. Blots were blocked with Bullet Blocking One solution by shaking for 5 min at room temperature and then incubated overnight at 4°C with the appropriate antibodies: anti-aryl hydrocarbon receptor antibody (1:1000) and anti-β-actin (1:5000) (Abcam). Blots were then washed three times with TBST and incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000) (Abcam). Signals were developed with Amersham ECL Select Western Blotting Detection Reagent, visualized on a ChemiDoc Imager, and quantified with Image Lab software.

<1-8> Analysis of AchE activity after pretreatment with AhR antagonist
PC12 cells (10,000 cells/mL/well) were cultured in collagen IV-coated 24-well culture plates as described above. The cells were then exposed to AhR receptor antagonists (ANF and CH223191) at a final concentration of 1 μM for 1 h, followed by treatment with NGF (25 ng/mL) and test compounds (positive control; ILA, IPA, or tryptophan at a final concentration of 100 nM) for 5 consecutive days. Control cells without ANF and CH223191 were also grown and treated under the same conditions. The medium and test compounds were replaced after 3 days. Following treatment, the cells were lysed with ice-cold NP-40 cell lysis buffer. AchE activity in the cell lysates was then determined using the Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer's instructions. AchE activity was measured as described above.

<1-9> Statistical analysis Results are shown as mean and standard deviation. All statistical analyses were performed using IBM SPSS Statistics, version 22.0, statistical software package (IBM Corp., Armonk, NY, USA). The statistical significance of differences between each treatment group was analyzed by unpaired Student's t-test. A value of P < 0.05 was considered to be statistically significant.

<2> Results <2-1> Dose Response of Tryptophan and Its Metabolites To examine the dose response, PC12 cells were treated with tryptophan and its metabolites at concentrations ranging from 1 nM to 1 μM. All test compounds (ILA, IPA, and tryptophan) had no morphological effect on neurite outgrowth of PC12 cells in the absence of NGF (data not shown). However, in the presence of 25 ng/mL NGF, ILA, IPA, and tryptophan were found to promote neurite outgrowth of PC12 cells in a dose-dependent manner (Panel (A) of Figure 1). Compared with the NGF control, cells treated with 10 nM and 100 nM ILA showed significantly higher neurite outgrowth (P < 0.05), with 100 nM showing the maximum activity, whereas cells treated with IPA did not show significantly higher neurite outgrowth. Thus, it was confirmed that ILA may be significantly superior to IPA in promoting neurite outgrowth, especially NGF-induced neurite outgrowth. In addition, cells treated with 100 nM ILA also showed significantly higher AchE activity (P < 0.01) (Fig. 1, Panel (B)). On the other hand, the maximally effective concentration of IPA was 100 nM, which showed significantly higher AchE activity (P < 0.05) upon treatment with this concentration, but not the percentage of cells with neurites. In cells treated with tryptophan, no significant increase was observed in the percentage of cells with neurites or AchE activity.

<2-2> Effects of tryptophan and its metabolites on neurite outgrowth in PC12 cells To confirm the enhancing effect of tryptophan and its metabolites, PC12 cells were treated with NGF and 100 nM of the test compounds, and quantitative analysis of neurite outgrowth and AchE assay were performed as described in Materials and Methods. As shown in Figure 2, panel (A), ILA and IPA induced significant neurite outgrowth, whereas tryptophan induced slight outgrowth. Interestingly, ILA treatment significantly enhanced the percentage of cells with neurites in NGF-induced PC12 cells (P < 0.01), whereas IPA treatment did not significantly enhance the percentage of cells with neurites (Figure 2, panel (B)). Thus, it was again confirmed that ILA may be significantly superior to IPA in promoting neurite outgrowth, especially NGF-induced neurite outgrowth. The percentages of cells bearing neurites in cells treated with 100 nM ILA, IPA, and tryptophan reached 15.07 ± 0.50%, 14.49 ± 1.19%, and 12.29 ± 3.06%, respectively. These were comparable to the NGF control (12.68 ± 0.23%) and significantly higher than NTC (0.05 ± 0.09%). In addition, AchE activity was significantly increased in NGF-induced PC12 cells treated with ILA (P < 0.05), IPA (P < 0.01), and tryptophan (P < 0.05) compared with the NGF control (panel (C) of Fig. 2). These data indicate that indole derivatives can promote NGF-induced neurite outgrowth in PC12 cells.

