Immunostimulation composition, and immunostimulation method

Abstract

Disclosed is a composition for immunostimulation, the composition comprising: a bacterium having an immunostimulating ability; and one or more compounds selected from the group consisting of amino acids, peptides, and proteins having the function of enhancing the immunostimulating ability of the bacterium.

Description

Title of Invention: IMMUNOSTIMULATION COMPOSITION, AND IMMUNOSTIMULATION METHOD Technical Field

[0001] The present invention relates to an immunostimulatory composition and an immunostimulatory method. Background Art

[0002] Immunity is a biological defense mechanism that prevents foreign substances from entering the body and senses and eliminates foreign substances that have entered the body, and plays a very important role in maintaining health. On the other hand, causes of reduced immune function include lack of exercise, overwork, stress, lack of sleep, smoking, and alcohol consumption and the like, and there is a daily risk of reduced immune function. For these reasons, development of technical means for stimulating immune function is being promoted.

[0003] As such technical means, for example, compositions and the like capable of stimulating immune function through ingestion of pharmaceuticals, foods, and the like have been developed. For example, Patent Literature 1 discloses an interferon (IFN) production inducer containing, as an active ingredient, a lactic acid bacterium or a culture or processed product thereof capable of activating plasmacytoid dendritic cells (pDCs) and inducing interferon (IFN) production. Patent Literature 2 discloses an immunostimulatory activity-enhancing composition containing, as active ingredients, a lactic acid bacterium having immunostimulatory activity and an ester-linked product of a polyhydric alcohol and a saturated fatty acid. Patent Literature 3 discloses a method for producing a lactic acid bacterium having enhanced immunostimulatory activity and free of an ester-linked product of a polyhydric alcohol and a fatty acid, the method comprising contacting an immunostimulatory activity-enhancing composition containing an ester-linked product of a polyhydric alcohol and a fatty acid as an active ingredient with a lactic acid bacterium having immunostimulatory activity, and then removing the above-described immunostimulatory activity-enhancing composition.

[0004] Here, in order to use a composition capable of stimulating immune function as a pharmaceutical, a food, or the like for which quality control is required, it is required to control the immune function-stimulating activity of the composition by enhancing or suppressing it. Citation List Patent Literature

[0005] [Patent Literature 1] International Publication No. 2012 / 091081 [Patent Literature 2] Japanese Unexamined Patent Application Publication No. 2016-005452 [Patent Literature 3] Japanese Unexamined Patent Application Publication No. 2017-085975 Summary of Invention

[0006] The   present   invention aims   to provide a  novel immunostimulatory composition.

[0007] The present inventors have found that one or more compounds selected from the group consisting of predetermined amino acids, peptides, and proteins enhance the activation of pDCs induced by predetermined bacteria having immunostimulatory activity and the accompanying IFN-a production, and have completed the present invention.

[0008] The present invention provides, for example, the following inventions. [1] An immunostimulatory composition comprising: a bacterium having immunostimulatory activity; and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium. [2] An immunostimulatory method comprising administering to a subject or causing a subject to ingest: a bacterium having immunostimulatory activity; and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium. [3] A method for enhancing an immunostimulatory action induced by a bacterium having immunostimulatory activity, the method comprising administering to a subject or causing a subject to ingest: a bacterium having immunostimulatory activity; and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium. [4] An enhancer of an immunostimulatory action induced by a bacterium having immunostimulatory activity, the enhancer comprising one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium having immunostimulatory activity. [5] An enhancer of an immunostimulatory action induced by a bacterium having immunostimulatory activity, the enhancer comprising: a bacterium having immunostimulatory activity; and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium. [6] A composition for immunostimulation induced by a bacterium having immunostimulatory activity, the composition comprising one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium having immunostimulatory activity. [7] Use of a bacterium having immunostimulatory activity and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium, for producing an immunostimulatory composition. [8] Use of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, for producing a composition for immunostimulation induced by the bacterium. [9] Use of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, for producing an immunostimulatory composition to be administered or ingested in combination with the bacterium having immunostimulatory activity.

[10] Use of a bacterium having immunostimulatory activity for producing an immunostimulatory composition to be administered or ingested in combination with one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium having immunostimulatory activity.

[11] One or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, for use in immunostimulation induced by the bacterium having immunostimulatory activity.

[12] A bacterium having immunostimulatory activity for use in a therapeutic method of immunostimulation, the bacterium being administered or ingested in combination with one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium.

[13] One or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, for use in a therapeutic method of immunostimulation, the one or more compounds being administered or ingested in combination with the bacterium having immunostimulatory activity.

[14] Use of a bacterium having immunostimulatory activity in a non-therapeutic method of immunostimulation, the bacterium being administered or ingested in combination with one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium.

[15] Use of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, in a non-therapeutic method of immunostimulation, the one or more compounds being administered or ingested in combination with the bacterium having immunostimulatory activity.

[16] The composition, method, enhancer, use, compound, or bacterium according to any one of [1] to

[15] , wherein the bacterium having immunostimulatory activity is a lactic acid bacterium and / or an acetic acid bacterium.

[17] The composition, method, enhancer, use, compound, or bacterium according to

[16] , wherein the lactic acid bacterium is one or more selected from bacteria of the genus Lactococcus, bacteria of the genus Weissella, bacteria of the genus Bifidobacterium, and bacteria of the genus Lactobacillus.

[18] The composition, method, enhancer, use, compound, or bacterium according to

[16] , wherein the lactic acid bacterium is one or more selected from bacteria of the genus Lactococcus, bacteria of the genus Weissella, and bacteria of the genus Bifidobacterium.

[19] The composition, method, enhancer, use, compound, or bacterium according to

[17] , wherein the bacterium of the genus Lactococcus is Lactococcus lactis subsp. lactis or Lactococcus curvatus, the bacterium of the genus Weissella is Weissella paramesenteroides, the bacterium of the genus Bifidobacterium is Bifidobacterium animalis subsp. lactis, and the bacterium of the genus Lactobacillus is Lactobacillus paraplantarum.

[20] The composition, method, enhancer, use, compound, or bacterium according to

[18] , wherein the bacterium of the genus Lactococcus is Lactococcus lactis subsp. lactis, the bacterium of the genus Weissella is Weissella paramesenteroides, and the bacterium of the genus Bifidobacterium is Bifidobacterium animalis subsp. lactis.

[21] The composition or use according to any one of [1], [6] to

[10] , and

[16] to

[20] , wherein the content of the bacterium having immunostimulatory activity is 0.001% by mass or more and less than 100% by mass relative to the total amount of the immunostimulatory composition.

[22] The composition or use according to any one of [1], [6] to

[10] , and

[16] to

[21] , wherein the content of the compound is 0.000001% by mass or more and 90.0% by mass or less relative to the total amount of the immunostimulatory composition.

[23] The composition, method, enhancer, use, compound, or bacterium according to any one of [1] to

[22] , wherein the ratio of the content (% by mass) of the bacterium having immunostimulatory activity to the content (% by mass) of the compound ((content of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein) / (content of the bacterium having immunostimulatory activity)) is 0.001 or more and 50000 or less.

[24] The composition, method, enhancer, use, compound, or bacterium according to any one of [1] to

[23] , wherein the immunostimulatory activity is an activity to induce interferon a production in an immune cell.

[25] The composition, method, enhancer, use, compound, or bacterium according to any one of [1] to

[24] , wherein the immune cell is a pDC.

[26] The composition, method, enhancer, use, compound, or bacterium according to any one of [1] to

[25] , wherein the immunostimulatory activity-enhancing action is an action of enhancing induction of interferon a production in the immune cell by 1.10-fold or more as compared with an immunostimulatory composition comprising only the bacterium having immunostimulatory activity.

[27] The composition, method, enhancer, use, compound, or bacterium according to any one of [1] to

[26] , wherein the compound is one or more compounds selected from the group consisting of alanine, asparagine, cysteine, glutamine, leucine, proline, threonine, tryptophan, valine, arginine, histidine, lysine, isoleucine, serine, ornithine, cystine, glutathione, imidazole dipeptide, silk peptide, sardine-derived peptide, casein hydrolysate, collagen peptide, wheat protein hydrolysate, whey protein hydrolysate, and elastin.

[28] The composition, method, enhancer, use, compound, or bacterium according to any one of [1] to

[27] , wherein the compound is glutathione.

[29] An immunostimulatory composition obtained by adding, to a bacterium having immunostimulatory activity, one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium.

[30] A method for producing an immunostimulatory composition, the method comprising adding, to a bacterium having immunostimulatory activity, one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium. Brief Description of Drawings

[0009] [FIG. 1] FIG. 1 is a graph showing the results of evaluating the enhancing effect of reduced glutathione on IFN-a production by plasmacytoid dendritic cells (pDC) induced by Lactococcus lactis subsp. lactis JCM 5805. [FIG. 2] FIG. 2 is a graph showing the results of evaluating the enhancing effect of reduced glutathione on IFN-a production by pDC induced by a lactic acid bacterium. [FIG. 3] FIG. 3 is a graph showing the results of evaluating the enhancing effect of reduced glutathione on IFN-a production by pDC induced by a bifidobacterium. Description of Embodiments

[0010] Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0011] According to the present invention, a novel immunostimulatory composition can be provided.

[0012] Although the present invention is described in connection with specific examples and various embodiments, it will be readily understood by those skilled in the art that many modifications and applications of the embodiments described in the present specification are possible without departing from the spirit and scope of the present invention.

[0013] The present application claims priority based on Japanese Patent Application No. 2024-068171 filed with the Japan Patent Office on April 19, 2024, the contents of which are incorporated herein by reference in their entirety.

[0014] In the present disclosure, the expression “one or more selected from the group consisting of” encompasses one element constituting the group or all combinations that can be formed by two or more elements, and may be, for example, one of the elements constituting the group, or a combination of any two, three, four, five, six, seven, eight, nine, or more elements of the elements constituting the group.

[0015] [Immunostimulatory Composition] The first embodiment of the present disclosure relates to an immunostimulatory composition. The immunostimulatory composition contains a bacterium having immunostimulatory activity and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action. The immunostimulatory composition according to one embodiment has an immunostimulatory action because it contains a bacterium having immunostimulatory activity.

[0016] An immunostimulatory action (immunoactivating action) is an action of activating the innate immune system in cells or a living body, and may be an action of increasing or maintaining (suppressing a decrease in) the function of the innate immune system in cells or a living body. The immunostimulatory action (immunostimulatory activity) according to one aspect may include an immune cell-activating action (immune cell-activating activity), may include a dendritic cell-activating action (dendritic cell-activating activity), and may be a dendritic cell-activating action (dendritic cell-activating activity), and when the immunostimulatory action includes a dendritic cell-activating action, or when the immunostimulatory action is a dendritic cell-activating action, the bacterium having immunostimulatory activity has dendritic cell-activating activity.

[0017] The dendritic cell-activating action (dendritic cell-activating activity) may be an action of increasing the expression level of a cytokine by dendritic cells as compared with a case where the bacterium having immunostimulatory activity is not administered, ingested, or the like. The immunostimulatory action (immunostimulatory activity) according to one aspect may include a plasmacytoid dendritic cell-activating action (pDC-activating action) (plasmacytoid dendritic cell-activating activity (pDC-activating activity)), may be a pDC-activating action (pDC-activating activity), and when the immunostimulatory action includes a pDC-activating action, or when the immunostimulatory action is a pDC-activating action, the above-described bacterium has pDC-activating activity. The pDC-activating action (pDC-activating activity) may be an action (activity) of increasing the expression level of a cytokine by pDCs as compared with a case where the bacterium having immunostimulatory activity is not administered, ingested, or the like.

[0018] Plasmacytoid dendritic cells are a type of dendritic cell constituting the innate immune system. pDCs are the major producer cells of type I interferon in the body. Type I interferon exhibits proliferation-inhibiting activity against viruses and the like. Interferon a (IFN-a) and interferon 0 (IFN-0) are known as representative type I interferons, and in addition to these, interferon X (IFN-X) as a type II interferon is also considered to have an immunostimulatory action. Specific examples of pDCs include CAL-1 cells and the like. CAL-1 cells are a cancer cell line of human plasmacytoid dendritic cells established from tumor cells in the peripheral blood of a patient (Japanese Unexamined Patent Application Publication No. 2007-044008), and are a cell line deposited with the Patent Microorganism Depositary, Biotechnology Center, National Institute of Technology and Evaluation (NPMD, Japan) under accession number FERM BP-10914.

[0019] The immunostimulatory action (immunostimulatory activity), dendritic cell-activating action (dendritic cell-activating activity), or pDC-activating action (pDC-activating activity) according to one aspect may include an action of promoting the production (activity to promote the production) of at least one or more IFNs selected from the group consisting of interferon a (IFN-a), interferon P (IFN-P), and interferon X (IFN-X), may include an interferon a (IFN-a) production-promoting action (IFN-a production-promoting activity), and may be an IFN-a production-promoting action (IFN-a production-promoting activity).

[0020] Whether or not the immunostimulatory composition according to one embodiment has an immunostimulatory action can be determined as appropriate. For example, when immune cells are cultured in the presence of the immunostimulatory composition according to one embodiment, if the function of the immune cells is enhanced as compared with a case where immune cells are cultured in the absence of the composition, it is determined that the composition has an immunostimulatory action. In addition, when immune cells are cultured in the presence of the immunostimulatory composition according to one embodiment and a substance that reduces the function of immune cells, if the function of the immune cells is maintained (suppression of decrease) as compared with a case where immune cells are cultured in the absence of the composition and in the presence of the substance that reduces the function of immune cells, it is determined that the composition has an immunostimulatory action. The function of immune cells may be evaluated using, for example, an activation marker molecule (a protein or the like) indicating that the immune cells are in an activated state as an index, or may be evaluated using a molecule (a protein or the like; for example, IFN and the like) produced when the immune cells are in an activated state as an index.

[0021] Whether or not the immunostimulatory composition according to one embodiment has an immunostimulatory action can be confirmed, as a more detailed example, by measuring the IFN-a concentration according to the following procedures (i) to (iv). (i) Mouse-derived bone marrow cells from which erythrocytes have been removed are suspended in RPMI medium prepared so that each component described in the following <Composition of medium> reaches the respective final concentration described in the following <Composition of medium>, to a concentration of 1*106 cells / mL, to prepare a cell suspension. <Composition of medium> • 10% by volume FBS • 100 U / mL penicillin / streptomycin • 1 mM sodium pyruvate • 2.5 mM HEPES • 1% by mass non-essential amino acids for MEM (NEAA) • 50 pM P—mercaptoethanol • 100 ng / mL Flt-3L (ii) The prepared cell suspension is seeded at 1 mL each, and cultured for one week at 37°C and 5% by volume CO2 in a CO2 incubator to induce pDCs. (iii) Bone marrow cells containing the induced pDCs are suspended to a concentration of 2*105 cells / mL, seeded at 200 pL each into a 96-well plate, and 2 pL each of a composition containing bacteria adjusted to a concentration of 1 mg / mL with PBS is added. (iv) After 24 hours, the culture supernatant is collected, and the IFN-a concentration is measured by ELISA using an IFN-a measurement kit.

[0022] In the present specification, “bacterium having immunostimulatory activity” means a bacterium that has an action of stimulating (activating) immune cells. The “immunostimulatory activity” may be any activity to stimulate immune cells, and may be, for example, an activity to induce IFN production in immune cells, an activity to induce production of at least one or more IFNs selected from the group consisting of IFN-a, IFN-P, and IFN-Z in immune cells, or an activity to induce IFN-a production in immune cells. The “bacterium having immunostimulatory activity” may be either a viable cell or a dead cell, and may be a disrupted product of a viable cell or a dead cell, a freeze-dried product of a viable cell or a dead cell, a disrupted product of the freeze-dried product, a culture solution, a culture solution extract, and the like, or a processed product thereof. Here, the processed product includes, for example, a product processed by enzyme treatment, heat treatment, and the like, or a product obtained by ethanol precipitation of the processed product and recovered. The “bacterium having immunostimulatory activity” is preferably a product obtained by subjecting a disrupted product of a dead cell, a freeze-dried product thereof, or a disrupted product of the freeze-dried product to heat treatment  (heat-killed  cell).     When the “bacterium having immunostimulatory activity” is a dead cell, the bacterium can be produced by, for example, killing viable bacteria by heat treatment, pressure treatment, high-pressure steam treatment, electromagnetic wave treatment, electron beam treatment, radiation treatment, ultraviolet treatment, alcohol treatment, or electrolyzed water treatment and the like, and then drying as necessary by freeze-drying, spray drying, drum drying, hot air drying, or vacuum drying and the like.

