Immunostimulatory composition

Abstract

Disclosed is an immunostimulatory composition containing, as an active ingredient, bacteria which bond to BDCA2 and / or bacteria which are phagocytosed by plasmacytoid dendritic cells (pDCs) to at least a prescribed degree.

Description

Immunostimulating composition

[0001] The present disclosure relates to immunostimulatory compositions.

[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, as well as lactic acid bacteria with immunomodulatory activity obtained by the method and an immunomodulatory composition containing the lactic acid bacteria.

[0003] JP 2017-201984 A JP 2014-217372 A

[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.

[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 to, for example, the following items. [A0] A composition that satisfies at least one selected from the group consisting of the following (1) to (4): (1) comprising, as an active ingredient, 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 (Lactococcus lactis.subsp.lactis) ATCC15577 phagocytosed by pDCs, and wherein the composition is an immunostimulatory composition; (2) comprising, as an active ingredient, a bacterium that binds to blood dendritic cell antigen 2 (BDCA2), and wherein the composition is an immunostimulatory composition; (3) comprising, as an active ingredient, a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the bacterium satisfies the following requirement X, and wherein the composition is an immunostimulatory composition; [Requirement X] final concentration of 2.0 x 10 ５ (3) a culture medium containing Lactococcus lactis subsp. lactis JCM 5805 as a bacterium, wherein pDCs at a final concentration of 1.0 × 10 cells / mL 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 pre-cultured in a serum-free medium; (4) a culture medium 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] a final concentration of 2.0 × 10 ５cells / 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 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. [A1] An immunostimulating composition comprising, as an active ingredient, a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacterium phagocytosed by pDCs is at least 2.0 times the amount of Lactococcus lactis subsp. lactis (Lactococcus lactis.subsp.lactis) ATCC 15577 phagocytosed by pDCs. [A2] An immunostimulating composition comprising, as an active ingredient, a bacterium that binds 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 (Lactococcus lactis.subsp.lactis) ATCC 15577 to BDCA2. [A4] The composition according to [A3], wherein the binding ability to BDCA2 is evaluated based on the percentage of bacteria bound to BDCA2 as assessed by a binding assay. [A5] An immunostimulatory composition comprising, as an active ingredient, bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the bacteria satisfy the following requirement X: [Requirement X] final concentration of 2.0 x 10 ５[A6] A composition containing Lactococcus lactis subsp. lactis JCM 5805 as the bacterium, wherein pDCs at a final concentration of 1.0 × 10 cells / mL 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, and the pDCs are pDCs that have been pre-cultured in a serum-free medium. ５lactis 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 contacted pDCs is 15.0% or more, provided that the pDCs have been pre-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 bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [A9] 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, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [A10] The composition according to any one of [A0] to [A5], wherein the bacterium is Lactococcus lactis subsp. lactis. [A11] 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 13064. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (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 bifidum JCM 1255, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11454,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 11007 and Lactobacillus rhamnosus CRL 1505. [A12] The composition according to any one of [A0] to [A11], wherein the bacterium comprises a killed bacterium. [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 according to [A13], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of the bacteria phagocytized via BDCA2 binding is 2.0-fold or more of the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosis mediated by BDCA2 binding. [A15] The composition according to [A14], wherein the amount of the bacteria phagocytized not mediated by BDCA2 binding is the amount of the bacteria phagocytosis by the pDCs when BDCA2 of the pDCs is in a state where it cannot bind to a ligand. [A16] The composition according to any one of [A13] to [A15], wherein the bacteria promotes IFN-α production 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] In the food composition, pharmaceutical composition, or feed, the number of bacteria of the bacterium per unit package is 1 x 10, ８[B0] A method for stimulating immunity in a subject, comprising having the subject ingest or administering to a subject in need thereof bacteria that satisfy 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) a bacterium that binds to blood dendritic cell antigen 2 (BDCA2); (3) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) and satisfies the following requirement X; [Requirement X] a final concentration of 2.0 x 10 ５ (3) a bacterium in which pDCs at a final concentration of 1.0 × 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been pre-cultured in a serum-free medium; (4) the bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805, and satisfies the following requirement Y; [Requirement Y] final concentration of 2.0 × 10 ５cells / 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 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. [B1] A method for stimulating the immunity of a subject, comprising having the subject ingest or administering to a subject in need thereof bacteria that can be phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacteria phagocytosed by the pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC 15577 that can be phagocytosed by the pDCs. [B2] A method for stimulating the immunity of a subject, comprising having the subject ingest 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 BDCA2-binding ability of the bacterium is greater than that of Lactococcus lactis subsp. lactis ATCC 15577. [B4] The method according to [B3], wherein the BDCA2-binding ability is evaluated based on the proportion of bacteria bound to BDCA2 as assessed in a binding assay. [B5] A method for stimulating the immunity of a subject, comprising ingesting or administering to a subject in need thereof bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the bacterium satisfies the following requirement X: [Requirement X] a final concentration of 2.0 x 10 ５[B6] A method for stimulating the immunity of a subject, comprising administering Lactococcus lactis subsp. lactis JCM 5805 as a bacterium to the subject or administering the same to a subject in need thereof, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration of 2.0 x 10 ５lactis 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, provided that the pDCs have been pre-cultured in a serum-free medium. [B7] The method according to any one of [B0] to [B5], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [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 Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [B9] The method according to any one of [B0] to [B5], wherein the bacterium is Lactococcus lactis subsp. lactis. [B10] 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 13526. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (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 bifidum JCM 1255, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11454,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 11007 and Lactobacillus rhamnosus CRL 1505. [B11] The method according to any one of [B0] to [B10], wherein the bacterium comprises 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 according to [B12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of the bacteria phagocytized via BDCA2 binding is 2.0-fold or more of the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosis mediated by BDCA2 binding. [B14] The method according to [B13], wherein the amount of the bacteria phagocytized not mediated by BDCA2 binding is the amount of the bacteria phagocytosis by the pDCs in a state in which BDCA2 of the pDCs cannot bind to a ligand. [B15] The method according to any one of [B12] to [B14], wherein the bacteria promotes IFN-α production 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 food composition, pharmaceutical composition, or feed contains the bacterium at a cell count of 1 x 10 per unit package. ８[C0] A method for stimulating immunity in a subject, comprising having the subject ingest or administering to a subject in need thereof a composition containing 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) a bacterium that binds to blood dendritic cell antigen 2 (BDCA2); (3) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) and satisfies the following requirement X; [Requirement X] a final concentration of 2.0 x 10 ５ (3) a bacterium in which pDCs at a final concentration of 1.0 × 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been pre-cultured in a serum-free medium; (4) the bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805, and satisfies the following requirement Y; [Requirement Y] final concentration of 2.0 × 10 ５cells / 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 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. [C1] 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 bacteria that can be 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 ATCC 15577 that is phagocytosed by pDCs. [C2] 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 bacteria that bind to blood dendritic cell antigen 2 (BDCA2). [C3] The method according to [C0] or [C2], wherein the BDCA2-binding ability of the bacterium is greater than that of Lactococcus lactis subsp. lactis ATCC 15577. [C4] The method according to [C3], wherein the BDCA2-binding ability is evaluated based on the proportion of bacteria bound to BDCA2 assessed in a binding assay. [C5] A method for stimulating the immunity of 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 bacterium satisfies the following requirement X: [Requirement X] A final concentration of 2.0 x 10 ５[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 the bacterium, wherein pDCs at a final concentration of 1.0 × 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, and the pDCs are pDCs that have been pre-cultured in a serum-free medium. ５lactis 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, provided that the pDCs have been pre-cultured in a serum-free medium. [C7] The method according to any one of [C0] to [C5], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [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 Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [C9] The method according to any one of [C0] to [C5], wherein the bacterium is Lactococcus lactis subsp. lactis. [C10] 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 13064. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (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 bifidum JCM 1255, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11454,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 11007 and Lactobacillus rhamnosus CRL 1505. [C11] The method according to any one of [C0] to [C10], wherein the bacterium comprises a killed bacterium. [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 according to [C12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of the bacteria phagocytosis mediated by BDCA2 binding is 2.0-fold or more of the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosis mediated by BDCA2 binding. [C14] The method according to [C13], wherein the amount of the bacteria phagocytosis not mediated by BDCA2 binding is the amount of the bacteria phagocytosis by the pDCs in a state in which BDCA2 of the pDCs cannot bind to a ligand. [C15] The method according to any one of [C12] to [C14], wherein the bacteria promotes IFN-α production in the pDCs via phagocytosis by the pDCs. [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 food composition, pharmaceutical composition, or feed contains the bacterium in an amount of 1 x 10 per unit package. ８[D0] Bacteria for use in immune stimulation, which satisfy at least one selected from the group consisting of the following (1) to (4): (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDC), 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; (2) a bacterium that binds to blood dendritic cell antigen 2 (BDCA2); (3) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDC) and satisfies the following requirement X; [Requirement X] a final concentration of 2.0 x 10 ５ (3) a bacterium in which pDCs at a final concentration of 1.0 × 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been pre-cultured in a serum-free medium; (4) the bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805, and satisfies the following requirement Y; [Requirement Y] final concentration of 2.0 × 10 ５cells / 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 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. [D1] A bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) for use in immune stimulation, wherein the amount of the bacterium phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosed by pDCs. [D2] A bacterium that binds to blood dendritic cell antigen 2 (BDCA2) for use in immune stimulation. [D3] The bacterium for use according to [D0] or [D2], wherein the BDCA2-binding ability of the bacterium is greater than the BDCA2-binding ability of Lactococcus lactis subsp. lactis ATCC 15577. [D4] The bacterium for use according to [D3], wherein the BDCA2-binding ability is evaluated based on the proportion of bacteria bound to BDCA2 as assessed in a binding assay. [D5] The 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 of 2.0 x 10 ５Lactococcus lactis subsp. lactis JCM 5805 for use in immune stimulation, wherein pDCs at a final concentration of 1.0 × 10 cells / mL 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, and the pDCs are pDCs that have been pre-cultured in a serum-free medium. [D6] Lactococcus lactis subsp. lactis JCM 5805 for use in immune stimulation, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0 × 10 ５lactis 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, provided that the pDCs have been pre-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 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. [D9] The bacterium for use according to any one of [D0] to [D5], wherein the bacterium is Lactococcus lactis subsp. lactis. [D10] 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 13526. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (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 bifidum JCM 1255, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11454,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 11007 and Lactobacillus rhamnosus CRL 1505. [D11] The bacterium for use according to any one of [D0] to [D10], which comprises killed bacteria. [D12] The bacterium for use according to any one of [D0] to [D11], which is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2). [D13] The bacterium for use according to [D12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of the bacteria phagocytized via BDCA2 binding is 2.0-fold or more the amount of the bacteria phagocytized via BDCA2 binding in Lactococcus lactis subsp. lactis ATCC 15577. [D14] The bacterium for use according to [D13], wherein the amount of the bacteria phagocytized not mediated by BDCA2 binding is the amount of the bacteria phagocytized by the pDCs in a state in which BDCA2 of the pDCs cannot bind to a ligand. [D15] The bacterium for use according to any one of [D12] to [D14], wherein the bacterium promotes IFN-α production in the pDCs via phagocytosis by the pDCs. [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 food composition, pharmaceutical composition, or feed contains the bacterium in an amount of 1 x 10 per unit package. ８[E0] A composition for use in immune stimulation, comprising bacteria that satisfy 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) a bacterium that binds to blood dendritic cell antigen 2 (BDCA2); (3) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) and satisfies the following requirement X; [Requirement X] a final concentration of 2.0 x 10 ５ (3) a bacterium in which pDCs at a final concentration of 1.0 × 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been pre-cultured in a serum-free medium; (4) the bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805, and satisfies the following requirement Y; [Requirement Y] final concentration of 2.0 × 10 ５cells / 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 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. [E1] A composition for use in immune stimulation, comprising bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of the bacteria phagocytosed by the pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosed by the pDCs. [E2] A composition for use in immune stimulation, comprising bacteria that bind to blood dendritic cell antigen 2 (BDCA2). [E3] The composition for use according to [E0] or [E2], wherein the BDCA2-binding ability of the bacterium is greater than the BDCA2-binding ability of Lactococcus lactis subsp. lactis ATCC 15577. [E4] The composition for use according to [E3], wherein the BDCA2-binding ability 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 that can be phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the bacteria satisfy the following requirement X: [Requirement X] final concentration of 2.0 x 10 ５[E6] A composition for use in immune stimulation, comprising Lactococcus lactis subsp. lactis JCM 5805 as the bacterium, wherein pDCs at a final concentration of 1.0 × 10 cells / mL 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 pre-cultured in a serum-free medium. [E7] A composition for use in immune stimulation, comprising Lactococcus lactis subsp. lactis JCM 5805 as the bacterium, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Final concentration 2.0 × 10 ５lactis 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 contacted pDCs is 15.0% or more, provided that the pDCs have been pre-cultured in a serum-free medium. [E7] The composition for use according to any one of [E0] to [E5], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [E8] 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, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus. [E9] The composition for use according to any one of [E0] to [E5], wherein the bacterium is Lactococcus lactis subsp. lactis. [E10] 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 13526. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (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 bifidum JCM 1255, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11454,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 11007 and Lactobacillus rhamnosus CRL 1505. [E11] The composition for use according to any one of [E0] to [E10], wherein the bacterium comprises killed bacteria. [E12] The composition for use according to 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] The composition for use according to [E12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of the bacteria phagocytosis mediated by BDCA2 binding is 2.0-fold or more the amount of the bacteria phagocytosis mediated by BDCA2 binding in Lactococcus lactis subsp. lactis ATCC 15577. [E14] The composition for use according to [E13], wherein the amount of the bacteria phagocytosis not mediated by BDCA2 binding is the amount of the bacteria phagocytosis by the pDCs in a state in which BDCA2 of the pDCs cannot bind to a ligand. [E15] The composition for use according to any one of [E12] to [E14], wherein the bacteria promotes IFN-α production in the pDCs via phagocytosis by the pDCs. [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 food composition, pharmaceutical composition, or feed contains the bacterium in an amount of 1 x 10 per unit package. ８[F0] Use of bacteria in immune stimulation, which 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) a bacterium that binds to blood dendritic cell antigen 2 (BDCA2); (3) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs) and satisfies the following condition X; [Requirement X] a final concentration of 2.0 x 10 ５ (3) a bacterium in which pDCs at a final concentration of 1.0 × 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been pre-cultured in a serum-free medium; (4) the bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805, and satisfies the following requirement Y; [Requirement Y] final concentration of 2.0 × 10 ５cells / 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 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. [F1] Use of bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) in immune stimulation, wherein the amount of the bacteria phagocytosed by pDCs is 2.0 times or more the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosed by pDCs. [F2] Use of bacteria that bind to blood dendritic cell antigen 2 (BDCA2) in immune stimulation. [F3] The use according to [F0] or [F2], wherein the binding ability of the bacteria to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC 15577 to BDCA2. [F4] The use according to [F3], wherein the binding ability to BDCA2 is evaluated based on the percentage of bacteria bound to BDCA2 as assessed by a binding assay. [F5] Use of bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) in immune stimulation, wherein the bacteria satisfy the following requirement X: [Requirement X] final concentration of 2.0 x 10 ５[F6] A use of the bacterium Lactococcus lactis subsp. lactis JCM 5805 in immune stimulation, wherein pDCs at a final concentration of 2.0 x 10 cells / mL 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, and the pDCs are pDCs that have been pre-cultured in a serum-free medium. ５lactis 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, provided that the pDCs have been pre-cultured in a serum-free medium. [F7] The use according to any one of [F0] to [F5], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [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 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 13526. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (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 bifidum JCM 1255, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11454,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 11007 and Lactobacillus rhamnosus CRL 1505. [F11] The use according to any one of [F0] to [F10], wherein the bacterium comprises a killed bacterium. [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 according to [F12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of the bacteria phagocytized via BDCA2 binding is 2.0-fold or more the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosis mediated by BDCA2 binding. [F14] The use according to [F13], wherein the amount of the bacteria phagocytized not mediated by BDCA2 binding is the amount of the bacteria phagocytosis by the pDCs in a state in which BDCA2 of the pDCs cannot bind to a ligand. [F15] The use according to any one of [F12] to [F14], wherein the bacteria promotes IFN-α production in the pDCs via phagocytosis by the pDCs. [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 food composition, pharmaceutical composition, or feed contains the bacterium at a cell count of 1 x 10 per unit package. ８[F16] The use according to [F16], wherein the bacterium is a bacterium containing at least one of the following bacteria: [F18] The use according to any one of [F0] to [F17], wherein the bacterium is a bacterium containing at least one of the following bacteria: [G0] Use of a composition containing a bacterium containing at least one of the following bacteria: [G16] The use according to [F16], wherein the bacterium is a bacterium containing at least one of the following bacteria: [F18] The use according to any one of [F0] to [F17], wherein the bacterium is a bacterium containing at least one of the following bacteria: [G16] The use according to [F16], wherein the bacterium is a bacterium containing at least one of the following bacteria: [F16] The use according to any one of [F0] to [F17], wherein the bacterium is a bacterium containing at least one of the following bacteria: [G16] The use according to [F16], wherein the bacterium is a bacterium containing at least one of the following bacteria: [G16] The use according to any one of [F0] to [F17 ... ５ (3) a bacterium in which pDCs at a final concentration of 1.0 × 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been pre-cultured in a serum-free medium; (4) the bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805, and satisfies the following requirement Y; [Requirement Y] final concentration of 2.0 × 10 ５cells / 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 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. [G1] Use in immune stimulation of a composition containing 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 ATCC 15577 phagocytosed by pDCs. [G2] Use in immune stimulation of a composition containing bacteria that binds to blood dendritic cell antigen 2 (BDCA2), wherein the binding ability of the bacteria to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC 15577. [G3] The use according to [G0] or [G2], wherein the binding ability of the bacteria to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC 15577 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 immune stimulation, wherein the bacteria satisfy the following requirement X: [Requirement X] final concentration of 2.0 x 10 ５[G6] Use of a composition containing the bacterium Lactococcus lactis subsp. lactis JCM 5805 in immune stimulation, wherein pDCs at a final concentration of 2.0 x 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, and the pDCs are pDCs that have been pre-cultured in a serum-free medium. ５Lactococcus lactis subsp. lactis JCM 5805 at a final concentration of 10 μg / mL was contacted with pDCs at a concentration of 10 μg / mL in a serum-free medium for 24 hours, and the ratio of the number of pDCs that phagocytosed Lactococcus lactis subsp. lactis JCM 5805 to the total number of contacted pDCs was 15.0% or more, provided that the pDCs had been pre-cultured in a serum-free medium. [G7] The use according to any one of [G0] to [G5], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [G8] 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, 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 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 13526. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (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 bifidum JCM 1255, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11454,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 11007 and Lactobacillus rhamnosus CRL1505. [G11] The use according to any one of [G0] to [G10], wherein the bacterium comprises a killed bacterium. [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 according to [G12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of the bacteria phagocytized via BDCA2 binding is 2.0-fold or more of the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosis mediated by BDCA2 binding. [G14] The use according to [G13], wherein the amount of the bacteria phagocytized not mediated by BDCA2 binding is the amount of the bacteria phagocytosis by the pDCs in a state in which BDCA2 of the pDCs cannot bind to a ligand. [G15] The use according to any one of [G12] to [G14], wherein the bacteria promotes IFN-α production in the pDCs via phagocytosis by the pDCs. [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 food composition, pharmaceutical composition, or feed contains the bacterium in an amount of 1 x 10 per unit package. ８[G16] The use according to [G16], wherein the bacterium is a bacterium containing 2 or more bacteria. [G18] The use according to any one of [G0] to [G17], wherein the bacterium is a bacterium containing 2 or more bacteria. [G18] The use according to any one of [G0] to [G17], wherein the bacterium is a bacterium containing 2 or more bacteria. [H0] Use of a bacterium that satisfies at least one of the following (1) to (4) in the manufacture of an immunostimulatory composition: (1) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDC), 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; (2) a bacterium that binds to blood dendritic cell antigen 2 (BDCA2); (3) a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDC) and satisfies the following requirement X; [Requirement X] a final concentration of 2.0 x 10 ５ (3) a bacterium in which pDCs at a final concentration of 1.0 × 10 cells / mL are contacted with the bacterium 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 bacterium to the total number of pDCs that have been contacted is 2.5% or more, provided that the pDCs have been pre-cultured in a serum-free medium; (4) the bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805, and satisfies the following requirement Y; [Requirement Y] final concentration of 2.0 × 10 ５cells / 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 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. [H1] Use of bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) in the production of an immunostimulatory composition, wherein the amount of the bacteria phagocytosed by pDCs is 2.0-fold or more the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosed by pDCs. [H2] Use of bacteria that bind to blood dendritic cell antigen 2 (BDCA2) in the production of an immunostimulatory composition. [H3] The use according to [H0] or [H2], wherein the binding ability of the bacteria to BDCA2 is greater than the binding ability of Lactococcus lactis subsp. lactis ATCC 15577 to BDCA2. [H4] The use according to [H3], wherein the binding ability to BDCA2 is evaluated based on the percentage of bacteria bound to BDCA2 as assessed in a binding assay. [H5] Use of bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) in the manufacture of an immunostimulatory composition, wherein the bacteria satisfy the following requirement X: [Requirement X] final concentration of 2.0 x 10 ５[H6] Use of the bacterium Lactococcus lactis subsp. lactis JCM 5805 in the manufacture of an immunostimulatory composition, wherein pDCs at a final concentration of 2.0 x 10 cells / mL 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, and the pDCs are pDCs that have been pre-cultured in a serum-free medium. ５lactis 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, provided that the pDCs have been pre-cultured in a serum-free medium. [H7] The use according to any one of [H0] to [H5], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [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 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 13064. 9695, Streptococcus salivarius (Streptococcus salivarius) JCM 5707, Enterococcus lactis (Enterococcus lactis) JCM 30200, Oenococcus oeni (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 bifidum JCM 1255, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium pseudolongum JCM 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11454,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 11007 and Lactobacillus rhamnosus CRL1505. [H11] The use according to any one of [H0] to [H10], wherein the bacterium comprises a killed bacterium. [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 according to [H12], wherein the phagocytosis of the bacteria by the pDCs includes phagocytosis mediated by BDCA2 binding and phagocytosis not mediated by BDCA2 binding, and the amount of the bacteria phagocytized via BDCA2 binding is 2.0-fold or more the amount of Lactococcus lactis subsp. lactis ATCC 15577 phagocytosis mediated by BDCA2 binding. [H14] The use according to [H13], wherein the amount of the bacteria phagocytized not mediated by BDCA2 binding is the amount of the bacteria phagocytosis by the pDCs in a state in which BDCA2 of the pDCs cannot bind to a ligand. [H15] The use according to any one of [H12] to [H14], wherein the bacteria promotes IFN-α production in the pDCs via phagocytosis by the pDCs. [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 food composition, pharmaceutical composition, or feed contains the bacterium in an amount of 1 x 10 per unit package. ８The use according to [H16], wherein the number of bacteria is 1 or more. [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 index. [I2] The method according to [I1], wherein the screening step is carried out by a binding assay between the candidate bacteria and BDCA2. [I3] The method according to [I1] or [I2], wherein the bacteria selected are 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 according to 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 pDC, characterized by binding of bacteria to BDCA2. [J2] A method for activating pDC, comprising ingesting bacteria that bind to BDCA2 into a subject or administering the same to a subject in need thereof. [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 a 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 bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [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 bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis.subsp. lactis).The method according to any one of [J1] to [J3] or the composition according to any one of [J4] to [J6]. [J8] 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 13064. 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 ... pseudolongum) JCM 1205,Lactococcus lactis subsp. lactis (Lactococcus lactis.subsp.lactis) ATCC 7963, Lactococcus lactis subsp. lactis (Lactococcus lactis.subsp.lactis) 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis.subsp.lactis) ATCC 11454, Lactococcus lactis subsp. lactis (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 bacterium is one or more bacteria selected from the group consisting of Lactobacillus sp. 11007, Lactobacillus rhamnosus CRL1505. [K1] A method according to any one of [K1] to [J3] or the composition according to any one of [K4] to [J8], wherein the bacterium comprises killed bacteria. [K2] The method according to [K1], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium. [K3] The bacteria are selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella,[K4] The method according to any one of [K1] to [K3], wherein the bacterium is one or more bacteria selected from the group consisting of Bifidobacterium and Lactobacillus. [K5] 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 13064. 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 1205, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, 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 lactis.subsp.lactis) ATCC 11955, Lactococcus lactis subsp. lactis (Lactococcus lactis.subsp.lactis) ATCC 11454, Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) ATCC 11007, and Lactobacillus rhamnosus CRL 1505. [K6] The method according to any one of [K1] to [K5], wherein the bacterium is Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805. [K7] The method according to any one of [K1] to [K6], wherein the immunostimulatory ability and / or the ability to be phagocytosed by plasmacytoid dendritic cells (pDC) is enhanced.Bacteria. [L1] A method for producing an immunostimulating composition containing Lactococcus lactis subsp. lactis JCM 5805 as an active ingredient, the method comprising culturing Lactococcus lactis subsp. lactis JCM 5805 in an M17 medium supplemented with glucose at a final concentration of 0.1 to 10% by mass. [L2] The production 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 produced by the production method according to [L1].

