Composition for immunostimulation

Abstract

To provide a composition for immunostimulation.SOLUTION: A composition for immunostimulation comprising, as an active ingredient, a bacterium phagocytosed by plasmacytoid dentritic cells (pDCs) to a predetermined degree or more and / or a bacterium binding to BDCA2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to immunostimulatory compositions. [Background technology]

[0002] Immunostimulating compositions containing bacteria are known. For example, Patent Document 1 discloses an immunostimulating food composition containing lactic acid bacteria that activate pDCs (plasmacytoid dendritic cells) and induce IFN-α (interferon α) production. Furthermore, Patent Document 2 discloses a screening method for lactic acid bacteria with immunomodulatory activity, which includes measuring the number of uromodulin (Umod) protein binding sites of test lactic acid bacteria, lactic acid bacteria with immunomodulatory activity obtained by the method, and an immunomodulatory composition containing the lactic acid bacteria. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2017-201984 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-217372 Summary of the Invention [Problem to be solved by the invention]

[0004] Although several species of bacteria have been reported to have immunostimulatory properties, the mechanisms by which these bacteria stimulate the immune system and the properties they share remain largely unknown.

[0005] The present disclosure aims to provide an immunostimulatory composition. [Means for solving the problem]

[0006] The present inventors have found that bacteria phagocytosed by pDCs have immunostimulatory activity, and that bacteria that bind to blood dendritic cell antigen 2 (BDCA2) have immunostimulatory activity.

[0007] The present disclosure relates, for example, to the following sections: [A0] A composition that satisfies at least one selected from the group consisting of the following (1) to (4): (1) A composition comprising, as an active ingredient, bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacteria phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs, and the composition is an immunostimulatory composition; (2) The composition contains, as an active ingredient, a bacterium that binds to blood dendritic cell antigen 2 (BDCA2), and the composition is an immunostimulatory composition; (3) The composition contains, as an active ingredient, a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), the bacterium satisfies the following requirement X, and the composition is an immunostimulatory composition; [Requirement X] Final concentration 2.0×10 5 cells / mL of pDCs are contacted with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in a serum-free medium; (4) The bacterium contains Lactococcus lactis subsp. lactis JCM 5805, and the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [A1] An immunostimulatory composition comprising, as an active ingredient, bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs), An immunostimulatory composition, wherein the amount of the bacterium phagocytosed by pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDC. [A2] An immunostimulatory composition containing, as an active ingredient, bacteria that bind to blood dendritic cell antigen 2 (BDCA2). [A3] The composition according to [A0] or [A2], wherein the binding ability of the bacterium to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2. [A4] The composition described in [A3], wherein the binding ability to BDCA2 is evaluated based on the percentage of bacteria bound to BDCA2 as assessed in a binding assay. [A5] An immunostimulatory composition comprising, as an active ingredient, a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the bacterium satisfies the following requirement X: [Requirement X] Final concentration 2.0×10 5 After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in serum-free medium. [A6] A composition containing Lactococcus lactis subsp. lactis JCM 5805 as a bacterium, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [A7] The composition according to [A0] or [A6], wherein the composition is an immunostimulating composition. [A8] The composition according to any one of [A0] to [A5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [A9] The composition described in any one of [A0] to [A5], wherein the bacteria are one or more bacteria selected from the group consisting of bacteria of the genus Lactococcus, bacteria of the genus Leuconostoc, bacteria of the genus Streptococcus, bacteria of the genus Enterococcus, bacteria of the genus Tetragenococcus, bacteria of the genus Oenococcus, bacteria of the genus Weissella, bacteria of the genus Bifidobacterium, and bacteria of the genus Lactobacillus. [A10] The composition according to any one of [A0] to [A5], wherein the bacterium is Lactococcus lactis subsp. lactis. [A11] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis subsp. Infantis) JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The composition according to any one of [A0] to [A5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [A12] The composition according to any one of [A0] to [A11], wherein the bacteria include killed bacteria. [A13] The composition according to any one of [A0] to [A12], wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [A14] The composition described in [A13], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis via binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of the bacteria phagocytosed via binding to BDCA2 is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed via binding to BDCA2. [A15] The composition described in [A14], wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of the bacteria phagocytosis by the pDC when BDCA2 in the pDC cannot bind to a ligand. [A16] The composition according to any one of [A13] to [A15], wherein the bacterium promotes the production of IFN-α in the pDCs via phagocytosis by the pDCs. [A17] The composition according to any one of [A0] to [A16], which is a food composition, a pharmaceutical composition, or a feed. [A18] The number of bacteria of the above-mentioned bacteria per unit package in the above-mentioned food composition, pharmaceutical composition or feed is 1 x 10 8 The composition according to [A17], wherein the number of the hydroxyl groups is 1 or more. [B0] A method for stimulating immunity in a subject, comprising ingesting or administering to a subject in need thereof a bacterium that satisfies at least one selected from the group consisting of the following (1) to (4): (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacterium phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs; (2) bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) and that meet the following requirement X: [Requirement X] Final concentration 2.0×10 5 cells / mL of pDCs are contacted with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in a serum-free medium; (4) The bacterium is Lactococcus lactis subsp. lactis JCM 5805 and satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [B1] A method for stimulating immunity in a subject, comprising ingesting bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) into the subject or administering bacteria to a subject in need thereof, A method in which the amount of the bacterium phagocytosed by the pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by the pDC. [B2] A method for stimulating the immunity of a subject, comprising ingesting or administering to a subject in need thereof bacteria that bind to blood dendritic cell antigen 2 (BDCA2). [B3] The method according to [B0] or [B2], wherein the binding ability of the bacterium to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2. [B4] The method according to [B3], wherein the binding ability to BDCA2 is evaluated based on the proportion of bacteria bound to BDCA2, as assessed in a binding assay. [B5] A method for stimulating immunity in a subject, comprising ingesting or administering to a subject in need thereof bacteria that can be phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the bacteria satisfy the following requirement X: [Requirement X] Final concentration 2.0×10 5After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in serum-free medium. [B6] A method for stimulating the immunity of a subject, comprising ingesting Lactococcus lactis subsp. lactis JCM 5805 as a bacterium to the subject or administering it to a subject in need thereof, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [B7] The method according to any one of [B0] to [B5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [B8] The method according to any one of [B0] to [B5], wherein the bacterium is one or more bacteria selected from the group consisting of bacteria of the genus Lactococcus, bacteria of the genus Leuconostoc, bacteria of the genus Streptococcus, bacteria of the genus Enterococcus, bacteria of the genus Tetragenococcus, bacteria of the genus Oenococcus, bacteria of the genus Weissella, bacteria of the genus Bifidobacterium, and bacteria of the genus Lactobacillus. [B9] The method according to any one of [B0] to [B5], wherein the bacterium is Lactococcus lactis subsp. lactis. [B10] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis subsp. Infantis) JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The method according to any one of [B0] to [B5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [B11] The method according to any one of [B0] to [B10], wherein the bacteria include killed bacteria. [B12] The method according to any one of [B0] to [B11], wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [B13] The method described in [B12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis mediated by binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of phagocytosis of the bacteria mediated by binding to BDCA2 is 2.0 times or more the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 mediated by binding to BDCA2. [B14] The method described in [B13], wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of the bacteria phagocytosis by the pDC in a state in which BDCA2 in the pDC cannot bind to a ligand. [B15] The method according to any one of [B12] to [B14], wherein the bacterium promotes the production of IFN-α in the pDCs via phagocytosis by the pDCs. [B16] The method according to any one of [B0] to [B15], wherein the bacterium is contained in a food composition, a pharmaceutical composition, or a feed. [B17] The number of bacteria of the bacteria per unit package in the food composition, pharmaceutical composition, or feed is 1 x 10 8 The method according to [B16], wherein the number of the plurality of electrodes is 1 or more. [C0] A method for stimulating immunity in a subject, comprising ingesting a composition containing bacteria that satisfy at least one selected from the group consisting of the following (1) to (4) to the subject or administering it to a subject in need thereof: (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacterium phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs; (2) bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) and that meet the following requirement X: [Requirement X] Final concentration 2.0×10 5 cells / mL of pDCs are contacted with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in a serum-free medium; (4) The bacterium is Lactococcus lactis subsp. lactis JCM 5805 and satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [C1] A method for stimulating immunity in a subject, comprising ingesting or administering to a subject in need thereof a composition containing bacteria that can be phagocytosed by plasmacytoid dendritic cells (pDCs), A method in which the amount of the bacterium phagocytosed by the pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by the pDC. [C2] A method for stimulating the immunity of a subject, comprising ingesting or administering to a subject in need thereof a composition containing bacteria that bind to blood dendritic cell antigen 2 (BDCA2). [C3] The method according to [C0] or [C2], wherein the binding ability of the bacterium to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2. [C4] The method according to [C3], wherein the binding ability to BDCA2 is evaluated based on the proportion of bacteria bound to BDCA2 as assessed in a binding assay. [C5] A method for stimulating immunity in a subject, comprising ingesting a composition containing bacteria that can be phagocytosed by plasmacytoid dendritic cells (pDCs) to the subject or administering the composition to a subject in need thereof, wherein the bacteria satisfy the following requirement X: [Requirement X] Final concentration 2.0×10 5After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in serum-free medium. [C6] A method for stimulating the immunity of a subject, comprising having the subject ingest or administering to a subject in need thereof a composition containing Lactococcus lactis subsp. lactis JCM 5805 as a bacterium, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [C7] The method according to any one of [C0] to [C5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [C8] The method according to any one of [C0] to [C5], wherein the bacterium is one or more bacteria selected from the group consisting of bacteria of the genus Lactococcus, bacteria of the genus Leuconostoc, bacteria of the genus Streptococcus, bacteria of the genus Enterococcus, bacteria of the genus Tetragenococcus, bacteria of the genus Oenococcus, bacteria of the genus Weissella, bacteria of the genus Bifidobacterium, and bacteria of the genus Lactobacillus. [C9] The method according to any one of [C0] to [C5], wherein the bacterium is Lactococcus lactis subsp. lactis. [C10] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis subsp. Infantis) JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The method according to any one of [C0] to [C5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [C11] The method according to any one of [C0] to [C10], wherein the bacteria include dead bacteria. [C12] The method according to any one of [C0] to [C11], wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [C13] The method described in [C12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of phagocytosis of the bacteria mediated by binding to BDCA2 is 2.0 times or more the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 mediated by binding to BDCA2. [C14] The method described in [C13], wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of the bacteria phagocytosis by the pDC in a state in which BDCA2 in the pDC cannot bind to a ligand. [C15] The method according to any one of [C12] to [C14], wherein the bacterium promotes the production of IFN-α in the pDC through phagocytosis by the pDC. [C16] The method according to any one of [C0] to [C15], wherein the composition is a food composition, a pharmaceutical composition, or a feed. [C17] The number of bacteria of the bacteria per unit package in the food composition, pharmaceutical composition, or feed is 1 x 10 8 The method according to [C16], wherein the number of the first and second electrodes is 1 or more. [D0] A bacterium satisfying at least one of the following conditions (1) to (4) for use in stimulating immunity: (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacterium phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs; (2) bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) and that meet the following requirement X: [Requirement X] Final concentration 2.0×10 5 cells / mL of pDCs are contacted with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in a serum-free medium; (4) The bacterium is Lactococcus lactis subsp. lactis JCM 5805 and satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [D1] A bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) for use in immune stimulation, The amount of the bacterium phagocytosed by pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDC. [D2] A bacterium that binds to blood dendritic cell antigen 2 (BDCA2) for use in stimulating immunity. [D3] A bacterium for use according to [D0] or [D2], wherein the binding ability of the bacterium to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2. [D4] Bacteria for use according to [D3], wherein the ability to bind to BDCA2 is evaluated based on the proportion of bacteria bound to BDCA2 as assessed in a binding assay. [D5] A bacterium for use in immune stimulation, which is phagocytosed by plasmacytoid dendritic cells (pDCs), and which satisfies the following requirement X: [Requirement X] Final concentration 2.0×10 5 After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in serum-free medium. [D6] Lactococcus lactis subsp. lactis JCM 5805, a bacterium, for use in stimulating immunity, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [D7] The bacterium for use according to any one of [D0] to [D5], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [D8] The bacterium for use according to any one of [D0] to [D5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [D9] A bacterium for use according to any one of [D0] to [D5], wherein the bacterium is Lactococcus lactis subsp. lactis. [D10] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis subsp. Infantis) JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The bacterium for use according to any one of [D0] to [D5], which is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [D11] The bacterium for use according to any one of [D0] to [D10], wherein the bacterium includes killed bacteria. [D12] A bacterium for use according to any one of [D0] to [D11], wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [D13] A bacterium for use according to [D12], wherein the phagocytosis of the bacterium by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of phagocytosis of the bacterium via binding to BDCA2 is 2.0 times or more the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 via binding to BDCA2. [D14] The bacterium for use described in [D13], wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of phagocytosis of the bacterium by the pDC in a state in which BDCA2 of the pDC cannot bind to a ligand. [D15] The bacterium for use according to any one of [D12] to [D14], wherein the bacterium promotes the production of IFN-α in the pDC through phagocytosis by the pDC. [D16] The bacterium for use according to any one of [D0] to [D15], wherein the bacterium is contained in a food composition, a pharmaceutical composition, or a feed. [D17] The number of bacteria in the food composition, pharmaceutical composition, or feed per unit package is 1 x 10 8 The bacterium for use according to [D16], wherein the number of bacteria is 1 or more. [E0] A composition for use in stimulating immunity, comprising bacteria that satisfy at least one of the following conditions (1) to (4): (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacterium phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs; (2) bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) and that meet the following requirement X: [Requirement X] Final concentration 2.0×10 5cells / mL of pDCs are contacted with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in a serum-free medium; (4) The bacterium is Lactococcus lactis subsp. lactis JCM 5805 and satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [E1] A composition containing bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) for use in immune stimulation, A composition for use, wherein the amount of the bacterium phagocytosed by pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDC. [E2] A composition containing bacteria that bind to blood dendritic cell antigen 2 (BDCA2) for use in immune stimulation, the composition for use. [E3] A composition for use according to [E0] or [E2], wherein the binding ability of the bacterium to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2. [E4] A composition for use according to [E3], wherein the binding ability to BDCA2 is evaluated based on the proportion of bacteria bound to BDCA2 as assessed in a binding assay. [E5] A composition for use in immune stimulation, comprising bacteria phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the bacteria satisfy the following requirement X: [Requirement X] Final concentration 2.0×10 5 After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in serum-free medium. [E6] A composition for use in stimulating immunity, comprising Lactococcus lactis subsp. lactis JCM 5805 as a bacterium, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [E7] The composition for use according to any one of [E0] to [E5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [E8] A composition for use according to any one of [E0] to [E5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [E9] A composition for use according to any one of [E0] to [E5], wherein the bacterium is Lactococcus lactis subsp. lactis. [E10] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis subsp. Infantis) JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The composition for use according to any one of [E0] to [E5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [E11] The composition for use according to any one of [E0] to [E10], wherein the bacteria include killed bacteria. [E12] A composition for use described in any one of [E0] to [E11], wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [E13] A composition for use described in [E12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis via binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of phagocytosis of the bacteria via binding to BDCA2 is 2.0 times or more the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 via binding to BDCA2. [E14] A composition for use described in [E13], wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of the bacteria phagocytosis by the pDC in a state in which BDCA2 in the pDC cannot bind to a ligand. [E15] A composition for use according to any one of [E12] to [E14], wherein the bacterium promotes the production of IFN-α in the pDC through phagocytosis by the pDC. [E16] The composition for use according to any one of [E0] to [E15], which is a food composition, a pharmaceutical composition, or a feed. [E17] The number of bacteria of the above-mentioned bacteria per unit package in the above-mentioned food composition, pharmaceutical composition, or feed is 1 x 10 8 The composition for use according to [E16], wherein the number of the compounds is 1 or more. [F0] Use of bacteria that satisfy at least one of the following conditions (1) to (4) in stimulating immunity: (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacterium phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs; (2) bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) and that meet the following requirement X: [Requirement X] Final concentration 2.0×10 5cells / mL of pDCs are contacted with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in a serum-free medium; (4) The bacterium is Lactococcus lactis subsp. lactis JCM 5805 and satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [F1] Use of bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in immune stimulation, The amount of the bacteria phagocytosed by pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDC, and used. [F2] Use of bacteria that bind to blood dendritic cell antigen 2 (BDCA2) in stimulating immunity. [F3] The use according to [F0] or [F2], wherein the binding ability of the bacterium to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2. [F4] The use according to [F3], wherein the binding ability to BDCA2 is evaluated based on the proportion of bacteria bound to BDCA2 as assessed in a binding assay. [F5] Use of bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in immune stimulation, wherein the bacteria satisfy the following requirement X: [Requirement X] Final concentration 2.0×10 5 After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in serum-free medium. [F6] Use of the bacterium Lactococcus lactis subsp. lactis JCM 5805 in immune stimulation, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [F7] The use according to any one of [F0] to [F5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [F8] The use according to any one of [F0] to [F5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [F9] The use according to any one of [F0] to [F5], wherein the bacterium is Lactococcus lactis subsp. lactis.[F10] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis subsp. Infantis) JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The use according to any one of [F0] to [F5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [F11] The use according to any one of [F0] to [F10], wherein the bacteria include killed bacteria. [F12] The use according to any one of [F0] to [F11], wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [F13] The use described in [F12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis mediated by binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of phagocytosis of the bacteria mediated by binding to BDCA2 is 2.0 times or more the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 mediated by binding to BDCA2. [F14] The use described in [F13], wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of the bacteria phagocytosis by the pDC in a state in which BDCA2 in the pDC cannot bind to a ligand. [F15] The use according to any one of [F12] to [F14], wherein the bacterium promotes the production of IFN-α in the pDC through phagocytosis by the pDC. [F16] The use according to any one of [F0] to [F15], wherein the bacterium is contained in a food composition, a pharmaceutical composition, or a feed. [F17] The number of bacteria of the above bacteria per unit package in the above food composition, pharmaceutical composition, or feed is 1 x 10 8 The use according to [F16], wherein the number of [F18] The use according to any one of [F0] to [F17], which is a non-therapeutic use. [G0] Use of a composition containing bacteria that satisfies at least one of the following (1) to (4) in stimulating immunity: (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacterium phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs; (2) bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) and that meet the following requirement X: [Requirement X] Final concentration 2.0×10 5 cells / mL of pDCs are contacted with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in a serum-free medium; (4) The bacterium is Lactococcus lactis subsp. lactis JCM 5805 and satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [G1] Use of a composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in stimulating immunity, The amount of the bacteria phagocytosed by pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDC, and used. [G2] Use of a composition containing bacteria that bind to blood dendritic cell antigen 2 (BDCA2) in stimulating immunity. [G3] The use according to [G0] or [G2], wherein the binding ability of the bacterium to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2. [G4] The use according to [G3], wherein the binding ability to BDCA2 is evaluated based on the percentage of bacteria bound to BDCA2 as assessed in a binding assay. [G5] Use of a composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in stimulating immunity, wherein the bacteria satisfy the following requirement X: [Requirement X] Final concentration 2.0×10 5After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in serum-free medium. [G6] Use of a composition containing the bacterium Lactococcus lactis subsp. lactis JCM 5805 in stimulating immunity, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [G7] The use according to any one of [G0] to [G5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [G8] The use described in any one of [G0] to [G5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [G9] The use according to any one of [G0] to [G5], wherein the bacterium is Lactococcus lactis subsp. lactis. [G10] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis subsp. Infantis) JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The use according to any one of [G0] to [G5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [G11] The use according to any one of [G0] to [G10], wherein the bacteria include killed bacteria. [G12] The use according to any one of [G0] to [G11], wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [G13] The use described in [G12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of phagocytosis of the bacteria via binding to BDCA2 is 2.0 times or more the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 via binding to BDCA2. [G14] The use described in [G13], wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of the bacteria phagocytosis by the pDC in a state in which BDCA2 in the pDC cannot bind to a ligand. [G15] The use according to any one of [G12] to [G14], wherein the bacterium promotes the production of IFN-α in the pDC through phagocytosis by the pDC. [G16] The use according to any one of [G0] to [G15], wherein the composition is a food composition, a pharmaceutical composition, or a feed. [G17] The number of bacteria of the above bacteria per unit package in the above food composition, pharmaceutical composition or feed is 1 x 10 8 The use according to [G16], wherein the number of [G18] A use according to any one of [G0] to [G17], which is a non-therapeutic use. [H0] Use of bacteria that satisfy at least one of the following conditions (1) to (4) in the production of an immunostimulatory composition: (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacterium phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs; (2) bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) and that meet the following requirement X: [Requirement X] Final concentration 2.0×10 5 cells / mL of pDCs are contacted with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in a serum-free medium; (4) The bacterium is Lactococcus lactis subsp. lactis JCM 5805 and satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [H1] Use of bacteria that can be phagocytosed by plasmacytoid dendritic cells (pDCs) in the manufacture of an immunostimulatory composition, The amount of the bacteria phagocytosed by pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDC, and used. [H2] Use of bacteria that bind to blood dendritic cell antigen 2 (BDCA2) in the manufacture of an immunostimulatory composition. [H3] The use according to [H0] or [H2], wherein the binding ability of the bacterium to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2. [H4] The use according to [H3], wherein the binding ability to BDCA2 is evaluated based on the proportion of bacteria bound to BDCA2 as assessed in a binding assay. [H5] Use of a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) in the manufacture of an immunostimulatory composition, wherein the bacterium satisfies the following requirement X: [Requirement X] Final concentration 2.0×10 5After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been previously cultured in serum-free medium. [H6] Use of the bacterium Lactococcus lactis subsp. lactis JCM 5805 in the production of an immunostimulatory composition, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [H7] The use according to any one of [H0] to [H5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [H8] The use according to any one of [H0] to [H5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [H9] The use according to any one of [H0] to [H5], wherein the bacterium is Lactococcus lactis subsp. lactis. [H10] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis subsp. Infantis) JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The use according to any one of [H0] to [H5], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [H11] The use according to any one of [H0] to [H10], wherein the bacteria include killed bacteria. [H12] The use according to any one of [H0] to [H11], wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [H13] The use described in [H12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of phagocytosis of the bacteria via binding to BDCA2 is 2.0 times or more the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 via binding to BDCA2. [H14] The use described in [H13], wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of the bacteria phagocytosis by the pDC in a state in which BDCA2 in the pDC cannot bind to a ligand. [H15] The use according to any one of [H12] to [H14], wherein the bacterium promotes the production of IFN-α in the pDC through phagocytosis by the pDC. [H16] The use according to any one of [H0] to [H15], wherein the composition is a food composition, a pharmaceutical composition, or a feed. [H17] The number of bacteria in the food composition, pharmaceutical composition, or feed per unit package is 1 x 10 8 The use according to [H16], wherein the number of [I1] A method for screening for bacteria having immunostimulatory activity, comprising a step of screening for the bacteria having immunostimulatory activity using binding to blood dendritic cell antigen 2 (BDCA2) as an indicator. [I2] The method according to [I1], wherein the screening step is carried out by a binding assay between the candidate bacterium and BDCA2. [I3] The method according to [I1] or [I2], wherein the bacterium selected is any one of [A0] to [A18], [B0] to [B17], [C0] to [C17], [D0] to [D17], [E0] to [E17], [F0] to [F18], [G0] to [G18], and [H0] to [H17]. [I4] The composition, method, bacterium for use, composition for use, or use described in any one of [A0] to [A18], [B0] to [B17], [C0] to [C17], [D0] to [D17], [E0] to [E17], [F0] to [F18], [G0] to [G18], and [H0] to [H17], wherein the bacterium is selected by the method described in [I1] or [I2]. [J1] A method for activating pDCs, characterized by binding bacteria to BDCA2. [J2] A method for activating pDCs, comprising ingesting or administering to a subject in need thereof bacteria that bind to BDCA2. [J3] The method according to [J1] or [J2], wherein the bacterium is phagocytosed by pDC via binding to BDCA2. [J4] A composition containing bacteria for use in the method according to any one of [J1] to [J3]. [J5] The method according to any one of [J1] to [J3] or the composition according to [J4], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [J6] The method according to any one of [J1] to [J3] or the composition according to [J4] or [J5], wherein the bacterium is one or more bacteria selected from the group consisting of bacteria of the genus Lactococcus, bacteria of the genus Leuconostoc, bacteria of the genus Streptococcus, bacteria of the genus Enterococcus, bacteria of the genus Tetragenococcus, bacteria of the genus Oenococcus, bacteria of the genus Weissella, bacteria of the genus Bifidobacterium, and bacteria of the genus Lactobacillus. [J7] The method according to any one of [J1] to [J3] or the composition according to any one of [J4] to [J6], wherein the bacterium is Lactococcus lactis subsp. lactis. [J8] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni oeni) JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The method according to any one of [J1] to [J3] or the composition according to any one of [J4] to [J7], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [J9] The method according to any one of [J1] to [J3] or the composition according to any one of [J4] to [J8], wherein the bacteria include killed bacteria. [K1] A method for enhancing the immunostimulatory activity and / or phagocytosis by plasmacytoid dendritic cells (pDCs) of bacteria, comprising a step of culturing bacteria in M17 medium supplemented with glucose at a final concentration of 0.1 to 10% by mass. [K2] The method according to [K1], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria. [K3] The method according to [K1] or [K2], wherein the bacterium is one or more bacteria selected from the group consisting of bacteria of the genus Lactococcus, bacteria of the genus Leuconostoc, bacteria of the genus Streptococcus, bacteria of the genus Enterococcus, bacteria of the genus Tetragenococcus, bacteria of the genus Oenococcus, bacteria of the genus Weissella, bacteria of the genus Bifidobacterium, and bacteria of the genus Lactobacillus. [K4] The method according to any one of [K1] to [K3], wherein the bacterium is Lactococcus lactis subsp. lactis. [K5] The above bacteria are Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni oeni) JCM 6125, Weissella paramesenteroides JCM 9890, Weissella viridescens JCM 1174, Bifidobacterium animalis subsp. animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactislactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 The method according to any one of [K1] to [K4], wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505. [K6] The method according to any one of [K1] to [K5], wherein the bacterium is Lactococcus lactis subsp. lactis JCM 5805. [K7] A bacterium whose immunostimulatory activity and / or ability to be phagocytosed by plasmacytoid dendritic cells (pDCs) has been enhanced by the method described in any one of [K1] to [K6]. [L1] A method for producing an immunostimulatory composition containing Lactococcus lactis subsp. lactis JCM 5805 as an active ingredient, the method comprising the step of culturing Lactococcus lactis subsp. lactis JCM 5805 in M17 medium to which glucose has been added at a final concentration of 0.1 to 10% by mass. [L2] The method according to [L1], in which the composition according to any one of [A0] to [A17], [C0] to [C17], [E0] to [E17], [G0] to [G18] and [H0] to [H17] is produced. [L3] The composition, method, composition for use, or use according to any one of [A0] to [A17], [C0] to [C17], [E0] to [E17], [G0] to [G18], and [H0] to [H17], wherein the composition is a composition manufactured by the manufacturing method according to [L1]. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide an immunostimulating composition. For example, the immunostimulating composition of the present invention can stimulate immunity through the phagocytosis of the bacteria, which is the active ingredient, by plasmacytoid dendritic cells (pDCs). Also, for example, the immunostimulating composition of the present invention can stimulate immunity through the binding of the bacteria, which is the active ingredient, to BDCA2 on the surface of plasmacytoid dendritic cells (pDCs). Also, for example, the immunostimulating composition of the present invention can stimulate immunity through the binding of the bacteria, which is the active ingredient, to BDCA2 on the surface of pDCs, which is phagocytosed by dendritic cells, activating pDCs.