[0023] Whether or not the bacterium according to the present embodiment has immunostimulatory activity is not particularly limited, and if the function of immune cells is enhanced when immune cells are cultured in the presence of the bacterium having immunostimulatory activity as compared with a case where immune cells are cultured in the absence of the bacterium, it may be determined that the bacterium has an immunostimulatory action. Whether or not the bacterium according to the present embodiment has immunostimulatory activity can be confirmed, as a more detailed example, by measuring the IFN-a concentration according to the above procedures (i) to (iv).

[0024] Whether or not the bacterium according to the present embodiment has immunostimulatory activity can be expressed by an index of producing 30 pg / mL or more, preferably 50 pg / mL or more, more preferably 60 pg / mL or more, more preferably 70 pg / mL or more, more preferably 80 pg / mL or more, more preferably 90 pg / mL or more, more preferably 100 pg / mL or more, more preferably 150 pg / mL or more, more preferably 200 pg / mL or more, more preferably 250 pg / mL or more, more preferably 300 pg / mL or more, more preferably 400 pg / mL or more, more preferably 500 pg / mL or more, more preferably 600 pg / mL or more, more preferably 700 pg / mL or more, and particularly preferably 800 pg / mL or more of IFN-a by co-culturing the bacterium at a final concentration of 10 ug / mL for 24 hours with bone marrow cells at a final concentration of 2*105 cells / mL, which contain pDCs and are obtained by collecting cells from mouse bone marrow and culturing them for 7 days in a cell culture medium containing 100 ng / mL Flt3-L.

[0025] The bacterium according to the present embodiment is not particularly limited, but may be a bacterium harmless to the human body. The bacterium according to one aspect may be a Gram-positive bacterium or a Gram-negative bacterium. The bacterium according to the present embodiment may be a lactic acid bacterium and / or an acetic acid bacterium, and may in particular be a lactic acid bacterium. The bacterium having immunostimulatory activity according to the present embodiment may be one type of bacterium or a mixture of two or more types of bacteria, and may be, for example, one or more types of lactic acid bacteria, one or more types of acetic acid bacteria, or a mixture of one or more types of lactic acid bacteria and one or more types of acetic acid bacteria.

[0026] Lactic acid bacteria are bacteria that produce lactic acid as a metabolite. Examples of lactic acid bacteria include bacteria of the genus Oenococcus, bacteria of the genus Bifidobacterium, bacteria of the genus Weissella, bacteria of the genus Tetragenococcus, bacteria of the genus Lactococcus, bacteria of the genus Leuconostoc, bacteria of the genus Pediococcus, bacteria of the genus Streptococcus, bacteria of the genus Enterococcus, bacteria of the genus Lactobacillus, bacteria of the genus Bacillus, and bacteria of the genus Heyndrickxia (formerly classified as bacteria of the genus Bacillus) and the like.

[0027] Note that bacteria of the genus Lactobacillus in the present invention include bacteria that were classified in the genus Lactobacillus before the reclassification of the genus Lactobacillus. For example, bacteria reclassified into the newly established genera of Acetilactobacillus, Apilactobacillus, Agrilactobacillus, Bombilactobacillus, Amylolactobacillus, Companilactobacillus, Dellaglioa, Fructilactobacillus, Furfurilactobacillus, Holzapfelia, Lacticaseibacillus, Latilactobacillus, Ligilactobacillus, Loigolactobacillus, Lactiplantibacillus, Lentilactobacillus, Limosilactobacillus, Paralactobacillus, Lapidilactobacillus, Levilactobacillus, Liquorilactobacillus, Paucilactobacillus, Schleiferilactobacillus, Secundilactobacillus, and the like in association with the reclassification of the genus Lactobacillus are included.

[0028] Among the above, the bacterium is preferably a bacterium of the genus Oenococcus, a bacterium of the genus Bifidobacterium, a bacterium of the genus Lentilactobacillus, a bacterium of the genus Weissella, a bacterium of the genus Tetragenococcus, a bacterium of the genus Lactococcus, a bacterium of the genus Leuconostoc, a bacterium of the genus Pediococcus, a bacterium of the genus Enterococcus, a bacterium of the genus Lactobacillus, a bacterium of the genus Lactiplantibacillus, a bacterium of the genus Lacticaseibacillus, or a bacterium of the genus Limosilactobacillus, and more preferably a bacterium of the genus Lactococcus, a bacterium of the genus Weissella, a bacterium of the genus Bifidobacterium, or a bacterium of the genus Lactobacillus.

[0029] Examples of the above-described bacteria of the genus Oenococcus include, but are not particularly limited to, Oenococcus oeni and the like. Specific examples of bacteria of the genus Oenococcus include Oenococcus oeni JCM 6125 and the like.

[0030] Examples of the above-described bacteria of the genus Bifidobacterium include, but are not particularly limited to, Bifidobacterium animalis subsp. lactis, Bifidobacterium longum subsp. infantis, and the like. Specific examples of bacteria of the genus Bifidobacterium include Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium longum subsp. infantis JCM 1222, Bifidobacterium longum subsp. longum BB536, and the like.

[0031] Examples of the above-described bacteria of the genus Weissella include, but are not particularly limited to, Weissella paramesenteroides, Weissella viridescens, and the like. Specific examples of bacteria of the genus Weissella include Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, and the like.

[0032] Examples of the above-described bacteria of the genus Tetragenococcus include, but are not particularly limited to, Tetragenococcus halophilus and the like. Specific examples of bacteria of the genus Tetragenococcus include Tetragenococcus halophilus NRIC 0098, Tetragenococcus halophilus No. 1, and the like.

[0033] Examples of the above-described bacteria of the genus Lactococcus include, but are not particularly limited to, Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus curvatus, Lactococcus plantarum, and the like.

[0034] Specific examples of the above-described bacteria of the genus Lactococcus include Lactococcus lactis subsp. lactis JCM 5805 (hereinafter sometimes referred to as JCM 5805), Lactococcus lactis subsp. lactis NBRC 12007, Lactococcus lactis subsp. lactis NRIC 1150, Lactococcus lactis subsp. lactis JCM 20101, Lactococcus lactis subsp. lactis JCM 7638, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, Lactococcus lactis subsp. lactis ATCC 29146, Lactococcus lactis subsp. lactis ATCC 27861, Lactococcus lactis subsp. lactis ATCC 19435, Lactococcus lactis subsp. lactis ATCC 15577, Lactococcus lactis subsp. lactis ATCC 15346, Lactococcus lactis subsp. lactis ATCC 13675, Lactococcus lactis subsp. lactis ATCC 12929, Lactococcus lactis subsp. lactis ATCC 11955, Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, Lactococcus garvieae NBRC 100934, Lactococcus lactis subsp. cremoris JCM 16167, Lactococcus lactis subsp. cremoris NBRC 100676, Lactococcus lactis subsp. hordniae JCM 1180, Lactococcus lactis subsp. hordniae JCM 11040, Lactococcus curvatus JCM 1096, Lactococcus plantarum JCM 11056, and the like.

[0035] Examples of the above-described bacteria of the genus Leuconostoc include, but are not particularly limited to, Leuconostoc carnosum, Leuconostoc lactis, and the like. Specific examples of bacteria of the genus Leuconostoc include Leuconostoc carnosum JCM 9695, Leuconostoc lactis NBRC 12455, and the like.

[0036] Examples of the above-described bacteria of the genus Pediococcus include, but are not particularly limited to, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus cellicola, Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parvulus, and Pediococcus stilesii and the like. Specific examples of bacteria of the genus Pediococcus include, for example, Pediococcus acidilactici JCM 8797, Pediococcus acidilactici K15, and Pediococcus damnosus JCM 5886 and the like.

[0037] Examples of the above-described bacteria of the genus Streptococcus include, but are not particularly limited to, Streptococcus thermophilus and the like. Specific examples of bacteria of the genus Pediococcus include, for example, Streptococcus thermophilus SBC 8781 and the like.

[0038] Examples of the above-described bacteria of the genus Enterococcus include, but are not particularly limited to, Enterococcus alcedinis and Enterococcus faecalis and the like. Specific examples of bacteria of the genus Enterococcus include Enterococcus faecalis EC-12 and the like.

[0039] Examples of the above-described bacteria of the genus Lactobacillus include, but are not particularly limited to, Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei, Lacticaseibacillus paracasei subsp. paracasei, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus rhamnosus, Lacticaseibacillus rhamnosus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus parakefiri, Lentilactobacillus parakefiri, Lactobacillus plantarum, Lactiplantibacillus plantarum, Lactobacillus plantarum subsp. plantarum, Lactiplantibacillus plantarum subsp. plantarum, Lactobacillus pentosus, Lactiplantibacillus pentosus, Lactobacillus helveticus, Limosilactobacillus reuteri subsp. reuteri, Lactobacillus crispatus, Lactobacillus paraplantarum, Lactiplantibacillus paraplantarum, and Lactobacillus johnsonii and the like.

[0040] Specific examples of bacteria of the genus Lactobacillus include Lactobacillus paracasei KW3110, Lactobacillus paracasei MCC1849, Lactobacillus paracasei K71, Lactobacillus paracasei K-2, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus CRL1505, Lactobacillus gasseri SBT2055, Lactobacillus acidophilus L-92, Lactobacillus casei subsp. casei 327, Lactobacillus (Lacticaseibacillus in the new classification) casei Shirota, Lactobacillus bulgaricus OLL1073R-1, Lactobacillus parakefiri (Lentilactobacillus parakefiri in the new classification) JCM 8573, Lactobacillus plantarum (Lactiplantibacillus plantarum in the new classification) L-137, Lactobacillus pentosus (Lactiplantibacillus pentosus in the new classification) ONRICb0240, Lactobacillus paraplantarum (Lactiplantibacillus paraplantarum in the new classification) JCM 1149, Lactobacillus acidophilus JCM 1021, Lactobacillus acidophilus JCM 1132, Lactobacillus helveticus JCM 1003, Limosilactobacillus reuteri subsp. reuteri JCM 1112, Lactobacillus crispatus JCM 1185, and Lactobacillus johnsonii JCM 2012 and the like.

[0041] Examples of the above-described bacteria of the genus Bacillus include, but are not particularly limited to, Bacillus coagulans and the like. Specific examples of bacteria of the genus Bacillus include, for example, Bacillus coagulans SANK 70258 strain and the like.

[0042] Examples of bacteria of the genus Heyndrickxia include, but are not particularly limited to, Heyndrickxia coagulans (also known as Bacillus coagulans) and the like. Specific examples of bacteria of the genus Heyndrickxia include, for example, Heyndrickxia (Bacillus) coagulans SANK 70258 and Heyndrickxia (Bacillus) coagulans BC99 and the like.

[0043] Acetic acid bacteria are bacteria that produce acetic acid as a metabolite. Examples of acetic acid bacteria include, but are not particularly limited to, bacteria of the genus Gluconacetobacter, bacteria of the genus Acetobacter, and bacteria of the genus Gluconobacter and the like, with bacteria of the genus Gluconacetobacter being preferred, Gluconacetobacter hansenii being more preferred, and Gluconacetobacter hansenii GK-1 being even more preferred.

[0044] In addition to the above, the lactic acid bacteria may be a bacterium of the genus Akkermansia, a bacterium of the genus Bacteroides, a bacterium of the genus Blautia, a bacterium of the genus Clostridium, a bacterium of the genus Collinsella, a bacterium of the genus Faecalibacterium, a bacterium of the genus Faecalicatena, a bacterium of the genus Lacrimispora, a bacterium of the genus Paeniclostridium, a bacterium of the genus Parabacteroides, or a bacterium of the genus Roseburia.

[0045] Specific examples of bacteria of the genus Akkermansia include, for example, Akkermansia muciniphila JCM 30893 and the like.

[0046] Specific examples of bacteria of the genus Bacteroides include, for example, Bacteroides caccae JCM 9498T, Bacteroides fragilis JCM 11019T, Bacteroides fragilis JCM 11017, Bacteroides fragilis JCM 17586, Bacteroides fragilis JCM 17587, Bacteroides ovatus JCM 5824T, Bacteroides thetaiotaomicron ATCC 29148T, Bacteroides thetaiotaomicron ATCC 29741, Bacteroides thetaiotaomicron ATCC 12290, Bacteroides uniformis JCM 5828T, Bacteroides uniformis JCM 13286, Bacteroides uniformis JCM 13287, and Bacteroides uniformis JCM 13288 and the like.

[0047] Specific examples of bacteria of the genus Blautia include, for example, Blautia acetigignens JCM 34803T, Blautia ammoniilytica JCM 34802T, Blautia argi JCM 31394T, Blautia caecimuris JCM 34498T, Blautia coccoides JCM 1395T, Blautia faecis JCM 17205T, Blautia glucerasea JCM 17039T, Blautia hansenii JCM 14655, Blautia hansenii JCM 35484, Blautia hominis JCM 32276T, Blautia hydrogenotrophica JCM 31266, Blautia liquoris JCM 34225T, Blautia luti JCM 17040T, Blautia obeum JCM 31340, Blautia producta JCM 1471T, Blautia pseudococcoides JCM 35243T, Blautia schinkii JCM 14657T, Blautia wexlerae JCM 31267, and Blautia wexlerae JCM 35486 and the like.

[0048] Specific examples of bacteria of the genus Clostridium include, for example, Clostridium butyricum JCM NT, Clostridium nexile JCM 31500T, and Clostridium symbiosum JCM 1297T and the like.

[0049] Specific examples of bacteria of the genus Collinsella include, for example, Collinsella aerofaciens JCM 10188T, Collinsella intestinalis JCM 10643T, Collinsella stercoris JCM 10641T, and Collinsella tanakaei JCM 16071T and the like.

[0050] Specific examples of bacteria of the genus Faecalibacterium include, for example, Faecalibacterium hattorii JCM 39210, Faecalibacterium longum JCM 39208, Faecalibacterium prausnitzii JCM 31915, Faecalibacterium prausnitzii JCM 39207, and Faecalibacterium prausnitzii JCM 39209 and the like.

[0051] Specific examples of bacteria of the genus Faecalicatena include, for example, Faecalicatena oroticum JCM 1429T and the like.

[0052] Specific examples of bacteria of the genus Lacrimispora include, for example, Lacrimispora celerecrescens JCM 15734T, Lacrimispora sphenoides JCM 1415T, and Lacrimispora xylanolytica JCM 15735T and the like.

[0053] Specific examples of bacteria of the genus Paeniclostridium include, for example, Paeniclostridium sordellii JCM 3814T and the like.

[0054] Specific examples of bacteria of the genus Parabacteroides include, for example, Parabacteroides merdae JCM 9497T and the like.

[0055] Specific examples of bacteria of the genus Roseburia include, for example, Roseburia hominis JCM 17582, Roseburia intestinalis JCM 17583, and Roseburia inulinivorans JCM 17584 and the like.

[0056] The bacterium according to one preferred aspect may be at least one bacterium selected from the group consisting of Lactobacillus rhamnosus CRL1505 or a mutant strain thereof, Gluconacetobacter hansenii or a mutant strain thereof, Lactobacillus acidophilus (GK-1) L-92 or a mutant strain thereof, Lactobacillus plantarum L-137 or a mutant strain thereof, Lactobacillus bulgaricus OLL1073R-1 or a mutant strain thereof, Lactococcus lactis subsp. lactis JCM 5805 or a mutant strain thereof, Lactococcus curvatus JCM 1096 or a mutant strain thereof, Lactococcus lactis subsp. lactis ATCC 7962 or a mutant strain thereof, and Lactobacillus paraplantarum JCM 1149 or a mutant strain thereof. The bacterium according to one more preferred aspect may be at least one bacterium selected from the group consisting of Lactococcus lactis subsp. lactis JCM 5805 or a mutant strain thereof, Lactococcus curvatus JCM 1096 or a mutant strain thereof, Lactococcus lactis subsp. lactis ATCC 7962 or a mutant strain thereof, and Lactobacillus paraplantarum JCM 1149 or a mutant strain thereof.

