Immune-activating compositions

Abstract

Disclosed is an immune-activating composition containing a bacterium that is phagocytosed by a plasmacytoid dendritic cell (pDC) to a prescribed degree or more and / or a bacterium that binds to BDCA2 as an effective ingredient.

Description

Technical Field

[0001] This disclosure relates to compositions for immune activation. Background Technology

[0002] Compositions for immune activation containing bacteria are known. For example, Patent Document 1 discloses a food composition for immune activation containing lactic acid bacteria that activate pDCs (plasmacytoid dendritic cells) and induce the production of IFN-α (interferon-α). Patent Document 2 discloses a method for screening immunomodulatory lactic acid bacteria, including a step of determining the binding number of urinary modulatory protein (Umod) by the test lactic acid bacteria, immunomodulatory lactic acid bacteria obtained by this method, and an immunomodulatory composition containing the lactic acid bacteria.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 2017-201984

[0006] Patent Document 2: Japanese Patent Application Publication No. 2014-217372 Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] Many bacteria with immune-activating capabilities have been reported. However, the mechanisms by which these bacteria activate immunity and their common properties remain largely unclear.

[0009] The purpose of this disclosure is to provide an immune-activating composition.

[0010] Methods for solving problems

[0011] The inventors have discovered that bacteria phagocytosed by pDCs possess immune-activating capabilities. Furthermore, the inventors have discovered that bacteria binding to blood dendritic cell antigen 2 (BDCA2) possess immune-activating capabilities.

[0012] This disclosure relates to, for example, the following items.

[0013] [A0] A composition that satisfies at least one of the following groups (1) to (4): (1) Containing bacteria as an active ingredient, the bacteria are phagocytosed by plasma cell-like dendritic cells (pDCs), and the amount of bacteria phagocytosed by pDCs is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs. The composition is an immune-activating composition. (2) Containing bacteria that bind to blood dendritic cell antigen 2 (BDCA2) as an active ingredient, the above composition is an immune-activating composition; (3) Containing bacteria as an active ingredient, wherein the bacteria are phagocytosed by plasma cell-like dendritic cells (pDCs) and the bacteria satisfy the following requirement X, the composition is an immune-activating composition; [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium. (4) Contains Lactococcus lactis subsp. lactis JCM 5805 as a bacterium, and the aforementioned Lactococcus lactis subsp. lactis JCM 5805 meets the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0014] [A1] An immune-activating composition comprising bacteria as an active ingredient, wherein the bacteria are phagocytosed by plasma cell-like dendritic cells (pDCs), wherein, The amount of bacteria that pDC phagocytosed was more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

[0015] [A2] An immune-activating composition comprising bacteria that bind to blood dendritic cell antigen 2 (BDCA2) as an active ingredient.

[0016] [A3] The composition according to [A0] or [A2], wherein the binding capacity of the above-mentioned bacteria to BDCA2 is greater than that of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2.

[0017] [A4] The composition according to [A3], wherein the binding ability with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as evaluated in the binding assay.

[0018] [A5] An immune-activating composition comprising bacteria as an active ingredient, wherein the bacteria are phagocytosed by plasma cell-like dendritic cells (pDCs), and wherein the bacteria satisfy the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium.

[0019] [A6] A composition comprising Lactococcus lactis subsp. lactis JCM 5805 as a bacterium, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0020] [A7] The composition according to [A0] or [A6], wherein the composition is an immune-activating composition.

[0021] [A8] The composition according to any one of [A0] to [A5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0022] [A9] The composition according to any one of [A0] to [A5], wherein the bacteria are one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0023] [A10] The composition according to any one of [A0] to [A5], wherein the bacteria is Lactococcus lactis subsp. lactis.

[0024] [A11] The composition according to any one of [A0] to [A5], wherein the bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, and Oenococcus oeni JCM. 6125. *Weissella paramesenteroides* JCM 9890, *Weissella viridescens* JCM 1174, *Bifidobacterium animalis subsp. animalis* JCM 1190, *Bifidobacterium animalis subsp. lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum subsp. Infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. lactis ATCC 7963, *Lactococcus lactis* subsp. lactis Lactococcus lactis subsp. lactis) ATCC 7962, Lactococcus lactis subsp. lactis) ATCC 15346, Lactococcus lactis subsp. lactis) ATCC 13675, Lactococcus lactis subsp. lactis.One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0025] [A12] The composition according to any one of [A0] to [A11], wherein the bacteria comprise dead bacteria.

[0026] [A13] The composition according to any one of [A0] to [A12], wherein the bacteria are bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

[0027] [A14] According to the composition of [A13], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, and the amount of bacteria phagocytized via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytized via binding to BDCA2.

[0028] [A15] According to the composition described in [A14], wherein the amount of phagocytosis not via binding with BDCA2 is the amount of phagocytosis of the bacteria by the pDC in a state where the BDCA2 of the pDC cannot bind to the ligand.

[0029] [A16] The composition according to any one of [A13] to [A15], wherein the bacteria promote the production of IFN-α in the pDC by phagocytosis of the pDC.

[0030] [A17] The composition according to any one of [A0] to [A16] is a food composition, a pharmaceutical composition or a feed.

[0031] [A18] According to the composition described in [A17], the bacterial count of the bacteria in each unit package of the above-mentioned food composition, pharmaceutical composition, or feed is 1 × 10⁻⁶. 8 More than one.

[0032] [B0] A method for activating the immunity of a subject, comprising the steps of ingesting bacteria from the group consisting of at least one of the following (1) to (4) or administering bacteria to the subject in need of such bacteria: (1) Bacteria phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of bacteria phagocytosed by pDCs is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs. (2) Bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDC) and meet the following requirement X; [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium. (4) The above-mentioned bacteria are Lactococcus lactis subsp. lactis JCM5805 and meet the following requirement Y; [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0033] [B1] A method for activating the immunity of a subject, comprising the steps of ingesting bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) or administering bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) to the subject in need, wherein, The amount of bacteria that pDC phagocytosed was more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

[0034] [B2] A method for activating the immunity of a subject, comprising the steps of causing the subject to take up bacteria that bind to blood dendritic cell antigen 2 (BDCA2) or administering bacteria that bind to blood dendritic cell antigen 2 (BDCA2) to the subject in need of them.

[0035] [B3] The method according to [B0] or [B2], wherein the binding capacity of the above-mentioned bacteria to BDCA2 is greater than that of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2.

[0036] [B4] The method according to [B3], wherein the binding capacity with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as evaluated in the binding assay.

[0037] [B5] A method for activating the immunity of a subject, comprising the step of administering to the subject either bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) or bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) to the subject in need, wherein the bacteria satisfy the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium.

[0038] [B6] A method for activating the immunity of a subject, comprising the steps of ingesting Lactococcus lactis subsp. lactis JCM 5805 as a bacterium into the subject or administering Lactococcus lactis subsp. lactis JCM 5805 as a bacterium to the subject in need, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0039] [B7] The method according to any one of [B0] to [B5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0040] [B8] The method according to any one of [B0] to [B5], wherein the bacteria are selected from one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0041] [B9] The method according to any one of [B0] to [B5], wherein the above-mentioned bacteria is Lactococcus lactis subsp. lactis.

[0042] [B10] The method according to any one of [B0] to [B5], wherein the bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, and Weissella. * *Weissella virides* JCM 9890, *Bifidobacterium animalis* JCM 1174, *Bifidobacterium animalis* subsp. *animalis* JCM 1190, *Bifidobacterium animalis* subsp. *lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum* subsp. *infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. *lactis* ATCC 7963, *Lactococcus lactis* subsp. *lactis* ATCC 7962, Lactococcus lactis. subsp. lactis ATCC 15346, Lactococcus lactis. subsp. lactis ATCC 13675, Lactococcus lactis. subsp.One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0043] [B11] The method according to any one of [B0] to [B10], wherein the bacteria comprise dead bacteria.

[0044] [B12] The method according to any one of [B0] to [B11], wherein the bacteria are bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

[0045] [B13] According to the method of [B12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, and the amount of bacteria phagocytized via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytized via binding to BDCA2.

[0046] [B14] According to the method described in [B13], wherein the amount of phagocytosis not via binding with BDCA2 is the amount of phagocytosis of the bacteria by the pDC in a state where the BDCA2 of the pDC cannot bind to the ligand.

[0047] [B15] The method according to any one of [B12] to [B14], wherein the bacteria promote the production of IFN-α in the pDC by phagocytosis of the pDC.

[0048] [B16] The method according to any one of [B0] to [B15], wherein the above-mentioned bacteria are contained in a food composition, a pharmaceutical composition or a feed.

[0049] [B17] According to the method described in [B16], the bacterial count of each unit package of the above-mentioned food composition, pharmaceutical composition, or feed is 1 × 10⁻⁶. 8 More than one.

[0050] [C0] A method for activating the immunity of a subject, comprising the steps of: administering to the subject a composition containing bacteria satisfying at least one of the group consisting of (1) to (4) below, or administering to the subject the composition containing bacteria satisfying at least one of the group consisting of (1) to (4) below, (1) Bacteria phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of bacteria phagocytosed by pDCs is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs. (2) Bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDC) and meet the following requirement X; [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium. (4) The above-mentioned bacteria are Lactococcus lactis subsp. lactis JCM5805 and meet the following requirement Y; [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0051] [C1] A method for activating the immunity of a subject, comprising the steps of ingesting a composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) or administering a composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) to a subject in need of it. The amount of bacteria that pDC phagocytosed was more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

[0052] [C2] A method for activating the immunity of a subject, comprising the steps of ingesting a composition containing bacteria that bind to blood dendritic cell antigen 2 (BDCA2) or administering a composition containing bacteria that bind to blood dendritic cell antigen 2 (BDCA2) to a subject in need of it.

[0053] [C3] The method according to [C0] or [C2], wherein the binding capacity of the above-mentioned bacteria to BDCA2 is greater than that of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2.

[0054] [C4] The method according to [C3], wherein the binding capacity with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as evaluated in the binding assay.

[0055] [C5] A method for activating the immunity of a subject, comprising the steps of ingesting a composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) or administering a composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) to the subject in need, wherein the bacteria satisfy the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium.

[0056] [C6] A method for activating the immunity of a subject, comprising the steps of administering to the subject a composition containing *Lactococcus lactis* subsp. *lactis* JCM 5805 as a bacterium, or administering to the subject a composition containing *Lactococcus lactis* subsp. *lactis* JCM 5805 as a bacterium, wherein the *Lactococcus lactis* subsp. *lactis* JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0057] [C7] The method according to any one of [C0] to [C5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0058] [C8] The method according to any one of [C0] to [C5], wherein the bacteria are selected from one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0059] [C9] The method according to any one of [C0] to [C5], wherein the bacteria is Lactococcus lactis subsp. lactis.

[0060] [C10] The method according to any one of [C0] to [C5], wherein the bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, and Weissella. * *Weissella virides* JCM 9890, *Bifidobacterium animalis* JCM 1174, *Bifidobacterium animalis* subsp. *animalis* JCM 1190, *Bifidobacterium animalis* subsp. *lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum* subsp. *infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. *lactis* ATCC 7963, *Lactococcus lactis* subsp. *lactis* ATCC 7962, Lactococcus lactis. subsp. lactis ATCC 15346, Lactococcus lactis. subsp. lactis ATCC 13675, Lactococcus lactis. subsp.One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0061] [C11] The method according to any one of [C0] to [C10], wherein the bacteria comprise dead bacteria.

[0062] [C12] The method according to any one of [C0] to [C11], wherein the bacteria are bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

[0063] [C13] According to the method of [C12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, and the amount of bacteria phagocytized via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytized via binding to BDCA2.

[0064] [C14] According to the method of [C13], wherein the amount of phagocytosis not via binding with BDCA2 is the amount of phagocytosis of the bacteria by the pDC in the state where the BDCA2 of the pDC cannot bind to the ligand.

[0065] [C15] The method according to any one of [C12] to [C14], wherein the bacteria promote the production of IFN-α in the pDC by phagocytosis of the pDC.

[0066] [C16] The method according to any one of [C0] to [C15], wherein the above composition is a food composition, a pharmaceutical composition or feed.

[0067] [C17] According to the method of [C16], wherein the bacterial count of the bacteria in each unit package of the above-mentioned food composition, pharmaceutical composition or feed is 1 × 10⁻⁶. 8 More than one.

[0068] [D0] A bacterium used in immune activation that satisfies at least one of the following groups (1) to (4): (1) Bacteria phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of bacteria phagocytosed by pDCs is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs. (2) Bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDC) and meet the following requirement X; [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium. (4) The above-mentioned bacteria are Lactococcus lactis subsp. lactis JCM5805 and meet the following requirement Y; [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0069] [D1] A bacterium phagocytosed by plasmacytoid dendritic cells (pDCs) for use in immune activation. The amount of bacteria that pDC phagocytosed was more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

[0070] [D2] A bacterium that binds to blood dendritic cell antigen 2 (BDCA2) for use in immune activation.

[0071] [D3] Bacteria for application as described in [D0] or [D2], wherein the binding capacity of the bacteria to BDCA2 is greater than that of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2.

[0072] [D4] The bacteria used for application as described in [D3], wherein the binding capacity with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as evaluated in the binding assay.

[0073] [D5] A bacterium phagocytosed by plasmacytoid dendritic cells (pDCs) for use in immune activation, wherein the bacterium meets the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium.

[0074] [D6] A type of Lactococcus lactis subsp. lactis JCM 5805 used as a bacterium for immune activation, wherein the Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0075] [D7] Bacteria for application according to any one of [D0] to [D5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0076] [D8] Bacteria for application according to any one of [D0] to [D5], wherein the bacteria are selected from one or more of the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0077] [D9] Bacteria for application according to any one of [D0] to [D5], wherein the bacteria are Lactococcus lactis subsp. lactis.

[0078] [D10] The bacteria for application according to any one of [D0] to [D5], wherein the bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, and Oenococcus salivarius. * *Weissella paramesenteroides* JCM 6125, *Weissella viridescens* JCM 9890, *Weissella viridescens* JCM 1174, *Bifidobacterium animalis subsp. animalis* JCM 1190, *Bifidobacterium animalis subsp. lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum subsp. Infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis subsp. lactis* ATCC 7963, *Lactococcus lactis* subsp. *lactis* ATCC 7962, *Lactococcus lactis* subsp. *lactis* ATCC 15346, *Lactococcus lactis* subsp. *lactis* ATCC 13675, *Lactococcus lactis* subsp. *lactis*One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0079] [D11] Bacteria for application according to any one of [D0] to [D10], wherein the bacteria comprise dead bacteria.

[0080] [D12] Bacteria for application according to any one of [D0] to [D11], wherein the bacteria are bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

[0081] [D13] According to [D12], the bacteria used for application, wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, and the amount of bacteria phagocytized via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytized via binding to BDCA2.

[0082] [D14] The bacteria for application according to [D13], wherein the amount of phagocytosis not via binding with BDCA2 is the amount of phagocytosis of the bacteria by the pDC in a state where the BDCA2 of the pDC cannot bind to the ligand.

[0083] [D15] Bacteria for application according to any one of [D12] to [D14], wherein the bacteria promote the production of IFN-α in the pDC via phagocytosis of the pDC.

[0084] [D16] Bacteria for application according to any one of [D0] to [D15], wherein the bacteria are contained in a food composition, a pharmaceutical composition or a feed.

[0085] [D17] The bacteria for application as described in [D16], wherein the bacterial count of each unit package of the aforementioned food composition, pharmaceutical composition, or feed is 1 × 10⁻⁶. 8More than one.

[0086] [E0] A composition for use in immune activation, comprising at least one of the bacteria selected from the group consisting of (1) to (4) below: (1) Bacteria phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of bacteria phagocytosed by pDCs is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs. (2) Bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDC) and meet the following requirement X; [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium. (4) The above-mentioned bacteria are Lactococcus lactis subsp. lactis JCM5805 and meet the following requirement Y; [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0087] [E1] A composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) for use in immune activation, wherein, The amount of bacteria that pDC phagocytosed was more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

[0088] [E2] A composition containing bacteria that bind to blood dendritic cell antigen 2 (BDCA2) for use in immune activation.

[0089] [E3] The composition for application according to [E0] or [E2], wherein the binding capacity of the bacteria to BDCA2 is greater than that of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2.

[0090] [E4] The composition for application according to [E3], wherein the binding ability with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as evaluated in a binding assay.

[0091] [E5] A composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) for use in immune activation, wherein the bacteria satisfy the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium.

[0092] [E6] A composition containing *Lactococcus lactis* subsp. *lactis* JCM 5805 as a bacterium for use in immune activation, wherein the *Lactococcus lactis* subsp. *lactis* JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0093] [E7] The composition for application according to any one of [E0] to [E5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0094] [E8] The composition for use according to any one of [E0] to [E5], wherein the bacteria are one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0095] [E9] The composition for use according to any one of [E0] to [E5], wherein the bacteria is Lactococcus lactis subsp. lactis.

[0096] [E10] The composition for application according to any one of [E0] to [E5], wherein the bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, and Oenococcus. * *Weissella paramesenteroides* JCM 6125, *Weissella viridescens* JCM 9890, *Weissella viridescens* JCM 1174, *Bifidobacterium animalis subsp. animalis* JCM 1190, *Bifidobacterium animalis subsp. lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum subsp. Infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis subsp. lactis* ATCC 7963, *Lactococcus lactis* subsp. *lactis* ATCC 7962, *Lactococcus lactis* subsp. *lactis* ATCC 15346, *Lactococcus lactis* subsp. *lactis* ATCC 13675, *Lactococcus lactis* subsp. *lactis*One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0097] [E11] The composition for application according to any one of [E0] to [E10], wherein the bacteria comprise dead bacteria.

[0098] [E12] The composition for application according to any one of [E0] to [E11], wherein the bacteria are bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

[0099] [E13] The composition for application according to [E12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, wherein the amount of bacteria phagocytosed via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed via binding to BDCA2.

[0100] [E14] The composition for application according to [E13], wherein the amount of phagocytosis not via binding with BDCA2 is the amount of phagocytosis of the bacteria by the pDC in a state where the BDCA2 of the pDC cannot bind to the ligand.

[0101] [E15] The composition for application according to any one of [E12] to [E14], wherein the bacteria promote the production of IFN-α in the pDC via phagocytosis of the pDC.

[0102] [E16] The composition for application according to any one of [E0] to [E15] is a food composition, a pharmaceutical composition or a feed.

[0103] [E17] The composition for application according to [E16], wherein the bacterial count of the bacteria in each unit package of the above-mentioned food composition, pharmaceutical composition or feed is 1 × 10⁻⁶. 8 More than one.

[0104] [F0] The application of bacteria that meet at least one of the following groups (1) to (4) in immune activation: (1) Bacteria phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of bacteria phagocytosed by pDCs is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs. (2) Bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDC) and meet the following requirement X; [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium. (4) The above-mentioned bacteria are Lactococcus lactis subsp. lactis JCM5805 and meet the following requirement Y; [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0105] [F1] The application of bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in immune activation, among which, The amount of bacteria that pDC phagocytosed was more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

[0106] [F2] Application of bacteria that bind to blood dendritic cell antigen 2 (BDCA2) in immune activation.

[0107] [F3] According to the application described in [F0] or [F2], wherein the binding capacity of the above-mentioned bacteria to BDCA2 is greater than that of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2.

[0108] [F4] According to the application described in [F3], the binding ability with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as evaluated in the binding assay.

[0109] [F5] Application of bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in immune activation, wherein the bacteria satisfy the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium.

[0110] [F6] The application of Lactococcus lactis subsp. lactis JCM5805 as a bacterium in immune activation, wherein the aforementioned Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0111] [F7] The application according to any one of [F0] to [F5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0112] [F8] The application according to any one of [F0] to [F5], wherein the bacteria are selected from one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0113] [F9] The application according to any one of [F0] to [F5], wherein the above-mentioned bacteria is Lactococcus lactis subsp. lactis.

[0114] [F10] The application according to any one of [F0] to [F5], wherein the aforementioned bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, and Weissella. * *Weissella virides* JCM 9890, *Bifidobacterium animalis* JCM 1174, *Bifidobacterium animalis* subsp. *animalis* JCM 1190, *Bifidobacterium animalis* subsp. *lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum* subsp. *infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. *lactis* ATCC 7963, *Lactococcus lactis* subsp. *lactis* ATCC 7962, Lactococcus lactis. subsp. lactis ATCC 15346, Lactococcus lactis. subsp. lactis ATCC 13675, Lactococcus lactis. subsp.One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0115] [F11] The application according to any one of [F0] to [F10], wherein the bacteria comprise dead bacteria.