[0009] According to the present invention, a method for screening for bacteria having immunostimulatory activity can be provided. For example, in the screening method of the present invention, bacteria that bind to BDCA2 can be selected as bacteria that may have immunostimulatory activity, using the binding of bacteria to BDCA2 as an indicator. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the fluorescence detection area 22 hours after addition of LC-Plasma in Example 1. [Figure 2] FIG. 1 shows the results of measuring the IFN-α concentration in the culture supernatant collected 22 hours after the addition of LC-Plasma in Example 1. [Figure 3] FIG. 1 shows the fluorescence detection area 22 hours after the addition of lactic acid bacteria for the eight types of lactic acid bacteria tested in Example 2. [Figure 4] FIG. 1 shows the results of measuring the IFN-α concentration in the culture supernatant collected 22 hours after the addition of lactic acid bacteria for the eight types of lactic acid bacteria tested in Example 2. [Figure 5] FIG. 1 shows the correlation between the fluorescence detection area on the horizontal axis and the IFN-α concentration in the culture supernatant on the vertical axis for the eight types of lactic acid bacteria tested in Example 2. [Figure 6] FIG. 10 shows the fluorescence detection area 22 hours after addition of LC-Plasma for the PL group, PL+Isotype group, and PL+BDCA2 anti group in Example 3. [Figure 7] FIG. 10 shows the results of measuring the IFN-α concentration in the culture supernatant collected 22 hours after the addition of LC-Plasma for the PL group, PL+Isotype group, and PL+BDCA2 anti group in Example 3. [Figure 8] FIG. 10 shows the fluorescence detection areas 22 hours after the addition of various lactic acid bacteria for the isotype group and the BDCA2 anti group in Example 4. [Figure 9] FIG. 10 shows the results of measuring the IFN-α concentration in the culture supernatant collected 22 hours after the addition of various lactic acid bacteria for the isotype group and the BDCA2 anti group in Example 4. [Figure 10] FIG. 1 shows the correlation between the amount of phagocytosis mediated by binding to BDCA2 on the horizontal axis and the IFN-α concentration in the culture supernatant on the vertical axis for various lactic acid bacteria. [Figure 11]FIG. 10 shows the percentage of LC-Plasma bound to BDCA2 at BDCA2 concentrations of 1, 5, 10, 20, or 50 μg / mL, as assessed by BDCA2 binding assay in Example 5. [Figure 12] FIG. 1 shows the results of adding DMSO or cytochalasin D+DMSO and measuring the number of viable cells 24 hours later using Cell Counting Kit-8 in Example 7. [Figure 13] FIG. 10 is a graph showing the Total Integrate Intensity (TII) representing the phagocytic activity 24 hours after the addition of lactic acid bacteria for the six types of lactic acid bacteria tested in Example 7. [Figure 14] FIG. 10 shows the IFN-α concentration in the supernatant 24 hours after the addition of lactic acid bacteria for the six types of lactic acid bacteria tested in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, an embodiment for carrying out the present invention will be described, but the present invention is not limited to the following embodiment.

[0012] A first embodiment of the present invention is an immunostimulating composition containing a bacterium as an active ingredient. In one aspect, the bacterium contained as an active ingredient in the immunostimulating composition is a bacterium that is phagocytosed by pDC. In another aspect, the bacterium contained as an active ingredient in the immunostimulating composition is a bacterium that binds to BDCA2.

[0013] Hereinafter, "an immunostimulating composition containing, as an active ingredient, bacteria that are phagocytosed by pDCs" and / or "an immunostimulating composition containing, as an active ingredient, bacteria that bind to BDCA2" may be referred to as "the immunostimulating composition of this embodiment."

[0014] The bacteria contained in the immunostimulatory composition of this embodiment (hereinafter sometimes referred to as "bacteria of this embodiment") may be bacteria that are particularly useful for the human body. The bacteria of this embodiment may be gram-positive or gram-negative bacteria. For example, the bacteria of this embodiment may be lactic acid bacteria or acetic acid bacteria, and in a preferred embodiment may be lactic acid bacteria, in a more preferred embodiment may be bacteria of the genus Lactococcus, and in an even more preferred embodiment may be Lactococcus lactis subsp. lactis.

[0015] Lactic acid bacteria are bacteria that produce lactic acid as a metabolite. Examples of lactic acid bacteria include bacteria of the genus Oenococcus, Bifidobacterium, Weissella, Tetragenococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Lactobacillus, acetic acid bacteria, and Bacillus. In one embodiment, the lactic acid bacteria may be bacteria of the genus Lactobacillus or Lactococcus.

[0016] In this specification, the term "Lactobacillus" includes bacteria that were classified into the genus Lactobacillus before the reclassification of the genus Lactobacillus. For example, with the reclassification of the Lactobacillus genus, the following new species have been added: Acetilactobacillus, Agrilactobacillus, Amylolactobacillus, Apilactobacillus, Bombilactobacillus, Companilactobacillus, Dellaglioa, Fructilactobacillus, Furfurilactobacillus, Holzapfelia, Lacticaseibacillus, Lactiplantibacillus, and Lapidilactobacillus. This includes bacteria classified into the genera Lapidilactobacillus, Latilactobacillus, Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolactobacillus, Paralactobacillus, Paucilactobacillus, Schleiferilactobacillus, and Secundilactobacillus.

[0017] Among the above, bacteria of the genus Oenococcus, Bifidobacterium, Lentilactobacillus, Weissella, Tetragenococcus, Lactococcus, Leuconostoc, Pediococcus, Enterococcus, Lactobacillus, and Lactiplantibacillus are preferred.

[0018] Examples of the Oenococcus include Oenococcus oeni, etc. Specific examples of the Oenococcus include Oenococcus oeni JCM 6125, etc.

[0019] Examples of bacteria of the genus Bifidobacterium include Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis. Specific examples of Bifidobacterium include Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium longum subsp. infantis JCM 1222, Bifidobacterium longum subsp. longum BB536, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis M-63, Bifidobacterium longum subsp. longum N61, Bifidobacterium bifidum OLB6378, Bifidobacterium breve M-16V, Bifidobacterium breve MCC1274, and Bifidobacterium pseudolongum JCM 1205.

[0020] Examples of Weissella include Weissella paramesenteroides and Weissella viridescens. Specific examples of Weissella include Weissella paramesenteroides JCM 9890 and Weissella viridescens JCM 1174.

[0021] Examples of bacteria of the genus Tetragenococcus include Tetragenococcus halophilus, etc. Specific examples of bacteria of the genus Tetragenococcus include Tetragenococcus halophilus NRIC 0098 and Tetragenococcus halophilus No. 1, etc.

[0022] Examples of Lactococcus bacteria include Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, and Lactococcus plantarum.

[0023] Specific examples of Lactococcus bacteria include Lactococcus lactis subsp. lactis 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, and Lactococcus lactis subsp. lactis ATCC 27861, Lactococcus lactis subsp. lactis ATCC 19435, 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. holdoniae JCM 1180, Lactococcus lactis subsp. holdoniae JCM 11040, and Lactococcus plantarum JCM 11056.

[0024] Examples of bacteria of the genus Leuconostoc include Leuconostoc carnosum and Leuconostoc lactis. Specific examples of bacteria of the genus Leuconostoc include Leuconostoc carnosum JCM 9695 and Leuconostoc lactis NBRC 12455.

[0025] Examples of bacteria of the genus Pediococcus include Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus cellicola, Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parvulus, and Pediococcus stilesii. Specific examples of bacteria of the genus Pediococcus include Pediococcus acidilactici JCM 8797, Pediococcus acidilactici K15, and Pediococcus damnosus JCM 5886.

[0026] Examples of bacteria of the genus Streptococcus include Streptococcus thermophilus, etc. Specific examples of bacteria of the genus Pediococcus include Streptococcus thermophilus SBC 8781, etc.

[0027] Examples of Enterococcus bacteria include Enterococcus alcedinis and Enterococcus faecalis. Specific examples of Enterococcus bacteria include Enterococcus faecalis EC-12.

[0028] Examples of Lactobacillus bacteria include Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus acidophilus, and Lactobacillus bulgaricus. Lactobacillus bulgaricus, Lactobacillus parakefiri, Lactobacillus plantarum, and Lactobacillus pentosus.

[0029] Specific examples of Lactobacillus bacteria include Lactobacillus paracasei KW3110, Lactobacillus paracasei MCC 1849, Lactobacillus paracasei K71, Lactobacillus paracasei K-2, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus CRL1505, Lactobacillus gasseri SBT2055, Lactobacillus gasseri OLL2716, Lactobacillus gasseri PA-3, Lactobacillus acidophilus L-92, Lactobacillus casei subsp. casei 327, Lactobacillus (newly classified as Lacticaseibacillus) casei Shirota, Lactobacillus bulgaricus OLL1073R-1, and Lactobacillus parakeophili (newly classified as Lentilactobacillus parakeophili) JCM. 8573, Lactobacillus plantarum (newly classified as Lactiplantibacillus plantarum) L-137, Lactobacillus pentosus (newly classified as Lactiplantibacillus pentosus) ONRICb0240, and Lactobacillus helveticus GCL1815.

[0030] The acetic acid bacteria are not particularly limited, but examples thereof include bacteria of the genus Gluconacetobacter, Acetobacter, and Gluconobacter, preferably bacteria of the genus Gluconacetobacter, more preferably Gluconacetobacter hansenii, and even more preferably Gluconacetobacter hansenii GK-1.

[0031] Bacteria of the genus Bacillus include, but are not limited to, Bacillus coagulans, etc. Specific examples of Bacillus include Bacillus coagulans SANK70258 strain, etc.

[0032] Among the above, the bacteria according to this embodiment are preferably selected from the group consisting of bacteria of the genus Lactobacillus, Lactococcus, and Gluconacetobacter, in terms of edible flavor.

[0033] In addition to the above, the bacteria according to this embodiment may be bacteria of the genus Akkermansia, Bacteroides, Blautia, Clostridium, Collinsella, Enterococcus, Faecalibacterium, Faecalicatena, Lacrimispora, Paeniclostridium, Parabacteroides, or Roseburia.

[0034] Examples of Akkermansia include Akkermansia muciniphila JCM 30893.

[0035] Bacteroides genus bacteria include 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 setaiotaomicron ATCC 29148T, Bacteroides setaiotaomicron ATCC 29741, Bacteroides setaiotaomicron ATCC 12290, Bacteroides uniformis JCM 5828T, Bacteroides uniformis JCM 13286, and Bacteroides uniformis JCM 13287, and Bacteroides uniformis JCM 13288.

[0036] Blautia genus fungi include Blautia acetigengens JCM 34803T, Blautia ammoniilytica JCM 34802T, Blautia algi JCM 31394T, Blautia caekimuris JCM 34498T, Blautia coccoides JCM 1395T, Blautia phecus JCM 17205T, Blautia glucellacea JCM 17039T, Blautia hansenii JCM 14655, Blautia hansenii JCM 35484, Blautia hominis JCM 32276T, Blautia hydrogenotrophica JCM 31266, Blautia liqualis JCM 34225T, and Blautia luti JCM 14655. 17040T, Blautia obeum JCM 31340, Blautia producta JCM 1471T, Blautia pseudococcoides JCM 35243T, Blautia cinquii JCM 14657T, Blautia wechslerae JCM 31267, and Blautia wechslerae JCM 35486.

[0037] Examples of Clostridium bacteria include Clostridium butyricum JCM NT, Clostridium nexile JCM 31500T, and Clostridium symbiosum JCM 1297T.

[0038] Examples of Collinsella species include Collinsella aerofaciens JCM 10188T, Collinsella intestinalis JCM 10643T, Collinsella stercoris JCM 10641T, and Collinsella tanakaei JCM 16071T.

[0039] Examples of Enterococcus include Enterococcus faecalis JCM 5803T, Enterococcus faecalis JCM 20307, ​​Enterococcus faecium JCM 5804T, and Enterococcus faecium JCM 8903.

[0040] Examples of bacteria of the genus Faecalibacterium include Faecalibacterium hattorii JCM 39210, Faecalibacterium longum JCM 39208, Faecalibacterium prausnitzii JCM 31915, Faecalibacterium prausnitzii JCM 39207, and Faecalibacterium prausnitzii JCM 39209.

[0041] Examples of Faecalicatena species include Faecalicatena oroticum JCM 1429T.

[0042] Examples of the genus Lacrimispora include Lacrimispora celereculescens JCM 15734T, Lacrimispora sphenoides JCM 1415T, and Lacrimispora xylanolytica JCM 15735T.

[0043] Examples of the genus Paeniclostridium include Paeniclostridium sordellii JCM 3814T.

[0044] Examples of Parabacteroides include Parabacteroides meldae JCM 9497T.

[0045] Examples of Roseburia species include Roseburia hominis JCM 17582, Roseburia intestinalis JCM 17583, and Roseburia inulinovorans JCM 17584.

[0046] Of the above lactic acid bacteria and acetic acid bacteria, JCM strains can be obtained from the Microbial Materials Development Laboratory, RIKEN BioResource Center (3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture), NBRC strains from the Biological Genetic Resources Division, National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture), NRIC strains from the Tokyo University of Agriculture and Technology Culture Collection (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo), and ATCC strains from the American Type Culture Collection (USA). In addition to being available from public institutions, the above lactic acid bacteria and acetic acid bacteria can also be obtained by isolation or purification using known methods from commercially available products containing lactic acid bacteria or acetic acid bacteria.

[0047] Lactobacillus paracasei KW3110 has been deposited with the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), an international depositary authority under the Budapest Treaty for the Deposit of Patent Microorganisms (currently the National Institute of Technology and Evaluation (NITE) Biotechnology Center Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan), under the accession number FERM BP-08634 (deposit date: February 20, 2004). A derivative of Lactobacillus paracasei KW3110 has been deposited with the same Patent Organism Depositary under the accession number FERM BP-08635 (deposit date: February 20, 2004).

[0048] Lactococcus lactis subsp. lactis JCM 5805 can be obtained from the Microbial Engineering Division of the RIKEN BioResource Center, as described above, but the present invention can also use the same strain of Lactococcus lactis subsp. lactis JCM 5805 stored in a collection institution other than the Microbial Engineering Division of the RIKEN BioResource Center. Specifically, the same strain of Lactococcus lactis subsp. lactis JCM 5805 can be obtained from the Biological Genetic Resources Division of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan), the Strain Collection of Tokyo University of Agriculture (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, Japan), the American Type Culture Collection (USA), and other sources. 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.

[0049] The bacterium according to this embodiment may be a mutant strain of a bacterial strain included in the above list. The bacterial mutant strain may have properties that can achieve the objective of the present technology (e.g., immunostimulatory activity when the composition is an immunostimulatory composition). Such a mutant strain may be constructed by non-artificially introducing a mutation into a bacterial strain included in the above list. Furthermore, such a mutant strain may be constructed by artificially introducing a mutation into a bacterial strain included in the above list, for example, by introducing a mutation into the bacterium by treatment with a mutagen such as ultraviolet (UV) or a DNA alkylating agent, or by introducing a mutation into the strain using a genetic engineering method such as gene editing, typified by CRISPR-Cas9.

[0050] The bacterium contained in the immunostimulatory composition of the present embodiment may be a single bacterium or a mixture of two or more types of bacteria, for example, a mixture of lactic acid bacteria and acetic acid bacteria.

