WO2021149806A1

WO2021149806A1 - Composition for improving neurite outgrowth

Info

Publication number: WO2021149806A1
Application number: PCT/JP2021/002271
Authority: WO
Inventors: Chyn Boon WONG; Tetsuya Kuhara
Original assignee: Morinaga Milk Industry Co., Ltd.
Priority date: 2020-01-24
Filing date: 2021-01-22
Publication date: 2021-07-29
Also published as: JP2024069369A; JP2023511678A

Abstract

A composition usable for a specific use, such as a composition for improving neurite outgrowth is provided. Such a composition is provided by using indole-3-lactic acid (ILA) or an indole-3-lactic acid-producing bacterium is used as an active ingredient.

Description

COMPOSITION FOR IMPROVING NEURITE OUTGROWTH

Field of the Invention

The present invention relates to a composition usable for a specific use, such as a composition for improving neurite outgrowth, improving neuronal differentiation, or obtaining an effect based thereon.

Description of the Related Art

Tryptophan is an essential amino acid bearing an indole ring, derived from dietary proteins. Tryptophan is mainly digested and absorbed in the small intestine, but significant amounts of tryptophan may persist to the colon, where they are metabolized by gut bacteria resulting in a variety of indole derivatives such as indole-3-lactic acid (ILA) and indole-3-propionic acid (IPA) (Non-patent document 1).

IPA was found to exhibit potent neuroprotective effects against the Alzheimer’s β-amyloid (Non-patent document 2) as well as neuronal damage and oxidative stress in the ischemic hippocampus (Non-patent document 3). Nonetheless, the effects of ILA on neuronal health await exploration.

ILA and IPA may be produced by certain gut bacterial species. For instance, strains of bifidobacterial species commonly isolated from the intestines of human infants, such as Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, and Bifidobacterium bifidum, can produce high levels of ILA (Non-patent document 4).

Non-patent document 1: Roager, H. M. & Licht, T. R. Microbial tryptophan catabolites in health and disease. Nat. Commun. 9, 1-10 (2018). Non-patent document 2: 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). Non-patent document 3: 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). 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).

Object to be Achieved by the Invention

An object of the present invention is to provide a composition usable for a specific use, such as a composition for improving neurite outgrowth, improving neuronal differentiation, or obtaining an effect based thereon. An object of the present invention can also be to provide means and methods for neuroprotection in vivo and in vitro.

Means for Achieving the Object

In order to achieve the aforementioned object, the inventors of the present invention conducted various researches. As a result, they found that indole-3-lactic acid (ILA) can improve neurite outgrowth, and thus they accomplished the present invention.

The present invention can be thus embodied, for example, as follows.
[1] A composition for promoting neuronal differentiation and/or improving neurite outgrowth, containing the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[2] A composition for preventing, improving, and/or treating a symptom related to a nerve disorder, containing the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[3] The composition mentioned above, wherein the composition at least contains the ingredient (B).

[4] The composition mentioned above, wherein the ingredient (B) is a Bifidobacterium bacterium.

[5] The composition mentioned above, wherein the ingredient (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.

[6] The composition mentioned above, wherein the composition is a food or drink composition.

[7] The composition mentioned above, wherein the composition is a pharmaceutical composition.

[8] A method for promoting neuronal differentiation and/or improving neurite outgrowth, comprising administering the ingredient(s) (A) and/or (B) shown below as an active ingredient to a subject:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[9] A method for preventing, improving, and/or treating a symptom related to a nerve disorder, comprising administering the ingredient(s) (A) and/or (B) shown below as an active ingredient to a subject:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[10] The method mentioned above, wherein at least the ingredient (B) is administered to the subject.

[11] The method mentioned above, wherein the ingredient (B) is a Bifidobacterium bacterium.

[12] The method mentioned above, wherein the ingredient (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.

[13] The method mentioned above, wherein the subject is a human subject.

[14] Use of the ingredient(s) (A) and/or (B) shown below for promoting neuronal differentiation and/or improving neurite outgrowth:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[15] Use of the ingredient(s) (A) and/or (B) shown below for preventing, improving, and/or treating a symptom related to a nerve disorder:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[16] Use of the ingredient(s) (A) and/or (B) shown below in manufacturing of the composition mentioned above:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[17] A composition for use in promoting neuronal differentiation and/or improving neurite outgrowth, said composition comprising the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[18] A composition for use in preventing, improving, and/or treating a symptom related to a nerve disorder, the composition comprising the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

[19] The composition for use according to the composition for use above, wherein the composition at least contains the ingredient (B).

[20] The composition for use according to the composition for use above, wherein the ingredient (B) is a Bifidobacterium bacterium.

[21] The composition for use according to the composition for use above, wherein the ingredient (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.

[22] The composition for use according to the composition for use above, wherein the composition is a food or drink composition.

[23] The composition for use according to the composition for use above, wherein the composition is a pharmaceutical composition.

A diagram showing effects of tryptophan and its metabolites on nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells at 1 nM, 10 nM, 100 nM and 1 μM. PC12 cells were treated with NGF (25 ng/mL) and the test compounds (ILA, IPA, or Trp) for five consecutive days. (A) Percentage of neurite-bearing cells in PC12 cells. (B) Acetylcholinesterase (AchE) activity in PC12 cells. The data represent the mean ± SD of three replicates. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NGF-treated control. NTC, non-treated control; PC, positive control; ILA, indole-3-lactic acid; IPA, indole-3-propionic acid; Trp, tryptophan. A diagram (photograph) showing effects of tryptophan and its metabolites on nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells. PC12 cells were treated with NGF (25 ng/mL) and ILA, IPA, or Trp (100 nM) for five consecutive days. (A) Percentage of neurite-bearing cells in PC12 cells. (A) Images of βIII-tubulin (green) immunostaining of PC12 cells at a magnification of x100. Scale bars: 100 μm. Nuclei were counterstained with DAPI (blue). (B) Percentage of neurite-bearing cells in PC12 cells. (C) Acetylcholinesterase (AchE) activity in PC12 cells. The data represent the mean ± SD of three replicates. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NGF-treated control. NTC, non-treated control; PC, positive control; ILA, indole-3-lactic acid; IPA, indole-3-propionic acid; Trp, tryptophan. A diagram (photograph) showing effects of tryptophan and its metabolites on the phosphorylation of TrkA, ERK1/2, and CREB in PC12 cells. Phosphorylation of TrkA, ERK1/2, and CREB in PC12 cells treated for 24 h with NGF (25 ng/mL) and ILA, IPA, or Trp (100 nM) was detected using western blot analysis. The data represent the mean ± SD of three replicates. *P < 0.05, **P < 0.01 vs. NGF-treated control. NTC, non-treated control; PC, positive control; ILA, indole-3-lactic acid; IPA, indole-3-propionic acid; Trp, tryptophan. A diagram (photograph) showing effects of tryptophan and its metabolites on the aryl hydrocarbon receptor (AhR) in PC12 cells. PC12 cells were pre-treated with the AhR antagonist, α-naphthoflavone (ANF; 1 μM), for 1 h and treated for five consecutive days with NGF (25 ng/mL) and ILA, IPA, or Trp (100 nM). Non-pre-treated PC12 cells served as a 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 β-actin blot served as a loading control. (B) Acetylcholinesterase (AchE) activity in PC12 cells. The data represent the mean ± SD of three replicates. *P < 0.05, **P < 0.01 vs. NGF-treated control. NTC, non-treated control; PC, positive control; ILA, indole-3-lactic acid; IPA, indole-3-propionic acid; Trp, tryptophan. A diagram (photograph) showing effects of tryptophan and its metabolites on the aryl hydrocarbon receptor (AhR) in PC12 cells. PC12 cells were pre-treated with the AhR antagonist, CH223191 (1 μM), for 1 h and treated for five consecutive days with NGF (25 ng/mL) and ILA, IPA, or Trp (100 nM). Non-pre-treated PC12 cells served as a 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 β-actin blot served as a loading control. (B) Acetylcholinesterase (AchE) activity in PC12 cells. The data represent the mean ± SD of three replicates. *P < 0.05, **P < 0.01 vs. NGF-treated control. NTC, non-treated control; PC, positive control; ILA, indole-3-lactic acid; IPA, indole-3-propionic acid; Trp, tryptophan.

Modes for Carrying out the Invention

Hereinafter, the present invention will be explained in detail.

<1> Active ingredient
In the present invention, the ingredient(s) (A) and/or (B) shown below is/are used as (an) active ingredient(s):
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

That is, the ingredient(s) (A) and/or (B) is/are also referred to as "active ingredient". As the active ingredient, for example, at least the ingredient (B) may be used. 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 being administered to a subject.