[0057] Among the above-described lactic acid bacteria and acetic acid bacteria, JCM strains can be obtained from the RIKEN BioResource Center, Microorganism Division (3-1-1 Koyadai, Tsukuba, Ibaraki), NBRC strains can be obtained from the Biological Resource Center, National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu, Chiba), NRIC strains can be obtained from the Culture Collection, Tokyo University of Agriculture (1-1-1 Sakuragaoka, Setagaya, Tokyo), and ATCC strains can be obtained from the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia, USA), respectively. In addition, the above-described lactic acid bacteria and acetic acid bacteria can be obtained not only from public institutions but also by isolating or purifying them from commercially available products containing lactic acid bacteria or acetic acid bacteria using known methods.

[0058] The above-described JCM strains can be obtained from the RIKEN BioResource Center, Microorganism Division as described above, but in the present invention, the same strains as the above-described JCM strains stored at storage institutions other than the RIKEN BioResource Center, Microorganism Division can be used. Specifically, the same strain as Lactococcus lactis subsp. lactis JCM 5805 can be obtained from the Biological Resource Center, National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu, Chiba), the Culture Collection, Tokyo University of Agriculture (1-1-1 Sakuragaoka, Setagaya, Tokyo), the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia, USA), and the like. Lactococcus lactis subsp. lactis JCM 5805 has been deposited with the American Type Culture Collection as Lactococcus lactis subsp. lactis ATCC 9936 and Lactococcus lactis subsp. lactis ATCC 19435.

[0059] The above-described mutant strains may be any strains having characteristics (for example, immunostimulatory activity and the like) capable of achieving the objectives of the present technology. In addition, the mutant strain is preferably a bacterium having the same bacteriological properties as the above-described strain and having immunostimulatory activity equal to or greater than that of the above-described strain. Whether or not a mutant strain has immunostimulatory activity equal to or greater than that of the above-described strain can be confirmed, for example, by the evaluation method described below or the method of the examples described below.

[0060] Such a mutant strain may be constructed by non-artificially introducing a mutation into the above-described strain. In addition, such a mutant strain may be constructed by artificially introducing a mutation into the above-described strain, and may be constructed, for example, by introducing a mutation into the above-described bacterium by treatment with a mutagen such as ultraviolet (UV) light or a DNA alkylating agent, or may be constructed by introducing a mutation into the above-described strain by a genetic manipulation method such as known genetic recombination or gene editing represented by CRISPR-Cas9.

[0061] The bacterium according to the present embodiment can be cultured by a conventional method according to its bacterial species. For example, when the above-described bacterium is a lactic acid bacterium or an acetic acid bacterium, it can be carried out by a known method using a known medium. The medium is not particularly limited as long as it is a medium capable of growing the above-described bacterium, but for example, a medium prepared by appropriately adding sugars to MRS medium, GAM medium, or M17 medium can be used, and inorganic salts, vitamins, and / or amino acids and the like may be added as appropriate. Culturing can be carried out at 25 to 40°C for several hours to several days.

[0062] The effective amount of bacteria contained in the composition according to the present embodiment is not particularly limited as long as it is an amount capable of exerting immunostimulatory activity, and may vary depending on the subject of application; when the subject of application is a mammal such as a human, the lower limit thereof can be, for example, 1*108 cells, 1*109 cells, or 1*1010 cells, and the upper limit thereof can be, for example, 1*1014 cells, 1*1013 cells, or 1*1012 cells. These upper and lower limits can each be combined arbitrarily, and the range of the above-described intake amount can be, for example, 1*108 or more and 1*1014 cells or less, 1*108 or more and 1*1013 cells or less, 1*108 or more and 1*1012 cells or less, 1*109 or more and 1*1014 cells or less, 1*109 or more and 1*1013 cells or less, 1*1010 or more and 1*1014 cells or less, 1*1010 or more and 1*1013 cells or less, or 1*1010 or more and 1*1012 cells or less. The number of bacteria can be measured by a known microscope, flow cytometer, or non-culture rapid microbial testing device (for example, ELESTA PixeeMo (AFI Technology Co., Ltd.)) and the like, but measurement by microscope is preferred from the viewpoint of versatility. The composition having immunostimulatory activity may be used, for example, in the form of a food composition, a pharmaceutical composition, a quasi-drug, a bacterial powder (a powder obtained by drying bacterial cells or a powder containing the same and the like), or a feed and the like.

[0063] The content of the bacterium having immunostimulatory activity in the immunostimulatory composition according to one embodiment is not particularly limited and may be any amount that satisfies the effective amount for immunostimulation, and therefore may vary depending on the form of the immunostimulatory composition according to the present embodiment. For example, the dry mass of the bacterium according to the present embodiment relative to the total mass of the immunostimulatory composition according to the present embodiment may be 0.0001% by mass or more, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.02% by mass or more, 0.05% by mass or more, 0.10% by mass or more, 0.30% by mass or more, 1.0% by mass or more, 1.5% by mass or more, 2.0% by mass or more, 2.5% by mass or more, 3.0% by mass or more, 5.0% by mass or more, 7.0% by mass or more, or 10.0% by mass or more, and may be less than 100% by mass, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, or 15% by mass or less.

[0064] These lower and upper limits can each be combined arbitrarily, and for example, the dry mass of the bacterium according to the present embodiment relative to the total mass of the immunostimulatory composition according to the present embodiment may be 0.0001% by mass or more and less than 100% by mass, 0.0001% by mass or more and 95% by mass or less, 0.0001% by mass or more and 90% by mass or less, 0.0001% by mass or more and 80% by mass or less, 0.0001% by mass or more and 70% by mass or less, 0.0001% by mass or more and 60% by mass or less, 0.0001% by mass or more and 50% by mass or less, 0.0001% by mass or more and 30% by mass or less, 0.0001% by mass or more and 25% by mass or less, 0.0001% by mass or more and 20% by mass or less, 0.0001% by mass or more and 15% by mass or less, 0.001% by mass or more and less than 100% by mass, 0.001% by mass or more and 95% by mass or less, 0.001% by mass or more and 90% by mass or less, 0.001% by mass or more and 80% by mass or less, 0.001% by mass or more and 70% by mass or less, 0.001% by mass or more and 60% by mass or less, 0.001% by mass or more and 50% by mass or less, 0.001% by mass or more and 30% by mass or less, 0.001% by mass or more and 25% by mass or less, 0.001% by mass or more and 20% by mass or less, 0.001% by mass or more and 15% by mass or less, 0.005% by mass or more and less than 100% by mass, 0.005% by mass or more and 95% by mass or less, 0.005% by mass or more and 90% by mass or less, 0.005% by mass or more and 80% by mass or less, 0.005% by mass or more and 70% by mass or less, 0.005% by mass or more and 60% by mass or less, 0.005% by mass or more and 50% by mass or less, 0.005% by mass or more and 30% by mass or less, 0.005% by mass or more and 25% by mass or less, 0.005% by mass or more and 20% by mass or less, 0.005% by mass or more and 15% by mass or less, 0.01% by mass or more and less than 100% by mass, 0.01% by mass or more and 95% by mass or less, 0.01% by mass or more and 90% by mass or less, 0.01% by mass or more and 80% by mass or less, 0.01% by mass or more and 70% by mass or less, 0.01% by mass or more and 60% by mass or less, 0.01% by mass or more and 50% by mass or less, 0.01% by mass or more and 30% by mass or less, 0.01% by mass or more and 25% by mass or less, 0.01% by mass or more and 20% by mass or less, 0.01% by mass or more and 15% by mass or less, 0.02% by mass or more and less than 100% by mass, 0.02% by mass or more and 95% by mass or less, 0.02% by mass or more and 90% by mass or less, 0.02% by mass or more and 80% by mass or less, 0.02% by mass or more and 70% by mass or less, 0.02% by mass or more and 60% by mass or less, 0.02% by mass or more and 50% by mass or less, 0.02% by mass or more and 30% by mass or less, 0.02% by mass or more and 25% by mass or less, 0.02% by mass or more and 20% by mass or less, 0.02% by mass or more and 15% by mass or less, 0.05% by mass or more and less than 100% by mass, 0.05% by mass or more and 95% by mass or less, 0.05% by mass or more and 90% by mass or less, 0.05% by mass or more and 80% by mass or less, 0.05% by mass or more and 70% by mass or less, 0.05% by mass or more and 60% by mass or less, 0.05% by mass or more and 50% by mass or less, 0.05% by mass or more and 30% by mass or less, 0.05% by mass or more and 25% by mass or less, 0.05% by mass or more and 20% by mass or less, 0.05% by mass or more and 15% by mass or less, 0.10% by mass or more and less than 100% by mass, 0.10% by mass or more and 95% by mass or less, 0.10% by mass or more and 90% by mass or less, 0.10% by mass or more and 80% by mass or less, 0.10% by mass or more and 70% by mass or less, 0.10% by mass or more and 60% by mass or less, 0.10% by mass or more and 50% by mass or less, 0.10% by mass or more and 30% by mass or less, 0.10% by mass or more and 25% by mass or less, 0.10% by mass or more and 20% by mass or less, 0.10% by mass or more and 15% by mass or less, 0.30% by mass or more and less than 100% by mass, 0.30% by mass or more and 95% by mass or less, 0.30% by mass or more and 90% by mass or less, 0.30% by mass or more and 80% by mass or less, 0.30% by mass or more and 70% by mass or less, 0.30% by mass or more and 60% by mass or less, 0.30% by mass or more and 50% by mass or less, 0.30% by mass or more and 30% by mass or less, 0.30% by mass or more and 25% by mass or less, 0.30% by mass or more and 20% by mass or less, 0.30% by mass or more and 15% by mass or less, 1.0% by mass or more and less than 100% by mass, 1.0% by mass or more and 95% by mass or less, 1.0% by mass or more and 90% by mass or less, 1.0% by mass or more and 80% by mass or less, 1.0% by mass or more and 70% by mass or less, 1.0% by mass or more and 60% by mass or less, 1.0% by mass or more and 50% by mass or less, 1.0% by mass or more and 30% by mass or less, 1.0% by mass or more and 25% by mass or less, 1.0% by mass or more and 20% by mass or less, 1.0% by mass or more and 15% by mass or less, 1.5% by mass or more and less than 100% by mass, 1.5% by mass or more and 95% by mass or less, 1.5% by mass or more and 90% by mass or less, 1.5% by mass or more and 80% by mass or less, 1.5% by mass or more and 70% by mass or less, 1.5% by mass or more and 60% by mass or less, 1.5% by mass or more and 50% by mass or less, 1.5% by mass or more and 30% by mass or less, 1.5% by mass or more and 25% by mass or less, 1.5% by mass or more and 20% by mass or less, 1.5% by mass or more and 15% by mass or less, 2.0% by mass or more and less than 100% by mass, 2.0% by mass or more and 95% by mass or less, 2.0% by mass or more and 90% by mass or less, 2.0% by mass or more and 80% by mass or less, 2.0% by mass or more and 70% by mass or less, 2.0% by mass or more and 60% by mass or less, 2.0% by mass or more and 50% by mass or less, 2.0% by mass or more and 30% by mass or less, 2.0% by mass or more and 25% by mass or less, 2.0% by mass or more and 20% by mass or less, 2.0% by mass or more and 15% by mass or less, 2.5% by mass or more and less than 100% by mass, 2.5% by mass or more and 95% by mass or less, 2.5% by mass or more and 90% by mass or less, 2.5% by mass or more and 80% by mass or less, 2.5% by mass or more and 70% by mass or less, 2.5% by mass or more and 60% by mass or less, 2.5% by mass or more and 50% by mass or less, 2.5% by mass or more and 30% by mass or less, 2.5% by mass or more and 25% by mass or less, 2.5% by mass or more and 20% by mass or less, 2.5% by mass or more and 15% by mass or less, 3.0% by mass or more and less than 100% by mass, 3.0% by mass or more and 95% by mass or less, 3.0% by mass or more and 90% by mass or less, 3.0% by mass or more and 80% by mass or less, 3.0% by mass or more and 70% by mass or less, 3.0% by mass or more and 60% by mass or less, 3.0% by mass or more and 50% by mass or less, 3.0% by mass or more and 30% by mass or less, 3.0% by mass or more and 25% by mass or less, 3.0% by mass or more and 20% by mass or less, 3.0% by mass or more and 15% by mass or less, 5.0% by mass or more and less than 100% by mass, 5.0% by mass or more and 95% by mass or less, 5.0% by mass or more and 90% by mass or less, 5.0% by mass or more and 80% by mass or less, 5.0% by mass or more and 70% by mass or less, 5.0% by mass or more and 60% by mass or less, 5.0% by mass or more and 50% by mass or less, 5.0% by mass or more and 30% by mass or less, 5.0% by mass or more and 25% by mass or less, 5.0% by mass or more and 20% by mass or less, 5.0% by mass or more and 15% by mass or less, 7.0% by mass or more and less than 100% by mass, 7.0% by mass or more and 95% by mass or less, 7.0% by mass or more and 90% by mass or less, 7.0% by mass or more and 80% by mass or less, 7.0% by mass or more and 70% by mass or less, 7.0% by mass or more and 60% by mass or less, 7.0% by mass or more and 50% by mass or less, 7.0% by mass or more and 30% by mass or less, 7.0% by mass or more and 25% by mass or less, 7.0% by mass or more and 20% by mass or less, 7.0% by mass or more and 15% by mass or less, 10.0% by mass or more and less than 100% by mass, 10.0% by mass or more and 95% by mass or less, 10.0% by mass or more and 90% by mass or less, 10.0% by mass or more and 80% by mass or less, 10.0% by mass or more and 70% by mass or less, 10.0% by mass or more and 60% by mass or less, 10.0% by mass or more and 50% by mass or less, 10.0% by mass or more and 30% by mass or less, 10.0% by mass or more and 25% by mass or less, 10.0% by mass or more and 20% by mass or less, or 10.0% by mass or more and 15% by mass or less. The above-described dry mass of the bacterium according to the present embodiment can be used, for example, as a daily dose for an adult weighing 60 kg. The dose can be set according to factors such as the health condition of the person receiving or ingesting the dose, the method of administration or ingestion, and the combination with other agents.

[0065] The number of bacteria having immunostimulatory activity in the immunostimulatory composition according to the present embodiment may be 1.0*103 cells / mL or more, 1.0*104 cells / mL or more, 1.0*105 cells / mL or more, 1.0*106 cells / mL or more, 1.0*107 cells / mL or more, or 4.0*107 cells / mL or more, and may be 1.0*1011 cells / mL or less, 1.0*1010 cells / mL or less, 3.0*109 cells / mL or less, or 1.0*109 cells / mL or less. These lower and upper limits can each be combined arbitrarily, and for example, the number of bacteria having immunostimulatory activity in the immunostimulatory composition according to the present embodiment may be 1.0*103 cells / mL or more and 1.0*1011 cells / mL or less, 1.0*103 cells / mL or more and 1.0*1010 cells / mL or less, 1.0*103 cells / mL or more and 3.0*109 cells / mL or less, 1.0*103 cells / mL or more and 1.0*109 cells / mL or less, 1.0*104 cells / mL or more and 1.0*1011 cells / mL or less, 1.0*104 cells / mL or more and 1.0*1010 cells / mL or less, 1.0*104 cells / mL or more and 3.0*109 cells / mL or less, 1.0*104 cells / mL or more and 1.0*109 cells / mL or less, 1.0*105 cells / mL or more and 1.0*1011 cells / mL or less, 1.0*105 cells / mL or more and 1.0*1010 cells / mL or less, 1.0*105 cells / mL or more and 3.0*109 cells / mL or less, 1.0*105 cells / mL or more and 1.0*109 cells / mL or less, 1.0*106 cells / mL or more and 1.0*1011 cells / mL or less, 1.0*106 cells / mL or more and 1.0*1010 cells / mL or less, 1.0*106 cells / mL or more and 3.0*109 cells / mL or less, 1.0*107 cells / mL or more and 1.0*1011 cells / mL or less, 1.0*107 cells / mL or more and 1.0*1010 cells / mL or less, 1.0*107 cells / mL or more and 1.0*109 cells / mL or less, 4.0*107 cells / mL or more and 1.0*1011 cells / mL or less, 4.0*107 cells / mL or more and 1.0*1010 cells / mL or less, or 4.0*107 cells / mL or more and 1.0*109 cells / mL or less. In this case, the daily intake of the liquid immunostimulatory composition may be 10 mL or more and 1,000 mL or less, 30 mL or more and 800 mL or less, 50 mL or more and 500 mL or less, or 100 mL or more and 250 mL or less.