[0051] In one aspect, the bacteria according to this embodiment are bacteria that are phagocytosed by pDCs. That is, one aspect of this embodiment is an immunostimulatory composition that contains, as an active ingredient, bacteria that are phagocytosed by pDCs.

[0052] Plasmacytoid dendritic cells (pDCs), also known as plasmacytoid dendritic cells, are a type of dendritic cell that constitutes the innate immune system. pDCs are the main producers of type I interferon in the body. Type I interferon exhibits growth inhibitory activity against viruses and other pathogens. Representative type I interferons are interferon α (IFN-α) and interferon β (IFN-β), which are thought to have immunostimulatory properties.

[0053] Phagocytosis is a process in which cells ingest relatively large objects, such as solid particles, other cells, or bacteria. During phagocytosis, the cell extends its plasma membrane to envelop the object, ingesting it, and then the vesicles containing the ingested object (phagosomes) fuse with lysosomes to degrade the contents.

[0054] Whether a bacterium is phagocytosed by pDCs can be assessed by an evaluation method using bacteria labeled with a fluorogenic or fluorescent dye, as described below. Furthermore, whether a bacterium is phagocytosed by pDCs can be assessed by the decrease in bacterial uptake by pDCs when pDC phagocytosis is inhibited. For example, if the efficiency of uptake by pDCs in which phagocytosis has been inhibited with an actin polymerization inhibitor (e.g., cytochalasin D or latrunculin B) is significantly lower than the efficiency of uptake by pDCs in which polymerization has not been inhibited, the bacterium can be assessed as being phagocytosed by pDCs. Furthermore, if the efficiency of uptake by pDCs in which phagocytosis has been inhibited with a ligand or antibody that binds to a pDC surface protein involved in bacterial phagocytosis, such as BDCA2, as described below, is significantly lower than the efficiency of uptake by pDCs in which binding to a pDC surface protein involved in bacterial phagocytosis has not been inhibited, the bacterium can be assessed as being phagocytosed by pDCs. The efficiency of bacterial uptake by pDCs can be measured, for example, by the amount of uptake at a predetermined time point after adding the bacterium to pDCs.

[0055] The bacterium of this embodiment may be a bacterium whose amount phagocytosis by pDC is 0.01 times or more, 0.1 times or more, 0.3 times or more, 0.5 times or more, 0.7 times or more, 0.9 times or more, or 1.0 times or more the amount phagocytosis by pDC of Lactococcus lactis subsp. lactis JCM 5805. Furthermore, the bacterium according to this embodiment may be a bacterium whose amount of phagocytosis by pDC is more than 1.0 times, 1.2 times or more, 1.5 times or more, 2.0 times or more, 3.0 times or more, 4.0 times or more, 5.0 times or more, 7.0 times or more, 10 times or more, 15 times or more, 20 times or more, 30 times or more, 40 times or more, 60 times or more, or 100 times or more than the amount of phagocytosis by pDC of Lactococcus lactis subsp. lactis ATCC 15577.

[0056] Furthermore, the amount of phagocytosis of the bacteria according to this embodiment by pDCs can be evaluated by an evaluation method using bacteria labeled with a fluorescent or fluorogenic dye, which will be described later. In the above evaluation method, for example, the bacteria according to this embodiment and control bacteria (Lactococcus lactis subsp. lactis JCM 5805 or Lactococcus lactis subsp. lactis ATCC15577) are contacted with pDCs having the same cell number (or cell concentration) for a certain period of time under the same conditions, and then the bacteria are removed from the medium, and the fluorescence area per unit area of ​​the observation field of a fluorescence microscope (for example, μm ) in the pDCs is measured. 2 / image) can be evaluated as the amount of phagocytosis. In this case, the amount of phagocytosis is preferably the average value of multiple (2 or more, 3 or more, 5 or more, 7 or more, or 10 or more) visual fields or multiple (2 or more, 3 or more, 5 or more, 7 or more, or 10 or more) samples. When the amount of phagocytosis by pDCs is evaluated by an evaluation method using bacteria labeled with a fluorogenic or fluorescent dye described below, the bacteria according to this embodiment have a phagocytosis amount of 50 μm 2 / image or more, 100μm 2 / image or more, 200μm 2 / image or more, 400μm 2 / image or more, 500μm 2 / image or more, 600μm 2 / image or more, 700μm 2 / image or more, 800μm 2 / image or more, or 900μm 2 / image or more, where "image" is 0.572 mm 2 This means the field of view.

[0057] Furthermore, when the amount of phagocytosis by pDC of Lactococcus lactis subsp. lactis ATCC 15577 was subtracted from the amount of phagocytosis by pDC of the bacterium according to this embodiment, the difference was 1 μm 2 / image or more, 20μm 2 / image or more, 50μm 2 / image or more, 70μm 2 / image or more, 100μm2 / image or more, 140μm 2 / image or more, 200μm 2 / image or more, 300μm 2 / image or more, 400μm 2 / image or more, 500μm 2 / image or more, 600μm 2 / image or more, 700μm 2 / image or more or 900μm 2 It can be more than / image.

[0058] The bacteria according to this embodiment were found to have a virulence of 0.572 mm by measurement using a protocol similar to that of the evaluation method using bacteria labeled with a fluorogenic or fluorescent dye described below. 2 Total Integrate Intensity (TII, OCU × μm) per field of view 2 When the amount of phagocytosis by pDCs was evaluated using the index of TII, the TII was 10,000 OCU × μm 2 / Image or more, 30000OCU×μm 2 / Image or more, 60000OCU×μm 2 / Image or more, 100000OCU×μm 2 / Image or more, 150000OCU×μm 2 / Image or more, 200000OCU×μm 2 / Image or above or 250,000 OCU×μm 2 It can be more than / Image.

[0059] In these cases, the amount of phagocytosis by pDCs is not particularly limited as long as it is an index value indicating the amount of phagocytosis by pDCs, and the amount of phagocytosis by pDCs can be evaluated using methods that are commonly used by those skilled in the art as a method for measuring the amount of phagocytosis. For example, the amount of phagocytosis by pDCs can be measured as the fluorescence intensity of pDCs measured after contact with bacteria labeled with a fluorogenic or fluorescent dye. As a more specific example, the amount of phagocytosis by pDCs can be evaluated using the fluorescence detected from pDCs as an index when bacteria labeled with a fluorescent dye that becomes highly fluorescent in the acidic environment of the endosome (e.g., pHrodo Red SE, Invitrogen, P36600) are contacted with pDCs for a sufficient period of time (e.g., 24 hours). Fluorescence indicators that are commonly used by those skilled in the art may be used, such as the area or percentage of areas in a fluorescent image where the fluorescence intensity is greater than a predetermined value, the value obtained by multiplying the area where the fluorescence intensity is greater than a predetermined value by the mean fluorescence intensity, the average, median, or histogram of the fluorescence intensity in a fluorescent image, a histogram of fluorescence intensity obtained by flow cytometry analysis or the percentage of cells contained within a gate when gating on fluorescence intensity is performed, or the fluorescence intensity of bulk cells contained in a cell suspension or a microwell for analysis with a microwell plate reader. Furthermore, the amount of phagocytosis by pDCs may be evaluated using the expression level of a biomarker whose expression level varies depending on the amount of phagocytosis by pDCs as an indicator. The values ​​described above can be used as the "amount of phagocytosis by pDCs" in this embodiment.

[0060] In this embodiment, the amount of phagocytosis by pDC may be the amount of phagocytosis evaluated using CAL-1 cells. CAL-1 cells are a human plasmacytoid dendritic cell (pDC) cancer cell line established from tumor cells in a patient's peripheral blood, and are a cell line deposited at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganisms Depositary (NPMD, Japan) under deposit number FERM BP-10914. CAL-1 cells are disclosed in, for example, Japanese Patent Application Laid-Open No. 2007-044008.

[0061] The amount of phagocytosis by pDC may be the amount of phagocytosis evaluated by the method described below as "Evaluation method using bacteria labeled with a fluorescent dye," as a detailed example. The amount of phagocytosis by pDC may be the amount of phagocytosis evaluated by the method described in the Examples, as a more detailed example.

[0062] [Evaluation method using bacteria labeled with fluorescent dyes] (phagocytosis experiment) CAL-1 cells were cultured at a density of 5.0 × 10 in serum-free medium (e.g., RPMI-1640 medium (Sigma, R8758) supplemented with penicillin / streptomycin (Gibco, 15140-12) at a final concentration of 1.0% by volume). 5 A cell suspension containing 2.0 × 10 cells / mL was prepared, and the cell suspension was seeded into a 10 cm diameter culture dish at 10 mL / well. The cells were cultured for 16 hours and then harvested by pipetting. The harvested CAL-1 cells were then transferred to the same medium at a concentration of 2.0 × 10 cells / mL. 5 The cells were resuspended at a concentration of 1000 cells / mL and seeded at 200 μL / well into a 96-well microplate. Bacteria stained with pHrodo Red SE (Invitrogen, P36600) as described below were added to a final concentration of 10 μg / mL, and the fluorescence of pHrodo Red SE in each well was measured over time using an Incucyte® SX5 Live-Cell Analysis System (Sartorius).

[0063] (Bacterial staining with pHrodo Red SE) pHrodo Red SE (Invitrogen, P36600) was dissolved in DMSO to prepare a 10.2 mM solution. Bacterial bulk powder was weighed into a 2 mL Eppendorf tube and 0.1 M sodium bicarbonate adjusted to pH 9.0 was added to prepare a bacterial solution at 20 mg / mL. 95 μL of the bacterial solution was transferred to a new 2 mL Eppendorf tube and 5 μL of 10.2 mM pHrodo Red SE prepared as described above was added. After vortexing to disperse the bacteria, the mixture was protected from light with aluminum foil and incubated at room temperature for 60 minutes. 750 μL of PBS was then added, vortexed, and centrifuged at 20,000 × g for 2 minutes at room temperature. 800 μL of the supernatant was discarded, and 1.5 mL of PBS was added. The precipitate was completely suspended by vortexing. The mixture was then centrifuged again at 20,000 × g for 2 minutes at room temperature, and 1.5 mL of the supernatant was discarded. Add 140 μL of PBS to suspend the bacteria and create a 10 mg / mL solution of stained bacteria. Store at 4°C, protected from light by aluminum foil, until use. Immediately before use, dilute the stained bacteria 10-fold with PBS to create a 1 mg / mL solution of bacteria. The stained bacterial solution should be used within 24 hours of creation.

[0064] (Evaluation of phagocytosis amount) The entire field of view (0.572 mm) to be evaluated was acquired from each well of the plate. 2 The amount of bacteria phagocytized by CAL-1 cells was quantified using the total area (fluorescence detection area) where pHrodo Red SE fluorescence was detected out of the total area (fluorescence detection area) as an index. Using the Incucyte® SX5 Live-Cell Analysis System (Sartorius), one image (0.572 mm) was selected according to the number of images to be evaluated. 2 Fluorescence detection area (e.g., μm) per 2 / image) can be analyzed to evaluate the amount of phagocytosis. In this case, since the stained bacteria will fluoresce slightly even if they are not phagocytosed, the threshold value of the Orange Channel is set to 5.0 to eliminate the influence of fluorescence not due to phagocytosis. In addition, depending on the type of bacteria, bacterial cells may form aggregates that exceed the threshold value even if they are not phagocytosed, so the Area of ​​Filters in the Orange Channel is set to 20 to eliminate the influence of excessively large aggregates.

[0065] According to one aspect of this embodiment, bacteria phagocytosed by pDCs may satisfy the following requirement X0. Note that the "proportion of the number of pDCs that phagocytosed bacteria to the total number of contacted pDCs" below corresponds to the "phagocytosis rate" evaluated in Examples 8 and 9 described below. In one aspect, pDCs satisfying requirement X0 may be CAL-1 cells (FERM BP-10914). [Requirement X0] Final concentration 2.0×10 5 After contacting 1000 cells / mL of pDCs with the bacteria at a final concentration of 10 μg / mL in serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is a predetermined ratio, where the pDCs are pDCs that have been previously cultured in serum-free medium.

[0066] The specified ratio for requirement X0 may be, for example, 2.5% or more, 3.0% or more, 4.0% or more, 5.0% or more, 5.5% or more, 6.5% or more, 7.0% or more, 7.5% or more, 8.0% or more, 9.0% or more, 9.5% or more, 10.0% or more, 10.5% or more, 11.0% or more, 11.5% or more, 12.0% or more, 12.5% ​​or more, 13.0% or more, 13.5% or more, 14.0% or more, 14.5% or more, 15.0% or more, 15.5% or more, 16.0% or more, 16.5% or more, 17.0% or more, or 17.5% or more, or may be less than 100%, 50.0% or less, 25.0% or less, or 18.0% or less. These lower and upper limits can be combined in any way. For example, the specified percentages related to requirement X0 are 2.5% or more and less than 100%, 2.5% or more and less than 50.0%, 2.5% or more and less than 25.0%, 2.5% or more and less than 18.0%, 3.0% or more and less than 100%, 3.0% or more and less than 50.0%, 3.0% or more and less than 25.0%, 3.0% or more and less than 18.0%, 4.0% or more and less than 100%, and 4.0% or more and less than 50.0%. 0% or less, 4.0% to 25.0% or less, 4.0% to 18.0% or less, 5.0% to less than 100%, 5.0% to 50.0% or less, 5.0% to 25.0% or less, 5.0% to 18.0% or less, 5.5% to less than 100%, 5.5% to 50.0% or less, 5.5% to 25.0% or less, 5.5% to 18.0% or less, 6.5% to less than 100%, 6.5% to 50.0% or less, 6.5% to 25.0% or less , 6.5% or more and 18.0% or less, 7.0% or more and less than 100%, 7.0% or more and 50.0% or less, 7.0% or more and 25.0% or less, 7.0% or more and 18.0% or less, 7.5% or more and less than 100%, 7.5% or more and 50.0% or less, 7.5% or more and 25.0% or less, 7.5% or more and 18.0% or less, 8.0% or more and less than 100%, 8.0% or more and 50.0% or less, 8.0% or more and 25.0% or less, 8.0% or more and 18.0% or less, 9.0 % or more but less than 100%, 9.0% or more but less than 50.0%, 9.0% or more but less than 25.0%, 9.0% or more but less than 18.0%, 9.5% or more but less than 100%, 9.5% or more but less than 50.0%, 9.5% or more but less than 25.0%, 9.5% or more but less than 18.0%, 10.0% or more but less than 100%, 10.0% or more but less than 50.0%, 10.0% or more but less than 25.0%, 10.0% or more but less than 18.0%, 10.5% or more but less than 100%, 10.5% to 50.0%, 10.5% to 25.0%, 10.5% to 18.0%, 11.0% to under 100%, 11.0% to 50.0%, 11.0% to 25.0%, 11.0% to 18.0%, 11.5% to under 100%, 11.5% to 50.0%, 11.5% to 25.0%, 11.5% to 18.0%, 12.0% to under 100%, 12.0% to 50.0%, 12.0% to 25.0%, 12.0% to 18.0% , 12.5% ​​or more and less than 100%, 12.5% ​​or more and less than 50.0%, 12.5% ​​or more and less than 25.0%, 12.5% ​​or more and less than 18.0%, 13.0% or more and less than 100%, 13.0% or more and less than 50.0%, 13.0% or more and less than 25.0%, 13.0% or more and less than 18.0%, 13.5% or more and less than 100%, 13.5% or more and less than 50.0%, 13.5% or more and less than 25.0%, 13.5% or more and less than 18.0%, 14.0% or more and less than 100%, 14.0% or more and less than 50.0%, 14.0% or more and less than 25.0% or less, 14.0% to 18.0% or less, 14.5% to less than 100%, 14.5% to 50.0%, 14.5% to 25.0% or less, 14.5% to 18.0% or less, 15.0% to less than 100%, 15.0% to 50.0%, 15.0% to 25.0% or less, 15.0% to 18.0% or less, 15.5% to less than 100%, 15.5% to 50.0%, 15.5% to 25.0% or less, 15.5% to 18.0% or less, 16.0% to less than 100%, 16.0% to 50. The ratio may be 0% or less, 16.0% to 25.0%, 16.0% to 18.0%, 16.5% to less than 100%, 16.5% to 50.0%, 16.5% to 25.0%, 16.5% to 18.0%, 17.0% to less than 100%, 17.0% to 50.0%, 17.0% to 25.0%, 17.0% to 18.0%, 17.5% to less than 100%, 17.5% to 50.0%, 17.5% to 25.0%, or 17.5% to 18.0%. When the specified ratio related to requirement X0 is equal to or greater than the above lower limit, the immunostimulatory ability of the composition is more likely to be enhanced than when the ratio is less than the above lower limit.

[0067] Whether a certain bacterium satisfies requirement X0 can be evaluated, for example, according to a method including pre-culturing pDCs in serum-free medium (pretreatment step), contacting the pDCs obtained in the pretreatment step with bacteria in serum-free medium for 24 hours (pDC contact step), and evaluating the ratio of the number of pDCs that phagocytose the bacteria to the total number of pDCs contacted in the contact step (phagocytosis evaluation step).

[0068] Serum related to requirement X0 generally refers to serum derived from humans or other animals used in the culture of animal cells, including humans, such as serum derived from humans, bovine serum, or equine serum, and more specifically, fetal bovine serum (FBS).

[0069] The medium satisfying requirement X0 can be the same as a medium (e.g., a basal medium) commonly used by those skilled in the art for cell culture. For example, the medium may be a medium containing water as a solvent, sugars such as D-glucose, and amino acids such as L-glutamine. The medium may further contain additives. The additives may be those commonly used by those skilled in the art for cell culture, such as at least one selected from the group consisting of antibiotics (penicillin, streptomycin, etc.), vitamins, inorganic salts, and buffers. In one aspect, the D-glucose content of the medium can be 500 mg / L or more, or 1200 mg / L or more, and the D-glucose content of the medium can be 6000 mg / L or less, or 3000 mg / L or less. These upper and lower limits can be combined arbitrarily, for example, the D-glucose content of the medium can be 500 mg / L to 6000 mg / L, 500 mg / L to 3000 mg / L, 1200 mg / L to 6000 mg / L, or 1200 mg / L to 3000 mg / L. In one aspect, the L-glutamine content of the medium can be 100 mg / L or more, or 250 mg / L or more, and the L-glutamine content of the medium can be 1000 mg / L or less, or 600 mg / L or less. These upper and lower limits can be arbitrarily combined. For example, the L-glutamine content of the medium may be 100 mg / L to 1000 mg / L, 100 mg / L to 600 mg / L, 250 mg / L to 1000 mg / L, or 250 mg / L to 600 mg / L. The medium can be prepared by a method commonly used by those skilled in the art. For example, it can be prepared by adding sugars, amino acids, and other components to water. For example, the medium can also be prepared by adding missing components to a basal medium that can be purchased or easily prepared by those skilled in the art, such as RPMI-1640 medium (Roswell Park Memorial Institute-1640 medium) or DMEM medium (Dulbecco's Modified Eagle Medium). The prepared medium may be sterilized using an autoclave or the like before use in the contact step. In a specific embodiment, the medium may be RPMI-1640 medium.

[0070] The culture in the pretreatment step may be carried out under an environment in which those skilled in the art normally culture cells, and in a specific embodiment, the culture may be carried out in an air atmosphere at 37°C, 5% CO2, and 100% humidity. pDCs are usually floating cells, but the pretreatment step causes them to take on an adherent cell-like appearance. Therefore, the pretreatment step is preferably carried out using a culture vessel to which pDCs can adhere. Examples of culture vessels to which pDCs can adhere include cell-adhesive well plates (e.g., 96-well microplates) and glass-bottom dishes. The concentration of pDCs in the pretreatment step is not particularly limited, and for example, the concentration before pDC adhesion can be 0.1 x 10 6 cells / mL or more 10.0×10 6 cells / mL or less, and in one particular embodiment, 5.0 x 10 5 It may also be cells / mL.

[0071] The culture time in the pretreatment step can be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, or 8 hours or more, and can be, for example, 72 hours or less, 48 ​​hours or less, 36 hours or less, 24 hours or less, 16 hours or less, 12 hours or less, 10 hours or less, or 8 hours or less. These upper and lower limits can be combined arbitrarily, and in a specific embodiment, the culture time in the pretreatment step can be 16 hours.

[0072] The medium used in the pDC contacting step can be the same as that used in the pretreatment step. The medium used in the pDC contacting step may have the same or different composition as the medium used in the pretreatment step, and in a specific embodiment, may have the same composition.

[0073] The ratio of the number of pDCs that phagocytosed bacteria to the total number of contacted pDCs is not particularly limited and can be evaluated according to methods that those skilled in the art may use to evaluate cell populations. Such evaluation methods may, for example, be a method of evaluating cells contained in a cell population individually or a method of evaluating cells contained in a cell population as a bulk, and in one embodiment, may be a method of evaluating cells contained in a cell population individually. In a method of evaluating cells contained in a cell population individually, for example, the ratio of the number of particles or solutions in which a bacterial signal is also detected to the number of particles or solutions in which a pDC-derived signal is detected can be evaluated as the ratio of the number of pDCs that phagocytosed bacteria to the total number of contacted pDCs. The pDC-derived signal and the bacterial-derived signal may be, for example, scattered light or fluorescence. The pDC-derived signal may be, for example, scattered light, and in a specific embodiment, may be forward scattered light (FSC) and side scattered light (SSC). The bacterial-derived signal may be, for example, a signal detected from bacteria or a protein contained therein, or a signal detected from nucleic acid amplified using DNA contained in bacteria as a template, and in one embodiment, may be, for example, a signal detected from bacteria or a protein contained therein.