Use of the active ingredient, specifically administration of the active ingredient to a subject, may improve neurite outgrowth in the subject, that is, may provide an effect of improving neurite outgrowth. This effect is also referred to as "neurite outgrowth-improving effect". Examples of neurite outgrowth include NGF-induced neurite outgrowth. NGF can be produced by a subject to be administered with the active ingredient. That is, NGF produced by the subject can function in combination with the active ingredient. Hence, it is not necessary to administer NGF to the subject in addition to the active ingredient provided herein. The neurite outgrowth-improving effect can be confirmed by, for example, confirming an increase in the amount of neurite bearing cells in a sample. In other words, the neurite outgrowth-improving effect can be confirmed when, for example, the amount of neurite bearing cells in a sample after administration of the active ingredient is higher than that before administration of the active ingredient. The phrase "the amount of neurite bearing cells" may refer to an absolute amount of neurite bearing cells or a relative amount of neurite bearing cells. The relative amount of neurite bearing cells in a sample can be calculated as, for example, a ratio of the number of neurite bearing cells to the total number of cells in the sample. In particular, the relative amount of neurite bearing cells can be calculated as the ratio of the number of neurite bearing cells after use of the active ingredient(s) compared to the total number of cells in the sample, wherein the "neurite outgrowth-improving effect" can be determined when comparing the relative amount of neurite bearing cells before and after the use of the active ingredient(s). In another embodiment, the absolute or relative amount of neurite bearing cells may be referenced using the absolute or relative number of neurite bearing cells from a reference subject, in particular a subject not having used the active ingredient(s). The reference subject may be a group of subjects for which the average or median is obtained to be used as a reference. The phrase "neurite bearing cell" may refer to a cell displaying projection(s) of at least 1.5 times longer than the length of the cell body. In particular, the phrase "neurite bearing cell" may refer to a nerve cell (i.e. neuron) displaying projection(s) of at least 1.5 times longer than the length of the cell body. Examples of the projection include neurite, axon, and dendrite. The sample is not particularly limited so long as it contains nerve cells. The sample can be obtained by, for example, biopsy from a subject.

An improved neurite outgrowth may be an indicator of neuronal differentiation. Therefore, use of the active ingredient, specifically administration of the active ingredient to a subject, may promote neuronal differentiation, that is, may provide an effect of promoting neuronal differentiation. This effect is also referred to as "neuronal differentiation-promoting effect". In other words, an improved neurite outgrowth may result from a promoted neuronal differentiation. That is, the neurite outgrowth-improving effect may be an example of the neuronal differentiation-promoting effect. The neuronal differentiation-promoting effect can be confirmed by, for example, confirming the neurite outgrowth-improving effect.

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

Furthermore, use of the active ingredient, specifically administration of the active ingredient to a subject, may provide an effect based on a promoted neuronal differentiation and/or an improved neurite outgrowth. A promoted neuronal differentiation and/or an improved neurite outgrowth may result in, for example, protection of neural function and/or improvement of neural function. That is, a promoted neuronal differentiation and/or an improved neurite outgrowth may result in, for example, prevention, improvement, and/or treatment of a symptom related to a nerve disorder. That is, examples of the effect based on a promoted neuronal differentiation and/or an improved neurite outgrowth include the effect of protecting neural function, effect of improving neural function, and effect of preventing, improving, and/or treating a symptom related to a nerve disorder. The symptom related to a nerve disorder may be or may not be a disease. In other words, the symptom related to a nerve disorder may be or may not be caused by a disease. Examples of the symptom related to a nerve disorder include neurodegenerative diseases, and in other words, include a symptom caused by neurodegenerative diseases. Examples of the neurodegenerative diseases include diseases associated with impaired neuronal differentiation or impaired neurite outgrowth. Specific examples of the neurodegenerative diseases include Alzheimer's disease (AD), dementia, like e.g. 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 (SCD), 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.). It is expected within the present invention that ILA as provided herein can cross the blood-brain barrier.

In context with the above and in light of the appended, illustrative examples, the present invention also relates to the use of indole-3-lactic acid and/or an indole-3-lactic acid-producing bacterium in the prevention, improvement, and/or treatment of (a) nerve disorder(s), like (a) neurodegenerative disease(s). These diseases may comprise, inter alia, Alzheimer's disease (AD), dementia, like e.g. 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 (SCD), 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. In a preferred embodiment of the present invention said prevention, improvement, and/or treatment of (a) nerve disorder(s) is related to the promotion of neuronal differentiation as surprisingly shown herein and in the appended examples for indole-3-lactic acid (ILA). Accordingly, in context of the present invention it was surprisingly be found that indole-3-lactic acid (and/or indole-3-lactic acid-producing bacterium) has a neuronal differentiation promoting effect which is considered to ameliorate and/or improve symptoms of nerve disorder(s), like of neurodegenerative disease(s). Such a neuronal differentiation-promoting effect is disclosed herein and illustrated in the appended technical examples, i.e. by the surprising effect of indole-3-lactic acid (ILA) on neuronal differentiation and/or on neurite outgrowth. Accordingly, and in context of the present invention, herein disclosed is also the medical use of indole-3-lactic acid (ILA) and/or of an indole-3-lactic acid-producing bacterium in the treatment and/or prevention of a nerve disorder, like a neurodegenerative disorder. Said indole-3-lactic acid (ILA) and/or of an indole-3-lactic acid-producing bacterium is, in this context, to be administered to a subject in need of such prevention, improvement, and/or treatment of the nerve disorder, like a neurodegenerative disease(s). Preferably said subject is a mammal, like primates such as humans, monkeys and chimpanzees or also mammals, like horse, cow, sheep, goat, pig, dog, and cat. Yet, most preferably the subject to be treated with the means and methods of the present invention is a human.

The aforementioned effects, such as the neurite outgrowth-improving effect, the neuronal differentiation-promoting effect, and the effects based thereon, are also collectively referred to as "neuro-promoting effect".

The present invention may provide use of the active ingredient(s) for obtaining the neuro-promoting effect. That is, the present invention may provide use of the active ingredient(s) for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improving, and/or treating a symptom related to a nerve disorder. Also, the present invention may provide use of the active ingredient(s) for manufacturing of a composition for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improving, and/or treating a symptom related to a nerve disorder.

The present invention may provide the active ingredient(s) for use in obtaining the neuro-promoting effect. That is, the present invention may provide the active ingredient(s) for use in promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improving, and/or treating a symptom related to a nerve disorder. Also, the present invention may provide the active ingredient(s) for use in manufacturing of a composition for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improving, and/or treating a symptom related to a nerve disorder.

The active ingredient(s) may be used for therapeutic purposes or non-therapeutic purposes. That is, the neuro-promoting effect may be obtained by a therapeutic purpose or a non-therapeutic purpose, unless otherwise stated.

The phrase "therapeutic purpose" may refer to, for example, a concept containing a medical practice, and may particularly refer to a concept containing a treatment practice through therapy for a human body. Therapy may be preventive and/or curative.

The phrase "non-therapeutic purpose" may refer to, for example, a concept not containing a medical practice, and may particularly refer to a concept not containing a treatment practice through therapy for a human body. Examples of the non-therapeutic purpose include purpose of health promotion and/or purpose of beauty.

The phrase "prevention of a symptom or a disease" may mean, for example, preventing and/or delaying onset of a symptom or a disease, or decreasing the possibility of onset of a symptom or a disease. The phrase "improvement of a symptom or a disease" or "treatment of a symptom or a disease" may mean, for example, changing of a symptom or a disease for the better, preventing or delaying deterioration of a symptom or a disease, or preventing or delaying progression of a symptom or a disease. The phrase "improvement of a symptom or a disease" may particularly refer to the aforementioned phenomenon/phenomena obtained by non-therapeutic purpose. The phrase "treatment of a symptom or a disease" may particularly refer to the aforementioned phenomenon/phenomena obtained by therapeutic purpose.

ILA can be significantly superior to indole-3-propionic acid (IPA) for obtaining neuro-promoting effect, such as the neurite outgrowth-improving effect, the neuronal differentiation-promoting effect, and the effects based thereon. Similarly, the ILA-producing bacterium can be significantly superior to IPA for obtaining neuro-promoting effect, such as the neurite outgrowth-improving effect, the neuronal differentiation-promoting effect, and the effects based thereon, since ILA, which can be produced by the ILA-producing bacterium, can be significantly superior to IPA for obtaining neuro-promoting effect. Similarly, ILA and the ILA-producing bacterium each can be significantly superior to an IPA-producing bacterium for obtaining neuro-promoting effect, such as the neurite outgrowth-improving effect, the neuronal differentiation-promoting effect, and the effects based thereon, since ILA can be significantly superior to IPA, which can be produced by IPA-producing bacterium, for obtaining neuro-promoting effect. The active ingredient can be superior to IPA or the IPA-producing bacterium, for example, for promoting neurite outgrowth, in particular, NGF-induced neurite outgrowth, for promoting NGF-induced neurite outgrowth, in particular, via promoting phosphorylation of TrkA, for promoting NGF-induced neurite outgrowth, in particular, via promoting ERK signaling, or for obtaining an effect based thereon. Such a superiority of the active ingredient can be based on, for example, a difference between ILA and IPA as a legend of aryl hydrocarbon receptor (AhR). That is, since the action of ILA and IPA seems to be exerted via binding to AhR, and ILA can be a more dominant AhR ligand than IPA, the active ingredient can exhibit such a superiority.