[0116] [F12] The application according to any one of [F0] to [F11], wherein the bacteria are bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

[0117] [F13] According to the application described in [F12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, and the amount of bacteria phagocytosed via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed via binding to BDCA2.

[0118] [F14] According to the application described in [F13], wherein the amount of phagocytosis not via binding with BDCA2 is the amount of phagocytosis of the bacteria by the pDC in the state where the BDCA2 of the pDC cannot bind to the ligand.

[0119] [F15] The application according to any one of [F12] to [F14], wherein the bacteria promote the production of IFN-α in the pDC by phagocytosis of the pDC.

[0120] [F16] The application according to any one of [F0] to [F15], wherein the bacteria are contained in a food composition, a pharmaceutical composition or a feed.

[0121] [F17] According to the application described in [F16], the bacterial count of the above-mentioned bacteria in each unit package of the above-mentioned food composition, pharmaceutical composition or feed is 1 × 10⁻⁶. 8 More than one.

[0122] [F18] The application according to any one of [F0] to [F17] is a non-therapeutic application.

[0123] [G0] The use of a composition containing at least one of the bacteria selected from the group consisting of (1) to (4) below in immune activation: (1) Bacteria phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of bacteria phagocytosed by pDCs is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs. (2) Bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDC) and meet the following requirement X; [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium. (4) The above-mentioned bacteria are Lactococcus lactis subsp. lactis JCM5805 and meet the following requirement Y; [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0124] [G1] The application of a composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in immune activation, wherein, The amount of bacteria that pDC phagocytosed was more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

[0125] [G2] The use of compositions containing bacteria that bind to blood dendritic cell antigen 2 (BDCA2) in immune activation.

[0126] [G3] According to the application described in [G0] or [G2], wherein the binding capacity of the above-mentioned bacteria to BDCA2 is greater than that of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2.

[0127] [G4] According to the application described in [G3], the binding ability with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as evaluated in the binding assay.

[0128] [G5] The use of a composition containing bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in immune activation, wherein the bacteria satisfy the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium.

[0129] [G6] The use of a composition containing *Lactococcus lactis* subsp. *lactis* JCM 5805 as a bacterium in immune activation, wherein the *Lactococcus lactis* subsp. *lactis* JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0130] [G7] The application according to any one of [G0] to [G5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0131] [G8] The application according to any one of [G0] to [G5], wherein the bacteria are selected from one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0132] [G9] The application according to any one of [G0] to [G5], wherein the above-mentioned bacteria is Lactococcus lactis subsp. lactis.

[0133] [G10] Application according to any one of [G0] to [G5], wherein the above-mentioned bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, and Weissella. * *Weissella virides* JCM 9890, *Bifidobacterium animalis* JCM 1174, *Bifidobacterium animalis* subsp. *animalis* JCM 1190, *Bifidobacterium animalis* subsp. *lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum* subsp. *infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. *lactis* ATCC 7963, *Lactococcus lactis* subsp. *lactis* ATCC 7962, Lactococcus lactis. subsp. lactis ATCC 15346, Lactococcus lactis. subsp. lactis ATCC 13675, Lactococcus lactis. subsp.One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0134] [G11] The application according to any one of [G0] to [G10], wherein the bacteria comprise dead bacteria.

[0135] [G12] The application according to any one of [G0] to [G11], wherein the bacteria are bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

[0136] [G13] According to the application described in [G12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, and the amount of bacteria phagocytosed via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed via binding to BDCA2.

[0137] [G14] According to the application described in [G13], wherein the amount of phagocytosis not via binding with BDCA2 is the amount of phagocytosis of the bacteria by the pDC in a state where the BDCA2 of the pDC cannot bind to the ligand.

[0138] [G15] The application according to any one of [G12] to [G14], wherein the bacteria promote the production of IFN-α in the pDC by phagocytosis of the pDC.

[0139] [G16] Application according to any one of [G0] to [G15], wherein the above composition is a food composition, a pharmaceutical composition or feed.

[0140] [G17] According to the application described in [G16], wherein the bacterial count of the above-mentioned bacteria in each unit package of the above-mentioned food composition, pharmaceutical composition or feed is 1 × 10⁻⁶. 8 More than one.

[0141] [G18] The application according to any one of [G0] to [G17] is a non-therapeutic application.

[0142] [H0] The use of bacteria that satisfy at least one of the groups (1) to (4) below in the manufacture of immune-activating compositions: (1) Bacteria phagocytosed by plasmacytoid dendritic cells (pDCs), wherein the amount of bacteria phagocytosed by pDCs is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed by pDCs. (2) Bacteria that bind to blood dendritic cell antigen 2 (BDCA2); (3) Bacteria that are phagocytosed by plasmacytoid dendritic cells (pDC) and meet the following requirement X; [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium. (4) The above-mentioned bacteria are Lactococcus lactis subsp. lactis JCM5805 and meet the following requirement Y; [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0143] [H1] The use of bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in the manufacture of immune-activating compositions, wherein, The amount of bacteria that pDC phagocytosed was more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

[0144] The use of [H2] bacteria that bind to blood dendritic cell antigen 2 (BDCA2) in the manufacture of immune-activating compositions.

[0145] [H3] According to the application described in [H0] or [H2], wherein the binding capacity of the above-mentioned bacteria to BDCA2 is greater than that of Lactococcus lactis subsp. lactis ATCC15577 to BDCA2.

[0146] [H4] According to the application described in [H3], the binding ability with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as assessed in the binding assay.

[0147] [H5] The use of bacteria phagocytosed by plasmacytoid dendritic cells (pDCs) in the manufacture of compositions for immune activation, wherein the bacteria satisfy the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and the aforementioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium, the number of pDCs that have engulfed the aforementioned bacteria is at least 2.5% of the total number of pDCs that have been contacted, wherein the pDCs are pDCs that have been pre-cultured in a serum-free culture medium.

[0148] [H6] The use of Lactococcus lactis subsp. lactis JCM5805, as a bacterium, in the manufacture of an immune-activating composition, wherein the aforementioned Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0149] [H7] The application according to any one of [H0] to [H5], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0150] [H8] The application according to any one of [H0] to [H5], wherein the bacteria are selected from one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0151] [H9] The application according to any one of [H0] to [H5], wherein the above-mentioned bacteria is Lactococcus lactis subsp. lactis.

[0152] [H10] The application according to any one of [H0] to [H5], wherein the above-mentioned bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, and Weissella. * *Weissella virides* JCM 9890, *Bifidobacterium animalis* JCM 1174, *Bifidobacterium animalis* subsp. *animalis* JCM 1190, *Bifidobacterium animalis* subsp. *lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum* subsp. *infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. *lactis* ATCC 7963, *Lactococcus lactis* subsp. *lactis* ATCC 7962, Lactococcus lactis. subsp. lactis ATCC 15346, Lactococcus lactis. subsp. lactis ATCC 13675, Lactococcus lactis. subsp.One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0153] [H11] The application according to any one of [H0] to [H10], wherein the bacteria comprise dead bacteria.

[0154] [H12] The application according to any one of [H0] to [H11], wherein the bacteria are bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to blood dendritic cell antigen 2 (BDCA2).

[0155] [H13] According to the application described in [H12], wherein the phagocytosis of the bacteria by the pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, and the amount of bacteria phagocytosed via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed via binding to BDCA2.

[0156] [H14] According to the application described in [H13], wherein the amount of phagocytosis not via binding with BDCA2 is the amount of phagocytosis of the bacteria by the pDC in a state where the BDCA2 of the pDC cannot bind to the ligand.

[0157] [H15] The application according to any one of [H12] to [H14], wherein the bacteria promote the production of IFN-α in the pDC via phagocytosis of the pDC.

[0158] [H16] Application according to any one of [H0] to [H15], wherein the above composition is a food composition, a pharmaceutical composition or feed.

[0159] [H17] According to the application described in [H16], the bacterial count of the above-mentioned bacteria in each unit package of the above-mentioned food composition, pharmaceutical composition or feed is 1 × 10⁻⁶. 8 More than one.

[0160] [I1] A method for screening bacteria with immune activation capabilities, comprising the step of screening the aforementioned bacteria with immune activation capabilities by using binding to blood dendritic cell antigen 2 (BDCA2) as an indicator.

[0161] [I2] According to the method described in [I1], the above screening step is performed by measuring the binding of candidate bacteria to BDCA2.

[0162] [I3] According to the method described in [I1] or [I2], wherein the bacteria mentioned above involved in any one of [A0]~[A18], [B0]~[B17], [C0]~[C17], [D0]~[D17], [E0]~[E17], [F0]~[F18], [G0]~[G18] and [H0]~[H17] are selected.

[0163] [I4] A composition, method, bacteria for application, composition for application, or application according to any one of [A0]~[A18], [B0]~[B17], [C0]~[C17], [D0]~[D17], [E0]~[E17], [F0]~[F18], [G0]~[G18], and [H0]~[H17], wherein the bacteria are selected by the method described in [I1] or [I2].

[0164] [J1] A method for activating pDC, characterized in that bacteria are bound to BDCA2.

[0165] [J2] A method for activating pDC, comprising the steps of ingesting bacteria that bind to BDCA2 by a subject or administering bacteria that bind to BDCA2 to a subject in need of them.

[0166] [J3] The method according to [J1] or [J2] includes the step of causing the bacteria to be phagocytosed by pDC via binding to BDCA2.

[0167] [J4] A composition containing bacteria, used in any one of [J1] to [J3], by means of bacteria.

[0168] [J5] The method according to any one of [J1] to [J3] or the composition according to [J4], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0169] [J6] The method according to any one of [J1] to [J3] or the composition according to [J4] or [J5], wherein the bacteria are one or more selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0170] [J7] The method according to any one of [J1] to [J3] or the composition according to any one of [J4] to [J6], wherein the bacteria is Lactococcus lactis subsp. lactis.

[0171] [J8] The method according to any one of [J1] to [J3] or the composition according to any one of [J4] to [J7], wherein the bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, and Oenococcus oeni JCM. 6125. *Weissella paramesenteroides* JCM 9890, *Weissella viridescens* JCM 1174, *Bifidobacterium animalis subsp. animalis* JCM 1190, *Bifidobacterium animalis subsp. lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum subsp. Infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. lactis ATCC 7963, *Lactococcus lactis* subsp. lactis Lactococcus lactis subsp. lactis) ATCC 7962, Lactococcus lactis subsp. lactis) ATCC 15346, Lactococcus lactis subsp. lactis) ATCC 13675, Lactococcus lactis subsp. lactis.One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL 1505.

[0172] [J9] The method according to any one of [J1] to [J3] or the composition according to any one of [J4] to [J8], wherein the bacteria comprise dead bacteria.

[0173] [K1] A method for enhancing the immune activation capacity and / or the ability of bacteria to be phagocytosed by plasmacytoid dendritic cells (pDCs), comprising culturing bacteria in M17 medium supplemented with glucose at a final concentration of 0.1 to 10% by mass.

[0174] [K2] The method according to [K1], wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

[0175] [K3] According to the method described in [K1] or [K2], wherein the bacteria are selected from one or more bacteria selected from the group consisting of Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium and Lactobacillus.

[0176] [K4] The method according to any one of [K1] to [K3], wherein the above-mentioned bacteria is Lactococcus lactis subsp. lactis.

[0177] [K5] The method according to any one of [K1] to [K4], wherein the bacteria are selected from Lactococcus plantarum JCM 11056, Lactococcus curvatus JCM 1096, Lactococcus murinus JCM 1717, Lactococcus parakefiri JCM 8573, Lactococcus paraplantarum JCM 12533, Leuconostoc carnosum JCM 9695, Streptococcus salivarius JCM 5707, Enterococcus lactis JCM 30200, Oenococcus oeni JCM 6125, and Weissella. * *Weissella virides* JCM 9890, *Bifidobacterium animalis* JCM 1174, *Bifidobacterium animalis* subsp. *animalis* JCM 1190, *Bifidobacterium animalis* subsp. *lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum* subsp. *infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. *lactis* ATCC 7963, *Lactococcus lactis* subsp. *lactis* ATCC 7962, Lactococcus lactis. subsp. lactis ATCC 15346, Lactococcus lactis. subsp. lactis ATCC 13675, Lactococcus lactis. subsp.One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL1505.

[0178] [K6] The method according to any one of [K1] to [K5], wherein the bacteria is Lactococcus lactis subsp. lactis JCM 5805.

[0179] [K7] A bacterium that enhances its immune activation capacity and / or its ability to be phagocytosed by plasmacytoid dendritic cells (pDCs) by any one of the methods described in [K1] to [K6].

[0180] [L1] A method for manufacturing an immune-activating composition containing Lactococcus lactis subsp. lactis JCM 5805 as an active ingredient, comprising the step of culturing Lactococcus lactis subsp. lactis JCM 5805 in M17 medium supplemented with glucose at a final concentration of 0.1 to 10% by mass.

[0181] [L2] The manufacturing method described in [L1] is used to manufacture the composition of any one of [A0] to [A17], [C0] to [C17], [E0] to [E17], [G0] to [G18] and [H0] to [H17].

[0182] [L3] A composition, method, composition for application, or application according to any one of [A0]~[A17], [C0]~[C17], [E0]~[E17], [G0]~[G18], and [H0]~[H17], wherein the composition is a composition manufactured by the manufacturing method described in [L1].

[0183] Invention Effects

[0184] According to the present invention, an immune-activating composition can be provided. For example, the immune-activating composition of the present invention can activate immunity by means of bacteria as the active ingredient being phagocytosed by plasmacytoid dendritic cells (pDCs). Alternatively, for example, the immune-activating composition of the present invention can activate immunity by means of bacteria as the active ingredient binding to BDCA2 on the surface of plasmacytoid dendritic cells (pDCs). Furthermore, for example, the immune-activating composition of the present invention can activate immunity by means of bacteria as the active ingredient being phagocytosed by dendritic cells through binding to BDCA2 on the surface of pDCs, thereby activating pDCs.

[0185] According to the present invention, a method for screening bacteria with immune-activating capabilities can be provided. For example, the screening method of the present invention can use the binding of bacteria to BDCA2 as an indicator to screen bacteria that bind to BDCA2 as potentially having immune-activating capabilities. Attached Figure Description

[0186] Figure 1 This is a graph showing the fluorescence detection area 22 hours after adding LC-Plasma in Example 1.

[0187] Figure 2 This is a graph showing the results of the determination of IFN-α concentration in the culture supernatant recovered 22 hours after adding LC-Plasma in Example 1.

[0188] Figure 3 This is a graph showing the fluorescence detection area of ​​the eight tested lactic acid bacteria 22 hours after the addition of the lactic acid bacteria in Example 2.

[0189] Figure 4 This is a graph showing the results of the determination of IFN-α concentration in the culture supernatant recovered 22 hours after the addition of lactic acid bacteria for the eight tested lactic acid bacteria in Example 2.

[0190] Figure 5 This is a graph showing the correlation between the fluorescence detection area on the horizontal axis and the IFN-α concentration in the culture supernatant for the eight tested lactic acid bacteria in Example 2.

[0191] Figure 6 This is a graph showing the fluorescence detection area of ​​the PL group, the PL+ isotype group, and the PL+BDCA2 anti group 22 hours after the addition of LC-Plasma in Example 3.

[0192] Figure 7 This is a graph showing the results of the determination of IFN-α concentration in the culture supernatant recovered 22 hours after the addition of LC-Plasma in Example 3 for the PL group, PL+ isotype group and PL+BDCA2 anti group.

[0193] Figure 8 This is a graph showing the fluorescence detection area of ​​the isotype group and the BDCA2 anti group 22 hours after the addition of various lactic acid bacteria in Example 4.

[0194] Figure 9 This is a graph showing the results of the determination of IFN-α concentration in the culture supernatant recovered 22 hours after the addition of various lactic acid bacteria in the isotype group and the BDCA2 anti group in Example 4.

[0195] Figure 10 This is a graph showing the correlation between various lactic acid bacteria, with the horizontal axis representing the amount of phagocytosis via binding to BDCA2 and the vertical axis representing the concentration of IFN-α in the culture supernatant.

[0196] Figure 11 This is a graph showing the proportion of LC-Plasma bound to BDCA2 under conditions where the concentration of BDCA2 was 1, 5, 10, 20, or 50 μg / mL, as evaluated by BDCA2 binding assay in Example 5.

[0197] Figure 12 This is a graph showing the results of adding DMSO or pinocembrin D+DMSO in Example 7 and measuring the number of viable cells 24 hours later using a cell counting kit-8.

[0198] Figure 13 This is a graph showing the total integral intensity (TII) of phagocytic activity for the six tested lactic acid bacteria in Example 7, representing the activity 24 hours after the addition of the lactic acid bacteria.

[0199] Figure 14 This is a graph showing the IFN-α concentration in the supernatant 24 hours after the addition of the six tested lactic acid bacteria in Example 7. Detailed Implementation

[0200] The following describes the methods for implementing the present invention, but the present invention is not limited to the following embodiments.

[0201] The first embodiment of the present invention is an immune-activating composition containing bacteria as an active ingredient. In one embodiment, the bacteria contained as an active ingredient in the immune-activating composition are bacteria phagocytosed by pDCs. In another embodiment, the bacteria contained as an active ingredient in the immune-activating composition are bacteria that bind to BDCA2.

[0202] Hereinafter, "an immune-activating composition containing bacteria phagocytosed by pDCs as an active ingredient" and / or "an immune-activating composition containing bacteria that bind to BDCA2 as an active ingredient" will sometimes be referred to as "the immune-activating composition of this embodiment".

[0203] The bacteria contained in the immune-activating composition of this embodiment (hereinafter sometimes referred to as "bacteria of this embodiment") are particularly beneficial to the human body. The bacteria of this embodiment can be Gram-positive or Gram-negative bacteria. For example, the bacteria of this embodiment can be lactic acid bacteria or acetic acid bacteria. Preferably, they can be lactic acid bacteria; more preferably, they can be *Lactococcus*; and even more preferably, they can be *Lactococcus lactis* subsp. *lactococcus*.

[0204] Lactic acid bacteria are bacteria that produce lactic acid as a metabolite. Examples of lactic acid bacteria include, for instance, bacteria of the genus *Oenococcus*, *Bifidobacterium*, *Weissella*, *Tetragenococcus*, *Lactococcus*, *Leuconostoc*, *Pediococcus*, *Streptococcus*, *Enterococcus*, *Lactobacillus*, acetic acid bacteria, and Bacillus species. In one embodiment, lactic acid bacteria can be bacteria of the genus *Lactobacillus* or *Lactococcus*.

[0205] It should be noted that the *Lactobacillus* genus used in this specification includes bacteria that were classified as *Lactobacillus* before the reclassification of the genus. For example, this includes bacteria newly classified as *Acetilactobacillus*, *Agrilactobacillus*, *Amylolactobacillus*, *Apilactobacillus*, *Bombilactobacillus*, *Companilactobacillus*, *Dellaglioa*, *Fructilactobacillus*, *Furfurilactobacillus*, *Holzapfelia*, *Lacticaseibacillus*, *Lactiplantibacillus*, and others that have been reclassified as *Lactobacillus*. Bacteria belonging to the genera *Lapidilactobacillus*, *Latilactobacillus*, *Lentilactobacillus*, *Levilactobacillus*, *Ligilactobacillus*, *Limosilactobacillus*, *Liquorilactobacillus*, *Loigolactobacillus*, *Paralactobacillus*, *Paucilactobacillus*, *Schleiferilactobacillus*, and *Secundilactobacillus*.

[0206] Among the above, the preferred bacteria are those of the genera *Oenococcus*, *Bifidobacterium*, *Lentilactobacillus*, *Weissella*, *Tetragenococcus*, *Lactococcus*, *Leuconostoc*, *Pediococcus*, *Enterococcus*, *Lactobacillus*, and *Lactiplantibacillus*.

[0207] Examples of bacteria in the genus *Oenococcus* include *Oenococcus oeni*. Specific examples of *Oenococcus* include *Oenococcus oeni* JCM 6125.

[0208] Examples of Bifidobacterium species include *Bifidobacterium animalis* subsp. *lactis* and *Bifidobacterium longum* subsp. *infantis*. Specific examples of Bifidobacterium species include *Bifidobacterium animalis* subsp. *lactis* JCM 10602, *Bifidobacterium longum* subsp. *infantis* JCM 1222, *Bifidobacterium longum* subsp. *BB536*, *Bifidobacterium animalis* subsp. *lactis* BB-12, *Bifidobacterium longum* subsp. *infantis* M-63, *Bifidobacterium longum* subsp. *N61*, *Bifidobacterium bifidum* OLB6378, *Bifidobacterium breve* M-16V, *Bifidobacterium breve* MCC1274, and *Bifidobacterium pseudolongum* JCM 1205.