[0066] In the immunostimulatory composition according to the present embodiment, the number of bacteria having immunostimulatory activity in the immunostimulatory composition according to one embodiment per unit package may be 1.0*107 cells or more, 1.0*108 cells or more, 3.0*108 cells or more, 5.0*108 cells or more, 1.0*109 cells or more, 5.0*109 cells or more, 1.0*1010 cells or more, 5.0*1010 cells or more, or 1.0*1011 cells or more, and may be 1.0*1014 cells or less, 1.0*1013 cells or less, or 1.0*1012 cells or less. These upper and lower limits can each be combined arbitrarily, and for example, the number of bacteria according to the present embodiment per unit package in the immunostimulatory composition according to the present embodiment may be 1.0*107 or more and 1.0*1014 cells or less, 1.0*107 or more and 1.0*1013 cells or less, 1.0*107 or more and 1.0*1012 cells or less, 1.0*108 or more and 1.0*1014 cells or less, 1.0*108 or more and 1.0*1013 cells or less, 1.0*108 or more and 1.0*1012 cells or less, 3.0*108 or more and 1.0*1014 cells or less, 3.0*108 or more and 1.0*1013 cells or less, 3.0*108 or more and 1.0*1012 cells or less, 5.0*108 or more and 1.0*1014 cells or less, 5.0*108 or more and 1.0*1013 cells or less, 5.0*108 or more and 1.0*1012 cells or less, 1.0*109 or more and 1.0*1014 cells or less, 1.0*109 or more and 1.0*1013 cells or less, 1.0*109 or more and 1.0*1012 cells or less, 5.0*109 or more and 1.0*1014 cells or less, 5.0*109 or more and 1.0*1013 cells or less, 5.0*109 or more and 1.0*1012 cells or less, 1.0*1010 or more and 1.0*1013 cells or less, 5.0*1010 or more and 5.0*1013 cells or less, 1.0*1011 or more and 1.0*1014 cells or less, 1.0*1010 or more and 1.0*1014 cells or less, 5.0*1010 or more and 1.0*1014 cells or less, 1.0*1010 or more and 1.0*1012 cells or less, 5.0*1010 or more and 1.0*1012 cells or less, 1.0*1011 or more and 1.0*1013 cells or less, or 1.0*1011 or more and 1.0*1012 cells or less.

[0067] “Amino acid” means a compound having an amino group and a carboxy group. However, proline and derivatives thereof have an imino group instead of an amino group but are included in amino acids. In the present specification, “peptide” means a compound in which 2 to 50 amino acids are linked by peptide bonds. In the present specification, “protein” means a compound in which 51 or more amino acids are linked by peptide bonds. “One or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity” means the above-described compound having an action of enhancing the immunostimulatory action by a bacterium having immunostimulatory activity, and may be a compound that, when the compound, a bacterium having immunostimulatory activity, and immune cells are co-cultured, results in a higher degree of immunostimulatory action as compared with a case where only the bacterium having immunostimulatory activity and immune cells are co-cultured.

[0068] The method for determining whether or not the compound according to the present embodiment has an immunostimulatory activity-enhancing action on bacteria is not particularly limited, and if the function of immune cells in the presence of a bacterium having immunostimulatory action is enhanced in the presence of the compound as compared with the absence of the compound, it is determined that the compound has an immunostimulatory activity-enhancing action on the above-described bacterium. Whether or not the compound according to the present embodiment has an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory action can be confirmed, as a more detailed example, by an increase in the IFN-a concentration measured according to the following procedures (i') to (iv') in the presence of the compound as compared with the absence of the compound. (i') Mouse-derived bone marrow cells from which erythrocytes have been removed are suspended in RPMI medium prepared so that each component described in the following (Composition of medium) reaches the respective final concentration described in the following (Composition of medium), to a concentration of 1*106 cells / mL, to prepare a cell suspension. <Composition of medium> • 10% by volume FBS • 100 U / mL penicillin / streptomycin • 1 mM sodium pyruvate • 2.5 mM HEPES • 1% by mass non-essential amino acids for MEM (NEAA) • 50 pM P—mercaptoethanol • 100 ng / mL Flt-3L (ii') The prepared cell suspension is seeded at 1 mL each, and cultured for one week at 37°C and 5% by volume CO2 in a CO2 incubator to induce pDCs. (iii') Bone marrow cells containing the induced pDCs are suspended to a concentration of 2*105 cells / mL, seeded at 200 gL each into a 96-well plate, and 2 gL each of a bacterial suspension adjusted to a concentration of 1 mg / mL with PBS is added, and further, a compound solution adjusted to a predetermined concentration is added. (iv') After 24 hours, the culture supernatant is collected, and the IFN-a concentration is measured by ELISA using an IFN-a measurement kit.

[0069] Amino acids include both free forms and salts. The amino acid may be either an L-amino acid or a D-amino acid. The amino acid may be any of an acidic amino acid (for example, aspartic acid and glutamic acid and the like), a neutral amino acid (for example, alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cystine, ornithine, Y-aminobutyric acid, hydroxyproline, selenomethionine, homoserine, P-alanine, S-aminolevulinic acid, and theanine and the like), and a basic amino acid (for example, arginine, histidine, and lysine and the like). The portion of the amino acid other than the amino group and the carboxy group (in the case of an a-amino acid, also referred to in particular as a side chain) may be hydrophilic or hydrophobic, may or may not have an aromatic group, may or may not have sulfur, and may or may not have an amide group. The amino acid may be an amino acid involved in ketone body metabolism (for example, isoleucine and leucine and the like), an amino acid having an antioxidant action (for example, cysteine, glycine, serine, glutamic acid, and Y-aminobutyric acid and the like), an amino acid having an immunostimulatory action (for example, cysteine, glycine, glutamic acid, arginine, cystine, and theanine and the like), an amino acid having a muscle synthesis action (for example, isoleucine, leucine, methionine, valine, and lysine and the like), an amino acid that is a neurotransmitter or has a synthesis action thereof (for example, glutamine, glycine, serine, tryptophan, glutamic acid, and Y-aminobutyric acid and the like), an amino acid having a collagen synthesis action (for example, glycine, proline, and hydroxyproline and the like), or an amino acid having an anti-anxiety action (for example, glycine, serine, tryptophan, glutamic acid, and Y-aminobutyric acid and the like). Specific examples of the amino acid include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cystine, ornithine, Y-aminobutyric acid, hydroxyproline, selenomethionine, homoserine, P-alanine, 8-aminolevulinic acid, and theanine and the like.

[0070] The amino acid having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity is preferably an acidic amino acid (for example, aspartic acid and glutamic acid and the like), a neutral amino acid (for example, alanine, asparagine, cysteine, glutamine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cystine, ornithine, Y-aminobutyric acid, hydroxyproline, homoserine, P-alanine, 5-aminolevulinic acid, and theanine and the like), or a basic amino acid (for example, arginine, histidine, and lysine and the like). The amino acid having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity is preferably an amino acid involved in ketone body metabolism (for example, isoleucine and leucine and the like), an amino acid having an antioxidant action (for example, cysteine, serine, glutamic acid, and Y-aminobutyric acid and the like), an amino acid having an immunostimulatory action (for example, cysteine, glutamic acid, arginine, cystine, and theanine and the like), an amino acid having a muscle synthesis action (for example, isoleucine, leucine, valine, and lysine and the like), an amino acid that is a neurotransmitter or has a synthesis action thereof (for example, glutamine, serine, tryptophan, glutamic acid, and Y-aminobutyric acid and the like), an amino acid having a collagen synthesis action (for example, proline and hydroxyproline and the like), or an amino acid having an anti-anxiety action (for example, serine, tryptophan, glutamic acid, and Y-aminobutyric acid and the like). The amino acid having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity is preferably one or more compounds selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cystine, ornithine, Y-aminobutyric acid, hydroxyproline, homoserine, P—alanine, S-aminolevulinic acid, and theanine, more preferably alanine, asparagine, cysteine, glutamine, leucine, proline, threonine, tryptophan, valine, arginine, histidine, lysine, isoleucine, serine, ornithine, and cystine, and is, as a more preferred amino acid, one or more compounds selected from the group consisting of L-alanine, L-asparagine,   L-cysteine, L-glutamine, L-leucine,   L-proline, L-threonine, L-tryptophan, L-valine, L-arginine, L-histidine, L-lysine, L-isoleucine, L-serine, L-ornithine, and L-cystine.

[0071] Peptides and proteins include both free forms and salts. The amino acids constituting peptides and proteins may be either L-amino acids or D-amino acids. The number of amino acids constituting a peptide may be 2 to 20 (oligopeptide) or may be 21 to 50. The number of amino acids of an oligopeptide may be any of 2 (dipeptide), 3 (tripeptide), 4 (tetrapeptide), 5 (pentapeptide), 6 (hexapeptide), 7 (heptapeptide), 8 (octapeptide), 9 (nonapeptide), 10 (decapeptide), 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Peptides and proteins may be plant-derived (for example, rice, wheat, barley, corn, soybean, and pea and the like) or may be animal-derived (for example, cattle, pig, chicken, sheep, sardine, herring, mackerel, bonito, and tuna and the like). In addition, the peptide may be a hydrolysate of a protein (for example, a hydrolysate by a hydrolase and the like). The peptide may be a peptide having an antioxidant action (for example, glutathione and imidazole peptide and the like), a peptide having a gelling action (for example, casein hydrolysate and whey protein hydrolysate and the like), a peptide having an action of promoting the decomposition of lactic acid (for example, imidazole peptide and the like), a peptide having an action of regulating the amount of uric acid (for example, imidazole peptide and the like), a peptide having a buffering action against a decrease in muscle pH (for example, imidazole peptide and the like), or a peptide that stimulates the transport of calcium in the body and Ca2+-ATPase activity (for example, imidazole peptide and the like). Specific examples of proteins (including, for example, proteins before decomposition from which peptides are derived) include elastin, collagen, silk protein (for example, silk fibroin and sericin and the like), wheat gluten, and milk protein (such as casein and whey protein) and the like. In addition, examples of peptides include glutathione and imidazole dipeptide and the like. Glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine, and is preferably reduced glutathione (GSH). Imidazole dipeptide is a general term for dipeptides containing histidine or methylhistidine, and specifically includes carnosine (P-alanyl-histidine), anserine             (P-alanyl-1-methylhistidine),             balenine (P-alanyl-3-methylhistidine), and homocarnosine (Y—aminobutyric acid-histidine) and the like. Preferred peptides include GSH, imidazole dipeptide, silk peptide, sardine-derived peptide, casein hydrolysate, collagen peptide, wheat protein hydrolysate, and whey protein hydrolysate and the like. Preferred proteins include elastin and milk protein and the like.

[0072] The peptide or protein having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity is preferably plant-derived (for example, rice, wheat, barley, corn, soybean, and pea and the like) or animal-derived (for example, cattle, pig, chicken, sheep, sardine, herring, mackerel, bonito, and tuna and the like). In addition, the peptide having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity is preferably a hydrolysate of a protein (for example, a hydrolysate by a hydrolase and the like). The peptide having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity may be a peptide having an antioxidant action (for example, glutathione and imidazole peptide and the like), a peptide having a gelling action (for example, casein hydrolysate and whey protein hydrolysate and the like), a peptide having an action of promoting the decomposition of lactic acid (for example, imidazole peptide and the like), a peptide having an action of regulating the amount of uric acid (for example, imidazole peptide and the like), a peptide having a buffering action against a decrease in muscle pH (for example, imidazole peptide and the like), or a peptide that stimulates the transport of calcium in the body and Ca2+-ATPase activity (for example, imidazole peptide and the like). The protein having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity (including, for example, proteins before decomposition from which peptides are derived) is preferably elastin, collagen, silk protein (for example, silk fibroin and sericin and the like), wheat gluten, casein, and whey protein and the like. In addition, the peptide having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity is preferably glutathione and imidazole dipeptide and the like. The imidazole dipeptide preferably includes carnosine (P-alanyl-histidine), anserine (P-alanyl-1-methylhistidine), balenine     (P—alanyl-3-methylhistidine),     and homocarnosine (Y—aminobutyric acid-histidine) and the like. The peptide having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity is preferably GSH, imidazole dipeptide, silk peptide, sardine-derived peptide, casein hydrolysate, collagen peptide, wheat protein hydrolysate, and whey protein hydrolysate and the like. The protein having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity is preferably elastin and the like.

[0073] The one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, are preferably one or more compounds selected from the group consisting of alanine, asparagine, cysteine, glutamine, leucine, proline, threonine, tryptophan, valine, arginine, histidine, lysine, ornithine, cystine, glutathione, imidazole dipeptide, silk peptide, sardine-derived peptide, casein hydrolysate, collagen peptide, wheat protein hydrolysate, whey protein hydrolysate, and elastin.

[0074] As the silk peptide, one obtained by hydrolyzing silk fibrous protein (silk fibroin) using an enzyme can be used, and for example, “Noukioku no Moto” manufactured by Japan-central-pharm Co., Ltd. can be used.

[0075] As the sardine-derived peptide, one obtained by enzymatic hydrolysis of sardine protein with alkaline protease can be used, and for example, “Peptide a-1000N” manufactured by Senmi Ekisu Co., Ltd. can be used.

[0076] The casein hydrolysate is sometimes referred to as casein peptide, and one obtained by hydrolysis using an enzyme can be used, and for example, “CE90GMM” manufactured by Nippon Shinyaku Co., Ltd. and the like can be used.

[0077] The collagen peptide may be, for example, a chicken collagen peptide. As the chicken collagen peptide, one extracted and purified using chicken feet (domestic or foreign) (momiji) as a raw material can be used, and for example, “C-LAP” manufactured by NH Foods Ltd. can be used.

[0078] As the imidazole dipeptide, one extracted and purified using chicken breast meat (domestic or foreign) as a raw material can be used, and for example, “Imidapeptide” manufactured by Japan Preventive Medicine Inc. can be used.

[0079] As the enzymatically decomposed wheat protein, one obtained by hydrolyzing gluten using an enzyme can be used, and for example, “Meripro 500” manufactured by Koyo Mercantile Co., Ltd. can be used.

[0080] As the whey protein hydrolysate, one obtained by hydrolyzing whey protein using an enzyme can be used, and for example, “LE80GF-US” manufactured by Nippon Shinyaku Co., Ltd. and “Whey Peptide HW-3” manufactured by Megmilk Snow Brand Co., Ltd. can be used.

[0081] As the elastin, one obtained by performing pretreatment and the like on animal biological tissue and then hydrolyzing it using an enzyme (in some cases further separated and purified) can be used, and for example, “Pure Elastin” manufactured by Vital Resource Applied Laboratory, Inc. can be used.

[0082] As the milk protein, milk protein itself and a whey protein concentrate produced from milk protein by ultrafiltration can be used, and for example, “PROGEL800” manufactured by Nippon Shinyaku Co., Ltd. can be used.