[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 bacterium, which is an 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 bacterium, which is an 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 bacterium, which is an active ingredient, to BDCA2 on the surface of pDCs, which is phagocytosed by dendritic cells, thereby 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 index.

[0010] 1 is a diagram showing the fluorescence detection area 22 hours after the addition of LC-Plasma in Example 1. FIG. 2 is a diagram showing the measurement results of IFN-α concentration in the culture supernatant recovered 22 hours after the addition of LC-Plasma in Example 1. FIG. 3 is a diagram showing the fluorescence detection area 22 hours after the addition of lactic acid bacteria for eight types of lactic acid bacteria tested in Example 2. FIG. 4 is a diagram showing the measurement results of IFN-α concentration in the culture supernatant recovered 22 hours after the addition of lactic acid bacteria for eight types of lactic acid bacteria tested in Example 2. FIG. 5 is a diagram showing 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 eight types of lactic acid bacteria tested in Example 2. FIG. 6 is a diagram showing the fluorescence detection area 22 hours after the addition of LC-Plasma for the PL group, PL + Isotype group, and PL + BDCA2 anti group in Example 3. 1 shows the results of measuring the IFN-α concentration in culture supernatants collected 22 hours after the addition of LC-Plasma for the PL group, PL + isotype group, and PL + BDCA2 anti group in Example 3. 2 shows the fluorescence detection area 22 hours after the addition of various lactic acid bacteria for the isotype group and BDCA2 anti group in Example 4. 3 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 BDCA2 anti group in Example 4. 4 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.

[0033] Figure 1 shows the percentage of LC-Plasma bound to BDCA2 at BDCA2 concentrations of 1, 5, 10, 20, or 50 μg / mL, evaluated by BDCA2 binding assay in Example 5. Figure 2 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 3 shows 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 4 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.

[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 a composition for immunostimulation comprising a bacterium as an active ingredient. In one aspect, the bacterium contained as an active ingredient in the composition for immunostimulation is a bacterium that is phagocytosed by pDC. In one aspect, the bacterium contained as an active ingredient in the composition for immunostimulation is a bacterium that binds to BDCA2.

[0013] Hereinafter, "an immunostimulating composition containing, as an active ingredient, bacteria that are phagocytosed by pDC" and / or "an immunostimulating composition containing, as an active ingredient, bacteria that bind to BDCA2" may be referred to as "the immunostimulating composition of the present 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, the bacteria 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 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 Lactobacillus or Lactococcus genus.

[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 genus Lactobacillus, the following genera have been newly 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, Secundilactobacillus, and the like.

[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 bacteria 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. Examples include 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 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 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 bacteria of the genus Enterococcus include Enterococcus alcedinis and Enterococcus faecalis. Specific examples of bacteria of the genus Enterococcus 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, Lactobacillus bulgaricus, and Lactobacillus parakefii. 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, and Lactobacillus bulgaricus. OLL1073R-1, Lactobacillus parakeefili (newly classified as Lentilactobacillus parakeefili) JCM 8573, Lactobacillus plantarum (newly classified as Lactipranchibacillus plantarum) L-137, Lactobacillus pentosus (newly classified as Lactipranchibacillus 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] The Bacillus bacteria are not particularly limited, but include, for example, Bacillus coagulans, etc. Specific examples of Bacillus bacteria 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 the genus Akkermansia include Akkermansia muciniphila JCM 30893.

[0035] Examples of the genus Bacteroides 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, Bacteroides uniformis JCM 13287, and Bacteroides uniformis JCM 13288.

[0036] Examples of Blautia include Blautia acetigens JCM 34803T, Blautia ammoniilytica JCM 34802T, Blautia algi JCM 31394T, Blautia caekimuris JCM 34498T, Blautia coccoides JCM 1395T, Blautia faeces JCM 17205T, Blautia glucellacea JCM 17039T, Blautia hansenii JCM 14655, Blautia hansenii JCM 35484, Blautia hominis JCM 32276T, Blautia hydrogenotrophica JCM 31266, and Blautia liquorice JCM 34498T. 34225T, Blautia luti JCM 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 bacteria of the genus Clostridium include Clostridium butyricum JCM NT, Clostridium neccille JCM 31500T, and Clostridium symbiosum JCM 1297T.

[0038] Examples of Collinsella bacteria 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 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 the genus Faecalicatena include Faecalicatena oroticum JCM 1429T.

[0042] Examples of the fungi of the genus Lacrimispora include Lacrimispora celerecrescens 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 the Parabacteroides genus include Parabacteroides meldae JCM 9497T.

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

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

[0047] Lactobacillus paracasei KW3110 is an international depositary authority under the Budapest Treaty for the Deposit of Patent Microorganisms, the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (Central No. 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, Japan) (currently the National Institute of Technology and Evaluation (NITE-IPOD) Patent Organism Depositary (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture, Japan)) under the accession number FERM BP-08634 (deposit date: February 20, 2004). A derivative strain of Lactobacillus paracasei KW3110 has been deposited at the same Patent Organism Depositary as FERM BP-08635 (deposit date: February 20, 2004).

[0048] As mentioned above, Lactococcus lactis subsp. lactis JCM 5805 can be obtained from the Microbial Materials Development Laboratory of the RIKEN BioResource Center, but the present invention can use the same strain of Lactococcus lactis subsp. lactis JCM 5805 stored in a collection institution other than the Microbial Materials Development Laboratory 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 City, Chiba Prefecture, Japan), the Strain Collection of Tokyo University of Agriculture (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, Japan), the American Type Culture Collection (USA), etc. 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 any property capable of achieving the objective of the present technology (e.g., immunostimulatory ability 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 the like. Interferon α (IFN-α) and interferon β (IFN-β) are known as representative type I interferons, and these are thought to have immunostimulatory activity.

[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 pDC 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 pDC can be assessed by observing a decrease in bacterial uptake by pDC when pDC phagocytosis is inhibited. For example, if the efficiency of uptake by pDC 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 pDC in which polymerization has not been inhibited, the bacterium can be assessed as being phagocytosed by pDC. Furthermore, if the efficiency of uptake by pDC 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 pDC 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 pDC. The efficiency of bacterial uptake by pDC can be measured, for example, by the amount of uptake at a predetermined time point after adding the bacterium to pDC.

[0055] The bacterium of this embodiment may be a bacterium whose amount of 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 of phagocytosis by pDC of Lactococcus lactis subsp. lactis (Lactococcus lactis. subsp. lactis) JCM 5805. Furthermore, the bacterium according to this embodiment may be a bacterium in which the 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 the amount of phagocytosis by pDC of Lactococcus lactis subsp. lactis (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, as described below. 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 ATCC 15577) are contacted with pDCs of 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 (e.g., μm ) per unit area of ​​the observation field of a fluorescence microscope 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 pDC of the bacteria according to this embodiment is evaluated by an evaluation method using bacteria labeled with a fluorogenic or fluorescent dye, which will be described later, the amount of phagocytosis by pDC is 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 900 μm ２ / image or more, where "image" is 0.572 mm ２ This means the field of view.

[0057] Furthermore, the difference between the amount of phagocytosis by the pDC of the bacterium according to this embodiment and the amount of phagocytosis by the pDC of Lactococcus lactis subsp. lactis ATCC 15577 was 1 μm ２ / image or more, 20μm ２ / image or more, 50μm 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 900 μm ２ It may be / image or more.

[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 ２ When the amount of phagocytosis by pDC was evaluated using the index of TII, the amount of phagocytosis by pDC 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 more or 250,000 OCU x μm ２ / Image or more.

[0059] In these cases, the amount of phagocytosis by pDC is not particularly limited as long as it is an index value indicating the amount of phagocytosis by pDC, and the amount of phagocytosis by pDC can be evaluated using methods commonly used by those skilled in the art that can measure the amount of phagocytosis. For example, the amount of phagocytosis by pDC can be measured as the fluorescence intensity of pDC after contacting bacteria labeled with a fluorescent or fluorescent dye with the bacteria. As a more detailed example, the amount of phagocytosis by pDC can be evaluated using the fluorescence detected from pDC when bacteria labeled with a fluorescent dye (e.g., pHrodo Red SE, Invitrogen, P36600) that becomes highly fluorescent in the acidic environment of the endosome are contacted with the pDC 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 the area 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 the fluorescence intensity obtained by flow cytometry analysis or the percentage of cells contained within a gate when gating on the 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 treated 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 a detailed example under the heading "Evaluation method using bacteria labeled with a fluorescent dye." 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 a serum-free medium (for example, a medium prepared by adding penicillin / streptomycin (Gibco, 15140-12) to a final concentration of 1.0% by volume to RPMI-1640 medium (Sigma, R8758)). 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, cultured for 16 hours, and then collected by pipetting. The collected CAL-1 cells were then transferred to the same medium at a concentration of 2.0 × 10 ５ The cells were resuspended at a concentration of 100 μg / mL and seeded into each well of a 96-well microplate at 200 μL / well. Bacteria stained with pHrodo Red SE (Invitrogen, P36600) by the method 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] (Staining of bacteria 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 the 10.2 mM pHrodo Red SE prepared as described above was added. The bacteria were dispersed by vortexing, then 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 room temperature for 2 minutes at 20,000 × g. After discarding 800 μL of the supernatant, add 1.5 mL of PBS and vortex to completely suspend the precipitate. After another centrifugation at 20,000 × g for 2 minutes at room temperature, discard 1.5 mL of the supernatant. Add 140 μL of PBS to suspend the bacteria, creating a 10 mg / mL bacterial solution. Store the solution at 4°C, shielded from light with aluminum foil, until use. Dilute the stained bacteria 10-fold with PBS immediately before use to create a 1 mg / mL bacterial solution. The stained bacterial solution should be used within 24 hours of preparation.