[0209] Examples of bacteria in the genus *Weissella* include *Weissella paramesenteroides* and *Weissella viridescens*. Specific examples of bacteria in the genus *Weissella* include *Weissella paramesenteroides* JCM 9890 and *Weissella viridescens* JCM 1174.

[0210] Examples of tetracocci include Tetragenococcus halophilus. Specific examples of tetracocci include Tetragenococcus NRIC 0098 and Tetragenococcus halophilus No. 1.

[0211] Examples of Lactococcus species include Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, and Lactococcus plantarum.

[0212] Specific examples of *Lactococcus* species include: *Lactococcus lactis* subsp. *lactococcus* JCM 5805, *Lactococcus lactis* subsp. *lactococcus* NBRC 12007, *Lactococcus lactis* subsp. *lactococcus* NRIC 1150, *Lactococcus lactis* subsp. *lactococcus* JCM 20101, *Lactococcus lactis* subsp. *lactococcus* JCM 7638, *Lactococcus lactis* subsp. *lactococcus* ATCC 7963, *Lactococcus lactis* subsp. *lactococcus* ATCC 7962, *Lactococcus lactis* subsp. *lactococcus* subsp. *lactococcus* ATCC 29146, *Lactococcus lactis* subsp. *lactococcus* subsp. *lactococcus* ATCC 27861, *Lactococcus lactis* subsp. *lactococcus* subsp. *lactococcus* ATCC 19435, *Lactococcus lactis* subsp. *lactococcus* subsp. *lactococcus* ATCC 15346, *Lactococcus lactis* subsp. *lactococcus* subsp. *lactococcus* ATCC 13675, *Lactococcus lactis* subsp. *lactococcus* subsp. *lactococcus* ATCC 12929, and so on. Lactococcus lactis subsp. milk ATCC 11454, Lactococcus lactis subsp. milk ATCC 11007, Lactococcus gasseri NBRC 100934, Lactococcus lactis subsp. milk fat JCM 16167, Lactococcus lactis subsp. milk fat NBRC 100676, Lactococcus lactis subsp. hominis JCM 1180, Lactococcus lactis subsp. hominis JCM 11040, and Lactococcus plantarum JCM 11056, etc.

[0213] Examples of Leuconostoc species include Leuconostoc carnosum and Leuconostoc lactis. Specific examples of Leuconostoc species include Leuconostoc carnosum JCM9695 and Leuconostoc lactis NBRC 12455.

[0214] Examples of *Pediococcus* species include *Pediococcus acidilactici*, *Pediococcus pentosaceus*, *Pediococcus cellicola*, *Pediococcus claussenii*, *Pediococcus damnosus*, *Pediococcus ethanolidurans*, *Pediococcus inopinatus*, *Pediococcus parvulus*, and *Pediococcus stilesii*. Specific examples of *Pediococcus* species include *Pediococcus acidilactici* JCM 8797, *Pediococcus acidilactici* K15, and *Pediococcus damnosus* JCM 5886.

[0215] Examples of Streptococcus species include Streptococcus thermophilus. Specific examples of Pediococcus species include Streptococcus thermophilus SBC 8781.

[0216] Examples of Enterococci include Enterococcus alcedinis and Enterococcus faecalis. Specific examples of Enterococci include Enterococcus faecalis EC-12.

[0217] Examples of Lactobacillus species include Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus parakefiri, Lactobacillus plantarum, and Lactobacillus pentosus.

[0218] Specific examples of Lactobacillus species 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 casei* (in the new classification, *Lactobacillus shirota*), *Lactobacillus bulgaricus* OLL1073R-1, *Lactobacillus parakefeldiana* (in the new classification, *Lactobacillus parakefeldiana* JCM 8573), *Lactobacillus plantarum* (in the new classification, *Lactobacillus plantarum*) L-137, *Lactobacillus pentosaccharide* (in the new classification, *Lactobacillus pentosaccharide*) ONRICb0240, and *Lactobacillus helveticus* GCL1815, among others.

[0219] The acetic acid bacteria are not particularly limited, and can be listed as, for example, bacteria of the genus Gluconacetobacter, bacteria of the genus Acetobacter, bacteria of the genus Gluconobacter, etc., preferably bacteria of the genus Gluconacetobacter, more preferably Gluconacetobacter hanni, and even more preferably Gluconacetobacter hanni GK-1.

[0220] As for the genus Bacillus, there is no particular limitation; examples include Bacillus coagulans. Specific examples of the genus Bacillus include Bacillus coagulans strain SANK70258.

[0221] From the viewpoint of flavor for consumption, the bacteria in this embodiment are preferably selected from the group consisting of Lactobacillus, Lactococcus, and Staphylococcus.

[0222] In addition to the above, the bacteria in this embodiment may also be Akkermansia, Bacteroides, Blautia, Clostridium, Collinsella, Enterococcus, Faecalibacterium, Faecalicatena, Lacrimispora, Paeniclostridium, Parabacteroides, or Roseburia.

[0223] As a member of the Akkermansia genus, Akkermansia myxophilus JCM 30893 can be cited as an example.

[0224] As members of the genus *Bacteroides*, examples include *Bacteroides foetida* JCM 9498T, *Bacteroides fragilis* JCM11019T, *Bacteroides fragilis* JCM 11017, *Bacteroides fragilis* JCM 17586, *Bacteroides fragilis* JCM 17587, *Bacteroides ovata* JCM 5824T, *Bacteroides pleomorpha* ATCC 29148T, *Bacteroides pleomorpha* ATCC 29741, *Bacteroides pleomorpha* ATCC 12290, *Bacteroides monomorpha* JCM 5828T, *Bacteroides monomorpha* JCM 13286, *Bacteroides monomorpha* JCM 13287, and *Bacteroides monomorpha* JCM13288.

[0225] As members of the genus *Blautia*, examples include *Blautia acetogenes* JCM 34803T, *Blautia ammonialytica* JCM 34802T, *Blautia argus* JCM 31394T, *Blautia mouse cecum* JCM 34498T, *Blautia spheroidae* JCM 1395T, *Blautia fecalis* JCM 17205T, *Blautia glucosaminea* JCM 17039T, *Blautia hannivarum* JCM 14655, *Blautia hannivarum* JCM 35484, *Blautia humanis* JCM 32276T, *Blautia hydrophila* JCM 31266, *Blautia baijiu* JCM 34225T, and *Blautia rhynchophylla* JCM. 17040T, Blautia ovalis JCM 31340, Blautia producta JCM 1471T, Blautia pseudococcus JCM 35243T, Blautia schenckii JCM 14657T, Blautia velutipes JCM 31267, and Blautia velutipes JCM 35486.

[0226] Examples of Clostridium species include Clostridium butyricum JCM NT, Clostridium medullaris JCM 31500T, and Clostridium symbioticum JCM 1297T.

[0227] Examples of bacteria belonging to the genus Collinsella include Collins aerogenes JCM 10188T, Collins enterica JCM 10643T, Collins feces JCM 10641T, and Collins tanakaei JCM 16071T.

[0228] As bacteria of the genus Enterococcus, examples include Enterococcus faecalis JCM 5803T, Enterococcus faecalis JCM20307, ​​Enterococcus faecium JCM 5804T, and Enterococcus faecium JCM 8903.

[0229] As members of the genus Faecalibacterium, examples include Faecalibacterium tsukuri JCM 39210, Faecalibacterium longum JCM 39208, Faecalibacterium pretonii JCM 31915, Faecalibacterium pretonii JCM 39207, and Faecalibacterium pretonii JCM 39209.

[0230] As a member of the genus *Faecalicatena*, *Faecalicatena orotica* JCM 1429T is an example.

[0231] Examples of species belonging to the genus *Lacrimispora* include *Lacrimispora vesiculata* JCM 15734T, *Lacrimispora pseudowedge-shaped* JCM 1415T, and *Lacrimispora xylana* JCM 15735T.

[0232] As a member of the genus Paeniclostridium, one example is Clostridium soxolense JCM 3814T.

[0233] As a member of the Parabacteroides genus, Parabacteroides fecalis JCM 9497T can be cited as an example.

[0234] Examples of Roseburia species include Rosburia humanis JCM 17582, Rosburia enterica JCM 17583, and Rosburia inulinica JCM 17584.

[0235] Of the aforementioned lactic acid bacteria and acetic acid bacteria, the JCM strain can be obtained from the Microbial Materials Development Laboratory of the RIKEN Center for Biological Resources (3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture, Japan); the NBRC strain can be obtained from the Biogenetic Resources Division of the Technical Base for Product Evaluation (2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan); the NRIC strain can be obtained from the Strains Preservation Laboratory of Tokyo University of Agriculture (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, Japan); and the ATCC strain can be obtained from the American Center for Type Culture Collection (USA). In addition to being obtained from public institutions, the aforementioned lactic acid bacteria and acetic acid bacteria can also be obtained by isolating or purifying them from commercially available products containing lactic acid bacteria or acetic acid bacteria using known methods.

[0236] Furthermore, *Lactobacillus paracasei* KW3110 is deposited under FERM BP-08634 at the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (NIST), an independent administrative agency that serves as an international collection institution based on the Budapest Treaty for the collection of patent microorganisms (1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan, Central No. 6) (now the Patent Biological Collection Center of the Biotechnology Center of the Technical Base for Product Evaluation (NITE-IPOD), Room 2-5-8120, Kazusa-Kamazutari, Kisarazu City, Chiba Prefecture, Japan) (Deposit date: February 20, 2004). Additionally, a derivative strain of *Lactobacillus paracasei* KW3110 is deposited at the same Patent Biological Collection Center under FERM BP-08635 (Deposit date: February 20, 2004).

[0237] As mentioned above, *Lactococcus lactis* subsp. *lactococcus* JCM 5805 can be obtained from the Microbial Materials Development Laboratory of the RIKEN Center for Biological Resources. However, in this invention, identical strains of *Lactococcus lactis* subsp. *lactococcus* JCM 5805 deposited in institutions other than the RIKEN Center for Biological Resources can be used. Specifically, identical strains of *Lactococcus lactis* subsp. *lactococcus* JCM 5805 can be obtained from the Biogenetic Resources Division of the Technical Base for Product Evaluation (2-5-8 Kazusa-kama-ashi, Kisarazu City, Chiba Prefecture, Japan), the Strains Preservation Laboratory of Tokyo University of Agriculture (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, Japan), and the American Type Culture Collection (USA). *Lactococcus lactis* subsp. *lactococcus* JCM 5805 is deposited at the American Type Culture Collection under the names *Lactococcus lactis* subsp. *lactococcus* ATCC 9936 and *Lactococcus lactis* subsp. *lactococcus* ATCC 19435.

[0238] The bacteria in this embodiment can be mutant strains of the strains listed above. The mutant bacterial strain must possess characteristics capable of achieving the purpose of this technology (e.g., if the composition is an immune-activating composition, it must have immune-activating ability). Such mutant strains can be constructed by non-artificially introducing mutations into the strains listed above. Alternatively, such mutant strains can be constructed by artificially introducing mutations into the strains listed above. For example, mutations can be introduced into the bacteria by treatment with mutagens such as ultraviolet light (UV) or DNA alkylating agents, or by gene manipulation methods such as gene editing, represented by CRISPR-Cas9.

[0239] The immune-activating composition of this embodiment may contain one type of bacteria or a mixture of two or more types of bacteria. For example, the bacteria may be a mixture of lactic acid bacteria and acetic acid bacteria.

[0240] In one embodiment, the bacteria are bacteria phagocytosed by pDCs. That is, one embodiment of this invention is an immune-activating composition containing bacteria phagocytosed by pDCs as an active ingredient.

[0241] Plasma-like dendritic cells (pDCs), also known as plasmacytoid dendritic cells, are one of the dendritic cells that make up the innate immune system. pDCs are the main type I interferon-producing cells in the body. Type I interferons exhibit proliferative inhibitory activity against viruses and other pathogens. Representative type I interferons include interferon-α (IFN-α) and interferon-β (IFN-β), which are believed to possess immune-activating capabilities.

[0242] Phagocytosis is a phenomenon that occurs when a cell ingests solid particles or other larger objects such as cells or bacteria. During phagocytosis, the cell expands its cell membrane to envelop the object and take it inside the cell. Subsequently, vesicles containing the ingested object (phagosomes) fuse with lysosomes and break down the contents.

[0243] The fact that a bacterium is phagocytosed by pDCs can be evaluated using the evaluation method described later, which utilizes bacteria labeled with fluorescent or fluorescent dyes. Furthermore, regarding whether a bacterium is phagocytosed by pDCs, in the case of inhibiting pDC phagocytosis, this can be evaluated by a reduction in pDC uptake by the bacteria. For example, when the uptake efficiency of phagocytosed pDCs inhibited by actin polymerization inhibitors (e.g., pinocytin D, erythrosporin B) is significantly lower than the uptake efficiency of pDCs without inhibition of polymerization, the bacterium can be evaluated as being phagocytosed by pDCs. Additionally, when the uptake efficiency of phagocytosed pDCs is significantly lower than the uptake efficiency of pDCs without inhibition of binding to pDC surface proteins associated with bacterial phagocytosis, for example, using ligands or antibodies such as BDCA2 (described later) that bind to pDC surface proteins associated with bacterial phagocytosis, the bacterium can be evaluated as being phagocytosed by pDCs. For example, regarding the uptake efficiency of pDCs by bacteria, the amount of pDCs added to the bacteria at a specified time can be used as an indicator.

[0244] The bacteria in this embodiment can be bacteria whose pDC phagocytic capacity is 0.01 times, 0.1 times, 0.3 times, 0.5 times, 0.7 times, 0.9 times, or 1.0 times that of the pDC of Lactococcus lactis subsp. lactis JCM 5805. Alternatively, the bacteria in this embodiment can be bacteria whose pDC phagocytic capacity is more than 1.0 times, 1.2 times, 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 7.0 times, 10 times, 15 times, 20 times, 30 times, 40 times, 60 times, or 100 times that of the pDC of Lactococcus lactis subsp. lactis ATCC 15577.

[0245] Furthermore, the amount of bacteria phagocytosed by pDC in this embodiment can be evaluated using the evaluation method described later, which utilizes bacteria labeled with fluorescent or fluorescent dyes. This evaluation method can be performed, for example, as follows: Under the same conditions, the bacteria of this embodiment and control bacteria (Lactococcus lactis subsp. JCM 5805 or Lactococcus lactis subsp. ATCC 15577) are contacted with pDC of the same cell number (or the same cell concentration) for a certain period of time. After removing the bacteria from the culture medium, the fluorescence area per unit area of ​​the observation field of a fluorescence microscope in the pDC is evaluated (e.g., μm²). 2 / image), as the phagocytosis amount. In this case, the phagocytosis amount is preferably the average of multiple (2 or more, 3 or more, 5 or more, 7 or more, or 10 or more) fields of view or multiple (2 or more, 3 or more, 5 or more, 7 or more, or 10 or more) samples. When evaluating the phagocytosis amount of pDC using the evaluation method for bacteria labeled with fluorescent or fluorescent pigments described later, the phagocytosis amount of pDC of the bacteria in this embodiment can be 50 μm. 2 / Image above, 100μm 2 / Image above, 200μm 2 / Image above, 400μm 2 / Image above, 500μm 2 / Image above, 600μm 2 / Image above, 700μm 2 / Image above, 800μm 2 / Image above or 900μm 2 / Image above. Here, "image" refers to 0.572mm. 2 The field of view.

[0246] Furthermore, the difference between the amount of pDC phagocytosed by the bacteria in this embodiment and the amount of pDC phagocytosed by *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 can be 1 μm. 2 / Image above, 20μm 2 / Image above, 50μm 2 / Image above, 70μm 2 / Image above, 100μm 2 / Image above, 140μm 2 / Image above, 200μm 2 / Image above, 300μm 2 / Image above, 400μm 2 / Image above, 500μm 2 / Image above, 600μm 2 / Image above, 700μm 2 / Image above or 900μm 2 / Image above.

[0247] The assay was performed using the same method as described below for evaluating bacteria labeled with fluorescent or fluorescent dyes, at a rate of 0.572 mm. 2 Total fluorescence in the field of view (total integrated intensity, TII, OCU×μm) 2 When using images as an indicator to evaluate the phagocytic capacity of pDCs, the TII of bacteria in this embodiment can be 10000 OCU × μm. 2 / Image above, 30000 OCU×μm 2 / Image above, 60000 OCU×μm 2 / Image above, 100000 OCU×μm 2 / Image above, 150000 OCU×μm 2 / Image above, 200000 OCU×μm 2 / Image above or 250000 OCU×μm 2 / Image above.

[0248] In these cases, the amount of pDC phagocytosed is not particularly limited as long as it is a value representing an indicator of the amount of pDC phagocytosed. The evaluation of the amount of pDC phagocytosed can be done using methods commonly used by those skilled in the art that can measure the amount of phagocytosed. For example, the amount of pDC phagocytosed can be measured by the fluorescence intensity of pDC after contact with bacteria labeled with fluorescent or fluorescent dyes. As a more detailed example, the amount of pDC phagocytosed can be evaluated by the fluorescence detected from pDC after contact with pDC for a sufficient period of time (e.g., 24 hours) using bacteria labeled with fluorescent dyes that are highly fluorescent in the acidic environment of the endosome (e.g., pHrodo Red SE, Invitrogen, P36600). As an indicator of fluorescence, indicators commonly used by those skilled in the art can be used. For example, the area or proportion of fluorescence intensity exceeding a specified value in a fluorescence image, the value obtained by multiplying the area exceeding the specified value by the average fluorescence intensity, the average, median, or histogram of fluorescence intensity in a fluorescence image, a histogram of fluorescence intensity obtained by flow cytometry analysis, the proportion of cells contained within a gate when fluorescence intensity is gated, or the fluorescence intensity of bulk cells contained in cell suspensions or microwells used for microplate reader analysis, etc. Additionally, the expression level of a biomarker whose expression level varies according to the amount of pDC phagocytosed can be used as an indicator to evaluate the amount of pDC phagocytosed. The values ​​described above can be considered as the "amount of pDC phagocytosed" in this embodiment.

[0249] In this embodiment, the phagocytic capacity of pDCs can be the phagocytic capacity evaluated using CAL-1 cells. CAL-1 cells are a cancer cell line of human plasmacytoid dendritic cells (pDCs) established from tumor cells in the peripheral blood of a patient. This cell line is deposited with accession number FERM BP-10914 at the Patent and Microbial Collection Center of the Biotechnology Center, Japan (NPMD). CAL-1 cells are disclosed in Japanese Patent Application Publication No. 2007-044008, etc.

[0250] As a detailed example, the amount of pDC phagocytosed can be evaluated using a method described below as "Evaluation method using bacteria labeled with fluorescent dyes". As a more detailed example, the amount of pDC phagocytosed can be evaluated using a method described in the examples.

[0251] [Evaluation methods using fluorescently labeled bacteria]

[0252] (Eating Experiment)

[0253] Prepare a medium containing 5.0 × 10⁻⁶ penicillin / streptomycin (Gibco, 15140-12) using a serum-free medium (e.g., RPMI-1640 medium (Sigma, R8758) supplemented with a final concentration of 1.0 v / v penicillin / streptomycin). 5 A cell suspension of CAL-1 cells / mL was prepared, and 10 mL of the cell suspension was seeded into 10 cm diameter culture dishes. After 16 hours of incubation, the cells were recovered by pipetting. The recovered CAL-1 cells were then incubated at 2.0 × 10⁶ cells / mL. 5 Resuspend the cells at a concentration of 10 cells / mL in the same medium and seed 200 μL / well into each well of a 96-well microplate. Add bacteria stained with pHrodo Red SE (Invitrogen, P36600) using the method described below at a final concentration of 10 μg / mL. Measure the fluorescence of pHrodo Red SE in each well over time using an Incucyte (registered trademark) SX5 live cell analysis system (Sartorius).