[0083] The immunostimulatory activity-enhancing action of the compound according to the present embodiment on bacteria is preferably such that the IFN-a concentration measured according to the above procedures (i') to (iv') in the presence of the compound as compared with the absence of the compound has, as a lower limit thereof, 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.05-fold or more, 1.08-fold or more, 1.10-fold or more, 1.13-fold or more, 1.15-fold or more, 1.18-fold or more, 1.20-fold or more, 1.23-fold or more, 1.25-fold or more, 1.28-fold or more, 1.30-fold or more, 1.40-fold or more, 1.50-fold or more, 1.60-fold or more, 1.70-fold or more, 1.80-fold or more, 1.90-fold or more, or 2.0-fold or more, and preferably has, as an upper limit thereof, 4.0-fold or less, 3.8-fold or less, 3.5-fold or less, 3.3-fold or less, 3.0-fold or less, 2.8-fold or less, 2.5-fold or less, 2.3-fold or less, 2.0-fold or less, 1.80-fold or less, or 1.50-fold or less. These upper and lower limits can each be combined arbitrarily. The immunostimulatory activity-enhancing action of the compound according to the present embodiment on bacteria is preferably such that the IFN-a concentration measured according to the above procedures (i') to (iv') in the presence of the compound as compared with the absence of the compound is, for example, 1.01-fold or more and 4.0-fold or less, 1.01-fold or more and 4.0-fold or less, 1.01-fold or more and 3.8-fold or less, 1.01-fold or more and 3.5-fold or less, 1.01-fold or more and 3.3-fold or less, 1.01-fold or more and 3.0-fold or less, 1.01-fold or more and 2.8-fold or less, 1.01-fold or more and 2.5-fold or less, 1.01-fold or more and 2.3-fold or less, 1.01-fold or more and 2.0-fold or less, 1.01-fold or more and 1.8-fold or less, 1.01-fold or more and 1.5-fold or less, 1.02-fold or more and 4.0-fold or less, 1.02-fold or more and 3.8-fold or less, 1.02-fold or more and 3.5-fold or less, 1.02-fold or more and 3.3-fold or less, 1.02-fold or more and 3.0-fold or less, 1.02-fold or more and 2.8-fold or less, 1.02-fold or more and 2.5-fold or less, 1.02-fold or more and 2.3-fold or less, 1.02-fold or more and 2.0-fold or less, 1.02-fold or more and 1.8-fold or less, 1.02-fold or more and 1.5-fold or less, 1.03-fold or more and 4.0-fold or less, 1.03-fold or more and 3.8-fold or less, 1.03-fold or more and 3.5-fold or less, 1.03-fold or more and 3.3-fold or less, 1.03-fold or more and 3.0-fold or less, 1.03-fold or more and 2.8-fold or less, 1.03-fold or more and 2.5-fold or less, 1.03-fold or more and 2.3-fold or less, 1.03-fold or more and 2.0-fold or less, 1.03-fold or more and 1.8-fold or less, 1.02-fold or more and 1.5-fold or less, 1.05-fold or more and 4.0-fold or less, 1.05-fold or more and 3.8-fold or less, 1.05-fold or more and 3.5-fold or less, 1.05-fold or more and 3.3-fold or less, 1.05-fold or more and 3.0-fold or less, 1.05-fold or more and 2.8-fold or less, 1.05-fold or more and 2.5-fold or less, 1.05-fold or more and 2.3-fold or less, 1.05-fold or more and 2.0-fold or less, 1.05-fold or more and 1.8-fold or less, 1.05-fold or more and 1.5-fold or less, 1.08-fold or more and 4.0-fold or less, 1.08-fold or more and 3.8-fold or less, 1.08-fold or more and 3.5-fold or less, 1.08-fold or more and 3.3-fold or less, 1.08-fold or more and 3.0-fold or less, 1.08-fold or more and 2.8-fold or less, 1.08-fold or more and 2.5-fold or less, 1.08-fold or more and 2.3-fold or less, 1.08-fold or more and 2.0-fold or less, 1.08-fold or more and 1.8-fold or less, 1.08-fold or more and 1.5-fold or less, 1.10-fold or more and 4.0-fold or less, 1.10-fold or more and 3.8-fold or less, 1.10-fold or more and 3.5-fold or less, 1.10-fold or more and 3.3-fold or less, 1.10-fold or more and 3.0-fold or less, 1.10 or more and 2.8-fold or less, 1.10-fold or more and 2.5-fold or less, 1.10-fold or more and 2.3-fold or less, 1.10-fold or more and 2.0-fold or less, 1.10-fold or more and 1.8-fold or less, 1.10-fold or more and 1.5-fold or less, 1.13-fold or more and 4.0-fold or less, 1.13-fold or more and 3.8-fold or less, 1.13-fold or more and 3.5-fold or less, 1.13-fold or more and 3.3-fold or less, 1.13-fold or more and 3.0-fold or less, 1.13 or more and 2.8-fold or less, 1.13 or more and 2.5-fold or less, 1.13-fold or more and 2.3-fold or less, 1.13-fold or more and 2.0-fold or less, 1.13-fold or more and 1.8-fold or less, 1.13-fold or more and 1.5-fold or less, 1.15-fold or more and 4.0-fold or less, 1.15-fold or more and 3.8-fold or less, 1.15-fold or more and 3.5-fold or less, 1.15-fold or more and 3.3-fold or less, 1.15-fold or more and 3.0-fold or less, 1.15-fold or more and 2.8-fold or less, 1.15-fold or more and 2.5-fold or less, 1.15-fold or more and 2.3-fold or less, 1.15-fold or more and 2.0-fold or less, 1.15-fold or more and 1.8-fold or less, 1.15-fold or more and 1.5-fold or less, 1.18-fold or more and 4.0-fold or less, 1.18-fold or more and 3.8-fold or less, 1.18-fold or more and 3.5-fold or less, 1.18-fold or more and 3.3-fold or less, 1.18-fold or more and 3.0-fold or less, 1.18-fold or more and 2.8-fold or less, 1.18-fold or more and 2.5-fold or less, 1.18-fold or more and 2.3-fold or less, 1.18-fold or more and 2.0-fold or less, 1.18-fold or more and 1.8-fold or less, 1.18-fold or more and 1.5-fold or less, 1.20-fold or more and 4.0-fold or less, 1.20-fold or more and 3.8-fold or less, 1.20-fold or more and 3.5-fold or less, 1.20-fold or more and 3.3-fold or less, 1.20-fold or more and 3.0-fold or less, 1.20-fold or more and 2.8-fold or less, 1.20-fold or more and 2.5-fold or less, 1.20-fold or more and 2.3-fold or less, 1.20-fold or more and 2.0-fold or less, 1.20-fold or more and 1.8-fold or less, 1.20-fold or more and 1.5-fold or less, 1.23-fold or more and 4.0-fold or less, 1.23-fold or more and 3.8-fold or less, 1.23-fold or more and 3.5-fold or less, 1.23-fold or more and 3.3-fold or less, 1.23-fold or more and 3.0-fold or less, 1.23-fold or more and 2.8-fold or less, 1.23-fold or more and 2.5-fold or less, 1.23-fold or more and 2.3-fold or less, 1.23-fold or more and 2.0-fold or less, 1.23-fold or more and 1.8-fold or less, 1.23-fold or more and 1.5-fold or less, 1.25-fold or more and 4.0-fold or less, 1.25-fold or more and 3.8-fold or less, 1.25-fold or more and 3.5-fold or less, 1.25-fold or more and 3.3-fold or less, 1.25-fold or more and 3.0-fold or less, 1.25-fold or more and 2.8-fold or less, 1.25-fold or more and 2.5-fold or less, 1.25-fold or more and 2.3-fold or less, 1.25-fold or more and 2.0-fold or less, 1.25-fold or more and 1.8-fold or less, 1.25-fold or more and 1.5-fold or less, 1.28-fold or more and 4.0-fold or less, 1.28-fold or more and 3.8-fold or less, 1.28-fold or more and 3.5-fold or less, 1.28-fold or more and 3.3-fold or less, 1.28-fold or more and 3.0-fold or less, 1.28-fold or more and 2.8-fold or less, 1.28-fold or more and 2.5-fold or less, 1.28-fold or more and 2.3-fold or less, 1.28-fold or more and 2.0-fold or less, 1.28-fold or more and 1.8-fold or less, 1.28-fold or more and 1.5-fold or less, 1.30-fold or more and 4.0-fold or less, 1.30-fold or more and 3.8-fold or less, 1.30-fold or more and 3.5-fold or less, 1.30-fold or more and 3.3-fold or less, 1.30-fold or more and 3.0-fold or less, 1.30-fold or more and 2.8-fold or less, 1.30-fold or more and 2.5-fold or less, 1.30-fold or more and 2.3-fold or less, 1.30-fold or more and 2.0-fold or less, 1.30-fold or more and 1.8-fold or less, 1.30-fold or more and 1.5-fold or less, 1.40-fold or more and 4.0-fold or less, 1.40-fold or more and 3.8-fold or less, 1.40-fold or more and 3.5-fold or less, 1.40-fold or more and 3.3-fold or less, 1.40-fold or more and 3.0-fold or less, 1.40-fold or more and 2.8-fold or less, 1.40-fold or more and 2.5-fold or less, 1.40-fold or more and 2.3-fold or less, 1.40-fold or more and 2.0-fold or less, 1.40-fold or more and 1.8-fold or less, 1.40-fold or more and 1.5-fold or less, 1.50-fold or more and 4.0-fold or less, 1.50-fold or more and 3.8-fold or less, 1.50-fold or more and 3.5-fold or less, 1.50-fold or more and 3.3-fold or less, 1.50-fold or more and 3.0-fold or less, 1.50-fold or more and 2.8-fold or less, 1.50-fold or more and 2.5-fold or less, 1.50-fold or more and 2.3-fold or less, 1.50-fold or more and 2.0-fold or less, 1.50-fold or more and 1.8-fold or less, 1.60-fold or more and 4.0-fold or less, 1.60-fold or more and 3.8-fold or less, 1.60-fold or more and 3.5-fold or less, 1.60-fold or more and 3.3-fold or less, 1.60-fold or more and 3.0-fold or less, 1.60-fold or more and 2.8-fold or less, 1.60-fold or more and 2.5-fold or less, 1.60-fold or more and 2.3-fold or less, 1.60-fold or more and 2.0-fold or less, 1.60-fold or more and 1.8-fold or less, 1.70-fold or more and 4.0-fold or less, 1.70-fold or more and 3.8-fold or less, 1.70-fold or more and 3.5-fold or less, 1.70-fold or more and 3.3-fold or less, 1.70-fold or more and 3.0-fold or less, 1.70-fold or more and 2.8-fold or less, 1.70-fold or more and 2.5-fold or less, 1.70-fold or more and 2.3-fold or less, 1.70-fold or more and 2.0-fold or less, 1.70-fold or more and 1.8-fold or less, 1.80-fold or more and 4.0-fold or less, 1.80-fold or more and 3.8-fold or less, 1.80-fold or more and 3.5-fold or less, 1.80-fold or more and 3.3-fold or less, 1.80-fold or more and 3.0-fold or less, 1.80-fold or more and 2.8-fold or less, 1.80-fold or more and 2.5-fold or less, 1.80-fold or more and 2.3-fold or less, 1.80-fold or more and 2.0-fold or less, 1.90-fold or more and 4.0-fold or less, 1.90-fold or more and 3.8-fold or less, 1.90-fold or more and 3.5-fold or less, 1.90-fold or more and 3.3-fold or less, 1.90-fold or more and 3.0-fold or less, 1.90-fold or more and 2.8-fold or less, 1.90-fold or more and 2.5-fold or less, 1.90-fold or more and 2.3-fold or less, 1.90-fold or more and 2.0-fold or less, 2.00-fold or more and 4.0-fold or less, 2.00-fold or more and 3.8-fold or less, 2.00-fold or more and 3.5-fold or less, 2.00-fold or more and 3.3-fold or less, 2.00-fold or more and 3.0-fold or less, 2.00-fold or more and 2.8-fold or less, 2.00-fold or more and 2.5-fold or less, or 2.00-fold or more and 2.3-fold or less.

[0084] The content of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein in the immunostimulatory composition according to one embodiment relative to the total amount of the immunostimulatory composition can have, for example, a lower limit of 0.000001% by mass or more, 0.00001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, 0.02% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, or 2% by mass or more, and an upper limit of 90.0% by mass or less, 85.0% by mass or less, 80.0% by mass or less, 75.0% by mass or less, 50.0% by mass or less, 25.0% by mass or less, 10.0% by mass or less, 5.0% by mass or less, 1.0% by mass or less, 0.80% by mass or less, 0.50% by mass or less, 0.30% by mass or less, 0.25% by mass or less, 0.20% by mass or less, 0.10% by mass or less, 0.05% by mass or less, 0.02% by mass or less, or 0.01% by mass or less. These upper and lower limits can each be combined arbitrarily. The content of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein in the immunostimulatory composition according to one embodiment relative to the total amount of the immunostimulatory composition may be, for example, 0.000001% by mass or more and 90.0% by mass or less, 0.000001% by mass or more and 85.0% by mass or less, 0.000001% by mass or more and 80.0% by mass or less, 0.000001% by mass or more and 75.0% by mass or less, 0.000001% by mass or more and 50.0% by mass or less, 0.000001% by mass or more and 25.0% by mass or less, 0.000001% by mass or more and 10.0% by mass or less, 0.000001% by mass or more and 5.0% by mass or less, 0.000001% by mass or more and 1.0% by mass or less, 0.000001% by mass or more and 0.80% by mass or less, 0.000001% by mass or more and 0.50% by mass or less, 0.000001% by mass or more and 0.30% by mass or less, 0.000001% by mass or more and 0.25% by mass or less, 0.000001% by mass or more and 0.20% by mass or less, 0.000001% by mass or more and 0.10% by mass or less, 0.000001% by mass or more and 0.05% by mass or less, 0.000001% by mass or more and 0.02% by mass or less, 0.000001% by mass or more and 0.01% by mass or less, 0.00001% by mass or more and 90.0% by mass or less, 0.00001% by mass or more and 85.0% by mass or less, 0.00001% by mass or more and 80.0% by mass or less, 0.00001% by mass or more and 75.0% by mass or less, 0.00001% by mass or more and 50.0% by mass or less, 0.00001% by mass or more and 25.0% by mass or less, 0.00001% by mass or more and 10.0% by mass or less, 0.00001% by mass or more and 5.0% by mass or less, 0.00001% by mass or more and 1.0% by mass or less, 0.00001% by mass or more and 0.80% by mass or less, 0.00001% by mass or more and 0.50% by mass or less, 0.00001% by mass or more and 0.30% by mass or less, 0.00001% by mass or more and 0.25% by mass or less, 0.00001% by mass or more and 0.20% by mass or less, 0.00001% by mass or more and 0.10% by mass or less, 0.00001% by mass or more and 0.05% by mass or less, 0.00001% by mass or more and 0.02% by mass or less, 0.00001% by mass or more and 0.01% by mass or less, 0.001% by mass or more and 90.0% by mass or less, 0.001% by mass or more and 85.0% by mass or less, 0.001% by mass or more and 80.0% by mass or less, 0.001% by mass or more and 75.0% by mass or less, 0.001% by mass or more and 50.0% by mass or less, 0.001% by mass or more and 25.0% by mass or less, 0.001% by mass or more and 10.0% by mass or less, 0.001% by mass or more and 5.0% by mass or less, 0.001% by mass or more and 1.0% by mass or less, 0.001% by mass or more and 0.80% by mass or less, 0.001% by mass or more and 0.50% by mass or less, 0.001% by mass or more and 0.30% by mass or less, 0.001% by mass or more and 0.25% by mass or less, 0.001% by mass or more and 0.20% by mass or less, 0.001% by mass or more and 0.10% by mass or less, 0.001% by mass or more and 0.05% by mass or less, 0.001% by mass or more and 0.02% by mass or less, 0.001% by mass or more and 0.01% by mass or less, 0.01% by mass or more and 90.0% by mass or less, 0.01% by mass or more and 85.0% by mass or less, 0.01% by mass or more and 80.0% by mass or less, 0.01% by mass or more and 75.0% by mass or less, 0.01% by mass or more and 50.0% by mass or less, 0.01% by mass or more and 25.0% by mass or less, 0.01% by mass or more and 10.0% by mass or less, 0.01% by mass or more and 5.0% by mass or less, 0.01% by mass or more and 1.0% by mass or less, 0.01% by mass or more and 0.80% by mass or less, 0.01% by mass or more and 0.50% by mass or less, 0.01% by mass or more and 0.30% by mass or less, 0.01% by mass or more and 0.25% by mass or less, 0.01% by mass or more and 0.20% by mass or less, 0.01% by mass or more and 0.10% by mass or less, 0.01% by mass or more and 0.05% by mass or less, 0.01% by mass or more and 0.02% by mass or less, 0.02% by mass or more and 90.0% by mass or less, 0.02% by mass or more and 85.0% by mass or less, 0.02% by mass or more and 80.0% by mass or less, 0.02% by mass or more and 75.0% by mass or less, 0.02% by mass or more and 50.0% by mass or less, 0.02% by mass or more and 25.0% by mass or less, 0.02% by mass or more and 10.0% by mass or less, 0.02% by mass or more and 5.0% by mass or less, 0.02% by mass or more and 1.0% by mass or less, 0.02% by mass or more and 0.80% by mass or less, 0.02% by mass or more and 0.50% by mass or less, 0.02% by mass or more and 0.30% by mass or less, 0.02% by mass or more and 0.25% by mass or less, 0.02% by mass or more and 0.20% by mass or less, 0.02% by mass or more and 0.10% by mass or less, 0.02% by mass or more and 0.05% by mass or less, 0.05% by mass or more and 90.0% by mass or less, 0.05% by mass or more and 85.0% by mass or less, 0.05% by mass or more and 80.0% by mass or less, 0.05% by mass or more and 75.0% by mass or less, 0.05% by mass or more and 50.0% by mass or less, 0.05% by mass or more and 25.0% by mass or less, 0.05% by mass or more and 10.0% by mass or less, 0.05% by mass or more and 5.0% by mass or less, 0.05% by mass or more and 1.0% by mass or less, 0.05% by mass or more and 0.80% by mass or less, 0.05% by mass or more and 0.50% by mass or less, 0.05% by mass or more and 0.30% by mass or less, 0.05% by mass or more and 0.25% by mass or less, 0.05% by mass or more and 0.20% by mass or less, 0.05% by mass or more and 0.10% by mass or less, 0.10% by mass or more and 90.0% by mass or less, 0.10% by mass or more and 85.0% by mass or less, 0.10% by mass or more and 80.0% by mass or less, 0.10% by mass or more and 75.0% by mass or less, 0.10% by mass or more and 50.0% by mass or less, 0.10% by mass or more and 25.0% by mass or less, 0.10% by mass or more and 10.0% by mass or less, 0.10% by mass or more and 5.0% by mass or less, 0.10% by mass or more and 1.0% by mass or less, 0.10% by mass or more and 0.80% by mass or less, 0.10% by mass or more and 0.50% by mass or less, 0.10% by mass or more and 0.30% by mass or less, 0.10% by mass or more and 0.25% by mass or less, 0.10% by mass or more and 0.20% by mass or less, 0.50% by mass or more and 90.0% by mass or less, 0.50% by mass or more and 85.0% by mass or less, 0.50% by mass or more and 80.0% by mass or less, 0.50% by mass or more and 75.0% by mass or less, 0.50% by mass or more and 50.0% by mass or less, 0.50% by mass or more and 25.0% by mass or less, 0.50% by mass or more and 10.0% by mass or less, 0.50% by mass or more and 5.0% by mass or less, 0.50% by mass or more and 1.0% by mass or less, 0.50% by mass or more and 0.80% by mass or less, 1% by mass or more and 90.0% by mass or less, 1% by mass or more and 85.0% by mass or less, 1% by mass or more and 80.0% by mass or less, 1% by mass or more and 75.0% by mass or less, 1% by mass or more and 50.0% by mass or less, 1% by mass or more and 25.0% by mass or less, 1% by mass or more and 10.0% by mass or less, 1% by mass or more and 5.0% by mass or less, 2% by mass or more and 90.0% by mass or less, 2% by mass or more and 85.0% by mass or less, 2% by mass or more and 80.0% by mass or less, 2% by mass or more and 75.0% by mass or less, 2% by mass or more and 50.0% by mass or less, 2% by mass or more and 25.0% by mass or less, 2% by mass or more and 10.0% by mass or less, or 2% by mass or more and 5.0% by mass or less.