[0064] (Evaluation of phagocytosis amount) The entire visual field (0.572 mm) to be evaluated was obtained from each well of the plate. ２ The amount of bacteria phagocytized by CAL-1 cells is quantified using the total area (fluorescence detection area) where the fluorescence of pHrodo Red SE is detected among the total area (fluorescence detection area) of the whole area. ２ Fluorescence detection area (e.g., μm ２ / image) can be analyzed to evaluate the amount of phagocytosis. At this time, since the stained bacteria 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 and fluoresce beyond the threshold value even if they are not phagocytosed. Therefore, the influence of excessively large aggregates is eliminated by setting the Area of ​​Filters in the Orange Channel to 20.

[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 x 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 a predetermined ratio, where the pDCs are pDCs that have been previously cultured in a serum-free medium.

[0066] The specified percentage related to 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 ratios for 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%, 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 more Below, 6.5% to 18.0%; 7.0% to less than 100%; 7.0% to 50.0%; 7.0% to 25.0%; 7.0% to 18.0%; 7.5% to less than 100%; 7.5% to 50.0%; 7.5% to 25.0%; 7.5% to 18.0%; 8.0% to less than 100%; 8.0% to 50.0%; 8.0% to 25.0%; 8.0% to 18.0%; 9 0% or more and less than 100%, 9.0% or more and less than 50.0%, 9.0% or more and less than 25.0%, 9.0% or more and less than 18.0%, 9.5% or more and less than 100%, 9.5% or more and less than 50.0%, 9.5% or more and less than 25.0%, 9.5% or more and less than 18.0%, 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 50.0% or less, 10.5% or more and 25.0% or less, 10.5% or more and 18.0% or less, 11.0% or more and less than 100%, 11.0% or more and 50.0% or less, 11.0% or more and 25.0% or less, 11.0% or more and 18.0% or less, 11.5% or more and less than 100%, 11.5% or more and 50.0% or less, 11.5% or more and 25.0% or less, 11.5% or more and 18.0% or less, 12.0% or more and less than 100%, 12.0% or more and 50.0% or less, 12.0% or more and 25.0% or less, 12.0% or more and 18.0% or less, 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 %, 14.0% or more and 18.0% or less, 14.5% or more and less than 100%, 14.5% or more and 50.0% or less, 14.5% or more and 25.0% or less, 14.5% or more and 18.0% or less, 15.0% or more and less than 100%, 15.0% or more and 50.0% or less, 15.0% or more and 25.0% or less, 15.0% or more and 18.0% or less, 15.5% or more and less than 100%, 15.5% or more and 50.0% or less, 15.5% or more and 25.0% or less, 15.5% or more and 18.0% or less, 16.0% or more and less than 100%, 16.0% or more and 50 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 a serum-free medium (pretreatment step), contacting the pDCs obtained in the pretreatment step with bacteria in a 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 satisfying requirement X0 generally refers to serum derived from humans or other animals that is used for culturing animal cells, including humans, and examples thereof include serum derived from humans, bovine serum, and equine serum, and more specifically, fetal bovine serum (FBS).

[0069] The medium satisfying requirement X0 can be the same as the medium (e.g., basal medium) typically 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 typically 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 in any desired manner, and 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 combined arbitrarily. 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) and DMEM medium (Dulbecco's Modified Eagle Medium). The prepared medium may be sterilized using an autoclave or the like before being used in the contact step. In one particular embodiment, the medium may be RPMI-1640 medium.

[0070] The culture in the pretreatment step may be performed under an environment in which a person skilled in the art would normally culture cells. In a specific embodiment, the culture may be performed at 37°C and 5% CO ２ The pretreatment step may be carried out in air with a humidity of 100%. pDCs are usually floating cells, but since the pretreatment step causes them to resemble adherent cells, 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 pDCs adhere 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 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 may 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 commonly used by those skilled in the art for evaluating 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 signal derived from bacteria is also detected to the number of particles or solutions in which a signal derived from pDC 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 a flow cytometry method, i.e., 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. More specifically, the ratio of the number of pDCs that phagocytosed bacteria to the total number of contacted pDCs may be determined, for example, according to the method performed in Example 8 of the present application. First, the bacteria used in the pDC contacting step are fluorescently labeled by contacting the bacteria with a fluorescent dye or by genetically expressing a fluorescent protein in a protein contained therein. 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). The fluorescent labeling of the bacteria or the proteins contained therein and the fluorescent labeling of the dead cells can be detected and distinguished from each other. Finally, the presence or absence of phagocytosis of bacteria by pDCs is evaluated using a flow cytometer. Measurements are performed according to standard methods. The evaluation of the measurement results is not particularly limited, but for example, first, clusters containing pDCs are selected using scattered light as an indicator, then, if necessary, clusters containing live pDCs are selected from among them as particles that have not been labeled as dead cells, and finally, the proportion of particles in the clusters in which fluorescence derived from the fluorescent dye that labeled the bacteria can be detected 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 bacteria relative to the total number of contacted pDCs" below corresponds to the "phagocytosis rate" evaluated in Example 9, which will be described later. 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 compositions for immunostimulation, but the compositions described in this embodiment can be used as the composition, and the composition according to this embodiment may also be, for example, a composition for immunostimulation. [Requirement Y0] Final concentration 2.0 x 10 ５ cells / mL of pDCs are contacted with 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 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.

[0077] The specified percentage related to 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 the requirement Y0 can be set to 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%, .0% or less, 11.5% or more and 25.0% or less, 11.5% or more and 18.0% or less, 12.0% or more and less than 100%, 12.0% or more and 50.0% or less, 12.0% or more and 25.0% or less, 12.0% or more and 18.0% or less, 12.5% ​​or more and less than 100%, 12.5% ​​or more and 50.0% or less, 12.5% ​​or more and 25.0% or less, 12.5% ​​or more and 18.0% or less, 13.0% or more and less than 100%, 13.0% or more and 50.0% or less, 13.0% or more and 25.0% or less, 13.0% or more and 18.0% or less, 13.5% or more and less than 100%, 13.5% or more and 50.0% or less, 13.5% or more and 2 5.0% or less, 13.5% to 18.0%, 14.0% to less than 100%, 14.0% to 50.0%, 14.0% to 25.0%, 14.0% to 18.0%, 14.5% to less than 100%, 14.5% to 50.0%, 14.5% to 25.0%, 14.5% to 18.0%, 15.0% to less than 100%, 15.0% to 50.0%, 15.0% to 25.0%, 15.0% to 18.0%, 15.5% to less than 100%, 15.5% to 50.0%, 15.5% to 25.0%, 15.5% or more 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 on 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. The bacterium's ability to bind to BDCA2 means that its BDCA2-binding ability is greater than that 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 in which various proteins are present on the cell surface). For example, this means that the binding efficiency to pDCs expressing BDCA2 on the cell surface is statistically significantly greater than the binding efficiency to pDCs not expressing BDCA2 or pDCs in which BDCA2 on the cell surface has been blocked with an anti-BDCA2 antibody or the like (e.g., p-value between the two groups is less than 0.05). "Specific binding of bacteria to BDCA2" means that the bacteria bind only to BDCA2 in a contaminated system containing BDCA2 (e.g., a system in which various proteins are present on the cell surface), excluding nonspecific adsorption; for example, this means that the binding efficiency to pDCs expressing BDCA2 on the cell surface is statistically significantly greater than the binding efficiency to pDCs not expressing BDCA2 or pDCs in which BDCA2 on the cell surface has been blocked with an anti-BDCA2 antibody or the like (e.g., p-value between the two groups is less than 0.01).

[0081] The binding of bacteria to BDCA2 can be evaluated by methods commonly used by those skilled in the art, such as a binding assay. The binding assay may be an in vitro method capable of evaluating the binding of bacteria to BDCA2 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 may be present in a free state in an aqueous medium, and such BDCA2 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. Furthermore, 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 for evaluating the binding ability of a substance (2), 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 the binding ability to BDCA2 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 based on the bacterial cell count may be determined to be bacteria that bind to BDCA2.

[0084] Furthermore, when the rate of bacteria bound to BDCA2 is evaluated as the BDCA2-binding ability by a binding assay of one embodiment described later, the difference when the BDCA2-binding ability of the bacterium according to this embodiment is subtracted from the BDCA2-binding ability of Lactococcus lactis subsp. lactis ATCC 15577 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. The binding assay of one embodiment includes a step of contacting BDCA2 with bacteria (contacting step) and a step of evaluating the binding of BDCA2 to the 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 the 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 the 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 may be 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 aspect, 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 contact 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 may be 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 combined in any desired manner. For example, the concentration of BDCA2 can be selected from the group consisting of 0.01 μg / mL to 5000 μg / mL, 0.01 μg / mL to 2000 μg / mL, 0.01 μg / mL to 1000 μg / mL, 0.01 μg / mL to 500 μg / mL, 0.01 μg / mL to 200 μg / mL, 0.1 μg / mL to 50 ... In a particular embodiment, the concentration of the bacteria in the contacting step may be 10 μg / mL or more.

[0089] The concentration ratio of the bacteria to BDCA2 in the contact step is not particularly limited as long as the binding between BDCA2 and the 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 aspect, these upper and lower limits can be combined in any desired manner. For example, the BDCA2 concentration [μg / mL] in the contacting step may 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 a specific embodiment, the concentration of BDCA2 [μg / mL] in the contacting step may be 1.0 times the concentration of the bacteria [μg / mL].

[0090] The contact conditions, such as time and temperature, in the contact step can be appropriately determined by those skilled in the art so that the binding between BDCA2 and bacteria can be evaluated in the subsequent contact step. For example, the contact time 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 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 to 24 hours or less, 1 minute to 6 hours or less, 1 minute to 2 hours or less, 10 minutes to 24 hours or less, 10 minutes to 6 hours or less, 10 minutes to 2 hours or less, 30 minutes to 24 hours or less, 30 minutes to 6 hours or less, or 30 minutes to 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 contacting step is the 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 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 origin of the bacteria).

[0092] The index of 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 (2), such as the index value, or may be a value of an index indicating the binding ability to BDCA2 for each bacterium (e.g., the percentage of bacteria bound to BDCA2, or the average or median number of labeled BDCA2s in a bacterium). In a specific embodiment, the index of binding between BDCA2 and bacteria 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 fluorescence at a wavelength corresponding to the fluorescent dye labeling the bacteria is observed, and in which a certain level of fluorescence intensity at a wavelength corresponding to the fluorescent dye labeling the BDCA2 is observed. As a criterion for exceeding a certain level, for example, when a chimeric protein of BDCA2 bound to an Fc region is used, a threshold can be set so that the percentage of a control having only the Fc region binding to each bacterium is 0.1% or less.