ILA may be used as a free form, a salt thereof, or a mixture thereof. That is, the phrase "indole-3-lactic acid (ILA)" may refer to ILA in a free form, a salt thereof, or a mixture thereof, unless otherwise stated. The salt is not particularly limited so long as the neuro-promoting effect is achieved. Examples of the salt include, for example, ammonium salt, sodium salt, and potassium salt. As the salt, a single kind of salt may be used, or two or more kinds of salts may be used in combination.

As ILA, a commercially-available product may be used, or one appropriately prepared and obtained may be used. Methods for producing ILA are not particularly limited, and, for example, known methods can be used. ILA can be produced by, for example, chemical synthesis, enzymatic reaction, extraction, or fermentation. ILA can be produced by, specifically, for example, culturing an ILA-producing bacterium. ILA may be or may not be purified to a desired extent. That is, as ILA, a purified product may be used, or a material containing ILA may be used. Examples of the material containing ILA include a culture broth obtained by culturing an ILA-producing bacterium, a culture supernatant separated from the culture broth, and/or processed products thereof such as concentrates (e.g. concentrated liquids) thereof, concentrated and dried products thereof, and fractionated products thereof. The concentration of ILA 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, such as the contained amount in the composition of the present invention and the administration amount in the method of the present invention, of ILA shall be calculated on the basis of the amount of the ILA itself contained in the material. When ILA is in the form of a salt, the amount, such as the contained amount in the composition of the present invention and the administration amount in the method of the present invention, of ILA shall be calculated on the basis of the amount of ILA in terms of a value obtained by converting the mass of the salt into the mass of ILA in a free form in an amount equimolar to the salt.

The phrase "indole-3-lactic acid-producing bacterium (ILA-producing bacterium)" refers to a bacterium that is able to produce ILA. The ILA-producing bacterium may produce ILA from, for example, a carbon source and/or tryptophan. The ILA-producing bacterium may specifically be a bacterium that is able to produce ILA in a subject when the bacterium is administered to the subject. The ILA-producing bacterium may more specifically be a bacterium that is able to produce ILA in an intestine of a subject when the bacterium is administered to the subject. Examples of the intestine include a small intestine and a large intestine.

Examples of the ILA-producing bacterium include Bifidobacterium bacteria. Examples of the Bifidobacterium bacteria include Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum. Particular examples of the Bifidobacterium bacteria include Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum. As the ILA-producing bacterium, a single kind of bacterium may be used, or two or more kinds of bacteria may be used in combination.

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

It is reported that infant-type human-residential bifidobacteria (HRB) produced significantly higher concentrations of ILA compared with 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, a particular example of the ILA-producing bacterium used herein may be infant-type HRB. Examples of infant-type HRB include Bifidobacterium longum such as B. longum subsp. longum and B. longum subsp. infantis, Bifidobacterium breve, and Bifidobacterium bifidum.

Specific examples of Bifidobacterium longum include BB536 (NITE BP-02621), 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). Particular examples of Bifidobacterium longum include BB536, ATCC 15697, ATCC 15707, and M-63. More particular examples of Bifidobacterium longum include BB536. As Bifidobacterium longum, a single kind of strain may be used, or two or more kinds of strains may be used 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. Particular examples of Bifidobacterium breve include M-16V, MCC1274, and ATCC 15700. More particular examples of Bifidobacterium breve include M-16V. As Bifidobacterium breve, a single kind of strain may be used, or two or more kinds of strains may be used in combination.

Specific examples of Bifidobacterium bifidum include ATCC 29521, NITE BP-02429, NITE BP-02431, OLB6378, and BF-1. Particular examples of Bifidobacterium bifidum include ATCC 29521, NITE BP-02429, and NITE BP-02431. Specific examples of Bifidobacterium adolescentis include ATCC 15703. Specific examples of Bifidobacterium dentium include DSM 20436. Specific examples of Bifidobacterium animalis include 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 deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD; #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on January 26, 2018 as an international deposit under the provisions of the Budapest Treaty, and assigned an accession number of NITE BP-02621.

Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623) was deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD; #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on January 26, 2018 as an international deposit under the provisions of the Budapest Treaty, and assigned an accession number of NITE BP-02623.

Bifidobacterium breve M-16V (NITE BP-02622) was deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD, Address: #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on January 26, 2018 as an international deposit under the provisions of the Budapest Treaty, and assigned an accession number of NITE BP-02622.

Bifidobacterium breve MCC1274 (FERM BP-11175) was deposited at the independent administrative agency, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (currently, independent administrative agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (IPOD), Address: #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on August 25, 2009 as an international deposit under the provisions of the Budapest Treaty, and assigned an accession number of FERM BP-11175.

Bifidobacterium bifidum NITE BP-02429 was deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD, Address: #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on February 21, 2017 as an international deposit under the provisions of the Budapest Treaty, and assigned an accession number of NITE BP-02429.

Bifidobacterium bifidum NITE BP-02431 was deposited at the independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD, Address: #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on February 21, 2017 as an international deposit under the provisions of the Budapest Treaty, and assigned an accession number of NITE BP-02431.

These strains are available from, for example, the American Type Culture Collection (ATCC, Address: 10801 University Boulevard Manassas, VA 20110, United States of America), the Belgian Coordinated Collections of Microorganisms (BCCM, Address: Rue de la Science 8, 1000 Brussels, Belgium), or the depositories at which the strains were deposited.

A strain specified with the strain name exemplified above is not limited to the strain itself that has been deposited or registered to a certain organization with that strain name (hereinafter, also referred to as a "deposited strain" for convenience of explanation), but also includes strains substantially the same as the deposited strain (hereinafter, each also referred to as a "derivative strain"). That is, for example, the phrase "Bifidobacterium longum BB536" is not limited to the strain itself that has been deposited with the depository with an accession number of NITE BP-02621, but also includes strains substantially the same as the deposited strain. The phrase "a strain substantially the same as a deposited strain" refers to a strain that belongs to the same species as that of the deposited strain, is able to produce ILA, has a 16SrRNA gene of which the nucleotide sequence is preferably 99.86% or more, more preferably 99.93% or more, still more preferably 100% identical to that of the deposited strain, and preferably has the same biological characteristic as that of the deposited strain. A strain substantially the same as a deposited strain may be, for example, a derivative strain obtained from the deposited strain as a parent strain. Examples of the derivative strain include strains obtained from the deposited strain by breeding and strains spontaneously occurring from the deposited strain. Examples of methods for breeding include modification by a genetic engineering technique and modification by a mutagenesis treatment. Examples of the mutagenesis treatment include irradiation of X-ray, irradiation of ultraviolet, and a treatment with a mutation agent such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Examples of the strains spontaneously occurring from the deposited strain include strains spontaneously occurring upon use of the deposited strain. Examples of the use of the deposited strain include culturing, such as sub-culturing, of the deposited strain. A derivative strain may be constructed via a single kind of modification, or two or more kinds of modifications.

As the ILA-producing bacterium, a commercially-available product may be used, or one appropriately prepared and obtained may be used. Examples of the commercially-available product include B. longum subsp. longum BB536, B. breve M-16V, and Bifidobacterium longum subsp. infantis M-63 available from Morinaga Milk Industry Co., Ltd.

The phrase "indole-3-lactic acid-producing bacterium (ILA-producing bacterium)" as the active ingredient may specifically refer to cells of the ILA-producing bacterium. Cells of the ILA-producing bacterium can be easily obtained by culturing the ILA-producing bacterium. Methods for culturing is not particularly limited, so long as the ILA-producing bacterium can proliferate. As methods for culturing, for example, a method usually used for culturing Bifidobacterium bacteria can be use as it is, or after being modified as required. The culture temperature may be, for example, 25 to 50°C, preferably 35 to 42°C. The culture can be carried out preferably under anaerobic conditions, and for example, can be carried out with supply of an anaerobic gas such as carbon dioxide gas. Alternatively, the culture can also be carried out under microaerobic conditions such as a liquid static culture. The culture can be carried out, for example, until the ILA-producing bacterium is grown to a desired extent.