[0254] (Bacterial staining using pHrodo Red SE)

[0255] Dissolve pHrodo Red SE (Invitrogen, P36600) in DMSO to prepare a 10.2 mM solution. Weigh the bacterial raw material powder into a 2 mL Eppendorf tube and add 0.1 M sodium bicarbonate to adjust the pH to 9.0, thus preparing a bacterial solution with a concentration of 20 mg / mL. Transfer 95 μL of the bacterial solution to a new 2 mL Eppendorf tube and add 5 μL of the prepared 10.2 mM pHrodo Red SE. After dispersing the bacteria by vortexing, cover with aluminum foil and incubate at room temperature for 60 minutes. Then add 750 μL of PBS, vortex, and centrifuge at 20000 × g for 2 minutes at room temperature. Discard 800 μL of supernatant, add 1.5 mL of PBS, and vortex to completely suspend the precipitate. Centrifuge again at 20000 × g for 2 minutes at room temperature, and discard 1.5 mL of supernatant. Suspend the stained bacteria in 140 μL of PBS to prepare a bacterial suspension with a concentration of 10 mg / mL. Store the suspension at 4°C under light-proof aluminum foil until use. Just before use, dilute the stained bacteria 10-fold with PBS to prepare a bacterial suspension with a concentration of 1 mg / mL. It should be noted that the stained bacterial suspension should be used within 24 hours of preparation.

[0256] (Evaluation of the amount of food consumed)

[0257] The entire field of view (0.572mm) obtained from each hole in the plate is used as the evaluation object. 2 The total area of ​​pHrodo RedSE fluorescence detected (fluorescence detection area) was used as an indicator to quantify the amount of bacteria phagocytosed by CAL-1 cells. The Incucyte (registered trademark) SX5 live cell analysis system (Sartorius) was used to analyze each image (0.572 mm²) based on the number of images used for evaluation. 2 The fluorescence detection area (e.g., μm) 2 The image ( / ) can be used to evaluate the amount of phagocytosis. At this point, stained bacteria emit weak fluorescence even if they are not phagocytosed; therefore, to eliminate the influence of fluorescence independent of phagocytosis, the threshold for the orange channel is set to 5.0. Additionally, depending on the bacterial species, bacteria may form aggregates, sometimes emitting fluorescence exceeding the threshold even if they are not phagocytosed. Therefore, the area of ​​the filters for the orange channel is set to 20 to exclude the influence of excessively large aggregates.

[0258] In one embodiment, the bacteria engulfed by pDCs can satisfy the following requirement X0. It should be noted that the following "ratio of the number of pDCs engulfing bacteria to the total number of pDCs that came into contact" corresponds to the "phagocytosis rate" evaluated in Examples 8 and 9 described later. In one embodiment, the pDCs involved in requirement X0 can be CAL-1 cells (FERM BP-10914).

[0259] [Requirement X0] Make the final concentration 2.0 × 10⁻⁶ 5 After pDCs of 10 cells / mL were contacted with the above-mentioned bacteria at a final concentration of 10 μg / mL in a serum-free culture medium for 24 hours, the ratio of the number of pDCs that had engulfed the above-mentioned bacteria to the total number of pDCs that had been contacted was a predetermined ratio, wherein the pDCs were pDCs that had been pre-cultured in a serum-free culture medium.

[0260] The specified percentage involved in requirement X0 can 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 less than 100%, less than 50.0%, less than 25.0%, or less than 18.0%. These lower and upper limits can be combined arbitrarily. For example, the specified proportions involved in requirement X0 can be 2.5% or more but less than 100%, 2.5% or more but less than 50.0%, 2.5% or more but less than 25.0%, 2.5% or more but less than 18.0%, 3.0% or more but less than 100%, 3.0% or more but less than 50.0%, 3.0% or more but less than 25.0%, 3.0% or more but less than 18.0%, 4.0% or more but less than 100%, 4.0% or more but less than 50.0%, and 4. 0% or more and less than 25.0%, 4.0% or more and less than 18.0%, 5.0% or more and less than 100%, 5.0% or more and less than 50.0%, 5.0% or more and less than 25.0%, 5.0% or more and less than 18.0%, 5.5% or more and less than 100%, 5.5% or more and less than 50.0%, 5.5% or more and less than 18.0%, 6.5% or more and less than 100%, 6.5% or more and less than 50.0%, 6.5% or more and less than 25.0%. Below %, 6.5% and above but below 18.0%, 7.0% and above but less than 100%, 7.0% and above but below 50.0%, 7.0% and above but below 25.0%, 7.0% and above but below 18.0%, 7.5% and above but less than 100%, 7.5% and above but below 50.0%, 7.5% and above but below 25.0%, 7.5% and above but below 18.0%, 8.0% and above but less than 100%, 8.0% and above but below 50.0%, 8.0% and above but below 25.0%, 8.0% and below Above and below 18.0%, above 9.0% and below 100%, above 9.0% and below 50.0%, above 9.0% and below 25.0%, above 9.0% and below 18.0%, above 9.5% and below 100%, above 9.5% and below 50.0%, above 9.5% and below 25.0%, above 9.5% and below 18.0%, above 10.0% and below 100%, above 10.0% and below 50.0%, above 10.0% and below 25.0%, above 10.0% and below 18.0%.Below 0%, 10.5% and below 100%, 10.5% and below 50.0%, 10.5% and below 25.0%, 10.5% and below 18.0%, 11.0% and below 100%, 11.0% and below 50.0%, 11.0% and below 25.0%, 11.0% and below 18.0%, 11.5% and below 100%, 11.5% and below 50.0%, 11.5% and below 25.0%, 11.5% and below 18.0%, 12.0% and below 100%, 12.0% and below 50.0%, 12.0% and above 25.0%. Below 0%, 12.0% and below 18.0%, 12.5% ​​and below 100%, 12.5% ​​and below 50.0%, 12.5% ​​and below 25.0%, 12.5% ​​and below 18.0%, 13.0% and below 100%, 13.0% and below 50.0%, 13.0% and below 25.0%, 13.0% and below 18.0%, 13.5% and below 100%, 13.5% and below 50.0%, 13.5% and below 25.0%, 13.5% and below 18.0%, 14.0% and below 100%, 14.0% and above 50.0%. Below 0%, 14.0% and below 25.0%, 14.0% and below 18.0%, 14.5% and below 100%, 14.5% and below 50.0%, 14.5% and below 25.0%, 14.5% and below 18.0%, 15.0% and below 100%, 15.0% and below 50.0%, 15.0% and below 25.0%, 15.0% and below 18.0%, 15.5% and below 100%, 15.5% and below 50.0%, 15.5% and below 25.0%, 15.5% and below 18.0%, 16.0% and below 100%, 16.0% and below 50.0%, 16.0% and below 25.0%, 16.0% and below 18.0%, 16.5% and below 100%, 16.5% and below 50.0%, 16.5% and below 25.0%, 16.5% and below 18.0%, 17.0% and below 100%, 17.0% and below 50.0%, 17.0% and below 25.0%, 17.0% and below 18.0%, 17.5% and below 100%, 17.5% and below 50.0%, 17.5% and below 25.0%, or 17.5% and below 18.0%.Below 0%. When the specified proportion related to element X0 is above the aforementioned lower limit, the immune activation ability of the composition is more easily improved compared to a proportion below the aforementioned lower limit.

[0261] Whether a certain bacterium meets requirement X0 can be evaluated, for example, by a method comprising the following steps: pre-culturing pDCs in a serum-free medium (pretreatment step); contacting the pDCs obtained by the pretreatment step with the bacteria in a serum-free medium for 24 hours (pDC contact step); and evaluating the ratio of the number of pDCs that have engulfed the bacteria to the total number of pDCs that have been contacted in the contact step (engulfing evaluation step).

[0262] The serum involved in Requirement X0 refers to serum from humans or other animals that is commonly used when culturing animal cells, including human cells. Examples include serum from humans, serum from cattle, serum from horses, and more specifically, fetal bovine serum (FBS).

[0263] The culture medium involved in requirement X0 can be the same as that commonly used by those skilled in the art in cell culture (e.g., basal medium). For example, the culture medium can be a medium with water as the solvent, containing water, sugars such as D-glucose, and amino acids such as L-glutamine. The culture medium may also contain additives. As additives, they can be additives commonly used by those skilled in the art in cell culture, such as at least one selected from the group consisting of antibiotics (penicillin, streptomycin, etc.), vitamins, inorganic salts, and buffers. In one embodiment, the D-glucose content of the culture medium can be set to 500 mg / L or more or 1200 mg / L or more, or the D-glucose content of the culture medium can be set to 6000 mg / L or less or 3000 mg / L or less. These upper and lower limits can be combined arbitrarily. For example, the D-glucose content of the culture medium can be above 500 mg / L and below 6000 mg / L, above 500 mg / L and below 3000 mg / L, above 1200 mg / L and below 6000 mg / L, or above 1200 mg / L and below 3000 mg / L. In one embodiment, the L-glutamine content of the culture medium can be set above 100 mg / L or above 250 mg / L, and the L-glutamine content of the culture medium can be set below 1000 mg / L or below 600 mg / L. These upper and lower limits can be combined arbitrarily. For example, the L-glutamine content of the culture medium can be above 100 mg / L and below 1000 mg / L, above 100 mg / L and below 600 mg / L, above 250 mg / L and below 1000 mg / L, or above 250 mg / L and below 600 mg / L. Culture media can be prepared using methods commonly employed by those skilled in the art. For example, they can be prepared by adding sugars, amino acids, and other components to water. Alternatively, culture media can be prepared by adding missing components to commercially available or readily prepared basal media such as RPMI-1640 (Roswell Park Memorial Institute-1640) and DMEM (Dulbecco's Modified Eagle Medium). The prepared culture media can be sterilized using an autoclave or similar device before use in contact steps. RPMI-1640 is a specific example of a suitable culture medium.

[0264] The culture in the pretreatment step can be performed in environments typically used by those skilled in the art for cell culture, and in one particular manner, in air at 37°C, 5% CO2, and 100% humidity. pDCs are usually suspension cells, but to achieve an adherent cell-like appearance through the pretreatment step, it is preferable to use a culture vessel with pDC adhesion. Examples of culture vessels with pDC adhesion include cell-adhesive plates (e.g., 96-well microplates) and glass-bottomed culture dishes. The concentration of pDCs in the pretreatment step is not particularly limited; for example, based on the concentration of pDCs before adhesion, it can be 0.1 × 10⁻⁶. 6 More than 10.0 × 10⁻⁶ cells / mL 6 Below 10 cells / mL, in a specific method, it can be 5.0 × 10⁻⁶. 5 Cells / mL.

[0265] The incubation time for the pretreatment step can be set to more than 1 hour, more than 2 hours, more than 4 hours, more than 6 hours, or more than 8 hours. Alternatively, it can be set to less than 72 hours, less than 48 hours, less than 36 hours, less than 24 hours, less than 16 hours, less than 12 hours, less than 10 hours, or less than 8 hours. These upper and lower limits can be combined arbitrarily. In a specific method, the incubation time in the pretreatment step can be 16 hours.

[0266] The culture medium used in the pDC contact step can be the same as that described in the pretreatment step. The culture medium in the pDC contact step can be the same as or different from the culture medium in the pretreatment step; in one particular embodiment, it can be the same as the culture medium.

[0267] The proportion of pDCs that have engulfed bacteria relative to the total number of pDCs that have come into contact with the bacteria is not particularly limited, and those skilled in the art can evaluate it according to the methods used in cell population evaluation. Such evaluation methods may be, for example, methods that evaluate the cells contained in a cell population cell by cell or methods that evaluate the cells contained in a cell population in block form; in one approach, it may be a method that evaluates the cells contained in a cell population cell by cell. In the method of evaluating the cells contained in a cell population cell by cell, for example, the proportion of particles or solutions that also detect signals from bacteria out of the number of particles or solutions that detect signals from pDCs can be evaluated as the proportion of pDCs that have engulfed bacteria relative to the total number of pDCs that have come into contact with the bacteria. The signals from pDCs and from bacteria may be, for example, scattered light or fluorescence. The signal from pDCs may be, for example, scattered light; in a particular approach, it may be forward scattered light (FSC) and side scattered light (SSC). The signal from bacteria may be, for example, a signal detected from bacteria or proteins contained therein, or a signal detected from nucleic acids amplified using bacterial DNA as a template; in one approach, it may be, for example, a signal detected from bacteria or proteins contained therein.

[0268] In a preferred embodiment, the method for evaluating the cells contained in a cell population on a cell-by-cell basis can be flow cytometry. That is, in a preferred embodiment, the ratio of the number of pDCs that have engulfed bacteria to the total number of pDCs that have come into contact can be a ratio determined by flow cytometry.

[0269] In detail, the ratio of the number of pDCs that have engulfed bacteria to the total number of pDCs that have come into contact with them can be determined, for example, by flow cytometry using the method described below. It should be noted that, more specifically, the ratio of the number of pDCs that have engulfed bacteria to the total number of pDCs that have come into contact with them can be determined, for example, by the method implemented in Example 8 of this application.

[0270] First, the bacteria used in the pDC contact step are fluorescently labeled by contact with a fluorescent dye for the bacteria or by the genetic expression of a fluorescent protein for the proteins contained therein.

[0271] Then, after the pDC contact step, the dead cells contained in the pDC are contacted with a fluorescent dye for fluorescent labeling as needed. The fluorescent dye used to label the dead cells is not particularly limited to any fluorescent dye commonly used by those skilled in the art; in one particular manner, it may be 7-amino-actinomycin D (7-AAD). It should be noted that the fluorescent labeling of bacteria or their contained proteins can be detected in a way that distinguishes them from the fluorescent labeling of dead cells.

[0272] Finally, flow cytometry was used to evaluate whether pDCs phagocytosed bacteria. Assays were performed according to standard methods. There were no particular limitations on the evaluation of the results; for example, populations containing pDCs were first screened using scattered light as an indicator, then, as needed, populations containing live pDCs were selected from these as particles not labeled as dead cells, and finally, the proportion of particles in this population that showed fluorescence from the fluorescent dyes of the labeled bacteria was evaluated.

[0273] In one particular embodiment, the composition may be a composition containing *Lactococcus lactis* subsp. *lactococcus* JCM 5805 as a bacterium, and *Lactococcus lactis* subsp. *lactococcus* JCM 5805 satisfying the following requirement Y0. It should be noted that the following "ratio of the number of pDCs that have engulfed the bacteria to the total number of pDCs that have come into contact" corresponds to the "phagocytosis rate" evaluated in Example 9 described later. In one embodiment, the pDC involved in requirement Y0 may be CAL-1 cells (FERM BP-10914). The composition of this embodiment is not limited to use for immune activation; the composition described for this embodiment may be used, and the composition of this embodiment may be, for example, an immune activation composition.

[0274] [Requirement Y0] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and Lactococcus lactis subsp. milki at a final concentration of 10 μg / mL in serum-free medium, the proportion of pDCs that engulfed Lactococcus lactis subsp. milki at a final concentration of 10 μg / mL to the total number of pDCs that were contacted was greater than 15.0%, where the pDCs were pDCs that had been pre-cultured in serum-free medium.

[0275] The specified percentage involved in requirement Y0 can 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 less than 100%, less than 50.0%, less than 25.0%, or less than 18.0%. These lower and upper limits can be combined arbitrarily. For example, the specified proportions involved in requirement Y0 can be 10.0% or more but less than 100%, 10.0% or more but less than 50.0%, 10.0% or more but less than 25.0%, 10.0% or more but less than 18.0%, 10.5% or more but less than 100%, 10.5% or more but less than 50.0%, 10.5% or more but less than 25.0%, 10.5% or more but less than 18.0%, 11.0% or more but less than 100%, 11.0% or more but less than 50.0%, and 11.0% or more but less than 25.0%. Below 0%, 11.0% and below 18.0%, 11.5% and below 100%, 11.5% and below 50.0%, 11.5% and below 25.0%, 11.5% and below 18.0%, 12.0% and below 100%, 12.0% and below 50.0%, 12.0% and below 25.0%, 12.0% and below 18.0%, 12.5% ​​and below 100%, 12.5% ​​and below 50.0%, 12.5% ​​and below 25.0%, 12.5% ​​and above 18.0%. Below 0%, 13.0% and below 100%, 13.0% and above 50.0%, 13.0% and above 25.0%, 13.0% and above 18.0%, 13.5% and above 100%, 13.5% and above 50.0%, 13.5% and above 25.0%, 13.5% and above 18.0%, 14.0% and above 100%, 14.0% and above 50.0%, 14.0% and above 25.0%, 14.0% and above 18.0%, 14.5% and above 100%. 100%, 14.5% and below 50.0%, 14.5% and below 25.0%, 14.5% and below 18.0%, 15.0% and below 100%, 15.0% and below 50.0%, 15.0% and below 25.0%, 15.0% and below 18.0%, 15.5% and below 100%, 15.5% and below 50.0%, 15.5% and below 25.0%, 15.5% and below 18.0%, 16.0% and below 100%, 16.0% and above 50.0%.The percentages specified in requirement Y0 are as follows: 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, 17.5% or more and 25.0% or less, or 17.5% or more and 18.0% or less. When the specified percentages involved in requirement Y0 are above the aforementioned lower limit, the immune activation ability of the composition is more easily improved compared to the case where the percentages are below the aforementioned lower limit. The prescribed proportions involved in requirement Y0 can be evaluated using the same evaluation method as the prescribed proportions involved in requirement X0 described above.

[0276] Lactococcus lactis subsp. JCM 5805, which meets requirement Y0, can be prepared, for example, by a manufacturing method according to one of the second embodiments described later.

[0277] One embodiment of the bacteria has the property of binding to BDCA2 (blood dendritic cell antigen 2).

[0278] BDCA2 is one of the receptor proteins, primarily expressed in plasmacytoid dendritic cells (pDCs). In one embodiment, the bacteria used in this embodiment may be bacteria that selectively bind to BDCA2. In another embodiment, the bacteria may be bacteria that specifically bind to BDCA2. Bacterial binding to BDCA2 means that the BDCA2 binding capacity is greater than that of *Lactococcus lactis* subsp. *lactis* ATCC15577. Selective binding of bacteria to BDCA2 means that in a mixed system containing BDCA2 (e.g., a system containing various proteins on the cell surface), the bacteria preferentially bind to BDCA2. For example, the binding efficiency to pDCs expressing BDCA2 on the cell surface is statistically significantly higher than that to pDCs not expressing BDCA2 or to pDCs whose cell surface BDCA2 is blocked by anti-BDCA2 antibodies (e.g., p-value less than 0.05 between the two groups). Bacterial BDCA2-specific binding refers to the binding of bacteria to BDCA2 only in a mixed system containing BDCA2 (e.g., a system containing various proteins on the cell surface) except for specific adsorption. For example, the binding efficiency of pDCs expressing BDCA2 on the cell surface is statistically significantly higher than that of pDCs that do not express BDCA2 or pDCs whose cell surface BDCA2 is blocked by anti-BDCA2 antibodies (e.g., p-value less than 0.01 between the two groups).

[0279] The binding of bacteria to BDCA2 can be evaluated using methods commonly employed by those skilled in the art, such as binding assays. Binding assays can be in vitro methods capable of evaluating the binding of bacteria to BDCA2 based on specified detection indicators. These in vitro methods can be cell-free (i.e., cell-free) in vitro methods. The BDCA2 used in the binding assay can be BDCA2 in a free state in an aqueous medium, BDCA2 expressed by a cell line, or BDCA2 expressed on an artificial lipid bilayer membrane. Preferably, it can be BDCA2 in a free state in an aqueous medium, which may be purified. Such BDCA2 can be commercially available, or it can be a substance isolated and purified after expression in host cells using a vector or the like. Such BDCA2 can be a chimeric protein with a separation tag such as a flag tag.

[0280] The bacteria in this embodiment can be bacteria whose binding capacity to BDCA2 is 0.3 times, 0.5 times, 0.7 times, 0.9 times, or 1.0 times greater than that of *Lactococcus lactis* subsp. *lactococcus* JCM 5805 with BDCA2. Alternatively, the bacteria in this embodiment can have a binding capacity to BDCA2 greater than 1.0 times, 1.2 times, 1.5 times, 2.0 times, 2.5 times, 3.0 times, 5.0 times, 6.0 times, 10.0 times, 20.0 times, or 30.0 times greater than that of *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 with BDCA2.

[0281] In these cases, the binding affinity with BDCA2 is not particularly limited as long as it is a value representing the binding affinity index; for example, it can be IC. 50 The values ​​of indicators commonly used by those skilled in the art to evaluate the binding ability of two substances can be the values ​​of the indicators evaluating the binding of bacteria to BDCA2 in a binding assay described later. In a particular method, the binding ability to BDCA2 can be the proportion of bacteria bound to BDCA2 evaluated in a binding assay described later. When the indicator of the binding ability to BDCA2 is the proportion of bacteria bound to BDCA2, for example, bacteria that bind to BDCA2 at a rate of 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 based on bacterial count can be judged as bacteria bound to BDCA2.