[0093] Thus, the binding assay of one embodiment can evaluate the binding ability of bacteria to BDCA2. Furthermore, the present inventors have found that bacteria that bind to BDCA2 have immunostimulatory activity. Thus, using BDCA2 binding as an index, the immunostimulatory activity of bacteria can be evaluated and bacteria having immunostimulatory activity can be screened, and such an index can be obtained by a binding assay. That is, one embodiment of the present invention is a method for screening bacteria having immunostimulatory activity, which includes a step of screening using BDCA2 binding as an index. 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 binding to BDCA2 is 0.1-fold or more, 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 than the amount of phagocytosis mediated by pDC binding to 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 binding to BDCA2 is 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 amount of phagocytosis mediated by pDC binding to BDCA2 of Lactococcus lactis subsp. lactis ATCC 15577.

[0096] Furthermore, when the amount of phagocytosis mediated by the binding of pDC to BDCA2 was evaluated by the evaluation method described later, the bacterium according to this embodiment had a phagocytosis amount of BDCA2 mediated by pDC binding of 10 μm 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 900 μm ２ As 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 mediated by BDCA2 binding to the pDC of the above bacteria and the amount of phagocytosis mediated by BDCA2 binding to the pDC of Lactococcus lactis subsp. lactis ATCC 15577 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 900 μm ２ As described above in (Evaluation of phagocytosis amount), the area per image is 0.572 mm ２ is set to

[0098] In one aspect, phagocytosis of bacteria by pDC includes phagocytosis mediated by binding to BDCA2 and phagocytosis not mediated by binding to BDCA2, and the amount of phagocytosis mediated by BDCA2 may be a predetermined factor or more of the amount of phagocytosis not mediated by binding to BDCA2. The 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 regard to phagocytosis of bacteria by pDC, the amount of phagocytosis mediated by BDCA2 of the total amount of phagocytosis may be, 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. Bacteria whose amount phagocytosis via binding to BDCA2 is equal to or greater than a predetermined multiple of the amount phagocytosis without binding to BDCA2 are easily phagocytosed by pDC via binding to BDCA2, and therefore have high pDC activation ability and high immunostimulatory ability.

[0099] In these cases, the amount of phagocytosis by pDC via binding to BDCA2 is not particularly limited as long as it is an index value indicating the amount of phagocytosis by pDC via binding to BDCA2. For example, the amount of phagocytosis by pDC via binding to BDCA2 and the amount of phagocytosis not mediated by binding to BDCA2 can be evaluated as follows. First, the amount of phagocytosis by untreated pDC (phagocytosis amount P) is evaluated. Next, the amount of phagocytosis by pDC 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 pDC 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 pDC via binding to BDCA2. That is, in one aspect, the amount of phagocytosis not mediated by binding to BDCA2 may be the amount of phagocytosis of bacteria by pDC in a state in which BDCA2 in pDC cannot bind to a ligand.

[0100] The treatment of suppressing BDCA2 in a state capable of binding to a ligand may be, for example, exposing pDC to an anti-BDCA2 antibody (blocking), exposing pDC to a BDCA ligand (competitive inhibition), knocking down or knocking out BDCA2 in pDC according to a conventional method, or introducing a mutation into BDCA2 in pDC according to a conventional method to lose its ligand-binding ability. In a preferred embodiment, the treatment of suppressing BDCA2 in a state capable of binding to a ligand may be exposing pDC to an anti-BDCA2 antibody. In this case, the state in which BDCA2 on the cell surface of pDC is bound to the anti-BDCA2 antibody corresponds to the state in which BDCA2 in pDC cannot bind to a 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 can be obtained from suppliers such as Miltenyi Biotec (product number: 130-090-690).

[0101] The amount of phagocytosis mediated by pDC binding to BDCA2 may be, as a detailed example, the amount of phagocytosis evaluated by the method described below as "Method for evaluating the amount of phagocytosis mediated by BDCA2 binding and the amount of phagocytosis not mediated by BDCA2 binding." As a more detailed example, the amount of phagocytosis mediated by pDC binding to BDCA2 may be the amount of phagocytosis evaluated by the method described in the Examples. [Method for evaluating the amount of phagocytosis mediated by binding with BDCA2 and the amount of phagocytosis not mediated by binding with BDCA2] CAL-1 cells were seeded in each well in the same manner as in the phagocytosis experiment described above in [Evaluation method using bacteria labeled with a fluorescent dye]. Bacteria and an anti-BDCA2 antibody (Miltenyi Biotec, 130-090-690), or bacteria and an isotype control of the same isotype and subclass as the anti-BDCA2 antibody (Miltenyi Biotec, 130-106-545), were added to the suspension at 10 μg / mL and incubated for 30 minutes. Thereafter, the fluorescence intensity of pHrodo Red SE was measured for each sample in the same manner as in the evaluation of the amount of phagocytosis described above in [Evaluation method using bacteria labeled with a fluorescent dye]. The fluorescence detection area per image (e.g., μm ２ Then, the fluorescence detection area when the anti-BDCA2 antibody is added is defined as the amount of phagocytosis Q, and the fluorescence detection area when the isotype control is added is defined as the amount of phagocytosis P, and the amount of phagocytosis mediated by binding to BDCA2 (P-Q) and the amount of phagocytosis not mediated by binding to BDCA2 (P) are 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 immunostimulating composition of this embodiment have high immunostimulating ability. The immunostimulating ability of the bacteria contained in the immunostimulating composition of this embodiment may be 0.01-fold or more, 0.03-fold or more, 0.05-fold or more, 0.07-fold or more, 0.09-fold or more, 0.1-fold or more, 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 of the immunostimulating ability of pDC of Lactococcus lactis subsp. lactis JCM 5805. Furthermore, the immunostimulatory ability 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 ability of Lactococcus lactis subsp. lactis ATCC 15577. In these cases, the immunostimulatory ability 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 ability, and in a preferred aspect, it may be the expression level of IFN-α.

[0104] As used herein, immunostimulatory ability (immunostimulatory ability) refers to the ability to stimulate cellular immunity and / or humoral immunity, and refers to the stimulatory (activating) effect of the bacteria according to this embodiment on the innate immune system of cells contacted with the bacteria or animals administered with the bacteria. In one aspect, the immunostimulatory ability may be a dendritic cell activation ability that activates the innate immune system of dendritic cells, and more specifically, may be a plasmacytoid dendritic cell activation ability (pDC activation ability). 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 pDC. 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 ability 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 obtained 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. Furthermore, 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 a pDC lysate and then measuring the amount of IFN-α in the lysate 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. For example, the Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science) can be used to measure the amount of IFN-α by ELISA. Alternatively, the amount of mRNA encoding IFN-α in pDCs (e.g., in CAL-1 cells) can be measured by preparing a pDC lysate by a method commonly used by those skilled in the art, and then measuring the amount of mRNA encoding IFN-α in the lysate by a method commonly used by those skilled in the art for quantifying mRNA. Quantification of mRNA may be performed, for example, by quantitative PCR (q-PCR) such as real-time PCR, direct digital counting (e.g., nCounter®), or a next-generation sequencer (NGS).

[0106] When the immunostimulatory ability of bacteria is evaluated using the expression level of IFN-α by pDCs after contact with the bacteria as an index, the index may be the expression level of IFN-α after a predetermined time (e.g., 3 to 168 hours, e.g., 22 hours) has elapsed since contacting pDCs (e.g., CAL-1 cells) with the bacteria in a serum-free medium (e.g., a medium prepared by adding penicillin / streptomycin (Gibco, 15140-12) to a final concentration of 1.0% by volume to RPMI-1640 medium (Sigma, R8758)), or preferably 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 index 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 where 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 promotion of IFN-α production in pDCs through phagocytosis by pDCs following binding to BDCA2 by bacteria can be determined by the fact that the amount of IFN-α produced by pDCs contacted with BDCA2 after treatment to suppress BDCA2 in a ligand-binding state is smaller than the amount of IFN-α produced by pDCs not subjected to such treatment. The bacteria according to this embodiment may be bacteria in which the amount of IFN-α produced by pDCs contacted with BDCA2 after treatment to suppress BDCA2 in a ligand-binding state is at most 1 / 2-fold, 1 / 3-fold, 1 / 4-fold, 1 / 5-fold, 1 / 6-fold, 1 / 7-fold, 1 / 8-fold, 1 / 9-fold, or 1 / 10-fold the amount of IFN-α produced by pDCs not subjected to such treatment. In these cases, the treatment to suppress BDCA2 in a ligand-binding state and the amount of IFN-α produced are as described above.

[0109] The promotion of IFN-α production in pDC through phagocytosis by pDC 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 BDCA2-mediated phagocytosis in pDC is more than 0-fold, 0.1-fold or more, 0.2-fold or more, 0.3-fold or more, 0.4-fold or more, 0.5-fold or more, 0.6-fold or more, 0.7-fold or more, 0.8-fold or more, 0.9-fold or more, 1.0-fold or more, 1.2-fold or more, 1.4-fold or more, 1.6-fold or more, 1.8-fold or more, or 2.0-fold or more. In the above case, the fold change in the amount of IFN-α production relative to the fold change in the amount of BDCA2-mediated phagocytosis in pDC is 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, a pharmaceutical composition, a quasi-drug, a bacterial bulk powder (such as a powder of dried bacterial cells or a powder containing the same), an additive, or a feed, and is preferably a food composition. Immunostimulatory ability (immunostimulatory ability) refers to the stimulating (activating) effect of the innate immune system on cells or living organisms. The immunostimulatory composition according to one aspect 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 according to the following description.