[0085] The ratio of the content (% by mass) of the bacterium having immunostimulatory activity to the content (% by mass) of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein in the immunostimulatory composition according to one embodiment ((content of the bacterium having immunostimulatory activity) / (content of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein)) can have, for example, a lower limit of 0.001 or more, 0.003 or more, 0.005 or more, 0.01 or more, 0.015 or more, 0.02 or more, 0.025 or more, or 0.03 or more, and an upper limit of 50000 or less, 10000 or less, 1000 or less, 500 or less, 100 or less, 50 or less, 10 or less, 5 or less, 1.0 or less, 0.75 or less, 0.5 or less, or 0.3 or less. These upper and lower limits can each be combined arbitrarily. The ratio of the content (% by mass) of the bacterium having immunostimulatory activity to the content (% by mass) of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein in the immunostimulatory composition according to one embodiment ((content of the bacterium having immunostimulatory activity) / (content of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein)) may be, for example, 0.001 or more and 50000 or less, 0.001 or more and 10000 or less, 0.001 or more and 1000 or less, 0.001 or more and 500 or less, 0.001 or more and 100 or less, 0.001 or more and 50 or less, 0.001 or more and 10 or less, 0.001 or more and 5 or less, 0.001 or more and 1.0 or less, 0.001 or more and 0.75 or less, 0.001 or more and 0.5 or less, 0.001 or more and 0.3 or less, 0.003 or more and 50000 or less, 0.003 or more and 10000 or less, 0.003 or more and 1000 or less, 0.003 or more and 500 or less, 0.003 or more and 100 or less, 0.003 or more and 50 or less, 0.003 or more and 10 or less, 0.003 or more and 5 or less, 0.003 or more and 1.0 or less, 0.003 or more and 0.75 or less, 0.003 or more and 0.5 or less, 0.003 or more and 0.3 or less, 0.005 or more and 50000 or less, 0.005 or more and 10000 or less, 0.005 or more and 1000 or less, 0.005 or more and 500 or less, 0.005 or more and 100 or less, 0.005 or more and 50 or less, 0.005 or more and 10 or less, 0.005 or more and 5 or less, 0.005 or more and 1.0 or less, 0.005 or more and 0.75 or less, 0.005 or more and 0.5 or less, 0.005 or more and 0.3 or less, 0.01 or more and 50000 or less, 0.01 or more and 10000 or less, 0.01 or more and 1000 or less, 0.01 or more and 500 or less, 0.01 or more and 100 or less, 0.01 or more and 50 or less, 0.01 or more and 10 or less, 0.01 or more and 5 or less, 0.01 or more and 1.0 or less, 0.01 or more and 0.75 or less, 0.01 or more and 0.5 or less, 0.01 or more and 0.3 or less, 0.015 or more and 50000 or less, 0.015 or more and 10000 or less, 0.015 or more and 1000 or less, 0.015 or more and 500 or less, 0.015 or more and 100 or less, 0.015 or more and 50 or less, 0.015 or more and 10 or less, 0.015 or more and 5 or less, 0.015 or more and 1.0 or less, 0.015 or more and 0.75 or less, 0.015 or more and 0.5 or less, 0.015 or more and 0.3 or less, 0.02 or more and 50000 or less, 0.02 or more and 10000 or less, 0.02 or more and 1000 or less, 0.02 or more and 500 or less, 0.02 or more and 100 or less, 0.02 or more and 50 or less, 0.02 or more and 10 or less, 0.02 or more and 5 or less, 0.02 or more and 1.0 or less, 0.02 or more and 0.75 or less, 0.02 or more and 0.5 or less, 0.02 or more and 0.3 or less, 0.025 or more and 50000 or less, 0.025 or more and 10000 or less, 0.025 or more and 1000 or less, 0.025 or more and 500 or less, 0.025 or more and 100 or less, 0.025 or more and 50 or less, 0.025 or more and 10 or less, 0.025 or more and 5 or less, 0.025 or more and 1.0 or less, 0.025 or more and 0.75 or less, 0.025 or more and 0.5 or less, 0.025 or more and 0.3 or less, 0.03 or more and 50000 or less, 0.03 or more and 10000 or less, 0.03 or more and 1000 or less, 0.03 or more and 500 or less, 0.03 or more and 100 or less, 0.03 or more and 50 or less, 0.03 or more and 10 or less, 0.03 or more and 5 or less, 0.03 or more and 1.0 or less, 0.03 or more and 0.75 or less, 0.03 or more and 0.5 or less, or 0.03 or more and 0.3 or less.

[0086] The immunostimulatory composition according to one embodiment may be a food (including beverages), a pharmaceutical, or a quasi-drug. The food, pharmaceutical, or quasi-drug can each be produced by a conventional method. The content of the bacterium having immunostimulatory activity and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein in the food, pharmaceutical, or quasi-drug is not particularly limited and can be freely set according to the purpose.

[0087] When the immunostimulatory composition according to one embodiment is a food, a pharmaceutical, or a quasi-drug, the food, pharmaceutical, or quasi-drug may contain, in addition to the bacterium having immunostimulatory activity and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, components that can be ordinarily used in foods, pharmaceuticals, or quasi-drugs. The food, pharmaceutical, or quasi-drug according to one embodiment may contain bases, carriers, additives, and the like ordinarily used in foods, pharmaceuticals, or quasi-drugs. Examples of additives include excipients, oils, powders, buffers, solubilizing agents, antioxidants, surfactants, thickeners, preservatives, pH adjusters, chelating agents, stabilizers, irritation-reducing agents, antiseptics, pigments, colorants, fragrances, gloss-imparting agents, gelling agents, alcohols, water-soluble polymers, film-forming agents, and resins. The bases, carriers, and the various additives described above can be used alone or in combination of two or more as necessary.

[0088] When the immunostimulatory composition according to one embodiment is a food, examples of the food include health food, functional food, nutritional composition, dietary supplement, supplement, food for health, food for specified health uses, food with nutrient function claims, and food with functional claims and the like. Such food compositions can be labeled with, for example, support for maintaining immune function in healthy people (immune care), for those concerned about decreased immune function, suppression of decreased immune function, and the like. In addition, the immunostimulatory composition according to one embodiment can also be used as a food additive.

[0089] Examples of the food include seasonings, processed meat products, processed agricultural products, beverages (lactic acid bacteria beverages, soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, nutritional drinks, and the like), powdered beverages (powdered juice, powdered soup, powdered milk, and the like), concentrated beverages, confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummies, chewables, tablets, chocolate, and the like), bread, cereals, and the like. In addition, in the case of food for specified health uses, food with nutrient function claims, food with functional claims, and the like, the form may be capsules, granules, powder, syrup, troches, and the like.

[0090] When the immunostimulatory composition according to one embodiment is a pharmaceutical or a quasi-drug, the dosage form of the pharmaceutical or quasi-drug may be, for example, a liquid preparation, a suspension, a capsule, a granule, a pill, a powder, a tablet, a syrup, a troche, and the like. Examples of indications as a pharmaceutical include allergy, obesity, and heart failure and the like.

[0091] The immunostimulatory composition according to one embodiment is preferably administered or ingested into the body. The mode of administration or ingestion may be oral administration or oral ingestion, or parenteral administration or parenteral ingestion, but oral administration or oral ingestion is preferred. The immunostimulatory composition according to one embodiment may be administered or ingested only once, or may be administered or ingested multiple times, and when sustained immunostimulation is desired, it is preferably administered or ingested continuously or intermittently over a certain period of time. The food composition according to one aspect may be ingested, for example, for 1 week or more, 2 weeks or more, 1 month or more, 3 months or more, 6 months or more, 1 year or more, 3 years or more, 5 years or more, or 10 years or more, at a frequency of 1 to 5 times per day, once every 2 days, once every 3 days, once every 4 days, or once per week.

[0092] The immunostimulatory composition according to one embodiment is a composition for immunostimulation induced by a bacterium having immunostimulatory activity, and contains one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium. “Immunostimulation induced by a bacterium having immunostimulatory activity” means immunostimulation induced depending on the action of stimulating (activating) immune cells that is inherently possessed by the bacterium having immunostimulatory activity.

[0093] The immunostimulatory composition according to one embodiment is an immunostimulatory composition or an immunostimulatory composition with enhanced immunostimulatory action, obtained by adding, to a bacterium having immunostimulatory activity, one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium. The immunostimulatory composition or the immunostimulatory composition with enhanced immunostimulatory action is preferred from the viewpoint of bringing about enhancement of the immunostimulatory activity of the bacterium by the addition of the compound.

[0094] [Enhancer of Immunostimulatory Action Induced by Bacterium Having Immunostimulatory Activity] The second embodiment of the present disclosure relates to an enhancer of an immunostimulatory action induced by a bacterium having immunostimulatory activity (hereinafter also simply referred to as “enhancer”). The enhancer according to one aspect contains a bacterium having immunostimulatory activity and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium. In addition, the enhancer according to another aspect is an enhancer of an immunostimulatory action induced by a bacterium having immunostimulatory activity, and contains one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium. The enhancer according to the present embodiment has an action of enhancing the immunostimulatory action induced by the above-described bacterium having immunostimulatory activity. That is, the enhancer according to the present embodiment has an enhanced immunostimulatory action as compared with an immunostimulatory composition comprising only the bacterium having immunostimulatory activity.

[0095] The enhancer according to one embodiment preferably has an activity to induce interferon a production in immune cells that is, as a lower limit thereof, for example, 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.05-fold or more, 1.08-fold or more, 1.10-fold or more, 1.13-fold or more, 1.15-fold or more, 1.18-fold or more, 1.20-fold or more, 1.23-fold or more, 1.25-fold or more, 1.28-fold or more, 1.30-fold or more, 1.40-fold or more, 1.50-fold or more, 1.60-fold or more, 1.70-fold or more, 1.80-fold or more, 1.90-fold or more, or 2.0-fold or more as compared with an immunostimulatory composition comprising only the bacterium having immunostimulatory activity, and that preferably is, as an upper limit thereof, 4.0-fold or less, 3.8-fold or less, 3.5-fold or less, 3.3-fold or less, 3.0-fold or less, 2.8-fold or less, 2.5-fold or less, 2.3-fold or less, 2.0-fold or less, 1.8-fold or less, or 1.50-fold or less. In addition, these upper and lower limits can each be combined arbitrarily, and may be the same range as described for the immunostimulatory activity-enhancing action of the compound according to the present embodiment on bacteria.

[0096] As other specific aspects of the enhancer according to the present embodiment, the aspects of the immunostimulatory composition according to the present embodiment can be appropriately applied.

[0097] [Immunostimulatory Method, Method for Enhancing Immunostimulatory Action Induced by Bacterium Having Immunostimulatory Activity] The third embodiment of the present disclosure relates to an immunostimulatory method and a method for enhancing an immunostimulatory action induced by a bacterium having immunostimulatory activity (hereinafter also simply referred to as “enhancement method”). The immunostimulatory method and the enhancement method include administering to a subject or causing a subject to ingest a bacterium having immunostimulatory activity and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium. The enhancement method according to the present embodiment has an enhanced immunostimulatory action as compared with a case where only the bacterium having immunostimulatory activity is administered or caused to be ingested.

[0098] Examples of subjects in the above-described method include humans and non-human mammals, with humans being preferred. Examples of non-human mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, and monkeys.

[0099] The subject can be a subject in need of immunostimulation, and the subject in need of immunostimulation is not particularly limited, but examples include a subject infected with a virus, a subject wishing to prevent viral infection, a subject having a cold, a subject wishing to prevent a cold, and a subject aged 65 years or older and the like.

[0100] The bacterium having immunostimulatory activity and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium, can be administered to or caused to be ingested by a subject, for example, in the form of the composition of the first embodiment described above. In general, the expression “administering to a subject” is used for pharmaceutical compositions, and the expression “causing a subject to ingest” is used for food compositions, but both expressions mean that the composition is taken into the body of the subject and are synonymous. Therefore, the two are interchangeable expressions and there is no essential difference.