[0074] In a preferred embodiment, the method for evaluating each cell contained in the cell population may be flow cytometry. That is, in a preferred embodiment, the ratio of the number of pDCs that have phagocytosed bacteria to the total number of contacted pDCs may be a ratio determined by flow cytometry.

[0075] Specifically, the ratio of the number of pDCs that phagocytosed bacteria to the total number of contacted pDCs may be determined by flow cytometry, for example, using the method described below. Even more specifically, the ratio of the number of pDCs that phagocytosed bacteria to the total number of contacted pDCs may be determined, for example, using the method described in Example 8 of the present application. First, the bacteria used in the pDC contact step are fluorescently labeled by contacting the bacteria with a fluorescent dye or by genetically expressing a fluorescent protein in a protein contained in the bacteria. Next, after the pDC contacting step, if necessary, dead cells contained in the pDCs are fluorescently labeled by contacting them with a fluorescent dye. The fluorescent dye used to label dead cells is not particularly limited as long as it is one commonly used by those skilled in the art, and in a specific embodiment, it may be 7-Amino-Actinomycin D (7-AAD). Note that the fluorescent label of the bacteria or proteins contained therein and the fluorescent label of the dead cells can be detected and sorted from each other. Finally, the presence or absence of bacterial phagocytosis by pDCs is evaluated using a flow cytometer. Measurements are performed according to standard methods. Evaluation of the measurement results is not particularly limited, but for example, first, clusters containing pDCs are selected using scattered light as an indicator, and then, if necessary, clusters containing live pDCs are selected from these as particles that are not labeled as dead cells. Finally, the proportion of particles in the clusters that emit fluorescence from the fluorescent dye that labeled the bacteria is evaluated.

[0076] In a specific aspect, the composition according to this embodiment may contain Lactococcus lactis subsp. lactis JCM 5805 as the bacterium, and the Lactococcus lactis subsp. lactis JCM 5805 may satisfy the following requirement Y0. Note that the "proportion of the number of pDCs that phagocytose the bacteria relative to the total number of contacted pDCs" below corresponds to the "phagocytosis rate" evaluated in Example 9 described below. In one aspect, the pDCs satisfying requirement Y0 may be CAL-1 cells (FERM BP-10914). The composition according to this embodiment is not limited to immunostimulatory use, but the compositions described in this embodiment can also be used, and the composition according to this embodiment may also be, for example, an immunostimulatory composition. [Requirement Y0] Final concentration 2.0×10 5After contacting pDCs at 1000 cells / mL with Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed Lactococcus lactis subsp. lactis JCM 5805 to the total number of contacted pDCs is 15.0% or more, provided that the pDCs are pDCs that have been previously cultured in a serum-free medium.

[0077] The specified percentage for requirement Y0 may be, for example, 10.0% or more, 10.5% or more, 11.0% or more, 11.5% or more, 12.0% or more, 12.5% ​​or more, 13.0% or more, 13.5% or more, 14.0% or more, 14.5% or more, 15.0% or more, 15.5% or more, 16.0% or more, 16.5% or more, 17.0% or more, or 17.5% or more, or may be less than 100%, 50.0% or less, 25.0% or less, or 18.0% or less.These lower limit values ​​and upper limit values ​​can be combined arbitrarily. For example, the predetermined ratio related to requirement Y0 is 10.0% or more and less than 100%, 10.0% or more and less than 50.0%, 10.0% or more and less than 25.0%, 10.0% or more and less than 18.0%, 10.5% or more and less than 100%, 10.5% or more and less than 50.0%, 10.5% or more and less than 25.0%, 10.5% or more and less than 18.0%, 11.0% or more and less than 100%, 11.0% or more and less than 50.0%, 11.0% or more and less than 25.0%, 11.0% or more and less than 18.0%, 11.5% or more and less than 100%, 11.5% or more and less than 50 .0% or less, 11.5% to 25.0% or less, 11.5% to 18.0% or less, 12.0% to less than 100%, 12.0% to 50.0% or less, 12.0% to 25.0% or less, 12.0% to 18.0% or less, 12.5% ​​to less than 100%, 12.5% ​​to 50.0% or less, 12.5% ​​to 25.0% or less, 12.5% ​​to 18.0% or less, 13.0% to less than 100%, 13.0% to 50.0% or less, 13.0% to 25.0% or less, 13.0% to 18.0% or less, 13.5% to less than 100%, 13.5% to 50.0% or less, 13.5% to 2 5.0% or less, 13.5% to 18.0% or less, 14.0% to less than 100%, 14.0% to 50.0% or less, 14.0% to 25.0% or less, 14.0% to 18.0% or less, 14.5% to less than 100%, 14.5% to 50.0% or less, 14.5% to 25.0% or less, 14.5% to 18.0% or less, 15.0% to less than 100%, 15.0% to 50.0% or less, 15.0% to 25.0% or less, 15.0% to 18.0% or less, 15.5% to less than 100%, 15.5% to 50.0% or less, 15.5% to 25.0% or less, 15.5% or more It may be 18.0% or less, 16.0% or more and less than 100%, 16.0% or more and 50.0% or less, 16.0% or more and 25.0% or less, 16.0% or more and 18.0% or less, 16.5% or more and less than 100%, 16.5% or more and 50.0% or less, 16.5% or more and 25.0% or less, 16.5% or more and 18.0% or less, 17.0% or more and less than 100%, 17.0% or more and 50.0% or less, 17.0% or more and 25.0% or less, 17.0% or more and 18.0% or less, 17.5% or more and less than 100%, 17.5% or more and 50.0% or less, 17.5% or more and 25.0% or less, or 17.5% or more and 18.0% or less.When the predetermined ratio according to requirement Y0 is equal to or greater than the above-mentioned lower limit, the immunostimulatory ability of the composition is likely to be higher than when the ratio is below the above-mentioned lower limit. The predetermined ratio according to requirement Y0 can be evaluated according to the above-mentioned method for evaluating the predetermined ratio according to requirement X0.

[0078] Lactococcus lactis subsp. lactis JCM 5805 that satisfies requirement Y0 can be prepared, for example, according to a production method according to one aspect of the second embodiment described below.

[0079] The bacterium according to one aspect of this embodiment has the property of binding to BDCA2 (blood dendritic cell antigen 2).

[0080] BDCA2 is a type of receptor protein and is mainly expressed in plasmacytoid dendritic cells (pDCs). In one aspect of this embodiment, the bacterium according to this embodiment may be a bacterium that selectively binds to BDCA2. In one aspect of this embodiment, the bacterium according to this embodiment may be a bacterium that specifically binds to BDCA2. "Bacteria that bind to BDCA2" means that their BDCA2-binding ability is greater than the BDCA2-binding ability of Lactococcus lactis subsp. lactis ATCC15577. "Selective binding of bacteria to BDCA2" means that the bacteria bind with significant preference to BDCA2 in a contaminated system containing BDCA2 (e.g., a system containing various cell surface proteins), for example, the binding efficiency to pDCs expressing BDCA2 on their cell surface is statistically significantly greater (e.g., a p-value between the two groups is less than 0.05) than the binding efficiency to pDCs not expressing BDCA2 or pDCs whose cell surface BDCA2 has been blocked with an anti-BDCA2 antibody or the like. "Specific binding of bacteria to BDCA2" means that the bacteria bind only to BDCA2, excluding nonspecific adsorption, in a contaminated system containing BDCA2 (e.g., a system containing various cell surface proteins), for example, the binding efficiency to pDCs expressing BDCA2 on their cell surface is statistically significantly greater (e.g., a p-value between the two groups is less than 0.01) than the binding efficiency to pDCs not expressing BDCA2 or pDCs whose cell surface BDCA2 has been blocked with an anti-BDCA2 antibody or the like.

[0081] The binding of bacteria to BDCA2 can be evaluated by methods commonly used by those skilled in the art, such as binding assays. The binding assay may be an in vitro method that allows the binding of bacteria to BDCA2 to be evaluated based on a predetermined detection index. The in vitro method may be a cell-free in vitro method. The BDCA2 used in the binding assay may be BDCA2 present in a free state in an aqueous medium, BDCA2 expressed in a cell line, or BDCA2 expressed in an artificial lipid bilayer membrane. In a preferred embodiment, BDCA2 present in a free state in an aqueous medium may be purified. Such BDCA2 may be commercially available or may be isolated and purified after expression in host cells using a vector or the like. Such BDCA2 may be a chimeric protein tagged with an isolation tag, such as a Flag tag.

[0082] The bacterium according to this embodiment may have a BDCA2-binding ability that is 0.3-fold or more, 0.5-fold or more, 0.7-fold or more, 0.9-fold or more, or 1.0-fold or more that of Lactococcus lactis subsp. lactis JCM 5805. The bacterium according to this embodiment may have a BDCA2-binding ability that is more than 1.0-fold, 1.2-fold or more, 1.5-fold or more, 2.0-fold or more, 2.5-fold or more, 3.0-fold or more, 5.0-fold or more, 6.0-fold or more, 10.0-fold or more, 20.0-fold or more, or 30.0-fold or more that of Lactococcus lactis subsp. lactis ATCC 15577.

[0083] In these cases, the binding ability to BDCA2 is not particularly limited as long as it is an index value showing the binding ability, and can be, for example, IC 50The index may be a value of an index commonly used by those skilled in the art to evaluate the binding ability of two substances, such as the above, or may be a value of an index of binding between bacteria and BDCA2 evaluated in a binding assay of one embodiment described below. In a specific embodiment, the binding ability to BDCA2 may be the proportion of bacteria bound to BDCA2 evaluated in a binding assay of one embodiment described below. When the index of BDCA2 binding ability is the proportion of bacteria bound to BDCA2, for example, bacteria in which 3% or more, 5% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 90% or more, or 95% or more of the bacteria are bound to BDCA2 may be determined to be bacteria that bind to BDCA2.

[0084] Furthermore, when the percentage of bacteria bound to BDCA2 in a binding assay of one embodiment described below is evaluated as the binding ability to BDCA2, the difference when subtracting the BDCA2 binding ability of Lactococcus lactis subsp. lactis ATCC 15577 from the BDCA2 binding ability of the bacterium of this embodiment may be 0.5% or more, 1.0% or more, 1.5% or more, 2.0% or more, 4.0% or more, 5.0% or more, 7.0% or more, 9.0% or more, 10.0% or more, 15.0% or more, 20.0% or more, 25.0% or more, 30.0% or more, or 40.0% or more.

[0085] One embodiment of a binding assay for evaluating the binding of bacteria to BDCA2 is described below. One embodiment of the binding assay includes a step of contacting BDCA2 with bacteria (contacting step) and a step of evaluating the binding of BDCA2 to bacteria after the contacting step (evaluating step).

[0086] In the contacting step, bacteria and BDCA2 are contacted in an aqueous medium. The aqueous medium may be a buffer that those skilled in the art may use in in vitro methods using proteins, such as PBS (phosphate-buffered saline), HBSS (Hank's balanced salt solution), HEPES buffer, Tris buffer, or media with appropriately adjusted salt concentrations and pH. The aqueous medium may further contain additives that those skilled in the art may use in in vitro methods using proteins, such as surfactants, organic solvents (e.g., dimethyl sulfoxide and ethanol), and reducing agents (e.g., glutathione). The BDCA2 used in the contacting step may be BDCA2 present in a free state in the aqueous medium.

[0087] The concentration of bacteria in the contact step is not particularly limited as long as the binding of BDCA2 to the bacteria can be evaluated in the subsequent contact step. In one embodiment, the concentration of bacteria in the contact step may be 0.01 μg / mL or more, 0.1 μg / mL or more, 1.0 μg / mL or more, or 3.0 μg / mL or more, or 10,000 μg / mL or less, 1,000 μg / mL or less, 100 μg / mL or less, or 30 μg / mL or less. In one embodiment, these upper and lower limits can be combined in any desired manner. For example, the concentration of bacteria in the contact step can be 0.01 μg / mL or more and 10,000 μg / mL or less, 0.01 μg / mL or more and 1,000 μg / mL or less, 0.01 μg / mL or more and 100 μg / mL or less, 0.01 μg / mL or more and 30 μg / mL or less, 0.1 μg / mL or more and 10,000 μg / mL or less, 0.1 μg / mL or more and 1,000 μg / mL or less, 0.1 μg / mL or more and 100 μg / mL or less. In a particular embodiment, the concentration of the bacteria in the contacting step may be 10 μg / mL or less, 0.1 μg / mL to 30 μg / mL, 1.0 μg / mL to 10,000 μg / mL, 1.0 μg / mL to 1,000 μg / mL, 1.0 μg / mL to 100 μg / mL, 1.0 μg / mL to 30 μg / mL, 3.0 μg / mL to 10,000 μg / mL, 3.0 μg / mL to 1,000 μg / mL, 3.0 μg / mL to 1000 μg / mL, or 3.0 μg / mL to 30 μg / mL.

[0088] The concentration of BDCA2 in the contact step is not particularly limited, as long as the binding of BDCA2 to bacteria can be evaluated in the subsequent contact step. In one embodiment, the concentration of BDCA2 may be 0.01 μg / mL or more, 0.1 μg / mL or more, or 0.3 μg / mL or more, or 5000 μg / mL or less, 2000 μg / mL or less, 1000 μg / mL or less, 500 μg / mL or less, or 200 μg / mL or less. In one embodiment, these upper and lower limits can be arbitrarily combined. For example, the concentration of BDCA2 can be 0.01 μg / mL or more and 5000 μg / mL or less, 0.01 μg / mL or more and 2000 μg / mL or less, 0.01 μg / mL or more and 1000 μg / mL or less, 0.01 μg / mL or more and 500 μg / mL or less, 0.01 μg / mL or more and 200 μg / mL or less, 0.1 μg / mL or more and 50 ... In one particular embodiment, the concentration of the bacteria in the contacting step may be 10 μg / mL or more.

[0089] The concentration ratio of bacteria to BDCA2 in the contact step is not particularly limited, as long as the binding between BDCA2 and bacteria can be evaluated in the subsequent contact step. In one embodiment, the BDCA2 concentration [μg / mL] in the contact step may be 0.01-fold or more, 0.03-fold or more, 0.1-fold or more, or 0.3-fold or more, or 100-fold or less, 30-fold or less, 10-fold or less, or 3-fold or less, of the bacteria concentration [μg / mL]. In one embodiment, these upper and lower limits can be combined in any way. For example, the BDCA2 concentration [μg / mL] in the contact step can be 0.01 to 100 times, 0.01 to 30 times, 0.01 to 10 times, 0.01 to 3 times, 0.03 to 100 times, 0.03 to 30 times, 0.03 to 10 times, 0.03 to 3 times, 0.1 to 100 times, 0.1 to 30 times, 0.1 to 10 times, 0.1 to 3 times, or 0.3 to 100 times, 0.3 to 30 times, 0.3 to 10 times, or 0.3 to 3 times the bacterial concentration [μg / mL]. In one particular embodiment, the concentration [μg / mL] of BDCA2 in the contacting step may be 1.0 times the concentration [μg / mL] of the bacteria.

[0090] The contact conditions, such as time and temperature, in the contact step can be determined by those skilled in the art as appropriate, so long as they allow for evaluation of the binding between BDCA2 and bacteria in the subsequent contact step. For example, the contact time in the contact step is not particularly limited, as long as it allows for evaluation of the binding between BDCA2 and bacteria in the subsequent contact step. In one embodiment, the contact time in the contact step may be 1 minute or more, 10 minutes or more, or 30 minutes or more, and may be 24 hours or less, 6 hours or less, or 2 hours or less. In another embodiment, these upper and lower limits can be arbitrarily combined. For example, the contact time in the contact step may be 1 minute or more and 24 hours or less, 1 minute or more and 6 hours or less, 1 minute or more and 2 hours or less, 10 minutes or more and 24 hours or less, 10 minutes or more and 6 hours or less, 10 minutes or more and 2 hours or less, 30 minutes or more and 24 hours or less, 30 minutes or more and 6 hours or less, or 30 minutes or more and 2 hours or less. In a specific embodiment, the contact time in the contact step may be 1 hour. A more specific example of the contact step is a step according to the BDCA2 binding assay described in the Examples of the present specification.

[0091] In the evaluation step, the binding between BDCA2 and bacteria after the contact step is evaluated. The method for evaluating binding in the evaluation step is not particularly limited as long as it can evaluate the binding between BDCA2 and bacteria, and may be, for example, flow cytometry, ELISA, radioimmunoassay, surface plasmon resonance, or isothermal titration calorimetry. In a specific embodiment, the method for evaluating binding in the evaluation step may be flow cytometry. When evaluating the binding between BDCA2 and bacteria by flow cytometry, for example, BDCA2 and bacteria can be labeled with fluorescent dyes of different colors, and the binding between BDCA2 and bacteria can be evaluated based on the fluorescence intensity at the wavelengths corresponding to the fluorescent dyes. BDCA2 and bacteria may be labeled before the contact step, or they may be labeled after the contact step and before the evaluation step using a BDCA2-selective or bacteria-selective labeling method (e.g., labeling using a protein tag, labeling using a specific antibody, or labeling with a secondary antibody selective for the bacterial origin).

[0092] The index of the binding between BDCA2 and bacteria evaluated in the evaluation step is not particularly limited as long as it is an index value showing the binding ability, and can be, for example, IC 50 The index may be a value of an index commonly used by those skilled in the art to evaluate the binding ability of a substance (e.g., a percentage of bacteria bound to BDCA2, or the average or median number of labeled BDCA2 molecules per bacterium). In a specific embodiment, the index of BDCA2-bacterial binding evaluated in the evaluation step may be the percentage of bacteria bound to BDCA2. The percentage of bacteria bound to BDCA2 can be calculated, for example, as the percentage of particles in which a certain level of fluorescence intensity at the wavelength corresponding to the fluorescent dye labeling BDCA2 is observed among particles in which fluorescence at the wavelength corresponding to the fluorescent dye labeling the bacteria is observed. For example, when using a BDCA2 chimeric protein bound to an Fc region, a threshold can be set so that the percentage of bacteria bound to a control having only the Fc region is 0.1% or less.

[0093] Thus, one embodiment of the binding assay can evaluate the binding ability of bacteria to BDCA2. Furthermore, the present inventors have discovered that bacteria that bind to BDCA2 have immunostimulatory activity. Therefore, using binding to BDCA2 as an indicator, it is possible to evaluate the immunostimulatory activity of bacteria and screen for bacteria with immunostimulatory activity, and such an indicator can be obtained by a binding assay. That is, one embodiment of the present invention is a method for screening for bacteria with immunostimulatory activity, which includes a step of screening using binding to BDCA2 as an indicator. In one embodiment, the screening step is carried out by a binding assay between candidate bacteria and BDCA2. The candidate bacteria can be the same as the bacteria of one embodiment of the present invention. The binding assay is as described above.

[0094] The bacteria according to this embodiment (i.e., bacteria that are phagocytosed by pDCs and / or bacteria that bind to BDCA2) may be bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to BDCA2.

[0095] The bacterium according to this embodiment may be a bacterium in which the amount of phagocytosis mediated by pDC with BDCA2 is at least 0.1-fold, at least 0.3-fold, at least 0.5-fold, at least 0.7-fold, at least 0.9-fold, or at least 1.0-fold greater than the amount of phagocytosis mediated by pDC with BDCA2 of Lactococcus lactis subsp. lactis JCM 5805. Furthermore, the bacterium according to this embodiment may be a bacterium in which the amount of phagocytosis mediated by pDC with BDCA2 is more than 1.0-fold, 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 7.0-fold, 10-fold, 20-fold, 30-fold, 40-fold, 60-fold, or 100-fold greater than the amount of phagocytosis mediated by pDC with BDCA2 of Lactococcus lactis subsp. lactis ATCC 15577.

[0096] Furthermore, when the amount of phagocytosis of the bacteria according to this embodiment via the binding of BDCA2 by pDC is evaluated by the evaluation method described below, the amount of phagocytosis of the bacteria according to this embodiment via the binding of BDCA2 by pDC is evaluated by the evaluation method described below. 2 / image or more, 20μm 2 / image or more, 30μm 2 / image or more, 50μm 2 / image or more, 100μm 2 / image or more, 200μm 2 / image or more, 400μm 2 / image or more, 500μm 2 / image or more, 600μm 2 / image or more, 700μm 2 / image or more, 800μm 2 / image or more, or 900μm 2As described above in (Evaluation of phagocytosis amount), the area per image is 0.572 mm 2 is set to

[0097] Furthermore, the difference between the amount of phagocytosis by pDC of the above bacteria mediated by BDCA2 binding and the amount of phagocytosis by pDC of Lactococcus lactis subsp. lactis ATCC 15577 mediated by BDCA2 binding was 1 μm 2 / image or more, 20μm 2 / image or more, 40μm 2 / image or more, 70μm 2 / image or more, 100μm 2 / image or more, 140μm 2 / image or more, 200μm 2 / image or more, 300μm 2 / image or more, 400μm 2 / image or more, 500μm 2 / image or more, 600μm 2 / image or more, 700μm 2 / image or more or 900μm 2 As described above in (Evaluation of phagocytosis amount), the area per image is 0.572 mm 2 is set to

[0098] In one embodiment, phagocytosis of bacteria by pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of phagocytosis mediated by BDCA2 binding may be a predetermined factor or more greater than the amount of phagocytosis not mediated by BDCA2 binding. This predetermined factor may be, for example, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold. Furthermore, with respect to the phagocytosis of bacteria by pDCs, the amount of phagocytosis mediated by BDCA2 binding may account for, for example, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of the total amount of bacteria phagocytosis. Bacteria whose phagocytosis rate via binding to BDCA2 is at least a certain multiple of the phagocytosis rate without binding to BDCA2 are more likely to be phagocytosed by pDCs via binding to BDCA2, and therefore have high pDC activation and immunostimulatory potential.