[0282] Furthermore, regarding the bacteria of this embodiment, when evaluating the binding capacity of bacteria bound to BDCA2 by a binding assay described later, the difference between the BDCA2 binding capacity of the bacteria and the BDCA2 binding capacity of Lactococcus lactis subsp. lactis ATCC 15577 can 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.

[0283] The following describes one method for evaluating the binding of bacteria to BDCA2. One binding assay includes: a step of contacting BDCA2 with bacteria (contact step); and a step of evaluating the binding of BDCA2 to bacteria after the contact step (evaluation step).

[0284] In the contact step, the bacteria and BDCA2 are brought into contact in an aqueous medium. The aqueous medium can be a buffer solution used by those skilled in the art in in vitro methods of using proteins, such as PBS (phosphate-buffered saline), HBSS (Henkes' balanced salt solution), HEPES buffer, and Tris buffer, with the salt concentration and pH appropriately adjusted. The aqueous medium may also contain surfactants, organic solvents (e.g., dimethyl sulfoxide and ethanol), and reducing agents (e.g., glutathione), additives used by those skilled in the art in in vitro methods of using proteins. The BDCA2 in the contact step can be BDCA2 present in a free state in the aqueous medium.

[0285] Regarding the concentration of bacteria in the contact step, there are no particular limitations as long as the binding of BDCA2 to bacteria can be evaluated in subsequent contact steps. In one method, the concentration of bacteria in the contact step can 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, and can be less than 10000 μg / mL, less than 1000 μg / mL, less than 100 μg / mL, or less than 30 μg / mL. In another approach, these upper and lower limits can be arbitrarily combined. For example, the bacterial concentration in the contact step can be 0.01 μg / mL or higher and 10000 μg / mL or lower, 0.01 μg / mL or higher and 1000 μg / mL or lower, 0.01 μg / mL or higher and 100 μg / mL or lower, 0.01 μg / mL or higher and 30 μg / mL or lower, 0.1 μg / mL or higher and 10000 μg / mL or lower, 0.1 μg / mL or higher and 100 μg / mL or lower. The concentrations are: below L, 0.1 μg / mL to 30 μg / mL, 1.0 μg / mL to 10000 μg / mL, 1.0 μg / mL to 1000 μg / mL, 1.0 μg / mL to 100 μg / mL, 1.0 μg / mL to 30 μg / mL, 3.0 μg / mL to 10000 μg / mL, 3.0 μg / mL to 100 μg / mL, or 3.0 μg / mL to 30 μg / mL. In one particular method, the bacterial concentration in the contact step can be 10 μg / mL.

[0286] Regarding the concentration of BDCA2 in the contact step, there are no particular limitations as long as the binding of BDCA2 to bacteria can be evaluated in subsequent contact steps. In one method, the concentration of BDCA2 can be 0.01 μg / mL or more, 0.1 μg / mL or more, or 0.3 μg / mL or more, and can 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 another approach, these upper and lower limits can be arbitrarily combined. For example, the concentration of BDCA2 can be above 0.01 μg / mL and below 5000 μg / mL, above 0.01 μg / mL and below 2000 μg / mL, above 0.01 μg / mL and below 1000 μg / mL, above 0.01 μg / mL and below 500 μg / mL, above 0.01 μg / mL and below 200 μg / mL, above 0.1 μg / mL and below 5000 μg / mL, or above 0.1 μg / mL. The concentrations are specified as follows: below 2000 μg / mL, above 0.1 μg / mL but below 1000 μg / mL, above 0.1 μg / mL but below 500 μg / mL, above 0.1 μg / mL but below 200 μg / mL, above 0.3 μg / mL but below 5000 μg / mL, above 0.3 μg / mL but below 2000 μg / mL, above 0.3 μg / mL but below 1000 μg / mL, above 0.3 μg / mL but below 500 μg / mL, or above 0.3 μg / mL but below 200 μg / mL. In one particular method, the bacterial concentration in the contact step can be 10 μg / mL.

[0287] Regarding the concentration ratio of bacteria to BDCA2 in the contact step, there are no particular limitations as long as the binding of BDCA2 to bacteria can be evaluated in subsequent contact steps. In one method, the concentration of BDCA2 [μg / mL] in the contact step can be more than 0.01 times, more than 0.03 times, more than 0.1 times, or more than 0.3 times the concentration of bacteria [μg / mL], and can be less than 100 times, less than 30 times, less than 10 times, or less than 3 times. In another approach, these upper and lower limits can be arbitrarily combined. For example, the concentration of BDCA2 [μg / mL] in the contact step can be more than 0.01 times and less than 100 times, more than 0.01 times and less than 30 times, more than 0.01 times and less than 10 times, more than 0.01 times and less than 3 times, more than 0.03 times and less than 100 times, more than 0.03 times and less than 30 times, more than 0.03 times and less than 10 times, more than 0.03 times and less than 3 times, more than 0.1 times and less than 100 times, more than 0.1 times and less than 30 times, more than 0.1 times and less than 10 times, more than 0.1 times and less than 3 times, or more than 0.3 times and less than 100 times, more than 0.3 times and less than 30 times, more than 0.3 times and less than 10 times, or more than 0.3 times and less than 3 times. In one particular method, the concentration of BDCA2 [μg / mL] in the contact step can be 1.0 times the concentration of bacteria [μg / mL].

[0288] Regarding contact conditions such as time and temperature in the contact step, they can be appropriately determined by those skilled in the art, provided that the binding of BDCA2 to bacteria can be evaluated in subsequent contact steps. For example, regarding the contact time in the contact step, there is no particular limitation as long as the binding of BDCA2 to bacteria can be evaluated in subsequent contact steps. In one embodiment, the contact time in the contact step can be more than 1 minute, more than 10 minutes, or more than 30 minutes, or less than 24 hours, less than 6 hours, or less than 2 hours. In another embodiment, these upper and lower limits can be arbitrarily combined, for example, the contact time in the contact step can be more than 1 minute and less than 24 hours, more than 1 minute and less than 6 hours, more than 1 minute and less than 2 hours, more than 10 minutes and less than 24 hours, more than 10 minutes and less than 6 hours, more than 10 minutes and less than 2 hours, or more than 30 minutes and less than 24 hours, more than 30 minutes and less than 6 hours, or more than 30 minutes and less than 2 hours. In a particular embodiment, the contact time in the contact step can be 1 hour. As a more specific example of the contact steps, the steps for BDCA2 binding determination described in the embodiments of this application can be cited.

[0289] In the evaluation step, the binding of BDCA2 to bacteria after the contact step is evaluated. The binding evaluation method in this step is not particularly limited as long as it can evaluate the binding of BDCA2 to bacteria; for example, it can be flow cytometry, ELISA, radioimmunoassay, surface plasmon resonance, or isothermal titration calorimetry. In one specific approach, the binding evaluation method in this step can be flow cytometry. When evaluating the binding of BDCA2 to bacteria by flow cytometry, for example, BDCA2 and bacteria can be labeled with different colored fluorescent dyes, thereby evaluating the binding of BDCA2 to bacteria based on the fluorescence intensity at the wavelengths corresponding to these fluorescent dyes. BDCA2 and bacteria can be labeled before the contact step, or after the contact step and before the evaluation step, using BDCA2-selective labeling methods or bacterial-selective labeling methods (e.g., labeling using protein tags, labeling using specific antibodies, or labeling using bacterial-derived selective secondary antibodies).

[0290] The evaluation criteria for assessing the binding of BDCA2 to bacteria are not particularly limited as long as they are values ​​representing binding ability; for example, they could be IC50. 50 Values ​​of indicators commonly used by those skilled in the art when evaluating the binding affinity of two substances can be values ​​representing the binding affinity of each bacterium to BDCA2 (e.g., the proportion of bacteria binding to BDCA2 or the average or median number of BDCA2-labeled bacteria in one bacterium). In a particular embodiment, the indicator of BDCA2 binding to bacteria evaluated in the evaluation step can be the proportion of bacteria binding to BDCA2. The proportion of bacteria binding to BDCA2 can be calculated as the proportion of particles that exhibit fluorescence at a wavelength corresponding to the wavelength of the fluorescent dye labeled with the bacteria, and of particles that exhibit fluorescence intensity at a certain or higher wavelength corresponding to the fluorescent dye labeled with BDCA2. As a certain or higher benchmark, for example, in the case of using a chimeric protein containing BDCA2 bound to the Fc region, a threshold can be set such that the proportion of controls containing only the Fc region binding to each bacterium is less than 0.1%.

[0291] Therefore, the binding ability of bacteria to BDCA2 can be evaluated using a binding assay. Furthermore, the inventors have discovered that bacteria binding to BDCA2 possess immune-activating capabilities. Thus, using BDCA2 binding as an indicator, the immune-activating capability of bacteria can be evaluated, and bacteria with immune-activating capabilities can be screened; such an indicator can be obtained through a binding assay. Specifically, one embodiment of the present invention is a method for screening bacteria with immune-activating capabilities, which includes a screening step using BDCA2 binding as an indicator. In one embodiment, the screening step is performed by a BDCA2 binding assay of candidate bacteria. The candidate bacteria can be the same bacteria as those in one embodiment of the present invention. The binding assay is as described above.

[0292] The bacteria in this embodiment (i.e., bacteria phagocytosed by pDCs and / or bacteria that bind to BDCA2) can be bacteria that are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to BDCA2.

[0293] The bacteria in this embodiment can be bacteria whose phagocytic amount via pDCs bound to BDCA2 is 0.1 times, 0.3 times, 0.5 times, 0.7 times, 0.9 times, or 1.0 times that of Lactococcus lactis subsp. lactis JCM 5805 via pDCs bound to BDCA2. Alternatively, the bacteria in this embodiment can have phagocytic amount via pDCs bound to BDCA2 exceeding 1.0 times, 1.2 times, 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 7.0 times, 10 times, 20 times, 30 times, 40 times, 60 times, or 100 times that of Lactococcus lactis subsp. lactis ATCC 15577 via pDCs bound to BDCA2.

[0294] Furthermore, regarding the bacteria of this embodiment, when evaluating the amount of pDCs bound to BDCA2 using the evaluation method described later, the amount of pDCs bound to BDCA2 used by the bacteria of this embodiment can be 10 μm. 2 / Image above, 20μm 2 / Image above, 30μm 2 / Image above, 50μm 2 / Image above, 100μm 2 / Image above, 200μm 2 / Image above, 400μm 2 / Image above, 500μm 2 / Image above, 600μm 2 / Image above, 700μm 2 / Image above, 800μm 2 / Image above or 900μm 2 / Image above. It should be noted that, as described above in the (Evaluation of Attrition), the area of ​​each image is set to 0.572 mm². 2 .

[0295] Furthermore, the difference between the amount of bacteria phagocytosed via pDCs bound to BDCA2 and the amount of *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 phagocytosed via pDCs bound to BDCA2 can be 1 μm. 2 / Image above, 20μm 2 / Image above, 40μm 2 / Image above, 70μm 2 / Image above, 100μm 2 / Image above, 140μm 2 / Image above, 200μm 2 / Image above, 300μm 2 / Image above, 400μm 2 / Image above, 500μm 2 / Image above, 600μm 2 / Image above, 700μm 2 / Image above or 900μm 2 / Image above. It should be noted that, as described above in the (Evaluation of Attrition), the area of ​​each image is set to 0.572 mm². 2 .

[0296] In one method, the phagocytosis of bacteria by pDC includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2. The amount of phagocytosis via binding to BDCA2 can be a predetermined multiple or greater than the amount of phagocytosis without binding to BDCA2. This predetermined multiple can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Furthermore, regarding the phagocytosis of bacteria by pDC, the amount of phagocytosis via binding to BDCA2 in the total phagocytosis can 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 that are engulfed by pDCs at a rate greater than that not engulfed by BDCA2 binding are more likely to be engulfed by pDCs by binding to BDCA2. Therefore, pDCs have high activation capacity and high immune activation capacity.

[0297] In these cases, the amount of pDC phagocytosed via BDCA2 binding is not particularly limited as long as it is a value representing the amount of pDC phagocytosed via BDCA2 binding. For example, the amount of pDC phagocytosed by bacteria via BDCA2 binding and the amount phagocytosed without BDCA2 binding can be evaluated as follows: First, evaluate the amount of untreated pDC phagocytosed by bacteria (phagocytosis P). Then, evaluate the amount of pDC phagocytosed by bacteria treated with BDCA2 in a state where ligand binding on the cell surface is inhibited (phagocytosis Q). Thus, phagocytosis Q represents the amount of pDC phagocytosed without BDCA2 binding. Furthermore, the value obtained by subtracting phagocytosis Q from phagocytosis P represents the amount of pDC phagocytosed via BDCA2 binding. That is, in one approach, the amount of phagocytosis without BDCA2 binding can be the amount of pDC phagocytosed by bacteria in a state where BDCA2 cannot bind to the ligand.

[0298] Treatments that inhibit the ligand-binding state of BDCA2 can include, for example, exposing pDCs to an anti-BDCA2 antibody (blocking), exposing pDCs to BDCA ligands (competitive inhibition), knocking down or eliminating BDCA2 in pDCs using conventional methods, or introducing a mutation into the BDCA2 of pDCs using conventional methods to eliminate ligand binding ability. In a preferred embodiment, treatment that inhibits the ligand-binding state of BDCA2 can involve exposing pDCs to an anti-BDCA2 antibody. In this case, the state of BDCA2 on the cell surface of pDCs bound to the anti-BDCA2 antibody is equivalent to the state where BDCA2 in pDCs cannot bind to the ligand. In these cases, the anti-BDCA2 antibody can be a monoclonal antibody or a polyclonal antibody; in a preferred embodiment, it can be a monoclonal antibody. For example, anti-BDCA2 antibodies can be obtained from suppliers such as Miltenyi Biotec (product number: 130-090-690).

[0299] As a detailed example, the amount of pDC phagocytosis via binding to BDCA2 can be the amount of phagocytosis evaluated by a method described below as "Evaluation Method of Amount of Pphagocytosis via Binding to BDCA2 and Amount of Pphagocytosis without Binding to BDCA2". As a more detailed example, the amount of pDC phagocytosis via binding to BDCA2 can be the amount of phagocytosis evaluated by a method described in the examples.

[0300] [Evaluation methods for phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2]

[0301] To each well containing a CAL-1 cell suspension in the same manner as in the phagocytosis assay described above [using the evaluation method for bacteria labeled with fluorescent pigments], add 10 μg / mL of bacteria and anti-BDCA2 antibody (Miltenyi Biotec, 130-090-690) or bacteria and an isotype control of the same type and subtype as the anti-BDCA2 antibody (Miltenyi Biotec, 130-106-545), and incubate for 30 minutes. Then, measure the fluorescence intensity of pHrodo Red SE in the same manner as in the evaluation of phagocytosis using the same method described above [using the evaluation method for bacteria labeled with fluorescent pigments], and obtain the fluorescence detection area (e.g., μm²) for each image. 2 (Image). Then, the fluorescence detection area when anti-BDCA2 antibody was added was taken as the phagocytosis Q mentioned above, and the fluorescence detection area when isotype control was added was taken as the phagocytosis P mentioned above. The phagocytosis via binding to BDCA2 (PQ) and the phagocytosis without binding to BDCA2 (P) were evaluated.

[0302] In the immune activation composition of this embodiment, the bacteria can be live bacteria, dead bacteria, or a mixture of live and dead bacteria. The bacteria in the immune activation composition of this embodiment can be composed of dead bacteria.

[0303] The immune-activating composition of this embodiment contains bacteria with high immune-activating ability. The immune-activating ability of the bacteria in the immune-activating composition of this embodiment can be 0.01 times, 0.03 times, 0.05 times, 0.07 times, 0.09 times, 0.1 times, 0.3 times, 0.5 times, 0.7 times, 0.9 times, or 1.0 times or more than the immune-activating ability of pDCs of *Lactococcus lactis* subsp. *lactococcus* ATCC 15577. Furthermore, the immune-activating ability of the bacteria in the immune-activating composition of this embodiment can be more than 1.0 times, 1.2 times, 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, 7.0 times, 10 times, 20 times, 30 times, 40 times, 60 times, or 100 times or more than the immune-activating ability of pDCs of *Lactococcus lactis* subsp. *lactococcus* ATCC 15577. In these cases, the immune activation capacity is not particularly limited as long as it is a parameter used by those skilled in the art as an indicator of immune activation capacity. In a preferred embodiment, it can be the expression level of IFN-α.

[0304] The immune activation capacity (immune activation ability) in this specification refers to the ability to activate cellular immunity and / or humoral immunity, which is the activation effect (activation) of the innate immune system possessed by the bacteria of this embodiment in the cells they contact or the animals to which the drug is administered. In one embodiment, the immune activation capacity can be the dendritic cell activation capacity of the innate immune system that activates dendritic cells; more specifically, it can be the plasma cell-like dendritic cell activation capacity (pDC activation capacity). In another embodiment, the immune activation capacity can be an increase in the expression levels of cytokines or cell surface markers secreted by dendritic cells, preferably an increase in the expression level of IFN-α, or an increase in the expression level of IFN-α produced in pDCs. In vivo, pDCs are the main producers of IFN-α, and IFN-α is one of the cytokines responsible for the biological defense mechanisms of the innate immune system. Therefore, a substance that promotes the expression of IFN-α in pDCs can also be called a substance with dendritic cell activation capacity and pDC activation capacity, and can also be called a substance with immune activation capacity.

[0305] For example, the immune activation capacity of bacteria can be evaluated by the expression level of INF-α in pDCs after contact with the bacteria. The expression level of IFN-α in pDCs is the amount of a biomolecule that can serve as an indicator of the amount of IFN-α expressed by the bacteria in pDCs. In one approach, the expression level of IFN-α can 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 approach, the expression level of IFN-α can be the ELISPOT method, in which IFN-α secreted extracellularly is adsorbed onto cell culture wells and measured, or the amount of IFN-α secreted extracellularly and present in the culture supernatant. In these cases, the culture supernatant is the culture medium after contacting the bacteria with pDCs in the contact step. The amount of IFN-α in the culture supernatant can be determined after recovering the culture supernatant using methods commonly used by those skilled in the art for quantifying specific proteins. Furthermore, regarding the amount of IFN-α in pDCs (e.g., CAL-1 cells), the amount of IFN-α in the lysate can be determined using methods commonly performed by those skilled in the art, such as flow cytometry to measure intracellular cytokine staining, after preparing pDC lysates, and then according to methods commonly used by those skilled in the art for quantifying specific proteins. In these cases, the determination of the amount of IFN-α can be performed, for example, by ELISA or Western blotting. When determining the amount of IFN-α using ELISA, for example, a human IFNα multi-subtype ELISA kit (PBL Assay Science) can be used. Additionally, the amount of IFN-α-encoding mRNA in pDCs (e.g., CAL-1 cells) can be determined by preparing pDC lysates using methods commonly performed by those skilled in the art, and then according to methods commonly used by those skilled in the art for quantifying mRNA. Quantification of mRNA can be performed, for example, by quantitative PCR (q-PCR) using real-time PCR, direct digital counting (e.g., nCounter (registered trademark)), or by using next-generation sequencing (NGS).

[0306] When evaluating the immune activation capacity of bacteria by using the expression level of INF-α in pDCs after contact with the bacteria, this indicator can be the expression level of IFN-α in pDCs (e.g., CAL-1 cells) after contact with the bacteria in a serum-free medium (e.g., RPMI-1640 medium (Sigma, R8758) supplemented with a final concentration of 1.0% by volume of penicillin / streptomycin (Gibco, 15140-12)) for a specified time (e.g., 3 to 168 hours, or 22 hours as an example), preferably the amount of IFN-α secreted extracellularly and present in the culture supernatant after treatment under the same conditions. As a more detailed example, the indicator of the immune activation capacity of the bacteria can be the amount of IFN-α in the culture supernatant after treatment and measurement using the same method as in the examples.

[0307] In one embodiment of this invention, the bacteria are phagocytosed by plasmacytoid dendritic cells (pDCs) via binding to BDCA2. In this embodiment, the bacteria can promote the production of IFN-α in pDCs through phagocytosis. That is, the bacteria in this embodiment can promote the production of IFN-α in pDCs through phagocytosis of pDCs after binding to BDCA2.

[0308] The fact that bacteria promote the production of IFN-α in pDCs by phagocytosis of pDCs after binding with BDCA2 can be determined by the following: after treatment to inhibit the ligand-binding state of BDCA2, the amount of IFN-α produced in pDCs exposed to BDCA2 is less than the amount of IFN-α produced in pDCs without such treatment. The bacteria in this embodiment can be those in which the amount of IFN-α produced in pDCs exposed to BDCA2 after treatment to inhibit the ligand-binding state is less than 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10 of the amount of IFN-α produced in pDCs without such treatment. In these cases, the treatment to inhibit the ligand-binding state of BDCA2 and the amount of IFN-α produced have been explained above.