[0101] [Method for Producing Composition] The fourth embodiment of the present disclosure relates to a method for producing an immunostimulatory composition. The production method is a method for producing an immunostimulatory composition or an immunostimulatory composition with enhanced immunostimulatory action, the method comprising adding, to a bacterium having immunostimulatory activity, one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium. The immunostimulatory composition or the immunostimulatory composition with enhanced immunostimulatory action is preferred from the viewpoint of bringing about enhancement of the immunostimulatory activity of the bacterium by the addition of the compound.

[0102] [Use of Bacterium and Compound for Producing Composition] The fifth embodiment of the present disclosure relates to the use of a bacterium and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium, for producing a composition. The use according to one aspect is the use of a bacterium having immunostimulatory activity and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium, for producing an immunostimulatory composition; the use according to another aspect is the use of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, for producing a composition for immunostimulation induced by the above-described bacterium; the use according to yet another aspect is the use of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, for producing an immunostimulatory composition to be administered or ingested in combination with the bacterium having immunostimulatory activity; and the use according to another aspect is the use of a bacterium having immunostimulatory activity for producing an immunostimulatory composition to be administered or ingested in combination with one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium having immunostimulatory activity. The specific aspects of the immunostimulatory composition, bacterium, and compound according to the present embodiment are as described above.

[0103] [Compound for Use] The sixth embodiment of the present disclosure relates to one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, for use in immunostimulation induced by the above-described bacterium. The specific aspects of the bacterium and compound according to the present embodiment are as described above.

[0104] [Bacterium or Compound for Use in Therapeutic Method] The seventh embodiment of the present disclosure relates to the use of a bacterium or a compound for use in a therapeutic method of immunostimulation. In one aspect, the bacterium is a bacterium having immunostimulatory activity for use in a therapeutic method of immunostimulation, the bacterium being administered or ingested in combination with one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium; and in another aspect, the compound is one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on a bacterium having immunostimulatory activity, for use in a therapeutic method of immunostimulation, the one or more compounds being administered or ingested in combination with the above-described bacterium having immunostimulatory activity. The specific aspects of the bacterium and compound according to the present embodiment are as described above. In the present disclosure, use in a “therapeutic” method indicates use in treating the body of a subject. Use in a therapeutic method according to the present disclosure may be, for example, use for the purpose of medical practice or involving medical practice. Use in a therapeutic method according to the present disclosure may involve, for example, a healthcare professional administering a substance to a human or animal, or causing a human or animal to ingest a substance, or instructing a human or animal to administer or ingest a substance. Use in a therapeutic method according to the present disclosure may be, for example, use for therapeutic purposes or preventive purposes, may be use for therapeutic purposes, or for preventive purposes involving ingestion of a food composition, or may be for therapeutic purposes. Use in a therapeutic method according to the present disclosure may be, for example, use for non-healthy individuals.

[0105] [Use of Bacterium or Compound in Non-Therapeutic Method] The eighth embodiment of the present disclosure relates to the use of a bacterium or a compound for use in a non-therapeutic method of immunostimulation. In one aspect, the use is the use of a bacterium having immunostimulatory activity in a non-therapeutic method of immunostimulation, the bacterium being administered or ingested in combination with one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the above-described bacterium; and in another aspect, the use is the use of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium having immunostimulatory activity, in a non-therapeutic method of immunostimulation, the one or more compounds being administered or ingested in combination with the above-described bacterium. The specific aspects of the bacterium and compound according to the present embodiment are as described above. In the present disclosure, use in a “non-therapeutic” method means use of a substance that does not fall under use in a therapeutic method. Use in a non-therapeutic method according to the present disclosure may be, for example, use that is not for the purpose of medical practice and / or does not involve medical practice. Use in a non-therapeutic method according to the present disclosure need not involve, for example, a healthcare professional administering a substance to a human or animal, or causing a human or animal to ingest a substance, and / or instructing a human or animal to administer or ingest a substance. Use in a non-therapeutic method according to the present disclosure may be, for example, use for preventive purposes or health promotion purposes, may be use for preventive purposes involving administration or ingestion of a pharmaceutical composition or quasi-drug, or for health promotion purposes. Use in a non-therapeutic method according to the present disclosure may be, for example, use for healthy individuals. Examples

[0106] Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

[0107] [Experiment 1: Study on the Effect of Glutathione on pDC Activation and IFN-a Production Induced by JCM 5805] In Experiment 1, it was examined whether glutathione enhances or suppresses the activation of pDCs and IFN-a production induced by JCM 5805.

[0108] <1. Preparation of Lactic Acid Bacterium Suspension> JCM 5805 bacterial cells were seeded in 10 mL of MRS medium (Oxoid) and cultured at 30°C for 24 hours. Thereafter, 1 mL of the cultured lactic acid bacterium cell suspension was seeded in 100 mL of MRS medium and cultured at 30°C for 24 hours. Subsequently, the lactic acid bacteria were collected, washed three times with sterile water, heat-sterilized at 80°C for 30 minutes, and then freeze-dried to obtain heat-killed cells. The heat-killed cells were then adjusted with PBS (Takara Bio Inc.) to a concentration of 1 mg / mL to prepare a lactic acid bacterium suspension.

[0109] <2. Preparation of Reduced Glutathione Solution> Reduced glutathione (Tokyo Chemical Industry Co., Ltd.: G0074) was adjusted with PBS to 4 mM and 40 mM, respectively, to prepare glutathione solutions.

[0110] <3. Test Procedure> The test was carried out according to the following procedures (i) to (iv). (i) Bone marrow cells derived from female BALB / c mice were suspended in RPMI medium (Sigma Co., Ltd.) prepared so that each component described in the following (Composition of medium) reaches the respective final concentration described in the following (Composition of medium), to a concentration of 1*106 cells / mL, to prepare a cell suspension. (Composition of medium) • 10% by volume FBS • 100 U / mL penicillin / streptomycin • 1 mM sodium pyruvate • 2.5 mM HEPES • 1% by mass non-essential amino acids for MEM (NEAA) • 50 pM P-mercaptoethanol • 100 ng / mL Flt-3L (ii) The prepared cell suspension was seeded at 1 mL each, and cultured for one week at 37°C and 5% CO2 in a CO2 incubator to induce pDCs. (iii) Bone marrow cells containing the induced pDCs were suspended to a concentration of 2*105 cells / mL, seeded at 200 pL each into a 96-well plate, and 2 pL each of the JCM 5805 suspension from 1. above was added simultaneously with 5 pL of the glutathione solution from 2. above (glutathione final concentration: 0.1 mM or 1.0 mM). In addition, similarly, only the glutathione solution was added to a 96-well plate seeded with bone marrow cells containing pDCs. Furthermore, similarly, only the JCM 5805 suspension was added to a 96-well plate seeded with bone marrow cells containing pDCs. (iv) After 24 hours, the culture supernatant was collected, and the IFN-a concentration was measured using an IFN-a measurement kit (PBL Assay Science Ltd.).

[0111] The results are shown in FIG. 1. FIG. 1 is a graph showing the results of evaluating the enhancing effect on IFN-a production by pDC induced by JCM 5805. In FIG. 1, the data when only the JCM 5805 suspension was added is shown as JCM 5805, the data when only the glutathione solution was added is shown as GSH 0.1 mM or GSH 1.0 mM (0.1 mM or 1.0 mM indicates the glutathione final concentration; the same applies hereinafter in the text), and the data when the JCM 5805 suspension and the glutathione solution were added is shown as JCM 5805+GSH 0.1 mM or JCM 5805+GSH 1.0 mM. Note that FIG. 2 and FIG. 3 are the same except that the lactic acid bacterium is changed from JCM 5805 to Weissella paramesenteroides JCM 9890 strain (hereinafter sometimes referred to as JCM 9890) or Bifidobacterium animalis subsp. lactis JCM 10602 strain (hereinafter sometimes referred to as JCM 10602).

[0112] When the glutathione solution was used alone, IFN-a production by pDC was not induced. In addition, when the JCM 5805 suspension was used alone, IFN-a production by pDC was induced by JCM 5805. When both the glutathione solution and the JCM 5805 suspension were used, IFN-a production by pDC induced by JCM 5805 was enhanced as compared with a case where the JCM 5805 suspension was used alone. Furthermore, in this case, IFN-a production by pDC induced by JCM 5805 was enhanced in a glutathione concentration-dependent manner.

[0113] [Experiment 2: Study on the Effect of Glutathione on pDC Activation and IFN-a Production Induced by Lactic Acid Bacteria and Bifidobacteria]

[0114] In Experiment 2, it was examined whether glutathione enhances or suppresses the activation of pDC and IFN-a production induced by lactic acid bacteria other than JCM 5805.

[0115] <1. Preparation of Lactic Acid Bacterium Suspension> A lactic acid bacterium (Weissella paramesenteroides JCM 9890 strain) was cultured under the same culture conditions as in 1. of Experiment 1 above. Subsequently, the lactic acid bacteria were collected, washed three times with sterile water, heat-sterilized at 80°C for 30 minutes, and then freeze-dried to obtain heat-killed cells. The heat-killed cells were then adjusted with PBS (Takara Bio Inc.) to a concentration of 1 mg / mL to prepare a lactic acid bacterium suspension.

[0116] <2. Preparation of Bifidobacterium Suspension> Bifidobacterium (Bifidobacterium animalis subsp. lactis JCM 10602 strain) bacterial cells were seeded in 10 mL of GAM medium (a medium prepared by adding 1% by mass lactose to GAM broth “Nissui” (Nissui Pharmaceutical Co., Ltd.)) and cultured at 37°C for 24 hours under anaerobic conditions. 1 mL of the cultured bacterial cell suspension was seeded in 100 mL of GAM medium and further cultured at 37°C for 24 hours under anaerobic conditions.

[0117] <3. Preparation of Reduced Glutathione Solution> The reduced glutathione solution was prepared according to the same procedure as described in 2. of Experiment 1.

[0118] <4. Test Procedure> The test was carried out according to the same procedure as described in 3. of Experiment 1. In this experiment, one to which only the glutathione solution was added was not used, and one to which only 2 pL of each lactic acid bacterium suspension was added was used as a comparison target.

[0119] The results are shown in FIG. 2 to FIG. 3. FIG. 2 is a graph showing the results of evaluating the enhancing effect on IFN-a production by pDC induced by a lactic acid bacterium (Weissella paramesenteroides JCM 9890 strain). FIG. 3 is a graph showing the results of evaluating the enhancing effect on IFN-a production by pDC induced by a bifidobacterium (Bifidobacterium animalis subsp. lactis JCM 10602 strain).

[0120] When the lactic acid bacterium suspension or the bifidobacterium suspension was used alone, IFN-a production by pDC was induced by the lactic acid bacterium or bifidobacterium. In addition, when the glutathione solution and the lactic acid bacterium suspension or the bifidobacterium suspension were used, it was confirmed that, in either case, glutathione enhanced IFN-a production by pDC induced by the lactic acid bacterium or the bifidobacterium in a concentration-dependent manner.

[0121] [Experiment 3: Study on the Effect of Amino Acids, Peptides, or Proteins on pDC Activation and IFN-a Production Induced by JCM 5805] Using an amino acid, a peptide, or a protein instead of glutathione, an experiment similar to Experiment 1 was carried out, and the enhancing action or suppressing action on IFN-a production by pDC induced by JCM 5805 was investigated. The relative values of the amount of IFN-a production in the presence of each substance, when the amount of IFN-a production in the case where the JCM 5805 suspension was used alone was set to 1, are summarized in the following tables. The concentration of each substance in the tables means the final concentration.

[0122] All amino acids used were products of FUJIFILM Wako Pure Chemical Corporation.   “Noukioku no Moto” manufactured by Japan-central-pharm Co., Ltd. was used as the silk peptide, “Peptide a-1000N” manufactured by Senmi Ekisu Co., Ltd. was used as the sardine-derived peptide, “CE90GMM” manufactured by Nippon Shinyaku Co., Ltd. was used as the casein hydrolysate, “C-LAP” manufactured by NH Foods Ltd. was used as the chicken collagen peptide, “Imidapeptide” manufactured by Japan Preventive Medicine Inc. was used as the imidazole dipeptide, “L-Cystine Kyowa” manufactured by Kyowa Hakko Bio Co., Ltd. was used as L-cystine, “Meripro 500” manufactured by Koyo Mercantile Co., Ltd. was used as the enzymatically decomposed wheat protein, “LE80GF-US” manufactured by Nippon Shinyaku Co., Ltd. was used as the whey protein hydrolysate, “Whey Peptide HW-3” manufactured by Megmilk Snow Brand Co., Ltd. was used as the whey peptide, “Pure Elastin” manufactured by Vital Resource Applied Laboratory, Inc. was used as elastin, and “PROGEL800” manufactured by Nippon Shinyaku Co., Ltd. was used as the milk protein.

[0123] [Table 1] Table 1: Amino acid 500 pg / mL (However, L-cystine is 10 pg / mL) L-alanine 1.43 L-asparagine 1.11 L-cysteine 2.47 L-glutamine 1.07 Glycine 0.86 L-leucine 1.01 L-methionine 0.41 L-proline 1.37 L-threonine 1.14 L-tryptophan 1.05 L-valine 1.01 L-arginine 1.42 L-histidine 2.27 L-lysine 1.42 L-ornithine 1.10 L-cystine 1.39

[0124] [Table 2] Table 2: Peptide and protein 10 pg / mL Silk peptide 1.61 Sardine-derived peptide 1.55 Casein hydrolysate 1.47 Chicken collagen peptide 1.42 Imidazole dipeptide 1.39 Enzymatically   decomposed wheat protein 1.45 Whey protein hydrolysate 1.42 Whey peptide 1.18 Elastin 1.29 Milk protein 0.63

[0125] [Table 3] Table 3: Amino acid 0.05 to 500 pg / mL Concentration (pg / mL) 0.05 0.5 5 50 500 L-cysteine 1.38 1.51 1.98 2.25 2.47 L-histidine 1.34 1.36 1.86 1.87 2.27 L-lysine No data 1.36 1.39 1.35 1.42 L-ornithine 1.22 1.39 1.50 1.36 1.10 Concentration (pg / mL) 0.1 1 10 L-cystine 1.18 1.10 1.39

[0126] When any of the substances was used alone, IFN-a production by pDCs was not induced. It was confirmed that glycine, L-methionine, and milk protein suppressed IFN-a production by pDCs induced by JCM 5805, and that substances other than glycine, L-methionine, and milk protein enhanced IFN-a production by pDCs induced by JCM 5805.

[0127] [Experiment 4: Study on the Effect of Amino Acids on pDC Activation and IFN-a Production Induced by JCM 5805] In subsequent experiments, using CAL-1 cells (accession number FERM BP-10914) as pDCs, it was examined whether amino acids enhance or suppress the activation of pDCs and IFN-a production induced by JCM 5805. The above-described CAL-1 cells (accession number FERM BP-10914) are a sample distributed from Nagasaki University, a national university corporation (hereinafter referred to as the present sample) (all rights to the present sample belong to Nagasaki University, a national university corporation, and a license to use the present sample was received from Nagasaki University, a national university corporation). Cell culture was carried out at 37°C and 5% CO2. As the basal medium for culturing CAL-1 cells and for tests using CAL-1 cells, RPMI-1640 medium (Sigma, R8758) supplemented with penicillin / streptomycin (Gibco, 15140-12) at a final concentration of 1.0% by volume was used (hereinafter also referred to as “serum-free medium”).

[0128] <1. Preparation of Lactic Acid Bacterium Suspension> The lactic acid bacterium suspension was prepared according to the same procedure as described in 2. of Experiment 1.

[0129] <2. Preparation of Amino Acid Solution> All amino acids used were products of FUJIFILM Wako Pure Chemical Corporation, and were diluted with PBS to 50 mg / mL.