[0099] In these cases, the amount of phagocytosis by pDCs via binding to BDCA2 is not particularly limited as long as it is an index value indicating the amount of phagocytosis by pDCs via binding to BDCA2. For example, the amount of phagocytosis by pDCs via binding to BDCA2 and the amount of phagocytosis not mediated by binding to BDCA2 can be evaluated as follows. First, the amount of bacteria phagocytosis by untreated pDCs (phagocytosis amount P) is evaluated. Next, the amount of bacteria phagocytosis by pDCs treated to suppress BDCA2 in a state capable of binding to a ligand on the cell surface (phagocytosis amount Q) is evaluated. In this way, the phagocytosis amount Q represents the amount of phagocytosis by pDCs not mediated by binding to BDCA2. Furthermore, the value obtained by subtracting the phagocytosis amount Q from the phagocytosis amount P represents the amount of phagocytosis by pDCs via binding to BDCA2. That is, in one embodiment, the amount of phagocytosis not mediated by binding to BDCA2 may be the amount of bacteria phagocytosis by pDCs in a state in which BDCA2 in the pDCs cannot bind to a ligand.

[0100] Treatment to suppress BDCA2 in a state capable of binding to its ligand may involve, for example, exposing pDCs to an anti-BDCA2 antibody (blocking), exposing pDCs to a BDCA ligand (competitive inhibition), knocking down or knocking out BDCA2 in pDCs using conventional methods, or introducing a mutation into BDCA2 in pDCs using conventional methods to eliminate its ability to bind to its ligand. In a preferred embodiment, treatment to suppress BDCA2 in a state capable of binding to its ligand may involve exposing pDCs to an anti-BDCA2 antibody. In this case, the state in which BDCA2 on the cell surface of pDCs is bound to the anti-BDCA2 antibody corresponds to the state in which BDCA2 in pDCs is unable to bind to its ligand. In these cases, the anti-BDCA2 antibody may be a monoclonal or polyclonal antibody, and in a preferred embodiment, it may be a monoclonal antibody. For example, anti-BDCA2 antibodies are available from suppliers such as Miltenyi Biotec (product number: 130-090-690).

[0101] The amount of phagocytosis by pDCs via binding to BDCA2 may be, for example, the amount of phagocytosis evaluated by the method described below as "Method for evaluating the amount of phagocytosis via binding to BDCA2 and the amount of phagocytosis not mediated by binding to BDCA2." As a more detailed example, the amount of phagocytosis by pDCs via binding to BDCA2 may be the amount of phagocytosis evaluated by the method described in the Examples. [Method for assessing the amount of phagocytosis mediated by BDCA2 binding and the amount of phagocytosis not mediated by BDCA2 binding] Similar to the phagocytosis experiment described above [Evaluation method using bacteria labeled with a fluorescent dye], CAL-1 cell suspensions were seeded in each well. Bacteria and anti-BDCA2 antibody (Miltenyi Biotec, 130-090-690), or bacteria and an isotype control (Miltenyi Biotec, 130-106-545) of the same isotype and subclass as the anti-BDCA2 antibody, were added to each well at a concentration of 10 μg / mL, and the cells were incubated for 30 minutes. Then, similar to the evaluation of phagocytosis amount described above [Evaluation method using bacteria labeled with a fluorescent dye], the fluorescence intensity of pHrodo Red SE was measured, and the fluorescence detection area per image (e.g., μm) was calculated. 2 The fluorescence detection area when anti-BDCA2 antibody was added was defined as the amount of phagocytosis Q, and the fluorescence detection area when an isotype control was added was defined as the amount of phagocytosis P. The amount of phagocytosis mediated by binding to BDCA2 (PQ) and the amount of phagocytosis not mediated by binding to BDCA2 (P) were then evaluated.

[0102] In the immunostimulating composition of this embodiment, the bacteria may be live or dead, or may be a mixture of live and dead bacteria. The bacteria in the immunostimulating composition of this embodiment may also be dead bacteria.

[0103] The bacteria contained in the immunostimulatory composition of this embodiment have high immunostimulatory activity, which may be at least 0.01-fold, at least 0.03-fold, at least 0.05-fold, at least 0.07-fold, at least 0.09-fold, at least 0.1-fold, at least 0.3-fold, at least 0.5-fold, at least 0.7-fold, at least 0.9-fold, or at least 1.0-fold that of pDCs of Lactococcus lactis subsp. lactis JCM 5805. Furthermore, the immunostimulatory activity of the bacterium contained in the immunostimulatory composition of this embodiment may be more than 1.0-fold, 1.2-fold or more, 1.5-fold or more, 2.0-fold or more, 3.0-fold or more, 4.0-fold or more, 5.0-fold or more, 7.0-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 60-fold or more, or 100-fold or more than the immunostimulatory activity of Lactococcus lactis subsp. lactis ATCC 15577. In these cases, the immunostimulatory activity is not particularly limited as long as it is a parameter that a person skilled in the art may use as an index of immunostimulatory activity, and in a preferred embodiment, it may be the expression level of IFN-α.

[0104] As used herein, the term "immunostimulatory ability" refers to the ability to stimulate cellular immunity and / or humoral immunity, and refers to the ability of the bacteria according to this embodiment to stimulate (activate) the innate immune system in cells contacted with the bacteria or in animals administered with the bacteria. In one aspect, the immunostimulatory ability may be the ability to activate dendritic cells, which activate the innate immune system of dendritic cells, and more specifically, the ability to activate plasmacytoid dendritic cells (pDCs). In one aspect, the immunostimulatory ability may be an increase in the expression level of cytokines or cell surface markers secreted from dendritic cells, preferably an increase in the expression level of IFN-α, or an increase in the expression level of IFN-α production by pDCs. In vivo, pDCs are the main producers of IFN-α, and IFN-α is a type of cytokine responsible for the host defense mechanism via the innate immune system. Therefore, a substance capable of promoting IFN-α expression by pDCs can be said to be a substance having dendritic cell activation ability and pDC activation ability, or a substance having immunostimulatory ability.

[0105] For example, the immunostimulatory activity of bacteria can be evaluated using the amount of IFN-α expressed by pDCs after contact with the bacteria as an indicator. The amount of IFN-α expressed by pDCs is the amount of a biomolecule that can serve as an indicator for evaluating the amount of IFN-α expressed by pDCs due to the bacteria. In one embodiment, the amount of IFN-α expressed may be the amount of IFN-α secreted extracellularly and / or the amount of IFN-α in pDCs, or the amount of mRNA encoding IFN-α in pDCs. In a preferred embodiment, the amount of IFN-α expressed may be measured by the ELISPOT method, in which extracellularly secreted IFN-α is adsorbed to a cell culture well and measured, or the amount of IFN-α secreted extracellularly and present in the culture supernatant. In these cases, the culture supernatant is the medium used after contacting bacteria with pDCs in the contact step. The amount of IFN-α in the culture supernatant can be measured after collecting the culture supernatant using a method commonly used by those skilled in the art to quantify a specific protein. The amount of IFN-α in pDCs (e.g., in CAL-1 cells) can be measured by methods commonly used by those skilled in the art, such as intracellular cytokine staining using a flow cytometer, or by preparing pDC lysates and then measuring the amount of IFN-α in the lysates using methods commonly used by those skilled in the art to quantify specific proteins. In these cases, the amount of IFN-α can be measured by, for example, ELISA or Western blotting, or by ELISA. The amount of IFN-α measured by ELISA can be measured using, for example, the Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science). The amount of mRNA encoding IFN-α in pDCs (e.g., in CAL-1 cells) can be measured by preparing pDC lysates using methods commonly used by those skilled in the art and then measuring the amount of mRNA encoding IFN-α in the lysates using methods commonly used by those skilled in the art to quantify mRNA. Quantification of mRNA may be carried out by, for example, quantitative PCR (q-PCR) such as real-time PCR, direct digital counting (for example, nCounter (registered trademark)), or a method using a next-generation sequencer (NGS).

[0106] When the immunostimulatory ability of bacteria is evaluated using the amount of IFN-α expressed by pDCs after contact with the bacteria as an indicator, the indicator may be the amount of IFN-α expressed in pDCs (e.g., CAL-1 cells) a predetermined time (e.g., 3 to 168 hours, e.g., 22 hours) after contacting the bacteria with the bacteria in serum-free medium (e.g., RPMI-1640 medium (Sigma, R8758) supplemented with penicillin / streptomycin (Gibco, 15140-12) at a final concentration of 1.0% by volume). Preferably, the indicator may be the amount of IFN-α secreted extracellularly and present in the culture supernatant after treatment under the same conditions. As a more specific example, the indicator of the immunostimulatory ability of bacteria may be the amount of IFN-α in the culture supernatant, treated and measured in the same manner as in the Examples.

[0107] In one aspect, when the bacterium according to the present embodiment is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to BDCA2, the bacterium according to the present embodiment may promote IFN-α production in pDCs via phagocytosis by pDCs. That is, the bacterium according to the present embodiment may promote IFN-α production in pDCs via binding to BDCA2 followed by phagocytosis by pDCs.

[0108] The ability of bacteria to promote IFN-α production in pDCs through binding to BDCA2 followed by phagocytosis by pDCs can be determined by observing that the amount of IFN-α produced by pDCs exposed to BDCA2 after treatment to suppress ligand-capable BDCA2 is less than the amount of IFN-α produced by pDCs not subjected to such treatment. The bacteria of this embodiment may be bacteria that produce IFN-α by pDCs exposed to BDCA2 after treatment to suppress ligand-capable BDCA2, at most 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10 times the amount of IFN-α produced by pDCs not subjected to such treatment. In these cases, the treatment to suppress ligand-capable BDCA2 and the amount of IFN-α produced are as described above.

[0109] The promotion of IFN-α production in pDCs through phagocytosis by pDCs following binding of bacteria to BDCA2 may be such that the fold change in the amount of IFN-α production relative to the fold change in the amount of phagocytosis mediated by BDCA2 in pDCs is greater than 0, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1.0 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, or 2.0 or more. In the above case, a fold change in the amount of IFN-α production relative to the fold change in the amount of BDCA2-mediated phagocytosis in pDC of X-fold or more means that when the amount of BDCA2-mediated phagocytosis in pDC increases by Y%, the amount of IFN-α production in pDC increases by X×Y% or more, and / or when the amount of BDCA2-mediated phagocytosis in pDC decreases by Z%, the amount of IFN-α production in pDC decreases by X×Z% or more.

[0110] The immunostimulatory composition of this embodiment may be a food composition, pharmaceutical composition, quasi-drug, bacterial bulk powder (such as a powder of dried bacterial cells or a powder containing the same), additive, or feed, and is preferably a food composition. Immunostimulatory ability (immunostimulatory ability) refers to the ability to stimulate (activate) the innate immune system of cells or living organisms. The immunostimulatory composition according to one embodiment may be a composition for activating dendritic cells, or a composition for activating plasmacytoid dendritic cells (pDCs). The immunostimulatory composition of this embodiment can be produced as described below.

[0111] The content of the bacteria according to this embodiment is not particularly limited as long as it is an amount that satisfies the effective immunostimulatory amount, and therefore may vary depending on the form of the composition for immunostimulation of this embodiment. For example, the dry weight of the bacteria according to this embodiment relative to the total dry weight of the composition for immunostimulation of this 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% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% by mass or more, 10% by mass or more, 11% by mass or more, 12% by mass or more, 13% by mass or more, 14% by mass or more, 15% by mass or more, 16% by mass or more, 17% by mass or more, 18% by mass or more, 19% by mass or more, 20% by mass or more, 21% by mass or more, 22% by mass or more, 23% by mass or more, 24% by mass or more, 25% by mass or more, 26% by mass or more, 27% by mass or more, 28% by mass or more, 29% by mass or more, 30% by mass or more, 31% by mass or more, 32% by mass or more, 33% by mass or more, 34% by mass or more, 35% by mass or more, 36% by mass or more, May be 0% by mass or more, 7.0% by mass or more, 10.0% by mass or more, 80.0% by mass or more, 90.0% by mass or more, 95.0% by mass or more, 99.0% by mass or more, or 100% by mass, and 100% by mass or less , 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. These lower and upper limits can be combined arbitrarily. For example, the dry weight of bacteria relative to the total dry weight of the composition of this embodiment can be set to 0.0001% by mass or more and 100% by mass or less, 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, 0.0001% by mass or more and ... 10 mass% or more, 0.001 mass% or more and 100 mass% or less, 0.001 mass% or more and 95 mass% or less, 0.001 mass% or more and 90 mass% or less, 0.001 mass% or more and 80 mass% or less, 0.001 mass% or more and 70 mass% or less, 0.001 mass% or more and 60 mass% or less, 0.001 mass% or more and 50 mass% or less, 0.001 0.001 mass% to 20 mass%, 0.001 mass% to 15 mass%, 0.001 mass% to 10 mass%, 0.005 mass% to 100 mass%, 0.005 mass% to 95 mass%, 0.005 mass% to 90 mass%, 0.0.005% to 80% of mass, 0.005% to 70% of mass, 0.005% to 60% of mass, 0.005% to 50% of mass, 0.005% to 30% of mass, 0.005% to 25% of mass, 0.005% to 20% of mass, 0.005% to 15% of mass, 0.005% to 10% of mass, 0.01% to 100% of mass, 0.01% to 95% of mass, 0.01% to 90% of mass, 0.01% to 80% of mass, 0.01% to 70% of mass, 0.01% of mass... The following weight percentages are listed: ≥60% and ≤50%; ≥30% and ≤0.01%; ≥25% and ≤20%; ≥15% and ≤10%; ≥100% and ≤0.02%; ≥95% and ≤0.02%; ≥80% and ≤70%; ≥60% and ≤0.02%; ≥50% and ≤0.02%; ≥30% and ≤0.02%. Below, 0.02% mass% to 25% mass%, 0.02% mass% to 20% mass%, 0.02% mass% to 15% mass%, 0.02% mass% to 10% mass%, 0.05% mass% to 100% mass%, 0.05% mass% to 95% mass%, 0.05% mass% to 90% mass%, 0.05% mass% to 80% mass%, 0.05% mass% to 70% mass%, 0.05% mass% to 60% mass%, 0.05% mass% to 50% mass%, 0.05% mass% to 30% mass%, 0.05% mass% to 25% mass%, 0.05% mass% to 20% mass%, 0.05% mass% and above. Less than 15% by mass, 0.05% to 10% by mass, 0.10% to 100% by mass, 0.10% to 95% by mass, 0.10% to 90% by mass, 0.10% to 80% by mass, 0.10% to 70% by mass, 0.10% to 60% by mass, 0.10% to 50% by mass, 0.10% to 30% by mass, 0.10% to 25% by mass, 0.10% to 20% by mass, 0.10% to 15% by mass, 0.10% to 10% by mass, 0.30% to 100% by mass, 0.30% to 95% mass, 0.30% to 90% mass, 0.30% to 80% mass, 0.30% to 70% mass, 0.30% to 60% mass, 0.30% to 50% mass, 0.30% to 30% mass, 0.30% to 25% mass, 0.30% to 20% mass, 0.30% to 15% mass, 0.30% to 10% mass, 1.0% to 100% mass, 1.0% to 95% mass, 1.0% to 90% mass, 1.0% to 80% mass and below Below, 1.0% mass percentage to 70% mass percentage, 1.0% mass percentage to 60% mass percentage, 1.0% mass percentage to 50% mass percentage, 1.0% mass percentage to 30% mass percentage, 1.0% mass percentage to 25% mass percentage, 1.0% mass percentage to 20% mass percentage, 1.0% mass percentage to 15% mass percentage, 1.0% mass percentage to 10% mass percentage, 1.5% mass percentage to 100% mass percentage, 1.5% mass percentage to 95% mass percentage, 1.5% mass percentage to 90% mass percentage, 1.5% mass percentage to 80% mass percentage, 1.5% mass percentage to 70% mass percentage, 1.5% mass percentage to 60% mass percentage, 1.5% mass percentage to 50% mass percentage, 1.5% mass percentage 1.5% to 30% mass, 1.5% to 25% mass, 1.5% to 20% mass, 1.5% to 15% mass, 1.5% to 10% mass, 2.0% to 100% mass, 2.0% to 95% mass, 2.0% to 90% mass, 2.0% to 80% mass, 2.0% to 70% mass, 2.0% to 60% mass, 2.0% to 50% mass, 2.0% to 30% mass, 2.0% to 25% mass, 2.0% to 20% mass, 2.0% to 15% mass Below 2.0%, 2.0% to 10%, 2.5% to 100%, 2.5% to 95%, 2.5% to 90%, 2.5% to 80%, 2.5% to 70%, 2.5% to 60%, 2.5% to 50%, 2.5% to 30%, 2.5% to 25%, 2.5% to 20%, 2.5% to 15%, 2.5% to 10%, 3.0% to 100%, 3.0% to 95%, 3.0% to 90% of mass, 3.0% to 80% of mass, 3.0% to 70% of mass, 3.0% to 60% of mass, 3.0% to 50% of mass, 3.0% to 30% of mass, 3.0% to 25% of mass, 3.0% to 20% of mass, 3.0% to 15% of mass, 3.0% to 10% of mass, 5.0% to 100% of mass, 5.0% to 95% of mass, 5.0% to 90% of mass, 5.0% of mass... Quantity % or more but less than 80% of mass, 5.0% or more but less than 70% of mass, 5.0% or more but less than 60% of mass, 5.0% or more but less than 50% of mass, 5.0% or more but less than 30% of mass, 5.0% or more but less than 25% of mass, 5.0% or more but less than 20% of mass, 5.0% or more but less than 15% of mass, 5.0% or more but less than 10% of mass, 7.0% or more but less than 100% of mass, 7.0% or more but less than 95% of mass, 7.0% or more but less than 90% of mass, 7.0% or more but less than 80% of mass, 7.0% of mass Above 70% mass percentage and below, 7.0% mass percentage and above 60% mass percentage, 7.0% mass percentage and above 50% mass percentage, 7.0% mass percentage and above 30% mass percentage, 7.0% mass percentage and above 25% mass percentage, 7.0% mass percentage and above 20% mass percentage, 7.0% mass percentage and above 15% mass percentage, 7.0% mass percentage and above 10% mass percentage, 10.0% mass percentage and above 10% mass percentage, 10.0% mass percentage and above 100% mass percentage, 10.0% mass percentage and above 95% mass percentage, 10.0% mass percentage and above 90% mass percentage, 10.0% mass percentage and above 80% mass percentage, 10.0% mass percentage and above 70% mass percentage, 10. 0% or more quality, 60% or less quality, 10.0% or more quality, 50% or less quality, 10.0% or more quality, 30% or less quality, 10.0% or more quality, 25% or less quality, 10.0% or more quality, 20% or less quality, 10.0% or more quality, 15% or less quality, 80.0% or more quality, 100% or more quality, 80.0% or more quality, 95% or less quality, 90.0% or more quality, 100% or less quality, 90.0% or more quality, 95% or less quality. (Also, 95.0% or more quality, 100% or less quality.)

[0112] When the immunostimulating composition of this application form is in liquid form, the composition of this application form contains bacteria, 1.0×10 3 Cells / mL or higher, 1.0 × 10 4 Cells / mL or higher, 1.0 × 10 5Cells / mL or more, 1.0×10 6 Cells / mL or more, 1.0×10 7 cells / mL or 4.0 x 10 7 cells / mL or greater, and may be 1.0 x 10 11 Cells / mL or less, 1.0×10 10 Cells / mL or less, 3.0×10 9 cells / mL or less or 1.0 x 10 9 The number of bacteria contained in the composition according to this embodiment may be 1.0 × 10 cells / mL or less. These lower and upper limits may be combined arbitrarily and are not particularly limited. For example, the number of bacteria contained in the composition according to this embodiment may be 1.0 × 10 cells / mL or less. 3 cells / mL or more 1.0×10 11 Cells / mL or less, 1.0×10 3 cells / mL or more 1.0×10 10 Cells / mL or less, 1.0×10 3 cells / mL or more 3.0×10 9 Cells / mL or less, 1.0×10 3 cells / mL or more 1.0×10 9 Cells / mL or less, 1.0×10 4 cells / mL or more 1.0×10 11 Cells / mL or less, 1.0×10 4 cells / mL or more 1.0×10 10 Cells / mL or less, 1.0×10 4 cells / mL or more 3.0×10 9 Cells / mL or less, 1.0×10 4 cells / mL or more 1.0×10 9 Cells / mL or less, 1.0×10 5 cells / mL or more 1.0×10 11 Cells / mL or less, 1.0×10 5 cells / mL or more 1.0×10 10 Cells / mL or less, 1.0×10 5 cells / mL or more 3.0×10 9 Cells / mL or less, 1.0×10 5 cells / mL or more 1.0×10 9 Cells / mL or less, 1.0×10 6 cells / mL or more 1.0×10 11 Cells / mL or less, 1.0×10 6 cells / mL or more 1.0×1010 Cells / mL or less, 1.0×10 6 cells / mL or more 3.0×10 9 Cells / mL or less, 1.0×10 6 cells / mL or more 1.0×10 9 Cells / mL or less, 1.0×10 7 cells / mL or more 1.0×10 11 Cells / mL or less, 1.0×10 7 cells / mL or more 1.0×10 10 Cells / mL or less, 1.0×10 7 cells / mL or more 3.0×10 9 Cells / mL or less, 1.0×10 7 cells / mL or more 1.0×10 9 Cells / mL or less, 4.0×10 7 cells / mL or more 1.0×10 11 Cells / mL or less, 4.0×10 7 cells / mL or more 1.0×10 10 Cells / mL or less, 4.0×10 7 cells / mL or more 3.0×10 9 cells / mL or less or 4.0 x 10 7 cells / mL or more 1.0×10 9 In this case, the daily intake or administration amount of the liquid immunostimulating composition may be 10 mL to 1,000 mL, 10 mL to 800 mL, 10 mL to 500 mL, 10 mL to 250 mL, 30 mL to 1,000 mL, 30 mL to 800 mL, 30 mL to 500 mL, 30 mL to 250 mL, 50 mL to 1,000 mL, 50 mL to 800 mL, 50 to 500 mL, 50 mL to 250 mL, 100 mL to 1,000 mL, 100 mL to 800 mL, 100 to 500 mL, or 100 to 250 mL.