[0111] The content of the bacteria according to this embodiment is not particularly limited as long as it satisfies the effective amount of immunostimulation, 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, 5.0% by mass or more, 6.0% by mass or more, 7.0% by mass or more, 8.0% by mass or more, 9.0% by mass or more, 10.0% by mass or more, 11.0% by mass or more, 12.0% by mass or more, 13.0% by mass or more, 14.0% by mass or more, 15.0% by mass or more, 16.0% by mass or more, 17.0% by mass or more, 18.0% by mass or more, 19.0% by mass or more, 20.0% by mass or more, 21.0% by mass or more, 22.0% by mass or more, 23.0% by mass or more, 24.0% by mass or more, 25.0% by mass or more, 26.0% by mass or more, 27.0% by mass or more, 28.0% by mass or more, 29.0% by mass or more, 30.0% by mass or more, 31.0% It 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 ... % to 10 mass%, 0.001 mass% to 100 mass%, 0.001 mass% to 95 mass%, 0.001 mass% to 90 mass%, 0.001 mass% to 80 mass%, 0.001 mass% to 70 mass%, 0.001 mass% to 60 mass%, 0.001 mass% to 50 mass%, 0.0 01 mass% or more and 30 mass% or less, 0.001 mass% or more and 25 mass% or less, 0.001 mass% or more and 20 mass% or less, 0.001 mass% or more and 15 mass% or less, 0.001 mass% or more and 10 mass% or less, 0.005 mass% or more and 100 mass% or less, 0.005 mass% or more and 95 mass% or less, 0.005 mass% or more and 90 mass% or less,0.005 mass% or more and less than 80 mass%, 0.005 mass% or more and less than 70 mass%, 0.005 mass% or more and less than 60 mass%, 0.005 mass% or more and less than 50 mass%, 0.005 mass% or more and less than 30 mass%, 0.005 mass% or more and less than 25 mass%, 0.005 mass% or more and less than 20 mass%, 0.005 mass% or more and less than 15 mass%, 0.005 mass% or more and less than 10 mass%, 0.01 mass% or more and less than 100 mass%, 0.01 mass% or more and less than 95 mass%, 0.01 mass% or more and less than 90 mass%, 0.01 mass% or more and less than 80 mass%, 0.01 mass% or more and less than 70 mass%, 0. 0.01% by mass or more and less than 60% by mass, 0.01% by mass or more and less than 50% by mass, 0.01% by mass or more and less than 30% by mass, 0.01% by mass or more and less than 25% by mass, 0.01% by mass or more and less than 20% by mass, 0.01% by mass or more and less than 15% by mass, 0.01% by mass or more and less than 10% by mass, 0.02% by mass or more and less than 100% by mass, 0.02% by mass or more and less than 95% by mass, 0.02% by mass or more and less than 90% by mass, 0.02% by mass or more and less than 80% by mass, 0.02% by mass or more and less than 70% by mass, 0.02% by mass or more and less than 60% by mass, 0.02% by mass or more and less than 50% by mass, 0.02% by mass or more and less than 30% by mass % or less, 0.02 mass% or more and 25 mass% or less, 0.02 mass% or more and 20 mass% or less, 0.02 mass% or more and 15 mass% or less, 0.02 mass% or more and 10 mass% or less, 0.05 mass% or more and 100 mass% or less, 0.05 mass% or more and 95 mass% or less, 0.05 mass% or more and 90 mass% or less, 0.05 mass% or more and 80 mass% or less, 0.05 mass% or more and 70 mass% or less, 0.05 mass% or more and 60 mass% or less, 0.05 mass% or more and 50 mass% or less, 0.05 mass% or more and 30 mass% or less, 0.05 mass% or more and 25 mass% or less, 0.05 mass% or more and 2 ... % or more than 15% by mass, 0.05% or more than 10% by mass, 0.10% or more than 100% by mass, 0.10% or more than 95% by mass, 0.10% or more than 90% by mass, 0.10% or more than 80% by mass, 0.10% or more than 70% by mass, 0.10% or more than 60% by mass, 0.10% or more than 50% by mass, 0.10% or more than 30% by mass, 0.10% or more than 25% by mass, 0.10% or more than 20% by mass, 0.10% or more than 15% by mass, 0.10% or more than 10% by mass, 0.30% or more than 100% by mass,0.30 mass% or more and less than 95 mass%, 0.30 mass% or more and less than 90 mass%, 0.30 mass% or more and less than 80 mass%, 0.30 mass% or more and less than 70 mass%, 0.30 mass% or more and less than 60 mass%, 0.30 mass% or more and less than 50 mass%, 0.30 mass% or more and less than 30 mass%, 0.30 mass% or more and less than 25 mass%, 0.30 mass% or more and less than 20 mass%, 0.30 mass% or more and less than 15 mass%, 0.30 mass% or more and less than 10 mass%, 1.0 mass% or more and less than 100 mass%, 1.0 mass% or more and less than 95 mass%, 1.0 mass% or more and less than 90 mass%, 1.0 mass% or more and less than 80 mass% % or less, 1.0% by mass or more and less than 70% by mass, 1.0% by mass or more and less than 60% by mass, 1.0% by mass or more and less than 50% by mass, 1.0% by mass or more and less than 30% by mass, 1.0% by mass or more and less than 25% by mass, 1.0% by mass or more and less than 20% by mass, 1.0% by mass or more and less than 15% by mass, 1.0% by mass or more and less than 10% by mass, 1.5% by mass or more and less than 100% by mass, 1.5% by mass or more and less than 95% by mass, 1.5% by mass or more and less than 90% by mass, 1.5% by mass or more and less than 80% by mass, 1.5% by mass or more and less than 70% by mass, 1.5% by mass or more and less than 60% by mass, 1.5% by mass or more and less than 50% by mass, 1.5% by mass or more and less than 100% by mass mass% or more and less than 30 mass%, 1.5 mass% or more and less than 25 mass%, 1.5 mass% or more and less than 20 mass%, 1.5 mass% or more and less than 15 mass%, 1.5 mass% or more and less than 10 mass%, 2.0 mass% or more and less than 100 mass%, 2.0 mass% or more and less than 95 mass%, 2.0 mass% or more and less than 90 mass%, 2.0 mass% or more and less than 80 mass%, 2.0 mass% or more and less than 70 mass%, 2.0 mass% or more and less than 60 mass%, 2.0 mass% or more and less than 50 mass%, 2.0 mass% or more and less than 30 mass%, 2.0 mass% or more and less than 25 mass%, 2.0 mass% or more and less than 20 mass%, 2.0 mass% or more and less than 15 mass% or less, 2.0 mass% or more and 10 mass% or less, 2.5 mass% or more and 100 mass% or less, 2.5 mass% or more and 95 mass% or less, 2.5 mass% or more and 90 mass% or less, 2.5 mass% or more and 80 mass% or less, 2.5 mass% or more and 70 mass% or less, 2.5 mass% or more and 60 mass% or less, 2.5 mass% or more and 50 mass% or less, 2.5 mass% or more and 30 mass% or less, 2.5 mass% or more and 25 mass% or less, 2.5 mass% or more and 20 mass% or less, 2.5 mass% or more and 15 mass% or less, 2.5 mass% or more and 10 mass% or less, 3.0 mass% or more and 100 mass% or less, 3.0 mass% or more and 95 mass% or less,3.0 mass% or more and 90 mass% or less, 3.0 mass% or more and 80 mass% or less, 3.0 mass% or more and 70 mass% or less, 3.0 mass% or more and 60 mass% or less, 3.0 mass% or more and 50 mass% or less, 3.0 mass% or more and 30 mass% or less, 3.0 mass% or more and 25 mass% or less, 3.0 mass% or more and 20 mass% or less, 3.0 mass% or more and 15 mass% or less, 3.0 mass% or more and 10 mass% or less, 5.0 mass% or more and 100 mass% or less, 5.0 mass% or more and 95 mass% or less, 5.0 mass% or more and 90 mass% or less, 5.0 mass% or more and 80 mass% or less, 5.0 mass% or more and 70 mass% or less, 5.0 mass% or more and 60 mass% or less, 5.0 mass% or more and 50 mass% or less, 5.0 mass% or more and 30 mass% or less, 5.0 mass% or more and 25 mass% or less, 5.0 mass% or more and 20 mass% or less, 5.0 mass% or more and 15 mass% or less, 5.0 mass% or more and 10 mass% or less, 7.0 mass% or more and 100 mass% or less, 7.0 mass% or more and 95 mass% or less, 7.0 mass% or more and 90 mass% or less, 7.0 mass% or more and 80 mass% or less, 7.0 mass% or more and 70 mass% or less, 7.0 mass% or more and 60 mass% or less, 7.0 mass% or more and 50 mass% or less, 7.0 mass% or more and 30 mass% or less, 7.0 mass% or more and 25 mass% or less, 7.0 mass% or more and 20 mass% or less, 7.0 mass% or more and 15 mass% or less, 7.0 mass% or more and 10 mass% or less, 10.0 mass% or more and 100 mass% or less, 10.0 mass% or more and 95 mass% or less, 10.0 mass% or more and 90 mass% or less, 10.0 mass% or more and 80 mass% or less, 10.0 mass% or more and 70 mass% or less, 10.0 mass% or more and 60 mass% or less, 10.0 mass% or more and 50 mass% or less, 10.0 mass% or more and 30 mass% or less, 10.0 mass% or more and 25 mass% or less, 10.0 mass% or more and 20 mass% or less, 10.0 mass% or more and 15 mass% or less, 80.0 mass% or more and 100 mass% or less, 80.0 mass% or more and 95 mass% or less, 80.0 mass% or more and 90 mass% or less, 90.0 mass% or more and 100 mass% or less, 90.0 mass% or more and 95 mass% or less or 95.0 mass% or more and 100 mass% or less may be used.,

[0112] When the immunostimulating composition of the present embodiment is liquid, the number of bacteria contained in the composition according to the present embodiment is 1.0 × 10 ３ cells / mL or more, 1.0 × 10 ４ cells / mL or more, 1.0 × 10 ５Cells / mL or more, 1.0 x 10 ６ Cells / mL or more, 1.0 x 10 ７ cells / mL or 4.0 x 10 ７ cells / mL or more, and may be 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 １０ Cells / mL or less, 3.0 x 10 ９ cells / mL or less or 1.0 x 10 ９ The lower limit and upper limit may be any combination of cells / mL or less. For example, the number of bacteria contained in the composition according to this embodiment may be 1.0 × 10 ３ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ３ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ３ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ３ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 1.0 x 10 ４ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ４ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ４ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ４ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 1.0 x 10 ５ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ５ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ５ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ５ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 1.0 x 10 ６ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ６ Cells / mL or more 1.0 x 10１０ Cells / mL or less, 1.0 x 10 ６ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ６ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 1.0 x 10 ７ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ７ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ７ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ７ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 4.0 x 10 ７ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 4.0 x 10 ７ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 4.0 x 10 ７ Cells / mL or more 3.0 x 10 ９ cells / mL or less or 4.0 x 10 ７ Cells / mL or more 1.0 x 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 immunostimulating 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 ６ pcs or more, 1.0×10７ 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 or 1.0 x 10 １１ may be 1.0 × 10 or more, １４ Less than or equal to 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 according to the present embodiment contained in the composition per unit package may be, for example, 1.0 × 10 ５ pcs or more 1.0×10 １４ Less than or equal to 1.0×10 ５ pcs or more 1.0×10 １３ Less than or equal to 1.0×10 ５ pcs or more 1.0×10 １２ Less than or equal to 3.0×10 ５ pcs or more 1.0×10 １４ Less than or equal to 3.0×10 ５ pcs or more 1.0×10 １３ Less than or equal to 3.0×10 ５ pcs or more 1.0×10 １２ Less than or equal to 1.0×10 ６ pcs or more 1.0×10 １４ Less than or equal to 1.0×10 ６ pcs or more 1.0×10 １３ Less than or equal to 1.0×10 ６ pcs or more 1.0×10 １２ Less than or equal to 3.0×10 ６ pcs or more 1.0×10 １４ Less than or equal to 3.0×10 ６ pcs or more 1.0×10 １３ Less than or equal to 3.0×10 ６ pcs or more 1.0×10 １２ Less than or equal to 1.0×10 ７ pcs or more 1.0×10 １４ Less than or equal to 1.0×10 ７More 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 １０ More than 1.0×10１２ 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 Less than or equal to 1.0×10 １１ pcs or more 1.0×10 １４ Less than or equal to 1.0×10 １１ pcs or more 1.0×10 １３ or less or 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), special-use foods (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, in addition to the above-described unit packaging form per meal or per day, it is also suitable to provide it in a unit packaging form per week, per two weeks, per month, or per two months. It is preferable that the latter unit packaging form, for example, display the intake amount per meal or per day, so that the consumer can ingest an effective amount of the active ingredient of the present invention by following the labeling.