[0309] Regarding the promotion of IFN-α production in pDCs by bacteria through phagocytosis of pDCs bound to BDCA2, the fold change in IFN-α production of pDCs relative to the change in the amount phagocytosed via BDCA2 can be greater than 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0. In the above cases, a fold change in IFN-α production of pDCs relative to the change in the amount phagocytosed via BDCA2 of X times or more means that when the amount phagocytosed via BDCA2 by pDCs increases by Y%, the amount of IFN-α production of pDCs increases by X×Y% or more, and / or when the amount phagocytosed via BDCA2 by pDCs decreases by Z% or more, the amount of IFN-α production of pDCs decreases by X×Z% or more.

[0310] The immune-activating composition of this embodiment can be a food composition, a pharmaceutical composition, a quasi-drug, a bacterial raw material powder (a powder formed by drying bacterial cells or a powder containing the same), an additive, or a feed, preferably a food composition. Immune activation capacity (immune activation ability) refers to the activation (activation) of the innate immune system of cells or organisms. One type of immune-activating composition can be a dendritic cell activation composition, or a plasma cell-like dendritic cell activation (pDC activation) composition. The immune-activating composition of this embodiment can be implemented according to the following description.

[0311] The bacterial content in this embodiment is not particularly limited, as long as it is an effective amount to achieve immune activation. Therefore, it may vary depending on the form of the immune activation composition of this embodiment. For example, relative to the total dry weight of the immune activation composition of this embodiment, the dry weight of the bacteria in 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, 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. It can be below 100% by mass, below 95% by mass, below 90% by mass, below 80% by mass, below 70% by mass, below 60% by mass, below 50% by mass, below 30% by mass, below 25% by mass, below 20% by mass, or below 15% by mass. These lower and upper limits can be arbitrarily combined. For example, relative to the total dry weight of the composition of this embodiment, the dry weight of bacteria can be 0.0001% by mass or more and 100% by mass or less, 0.0001% by mass or more and 95% by mass or less, 0.0001% by mass or more and 90% by mass or less, 0.0001% by mass or more and 80% by mass or less, 0.0001% by mass or more and 70% by mass or less, 0.0001% by mass or more and 60% by mass or less, 0.0001% by mass or more and 50% by mass or less, 0.0001% by mass or more and 30% by mass or less, 0.0001% by mass or more and 25% by mass or less, 0.0001% by mass or more and 20% by mass or less, 0.0001% by mass or more and 15% by mass or less, 0.0001% by mass or more and 10% by mass or less. Below, 0.001% by mass and below 100% by mass, 0.001% by mass and below 95% by mass, 0.001% by mass and below 90% by mass, 0.001% by mass and below 80% by mass, 0.001% by mass and below 70% by mass, 0.001% by mass and below 60% by mass, 0.001% by mass and below 50% by mass, 0.001% by mass and below 30% by mass, 0.001% by mass and below 25% by mass, 0.001% by mass and below 20% by mass, 0.001% by mass and below 15% by mass, 0.001% by mass and below 10% by mass, 0.005% by mass and below 100% by mass, 0.005% by mass and below 95% by mass, 0.005% by mass and below 90% by mass, 0.0.005% or more and less than 80% by mass, 0.005% or more and less than 70% by mass, 0.005% or more and less than 60% by mass, 0.005% or more and less than 50% by mass, 0.005% or more and less than 30% by mass, 0.005% or more and less than 25% by mass, 0.005% or more and less than 20% by mass, 0.005% or more and less than 15% by mass, 0.005% or more and less than 10% by mass, 0.01% or more and less than 100% by mass, 0.01% or more and less than 95% by mass, 0.01% or more and less than 90% by mass, 0.01% or more and less than 80% by mass, 0.01% or more... The following are the product percentages: ≥70% and ≥60%; ≥50% and ≥30%; ≥25%; ≥20%; ≥15%; ≥10%; ≥100%; ≥95%; ≥90%; ≥80%; ≥70%; ≥60% Below, 0.02% by mass and below 50% by mass, 0.02% by mass and below 30% by mass, 0.02% by mass and below 25% by mass, 0.02% by mass and below 20% by mass, 0.02% by mass and below 15% by mass, 0.02% by mass and below 10% by mass, 0.05% by mass and below 100% by mass, 0.05% by mass and below 95% by mass, 0.05% by mass and below 90% by mass, 0.05% by mass and below 80% by mass, 0.05% by mass and below 70% by mass, 0.05% by mass and below 60% by mass, 0.05% by mass and below 50% by mass, 0.05% by mass and below Above and below 30% by mass, above 0.05% by mass and below 25% by mass, above 0.05% by mass and below 20% by mass, above 0.05% by mass and below 15% by mass, above 0.05% by mass and below 10% by mass, above 0.10% by mass and below 100% by mass, above 0.10% by mass and below 95% by mass, above 0.10% by mass and below 90% by mass, above 0.10% by mass and below 80% by mass, above 0.10% by mass and below 70% by mass, above 0.10% by mass and below 60% by mass, above 0.10% by mass and below 50% by mass, above 0.10% by mass and below 30% by mass, above 0.10% by mass and below 25% by mass, 0.10% or more and 20% or less by mass, 0.10% or more and 15% or less by mass, 0.10% or more and 10% or less by mass, 0.30% or more and 100% or less by mass, 0.30% or more and 95% or less by mass, 0.30% or more and 90% or less by mass, 0.30% or more and 80% or less by mass, 0.30% or more and 70% or less by mass, 0.30% or more and 60% or less by mass, 0.30% or more and 50% or less by mass, 0.30% or more and 30% or less by mass, 0.30% or more and 25% or less by mass, 0.30% or more and 20% or less by mass, 0.30% or more and 15% or less by mass Less than %, 0.30% or more and less than 10% of mass, 1.0% or more and less than 100% of mass, 1.0% or more and less than 95% of mass, 1.0% or more and less than 90% of mass, 1.0% or more and less than 80% of mass, 1.0% or more and less than 70% of mass, 1.0% or more and less than 60% of mass, 1.0% or more and less than 50% of mass, 1.0% or more and less than 30% of mass, 1.0% or more and less than 25% of mass, 1.0% or more and less than 20% of mass, 1.0% or more and less than 15% of mass, 1.0% or more and less than 10% of mass, 1.5% or more and less than 100% of mass, 1. 5% or more and less than 95% of mass, 1.5% or more and less than 90% of mass, 1.5% or more and less than 80% of mass, 1.5% or more and less than 70% of mass, 1.5% or more and less than 60% of mass, 1.5% or more and less than 50% of mass, 1.5% or more and less than 30% of mass, 1.5% or more and less than 25% of mass, 1.5% or more and less than 20% of mass, 1.5% or more and less than 15% of mass, 1.5% or more and less than 10% of mass, 2.0% or more and less than 100% of mass, 2.0% or more and less than 95% of mass, 2.0% or more and less than 90% of mass, 2.0% or more and less than 80% of mass. Less than 0% of mass, 2.0% or more and less than 70% of mass, 2.0% or more and less than 60% of mass, 2.0% or more and less than 50% of mass, 2.0% or more and less than 30% of mass, 2.0% or more and less than 25% of mass, 2.0% or more and less than 20% of mass, 2.0% or more and less than 15% of mass, 2.0% or more and less than 10% of mass, 2.5% or more and less than 100% of mass, 2.5% or more and less than 95% of mass, 2.5% or more and less than 90% of mass, 2.5% or more and less than 80% of mass, 2.5% or more and less than 70% of mass, 2.5% or more and less than 60% of mass, 2.5% or more and less than 50% by mass, 2.5% or more and less than 30% by mass, 2.5% or more and less than 25% by mass, 2.5% or more and less than 20% by mass, 2.5% or more and less than 15% by mass, 2.5% or more and less than 10% by mass, 3.0% or more and less than 100% by mass, 3.0% or more and less than 95% by mass, 3.0% or more and less than 90% by mass, 3.0% or more and less than 80% by mass, 3.0% or more and less than 70% by mass, 3.0% or more and less than 60% by mass, 3.0% or more and less than 50% by mass, 3.0% or more and less than 30% by mass, 3.0% or more and less than 2 ...3.0% or more and less than 20% by mass, 3.0% or more and less than 20% by mass, Less than 5% by mass, 3.0% or more but less than 20% by mass, 3.0% or more but less than 15% by mass, 3.0% or more but less than 10% by mass, 5.0% or more but less than 100% by mass, 5.0% or more but less than 95% by mass, 5.0% or more but less than 90% by mass, 5.0% or more but less than 80% by mass, 5.0% or more but less than 70% by mass, 5.0% or more but less than 60% by mass, 5.0% or more but less than 50% by mass, 5.0% or more but less than 30% by mass, 5.0% or more but less than 25% by mass, 5.0% or more but less than 20% by mass, 5.0% or more but less than 15% by mass, 5. 0% or more and less than 10% of mass, 7.0% or more and less than 100% of mass, 7.0% or more and less than 95% of mass, 7.0% or more and less than 90% of mass, 7.0% or more and less than 80% of mass, 7.0% or more and less than 70% of mass, 7.0% or more and less than 60% of mass, 7.0% or more and less than 50% of mass, 7.0% or more and less than 30% of mass, 7.0% or more and less than 25% of mass, 7.0% or more and less than 20% of mass, 7.0% or more and less than 15% of mass, 7.0% or more and less than 10% of mass, 10.0% or more and less than 100% of mass, 10.0% or more of mass And below 95% quality, above 10.0% quality and below 90% quality, above 10.0% quality and below 80% quality, above 10.0% quality and below 70% quality, above 10.0% quality and below 60% quality, above 10.0% quality and below 50% quality, above 10.0% quality and below 30% quality, above 10.0% quality and below 25% quality, above 10.0% quality and below 20% quality, above 10.0% quality and below 15% quality, above 80.0% quality and below 100% quality, above 80.0% quality and below 95% quality, above 80.0% quality and below 90% quality, above 90.0% quality and below 100% quality, 90.0% or more and 95% or less by mass, or 95.0% or more and 100% or less by mass.

[0312] When the immune activation composition of this embodiment is in liquid form, the number of bacteria contained in the composition of this embodiment can be 1.0 × 10⁻⁶. 3 1.0 × 10⁶ cells / mL or more 4 1.0 × 10⁶ cells / mL or more 5 1.0 × 10⁶ cells / mL or more 6 1.0 × 10⁶ cells / mL or more 7 10 cells / mL or more or 4.0 × 10 7 A cell / mL or higher is acceptable, up to 1.0 × 10⁻⁶ cells / mL. 11 Less than 10 cells / mL, 1.0 × 10 10 Less than 10 cells / mL, 3.0 × 10 9 Less than 1.0 × 10⁻⁶ cells / mL or 1.0 × 10⁻⁶ cells / mL 9 The number of cells / mL may be less than [number] cells / mL. These lower and upper limits can be combined arbitrarily without particular limitation. For example, the composition of this embodiment may contain 1.0 × 10⁻⁶ bacteria. 3 More than 1.0 × 10⁻⁶ cells / mL 11 Less than 10 cells / mL, 1.0 × 10 3 More than 1.0 × 10⁻⁶ cells / mL 10 Less than 10 cells / mL, 1.0 × 10 3 More than 3.0 × 10⁶ cells / mL and 3.0 × 10⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 3 More than 1.0 × 10⁻⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 4 More than 1.0 × 10⁻⁶ cells / mL 11 Less than 10 cells / mL, 1.0 × 10 4 More than 1.0 × 10⁻⁶ cells / mL 10 Less than 10 cells / mL, 1.0 × 10 4 More than 3.0 × 10⁶ cells / mL and 3.0 × 10⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 4 More than 1.0 × 10⁻⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 5 More than 1.0 × 10⁻⁶ cells / mL 11 Less than 10 cells / mL, 1.0 × 10 5 More than 1.0 × 10⁻⁶ cells / mL 10 Less than 10 cells / mL, 1.0 × 105 More than 3.0 × 10⁶ cells / mL and 3.0 × 10⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 5 More than 1.0 × 10⁻⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 6 More than 1.0 × 10⁻⁶ cells / mL 11 Less than 10 cells / mL, 1.0 × 10 6 More than 1.0 × 10⁻⁶ cells / mL 10 Less than 10 cells / mL, 1.0 × 10 6 More than 3.0 × 10⁶ cells / mL and 3.0 × 10⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 6 More than 1.0 × 10⁻⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 7 More than 1.0 × 10⁻⁶ cells / mL 11 Less than 10 cells / mL, 1.0 × 10 7 More than 1.0 × 10⁻⁶ cells / mL 10 Less than 10 cells / mL, 1.0 × 10 7 More than 3.0 × 10⁶ cells / mL and 3.0 × 10⁶ cells / mL 9 Less than 10 cells / mL, 1.0 × 10 7 More than 1.0 × 10⁻⁶ cells / mL 9 Less than 10 cells / mL, 4.0 × 10 7 More than 1.0 × 10⁻⁶ cells / mL 11 Less than 10 cells / mL, 4.0 × 10 7 More than 1.0 × 10⁻⁶ cells / mL 10 Less than 10 cells / mL, 4.0 × 10 7 More than 3.0 × 10⁶ cells / mL and 3.0 × 10⁶ cells / mL 9 Less than 10 cells / mL or 4.0 × 10⁻⁶ 7 More than 1.0 × 10⁻⁶ cells / mL 9 Cells / mL. In this case, the daily intake or dosage of the liquid immune-activating composition can be 10mL-1000mL, 10mL-800mL, 10mL-500mL, 10mL-250mL, 30mL-1000mL, 30mL-800mL, 30mL-500mL, 30mL-250mL, 50mL-1000mL, 50mL-800mL, 50-500mL, 50mL-250mL, 100mL-1000mL, 100mL-800mL, 100-500mL, or 100-250mL.

[0313] In the immune activation composition of this embodiment, the number of bacteria of this embodiment contained in each unit package of the composition may, for example, be 1.0 × 10⁻⁶. 4 More than 5.0 × 10 4 Above, 1.0×10 5 More than 3.0 × 10 5 More than 1.0 × 10 6 More than 3.0 × 10 6 More than 1.0 × 10 7 More than 3.0 × 10 7 More than 1.0 × 10 8 More than 3.0 × 10 8 More than 5.0 × 10 8 More than 1.0 × 10 9 More than 5.0 × 10 9 More than one, 1.0 × 10 10 More than 5.0 × 10 10 More than one or 1.0 × 10 11 More than one, can be 1.0 × 10 14 Less than 1.0 × 10 13 Less than 1.0 × 10 12 The number of bacteria in each unit package of the immune activation composition of this embodiment may be less than 1.0 × 10⁻⁶. Furthermore, these upper and lower limits can be combined arbitrarily. For example, the number of bacteria in each unit package of the composition in this embodiment may be 1.0 × 10⁻⁶. 5 More than 1.0 × 10 14 Less than 1.0 × 10 5 More than 1.0 × 10 13 Less than 1.0 × 10 5 More than 1.0 × 10 12 Less than 3.0 × 10 5 More than 1.0 × 10 14 Less than 3.0 × 10 5 More than 1.0 × 10 13 Less than 3.0 × 10 5 More than 1.0 × 10 12 Less than 1.0 × 10 6 More than 1.0 × 10 14 Less than 1.0 × 10 6 More than 1.0 × 10 13 Less than 1.0 × 10 6 More than 1.0 × 10 12 Less than 3.0 × 10 6More than 1.0 × 10 14 Less than 3.0 × 10 6 More than 1.0 × 10 13 Less than 3.0 × 10 6 More than 1.0 × 10 12 Less than 1.0 × 10 7 More than 1.0 × 10 14 Less than 1.0 × 10 7 More than 1.0 × 10 13 Less than 1.0 × 10 7 More than 1.0 × 10 12 Less than 3.0 × 10 7 More than 1.0 × 10 14 Less than 3.0 × 10 7 More than 1.0 × 10 13 Less than 3.0 × 10 7 More than 1.0 × 10 12 Less than 1.0 × 10 8 More than 1.0 × 10 14 Less than 1.0 × 10 8 More than 1.0 × 10 13 Less than 1.0 × 10 8 More than 1.0 × 10 12 Less than 3.0 × 10 8 More than 1.0 × 10 14 Less than 3.0 × 10 8 More than 1.0 × 10 13 Less than 3.0 × 10 8 More than 1.0 × 10 12 Less than 5.0 × 10 8 More than 1.0 × 10 14 Less than 5.0 × 10 8 More than 1.0 × 10 13 Less than 5.0 × 10 8 More than 1.0 × 10 12 Less than 1.0 × 10 9 More than 1.0 × 10 14 Less than 1.0 × 10 9 More than 1.0 × 10 13 Less than 1.0 × 10 9 More than 1.0 × 10 12 Less than 5.0 × 10 9 More than 1.0 × 10 14 Less than 5.0 × 109 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 × 10 12 Less than 5.0 × 10 10 More than 1.0 × 10 14 Less than 5.0 × 10 10 More than 1.0 × 10 13 Less than 5.0 × 10 10 More than 1.0 × 10 12 Less than 1.0 × 10 11 More than 1.0 × 10 14 Less than 1.0 × 10 11 More than 1.0 × 10 13 Less than 1.0 × 10 11 More than 1.0 × 10 12 The number is below 1.

[0314] From the viewpoint of reducing the burden of intake or administration, the immune-activating composition of this embodiment is preferably used by oral intake, oral administration, nasal intake, or nasal administration, more preferably by oral intake or oral administration. When used by oral intake, oral administration, nasal intake, or nasal administration, the bacteria or the composition containing bacteria preferably has high resistance to gastric juice and intestinal juice, for example, it has strong acid resistance. There is no particular limitation on the bacteria, and either live bacteria or dead bacteria can be used. From the perspective of immune activation effect, stability, and manufacturing efficiency, dead bacteria are preferred, and heat-killed bacterial cells are more preferred.

[0315] The immune-activating composition of this embodiment can be orally ingested by humans and non-human mammals. As a representative method of ingestion, food compositions can be cited. The provided food compositions are those containing the bacteria of this embodiment in an effective amount. Here, "containing in an effective amount" means that when each food composition is ingested in a normal dietary amount, the effective ingredient of the present invention is ingested to the extent that it exerts effects such as immune activation. Furthermore, the term "food composition" is used to encompass health foods, functional foods, nutritional supplements, health-functional foods (e.g., foods for specific health purposes, nutritional functional foods, functionally labeled foods), foods for special purposes (e.g., infant foods, foods for pregnant and postpartum women, foods for patients), and supplements. It should be noted that when the effective ingredient of the present invention is ingested by mammals other than humans, the food referred to in this invention is of course used as animal feed.

[0316] The immune-activating composition of this embodiment has effects such as immune activation, and therefore can be provided as part of everyday food intake. In this case, the immune-activating composition of this embodiment can be provided in a pre-defined unit package format with a predetermined intake per meal. Examples of pre-defined unit packages per meal include bags, packs, cans, or bottles. To better utilize the various effects of the immune-activating composition of this embodiment, the intake per meal can be determined according to the daily intake of the active ingredient of the present invention, as described later. The food product of this embodiment can be provided with instructions regarding intake on the packaging or along with documents containing such instructions.

[0317] The intake per meal specified in the unit packaging can be the effective daily intake, or it can be an intake that divides the effective daily intake into two or more meals (preferably two to six meals). Therefore, the unit packaging of the immune-activating composition of this embodiment can contain the active ingredient of the present invention at the daily intake amount described later, or it can contain the active ingredient of the present invention at one-half to one-sixth of the daily intake amount described later. For ease of intake, the immune-activating composition of this embodiment is preferably provided in a unit packaging form per meal (i.e., a daily unit packaging form) where the intake per meal is the effective daily intake amount.

[0318] The immune-activating composition of this embodiment can be administered to individuals who require immune activation. There are no particular limitations on the individuals requiring immune activation; examples include individuals infected with a virus, individuals with a cold, and individuals over 65 years of age.

[0319] The form of the "food composition" in the immune-activating composition of this embodiment is not particularly limited. For example, it can be in the form of a beverage, a semi-liquid or gel form, or a solid or powder form. Furthermore, examples of "supplements" include tablets manufactured by adding excipients, binders, etc. to the active ingredient of the immune-activating composition of this embodiment and then compressing the mixture; granules manufactured by adding excipients, binders, etc. to the active ingredient of this invention and then granulating the mixture; intraorally disintegrating tablets; and capsules formed by encapsulating the active ingredient of this invention into capsules. When provided as a supplement, in addition to the above-mentioned per-meal or daily unit packaging form, it is preferable to provide it in weekly, bi-weekly, monthly, or bi-monthly unit packaging forms. It should be noted that the latter unit packaging form preferably indicates the intake per meal or daily, so that the user can ingest an effective amount of the active ingredient of this invention according to the indicated amount.