[0130] <3. Test Procedure> A cell suspension containing CAL-1 cells at 5.0*105 cells / mL was prepared using serum-free medium, and the cell suspension was seeded at 10 mL / well in a culture dish with a diameter of 10 cm and cultured for 16 hours (pretreatment step). CAL-1 cells were collected by pipetting. The collected CAL-1 cells were resuspended in serum-free medium at a concentration of 2.0*105 cells / mL and seeded at 200 pL / well into each well of a 96-well microplate. Thereafter, 2 pL each of the JCM 5805 suspension from 1. above was added simultaneously with 2 pL of the amino acid solution from 2. above. In addition, similarly, only the amino acid solution was added to a 96-well plate seeded with CAL-1 cells. Furthermore, similarly, only the JCM 5805 suspension was added to a 96-well plate seeded with CAL-1 cells. After 24 hours, the culture supernatant was collected, and the IFN-a concentration in the culture supernatant was measured by ELISA using a Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science).

[0131] The relative values of the amount of IFN-a production in the presence of each substance, when the amount of IFN-a production in the case where the JCM 5805 suspension was used alone was set to 1, are summarized in the following table.

[0132] [Table 4] Table 4: Amino acid 500 pg / mL Asparagine 1.84 Glutamine 1.22 Glycine 0.17 Histidine 1.63 Isoleucine 1.16 Lysine 1.10 Serine 1.11 All are the L-form except for glycine.

[0133] When any of the substances was used alone, IFN-a production by pDCs was not induced. It was confirmed that glycine suppressed IFN-a production by pDCs induced by JCM 5805, and that substances other than glycine enhanced IFN-a production by pDCs induced by JCM 5805.

[0134] [Experiment 5: Study on the Effect of Glutathione on pDC Activation and IFN-a Production Induced by JCM 5805] In Experiment 5, using CAL-1 cells (accession number FERM BP-10914) as pDCs, it was examined whether glutathione or DL-buthionine-(S,R)-sulfoximine (hereinafter referred to as BSO) enhances or suppresses the activation of pDCs and IFN-a production induced by JCM 5805. BSO is an amino acid analog that functions as an inhibitor of Y—glutamylcysteine synthetase and has an effect of reducing the intracellular glutathione concentration. The above-described CAL-1 cells (accession number FERM BP-10914) are a sample distributed from Nagasaki University, a national university corporation (hereinafter referred to as the present sample) (all rights to the present sample belong to Nagasaki University, a national university corporation, and a license to use the present sample was received from Nagasaki University, a national university corporation). Cell culture was carried out at 37°C and 5% CO2. As the basal medium for culturing CAL-1 cells and for tests using CAL-1 cells, RPMI-1640 medium (Sigma, R8758) supplemented with penicillin / streptomycin (Gibco, 15140-12) at a final concentration of 1.0% by volume was used (hereinafter also referred to as “serum-free medium”).

[0135] <1. Preparation of Lactic Acid Bacterium Suspension> The lactic acid bacterium suspension was prepared according to the same procedure as described in 2. of Experiment 1.

[0136] <2. Preparation of Glutathione Solution> All glutathione used was that of Kyowa Hakko Bio Co., Ltd., and was diluted with PBS to 10 mM.

[0137] <3. Preparation of BSO Solution> All BSO used was that of Sigma (B2640), and was diluted with PBS to 40 mM.

[0138] <4. Preparation of N-ethylmorpholine Solution> N-ethylmorpholine (FUJIFILM Wako Pure Chemical Corporation, 055-02113) (hereinafter referred to as NEM) was diluted with ultrapure water to 50 mM and adjusted to pH 8.0 using 1 M hydrochloric acid (Wako Pure Chemical Industries, 083-01095).

[0139] <5. Preparation of Monobromobimane Solution> Monobromobimane (Tokyo Chemical Industry, B4220) (hereinafter referred to as mBBr) was dissolved by adding 500 p,L of acetonitrile (Nacalai Tesque, 00430-83), and then diluted to 8 mM with the 50 mM NEM adjusted in 4. above.

[0140] <6. Test Procedure> A cell suspension containing CAL-1 cells at 3.7*105 cells / mL was prepared using serum-free medium, and for the BSO 0.25 mM group, after adding the BSO solution from 3. above at 62.5 pL / well, the cell suspension was seeded at 10 mL / well in a culture dish with a diameter of 10 cm and cultured for 16 hours. Nothing was added to the BSO-free group, and the cell suspension at the same concentration was seeded and cultured for the same time (pretreatment step). CAL-1 cells were collected by pipetting. The collected CAL-1 cells were resuspended in serum-free medium at a concentration of 4.0*105 cells / mL and seeded at 200 pL / well into each well of a 96-well microplate. Thereafter, 5 pL each of the JCM 5805 suspension from 1. above was added simultaneously with 5 pL of the glutathione solution from 2. above. In addition, similarly, only the glutathione solution was added to a 96-well plate seeded with CAL-1 cells. Furthermore, similarly, only the JCM 5805 suspension was added to a 96-well plate seeded with CAL-1 cells. After 24 hours, the culture supernatant was collected, and the IFN-a concentration in the culture supernatant was measured by ELISA using a Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science). After washing the 96-well plate after supernatant collection with PBS, the NEM solution from 4. above and the mBBr solution from 5. above were added at 100 pL / well and allowed to stand at room temperature in the dark for 10 minutes. 10 pL of trichloroacetic acid was added, and after centrifugation, the supernatant was collected. The solution was passed through using a 0.20 pm filter (ADVANTEC, 13HP020AN05), and the glutathione concentration was measured using an HPLC method (apparatus:  Chromaster manufactured by HITACHI, column: COSMOSIL Packed Column 5C18-MS-2 4.6 mm I.D.X100 mm manufactured by Nacalai Tesque).

[0141] The relative values of the glutathione concentration of CAL-1 5       cells in the addition of glutathione and the addition of BSO, when the glutathione concentration of CAL-1 cells in the absence of JCM 5805, glutathione, and BSO was set to 1, are summarized in the following table. [Table 5] Table 5: Glutathione concentration of CAL-1 cells Glutathione-free Glutathione 0.25 mM BSO-free 1 2.42 BSO 0.25 mM 0.52 1.02 10

[0142] The relative values of the amount of IFN-a production in the presence of the JCM 5805 suspension and glutathione, when the amount of IFN-a production in the case where the JCM 5805 suspension was used alone was set to 1, are summarized in the following table.

[0143] [Table 6] Table 6: Amount of IFN-a production in the presence of the JCM 5805 suspension and glutathione Glutathione-free Glutathione 0.25 mM BSO-free 1 1.72 BSO 0.25 mM 0.62 1.11 15

[0144] The relative values of the amount of IFN-a production with glutathione alone, when the amount of IFN-a production in the case where the JCM 5805 suspension was used alone was set to 1, are summarized in the following table.

[0145] [Table 7] Table 7: Amount of IFN-a production in the case where glutathione was used alone Glutathione-free Glutathione 0.25 mM BSO-free 0.05 0.04 BSO 0.25 mM 0.05 0.04

[0146] The addition of 0.25 mM BSO reduced the glutathione concentration of CAL-1 cells. In contrast, it was confirmed that the addition of glutathione at 0.25 mM increased the glutathione concentration of CAL-1 cells to a level equivalent to that in the absence of BSO.

[0147] The addition of 0.25 mM BSO attenuated the amount of IFN-a production by CAL-1 cells induced by the addition of JCM 5805 as compared with the absence of BSO. In contrast, it was confirmed that the addition of 0.25 mM glutathione increased the amount of IFN-a production by CAL-1 cells induced by the addition of JCM 5805. In addition, it was confirmed that the addition of glutathione increased IFN-a production by CAL-1 cells even in the BSO-free group. Note that glutathione alone did not induce IFN-a production.

[0148] From the above results, it was found that glutathione alone does not affect the IFN-a production by CAL-1 cells, but that combining JCM 5805 and glutathione synergistically enhances the IFN-a production by CAL-1 cells. Furthermore, in the case where the intracellular glutathione concentration of CAL-1 cells was changed by adding BSO, which reduces the intracellular glutathione concentration, since the amount of IFN-a production by CAL-1 cells also decreased when the glutathione concentration in CAL-1 cells decreased, it was found that the intracellular glutathione concentration is important for IFN-a production.

[0149] [Experiment 6: Study on the Effect of Amino Acids, Peptides, or Proteins on pDC Activation and IFN-a Production Induced by Various Lactic Acid Bacteria or Bifidobacteria]

[0150] In Experiment 6, it was examined whether amino acids, peptides, or proteins enhance the activation of pDCs and IFN-a production induced by lactic acid bacteria or bifidobacteria other than JCM 5805.

[0151] <1. Preparation of Lactic Acid Bacterium Suspension> Lactococcus curvatus JCM 1096, Lactococcus lactis subsp. lactis ATCC 7962, and Lactobacillus paraplantarum JCM 1149 (Lactiplantibacillus paraplantarum JCM 1149) were seeded in 10 mL of MRS medium (Becton Dickinson and Company) and cultured at 30°C for 24 hours. Thereafter, 1 mL of the cultured lactic acid bacterium cell culture solution was seeded in 100 mL of MRS medium and cultured at 30°C for 24 hours. Subsequently, the lactic acid bacteria were collected, washed three times with sterile water, heat-sterilized at 80°C for 30 minutes, and then freeze-dried to obtain heat-killed cells. The heat-killed cells were then adjusted with PBS (Takara Bio Inc.) to a concentration of 1 mg / mL to prepare a lactic acid bacterium suspension.

[0152] <2. Preparation of Bifidobacterium Suspension> Bifidobacterium animalis subsp. lactis JCM 10602 was seeded in 10 mL of MRS medium and cultured at 30°C for 24 hours. Thereafter, 1 mL of the cultured lactic acid bacterium (bifidobacterium) cell culture solution was seeded in 100 mL of MRS medium and cultured at 30°C for 24 hours. Subsequently, the lactic acid bacteria (bifidobacteria) were collected, washed three times with sterile water, heat-sterilized at 80°C for 30 minutes, and then freeze-dried to obtain heat-killed cells. The heat-killed cells were then adjusted with PBS (Takara Bio Inc.) to a concentration of 1 mg / mL to prepare a lactic acid bacterium (bifidobacterium) suspension.

[0153] <3. Preparation of Amino Acid, Peptide, or Protein Solution> All amino acids used were products of FUJIFILM Wako Pure Chemical Corporation. A product of Tokyo Chemical Industry Co., Ltd. was used as glutathione. These were each prepared with PBS to 50 mg / mL.

[0154] <4. Test Procedure> The test was carried out according to the following procedures (i) to (iv). (i) Bone marrow cells derived from female BALB / c mice were suspended in RPMI medium (Sigma Co., Ltd.) prepared so that each component described in the following (Composition of medium) reaches the respective final concentration described in the following (Composition of medium), to a concentration of 1x106 cells / mL, to prepare a cell suspension. (Composition of medium) • 10% by volume FBS • 100 U / mL penicillin / streptomycin • 1 mM sodium pyruvate • 2.5 mM HEPES • 1% by mass non-essential amino acids for MEM (NEAA) • 50 pM p-mercaptoethanol • 100 ng / mL Flt-3L (ii) The prepared cell suspension was seeded at 1 mL each, and cultured for one week at 37°C and 5% CO2 in a CO2 incubator to induce pDCs. (iii) Bone marrow cells containing the induced pDCs were suspended to a concentration of 2*105 cells / mL, seeded at 200 pL each into a 96-well plate, and 2 pL each of the lactic acid bacterium suspension from 1. above or the bifidobacterium suspension from 2. above was added simultaneously with 2 pL of the amino acid or peptide solution from 3. above (final concentration: 0.5 mg / mL). Furthermore, similarly, only the lactic acid bacterium suspension or the bifidobacterium suspension was added to a 96-well plate seeded with bone marrow cells containing pDCs. (iv) After 24 hours, the culture supernatant was collected, and the IFN-a concentration was measured using an IFN-a measurement kit (PBL Assay Science Ltd.).

[0155] <5. Results> The relative values of the amount of IFN-a production in the case where various lactic acid bacteria or bifidobacteria were used in combination with amino acids, when the amount of IFN-a production in the case where the suspension of each lactic acid bacterium or bifidobacterium was used alone was set to 1, are summarized in the following table.

[0156] [Table 8] Genus Species Strain Ala Asn Cys His Ornithine Glutathione Lactococcus curvatus JCM1096 6.57 2.62 7.79 2.47 5.79 5.47 Lactococcus lactis lactis ATCC7962 1.55 1.46 4.75 2.21 1.47 1.95 Lactobacillus paraplantarum JCM1149 1.64 1.78 1.78 1.36 1.40 4.34 Bifidobacterium animalis lactis JCM10602 1.21 1.64 2.51 1.53 1.56 2.02 All amino acids are in L-form.

[0157] From the above results, it was confirmed that alanine, asparagine, cysteine, histidine, ornithine, and glutathione enhance IFN-a production by pDCs induced by various lactic acid bacteria and bifidobacteria.

Claims

1. An immunostimulatory composition comprising:a bacterium having immunostimulatory activity; and one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium.

2. The immunostimulatory composition according to claim1, wherein the bacterium having immunostimulatory activity is a lactic acid bacterium and / or an acetic acid bacterium.

3. The immunostimulatory composition according to claim2, wherein the lactic acid bacterium is one or more selected from bacteria of the genus Lactococcus, bacteria of the genus Weissella, bacteria of the genus Bifidobacterium, and bacteria of the genus Lactobacillus.

4. The immunostimulatory composition according to claim3, wherein the bacterium of the genus Lactococcus is Lactococcus lactis subsp. lactis or Lactococcus curvatus, the bacterium of the genus Weissella is Weissella paramesenteroides, the bacterium of the genus Bifidobacterium is Bifidobacterium animalis subsp. lactis, and the bacterium of the genus Lactobacillus is Lactobacillus paraplantarum.

5. The immunostimulatory composition according to claim1, wherein the content of the bacterium having immunostimulatory activity is 0.001% by mass or more and less than 100% by mass relative to the total amount of the immunostimulatory composition.

6. The immunostimulatory composition according to claim1, wherein the content of the compound is 0.000001% by mass or moreand 90.0% by mass or less relative to the total amount of the immunostimulatory composition.

7. The immunostimulatory composition according to claim1, wherein the ratio of the content (% by mass) of the bacterium having immunostimulatory activity to the content (% by mass) of the compound ((content of one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein) / (content of the bacterium having immunostimulatory activity)) is 0.001 or more and 50000 or less.

8. The immunostimulatory composition according to anyone of claims 1 to 7, wherein the immunostimulatory activity is an activity to induce interferon a production in an immune cell.

9. The immunostimulatory composition according to claim8, wherein the immune cell is a plasmacytoid dendritic cell.

10. The immunostimulatory composition according to any one of claims 1 to 7, wherein the immunostimulatory activity-enhancing action is an action of enhancing the immunostimulatory activity by 1.10-fold or more.

11. The immunostimulatory composition according to claim 8, wherein the compound is one or more compounds selected from the group consisting of alanine, asparagine, cysteine, glutamine, leucine, proline, threonine, tryptophan, valine, arginine, histidine, lysine, isoleucine, serine, ornithine, cystine, glutathione, imidazole dipeptide, silk peptide, sardine-derived peptide, casein hydrolysate, collagen peptide, wheat protein hydrolysate, whey protein hydrolysate, and elastin.

12. The immunostimulatory composition according to claim8, wherein the compound is glutathione.

13. An immunostimulatory method comprising administering to a subject or causing a subject to ingest:a bacterium having immunostimulatory activity; andone or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium.

14. A method for enhancing an immunostimulatory action induced by a bacterium having immunostimulatory activity, the method comprising administering to a subject or causing a subject to ingest:a bacterium having immunostimulatory activity; andone or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium.

15. The method according to claim 13 or 14, wherein the immunostimulatory activity is an activity to induce interferon a production in an immune cell.

16. An enhancer of an immunostimulatory action induced by a bacterium having immunostimulatory activity, the enhancer comprising one or more compounds selected from the group consisting of an amino acid, a peptide, and a protein, the one or more compounds having an immunostimulatory activity-enhancing action on the bacterium having immunostimulatory activity.

17. The enhancer according to claim 16, wherein the immunostimulatory activity is an activity to induce interferon a production in immune cells.

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