Culture media used for the culture are not particularly limited, so long as the ILA-producing bacterium can produce ILA, preferably produce ILA and proliferate. As culture media, for example, a culture medium usually used for culturing Bifidobacterium bacteria can be use as it is, or after being modified as required. The culture 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 the carbon source include saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and molasses, and these saccharides can each be used depending on the assimilability of the ILA-producing bacterium. Examples of the nitrogen source include ammonia, and ammonium or nitrate salts such as ammonium sulfate, ammonium chloride, and ammonium nitrate. Examples of the inorganic salt include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Examples of the organic component include peptone, soybean powder, defatted soybean meal, meat extract, and yeast extract. Examples of the milk component include milk proteins. Examples of the milk proteins include casein, whey, and degradation products thereof. These components may be used alone or in an appropriate combination. Specific examples of the culture medium usable for culturing the ILA-producing bacterium include Reinforced Clostridial medium, MRS medium (de Man, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium), TOSP medium (TOS propionate medium), and TOSP Mup medium (TOS propionate mupirocin medium).

As the ILA-producing bacterium, cells of the ILA-producing bacterium themselves or a fraction containing the same can be used. That is, as the ILA-producing bacterium, for example, a culture broth of the ILA-producing bacterium may be used as it is, or cells collected from the culture broth may be used. Furthermore, the cells or a fraction containing the same may also be used after being subject to a processing. The processing is not particularly limited, so long as the neuro-promoting effect is not spoiled. The processing may preferably be one by which the viability of cells is not decreased so much. Examples of the processing include dilution, concentration, freezing, and drying. That is, examples of the ILA-producing bacterium, specifically, examples of cells of the ILA-producing bacterium, include a culture broth of the ILA-producing bacterium, cells collected from the culture broth, and processed product thereof such as diluted products, concentrated products, frozen products, and dried products thereof. Specific examples of the processing include freezing of the culture broth, spray-drying, freeze-drying, and oil drop method. The cells contain viable cells so that ILA is produced after administration. The cells may or may not contain dead cells.

As the active ingredient, for example, a fraction containing both ILA and the ILA-producing bacterium may also be used. For example, a culture broth of the ILA-producing bacterium or a processed product thereof may contain both ILA and the ILA-producing bacterium.

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

That is, the composition of the present invention is a composition containing the ingredient(s) (A) and/or (B) shown below:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

The composition of the present invention can be used by being 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, for example, obtaining the neuro-promoting effect.

Use of the composition of the present invention, specifically administration of the composition of the present invention to a subject, may improve neurite outgrowth in the subject, that is, may provide the neurite outgrowth-improving effect. That is, the composition of the present invention may be, for example, a composition for improving neurite outgrowth.

Use of the composition of the present invention, specifically administration of the composition of the present invention to a subject, may promote neuronal differentiation, that is, may provide the neuronal differentiation-promoting effect. That is, the composition of the present invention may be, for example, a composition for promoting neuronal differentiation. The composition for improving neurite outgrowth may be an example of the composition for promoting neuronal differentiation.

Use of the composition of the present invention, specifically administration of the composition of the present invention to a subject, may provide an effect based on a promoted neuronal differentiation and/or an improved neurite outgrowth. That is, the composition of the present invention may be, for example, a composition for obtaining an effect based on a promoted neuronal differentiation and/or an improved neurite outgrowth. The composition of the present invention may be, specifically, for example, a composition for preventing, improving, and/or treating a symptom related to nerve disorder. The composition for obtaining an effect based on a promoted neuronal differentiation and/or an improved neurite outgrowth, such as the composition for preventing, improving, and/or treating a symptom related to nerve disorder, may be an example of the composition for promoting neuronal differentiation and/or the composition for improving neurite outgrowth.

The descriptions concerning a subject to which the active ingredient is administered mentioned in the method of the present invention 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 or drink composition, a pharmaceutical composition, or a feed composition. The composition of the present invention may particularly be a food or drink composition or a pharmaceutical composition. That is, the present invention may provide, for example, a food or drink composition for obtaining the neuro-promoting effect, such as for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improving, and/or treating a symptom related to a nerve disorder. The present invention may also provide, for example, a pharmaceutical composition for obtaining the neuro-promoting effect, such as for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improving, or treating a symptom related to a nerve disorder. The present invention may also provide, for example, a feed composition for obtaining the neuro-promoting effect, such as for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improving, and/or treating a symptom related to a nerve disorder. The composition of the present invention that is a food or drink composition, a pharmaceutical composition, or a feed composition is also referred to as a "food or drink composition of the present invention", a "pharmaceutical composition of the present invention", or a "feed composition of the present invention", respectively.

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

The additional ingredient is not particularly limited, so long as the neuro-promoting effect is not spoiled. As the additional ingredient, an ingredient acceptable depending on the use mode of the composition of the present invention can be adopted. Examples of the additional ingredient include ingredients that can be used by being blended in a food or drink, a pharmaceutical, or a feed. Specific examples of the additional ingredient include ingredients exemplified for the food or drink composition, the pharmaceutical composition, or the feed composition described below. As the additional ingredient, a single kind of ingredient may be used, or two or more kinds of ingredients may be used in combination.

The contained amount(s) and ratio thereof of ingredient(s) (i.e. the active ingredient and, optionally, additional ingredient(s)) in the composition of the present invention is not particularly limited, so long as the neuro-promoting effect is achieved. The contained amount(s) and ratio thereof of ingredient(s) in the composition of the present invention can be set depending on various conditions such as the type of the active ingredient, the type of the additional ingredient(s), the type, dosage form, and usage of the composition of the present invention, and the type, age, and health conditions of administration subject.

The amount of the active ingredient in the composition of the present invention, for example, may be 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, may be 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 may be within a range defined by a non-contradictory combination thereof, in terms of the amount of ILA.

The amount of the active ingredient in the composition of the present invention, for example, may be 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, may be 1x10¹³ cells/g or less, 1x10¹² cells/g or less, or 1x10¹¹ cells/g or less, or may be within a range defined by a combination thereof, in terms of the amount of cells of the ILA-producing bacterium. Also, the amount of the active ingredient in the composition of the present invention, for example, may be 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, may be 1x10¹³ cells/mL or less, 1x10¹² cells/mL or less, or 1x10¹¹ cells/mL or less, or may be within a range defined by a combination thereof, in terms of the amount of cells of the ILA-producing bacterium. The amount of the active ingredient in the composition of the present invention may be, specifically, for example, 1x10⁴ to 1x10¹³ cells/g, 1x10⁵ to 1x10¹³ cells/g, 1x10⁶ to 1x10¹² cells/g, preferably 1x10⁷ to 1x10¹¹ cells/g, more preferably 1x10⁸ to 1x10¹⁰ cells/g, in terms of the amount of cells of the ILA-producing bacterium. Also, the amount of the active ingredient in the composition of the present invention may be, specifically, for example, 1x10⁴ to 1x10¹³ cells/mL, 1x10⁵ to 1x10¹³ cells/mL, 1x10⁶ to 1x10¹² cells/mL, preferably 1x10⁷ to 1x10¹¹ cells/mL, more preferably 1x10⁸ to 1x10¹⁰ cells/mL, in terms of the amount of cells of the ILA-producing bacterium. The phrase "cells" may be read as "cfu". The phrase "cfu" refers to colony forming unit.

The amount of the active ingredient in the composition of the present invention may also be set, for example, so that the administration amount of the active ingredient mentioned in the method of the present invention is achieved.

The form of the composition of the present invention is not particularly limited. As the form of the composition of the present invention, a form acceptable depending on the use mode of the composition of the present invention can be adopted. Specific examples of the form of the composition of the present invention include forms exemplified for the food or drink composition, the pharmaceutical composition, or the feed composition described below.

In certain embodiments, the invention relates to a composition comprising the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium;
for use in medicine.

In one embodiment, the invention relates to a composition comprising the ingredient(s) (A) and/or (B) shown below used in the presence of nerve growth factor (NGF) as (an) active ingredient(s):
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium;
for use in medicine.

In a particular embodiment, the invention relates to a composition for use in preventing, improving, and/or treating a symptom related to a nerve disorder, the composition comprising indole-3-lactic acid or an indole-3-lactic acid-producing bacterium. In a particular embodiment, the invention relates to a composition for use in obtaining a neuro-promoting effect in a subject, the composition comprising indole-3-lactic acid or an indole-3-lactic acid-producing bacterium. In a particular embodiment, the invention relates to a composition for use in promoting neuronal differentiation or improving neurite outgrowth in a subject, the composition comprising indole-3-lactic acid or an indole-3-lactic acid-producing bacterium. In a particular embodiment, the invention relates to a composition for use in preventing, improving, and/or treating a symptom related to a disease that is associated with impaired neuronal differentiation or impaired neurite outgrowth, the composition comprising indole-3-lactic acid or an indole-3-lactic acid-producing bacterium. In certain embodiments, the composition for use according to the invention is a pharmaceutical composition or a food or drink composition. In certain embodiments, the composition for use according to the invention is a pharmaceutical composition.