[0113] In the immunostimulatory composition of this embodiment, the number of bacteria of this embodiment contained in the composition per unit package is, for example, 1.0 × 10 4 pcs or more, 5.0×10 4 That's it, 1.0 x 10 5 pcs or more, 3.0×10 5 pcs or more, 1.0×10 6 pcs or more, 3.0×10 6pcs or more, 1.0×10 7 pcs or more, 3.0×10 7 pcs or more, 1.0×10 8 pcs or more, 3.0×10 8 pcs or more, 5.0×10 8 pcs or more, 1.0×10 9 pcs or more, 5.0×10 9 pcs or more, 1.0×10 10 pcs or more, 5.0×10 10 or more than 1.0 x 10 11 may be 1.0 x 10 or more, 14 pcs or less, 1.0×10 13 or less, or 1.0 x 10 12 These upper and lower limits can be combined in any desired manner. For example, in the immunostimulating composition of the present embodiment, the number of bacteria of the present embodiment contained in the composition per unit package can be, for example, 1.0 × 10 5 pcs or more 1.0×10 14 pcs or less, 1.0×10 5 pcs or more 1.0×10 13 pcs or less, 1.0×10 5 pcs or more 1.0×10 12 pcs or less, 3.0×10 5 pcs or more 1.0×10 14 pcs or less, 3.0×10 5 pcs or more 1.0×10 13 pcs or less, 3.0×10 5 pcs or more 1.0×10 12 pcs or less, 1.0×10 6 pcs or more 1.0×10 14 pcs or less, 1.0×10 6 pcs or more 1.0×10 13 pcs or less, 1.0×10 6 pcs or more 1.0×10 12 pcs or less, 3.0×10 6 pcs or more 1.0×10 14 pcs or less, 3.0×10 6 pcs or more 1.0×10 13 pcs or less, 3.0×10 6 pcs or more 1.0×10 12 pcs or less, 1.0×10 7 pcs or more 1.0×10 14 pcs or less, 1.0×107 More than 1.0 × 10 13 Less than 1.0 × 10 7 More than 1.0 × 10 12 Less than 3.0 × 10 7 More than 1.0 × 10 14 Less than 3.0 × 10 7 More than 1.0 × 10 13 Less than 3.0 × 10 7 More than 1.0 × 10 12 Less than 1.0 × 10 8 More than 1.0 × 10 14 Less than 1.0 × 10 8 More than 1.0 × 10 13 Less than 1.0 × 10 8 More than 1.0 × 10 12 Less than 3.0 × 10 8 More than 1.0 × 10 14 Less than 3.0 × 10 8 More than 1.0 × 10 13 Less than 3.0 × 10 8 More than 1.0 × 10 12 Less than 5.0 × 10 8 More than 1.0 × 10 14 Less than 5.0 × 10 8 More than 1.0 × 10 13 Less than 5.0 × 10 8 More than 1.0 × 10 12 Less than 1.0 × 10 9 More than 1.0 × 10 14 Less than 1.0 × 10 9 More than 1.0 × 10 13 Less than 1.0 × 10 9 More than 1.0 × 10 12 Less than 5.0 × 10 9 More than 1.0 × 10 14 Less than 5.0 × 10 9 More than 1.0 × 10 13 Less than 5.0 × 10 9 More than 1.0 × 10 12 Less than 1.0 × 10 10 More than 1.0 × 10 14 Less than 1.0 × 10 10 More than 1.0 × 10 13 Less than 1.0 × 10 10pcs or more 1.0×10 12 Less than or equal to 5.0×10 10 pcs or more 1.0×10 14 Less than or equal to 5.0×10 10 pcs or more 1.0×10 13 Less than or equal to 5.0×10 10 pcs or more 1.0×10 12 pcs or less, 1.0×10 11 pcs or more 1.0×10 14 pcs or less, 1.0×10 11 pcs or more 1.0×10 13 or less than 1.0 x 10 11 pcs or more 1.0×10 12 It may be less than one.

[0114] In terms of reducing the burden of ingestion or administration, the immunostimulating composition of this embodiment is preferably used by oral ingestion, oral administration, nasal ingestion, or nasal administration, with oral ingestion or oral administration being more preferred. When used by oral ingestion, oral administration, nasal ingestion, or nasal administration, the bacteria or a composition containing bacteria preferably has high resistance to gastric fluid, intestinal fluid, etc., and for example, strong acid resistance. The bacteria are not particularly limited, and either live or killed bacteria can be used, but killed bacteria are preferred, with heat-killed bacteria being more preferred, in terms of immunostimulating effect, stability, production efficiency, etc.

[0115] The immunostimulatory composition of this embodiment can be orally ingested by humans and non-human mammals, and a typical intake form is a food composition. The provided food composition contains an effective amount of the bacterium of this embodiment. Here, "containing an effective amount" refers to a content such that the active ingredient of the present invention is ingested to an extent that the effect of immunostimulation, etc. is exerted when ingested in an amount normally consumed in each food composition. The term "food composition" is used to include health foods, functional foods, nutritional supplements, health-promoting foods (e.g., foods for specified health uses, nutritionally functional foods, and foods with functional claims), foods for special dietary uses (e.g., foods for infants, foods for pregnant women, and foods for the sick), and supplements. It goes without saying that when the active ingredient of the present invention is ingested by mammals other than humans, the food referred to in this invention is used as feed.

[0116] Because the immunostimulating composition of this embodiment has immunostimulating effects, it can be provided by being contained in foods that are consumed daily. In this case, the immunostimulating composition of this embodiment can be provided in a unit package form in which the amount to be taken per meal is predetermined. Examples of unit package forms per meal include forms in which a fixed amount is specified, such as a pack, package, can, or bottle. In order to better exert the various effects of the immunostimulating composition of this embodiment, the intake amount per meal can be determined according to the daily intake amount of the active ingredient of the present invention, which will be described later. The food of this embodiment may be provided with instructions regarding the intake amount displayed on the package, or may be provided together with a document or the like containing the instructions.

[0117] The predetermined intake amount per meal in the unit package form may be an effective daily intake amount, or an intake amount obtained by dividing the effective daily intake amount into two or more (preferably 2 to 6) doses. Therefore, the unit package form of the immunostimulating composition of this embodiment may contain the active ingredient of the present invention at the daily intake amount described below, or may contain the active ingredient of the present invention in an amount that is one-half to one-sixth of the daily intake amount described below. For convenience of intake, the immunostimulating composition of this embodiment is preferably provided in a unit package form per meal (i.e., a unit package form per day) in which the intake amount per meal is the effective daily intake amount.

[0118] The immunostimulatory composition of the present embodiment can be administered to a subject in need of immunostimulation. The subject in need of immunostimulation is not particularly limited, but examples thereof include subjects infected with a virus, subjects suffering from a cold, and subjects aged 65 or older.

[0119] The form of the "food composition" in the immunostimulating composition of this embodiment is not particularly limited, and may be, for example, a beverage, a semi-liquid or gel form, or a solid or powder form. Examples of "supplements" include tablets prepared by kneading the active ingredient of the immunostimulating composition of this embodiment with excipients, binders, etc., followed by tableting; granules prepared by granulating the active ingredient of the present invention with excipients, binders, etc.; orally disintegrating tablets; and capsules in which the active ingredient of the present invention is encapsulated in a capsule or the like. When provided as a supplement, the supplement may be provided in the above-described unit package per meal or per day, or in a unit package per week, per two weeks, per month, or per two months. It is preferable that the latter unit package be labeled with the amount of intake per meal or per day, so that the consumer can ingest an effective amount of the active ingredient of the present invention according to the label.

[0120] Examples of food compositions provided as the immunostimulating composition of this embodiment include health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, and foods with functional claims, all of which have immunostimulating properties. Such food compositions can be labeled, for example, as being for supporting the maintenance of immune function in healthy people (immune care), for those concerned about a decline in immune function, for suppressing a decline in immune function, for those concerned about sunburn, for those concerned about skin damage in daily life, for those concerned about dry skin, for those concerned about hot flashes, for those concerned about erythema, for those concerned about redness of the skin, for those concerned about rosy faces, for those concerned about rough hands, etc.

[0121] The food composition provided as the immunostimulating composition of this embodiment is not particularly limited as long as it contains the active ingredient of the present invention, and examples thereof include soft drinks, carbonated drinks, fruit juice drinks, vegetable juice drinks, fruit and vegetable juice drinks, livestock milk such as cow's milk, soy milk, dairy drinks, drink-type yogurt, drink-type or stick-type jelly, coffee, cocoa, tea drinks, nutritional drinks, energy drinks, sports drinks, mineral water (including both sparkling and non-sparkling), near-water, non-alcoholic beer-flavored drinks and other non-alcoholic drinks; carbohydrate-containing foods and drinks such as rice, noodles, bread, or pasta; cheeses, hard or soft yogurt, livestock milk and other raw foods made from fats and oils. Dairy products such as cream and ice cream; Western sweets such as cookies, cakes, and chocolate, Japanese sweets such as manju or yokan, tablet sweets (refreshing sweets) such as ramune, candies, gum, gummy candies, frozen desserts and ice creams such as jelly or pudding, snacks, and other sweets; alcoholic beverages such as whiskey, bourbon, spirits, liqueur, wine, fruit wine, sake, Chinese sake, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, sparkling wine, other miscellaneous alcoholic drinks, and chuhai; processed foods using eggs, processed seafood or meat (including organs such as liver) products (including delicacies), processed foods such as miso soup and other soups, condiments such as miso, soy sauce, furikake, and other seasonings, or liquid foods such as concentrated liquid foods.

[0122] Tea beverages include all types of fermented tea, semi-fermented tea, and non-fermented tea, such as black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, roasted green tea, oolong tea, turmeric tea, pu-erh tea, rooibos tea, rose tea, chrysanthemum tea, ginkgo leaf tea, and herbal tea (e.g., mint tea, jasmine tea).

[0123] Examples of fruits used in fruit juice beverages and fruit and vegetable juice beverages include apples, mandarin oranges, grapes, bananas, pears, peaches, mangoes, acai, blueberries, and plums. Examples of vegetables used in vegetable juice beverages and fruit and vegetable juice beverages include tomatoes, carrots, celery, pumpkins, cucumbers, and watermelons.

[0124] When the immunostimulatory composition of this embodiment is provided as feed, it can be provided in accordance with the description regarding food products above.

[0125] When the immunostimulating composition of this embodiment is provided as a pharmaceutical composition or quasi-drug, it can be formulated into an oral or parenteral formulation. Oral formulations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, liquids, jellies, emulsions, and suspensions. Parenteral formulations include injections suitable for local administration (including intradermal, subcutaneous, intramuscular, and intravenous injections), inhalants (e.g., inhalation aerosols, inhalation powders, and inhalation liquids), nasal drops (e.g., nasal powders and nasal liquids), ointments, creams, gels, suppositories, patches, and poultices, and in one embodiment, nasal drops. These formulations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, coloring agents, stabilizers, emulsifiers, dispersing agents, suspending agents, preservatives, and the like.

[0126] When the immunostimulatory composition of this embodiment is used as a pharmaceutical composition or quasi-drug, examples of the target diseases include those already known to be suitable for Type I IFN, such as cancers including renal cancer, multiple myeloma, chronic myeloid leukemia, hairy cell leukemia, glioblastoma, medulloblastoma, astrocytoma, malignant melanoma, mycosis fungoides, and adult T-cell leukemia; viral infections including subacute sclerosing panencephalitis, HTLV-1 myelopathy, hepatitis B, and hepatitis C; bacterial infections such as chlamydia (sexually transmitted disease), mycobacterium (tuberculosis), listeria (sepsis, etc.), staphylococcus (food poisoning), and helicobacter (gastritis); and autoimmune diseases including multiple sclerosis. The immunostimulatory composition of this embodiment can be used to prevent or treat the above diseases. Furthermore, since Type I IFN is known to have the activity of inhibiting differentiation from osteoblasts to osteoclasts, the immunostimulatory composition of this embodiment can also be used to prevent or treat osteoporosis and the like.

[0127] The immunostimulatory composition of the present embodiment can be used as a vaccine by expressing an antigen corresponding to a specific disease in or on the surface of the bacteria, which is the active ingredient, or secreting it outside the bacteria using genetic engineering techniques. In particular, because the bacterial cell wall has the function of protecting the antigen from gastric acid, a heterologous antigen-expressing bacterial strain in which the antigen is expressed in or on the surface of the bacteria is suitable as a host for an oral vaccine.

[0128] The immunostimulating composition of this embodiment may be a bulk bacterial powder of the bacterium of this embodiment (such as a powder of dried bacterial cells or a powder containing the same). Such bulk bacterial powder can be obtained, for example, by appropriately culturing, sterilizing, and drying the bacteria according to a conventional manufacturing method. Furthermore, the immunostimulating composition of this embodiment may be, for example, a composition for immunostimulation produced according to a manufacturing method according to one aspect of the second embodiment.

[0129] The bacterial bulk powder of this embodiment may contain components acceptable for use as food, medicine, or feed, in addition to the bacteria of this embodiment and components derived from the medium used to culture the bacteria, and may further contain at least one component selected from the group consisting of, for example, sugars, proteins, lipids, amino acids, vitamins, minerals, flavonoids, quinones, polyphenols, nucleic acids, fatty acids, acidulants, sweeteners, colorings, fragrances, seasonings, salt, emulsifiers, stabilizers, cooling agents, binders, disintegrants, lubricants, colorings, preservatives, sustained-release regulators, surfactants, and solubilizers.

[0130] The bacterial bulk powder of this embodiment can be used as a raw material for the food composition, pharmaceutical composition, or feed of this embodiment.

[0131] When the immunostimulating composition of this embodiment is provided as an additive, it can be carried out according to the description regarding the food composition, feed, quasi-drug, pharmaceutical composition, or bacterial bulk powder above. When the immunostimulating composition of this embodiment is provided as a food additive, it can be used as a functional ingredient in a functional food product having an immunostimulating effect.

[0132] The immunostimulatory composition of this embodiment can be determined depending on the recipient's sex, age, and weight, symptoms, intake time or administration time, dosage form, intake route or administration route, and materials or drugs to be combined, etc. The daily intake or administration amount of the immunostimulatory composition of this embodiment for an adult can be determined, for example, by the number of bacteria that are the active ingredient, and is generally 1×10 8 pcs or more, 1×10 9 1 x 10 or more 10 It can be more than 1 x 10 14 pcs or less, 1×10 13 1 x 10 or less 12 These upper and lower limits can be arbitrarily combined, and the range of the intake or administration amount can be, for example, 1 × 10 8 ~1×10 14 pieces, 1×10 9 ~1×10 14 1 x 1010 ~1×10 14 pieces, 1×10 8 ~1×10 13 pieces, 1×10 9 ~1×10 13 pieces, 1×10 10 ~1×10 13 pieces, 1×10 8 ~1×10 12 pieces, 1×10 9 ~1×10 12 1 x 10 10 ~1×10 12 The number of bacteria can be measured using a known microscope, flow cytometer, or non-culture rapid microorganism testing device (for example, ELESTA PixeeMo (AFI Technology Co., Ltd.)), but measurement using a microscope is preferred from the viewpoint of versatility. The subject to which the composition according to this embodiment is administered or ingested may be a human and / or a non-human mammal, and may also be a human.

[0133] The daily intake or administration amount of the immunostimulatory composition of this embodiment for an adult can also be determined by the dry cell mass of the bacterium that is the active ingredient, and is, for example, 2.5 × 10 -2 mg or more, 2.5×10 -1 mg or more, or 2.5 mg or more, and 4 mg or less, 2.5×10 3 mg or less or 2.5 x 10 2 These upper and lower limits can be arbitrarily combined, and the range of the intake or administration amount can be, for example, 2.5 × 10 -2 mg~2.5×10 4 mg, 2.5 × 10 -1 mg~2.5×10 4 mg, 2.5mg to 2.5×10 4 mg, 2.5 × 10 -2 mg~2.5×10 3 mg, 2.5 × 10 -1 mg~2.5×10 3 mg, 2.5×10mg to 2.5×10 3 mg, 2.5 × 10 -2 mg~2.5×102 mg, 2.5 × 10 -1 mg~2.5×10 2 mg or 2.5 mg to 2.5 × 10 2 It can be expressed as mg.

[0134] The intake amount of the immunostimulating composition of this embodiment and the intake timing and intake period described below are applicable whether the immunostimulating composition of this embodiment is used for non-therapeutic purposes or therapeutic purposes, and in the case of therapeutic purposes, intake can be read as administration.

[0135] It is preferable to continue taking or administering the immunostimulating composition of this embodiment for the period during which the immunostimulating effect is expected. From the viewpoint of better exerting the immunostimulating effect, the intake period or administration period of the active ingredient of the present invention can be, for example, one week or more, two weeks or more, three weeks or more, and preferably one month or more (four weeks or more) at the above-mentioned daily dose. The intake interval or administration interval of the active ingredient of the present invention can be once every three days, once every two days, or once a day at the above-mentioned daily dose, and is preferably once a day.

[0136] The immunostimulating composition of this embodiment may also be started ingestion or administration before an event or time when an immunostimulating effect is expected. Examples of events when an immunostimulating effect is expected include behaviors that may result in viral infection (e.g., participation in an event with a high risk of viral infection, traveling to an endemic area), and examples of times when an immunostimulating effect is expected include the period when a viral infection is prevalent. Examples of timing of ingestion or administration before an event when an immunostimulating effect is expected include at least one day, three days, one week, two weeks, three weeks, one month (four weeks), or two months (eight weeks) before. Furthermore, although not particularly limited, ingestion or administration may be continued, with an ingestion interval or administration interval between ingestion or administration, depending on the case, until the event when an immunostimulating effect is expected. The active ingredient of the present invention may also be started ingestion or administration after the event or time when an immunostimulating effect is expected. Examples of the timing of ingestion or administration after an event when an immunostimulating effect is expected include, for example, one day or more, three days or more, one week or more, or two weeks or more. Furthermore, although not particularly limited, when ingestion or administration is performed after an event when an immunostimulating effect is expected, in some cases, ingestion or administration can be continued with an ingestion interval or administration interval. In the present invention, it is particularly preferred that ingestion or administration of the active ingredient of the present invention is started before an event when an immunostimulating effect is expected, and ingestion or administration can be continued until after the event.

[0137] In addition, in this embodiment, the aspect in which the composition is an immunostimulatory composition can also be said to be, in another aspect, a method for stimulating the immunity of a subject, which includes having a subject ingest or administering the composition according to one aspect of this embodiment to a subject. The subject to which the composition is ingested or administered may be a subject in need thereof, such as a subject in need of the above-mentioned immunostimulation.

[0138] Furthermore, in this embodiment, the aspect in which the composition is an immunostimulatory composition can also be said to be, in another aspect, the use of the composition according to one aspect of this embodiment in stimulating the immune system of a subject. In this case, the use may be non-therapeutic and / or therapeutic, preferably non-therapeutic.

[0139] In the present disclosure, "therapeutic" use refers to use in treating the human or animal body. A therapeutic use according to the present disclosure may be, for example, a use for the purpose of or in conjunction with medical treatment. A therapeutic use according to the present disclosure may involve, for example, a medical professional administering or ingesting a substance to a human or animal, or instructing a human or animal to administer or ingest a substance. A therapeutic use according to the present disclosure may be, for example, a use for therapeutic purposes or a preventative purpose, or may be a preventative use involving the ingestion of a food composition, or may be a therapeutic purpose. A therapeutic use according to the present disclosure may be, for example, a use on an unhealthy individual.

[0140] In the present disclosure, "non-therapeutic" use refers to use of a substance that does not fall under therapeutic use. Non-therapeutic use according to the present disclosure may, for example, be use that is not intended for and / or does not involve medical treatment. Non-therapeutic use according to the present disclosure may not, for example, involve a medical professional administering or having a human or animal ingest the substance and / or instructing a human or animal to administer or ingest the substance. Non-therapeutic use according to the present disclosure may, for example, be use for preventive or health promotion purposes, and may be use for preventive or health promotion purposes involving the administration or ingestion of a pharmaceutical composition or quasi-drug. Non-therapeutic use according to the present disclosure may, for example, be use in healthy individuals.

[0141] Furthermore, in another aspect of the present embodiment, the aspect in which the composition is an immunostimulatory composition can also be said to be a composition according to one aspect of the present embodiment for use in stimulating the immune system of a subject. In this case, the use is, for example, therapeutic use.