[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, 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 non-alcoholic beverages such as 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 water), near-water, and non-alcoholic beer-flavored drinks; carbohydrate-containing foods and beverages such as rice, noodles, bread, and pasta; cheeses, hard or soft yogurt, livestock milk, and other raw foods and beverages made from fats and oils. dairy products such as cream and ice cream; Western sweets such as cookies, cakes and chocolates, Japanese sweets such as manju or yokan, tablet sweets (refreshing sweets) such as ramune, candies, chewing gum, gummy candies, frozen desserts and frozen confections 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, happoshu, other miscellaneous alcoholic drinks, and chuhai; processed foods such as processed products using eggs, processed seafood or meat (including liver and other offal) products (including delicacies), soups such as miso soup, condiments such as miso, soy sauce, furikake, and other seasonings, and 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 immunostimulating composition of this embodiment is used as a pharmaceutical composition or a quasi-drug, examples of the target diseases include cancers already known to be suitable for Type I IFN, 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 immunostimulating 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 of osteoblasts into 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, an immunostimulating composition 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 1 x 10 ８ pcs or more, 1×10 ９ or more or 1 x 10 １０ It can be more than 1×10 １４ Less than or equal to 1 x 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 x 10 １４ pieces, 1×10 ９ ~1 x 10 １４ pcs or 1 x 10 １０ ~1 x 10 １４ pieces, 1×10 ８ ~1 x 10 １３ pieces, 1×10 ９ ~1 x 10 １３ pieces, 1×10 １０ ~1 x 10 １３ pieces, 1×10 ８ ~1 x 10 １２ pieces, 1×10 ９ ~1 x 10 １２ pcs or 1 x 10 １０ ~1 x 10 １２ The number of bacteria can be measured using a known microscope, flow cytometer, or non-culture rapid microorganism testing device (e.g., 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 immunostimulating composition of this embodiment for an adult can also be determined by the dry cell mass of the bacterium, which is the active ingredient. For example, 2.5 × 10 －２ mg or more, 2.5 x 10 －１mg 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 to 2.5 x 10 ４ mg, 2.5 x 10 －１ mg to 2.5 x 10 ４ mg, 2.5 mg ~ 2.5 x 10 ４ mg, 2.5 x 10 －２ mg to 2.5 x 10 ３ mg, 2.5 x 10 －１ mg to 2.5 x 10 ３ mg, 2.5 x 10 mg ~ 2.5 x 10 ３ mg, 2.5 x 10 －２ mg to 2.5 x 10 ２ mg, 2.5 x 10 －１ mg to 2.5 x 10 ２ mg or 2.5 mg to 2.5 x 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., participating 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 a viral infection epidemic period. Examples of ingestion or administration timing 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 can be continued, with an ingestion interval or administration interval between ingestion or administration, as the case may be, 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 use and / or therapeutic use, and is preferably non-therapeutic use.

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

[0144] In one aspect, the biological characteristics, classification, and strain of the 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 the present embodiment ("bacteria according to the present 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 includes 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 immunostimulation. The subject in need of immunostimulation 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 to be used in the above-mentioned "method for activating pDCs, comprising binding bacteria to BDCA2" and "method for activating pDCs, comprising 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 by reference to the description of the bacteria contained in the immunostimulatory composition of this embodiment ("bacteria according to this 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 (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 by reference to the description of the "immunostimulatory composition of this 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), the method including a step of culturing bacteria in an M17 medium supplemented with glucose at a predetermined concentration (culturing step). In the method of the second embodiment, by using an 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 may be obtained from a supplier 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, for example, 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 concentration of glucose in the medium used in the culture step may be, for example, 0.1% by mass to 10% by mass, 0.3% by mass to 10% by mass, 0.5% by mass to 10% by mass, 1.0% by mass to 10% by mass, 0.1% by mass to 5.0% by mass, 0.3% by mass to 5.0% by mass, 0.5% by mass to 5.0% by mass, 1.0% by mass to 5.0% by mass, 0.1% by mass to 3.0% by mass, 0.3% by mass to 3.0% by mass, 0.5% by mass to 3.0% by mass, 1.0% by mass to 3.0% by mass, 0.1% by mass to 1.0% by mass, 0.3% by mass to 1.0% by mass, 0.5% by mass to 1.0% by mass, or 1.0% by mass. In this case, glucose is, 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 pDC 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 pDC 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 performed 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, but 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. 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 the bacteria 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 Lactococcus lactis subsp. lactis JCM 5805 as an active ingredient. In this case, Lactococcus lactis subsp. lactis JCM 5805 may satisfy, for example, the following requirement Y, and satisfaction of requirement Y may be confirmed according to the method described in relation to the first embodiment. [Requirement Y] Final concentration 2.0 x 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 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.

[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 (deposit number FERM BP-10914) provided by Nagasaki University (National University Corporation) 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.) The cells were cultured at 37°C and 5% CO. ２ The results are shown as the mean ± standard deviation. The basal medium used for culturing CAL-1 cells and for tests using 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 also referred to as "serum-free medium").

[0162] The lactic acid bacteria strains used in the following examples are shown 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 shown in Table 1, and then used as bacterial bulk powder (dried powder of bacterial cells or powder containing the same, etc.) prepared as follows, unless otherwise specified. 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, maintaining the temperature 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]

[0164]

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

[0166] [Staining of bacteria 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 dispersion by vortexing, the mixture was shielded from light with aluminum foil and incubated at room temperature for 60 minutes. 750 μL of PBS (Takara Bio Inc.) was then added, vortexed, and centrifuged at 20,000 g for 2 minutes at room temperature. After discarding 800 μL of the supernatant, 1.5 mL of PBS was added and the precipitate was completely suspended using a vortex mix. After another centrifugation at 20,000 g for 2 minutes at room temperature, 1.5 mL of the supernatant was discarded. Finally, 140 μL of PBS was added and suspended to prepare a 10 mg / mL bacterial solution of stained bacteria. The stained bacterial solution was protected from light with aluminum foil and stored at 4°C until use. Immediately before use, the solution was diluted 10-fold with PBS and used as a 1 mg / mL bacterial solution. The stained bacterial solution 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 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 recovered by pipetting. The recovered 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 μM cells / mL and seeded at 200 μL / well into each well of 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 the cells were 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) according to the method described above for staining bacteria with pHrodo Red SE was added to each well to a final concentration of 10 μg / mL. The fluorescence of pHrodo Red SE in each well was then measured over time using an Incucyte® SX5 Live-Cell Analysis System (Sartorius). After further culturing for 22 hours (contact step), the culture supernatant was collected from each well, 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 is enhanced 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 amount of bacteria phagocytosed by CAL-1 cells can be quantified using the total area (fluorescence detection area) in which pHrodo Red SE fluorescence is detected within the entire field of view as an index. The fluorescence intensity analysis conditions for the Incucyte (registered trademark) SX5 Live-Cell Analysis System (Sartorius) were as described in [Incucyte Fluorescence Intensity Analysis Conditions 1] below. The field of view area per image is 0.572 mm ２ It was.

[0169] [Incucyte Fluorescence Intensity Analysis Condition 1] For incucyte fluorescence intensity analysis, two channels, "Phase channel" and "Orange channel," were set. 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 ): 60.000 or more - Eccentricity: No setting 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] FIG. 1 shows the fluorescence detection area 22 hours after the addition of LC-Plasma. FIG. 2 shows the results of measuring the IFN-α concentration in the culture supernatant collected 22 hours after the addition of LC-Plasma. According to the results of FIG. 1, the amount of phagocytosis was significantly smaller in the LC-Plasma + Cyt_D group than in the LC-Plasma + DMSO group. Furthermore, according to the results of FIG. 2, the IFN-α concentration was significantly smaller in the LC-Plasma + Cyt_D group than in the LC-Plasma + DMSO group, and pDC activation by LC-Plasma was significantly suppressed. From these results, it became clear that activation was significantly suppressed in pDCs whose phagocytosis was inhibited.

[0171] Example 2: Relationship between phagocytosis by pDC and IFN-α production in various lactic acid bacteria. CAL-1 cells were cultured at 5.0 × 10 ５A 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 recovered by pipetting. The recovered CAL-1 cells were transferred to serum-free medium at a concentration of 2.0 × 10 cells / mL. ５ The 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 the cell suspension in each well to a final concentration of 10 μg / mL. A negative group was also prepared without the addition of bacteria. Thereafter, time-course measurement of pHrodo Red SE fluorescence and measurement of the IFN-α concentration in the culture supernatant were performed in the same manner as in Example 1, except for the fluorescence intensity analysis conditions of the Incucyte (registered trademark) SX5 Live-Cell Analysis System (Sartorius). The fluorescence intensity analysis conditions of the Incucyte (registered trademark) SX5 Live-Cell Analysis System (Sartorius) were as described in [Incucyte Fluorescence Intensity Analysis 2] below. The field area per image was 0.572 mm. ２ It was.

[0172] [Incucyte Fluorescence Intensity Analysis 2] Incucyte fluorescence intensity analysis, two channels, "Phase channel" and "Orange channel," were set. 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 ): 60.000 or more - Eccentricity: No setting 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: No setting - Mean Intensity: No setting - Mean Intensity: No setting

[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 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 LC-Plasma phagocytosis by pDC via binding to BDCA2 and IFN-α production> CAL-1 cells were seeded in each well as in Example 1. To the CAL-1 cell suspension, an 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 (to evaluate the effect of nonspecific binding by the anti-BDCA2 antibody) was added to a concentration of 10 μg / mL, followed by incubation for 30 minutes. No antibody was added to the control LC-Plasma group. Thereafter, for each of the LC-Plasma group, the LC-Plasma + Isotype group, and the LC-Plasma + BDCA2 anti group, exposure to LC-Plasma stained with pHrodo Red SE, evaluation of phagocytosis, and measurement of IFN-α concentration in the culture supernatant were performed in the same manner as in Example 2.

[0175] Figure 6 shows the fluorescence detection area 22 hours after the 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 results of measuring the IFN-α concentration in the culture supernatant collected 22 hours after the addition of LC-Plasma for the LC-Plasma group, the LC-Plasma + Isotype group, and the LC-Plasma + BDCA2 anti group. According to the results of Figures 6 and 7, in the LC-Plasma + BDCA2 anti group, in which the binding of BDCA2 to CAL-1 cells was inhibited by blocking with anti-BDCA2 antibodies, the amount of phagocytosis and IFN-α concentration were smaller compared to the LC-Plasma group and the LC-Plasma + Isotype group, in which the binding was not inhibited. These results revealed that the binding of LC-Plasma to BDCA on the surface of pDC is one of the steps that determine the degree of immune activation by LC-Plasma. Furthermore, these results revealed that the phagocytosis of LC-Plasma by pDC via binding to BDCA is one of the steps that determine the degree of immune activation by LC-Plasma.

[0176] Example 4: Relationship between phagocytosis of various lactic acid bacteria via binding to BDCA2 by pDC and IFN-α production> CAL-1 cell suspensions in each well were seeded in the same manner as in Example 1. In the BDCA2 anti-group, an anti-BDCA2 antibody (Miltenyi Biotec, 130-090-690) was added to 10 μg / mL, and in the isotype group, its isotype control antibody (Miltenyi Biotec, 130-106-545) was added to 10 μg / mL, and the cells were incubated for 30 minutes. Thereafter, in the isotype group and the BDCA2 anti-group, cells were exposed to the same various lactic acid bacteria as those tested in Example 2, stained with pHrodo Red SE, as in Example 2, and phagocytosis evaluation and measurement of IFN-α concentration in the culture supernatant were performed. The amount of phagocytosis mediated by binding to BDCA2 was calculated by the method shown below as "Method for calculating the amount of phagocytosis mediated by binding to BDCA2."