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

The food or drink composition may be a food or drink itself, or may be a material used for manufacturing of a food or drink. Examples of such a material include seasonings, food additives, and other various food raw materials. Specific examples of the food or drink composition include wheat flour products, instant foods, processed agricultural products, processed fishery products, processed livestock products, milk products (such as fermented milk, cheese, and infant formula), fats and oils, basic seasonings, composite seasonings, frozen foods, confectionery, beverages, and other commercially available foods and drinks. Specific examples of the food or drink composition also include health foods, functional foods, enteral nutritive foods, foods for special dietary uses, foods with health claims (such as foods for specified health uses, foods with nutrient function claims, and foods with function claims), nutritional supplements, and quasi drugs. The food or drink composition may be, for example, a supplement such as a tablet-like supplement.

The food or drink composition of the present invention can be produced by, for example, combining the active ingredient and the additional ingredient(s). An operation for combining the active ingredient and the additional ingredient(s) is also referred to as "addition of the active ingredient". Methods for producing the food or drink composition of the present invention are not particularly limited. The food or drink composition of the present invention can be produced by, for example, the same method using the same raw material(s) as those used for producing usual foods and drinks, provided that the active ingredient is added. The same shall apply to cases of producing the food or drink composition of the present invention as a material used for manufacturing of a food or drink. Addition of the active ingredient may be carried out at any stage of the manufacturing process of the food or drink composition. Addition of the active ingredient may be carried out at, for example, during or after the manufacturing process of the food or drink composition. That is, for example, a preliminarily prepared food or drink may be added with the active ingredient to thereby produce the food or drink composition of the present invention. Furthermore, the food or drink composition of the present invention may also be produced through, for example, a fermentation step by the active ingredient, specifically a fermentation step by the ILA-producing bacterium. Examples of the food or drink composition produced through a fermentation step include fermentation products such as fermented milk and probiotic drink. That is, the active ingredient, specifically the ILA-producing bacterium, may be used as, for example, a starter for manufacturing of a fermentation product. Of course, the active ingredient can also be added to a preliminarily prepared fermentation product.

Furthermore, the food or drink composition of the present invention may be used to produce another food or drink composition. That is, for example, when the food or drink composition of the present invention is provided as a material used for manufacturing of a food or drink, such as seasonings, food additives, and other various food raw materials, addition of the food or drink composition of the present invention may result in production of another food or drink composition. Such another food or drink composition is also an example of the food or drink composition of the present invention. The descriptions concerning addition of the active ingredient in manufacturing of a food or drink composition can be applied mutatis mutandis to addition of the composition of the present invention in manufacturing of a food or drink composition.

The food or drink composition of the present invention may be provided and sold as a food or drink on which an intended application including health applications, such as an application for promoting neuronal differentiation, improving neurite outgrowth, and/or preventing, improving, and/or treating a symptom related to a nerve disorder, is indicated. The food or drink composition of the present invention may be provided and sold as a food or drink on which an intended subject is indicated.

The phrase "indication" includes all actions for informing consumers about the aforementioned application, and all expressions that can evoke or bring into mind the aforementioned application fall into the phrase "indication" regardless of the purpose, content, subject, and medium of the indication. The indication may be carried out particularly by using an expression that enables consumers to directly recognize the aforementioned application.

Specific examples of the indication include: an action to deliver, pass, exhibit for delivery or passing, or import a commodity with respect to the food or drink composition of the present invention or a package of such a commodity with the aforementioned application written thereon; and an action to exhibit or distribute an advertisement, a price list, or a transaction document of a commodity with the aforementioned application written thereon, or to provide information of such an advertisement, a price list, or a transaction document with the aforementioned application included therein by an electromagnetic method such as the Internet. Particular examples of the indication include indications on a package, a container, a catalog, a pamphlet, an advertising material in a selling site, such as POP, or other documents.

Examples of the indication include indications of health foods, functional foods, enteral nutritive foods, foods for special dietary uses, foods with health claims (such as foods for specified health uses, foods with nutrient function claims, and foods with function claims), nutritional supplements, and quasi drugs. The indication may preferably be an indication approved by the government or the like, such as, for example, an indication approved under various institutions established by the government and put in a manner based on the approval. Examples of the indication approved by the government or the like include indications approved by Consumer Affairs Agency. Examples of the indications approved by Consumer Affairs Agency include, indications approved by institutions about foods with health claims (such as foods for specified health uses, foods with nutrient function claims, and foods with function claims) and similar institutions thereto. Specific examples of the indications approved by Consumer Affairs Agency include indications of foods for specified health uses, indications of conditional foods for specified health uses, indications that the composition may influence on the body structure or function, indications about reduction in a disease risk, and indications about evidence-based functionality. More specific examples of the indications approved by Consumer Affairs Agency include indications of foods for specified health uses, established in Cabinet Office Ordinance on Approval, etc. of Indication of Special use provided in Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009) (especially an indication of a health application), and similar indications thereto.

The amount of the active ingredient in the food or drink composition of the present invention may be, for example, within any of the aforementioned ranges. The amount of the active ingredient in the food or drink composition of the present invention may be, particularly, for example, 1x10⁴ to 1x10¹³ cells/g, 1x10⁵ to 1x10¹³ cells/g, 1x10⁶ to 1x10¹² cells/g, preferably 1x10⁷ to 1x10¹¹ cells/g, more preferably 1x10⁸ to 1x10¹⁰ cells/g, in terms of the amount of cells of the ILA-producing bacterium. Also, the amount of the active ingredient in the food or drink composition of the present invention may be, particularly, for example, 1x10⁴ to 1x10¹³ cells/mL, 1x10⁵ to 1x10¹³ cells/mL, 1x10⁶ to 1x10¹² cells/mL, preferably 1x10⁷ to 1x10¹¹ cells/mL, more preferably 1x10⁸ to 1x10¹⁰ cells/mL, in terms of the amount of cells of the ILA-producing bacterium.

<Pharmaceutical composition>
The pharmaceutical composition of the present invention is not particularly limited, so long as it contains the 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 can be 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. Examples of the dosage form in cases of oral administration include solid preparations such as powders, granules, tablets, and capsules; and liquid preparations such as solutions, syrups, suspensions, and emulsions. Examples of the dosage form in cases of parenteral administration include suppositories and ointments.

Methods for formulation are not particularly limited. The formulation can be carried out by, for example, a known method depending on the dosage form. In formulation, a physiologically acceptable additive can be used. Examples of the additive include various organic and inorganic ingredients. Specific examples of the additive include excipients, binders, disintegrators, lubricants, stabilizers, corrigents, pH regulators, colorants, diluents, surfactants, and solvents. These additives can be appropriately selected depending on various conditions such as the dosage form.

Examples of excipients include saccharide derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, and carboxymethylcellulose calcium; Arabic rubbers; 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.

Examples of binders include gelatin; polyvinylpyrrolidone; and macrogol, as well as the aforementioned excipients.

Examples of disintegrators include chemically-modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethylstarch sodium, and crosslinked polyvinyl pyrrolidone, as well as the aforementioned excipients.

Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as veecum and spermaceti; boric acid; glycols; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfuric acid salts such as sodium sulfate; leucine; lauryl sulfate salts such as sodium lauryl sulfate and magnesium lauryl sulfate; silicates such as silicic anhydride and silicic acid hydrate; and starch derivatives.

Examples of stabilizers include p-hyroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.

Examples of corrigents include sweeteners, acidulants, and fragrances.

The amount of the active ingredient in the pharmaceutical composition of the present invention may be, for example, within any of the aforementioned ranges. The amount of the active ingredient in the pharmaceutical composition of the present invention may be, particularly, for example, 1x10⁴ to 1x10¹³ cells/g, 1x10⁵ to 1x10¹³ cells/g, 1x10⁶ to 1x10¹² cells/g, preferably 1x10⁷ to 1x10¹¹ cells/g, more preferably 1x10⁸ to 1x10¹⁰ cells/g, in terms of the amount of cells of the ILA-producing bacterium. Also, the amount of the active ingredient in the pharmaceutical composition of the present invention may be, particularly, for example, 1x10⁴ to 1x10¹³ cells/mL, 1x10⁵ to 1x10¹³ cells/mL, 1x10⁶ to 1x10¹² cells/mL, preferably 1x10⁷ to 1x10¹¹ cells/mL, more preferably 1x10⁸ to 1x10¹⁰ cells/mL, in terms of the amount of cells of the ILA-producing bacterium.

<Feed composition>
The feed composition of the present invention is not particularly limited, so long as it contains the active ingredient. Examples of the feed composition include pet foods and livestock feeds. The feed composition may be provided in any form such as powder, granule, crumble, pellet, cube, paste, and liquid.