[0142] Furthermore, in this embodiment, the aspect in which the composition is an immunostimulatory composition can also be said to be, in another aspect, the use of the above-mentioned bacterium in the production of a composition according to one aspect of this embodiment.

[0143] Furthermore, one aspect of this embodiment is a method for activating pDCs, which comprises binding bacteria to BDCA2.

[0144] In one aspect, the biological characteristics, classification, and strain of bacteria that can be used in the above-mentioned method are the same as those described above for the bacteria contained in the immunostimulatory composition of this embodiment ("bacteria of this embodiment"). For example, the bacteria may be lactic acid bacteria and / or acetic acid bacteria. Furthermore, the bacteria may be bacteria that are phagocytosed by pDCs via binding to BDCA2.

[0145] Furthermore, one aspect of this embodiment is a method for activating pDCs, which comprises administering bacteria that bind to BDCA2 to a subject.

[0146] In one aspect, the biological characteristics, classification, and strain of bacteria that can be used in the above-mentioned method are described in the above-mentioned description of the bacteria contained in the immunostimulatory composition of this embodiment ("bacteria of this embodiment"). For example, the bacteria may be lactic acid bacteria and / or acetic acid bacteria. Furthermore, the bacteria may be bacteria that are phagocytosed by pDCs via binding to BDCA2.

[0147] In one embodiment, the subject of the above method may be a human. The human may be, but is not limited to, a subject in need of immunopotentiation. The subject in need of immunopotentiation may be, but is not limited to, a subject infected with a virus, a subject suffering from a cold, a subject aged 65 or over, or the like.

[0148] Furthermore, one aspect of this embodiment may be a composition containing bacteria for use in the above-mentioned "method for activating pDCs, characterized by binding bacteria to BDCA2" and "method for activating pDCs, including administering bacteria that bind to BDCA2 to a subject."

[0149] In one aspect, the biological characteristics, classification, and strain of the bacteria that can be used in the composition are described above with reference to the description of the bacteria contained in the immunostimulatory composition of the present embodiment ("bacteria of the present embodiment"). For example, the bacteria may be lactic acid bacteria and / or acetic acid bacteria. Furthermore, for example, the bacteria may be Lactococcus lactis subsp. lactis JCM 5805. Furthermore, the bacteria may be bacteria that are phagocytosed by pDCs via binding to BDCA2. Furthermore, the description of the composition is described above with reference to the description of the "immunostimulatory composition of the present embodiment" described as one embodiment of the present invention.

[0150] A second embodiment of the present disclosure relates to a method for enhancing the immunostimulatory ability of bacteria and / or their ability to be phagocytosed by plasmacytoid dendritic cells (pDCs), which includes a step (culturing step) of culturing bacteria in M17 medium supplemented with glucose at a predetermined concentration. In the method of the second embodiment, by using M17 medium supplemented with glucose at a predetermined concentration as the medium for culturing bacteria, the immunostimulatory ability of bacteria and / or their ability to be phagocytosed by pDCs is likely to be enhanced compared to when the medium is not used.

[0151] M17 medium can be obtained from suppliers such as Becton, Dickinson and Company. M17 medium contains, for example, water as a solvent and yeast extract, peptone, tryptone, meat extract, lactose, ascorbic acid, magnesium sulfate, and sodium glycerophosphate as solutes. The pH of M17 medium may be adjusted to 7.1±0.2 before use.

[0152] The final concentration of glucose in the medium used in the culture step may be, for example, 0.1% by mass or more, 0.3% by mass or more, 0.5% by mass or more, or 1.0% by mass or more, or 10% by mass or less, 5.0% by mass or less, 3.0% by mass or less, or 1.0% by mass or less. These lower and upper limits can be arbitrarily combined, and the final glucose concentration in the medium used in the culture step may be, for example, 0.1% to 10% by mass, 0.3% to 10% by mass, 0.5% to 10% by mass, 1.0% to 10% by mass, 0.1% to 5.0% by mass, 0.3% to 5.0% by mass, 0.5% to 5.0% by mass, 1.0% to 5.0% by mass, 0.1% to 3.0% by mass, 0.3% to 3.0% by mass, 0.5% to 3.0% by mass, 1.0% to 3.0% by mass, 0.1% to 1.0% by mass, 0.3% to 1.0% by mass, 0.5% to 1.0% by mass, or 1.0% by mass. In this case, glucose refers to, for example, D-glucose.

[0153] The culture time in the culture step is not particularly limited, and may be, for example, 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, or 18 hours or more, and may be, for example, 336 hours or less, 168 hours or less, 72 hours or less, 48 ​​hours or less, or 36 hours or less, and may be, for example, 24 hours. The culture temperature in the culture step is not particularly limited, and may be, for example, 20°C or more, 22°C or more, 25°C or more, or 30°C or more, and may be 45°C or less, 40°C or less, 38°C or less, 35°C or less, or 30°C or less, and may be, for example, 30°C.

[0154] The immunostimulatory ability and / or ability to be phagocytosed by pDCs of the bacteria may be evaluated in the same manner as described in Embodiment 1. The immunostimulatory ability and / or ability to be phagocytosed by pDCs of the bacteria after the culture step may be, for example, 1.05-fold or more, 1.10-fold or more, 1.15-fold or more, 1.20-fold or more, 1.25-fold or more, 1.30-fold or more, 1.35-fold or more, 1.40-fold or more, 1.45-fold or more, or 1.50-fold or more compared to the bacteria when the culture step is carried out using MRS medium as the medium.

[0155] One aspect of the second embodiment may also be a method for producing a composition for immunostimulation, which includes a culture step. The composition for immunostimulation produced by the production method of this aspect may be the composition of the first embodiment, and for example, its use and composition may satisfy the requirements of the composition of the first embodiment. The production method of this aspect may further include a step of sterilizing the bacteria and / or a step of drying the bacteria after the culture step.

[0156] The sterilization method in the step of sterilizing bacteria may be a method commonly used by those skilled in the art, such as 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 in one embodiment may be heat treatment. When the sterilization method is heat treatment, the heating temperature is not particularly limited, and may be, for example, 60°C or higher, 65°C or higher, 70°C or higher, or 75°C or higher, or 110°C or lower, 100°C or lower, 90°C or lower, 85°C or lower, or 80°C or lower. These lower and upper limits can be combined arbitrarily, and for example, the heating temperature may be 60 ° C. or higher and 110 ° C. or lower, 60 ° C. or higher and 100 ° C. or lower, 60 ° C. or higher and 90 ° C. or lower, 60 ° C. or higher and 85 ° C. or lower, 60 ° C. or higher and 80 ° C. or lower, 65 ° C. or higher and 100 ° C. or lower, 65 ° C. or higher and 90 ° C. or lower, 65 ° C. or higher and 85 ° C. or lower, 65 ° C. or higher and 80 ° C. or lower, 70 ° C. or higher and 90 ° C. or lower, 70 ° C. or higher and 85 ° C. or lower, 70 ° C. or higher and 80 ° C. or lower, 75 ° C. or higher and 90 ° C. or lower, or 75 ° C. or higher and 85 ° C. or lower. In addition, when the sterilization method is heat treatment, the heating time is not particularly limited, but may be, for example, 1 minute or more, 3 minutes or more, 5 minutes or more, or 10 minutes or more, and may be 24 hours or less, 4 hours or less, 60 minutes or less, 45 minutes or less, or 30 minutes or less. These lower and upper limits can be combined in any desired manner. For example, the heat treatment time may be 1 minute or more and 24 hours or less, 3 minutes or more and 4 hours or less, or 5 minutes or more and 60 minutes or less.

[0157] The drying method in the step of drying the bacteria may be a method that is commonly used by those skilled in the art, such as freeze drying, spray drying, drum drying, hot air drying, or vacuum drying, and in one embodiment may be spray drying.

[0158] Furthermore, the production method of this embodiment may include a step of washing the bacteria between and / or during each step, as necessary. Such washing may involve, for example, suspending the bacteria in a fresh medium or buffer solution and then isolating them by centrifugation or the like.

[0159] The immunostimulatory composition produced by the production method of this embodiment may be, for example, an immunostimulatory composition containing as an active ingredient Lactococcus lactis subsp. lactis JCM 5805. In this case, Lactococcus lactis subsp. lactis JCM 5805 may satisfy, for example, the following requirement Y, and whether requirement Y is satisfied may be confirmed according to the method described in relation to the first embodiment. [Requirement Y] Final concentration 2.0×10 5 After contacting pDCs at 1000 cells / mL with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of pDCs that have been contacted is 15.0% or more, with the proviso that the pDCs have been previously cultured in a serum-free medium. [Example]

[0160] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0161] In the following examples, CAL-1 cells (accession number FERM BP-10914) provided by Nagasaki University were used as a sample (hereinafter referred to as the "Sample"). (All rights to the Sample belong to Nagasaki University, and permission to use the Sample has been obtained from Nagasaki University.) Cell culture was performed at 37°C and 5% CO2. Results are shown as mean ± standard deviation. The basal medium for culturing and testing CAL-1 cells was RPMI-1640 medium (Sigma, R8758) supplemented with penicillin / streptomycin (Gibco, 15140-12) at a final concentration of 1.0% by volume (hereinafter referred to as "serum-free medium").

[0162] The lactic acid bacteria strains used in the following examples are listed in Table 1. The culture conditions indicated as A to G in Table 1 are shown in Table 2. In the examples, except for Lactobacillus rhamnosus CRL1505, lactic acid bacteria were cultured according to the culture method described in Table 2 according to the correspondence between A to G in Table 1, and then used as bacterial bulk powder (dried bacterial powder or powder containing the same, etc.) prepared as follows. Bacteria cultured in MRS medium (MRS BROTH, CODE: CM0359, Oxoid) were centrifuged (5000 rpm) and the supernatant was removed to obtain a bacterial solution. The resulting bacterial solution was then mixed with phosphate-buffered saline (Bio-Pharmaceuticals) at a ratio of 1:19 and washed twice by centrifugation (5000 rpm). The washed bacterial solution was heat-treated to kill the lactic acid bacteria in the bacterial solution. The heat treatment involved raising the temperature from room temperature to 80°C over 30 minutes, holding at 80°C for 30 minutes, and then lowering the temperature to room temperature over 30 minutes. After lowering the temperature, the mixture was freeze-dried to obtain a bacterial bulk powder.

[0163] [Table 1] TIFF2026027290000002.tif227147 TIFF2026027290000003.tif215147

[0164] [Table 2] TIFF2026027290000005.tif72149

[0165] For Lactobacillus rhamnosus CRL1505, the bacterial bulk powder prepared as follows was used. [Preparation method of Lactobacillus rhamnosus CRL1505 bacterial bulk powder] A solution containing Lactobacillus rhamnosus CRL1505 was prepared by dissolving FLORASSIST Probiotic Immune & Nasal Defense (Life Extension, #02208), a supplement containing Lactobacillus rhamnosus CRL1505, in 10 mL of MRS medium. The solution was then spread onto MRS agar medium and streaked to obtain single colonies of Lactobacillus rhamnosus CRL1505. A colony of Lactobacillus rhamnosus CRL1505 was added to MRS medium and incubated at 37°C for 24 hours. 700 μL of the culture was then mixed with 300 μL of 80% glycerol to prepare a glycerol stock of Lactobacillus rhamnosus CRL1505 and stored at -80°C. 100 μL of glycerol stock of Lactobacillus rhamnosus CRL1505 was added to 100 mL of MRS medium and incubated at 37°C for 24 hours. The culture medium was collected, centrifuged, and washed twice with ultrapure water. The suspension was then suspended in 20 mL of ultrapure water, sterilized at 80°C for 30 minutes, and lyophilized. This was used as the bulk bacterial powder of Lactobacillus rhamnosus CRL1505 in the following examples.

[0166] [Bacterial staining with pHrodo Red SE] pHrodo Red SE (Invitrogen, P36600) was dissolved in DMSO to prepare a 10.2 mM solution. Bacterial bulk powder was weighed into a 2 mL Eppendorf tube, and 0.1 M sodium bicarbonate adjusted to pH 9.0 was added to prepare a 20 mg / mL bacterial solution. 95 μL was then transferred to a new 2 mL Eppendorf tube, and 5 μL of the prepared 10.2 mM pHrodo Red SE was added. After vortexing, the mixture was protected from light with aluminum foil and incubated at room temperature for 60 minutes. 750 μL of PBS (Takara Bio) was then added, vortexed, and centrifuged at 20,000 g for 2 minutes at room temperature. 800 μL of the supernatant was discarded, and 1.5 mL of PBS was added. The precipitate was completely suspended by vortexing. The mixture was centrifuged again at 20,000 g for 2 minutes at room temperature, and 1.5 mL of the supernatant was discarded. Finally, 140 μL of PBS was added and suspended to create a 10 mg / mL bacterial suspension of stained bacteria. The stained bacterial suspension was protected from light with aluminum foil and stored at 4°C until use. Just before use, the suspension was diluted 10-fold with PBS to create a 1 mg / mL bacterial suspension. The stained bacterial suspension was used within 24 hours of preparation.

[0167] Example 1: Evaluation of the contribution of phagocytosis to IFN-α production by pDCs CAL-1 cells were cultured in serum-free medium at 5.0 × 10 5 A cell suspension containing 2.0 × 10 cells / mL was prepared, and the cell suspension was seeded at 10 mL / well in a 10 cm diameter culture dish and cultured for 16 hours (pretreatment step). CAL-1 cells were collected by pipetting. The collected CAL-1 cells were transferred to serum-free medium at a concentration of 2.0 × 10 cells / mL. 5The cells were resuspended at a concentration of 100 cells / mL and seeded at 200 μL per well into a 96-well microplate. For the LC-Plasma+Cyt_D group, 400 μM cytochalasin D (Fujifilm Wako, 037-17561) dissolved in 4% dimethyl sulfoxide (DMSO) was added to the cell suspension in each well to a final concentration of 10 μM and 0.1% DMSO, respectively, and incubated for 30 minutes. For the LC-Plasma+DMSO group, 4% DMSO was added to a final concentration of 0.1% DMSO, and incubated for 30 minutes. After incubation, Lactococcus lactis subsp. lactis JCM 5805 (Kirin Holdings Co., Ltd., hereafter also referred to as "LC-Plasma") stained with pHrodo Red SE (Invitrogen, P36600) as described above in "Staining bacteria with pHrodo Red SE" was added to each well to a final concentration of 10 μg / mL. The pHrodo Red SE fluorescence in each well was then measured over time using an Incucyte® SX5 Live-Cell Analysis System (Sartorius). After 22 hours of incubation (contact step), the culture supernatant from each well was collected, and the IFN-α concentration in the culture supernatant was measured by ELISA using a Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science).

[0168] Cytochalasin D is an actin polymerization inhibitor, and it is known that phagocytosis is suppressed in cells to which cytochalasin D has been added. pHrodo Red SE is a staining reagent whose fluorescence intensity increases in a low pH environment. When bacteria stained with pHrodo Red SE are phagocytosed by CAL-1 cells, the bacteria enter endosomes, creating a low pH environment, which increases the fluorescence intensity of pHrodo Red SE. Therefore, the total area in the entire field of view where pHrodo Red SE fluorescence is detected (fluorescence detection area) can be used as an index to quantify the amount of bacteria phagocytosed by CAL-1 cells. The fluorescence intensity analysis conditions for the Incucyte® SX5 Live-Cell Analysis System (Sartorius) were as described in [Incucyte Fluorescence Intensity Analysis Condition 1] below. The field of view area per image was 0.572 mm. 2 It was.

[0169] [Incucyte fluorescence intensity analysis condition 1] For the Incucyte fluorescence intensity analysis, two channels were set: the "Phase channel" and the "Orange channel." CAL-1 cells were detected in the Phase channel based on phase contrast. pHrodo Red SE fluorescence was detected in the Orange channel. Detailed detection conditions are as shown in the Scan Settings and Analysis Settings below. Scan Settings Scan Type:Non-Adherent Cell-by-Cell Vessel Type: 96-well Corning Image Channels:Phase, Orange (Acquisition Time: 400 ms) Objective: 20× Images per Well: 9 Analysis Settings Analysis Type: Basic Analyzer Phase Channel Segmentation:AI Confluence Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): Over 60,000 - Eccentricity: Not set Orange Channel Segmentation: Surface Fit - Threshold (OCU): 5.000 - Edge Split: On - Edge Sensitivity: 0 Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): Not set - Eccentricity: Not set - Mean Intensity: Not set - Mean Intensity: Not set

[0170] Figure 1 shows the fluorescence detection area 22 hours after the addition of LC-Plasma. Figure 2 shows the results of measuring the IFN-α concentration in the culture supernatant collected 22 hours after the addition of LC-Plasma. The results in Figure 1 indicate that the LC-Plasma + Cyt_D group had significantly lower phagocytosis levels than the LC-Plasma + DMSO group. Furthermore, the results in Figure 2 indicate that the LC-Plasma + Cyt_D group had significantly lower IFN-α concentrations than the LC-Plasma + DMSO group, indicating that pDC activation by LC-Plasma was significantly suppressed. These findings demonstrate that pDC activation is significantly suppressed when phagocytosis is inhibited.

[0171] Example 2: Relationship between pDC phagocytosis and IFN-α production in various lactic acid bacteria CAL-1 cells were cultured in serum-free medium at 5.0 × 10 5 A cell suspension containing 2.0 × 10 cells / mL was prepared, and the cell suspension was seeded at 10 mL / well in a 10 cm diameter culture dish and cultured for 16 hours (pretreatment step). CAL-1 cells were collected by pipetting. The collected CAL-1 cells were transferred to serum-free medium at a concentration of 2.0 × 10 cells / mL. 5The cells were resuspended at a concentration of 100 μg / mL and seeded into each well of a 96-well microplate at 200 μL / well. Various lactic acid bacteria (ATCC 15577, ATCC 7963, ATCC 11955, JCM 1096, JCM 12533, JCM 9695, JCM 6125, or LC-Plasma) stained with pHrodo Red SE (Invitrogen, P36600) were added to each well to a final concentration of 10 μg / mL. A negative group was also prepared without bacteria. Subsequently, pHrodo Red SE fluorescence was measured over time and the IFN-α concentration in the culture supernatant was measured using the same method as in Example 1, except for the fluorescence intensity analysis conditions using the Incucyte® SX5 Live-Cell Analysis System (Sartorius). The fluorescence intensity analysis conditions for the Incucyte® SX5 Live-Cell Analysis System (Sartorius) were as described in [Incucyte Fluorescence Intensity Analysis 2] below. The field of view area per image was 0.572 mm. 2 It was.

[0172] [Incucyte Fluorescence Intensity Analysis 2] For the Incucyte fluorescence intensity analysis, two channels were set: the "Phase channel" and the "Orange channel." CAL-1 cells were detected in the Phase channel based on phase contrast. pHrodo Red SE fluorescence was detected in the Orange channel. Detailed detection conditions are as shown in the Scan Settings and Analysis Settings below. Scan Settings Scan Type:Non-Adherent Cell-by-Cell Vessel Type: 96-well Corning Image Channels:Phase, Orange (Acquisition Time: 400 ms) Objective: 20× Images per Well: 9 Analysis Settings Analysis Type: Basic Analyzer Phase Channel Segmentation:AI Confluence Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): Over 60,000 - Eccentricity: Not set Orange Channel Segmentation: Surface Fit - Threshold (OCU): 5.000 - Edge Split: On - Edge Sensitivity: 0 Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): 20 - Eccentricity: Not set - Mean Intensity: Not set - Mean Intensity: Not set

[0173] Figure 3 shows the fluorescence detection area 22 hours after the addition of lactic acid bacteria for the eight types of lactic acid bacteria tested. Figure 4 shows the measurement results of the IFN-α concentration in the culture supernatant collected 22 hours after the addition of lactic acid bacteria for the 13 types of lactic acid bacteria tested. Figure 5 shows the correlation between the fluorescence detection area on the horizontal axis and the IFN-α concentration on the vertical axis for the eight types of lactic acid bacteria tested. The results of Figures 3 to 5 show that there is a strong positive correlation (R 2 This indicates that the immunostimulatory capacity of bacteria is determined by the amount of phagocytosis.

[0174] Example 3: Relationship between phagocytosis of LC-Plasma by pDCs via binding to BDCA2 and IFN-α production CAL-1 cells were seeded in each well as in Example 1. Anti-BDCA2 antibody (Miltenyi Biotec, 130-090-690) was added to the LC-Plasma + BDCA2 anti group, and an isotype control (Miltenyi Biotec, 130-106-545) of the same isotype and subclass as the anti-BDCA2 antibody was added to the LC-Plasma + Isotype group at 10 μg / mL to evaluate the effect of nonspecific binding by the anti-BDCA2 antibody. The cells were then incubated for 30 minutes. No antibody was added to the control LC-Plasma group. Subsequently, the LC-Plasma, LC-Plasma + Isotype, and LC-Plasma + BDCA2 anti groups were exposed to pHrodo Red SE-stained LC-Plasma, phagocytosis was assessed, and IFN-α concentrations in the culture supernatants were measured, as in Example 2.