[0320] Examples of food compositions provided in the form of immune-activating compositions according to this embodiment include health foods, functional foods, nutritional compositions, nutritional supplements, supplements, health foods, specific health foods, nutritional functional foods, or functionally labeled foods, etc., that have immune-activating capabilities. Such food compositions may be labeled as follows: assisting healthy individuals in maintaining immune function (immune care), for those concerned about decreased immune function, suppressing decreased immune function, for those concerned about sun protection, for those concerned about skin damage in daily life, for those concerned about dry skin, for those concerned about skin heat, for those concerned about skin erythema, for those concerned about skin redness, for those concerned about facial redness, for those concerned about rough hands, etc.

[0321] The food composition provided in this embodiment as an immune-activating composition is not particularly limited as long as it contains the active ingredient of the present invention. Examples include, for instance, soft drinks, carbonated drinks, fruit juice drinks, vegetable juice drinks, drinks containing both fruit and vegetable juices, animal milk such as milk, soy milk, milk drinks, beverage-type yogurt, beverage-type or stick-shaped jellies, coffee, cocoa, tea drinks, nutritional drinks, energy drinks, sports drinks, mineral water (including both foamed and non-foamed types), water-based drinks, non-alcoholic beer-flavored drinks, and other non-alcoholic beverages; carbohydrate-containing foods such as rice dishes, noodles, bread, or pasta; dairy products such as cheese, hard or soft yogurt, fresh cream made from oils other than animal milk, and ice cream; cookies, etc. Western-style desserts such as cakes and chocolates; Japanese-style desserts such as steamed buns or yokan; compressed candies such as lemon candy (cooling candies); candies; chewing gum; gummy candies; frozen desserts or iced foods such as jelly or pudding; fast food snacks and other types of desserts; whiskey, bourbon, spirits, liqueurs, wine, fruit wine, Japanese sake, Chinese sake, shochu, beer, non-alcoholic beer with an alcohol content of less than 1%, sparkling wine, other mixed wines, shochu mixed with soda water and other alcoholic beverages; processed products using eggs, processed products of fish, shellfish or animal meat (including offal such as liver) (including gourmet food), soups such as miso soup and other processed foods; seasonings such as miso, soy sauce, rice seasoning (furikake), and other compound seasonings; or liquid foods such as thick liquid foods, etc.

[0322] As a tea beverage, it includes any of the following: fermented tea, semi-fermented tea, and non-fermented tea. Examples include black tea, green tea, barley tea, genmaicha, sencha, gyokurocha, hojicha, oolong tea, turmeric tea, pu-erh tea, rooibos tea, rose tea, chrysanthemum tea, ginkgo leaf tea, and herbal teas (such as peppermint tea and jasmine tea).

[0323] Examples of fruits that can be used in fruit juice drinks, or drinks containing both fruit and vegetable juices, include apples, citrus fruits, grapes, bananas, pears, peaches, mangoes, acai berries, blueberries, and plums. Examples of vegetables that can be used in vegetable juice drinks, or drinks containing both fruit and vegetable juices, include tomatoes, carrots, celery, pumpkins, cucumbers, and watermelons.

[0324] When the immune-activating composition of this embodiment is provided as feed, it can be implemented in accordance with the above description of food.

[0325] When the immune-activating composition of this embodiment is provided as a pharmaceutical composition or a quasi-drug, it can be formulated into an oral or non-oral formulation. Examples of oral formulations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, liquids, gels, emulsions, and suspensions. Examples of non-oral formulations include injectables suitable for local administration (including intradermal, subcutaneous, intramuscular, and intravenous injections), inhalers (e.g., inhaled aerosols, inhaled powders, and inhaled liquids), nasal drops (e.g., nasal powders and nasal solutions), ointments, creams, gels, suppositories, patches, and wet wipes; one possible formulation is a nasal drop. These formulations can be formulated using pharmaceutically acceptable carriers through methods commonly employed in the art. Examples of pharmaceutically acceptable carriers include excipients, binders, diluents, additives, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives.

[0326] When the immune-activating composition of this embodiment is used as a pharmaceutical composition or a quasi-drug, the diseases that can be targeted include, for example, diseases known to be indicated by type I IFNs, cancers including renal cell carcinoma, multiple myeloma, chronic myeloid leukemia, hairy cell leukemia, glioblastoma, medulloblastoma, astrocytoma, malignant melanoma, mycosis fungoides, and adult T-cell leukemia; infectious diseases including subacute sclerosing panencephalitis, HTLV-1 myelopathy, hepatitis B, and hepatitis C; infections caused by bacteria such as chlamydia (sexually transmitted diseases), mycobacteria (tuberculosis), Listeria (septicemia, etc.), Staphylococcus (food poisoning), and Helicobacter pylori (gastritis); and autoimmune diseases including multiple sclerosis. Furthermore, since type I IFNs are known to inhibit the differentiation of osteoblasts into osteoclasts, the immune-activating composition of this embodiment can also be used for the prevention or treatment of osteoporosis, etc.

[0327] By using genetic engineering methods to express or secrete antigens corresponding to specific diseases in bacteria, which are the active ingredient, or onto the surface of bacteria, the immune-activating composition of this embodiment can be used as a vaccine. In particular, the bacterial cell wall has the function of protecting antigens from the decomposition of gastric acid, therefore, heterologous antigen-expressing strains that express antigens in or on the surface of bacteria are suitable as hosts for oral vaccines.

[0328] The immune-activating composition of this embodiment can be a bacterial cell raw material powder of this embodiment (a powder formed by drying bacterial cells or a powder containing the bacterial cells, etc.). Such a bacterial cell raw material powder can be obtained, for example, by appropriately culturing bacteria, sterilizing and drying them according to conventional manufacturing methods. Alternatively, the immune-activating composition of this embodiment can be, for example, an immune-activating composition manufactured according to a manufacturing method of one aspect of the second embodiment.

[0329] In addition to the bacteria of this embodiment and the components of the culture medium from which the bacteria were cultured, the bacterial raw material powder may also contain components acceptable as food, pharmaceutical, or feed. For example, it may also contain at least one component selected from the group consisting of sugars, proteins, lipids, amino acids, vitamins, minerals, flavonoids, quinones, polyphenols, nucleic acids, fatty acids, acidulants, sweeteners, colorants, flavorings, seasonings, salt, emulsifiers, stabilizers, cooling agents, binders, disintegrants, lubricants, colorants, preservatives, slow-release regulators, surfactants, and solvents.

[0330] The bacterial raw material powder of this embodiment can be used as a raw material for the food composition, pharmaceutical composition or feed of this embodiment.

[0331] When the immune-activating composition of this embodiment is provided as an additive, it can be implemented according to the description above regarding food compositions, feed, quasi-drugs, pharmaceutical compositions, or microbial raw material powders. When the immune-activating composition of this embodiment is provided as a food additive, it can be used as a functional-related ingredient in functionally labeled foods with immune-activating effects.

[0332] The immune-activating composition of this embodiment can be determined based on the recipient's sex, age and weight, symptoms, time of intake or administration, dosage form, route of intake or administration, and the materials or agents combined. The daily intake or dosage of the immune-activating composition of this embodiment for adults can be determined, for example, by the number of bacteria as the active ingredient, and can be set to 1 × 10⁻⁶. 8 More than one, 1×10 9 More than one or 1×10 10 More than one, can also be set to 1×10 14 Less than one, 1×10 13 Less than one or 1×10 12 Below 10. These upper and lower limits can be combined arbitrarily, and the range of the above intake or dosage can be set, for example, 1 × 10. 8 ~1×10 14 1×10 9 ~1×10 14 One or 1×10 10 ~1×10 14 1×10 8 ~1×10 13 1×10 9 ~1×10 13 1×10 10 ~1×10 131×10 8 ~1×10 12 1×10 9 ~1×10 12 One or 1×10 10 ~1×10 12 The bacterial count can be determined using known microscopes, flow cytometers, or rapid non-culture microbial detection devices (such as ELESTA PixeeMo (AFI Technologie Co., Ltd.)). From the viewpoint of high versatility, microscope measurement is preferred. The target of administration or ingestion of the composition of this embodiment can be humans or non-human mammals, and can be humans.

[0333] The daily intake or dosage of the immune-activating composition for adults in this embodiment can also be determined by the dried bacterial cell mass of the active ingredient, for example, it can be set to 2.5 × 10⁻⁶. -2 mg or more, 2.5 × 10 -1 For doses of 1 mg or more, or 2.5 mg or more, a value of 2.5 × 10 mg can be set. 4 Less than mg, 2.5×10 3 mg or less or 2.5 × 10 2 Below mg. These upper and lower limits can be combined arbitrarily, and the range of the above intake or dosage can be set, for example, as 2.5 × 10 mg. -2 mg~2.5×10 4 mg, 2.5×10 -1 mg~2.5×10 4 mg, 2.5mg~2.5×10 4 mg, 2.5×10 -2 mg~2.5×10 3 mg, 2.5×10 -1 mg~2.5×10 3 mg, 2.5×10 mg~2.5×10 mg 3 mg, 2.5×10 -2 mg~2.5×10 2 mg, 2.5×10 -1 mg~2.5×10 2 mg or 2.5 mg to 2.5 × 10 mg 2 mg.

[0334] The intake amount and the following intake timing and period of the immune-activating composition of this embodiment are applicable to cases where the immune-activating composition of this embodiment is used for either non-therapeutic or therapeutic purposes. In the case of therapeutic purposes, intake may be replaced by administration.

[0335] The immune-activating composition of this embodiment is preferably continuously ingested or administered during the period during which the immune-activating effect is expected. Regarding the ingestion or administration period of the active ingredient of the present invention, from the viewpoint of better exerting the immune-activating effect, for example, the above-mentioned daily dose can be ingested or administered for more than one week, two weeks, three weeks, or preferably more than one month (four weeks). Regarding the ingestion or administration interval of the active ingredient of the present invention, the above-mentioned daily dose can be ingested or administered once every three days, once every two days, or once a day, preferably once a day.

[0336] The immune-activating composition of this embodiment can also be taken or administered before the event or period for which the immune-activating effect is expected. Examples of events for which the immune-activating effect is expected include behaviors with a possibility of viral infection (e.g., participating in activities with a high risk of viral infection, traveling to an epidemic area), and examples of periods for which the immune-activating effect is expected include the epidemic period of a viral infection. Examples of intake or administration times before the event for which the immune-activating effect is expected include, for example, more than 1 day before, more than 3 days before, more than 1 week before, more than 2 weeks before, more than 3 weeks before, more than 1 month before (more than 4 weeks before), or more than 2 months before (more than 8 weeks before). Alternatively, intake or administration can be continuously carried out from the start of intake or administration until the event for which the immune-activating effect is expected, depending on the circumstances, but there is no particular limitation. In addition, the active ingredient of the present invention can also be taken or administered after the event or period for which the immune-activating effect is expected. Examples of intake or administration times after the event for which the immune-activating effect is expected include, for example, more than 1 day after, more than 3 days after, more than 1 week after, and more than 2 weeks after. Furthermore, when ingested or administered after an event for which an immune-activating effect is expected, ingestion or administration may be continued at ingestion or administration intervals as appropriate, but there are no particular limitations. In this invention, it is particularly preferred to begin ingestion or administration of the active ingredient of this invention before the event for which an immune-activating effect is expected, and to continue ingestion or administration until after that event.

[0337] It should be noted that, as another aspect, the composition in this embodiment, being an immune-activating composition, can also be referred to as a method of activating the immune system of a subject, including the step of ingesting the composition of this embodiment or administering it to the subject. The subject ingesting or administering the composition can be a subject that requires it, for example, a subject that requires the aforementioned immune activation.

[0338] Furthermore, as another aspect, the composition in this embodiment, being an immune-activating composition, can also be referred to as the application of the composition in one aspect of this embodiment to activate the immunity of a target. In this case, the application can be non-therapeutic and / or therapeutic, preferably non-therapeutic.

[0339] In this disclosure, "therapeutic" application refers to application in the treatment of a human or animal body. Therapeutic applications of this disclosure may include, for example, applications for or in conjunction with medical procedures. Therapeutic applications of this disclosure may include, for example, applications administered by a medical practitioner to a human or animal or ingesting a substance, or instructions to administer or ingest a substance to a human or animal. Therapeutic applications of this disclosure may include applications for therapeutic or preventative purposes, applications for therapeutic purposes or applications for preventative purposes involving the ingestion of a food composition, and applications for therapeutic purposes. Therapeutic applications of this disclosure may include applications for non-healthy individuals.

[0340] In this disclosure, "non-therapeutic" application refers to the application of a substance that is not a therapeutic application. Examples of non-therapeutic applications of this disclosure include applications that are not intended for medical treatment and / or do not involve medical treatment. Examples of non-therapeutic applications of this disclosure include administering the substance to a person or animal or ingesting the substance without the involvement of a medical practitioner and / or instructing the person or animal to administer or ingest the substance. Examples of non-therapeutic applications of this disclosure include applications for preventative or health-enhancing purposes, and applications for preventative or health-enhancing purposes involving the administration or ingestion of a pharmaceutical composition or quasi-drug. Examples of non-therapeutic applications of this disclosure include applications for healthy individuals.

[0341] Furthermore, as another aspect, the composition in this embodiment, being an immune-activating composition, can also be referred to as a composition of this embodiment for activating the immunity of a subject. In this case, the application is, for example, a therapeutic application.

[0342] In addition, as another aspect, the composition in this embodiment is an immune-activating composition, which may also be referred to as the application of the above-mentioned bacteria in the composition of one embodiment.

[0343] Another aspect of this embodiment is characterized by a method for activating pDCs that bind bacteria to BDCA2.

[0344] In one approach, the biological characteristics, classification, and strain of bacteria that can be used in the above-described methods are referenced to the description of the bacteria contained in the immune-activating composition of this embodiment ("bacteria of this embodiment"). For example, the bacteria may be lactic acid bacteria and / or acetic acid bacteria. Alternatively, the bacteria may be bacteria that are phagocytosed by pDCs via binding to BDCA2.

[0345] Another embodiment of this invention includes an activation method that administers bacteria that bind to BDCA2 to the target's pDC.

[0346] In one embodiment, the biological characteristics, classification, and strains of the bacteria used in the above-described methods are described in reference to the bacteria contained in the immune-activating composition of this embodiment ("bacteria of this embodiment"). For example, the bacteria may be lactic acid bacteria and / or acetic acid bacteria. Alternatively, the bacteria may be bacteria that are phagocytosed by pDCs via binding to BDCA2.

[0347] In one approach, the object of the above method can be a person. There are no particular limitations on the term "person," for example, it could be an object requiring immune activation. There are no particular limitations on the object requiring immune activation; for example, objects infected with a virus, objects with a cold, and objects over 65 years of age could be listed.

[0348] Alternatively, one embodiment may be a composition containing bacteria, used in the above-described "activation method of pDC characterized by binding bacteria to BDCA2" and "activation method of pDC including the step of administering bacteria bound to BDCA2 to a subject".

[0349] In one embodiment, the biological characteristics, classification, and strains of the bacteria used in the above-described composition are described in reference to the bacteria contained in the immune-activating composition of this embodiment ("the bacteria of this embodiment"). For example, the bacteria may be lactic acid bacteria and / or acetic acid bacteria. Alternatively, the bacteria may be, for example, *Lactococcus lactis* subsp. *lactis* JCM 5805. Furthermore, the bacteria may be bacteria that are phagocytosed by pDCs via binding to BDCA2. The description of the above composition is further referenced to the description of the "immune-activating composition of this embodiment" as an embodiment of the present invention.

[0350] A second embodiment of this disclosure relates to a method for enhancing the immune activation capacity and / or the ability of bacteria to be phagocytosed by plasmacytoid dendritic cells (pDCs), comprising the step of culturing bacteria in an M17 medium supplemented with a predetermined concentration of glucose (culturing step). The method of the second embodiment, by using an M17 medium supplemented with a predetermined concentration of glucose as the culture medium for bacterial culture, facilitates the enhancement of the immune activation capacity and / or the ability of bacteria to be phagocytosed by pDCs compared to when this medium is not used.

[0351] M17 medium can be obtained, for example, from suppliers such as Becton, Dickinson and Company. M17 medium is, for example, a water-soluble medium containing yeast extract, peptone, tryptone, meat extract, lactose, ascorbic acid, magnesium sulfate, and sodium glycerophosphate as solutes. For example, when using it, the pH of the M17 medium can be adjusted to 7.1 ± 0.2.

[0352] The final concentration of glucose in the culture medium used in the culturing step can 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 less, or less than 10% by mass, 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 combined arbitrarily. The final concentration of glucose in the culture medium used in the culturing step can be, for example, 0.1% by mass or more and 10% by mass or less, 0.3% by mass or more and 10% by mass or less, 0.5% by mass or more and 10% by mass or less, 1.0% by mass or more and 10% by mass or less, 0.1% by mass or more and 5.0% by mass or less, 0.3% by mass or more and 5.0% by mass or less, 0.5% by mass or more and 5.0% by mass or less, 1.0% by mass or more and 5.0% by mass or less, 0.1% by mass or more and 3.0% by mass or less, 0.3% by mass or more and 3.0% by mass or less, 0.5% by mass or more and 1.0% by mass or less, or 1.0% by mass or less. It should be noted that glucose in this case refers, for example, D-glucose.

[0353] There are no specific limitations on the cultivation time in the cultivation step. For example, it can be more than 1 hour, more than 3 hours, more than 6 hours, more than 12 hours, or more than 18 hours, or less than 336 hours, 168 hours, 72 hours, 48 ​​hours, or 36 hours. As an example, it can be 24 hours. There are no specific limitations on the cultivation temperature in the cultivation step. For example, it can be above 20℃, above 22℃, above 25℃, or above 30℃, or less than 45℃, 40℃, 38℃, 35℃, or 30℃. As an example, it can be 30℃.

[0354] The immune activation capacity and / or the ability to be phagocytosed by pDCs of the bacteria can be evaluated in the same manner as described in the first embodiment. The immune activation capacity and / or the ability to be phagocytosed by pDCs of the bacteria after the culture step, compared to the bacteria cultured using MRS medium as the culture medium, can be, for example, 1.05 times or more, 1.10 times or more, 1.15 times or more, 1.20 times or more, 1.25 times or more, 1.30 times or more, 1.35 times or more, 1.40 times or more, 1.45 times or more, or 1.50 times or more.

[0355] One embodiment of the second method may be a method for manufacturing an immune-activating composition that includes a culture step. The immune-activating composition manufactured by this method may be the composition of the first embodiment, for example, its use and composition may satisfy the requirements of the composition of the first embodiment. The manufacturing method of this method may further include a sterilization step and / or a drying step of the bacteria after the culture step.

[0356] The sterilization method used in the sterilization process can be any 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 water electrolysis. Heating treatment may be one of these methods. When the sterilization method is heat treatment, the heating temperature is not particularly limited; for example, it can be above 60°C, above 65°C, above 70°C, or above 75°C, or below 110°C, below 100°C, below 90°C, below 85°C, or below 80°C. These lower and upper limits can be combined arbitrarily. For example, the heating temperature can be above 60°C and below 110°C, above 60°C and below 100°C, above 60°C and below 90°C, above 60°C and below 85°C, above 60°C and below 80°C, above 65°C and below 100°C, above 65°C and below 90°C, above 65°C and below 85°C, above 65°C and below 80°C, above 70°C and below 90°C, above 70°C and below 85°C, above 70°C and below 80°C, above 75°C and below 90°C, or above 75°C and below 85°C. Furthermore, when the sterilization method is heat treatment, the heating time is not particularly limited; for example, it can be more than 1 minute, more than 3 minutes, more than 5 minutes, or more than 10 minutes, and can be less than 24 hours, less than 4 hours, less than 60 minutes, less than 45 minutes, or less than 30 minutes. These lower and upper limits can be combined arbitrarily. For example, the heating time can be more than 1 minute and less than 24 hours, more than 3 minutes and less than 4 hours, or more than 5 minutes and less than 60 minutes.

[0357] In the step of drying bacteria, the drying method can be any method commonly used by those skilled in the art, such as freeze drying, spray drying, drum drying, hot air drying, or vacuum drying. In one method, spray drying may be used.