The feed composition of the present invention can be produced by, for example, combining the active ingredient and the additional ingredient(s). An operation for combining the active ingredient and the additional ingredient(s) is also referred to as "addition of the active ingredient". Methods for producing the feed composition of the present invention are not particularly limited. The feed composition of the present invention can be produced by, for example, the same method using the same raw material(s) as those used for producing usual feeds, provided that the active ingredient is added. Addition of the active ingredient may be carried out at any stage of the manufacturing process of the feed composition. Addition of the active ingredient may be carried out at, for example, during or after the manufacturing process of the feed composition. That is, for example, a preliminarily prepared feed may be added with the active ingredient to thereby produce the feed composition of the present invention. Furthermore, the feed composition of the present invention may also be produced through, for example, a fermentation step by the active ingredient, specifically a fermentation step by the ILA-producing bacterium. Examples of the feed composition produced through a fermentation step include silage.

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

That is, the method of the present invention is a method comprising administering the ingredient(s) (A) and/or (B) shown below to a subject:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.

The method of the present invention can be carried out for, for example, obtaining the neuro-promoting effect.

Carrying out the method of the present invention, specifically administration of the active ingredient to a subject, may improve neurite outgrowth in the subject, that is, may provide the neurite outgrowth-improving effect. That is, the method of the present invention may be, for example, a method for improving neurite outgrowth.

Carrying out the method of the present invention, specifically administration of the method of the active ingredient to a subject, may promote neuronal differentiation, that is, may provide the neuronal differentiation-promoting effect. That is, the method of the present invention may be, for example, a method for promoting neuronal differentiation. The method for improving neurite outgrowth may be an example of the method for promoting neuronal differentiation.

Carrying out the method of the present invention, specifically administration of the active ingredient to a subject, may provide an effect based on a promoted neuronal differentiation and/or an improved neurite outgrowth. That is, the method of the present invention may be, for example, a method for obtaining an effect based on a promoted neuronal differentiation and/or an improved neurite outgrowth. The method of the present invention may be, specifically, for example, a method for preventing, improving, and/or treating a symptom related to nerve disorder. The method for obtaining an effect based on a promoted neuronal differentiation and/or an improved neurite outgrowth, such as the method for preventing, improving, and/or treating a symptom related to nerve disorder, may be an example of the method for promoting neuronal differentiation or the method for improving neurite outgrowth.

The phrase "administering the active ingredient to a subject" can be used interchangeably or equivalently to the phrase "allowing a subject to take in the active ingredient". Intake may be initiative one (i.e. free intake) or may be forced one (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 a food or drink or a feed, to a subject, to thereby allow the subject to initiatively take in the active ingredient. Administration may be oral administration or may be parenteral administration. Administration may typically be oral administration. Examples of the parenteral administration include tube administration, intrarectal administration, and Nasal administration.

Conditions for administering the active ingredient, such as the administration subject, the administration period, the number of times of administration, the administration amount, and other conditions rerated to administration, are not particularly limited, so long as the neuro-promoting effect is achieved. Conditions for administering the active ingredient can be set depending on various conditions such as the type of the active ingredient, and the type, age, and health conditions of the administration subject.

The administration subject is not particularly limited, so long as the neuro-promoting effect is achieved. Examples of the administration subject include mammals. Examples of the mammals include primates such as human, monkey, and chimpanzee; rodents such as mouse, rat, hamster, and guinea pig; and other various mammals such as rabbit, horse, cow, sheep, goat, pig, dog, and cat. Particular examples of mammals include human. The administration subject, such as mammals, may be, for example, a pet animal, a domestic animal, or an experimental animal. The administration subject may be a female subject or may be a male subject. The administration subject may be, for example, a subject in any age, such as infant, child, adult, middle-aged subject, or elderly subject. The administration subject may be, for example, a healthy subject or may be a non-healthy subject. Examples of the non-healthy subject include subjects having a symptom related to a nerve disorder.

The administration amount of the active ingredient, for example, may be 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, may be 10000 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 may be within a range defined by a non-contradictory combination thereof, in terms of the administration amount of ILA. The administration amount of the active ingredient may be, specifically, for example, 0.001 to 10000 mg/kg-body weight/day, preferably 0.1 to 1000 mg/kg-body weight/day, more preferably 1 to 100 mg/kg-body weight/day, still more preferably 10 to 100 mg/kg-body weight/day.

The administration amount of the active ingredient, for example, may be 1x10⁶ cells/kg-body weight/day or more, 1x10⁷ cells/kg-body weight/day or more, or 1x10⁸ cells/kg-body weight/day or more, may be 1x10¹² cells/kg-body weight/day or less, or 1x10¹¹ cells/kg-body weight/day or less, or 1x10¹¹ cells/kg-body weight/day or less, or may be within a range defined by a combination thereof, in terms of the administration amount of cells of the ILA-producing bacterium. The administration amount of the active ingredient may be, specifically, for example, 1x10⁶ to 1x10¹² cells/kg-body weight/day, preferably 1x10⁷ to 1x10¹¹ cells/kg-body weight/day, more preferably 1x10⁸ to 1x10¹⁰ cells/kg-body weight/day, in terms of the administration amount of cells of the ILA-producing bacterium. The phrase "cells" may be read as "cfu".

The administration period of the active ingredient, for example, may be 1 day or longer, 3 days or longer, 1 week or longer, 2 weeks or longer, 4 weeks or longer, 2 months or longer, 3 months or longer, 4 months or longer, 6 months or longer, 9 months or longer, 12 months or longer, may be 10 years or shorter, 5 years or shorter, 1 year or shorter, or 6 months or shorter, or may be within a range defined by a non-contradictory combination thereof. The active ingredient may be administered, for example, over the whole period of the life of the subject or during a partial period of the life of the subject. The active ingredient may be administered, for example, at least until the neuro-promoting effect is achieved. The active ingredient may be administered, for example, every day or once per several days. The active ingredient may be administered, particularly, every day. The administration amount of the active ingredient may be or may not be constant for each administration.

The active ingredient, for example, may be administered to the subject as it is, or may be prepared as a composition containing the active ingredient, such as a food or drink, pharmaceutical, or feed composition, and then administered to the subject. The descriptions concerning the composition of the present invention can be applied mutatis mutandis to the composition containing the active ingredient. The active ingredient may be administered solely or in combination with an additional ingredient. Examples of the additional ingredient include foods and drinks, pharmaceuticals, feeds, and ingredients contained therein. In addition, the descriptions concerning the additional ingredient contained in the composition of the present invention can be applied mutatis mutandis to the additional ingredient used in the method of the present invention.

The active ingredient can also be administered to the subject by, for example, using the composition of the present invention, specifically, by administering the composition of the present invention to the subject. That is, an embodiment of the method of the present invention may be a method comprising administering the composition of the present invention to the subject. That is, the phrase "administration of the active ingredient" also includes administration of the composition of the present invention. Conditions for administering the composition of the present invention, such as the administration subject, the administration period, the number of times of administration, the administration amount, and other conditions rerated to administration, are not particularly limited, so long as the neuro-promoting effect is achieved. Conditions for administering the composition of the present invention can be set depending on various conditions such as the type and contained amount of the active ingredient, the type and contained amount of the additional ingredient(s), the type and dosage form of the composition, and the type, age, and health conditions of the administration subject. The descriptions concerning the conditions for administering the active ingredient can be applied mutatis mutandis to the conditions for administering the composition of the present invention. That is, the composition of the present invention may be administered to, for example, such a subject as exemplified above. In addition, the administration amount of the composition of the present invention can be set, for example, so that such an administration amount of the active ingredient exemplified above is obtained. In addition, the composition of the present invention may be administered solely or in combination with an additional ingredient.

Examples

Hereinafter, the present invention will be more specifically explained with reference to 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 as well as 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 at the concentration of 10 mM in DMSO. The stock solutions were serially diluted in sterile MilliQ water to prepare the analytical samples. Nerve growth factor (NGF; 2.5S) was purchased from Alomone Labs (Jerusalem, Israel).

<1-1> Cell culture
PC12 cell, a rat adrenal pheochromocytoma cell line, was purchased from the European Collection of Authenticated Cell Cultures (ECACC 88022401; Salisbury, UK). The floating 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) in an atmosphere of 5% CO₂ at 37°C. The medium was replaced every three days.