[0175] Figure 6 shows the fluorescence detection area 22 hours after addition of LC-Plasma for the LC-Plasma group, the LC-Plasma + Isotype group, and the LC-Plasma + BDCA2 anti group. Figure 7 shows the IFN-α concentration in the culture supernatants collected 22 hours after addition of LC-Plasma for the LC-Plasma group, the LC-Plasma + Isotype group, and the LC-Plasma + BDCA2 anti group. The results in Figures 6 and 7 indicate that the LC-Plasma + BDCA2 anti group, in which the binding of BDCA2 to CAL-1 cells was inhibited by blocking with anti-BDCA2 antibodies, had lower phagocytosis and IFN-α concentrations than the LC-Plasma group and the LC-Plasma + Isotype group, in which the binding was not inhibited. These results demonstrate that the binding of LC-Plasma to BDCA on the surface of pDCs is one of the steps that determines the degree of immune activation by LC-Plasma. These results also revealed that phagocytosis of LC-Plasma by pDCs via binding to BDCA is one of the steps that determines the degree of immune activation by LC-Plasma.

[0176] Example 4: Relationship between phagocytosis of various lactobacilli by pDCs via binding to BDCA2 and IFN-α production CAL-1 cell suspensions were seeded in each well as in Example 1. Anti-BDCA2 antibody (Miltenyi Biotec, 130-090-690) was added to the BDCA2 anti group, and its isotype control antibody (Miltenyi Biotec, 130-106-545) was added to the isotype group at 10 μg / mL, followed by incubation for 30 minutes. Then, as in Example 2, the isotype and BDCA2 anti groups were exposed to the same various lactic acid bacteria stained with pHrodo Red SE as those used in Example 2, and phagocytosis was evaluated, and the IFN-α concentration in the culture supernatant was measured. The amount of phagocytosis mediated by binding to BDCA2 was calculated using the method described below as "Method for calculating the amount of phagocytosis mediated by binding to BDCA2."

[0177] [Method for calculating the amount of phagocytosis mediated by BDCA2 binding] First, to consider the influence of nonspecific binding by anti-BDCA2 antibodies, we evaluated the amount of bacteria engulfed by pDCs (phagocytosis amount P) in the isotype group treated with an isotype control antibody with the same isotype and subclass as the anti-BDCA2 antibody. Next, we evaluated the amount of bacteria engulfed by pDCs (phagocytosis amount Q) in the BDCA2 anti group treated with an anti-BDCA2 antibody that inhibits the binding of bacteria to BDCA2. We then calculated the amount of bacteria engulfed by pDCs via BDCA2 binding by subtracting the phagocytosis amount Q from the phagocytosis amount P.

[0178] Figure 8 shows the fluorescence detection area 22 hours after the addition of various lactic acid bacteria for the isotype group and the BDCA2 anti group. Figure 9 shows the results of measuring the IFN-α concentration in culture supernatants collected 22 hours after the addition of various lactic acid bacteria for the isotype group and the BDCA2 anti group. Figure 10 shows the correlation between the amount of phagocytosis via BDCA2 binding, calculated according to the above-mentioned method for calculating the amount of phagocytosis via BDCA2 binding, on the horizontal axis and the IFN-α concentration on the vertical axis for various lactic acid bacteria.

[0179] The results in Figures 8 and 9 show that the BDCA2 anti group, in which the binding of BDCA2 to CAL-1 cells was inhibited by blocking with anti-BDCA2 antibodies, had lower phagocytosis levels and IFN-α concentrations than the isotype group, in which binding was not inhibited. These results demonstrate that, even for bacteria other than LC-Plasma, the binding of bacteria to BDCA on the surface of pDCs is one of the steps that determines the degree of bacterial immune activation. These results also demonstrate that the phagocytosis of bacteria by pDCs via binding to BDCA is one of the steps that determines the degree of bacterial immune activation.

[0180] According to the results in Figure 10, there is a strong positive correlation (R 2= 0.86). This indicates that the immunostimulatory potential of bacteria is not only determined by the amount of phagocytosis, but also that one of the major pathways of bacterial immunostimulation is phagocytosis by pDCs via binding to BDCA2. It also suggests that the binding ability to BDCA2 is a useful parameter for evaluating the immunostimulatory potential of bacteria.

[0181] Example 5: Development of an assay to evaluate the ability of bacteria to bind to BDCA2 An assay to evaluate the ability of bacteria to bind to BDCA2 (BDCA2 binding assay) was established, as follows: Phosphate-buffered saline (PBS, Nacalai Tesque, 11482-15) containing 0.5 mM CaCl2 (hereinafter simply referred to as "buffer") was added to each well of a low-binding 96-well plate (Corning, 3474) in an amount of 100 μL. Recombinant human BDCA-2 Fc chimera protein (R&D Systems, 1376-DL; hereinafter simply referred to as "BDCA2") or recombinant human IgG1 Fc protein (R&D Systems, 110-HG) was added to each well to a final concentration of 1–50 μg / mL. To distinguish between contaminants and bacteria, LC-Plasma, stained with FITC according to standard methods, was added to each well to a final concentration of 10 μg / mL. The plate was then incubated at room temperature for 1 hour, shielded from light with aluminum foil. After adding 150 μL of buffer to each well, the 96-well plate was centrifuged at 400×g for 5 minutes at room temperature, and the supernatant was discarded. 100 μL of a 100-fold diluted solution of PE-conjugated F(ab')2-Goat anti-Human IgG Fc Secondary Antibody (ThermoFisher Scientific, H10104) in buffer (hereinafter referred to as "secondary antibody dilution") was added to each well and incubated for 15 minutes at room temperature. The solution in each well was subjected to flow cytometry (FACS) using a flow cytometer (BD Biosciences, BD LSRFortessa® X-20 Flow Cytometer).

[0182] The FACS results were analyzed as follows: First, the population containing lactic acid bacteria was gated using FSC / SSC. Next, gating was performed using wavelengths corresponding to SSC / FITC, and only the FITC-stained population (FITC+ population) was extracted. This FITC+ population was then gated using wavelengths corresponding to SSC / PE, and the proportion of the population that fluoresced at the wavelength corresponding to PE (PE+ population) was extracted. The threshold for the PE+ population was set so that the PE+ population was approximately 0.1% in the groups to which huIgfc was added at the same concentration as each BDCA2. The proportion of the PE+ population within the FITC+ population was calculated as the proportion of lactic acid bacteria that bound to BDCA2.

[0183] Figure 11 shows the relative values ​​evaluated by the BDCA2 binding assay when BDCA2 concentrations were 1, 5, 10, 20, or 50 μg / mL relative to 10 μg / mL LC-Plasma, with the percentage of LC-Plasma bound to BDCA2 at a BDCA2 concentration of 1 μg / mL set to 1. Figure 11 shows that the percentage of LC-Plasma bound to BDCA2 increased in a BDCA2 concentration-dependent manner. This demonstrates that the BDCA2 binding assay can be used to evaluate the binding ability of bacteria to BDCA2.

[0184] Example 6: Phagocytosis of bacteria by pDCs, phagocytosis via binding to BDCA2, and ability to bind to BDCA2 The lactic acid bacteria listed in Tables 3 and 4 were evaluated for their pDC-mediated phagocytosis and BDCA2-mediated phagocytosis, and the BDCA2-binding ability of the lactic acid bacteria listed in Table 5 was evaluated. The amount of pDC-mediated phagocytosis was evaluated using the same method as in Example 2, and is shown in Table 3 as (A-1) the fluorescence detection area 22 hours after the addition of the lactic acid bacteria. Table 4 also shows (A-2) the fluorescence detection area of ​​each bacterium minus the fluorescence detection area 22 hours after the addition of Lactococcus lactis subsp. lactis ATCC15577, and (A-3) the ratio of the fluorescence detection area of ​​each bacterium divided by the fluorescence detection area 22 hours after the addition of Lactococcus lactis subsp. lactis ATCC15577. The amount of pDC-mediated BDCA2-mediated phagocytosis was evaluated using the same method as in Example 4, and the results are shown in Table 3 as (B-1). Furthermore, the values ​​obtained by subtracting the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 from the amounts phagocytosis of these bacteria are shown in Table 4 as (B-2), and the values ​​(ratios) obtained by dividing the amounts phagocytosis of these bacteria by the amount of phagocytosis of Lactococcus lactis subsp. lactis ATCC15577 are shown in Table 4 as (B-3). The ability to bind to BDCA2 was evaluated by a BDCA2 binding assay similar to that described in Example 5. Specifically, the test was performed using BDCA2 or Recombinant Human IgG1 Fc Protein (R&D Systems, 110-HG) at a final concentration of 10 μg / mL, with a lactic acid bacteria concentration of 10 μg / mL, and the proportion of lactic acid bacteria bound to BDCA2 was measured as an index. Table 5 shows the percentage of binding to BDCA2 as (C-1), the value obtained by subtracting the percentage of Lactococcus lactis subsp. lactis ATCC15577 from that percentage as (C-2), and the value (ratio) obtained by dividing that percentage by the percentage of Lactococcus lactis subsp. lactis ATCC15577 as (C-3).Since these measurements were carried out over three or four days, the relative values ​​shown as (A-2), (A-3), (B-2), (B-3), (C-2), and (C-3) to the results of Lactococcus lactis subsp. lactis ATCC 15577 (results of (A-1), (B-1), or (C-1)) were calculated by subtracting or dividing the values ​​obtained using Lactococcus lactis subsp. lactis ATCC 15577 on each measurement day. In addition, for (A-2) the value obtained by subtracting the fluorescence detection area 22 hours after the addition of Lactococcus lactis subsp. lactis ATCC 15577 from the fluorescence detection area 22 hours after the addition of each lactic acid bacterium, and (B-2) the value obtained by subtracting the amount of phagocytosis via binding to BDCA2 of Lactococcus lactis subsp. lactis ATCC 15577 from the amount of phagocytosis via binding to BDCA2 of each lactic acid bacterium, values ​​below 0 were indicated as 0.

[0185] [Table 3]

[0186] [Table 4]

[0187] [Table 5]

[0188] Lactic acid bacteria with higher pDC phagocytosis levels, higher pDC phagocytosis levels via binding to BDCA2, and / or higher BDCA2 binding ability than at least Lactococcus lactis subsp. lactis ATCC 15577 can be said to have immunostimulatory activity. Therefore, based on the results in Tables 3 to 5, lactic acid bacteria with higher pDC phagocytosis levels, higher pDC phagocytosis levels via binding to BDCA2, and / or higher BDCA2 binding ability than Lactococcus lactis subsp. lactis ATCC 15577 can be said to have immunostimulatory activity.

[0189] Example 7: Importance of phagocytosis in IFN-α production by pDCs CAL-1 cells were seeded in each well as in Example 1. For the cytochalasin D (referred to as CytD in the graph) group, 400 μM cytochalasin D (Fujifilm Wako, 037-17561) dissolved in 4% dimethyl sulfoxide (DMSO) was added to the wells to a final concentration of 10 μM cytochalasin D and 0.1% DMSO, respectively, and incubated for 30 minutes. As a control, 4% DMSO alone was added to the wells to a final concentration of 0.1% DMSO, and the wells were incubated for 30 minutes. After incubation, lactic acid bacteria listed in Table 1, stained with pHrodo Red SE (Invitrogen, P36600) as described above in [Staining bacteria with pHrodo Red SE], were added to each well to a final concentration of 10 μg / mL, and the wells were cultured for 24 hours. Phagocytic activity and IFN-α concentration in each well were measured as in Example 1.

[0190] To confirm the lack of cytotoxicity of DMSO and cytochalasin D, cytochalasin D or DMSO, or the same volume of PBS as a control, was added as described above, and the cells were incubated for 30 minutes and then cultured for 24 hours. The cytotoxicity was evaluated by measuring the number of viable cells using Cell Counting Kit-8 (Dojindo Laboratories, CK04).

[0191] The Cell Counting Kit-8 is a kit for measuring cell number in cell proliferation or chemical sensitivity tests. By using the novel tetrazolium salt WST-8, which generates highly sensitive water-soluble formazan, as a colorimetric substrate, it enables more sensitive measurements than conventional Cell Counting Kits. WST-8 is reduced by intracellular dehydrogenases to generate water-soluble formazan. The number of viable cells can be easily determined by directly measuring the absorbance of this formazan at 450 nm. There is a linear proportional relationship between the number of cells and the amount of formazan generated. Cytotoxicity can be evaluated by observing fluctuations in the number of viable cells.

[0192] Furthermore, as described in detail in Example 1, cytochalasin D is an actin polymerization inhibitor, and it is known that phagocytosis is suppressed in cells to which cytochalasin D has been added.

[0193] Figure 12 shows the results of adding DMSO or cytochalasin D + DMSO (referred to as CytD in the graph) and measuring the number of viable cells 24 hours later using Cell Counting Kit-8. Figure 13 shows the results of measuring the total amount of fluorescence (Total Integrate Intensity, T1I, OCU × μm), which is an index of phagocytic activity, for the six types of lactic acid bacteria tested 24 hours after the addition of lactic acid bacteria. 2 14 shows the IFN-α concentration in the supernatant 24 hours after the addition of lactic acid bacteria for the six types of lactic acid bacteria tested. The results in FIG. 12 show that the addition of DMSO or cytochalasin D did not change the number of viable cells compared to the PBS-added group, indicating that the inhibitory reagent used in this test does not cause cytotoxicity. The results in FIG. 13 show that the phagocytic activity of various lactic acid bacteria was completely suppressed by treatment with cytochalasin D. The results in FIG. 14 also show that IFN-α production was completely abolished by inhibiting phagocytic activity. These results demonstrate that phagocytosis is essential for pDC immunostimulation by lactic acid bacteria, and considering the results in Example 1, it was shown that phagocytosis determines pDC immunostimulation.

[0194] <Example 8 Measurement of phagocytosis rate using FACS> Using the same protocol as in Example 2, various lactic acid bacteria (ATCC 15577, ATCC 7963, ATCC 11955, JCM 1096, JCM 12533, JCM 9695, ATCC 7962, JCM 1185, JCM 1149, or LC-Plasma) stained with pHrodo Red SE were added to CAL-1 cells. After 24 hours of incubation at 37°C, the cells were harvested using FACS buffer and stained with 7-AAD (manufacturer: BD biosciences, BD LSRFortessa). After washing the cells with FACS buffer, the washed cells were subjected to flow cytometry (FACS) using a flow cytometer (BD biosciences, BD LSRFortessa® X-20 Flow Cytometer). IFN-α in the supernatant obtained upon cell harvesting was evaluated as in Example 1.

[0195] The FACS results were analyzed as follows: First, a population containing CAL-1 cells was gated using FSC / SSC. Next, gating was performed using wavelengths corresponding to SSC / 7-AAD to extract only the population not stained with 7-AAD (7-AAD- population: viable CAL-1 cell population). The 7-AAD- population was then gated using wavelengths corresponding to PE / SSC to extract the proportion of the population in which fluorescence at wavelengths corresponding to PE (i.e., fluorescence from pHrodo Red SE, which labels lactate) was detected (PE+ population). The ratio of the PE+ population to the 7-AAD- population was used as the phagocytosis rate. The threshold for the PE+ population was determined as the lowest value of the population with the strongest PE intensity when plotting PE / SSC.

[0196] Table 6 summarizes the results of measuring the phagocytosis rate and IFN-α of various lactic acid bacteria. The results in Table 6 confirmed that there is a positive correlation between the phagocytosis rate obtained by flow cytometer and IFN-α. There was a positive correlation (R 2= 0.69). These results revealed that the immunostimulatory capacity of bacteria is determined by the amount of phagocytosis. [Table 6]

[0197] Example 9: Isolation of lactic acid bacteria strains with high phagocytosis rate In this example, LC-Plasma was cultured in two different media and evaluated for phagocytosis rate and IFN-α. One of the LC-Plasma cultured in the two different media was LC-Plasma cultured according to B in Table 1. The other was cultured according to the following method. The cells were suspended in MRS medium (product name: Difco Lactobacilli MRS Broth, manufacturer: Becton, Dickinson and Company; the same applies below) and plated on MRS agar medium. After 48 hours of culture at 30°C, colonies were picked and plated on 10 mL of M17 medium (product name: M17 Broth, manufacturer: Becton, Dickinson and Company) supplemented with glucose (product name: D(+)-Glucose, manufacturer: Fujifilm Wako Pure Chemical Industries) to a final concentration of 1%, and cultured for 24 hours at 30°C. 1 mL of the cultured cell suspension was inoculated into 100 mL of M17 medium and cultured at 30°C for 24 hours.

[0198] The phagocytosis rate and IFN-α of LC-Plasma cultured in the two types of media were evaluated in the same manner as in Example 8.

[0199] Table 7 summarizes the results of measuring the phagocytosis rate and IFN-α of the two strains. According to the results in Table 7, LC-Plasma cultured in M17 medium supplemented with 1% glucose had a higher phagocytosis rate than LC-Plasma cultured in MRS medium, making it possible to obtain lactic acid bacteria with enhanced immunostimulatory activity. [Table 7]

Claims

1. An immunostimulatory composition comprising, as an active ingredient, a bacterium that binds to blood dendritic cell antigen 2 (BDCA2).

2. 2. The composition of claim 1, wherein the bacterium has a binding ability to BDCA2 that is greater than the binding ability to BDCA2 of Lactococcus lactis subsp. lactis ATCC 15577.

3. The composition according to claim 2, wherein the ability to bind to BDCA2 is evaluated based on the percentage of bacteria bound to BDCA2 as assessed in a binding assay.

4. An immunostimulatory composition comprising, as an active ingredient, a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the bacterium satisfies the following requirement X: [Requirement X] Final concentration 2.0×10 ５ After contacting pDCs at a final concentration of 10 μg / mL with the bacteria at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, the ratio of the number of pDCs that have phagocytosed the bacteria to the total number of pDCs that have been contacted is 2.5% or more, with the proviso that the pDCs have been pre-cultured in a serum-free medium.

5. An immunostimulatory composition comprising, as an active ingredient, bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs), An immunostimulatory composition, wherein the amount of the bacterium phagocytosed by pDC is 2.0 times or more the amount of Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC15577 phagocytosed by pDC.

6. A composition comprising Lactococcus lactis subsp. lactis JCM 5805 as a bacterium, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0×10 ５ cells / mL of pDCs are contacted with the Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium for 24 hours, and then the ratio of the number of pDCs that have phagocytosed the Lactococcus lactis subsp. lactis JCM 5805 to the total number of contacted pDCs is 15.0% or more, with the proviso that the pDCs are pDCs that have been cultured in advance in a serum-free medium.

7. The composition of claim 6 , wherein the composition is an immunostimulating composition.

8. The composition according to any one of claims 1 to 5, wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

9. The composition according to any one of claims 1 to 5, wherein the bacteria are one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus.

10. The composition according to any one of claims 1 to 5, wherein the bacterium is Lactococcus lactis subsp. lactis.

11. The bacterium is selected from the group consisting of Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, and Leuconostoc carnosum JCM 13666. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (Oenococcus oeni) JCM 6125, Weissella paramesenteroides (Weissella paramesenteroides) JCM 9890, Weissella viridescens (Weissella viridescens) JCM 1174, Bifidobacterium animalis subsp. animalis (Bifidobacterium animalis JCM 1190, Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium bifidum JCM 1255, Bifidobacterium longum subsp. infantis JCM 1222, Bifidobacterium pseudolongum JCM 1255, Bifidobacterium longum subsp. infantis JCM 1222, Bifidobacterium pseudolongum JCM 1255, Bifidobacterium longum subsp. infantis JCM 1222, Bifidobacterium long ... Lactococcus lactis subsp. lactis (Lactococcus pseudolongum) JCM 1205, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 7963, Lactococcus lactis subsp. lactis (Lactococcuslactis ATCC 7962, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 15346, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 13675, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 12929, Lactococcus lactis subsp. lactis (Lactococcus The composition according to any one of claims 1 to 5, wherein the bacterium is one or more bacteria selected from the group consisting of Lactococcus lactis subsp. lactis ATCC 11955, Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, and Lactobacillus rhamnosus CRL1505.

12. The composition according to any one of claims 1 to 7, wherein the bacteria comprises killed bacteria.

13. The composition of any one of claims 1 to 7, wherein the bacteria are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

14. The composition of claim 13, wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of the bacteria phagocytosed via BDCA2 binding is 2.0 times or more greater than the amount of Lactococcus lactis subsp. lactis ATCC15577 mediated by BDCA2 binding.

15. The composition according to claim 14, wherein the amount of phagocytosis not mediated by binding to BDCA2 is the amount of the bacteria phagocytosis by the pDC in a state in which BDCA2 of the pDC cannot bind to a ligand.

16. The composition according to claim 13, wherein the bacteria promote the production of IFN-α in the pDCs through phagocytosis by the pDCs.

17. The composition according to any one of claims 1 to 7, which is a food composition, a pharmaceutical composition or a feed.

18. The number of bacteria in the food composition, pharmaceutical composition, or feed per unit package is 1 x 10 ８ 18. The composition of claim 17, wherein the number of

19. A method for screening for bacteria having immunostimulatory activity, the method comprising a step of screening for the bacteria having immunostimulatory activity using binding to blood dendritic cell antigen 2 (BDCA2) as an index.

20. 20. The method of claim 19, wherein the screening step is carried out by a binding assay between the candidate bacterium and BDCA2.

21. A method for enhancing the immunostimulatory ability and / or phagocytosis ability of bacteria by plasmacytoid dendritic cells (pDC), comprising a step of culturing bacteria in M17 medium to which glucose has been added at a final concentration of 0.1 to 10% by mass.

22. A method for producing an immunostimulatory composition containing Lactococcus lactis subsp. lactis JCM 5805 as an active ingredient, the method comprising the step of culturing Lactococcus lactis subsp. lactis JCM 5805 in an M17 medium to which glucose has been added at a final concentration of 0.1 to 10% by mass.

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