<2-3> Effects of tryptophan and its metabolites on the Ras/ERK pathway We next investigated whether activation of the TrkA receptor and extracellular signal-regulated kinase 1/2 (ERK1/2) is essential for the NGF-induced neurite outgrowth enhanced by indole derivatives in PC12 cells. Treatment of PC12 cells with ILA and IPA (100 nM) in the presence of NGF (25 ng/mL) induced phosphorylation of TrkA for 24 h (Fig. 3, panel (A)). The relative phosphorylation level of TrkA in ILA-treated cells (1.92 ± 0.15) was significantly increased (P < 0.05) compared with the NGF control (1.53 ± 0.17), whereas the relative phosphorylation level of TrkA in IPA-treated cells (1.85 ± 0.41) tended to increase but was not significantly different from the NGF control (1.53 ± 0.17). These results suggest that ILA may be significantly more effective than IPA in promoting NGF-induced neurite outgrowth, especially through promoting the phosphorylation of TrkA. The phosphorylation levels of ILA and IPA were proportional to their respective neurite outgrowth-promoting effects. Meanwhile, treatment of PC12 cells with tryptophan had no effect on inducing the phosphorylation of TrkA (1.62 ± 0.23).

In addition, the indole derivatives ILA (P < 0.01) and IPA (P < 0.05) strongly induced the phosphorylation (Thr202/Tyr204) of ERK1 (44 kDa) and ERK2 (42 kDa) after 24 h of treatment (panel (B) of Fig. 3). The P values were smaller for ILA than for IPA. This confirmed that ILA may be significantly superior to IPA in promoting NGF-induced neurite outgrowth, especially through promoting ERK signaling. The phosphorylation levels of ERK1/2 in ILA- and IPA-treated cells reached 1.98 ± 0.11 and 1.82 ± 0.10. Nevertheless, a significant increase in the phosphorylation level of ERK1/2 was also observed in tryptophan-treated cells (1.75 ± 0.06; P < 0.05). These results suggest that ERK signaling is involved in NGF-induced neuronal differentiation of PC12 cells, which is enhanced by indole derivatives.

The inventors continued to investigate the possibility that CREB (cAMP response element-binding protein) may be involved in the indole derivative-enhanced NGF-induced neurite outgrowth of PC12 cells. As shown in panel (C) of Figure 3, treatment of PC12 cells with indole derivatives (ILA or IPA at 100 nM) significantly increased CREB phosphorylation over 24 h compared with NGF control (P < 0.05). The phosphorylation levels of CREB in ILA- and IPA-treated PC12 cells reached 2.57 ± 0.48 and 2.51 ± 0.56, respectively. However, treatment of PC12 cells with tryptophan did not significantly induce CREB phosphorylation. These results strongly suggested that indole derivatives (ILA and IPA) induce phosphorylation of TrkA and ERK1/2, which in turn activate the transcription of CREB during the process of neurite outgrowth of PC12 cells.

<2-4> Potential role of tryptophan and its metabolites as AhR ligands The present inventors further investigated the possibility of indole derivatives as AhR ligands by Western blot analysis, and the possibility of the involvement of AhR signaling in the process of neurite outgrowth by measuring AchE activity. As a result, ILA and IPA may act as AhR ligands, and the relative expression level of AhR was significantly increased upon treatment (Figures 4 and 5).

Although there were some differences in sensitivity to AhR antagonists (ANF and CH223191),
The effects of ILA and IPA on AhR activation were suppressed in the presence of ANF and/or CH223191. As shown in Fig. 4 (A) and Fig. 5 (A), pretreatment with ANF (1 μM) significantly inhibited ILA-induced AhR activity in PC12 cells (P < 0.05), and AchE activity in ILA-treated cells also tended to decrease. Similarly, ILA-induced AhR activation and AchE activity were also significantly inhibited by pretreatment with CH223191 (1 μM), suggesting the specific involvement of AhR in the process of ILA-induced neuronal differentiation.