<1-2> Dose-response of tryptophan and its metabolites
PC12 cells (passage number <13) were seeded in collagen type IV-coated 24-well culture plates (Iwaki, Shizuoka, Japan) at a density of 10,000 cells/mL per well with the complete growth medium (RPMI supplemented with 10% (v/v) HS, 5% (v/v) FBS, and 1% (v/v) penicillin/streptomycin) for 24 h. The cells were changed to low serum (1% v/v HS) medium for 24 h. Thereafter, the cells were treated with NGF (25 ng/mL) and the test compounds (ILA, IPA, or tryptophan) at a wide range of final concentration (1 μM, 100 nM, 10 nM and 1 nM) for five consecutive days. The low serum medium and test compounds were replaced at day 3. Papaverine hydrochloride added under the same conditions was used as a positive control. Non-treated control (cells without NGF) and NGF control (cells treated with NGF only) were also grown under the same conditions. The cells were then subjected to immunofluorescence staining for quantification of neurite outgrowth and acetylcholinesterase assay. Thereafter, the maximal effective concentration of the test compounds was selected for further analyses.

<1-3> Immunofluorescence staining
Following treatment for five days, cells in transparent collagen IV-coated plates were washed twice with phosphate-buffered saline (PBS) at room temperature and fixed for 5 min using the absolute methanol (precooled to -20°C). Cells were washed thrice with ice-cold PBS and incubate with blocking buffer PBST (1% (w/v) bovine serum albumin/10% (v/v) normal goat serum/0.3 M glycine in PBS containing 0.1% (v/v) Tween-20) for 1 h. Cell body and processes were then labelled with an anti-βIII-tubulin mouse primary antibody (1 μg/mL; Abcam, Cambridge, UK) diluted in PBST containing 1% (w/v) bovine serum albumin at 4°C overnight, followed by an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (2μg/mL; Abcam) for 1 h at room temperature, protected from light. For nuclear staining, cells were counterstained with Cellstain(R) 4’,6’-Diamidino-2-phenylindole (DAPI; Dojindo Molecular Technologies, Kumamoto, Japan) for 10 min at 37 °C. Plates were then subjected to image acquisition and analysis of neurite outgrowth.

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

<1-5> Analysis of Acetylcholinesterase (AchE) activity
To examine the effect of tryptophan and its metabolites on AchE activity, which is a biochemical marker for neuronal differentiation in PC12 cells, the thiocholine produced from the hydrolysis of acetylthiocholine by the endogenous AchE enzyme in each sample was quantified by a fluorescence colourimetric assay. In brief, PC12 cells were grown and treated for five consecutive days as aforementioned. The cells were then 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 of the cell lysates was determined by the Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instructions. Assay signals were read with a fluorescence absorbance microplate reader at Ex/Em = 490/520 nm. The AchE activity was determined from the standard curve and normalized with the protein concentration in each sample. The protein concentration was determined by Pierce bicinchoninic acid (BCA) protein assay kit (Invitrogen, Paisley, UK) with bovine serum albumin as a standard.

<1-6> Western blot analysis of phospho-TrkA, ERK, phospho-ERK, CREB, and phospho-CREB proteins
PC12 cells (passage number <13) were seeded in collagen type IV-coated 24-well culture plates at a density of 50,000 cells/mL per well with the complete growth medium for 24 h, then shifted to low serum (1% v/v HS) medium for 24 h prior to exposure to NGF (25 ng/mL) and the test compounds (positive control, ILA, IPA or tryptophan at a final concentration of 100 nM) for 24 h. Non-treated control (cells without NGF) and NGF control (cells treated with NGF only) were also grown 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 for 15 min) at 4°C and the protein concentration was determined using a BCA kit with bovine serum albumin as a standard.

After boiling for 5 min, the cell lysate (20 μg) was separated on 10% SDS-PAGE and then transferred onto the polyvinylidene difluoride (PVDF) membrane on an iBlot dry blotting system (Invitrogen, Paisley, UK). Non-specific reactivity was blocked by Bullet Blocking One solution (Nacalai Tesque Inc., Kyoto, Japan) at room temperature for 5 min with shaking. Blots were incubated 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) overnight at 4°C. After three washes with Tris Buffered Saline containing 0.1% Tween-20 (TBST), the blots were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000) (Abcam) for 1 h. The blots were washed with TBST, and the proteins were detected by Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare, Chicago, Illinois, USA) according to the manufacturer’s instructions, and then the chemiluminescence signal was visualized using 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 per well) were cultured in collagen type IV-coated 24-well culture plates as aforementioned. The cells were then exposed to the antagonists of AhR receptor (ANF and CH223191) at a final concentration of 1 μM for 1 h prior to the treatment with NGF (25 ng/mL) and the test compounds (positive control, ILA, IPA or tryptophan at a final concentration of 100 nM) for five 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 three days. Following treatment, cell lysate containing 20 μg of total protein were then collected, separated on 8% SDS-PAGE and transferred onto the PVDF membrane on an iBlot dry blotting system. The blots were blocked in Bullet Blocking One solution at room temperature for 5 min with shaking, followed by overnight incubation at 4°C with the appropriate antibodies: anti-aryl hydrocarbon receptor antibody (1:1000) and anti-β-actin (1:5000) (Abcam). Next, the blots were washed thrice with TBST and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000) (Abcam) for 1 h. Signals were developed by Amersham ECL Select Western Blotting Detection Reagent, visualized using ChemiDoc Imager and quantified using Image Lab software.

<1-8> Analysis of AchE activity upon pre-treatment with AhR antagonists
PC12 cells (10,000 cells/mL per well) were cultured in collagen type IV-coated 24-well culture plates as aforementioned. The cells were then exposed to the antagonists of AhR receptor (ANF and CH223191) at a final concentration of 1 μM for 1 h prior to the treatment with NGF (25 ng/mL) and the test compounds (positive control, ILA, IPA or tryptophan at a final concentration of 100 nM) for five 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 three days. Following treatment, the cells were lysed with ice-cold NP-40 cell lysis buffer. AchE activity of the cell lysates was then determined by the Amplite Fluorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instructions. The AchE activity was determined as described above.

<1-9> Statistical analyses
Results are presented as the mean with standard deviation. All statistical analyses were performed using IBM SPSS Statistics, version 22.0, statistical software package (IBM Corp., Armonk, NY, USA). Statistical significance of differences between each treatment group was analyzed by independent 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) did not morphologically affect neurite outgrowth of PC12 cells in the absence of NGF (data not shown). However, when in the presence of 25 ng/mL NGF, it was found that ILA, IPA, and tryptophan promoted neurite outgrowth of PC12 cells in a dose-independent manner (Figure 1, panel(A)). Comparing with the NGF control, significant higher neurite outgrowth (P < 0.05) was observed in cells treated with ILA at 10 nM and 100 nM, with maximal activity seen at 100 nM, whereas no significant higher neurite outgrowth was observed in cells treated with IPA. That is, it was confirmed that ILA can be significantly superior to IPA for promoting neurite outgrowth, in particular, NGF-induced neurite outgrowth. In addition, significant higher AchE activity (P < 0.01) was also observed in cells treated with ILA at 100 nM (Figure 1, panel(B)). Meanwhile, the maximal effective concentration of IPA was seen at 100 nM where a significant higher AchE activity (P < 0.05) but not the percentage of neurite bearing cells was observed upon treatment. No significant increases in the percentage of neurite bearing cells and AchE activity were observed in tryptophan-treated cells.

<2-2> Effects of tryptophan and its metabolites on neurite outgrowth of PC12 cells
To verify the potentiating effects of tryptophan and its metabolites, PC12 cells were treated with NGF and the test compounds at 100 nM, and the neurite outgrowth quantification assay and AchE assay were performed as described in Materials and Methods. As shown in Figure 2, panel (A), prominent neurite outgrowth was elicited by ILA and IPA and slight outgrowth by tryptophan. Interestingly, treatment with ILA significantly enhanced the percentage of neurite bearing cells (P < 0.01) in NGF-induced PC12 cells, whereas treatment with IPA did not significantly enhance the percentage of neurite bearing cells (Figure 2, panel (B)). That is, again, it was confirmed that ILA can be significantly superior to IPA for promoting neurite outgrowth, in particular, NGF-induced neurite outgrowth. The percentage of neurite bearing cells for 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 with the NGF control (12.68 ± 0.23 %) and significantly higher than those of NTC (0.05 ± 0.09 %). In addition, the AchE activity was markedly increased in the NGF-induced PC12 cells treated with ILA (P < 0.05), IPA (P < 0.01), and tryptophan (P < 0.05), as compared to the NGF control (Figure 2, panel (C)). These data indicate that the indole derivatives could potentially enhance NGF-induced neurite outgrowth in PC12 cells.