[0177] [Method for calculating the amount of phagocytosis via binding to BDCA2] First, to consider the influence of nonspecific binding by the anti-BDCA2 antibody, the amount of bacteria phagocytosis by pDC (phagocytosis amount P) was evaluated in an isotype group treated with an isotype control antibody having the same isotype and subclass as the anti-BDCA2 antibody. Next, the amount of bacteria phagocytosis by pDC (phagocytosis amount Q) was evaluated in a BDCA2 anti group treated with an anti-BDCA2 antibody that inhibits the binding of bacteria to BDCA2. The value obtained by subtracting the phagocytosis amount Q from the phagocytosis amount P was calculated as the amount of phagocytosis by pDC via the binding to BDCA2.

[0178] Fig. 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. Fig. 9 shows the measurement results of IFN-α concentrations in culture supernatants collected 22 hours after the addition of various lactic acid bacteria for the isotype group and the BDCA2 anti group. Fig. 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] According to the results of Figures 8 and 9, the BDCA2 anti-group, in which the binding of BDCA2 to CAL-1 cells was inhibited by blocking with an anti-BDCA2 antibody, had lower phagocytosis levels and IFN-α concentrations than the isotype group, in which binding was not inhibited. These results demonstrate that, even in bacteria other than LC-Plasma, the binding of bacteria to BDCA on the surface of pDC is one of the steps that determines the degree of bacterial immunostimulation. These results also demonstrate that the phagocytosis of bacteria by pDC via binding to BDCA is one of the steps that determines the degree of bacterial immunostimulation.

[0180] According to the results of Figure 10, there is a strong positive correlation (R ２This indicates that the immunostimulatory ability 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 pDC via binding to BDCA2, and further suggests that the binding ability to BDCA2 is a useful parameter for evaluating the immunostimulatory ability of bacteria.

[0181] Example 5: Construction of an assay to evaluate the binding ability of bacteria to BDCA2 An assay to evaluate the binding ability of bacteria to BDCA2 (BDCA2 binding assay) was constructed, the protocol of which is shown below. ２ To each well of a low-adsorption 96-well plate (Corning, 3474), 100 μL of phosphate-buffered saline (PBS, Nacalai Tesque, 11482-15) (hereinafter also referred to simply as "buffer") containing 0.5 mM of BDCA-2 Fc Chimera Protein (R&D Systems, 1376-DL; hereinafter also referred to simply as "BDCA2") or Recombinant Human IgG1 Fc Protein (R&D Systems, 110-HG) was added to each well to a final concentration of 1 to 50 μg / mL. To distinguish between contaminants and bacteria, FITC-stained LC-Plasma was added to each well to a final concentration of 10 μg / mL according to standard methods, and the plate was incubated at room temperature for 1 hour while 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. Each well was filled with PE-conjugated F(ab') ２100 μL of a solution prepared by diluting Goat anti-Human IgG Fc Secondary Antibody (ThermoFisher Scientific, H10104) 100-fold with buffer (hereinafter also referred to as "secondary antibody dilution") was added and incubated at room temperature for 15 minutes. 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, a population containing lactic acid bacteria was gated using FSC / SSC. Next, gating was performed using a wavelength corresponding to SSC / FITC, and only the FITC-stained population (FITC+ population) was extracted. The FITC+ population was then gated using a wavelength corresponding to SSC / PE, and the proportion of the population that fluoresced at a 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 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 bound to BDCA2.

[0183] 11 is a graph showing the relative values ​​evaluated by the BDCA2 binding assay under conditions where 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. As shown in FIG. 11, the percentage of LC-Plasma bound to BDCA2 increased in a BDCA2 concentration-dependent manner. This demonstrates that the BDCA2 binding assay can evaluate the binding ability of bacteria to BDCA2.

[0184] Example 6: Bacterial phagocytosis by pDC, phagocytosis via binding to BDCA2, and ability to bind to BDCA2 The lactic acid bacteria listed in Tables 3 and 4 were evaluated for the amount of phagocytosis by pDC and the amount of phagocytosis by pDC via binding to BDCA2, and the lactic acid bacteria listed in Table 5 were evaluated for their ability to bind to BDCA2. The amount of phagocytosis by pDC 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 lactic acid bacteria. In addition, (A-2) the value obtained by subtracting the fluorescence detection area 22 hours after the addition of Lactococcus lactis subsp. lactis ATCC15577 from the fluorescence detection area, and (A-3) the fluorescence detection area obtained by subtracting the fluorescence detection area from Lactococcus lactis subsp. The value (ratio) obtained by dividing the area by the fluorescence detection area 22 hours after the addition of Lactococcus lactis ATCC15577 is shown in Table 4. The amount of phagocytosis by pDC via binding to BDCA2 was evaluated according to the same method as in Example 4, and the results are shown in Table 3 as (B-1). In addition, the value obtained by subtracting the amount of phagocytosis of Lactococcus lactis. subsp. lactis ATCC15577 from the amount of phagocytosis is shown in Table 4 as (B-2), and the value (ratio) obtained by dividing the amount of phagocytosis by the amount of phagocytosis of Lactococcus lactis. subsp. lactis ATCC15577 is shown in Table 4 as (B-3). The binding ability to BDCA2 was evaluated by the same BDCA2 binding assay as in Example 5. Specifically, BDCA2 or Recombinant Human IgG1 Fc Protein (R&D Systems, 110-HG) was used at a final concentration of 10 μg / mL, and the lactic acid bacteria concentration was 10 μg / mL. The percentage of lactic acid bacteria bound to BDCA2 was measured as an index. Table 5 shows the percentage bound to BDCA2 (C-1), the value obtained by subtracting the percentage of Lactococcus lactis subsp. lactis ATCC15577 from that percentage (C-2), and the value (ratio) obtained by dividing that percentage by the percentage of Lactococcus lactis subsp. lactis ATCC15577 (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 (the results of (A-1), (B-1) or (C-1)) were calculated by subtracting or dividing the values ​​obtained by testing Lactococcus lactis subsp. lactis ATCC 15577 on the respective measurement days. Furthermore, in (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 ​​of 0 or less were represented as 0.

[0185]

[0186]

[0187]

[0188] Lactic acid bacteria having a higher amount of phagocytosis by pDC, a higher amount of phagocytosis by pDC via binding to BDCA2, and / or a higher binding ability to BDCA2 than at least Lactococcus lactis subsp. lactis ATCC 15577 can be said to have immunostimulatory activity. Therefore, according to the results in Tables 3 to 5, lactic acid bacteria having a higher amount of phagocytosis by pDC, a higher amount of phagocytosis by pDC via binding to BDCA2, and / or a higher binding ability to BDCA2 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 in the same manner as in Example 1. In the cytochalasin D (referred to as CytD in the graph) addition group, 400 μM cytochalasin D (Fujifilm Wako, 037-17561) dissolved in 4% by volume dimethyl sulfoxide (DMSO) was added to the suspension, so that the cytochalasin D and DMSO were at final concentrations of 10 μM and 0.1% by volume, respectively, and the cells were incubated for 30 minutes. As a control, in the DMSO group, 4% by volume DMSO alone was added to a final concentration of 0.1% by volume, and the cells were similarly incubated for 30 minutes. After incubation, lactic acid bacteria listed in Table 1 were stained with pHrodo Red SE (Invitrogen, P36600) using the method described above in [Staining of bacteria with pHrodo Red SE] and added to each well to a final concentration of 10 μg / mL, followed by incubation for 24 hours. The phagocytic activity and IFN-α concentration in each well were measured using the same methods as in Example 1.

[0190] To confirm the lack of cytotoxicity of DMSO and cytochalasin D, cytochalasin D or DMSO, or an equal 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 Integrated Intensity, TII, 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 / Image). Figure 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. According to the results in Figure 12, the addition of DMSO or cytochalasin D did not change the number of viable cells compared to the group to which PBS was added, indicating that the inhibitory reagent used in this test does not cause cytotoxicity. According to the results in Figure 13, the phagocytic activity of various lactic acid bacteria was completely suppressed by treatment with cytochalasin D. According to the results in Figure 14, IFN-α production was completely eliminated by inhibiting phagocytic activity. This indicates that phagocytosis is essential for pDC immunostimulation in 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 incubation at 37°C for 24 hours, the cells were collected using FACS buffer and stained with 7-AAD (manufacturer: BD biosciences, BD LSRFortessa). The cells were washed with FACS buffer and then subjected to flow cytometry (FACS) using a flow cytometer (BD biosciences, BD LSRFortessa (registered trademark) X-20 flow cytometer). IFN-α in the supernatant obtained upon cell recovery was evaluated in the same manner as in Example 1.

[0195] Analysis of FACS results was performed using the following procedure. First, a population containing CAL-1 cells was gated using FSC / SSC. Next, gating was performed using a wavelength corresponding to SSC / 7-AAD, and only the population not stained with 7-AAD (7-AAD- population: a population of live CAL-1 cells) was extracted. The 7-AAD- population was then gated using a wavelength corresponding to PE / SSC, and the proportion of the population in which fluorescence at a wavelength corresponding to PE (i.e., fluorescence derived from pHrodo Red SE, which labels lactate) was detected (PE+ population) was extracted. The proportion of the PE+ population relative to the 7-AAD- population was taken as the phagocytosis rate. The threshold for the PE+ population was determined as the lowest value of the population with the strongest PE intensity between the two populations when PE / SSC was plotted.

[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 This result revealed that the immunostimulatory capacity of bacteria is determined by the amount of phagocytosis.

[0197] Example 9: Obtaining a lactic acid bacteria strain with a high phagocytosis rate In this example, LC-Plasma was cultured in two different media, and the phagocytosis rate and IFN-α were evaluated. 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 bacterial 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 culturing for 48 hours at 30°C, colonies were picked and inoculated into 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 bacterial cell suspension was inoculated into 100 mL of M17 medium and cultured for 24 hours at 30°C.

[0198] The phagocytosis rate and IFN-α of the 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 has a higher phagocytosis rate than LC-Plasma cultured in MRS medium, making it possible to obtain lactic acid bacteria with enhanced immunostimulatory activity.

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. The composition according to any one of claims 1 to 3, wherein the bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), and 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. The bacterium is a bacterium that is phagocytosed by plasmacytoid dendritic cells (pDCs), The composition according to any one of claims 1 to 3, wherein the amount of the bacteria 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. The composition according to any one of claims 1 to 3, wherein the bacterium is Lactococcus lactis subsp. lactis (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 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 according to any one of claims 1 to 3, wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

8. The composition according to any one of claims 1 to 3, wherein the bacteria are one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetragenococcus, Oenococcus, Weissella, Bifidobacterium, and Lactobacillus.

9. The composition according to any one of claims 1 to 3, wherein the bacterium is Lactococcus lactis subsp. lactis.

10. 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 3, 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.

11. The composition according to any one of claims 1 to 3, wherein the bacteria comprises killed bacteria.

12. The composition of any one of claims 1 to 3, wherein the bacteria are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

13. The composition of claim 12, 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.

14. The composition according to claim 13, 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.

15. The composition according to claim 12, wherein the bacteria promote the production of IFN-α in the pDCs through phagocytosis by the pDCs.

16. The composition according to any one of claims 1 to 3, which is a food composition, a pharmaceutical composition or a feed.

17. The number of bacteria per unit package in the food composition, pharmaceutical composition, or feed is 1 x 10 ８ 17. The composition of claim 16, wherein the number of

18. 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.

19. 19. The method of claim 18, wherein the screening step is carried out by a binding assay between the candidate bacterium and BDCA2.