On the other hand, ANF and CH223191 inhibited IPA-induced AhR activation and AchE activity with different potencies. In IPA-treated cells, AhR activity was significantly inhibited by CH223191 (P < 0.05), but not by ANF. Conversely, in IPA-treated cells, AchE activity was significantly inhibited by ANF (P < 0.05), but not by CH223191. These data suggested that IPA may be a less potent AhR ligand than ILA. Since the effects of ILA and IPA appear to be exerted through binding to the aryl hydrocarbon receptor (AhR), a more potent AhR ligand (which may be ILA) may promote neurite outgrowth more strongly than IPA. Thus, it was again confirmed that ILA may be significantly superior to IPA in promoting neurite outgrowth (especially NGF-induced neurite outgrowth). Neither ANF nor CH223191 affected the role of tryptophan in AhR activation and AchE activity, except in AchE activity in cells pretreated with ANF.

<Test Example>
1. Bacterial strains and culture supernatants (CS)
The bifidobacterial strains shown in Table 1 were cultured under anaerobic conditions at 37°C for 16 hours in MRS broth (Becton Dickinson, MD, USA) (MRS-C) supplemented with 0.05% L-cysteine (Kanto Chemical Co., Ltd., Chuo-ku, Tokyo, Japan) using Anaeropack (Mitsubishi Gas Chemical Co., Tokyo, Japan).

The vegetative cells were then harvested by centrifugation at 5000 g (4°C for 10 min) using a high-speed centrifugal refrigeration device HIMAC SCR20B (Hitachi Koki Co., Ltd., Tokyo, Japan) and washed twice with phosphate-buffered saline (PBS) and Dulbecco’s Formula (DS Pharma Biomedical Co., Ltd., Osaka, Japan). The whole cell pellet was then suspended in PBS containing 0.05% L-cysteine (PBS-C). The optical density (at 600 nm) of each cell suspension was adjusted to the same value (OD600 = 0.2) using PBS-C. The cell suspension (100 L) was added to MRS-C (3 mL) and cultured at 37°C for 24 h under anaerobic conditions. The culture suspension was centrifuged at 5000 g (4°C for 10 min) to obtain CS. After filtration (pore size 0.22 m; Millipore, MA, USA), the samples were stored at 80°C until use. All cultures were performed independently three times, and the data presented are the average values of the tests.

2. Quantification of ILA concentration in CS
The ILA concentrations in CS were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS; TSQ Quantum Discovery Max, Thermo Electron Corp., San Jose, CA, USA). Chromatographic separation was performed using an InertSustain C18 column (GL Sciences, Tokyo, Japan) (particle size 2.1 μm, column size 150 mm × 2 m). Mobile phase A (1 g/L AA in water) and mobile phase B (1 g/L AA in acetonitrile) were applied at a flow rate of 0.2 mL/min. Gradient elution started with 10% B, and went from 10% to 90% B in 0.1–18 min, 90% in 18.1–25 min, 90% to 10% in 25.1–28 min, and 10% in 28–40 min. Quantification was performed by comparing the metabolite concentrations in CS with those of the corresponding synthetic ILA standards and internal standards (MOIs). LC-MS/MS spectra (product ion data) of the positive precursor ions were evaluated to determine their final abundance.

As a result, the bifidobacterial strains produced ILA in the amounts shown in Table 1. It was confirmed that infant-type HRB (strains No. 1-10, etc.) can produce a larger amount of ILA than adult-type HRB and non-HRB (strains No. 11-19, etc.). It has been reported that none of these strains No. 1-19 produces other tryptophan metabolites such as IPA (Non-Patent Document 4: Sakurai T. et al. Production of Indole-3-Lactic Acid by Bifidobacterium Strains Isolated from Human Infants. 2019 Sep 11;7(9)).

The present invention provides a composition that can be used for specific purposes, such as a composition for improving neurite outgrowth.

Claims (1)

A composition for promoting neuronal differentiation and/or improving neurite outgrowth, comprising the following component (A) and/or (B) as active ingredients:
(A) indole-3-lactic acid;
(B) Indole-3-lactic acid producing bacteria.

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