<2-3> Effects of tryptophan and its metabolites on the Ras/ERK pathway
The inventors next investigated whether TrkA receptor and extracellular signal-regulated kinase 1/2 (ERK1/2) activation are 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 the phosphorylation of TrkA for 24 h (Figure 3, panel (A)). The relative phosphorylation level of TrkA in ILA-treated cells (1.92 ± 0.15) increased significantly (P < 0.05) comparing to NGF control (1.53 ± 0.17), while the phosphorylation level of TrkA in IPA-treated cells (1.85 ± 0.41) was tended to increase but not significantly different as compared to the NGF control (1.53 ± 0.17). That is, it was confirmed that ILA can be significantly superior to IPA for promoting NGF-induced neurite outgrowth, in particular, via promoting phosphorylation of TrkA. The phosphorylation levels of ILA and IPA were in proportion to their neurite outgrowth promoting activities. On the contrary, treatment of PC12 cells with tryptophan had no induction effect on 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 of ERK1 (44 kDa) and ERK2 (42 kDa) (Thr202/Tyr204) after 24 h treatment (Figure 3, panel (B)). The P value is smaller for ILA than for IPA. That is, it was confirmed that ILA can be significantly superior to IPA for promoting NGF-induced neurite outgrowth, in particular, via promoting ERK signaling. The phosphorylation level 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 indicate that ERK signaling is involved in the indole derivatives-enhanced NGF-induced neuronal differentiation of PC12 cells.

The inventors continued to investigate the possible involvement of CREB (cAMP response element-binding protein) in indole derivatives-enhanced NGF-induced neurite outgrowth of PC12 cells. As shown in Figure 3, panel (C), treatment of PC12 cells with the indole derivatives (100 nM ILA or IPA) significantly increased the phosphorylation of CREB (P < 0.05) for 24 h, comparing to NGF control. The phosphorylation level 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 indicate that indole derivatives (ILA and IPA) induced TrkA and ERK1/2 phosphorylation and in turn activated CREB transcription in the process of neurite outgrowth of PC12 cells.

<2-4> Potential role of tryptophan and its metabolites as AhR ligands
The inventors further investigated the potential role of indole derivatives as AhR ligands by western blot analysis and the possible involvement of AhR signaling in the process of neurite outgrowth by the measurement of AchE activity. The results showed that ILA and IPA could potentially act as AhR ligands, with significant increases in the relative expression of AhR upon the treatment (Figure 4 and 5).

Despite certain differences in susceptibility to the AhR antagonists (ANF and CH223191), the effects of ILA and IPA on AhR activation were abrogated in the presence of ANF and/or CH223191. As shown in Figures 4, panel (A) and 5, panel (A), pre-treatment of ANF (1μM) significantly inhibited ILA-induced AhR activity (P < 0.05) in PC12 cells, while the AchE activity of ILA-treated cells was also tended to decrease. Similarly, ILA-induced AhR activation and AchE activity were also greatly inhibited by CH223191 (1μM) pre-treatment, indicating the specific involvement of AhR in the process of ILA-induced neuronal differentiation.

Meanwhile, ANF and CH223191 inhibited IPA-induced AhR activation and AchE activity with different intensities. The AhR activity was significantly suppressed by CH223191 (P < 0.05) but not ANF in IPA-treated cells. In contrary, the AchE activity of IPA-treated cells was significantly inhibited by ANF (P < 0.05) but not CH223191. These data indicate that IPA might be a less dominant AhR ligand than ILA. Since the action of ILA and IPA seems to be exerted via binding to aryl hydrocarbon receptor (AhR), a more dominant AhR ligand, which can be ILA, can promote neurite outgrowth more strongly than IPA. Hence, again, it was confirmed that ILA can be significantly superior to IPA for promoting neurite outgrowth, in particular, NGF-induced neurite outgrowth. Both ANF and CH223191 did not affect the activity of tryptophan on AhR activation and AchE activity, with the exception in the AchE activity of ANF-pre-treated cells.

<Test Example>
1.Bacterial Strains and Culture Supernatants (CSs)
Bifidobacterial strains shown in Table 1 were each cultured at 37^oC for 16 h under anaerobic conditions in MRS broth (Becton Dickinson, MD, USA) supplemented with 0.05% L-cysteine (Kanto Chemical Co., Ltd., Chuo-ku, Tokyo, Japan))(MRS-C) using an Anaero Pack (Mitsubishi Gas Chemical, Tokyo, Japan).

The growth-phase bacterial cells were then harvested by centrifugation using a high-speed centrifugal refrigerating machine, HIMAC SCR20B (Hitachi Koki Co., Ltd., Tokyo, Japan) at 5000 g (4^oC for 10 min) and washed twice with phosphate buffered saline (PBS) and Dulbecco’s Formula (DS Pharma Biomedical Co., Ltd., Osaka, Japan). Subsequently, whole-cell pellets were suspended in PBS containing 0.05% L-cysteine (PBS-C). The optical density (at 600 nm) of each bacterial cell suspension was adjusted to the same value (OD600 = 0.2) using PBS-C. Cell suspensions (100 L) were added to MRS-C (3 mL) and cultured at 37^oC for 24 h under anaerobic conditions. The CSs were obtained by centrifuging the culture suspensions at 5000 g (4^oC for 10 min). Following filtration (pore size 0.22 m; Millipore, MA, USA), the samples were stored at 80^oC until use. All cultures were grown in independent triplicates, and the resulting data were expressed as the mean of these replicates.

2. Quantification of ILA Concentrations in CSs
The concentration of the ILA in CSs was analyzed using 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 Science Inc., Tokyo, Japan) (particle diameter 2.1 μm, column size 150 mm×2 m). Mobile phase A (containing 1 g/L AA in water) and mobile phase B (containing 1 g/L AA in acetonitrile) were applied at a flow rate of 0.2 mL/min. The gradient elution was started at 10% B. At 0.1-18 min, 10%-90% B; 18.1-25 min, 90%; 25.1-28 min, 90%-10%; 28-40 min, 10%. Quantitation was performed by comparing metabolite concentrations in CSs with those of the corresponding synthetic ILA standards and the internal standard (MOI). The LC-MS/MS spectrum (product ion data) of the positive precursor ion was evaluated to determine their final content.

As a result, the Bifidobacterial strains produced ILA in amounts shown in Table 1. It was confirmed that infant-type HRB, such as the strains Nos. 1 to 10, can produce a higher amount of ILA than adult-type HRB and non-HRB, such as the strains Nos. 11 to 19. It has been reported that none of these strains Nos. 1 to 19 produced 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)).

According to the present invention, a composition usable for a specific use, such as a composition for improving neurite outgrowth can be provided.

Claims

A composition for promoting neuronal differentiation and/or improving neurite outgrowth, containing the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
A composition for preventing, improving, and/or treating a symptom related to a nerve disorder, containing the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
The composition according to claim 1 or 2, wherein the composition at least contains the ingredient (B).
The composition according to any one of claims 1 to 3, wherein the ingredient (B) is a Bifidobacterium bacterium.
The composition according to any one of claims 1 to 4, wherein the ingredient (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.
The composition according to any one of claims 1 to 5, wherein the composition is a food or drink composition.
The composition according to any one of claims 1 to 5, wherein the composition is a pharmaceutical composition.
A method for promoting neuronal differentiation and/or improving neurite outgrowth, comprising administering the ingredient(s) (A) and/or (B) shown below as an active ingredient to a subject:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
A method for preventing, improving, and/or treating a symptom related to a nerve disorder, comprising administering the ingredient(s) (A) and/or (B) shown below as an active ingredient to a subject:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
The method according to claim 8 or 9, wherein at least the ingredient (B) is administered to the subject.
The method according to any one of claims 8 to 10, wherein the ingredient (B) is a Bifidobacterium bacterium.
The method according to any one of claims 8 to 11, wherein the ingredient (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.
The method according to any one of claims 8 to 12, wherein the subject is a human subject.
Use of the ingredient(s) (A) and/or (B) shown below for promoting neuronal differentiation and/or improving neurite outgrowth:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
Use of the ingredient(s) (A) and/or (B) shown below for preventing, improving, and/or treating a symptom related to a nerve disorder:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
Use of the ingredient(s) (A) and/or (B) shown below in manufacturing of the composition according to any one of claims 1 to 7:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
A composition for use in promoting neuronal differentiation and/or improving neurite outgrowth, the composition comprising the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
A composition for use in preventing, improving, and/or treating a symptom related to a nerve disorder, the composition comprising the ingredient(s) (A) and/or (B) shown below as an active ingredient:
(A) indole-3-lactic acid;
(B) an indole-3-lactic acid-producing bacterium.
The composition for use according to claim 17 or 18, wherein the composition at least contains the ingredient (B).
The composition for use according to any one of claims 17 to 19, wherein the ingredient (B) is a Bifidobacterium bacterium.
The composition for use according to any one of claims 17 to 20, wherein the ingredient (B) is Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium bifidum.
The composition for use according to any one of claims 17 to 21, wherein the composition is a food or drink composition.
The composition for use according to any one of claims 17 to 22, wherein the composition is a pharmaceutical composition.