[0358] In addition, the manufacturing method of this method may include a step of washing bacteria as needed between and / or during each step. Such washing may be, for example, separating the bacteria by suspending them in a fresh culture medium or buffer and then separating them by centrifugation.

[0359] The immune-activating composition manufactured by the method of this embodiment can be, for example, an immune-activating composition containing *Lactococcus lactis* subsp. *lactococcus* JCM 5805 as an active ingredient. In this case, *Lactococcus lactis* subsp. *lactococcus* JCM 5805 can, for example, satisfy requirement Y, which can be confirmed by the method described with respect to the first embodiment.

[0360] [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contact between pDCs at a concentration of 10 μg / mL and *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 at a final concentration of 10 μg / mL in a serum-free medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *Lactococcus lactis* JCM 5805 was at least 15.0% of the total number of pDCs that had been contacted, wherein the pDCs were pDCs that had been pre-cultured in a serum-free medium.

[0361] Example

[0362] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0363] In the following examples, CAL-1 cells (accession number FERM BP-10914) provided by Nagasaki University were used as the sample (hereinafter referred to as the "sample") (all rights to the sample belong to Nagasaki University, and a license to use the sample has been obtained from Nagasaki University). Cell culture was performed at 37°C and 5% CO2. Results are presented as mean ± standard deviation. The basal medium for culturing CAL-1 cells and for experiments using CAL-1 cells was a medium prepared by adding 1.0% (v / v) of penicillin / streptomycin (Gibco, 15140-12) to RPMI-1640 medium (Sigma, R8758) to a final concentration (hereinafter also referred to as "serum-free medium").

[0364] The strains of lactic acid bacteria used in the following examples are shown in Table 1. The culture conditions designated A through G in Table 1 are shown in Table 2. Except for *Lactobacillus rhamnosus* CRL1505, the lactic acid bacteria in the examples were cultured according to the correspondence between A and G shown in Table 1 using the culture methods described in Table 2, unless otherwise specified. The bacteria were then used in the form of bacterial raw material powder (powder formed by drying bacterial cells or powder containing the bacterial cells, etc.) prepared as follows. For bacteria cultured in MRS medium (MRS BROTH, CODE: CM0359, Oxoid), the culture was centrifuged (5000 rpm), and the supernatant was removed to obtain a bacterial culture. Next, the obtained bacterial culture was mixed with phosphate-buffered saline (Bio Biotechnology) at a ratio of 1:19 and washed twice by centrifugation (5000 rpm). The washed bacterial culture was then heated to kill the lactic acid bacteria. In the heat treatment, the temperature is raised from room temperature to 80°C over 30 minutes, held at 80°C for 30 minutes, and then cooled to room temperature over 30 minutes. After cooling, the mixture is freeze-dried to obtain bacterial raw material powder.

[0365] [Table 1]

[0366] [Table 2]

[0367] For Lactobacillus rhamnosus CRL1505, the bacterial raw material powder was prepared as follows.

[0368] [Preparation method of Lactobacillus rhamnosus CRL1505 bacterial raw material powder]

[0369] A solution containing *Lactobacillus rhamnosus* CRL1505 was prepared by dissolving FLORASSIST Probiotic Immune & Nasal Defense (Life Extension, #02208) in 10 mL of MRS medium. This solution was then spread onto MRS agar and streaked to obtain single colonies of *Lactobacillus rhamnosus* CRL1505. The *Lactobacillus rhamnosus* CRL1505 colonies were added to MRS medium and incubated at 37°C for 24 hours. 700 μL of the resulting culture was mixed with 300 μL of 80% glycerol to prepare a glycerol stock solution of *Lactobacillus rhamnosus* CRL1505, which was stored at -80°C.

[0370] Add 100 μL of Lactobacillus rhamnosus CRL1505 glycerol stock solution to 100 mL of MRS medium and incubate at 37 °C for 24 hours. The culture medium is then recovered, centrifuged twice, washed with ultrapure water, resuspended in 20 mL of ultrapure water, sterilized at 80 °C for 30 minutes, and then freeze-dried. This product is used as the bacterial raw material powder of Lactobacillus rhamnosus CRL1505 in the following examples.

[0371] [Bacterial staining using pHrodo Red SE]

[0372] Dissolve pHrodo Red SE (Invitrogen, P36600) in DMSO to prepare a 10.2 mM solution. Weigh the bacterial raw material powder into a 2 mL Eppendorf tube and add 0.1 M sodium bicarbonate to adjust the pH to 9.0, thus preparing a bacterial concentration of 20 mg / mL. Then, transfer 95 μL to a new 2 mL Eppendorf tube and add 5 μL of the prepared 10.2 mM pHrodo Red SE. After dispersing by vortexing, cover with aluminum foil and incubate at room temperature for 60 minutes. Then add 750 μL of PBS (Takara Bio), vortex, and centrifuge at 20000 g × 2 min at room temperature. Discard 800 μL of supernatant, add 1.5 mL of PBS, and vortex to completely suspend the precipitate. Centrifuge again at 20000 g × 2 min at room temperature, and discard 1.5 mL of supernatant. Finally, add 140 μL of PBS to suspend the stained bacterial suspension, thus preparing a bacterial suspension with a concentration of 10 mg / mL. The stained bacterial suspension is stored at 4°C under light-proof aluminum foil until use. Just before use, dilute 10-fold with PBS to prepare a bacterial suspension with a concentration of 1 mg / mL. It should be noted that the stained bacterial suspension should be used within 24 hours of preparation.

[0373] <Example 1: Evaluation of the contribution of phagocytosis to IFN-α production in pDC>

[0374] Prepare a solution containing 5.0 × 10⁻⁶ mg / L of serum-free culture medium. 5 A cell suspension of CAL-1 cells / mL was prepared, and 10 mL of the cell suspension was seeded into 10 cm diameter culture dishes and cultured for 16 hours (pretreatment step). CAL-1 cells were then recovered by pipetting. The recovered CAL-1 cells were then incubated at 2.0 × 10⁶ cells / mL. 5Cells were resuspended in serum-free medium at a concentration of 10 cells / mL and seeded at 200 μL / well in each well of a 96-well microplate. For the cell suspension in these wells, in the LC-Plasma+Cyt_D group, 400 μM pinocembrin D (Fujifilm Wako, 037-17561) dissolved in 4 v / v dimethyl sulfoxide (DMSO) was prepared, and pinocembrin D and DMSO were added to final concentrations of 10 μM and 0.1 v / v, respectively, and incubated for 30 min. In the LC-Plasma+DMSO group, 4 v / v DMSO was added to a final concentration of 0.1 v / v, and incubated for 30 min as well. After incubation, *Lactococcus lactis* subsp. *lactococcus* JCM 5805 (Kirin Holdings Co., Ltd., hereinafter also referred to as "LC-Plasma") stained with pHrodo Red SE (Invitrogen, P36600) using the method described above [bacterial staining with pHrodo Red SE] was added to each well at a final concentration of 10 μg / mL. Then, the fluorescence of pHrodo Red SE in each well was measured over time using an Incucyte (registered trademark) SX5 live cell analysis system (Sartorius). Subsequently, after 22 hours of incubation (contact step), the culture supernatant from each well was recovered, and the IFN-α concentration in the culture supernatant was determined using an ELISA method with a human IFN-α multi-subtype ELISA kit (PBL Assay Science).

[0375] It should be noted that pinocytin D is an inhibitor of actin polymerization, and phagocytosis is known to be inhibited in cells with added pinocytin D. Additionally, pHrodo Red SE is a staining reagent that enhances fluorescence intensity in low-pH environments. When bacteria stained with pHrodo Red SE are phagocytosed by CAL-1 cells, the bacteria enter the endosome, creating a low-pH environment, which increases the fluorescence intensity of pHrodo Red SE. Therefore, the total area of ​​pHrodo Red SE fluorescence detected across the entire field of view (fluorescence detection area) can be used to quantify the amount of bacteria phagocytosed by CAL-1 cells. It should be noted that the fluorescence intensity analysis conditions of the Incucyte (registered trademark) SX5 live cell analysis system (Sartorius) are as described in [Incucyte Fluorescence Intensity Analysis Conditions 1] below. Furthermore, the field of view area for each image is 0.572 mm². 2 .

[0376] [Incucyte Fluorescence Intensity Analysis Conditions 1]

[0377] In Incucyte fluorescence intensity analysis, two channels were set: a "phase channel" and an "orange channel." The phase channel detects CAL-1 cells based on phase difference. The orange channel detects the fluorescence of pHrodo Red SE. Detailed detection conditions are shown in the scan and analysis settings below.

[0378] Scan settings

[0379] Scan type: Non-adhesive cell-by-cell scan

[0380] Container type: 96-hole Corning plate

[0381] Image channels: Phase contrast, Orange (Acquisition time: 400 ms)

[0382] Objective lens: 20×

[0383] Number of images per hole: 9

[0384] Analysis settings

[0385] Analysis type: Basic analyzer

[0386] Phase difference channel

[0387] Segmentation: AI convergence

[0388] Cleaning: - Pore filling (μm) 2 ): 0.000 - Resize (pixels): 0 Filtering conditions: - Area (μm) 2 ): 60.000 and above - Eccentricity: Not set Orange Channel Segmentation: Surface Fitting - Threshold (OCU): 5.000 - Edge segmentation: Enabled - Edge sensitivity: 0 Cleaning: - Pore filling (μm) 2 ): 0.000 - Resize (pixels): 0 Filtering conditions: - Area (μm) 2 ): Not set - Eccentricity: Not set - Average intensity: Not set - Average intensity: Not set Figure 1 A graph showing the fluorescence detection area 22 hours after adding LC-Plasma. Figure 2 A graph showing the results of IFN-α concentration determination in the culture supernatant recovered 22 hours after LC-Plasma addition. Based on... Figure 1 The results showed that the LC-Plasma+Cyt_D group had significantly smaller phagocytosis compared to the LC-Plasma+DMSO group. Furthermore, according to... Figure 2 The results showed that the IFN-α concentration was significantly lower in the LC-Plasma+Cyt_D group compared to the LC-Plasma+DMSO group, indicating that LC-Plasma significantly inhibited the activation of pDCs. These results suggest that the activation of pDCs with inhibited phagocytosis is significantly suppressed.

[0389] <Example 2: The Relationship Between Phagocytosis of Various Lactic Acid Bacteria and pDC and IFN-α Production>

[0390] Prepare a solution containing 5.0 × 10⁻⁶ mg / L of serum-free culture medium. 5 A cell suspension of CAL-1 cells / mL was prepared, and 10 mL of the cell suspension was seeded into 10 cm diameter culture dishes and cultured for 16 hours (pretreatment step). CAL-1 cells were then recovered by pipetting. The recovered CAL-1 cells were then incubated at 2.0 × 10⁶ cells / mL. 5 Cells / mL were resuspended in serum-free medium and seeded at 200 μL / well in each well of a 96-well microplate. 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 these wells at a final concentration of 10 μg / mL. A group without added bacteria was prepared as a negative control. Then, except for the fluorescence intensity analysis conditions of the Incucyte (registered trademark) SX5 live cell analysis system (Sartorius), the fluorescence of pHrodo Red SE over time and the concentration of IFN-α in the culture supernatant were determined using the same method as in Example 1. It should be noted that the fluorescence intensity analysis conditions of the Incucyte (registered trademark) SX5 live cell analysis system (Sartorius) are as described in [Incucyte Fluorescence Intensity Analysis 2] below. Additionally, the field of view for each image is 0.572 mm². 2 .

[0391] [Incucyte fluorescence intensity analysis 2]

[0392] In Incucyte fluorescence intensity analysis, two channels were set up: a "phase difference channel" and an "orange channel." The phase difference channel detects CAL-1 cells based on phase difference. The orange channel detects the fluorescence of pHrodo Red SE. Detailed detection conditions are shown in the scan and analysis settings below.

[0393] Scan settings

[0394] Scan type: Non-adhesive cell-by-cell scan

[0395] Container type: 96-hole Corning plate

[0396] Image channels: Phase contrast, Orange (Acquisition time: 400 ms)

[0397] Objective lens: 20×

[0398] Number of images per hole: 9

[0399] Analysis settings

[0400] Analysis type: Basic analyzer

[0401] Phase difference channel

[0402] Segmentation: AI convergence

[0403] Cleaning: - Pore filling (μm) 2 ): 0.000 - Resize (pixels): 0 Filtering conditions: - Area (μm) 2 ): 60.000 and above -Eccentricity: Not set Orange Channel Segmentation: Surface Fitting - Threshold (OCU): 5.000 - Edge segmentation: Enabled - Edge sensitivity: 0 Cleaning: - Pore filling (μm) 2 ): 0.000 - Resize (pixels): 0 Filtering conditions: - Area (μm) 2 ): 20 - Eccentricity: Not set - Average intensity: Not set - Average intensity: Not set Figure 3This is a graph showing the fluorescence detection area of ​​the eight tested lactic acid bacteria 22 hours after the addition of the lactic acid bacteria. Figure 4 This is a graph showing the results of the determination of IFN-α concentration in the culture supernatant recovered 22 hours after adding lactic acid bacteria to 13 tested lactic acid bacteria. Figure 5 This is a graph showing the correlation between the fluorescence detection area on the horizontal axis and the IFN-α concentration on the vertical axis for the eight tested lactic acid bacteria. According to... Figures 3-5 The results showed that the phagocytic behavior of each type of lactic acid bacteria was strongly positively correlated with its immune activation capacity (R0). 2 =0.91). This result shows that the immune activation capacity of bacteria is determined by the amount of food phagocytosed.

[0404] <Example 3: Relationship between pDC phagocytosis of LC-Plasma via BDCA2 binding and IFN-α generation>

[0405] For the CAL-1 cell suspensions seeded in the same manner as in Example 1, anti-BDCA2 antibody (Miltenyi Biotec, 130-090-690) was added at 10 μg / mL to the LC-Plasma + BDCA2 anti group. In the LC-Plasma + isotype group, to evaluate the effect of non-specific binding of the anti-BDCA2 antibody, an isotype control of the same isotype and subtype as the anti-BDCA2 antibody (Miltenyi Biotec, 130-106-545) was added at 10 μg / mL, and incubated for 30 minutes. No antibody was added to the LC-Plasma group as a control. Then, for the LC-Plasma group, LC-Plasma + isotype group, and LC-Plasma + BDCA2 anti group, the exposure and phagocytosis evaluation of pHrodo Red SE-stained LC-Plasma cells and the determination of IFN-α concentration in the culture supernatant were performed in the same manner as in Example 2.

[0406] Figure 6 This is a graph showing the fluorescence detection area 22 hours after adding LC-Plasma for the LC-Plasma group, LC-Plasma + isotype group, and LC-Plasma + BDCA2 anti group. Figure 7 This is a graph showing the results of IFN-α concentration measurements in the culture supernatant recovered 22 hours after LC-Plasma addition for the LC-Plasma group, LC-Plasma + isotype group, and LC-Plasma + BDCA2 anti group. Based on... Figure 6 and 7The results showed that the LC-Plasma+BDCA2 anti group, which inhibited the binding of BDCA2 to CAL-1 cells by blocking with anti-BDCA2 antibody, had lower phagocytosis and IFN-α concentrations compared to the LC-Plasma group and the LC-Plasma+ isotype group, whose binding was not inhibited. These results indicate that the binding of LC-Plasma to BDCA on the surface of pDCs is one of the stages that determines the degree of immune activation of LC-Plasma. Furthermore, these results suggest that the phagocytosis of LC-Plasma by pDCs via BDCA binding is one of the stages that determines the degree of immune activation of LC-Plasma.

[0407] <Example 4: Relationship between phagocytosis of various lactic acid bacteria and IFN-α production via pDC bound to BDCA2>

[0408] For CAL-1 cell suspensions inoculated in the same manner as in Example 1, anti-BDCA2 antibody (Miltenyi Biotec, 130-090-690) was added at 10 μg / mL in the BDCA2 anti group, and the same isotype control antibody (Miltenyi Biotec, 130-106-545) was added at 10 μg / mL in the isotype group, and incubation was performed for 30 minutes. Then, for both the isotype group and the BDCA2 anti group, exposure, phagocytosis evaluation, and determination of IFN-α concentration in the culture supernatant of various lactic acid bacteria (the same strains tested in Example 2) stained with pHrodo Red SE were performed, as in Example 2. It should be noted that the amount of phagocytosis via BDCA2 binding was calculated using the method shown below as "Calculation Method of Phagocytosis via BDCA2 Binding".

[0409] [Method for calculating phagocytosis via binding with BDCA2]

[0410] First, to account for the effect of non-specific binding of anti-BDCA2 antibodies, the amount of bacteria phagocytosed by pDCs in the isotype group treated with an isotype control antibody having the same isotype and subclass as the anti-BDCA2 antibody (phagocytosis P) was evaluated. Then, the amount of bacteria phagocytosed by pDCs in the BDCA2 anti group treated with an anti-BDCA2 antibody that inhibits bacterial binding to BDCA2 (phagocytosis Q) was evaluated. Finally, the value obtained by subtracting phagocytosis Q from phagocytosis P was calculated as the amount of bacteria phagocytosed via pDC binding to BDCA2.

[0411] Figure 8 This is a graph showing the fluorescence detection area of ​​various lactic acid bacteria 22 hours after addition for the isotype group and the BDCA2 anti group. Figure 9This is a graph showing the results of IFN-α concentration measurements in the culture supernatant recovered 22 hours after the addition of various lactic acid bacteria to the isotype group and the BDCA2 anti group. Figure 10 This is a graph showing the correlation between various lactic acid bacteria, with the horizontal axis set as the amount of phagocytosis via BDCA2 binding calculated according to the above-described method, and the vertical axis set as the IFN-α concentration.

[0412] according to Figure 8 and 9 The results showed that the BDCA2 anti-group, which inhibited the binding of BDCA2 to CAL-1 cells by blocking with anti-BDCA2 antibody, had lower phagocytic volume and IFN-α concentration compared to the isotype group whose binding was not inhibited. These results indicate that, for bacteria other than LC-Plasma, the binding of bacteria to BDCA on the surface of pDCs is also one of the stages that determines the degree of bacterial immune activation. Furthermore, these results suggest that phagocytosis of bacteria via BDCA-bound pDCs is one of the stages that determines the degree of bacterial immune activation.

[0413] according to Figure 10 The results showed a strong positive correlation between the phagocytosis of each lactic acid bacteria via BDCA2 binding and its immune activation capacity (R0). 2 =0.86). This result indicates that bacterial immune activation capacity is not only determined by the amount of phagocytosis, but also that one of the main pathways of bacterial immune activation is mediated by phagocytosis of pDCs via binding to BDCA2. Furthermore, it suggests that the binding capacity to BDCA2 is useful as a parameter for evaluating bacterial immune activation capacity.

[0414] <Example 5: Construction of an analysis method for evaluating the binding ability of bacteria to BDCA2>

[0415] An analytical method for evaluating the binding ability of bacteria to BDCA2 (BDCA2 binding assay) was constructed as shown below. Phosphate-buffered saline (PBS, Nacalitesk, 11482-15) (hereinafter referred to as "buffer") containing 0.5 mM CaCl2 was added at 100 μL to each well of a low-adsorption 96-well plate (Corning, 3474). Recombinant human BDCA-2 Fc chimeric protein (R&D Systems, 1376-DL, hereinafter referred to as "BDCA2") or recombinant human IgG1 Fc protein (R&D Systems, 110-HG) was added to these wells at a final concentration of 1–50 μg / mL. Then, LC-Plasma stained with FITC according to standard methods was added to these wells at a final concentration of 10 μg / mL to distinguish impurities from bacteria, and the plates were incubated at room temperature for 1 hour under light-shielding conditions with aluminum foil. After adding 150 μL of buffer to each well, the 96-well plate was centrifuged at 400 g for 5 minutes at room temperature, and the supernatant was discarded. 100 μL of a buffer solution (hereinafter referred to as "secondary antibody dilution buffer") containing PE-conjugated F(ab')2-goat anti-human IgG Fc secondary antibody (ThermoFisher Scientific, H10104) was added to each well and incubated at room temperature for 15 minutes. The solutions from each well were then subjected to flow cytometry (FACS) using a BD Biosciences X-20 flow cytometer (BD LSRFortessa).

[0416] The analysis of FACS results was performed using the following steps. First, the lactic acid bacteria population was gated using FSC / SSC. Then, using the wavelength corresponding to SSC / FITC, only the FITC-stained population was extracted (FITC+ population). For this FITC+ population, the proportion of the population emitting fluorescence at the wavelength corresponding to PE was extracted using the wavelength corresponding to SSC / PE (PE+ population). It should be noted that the PE+ population threshold was set such that the PE+ population in each group supplemented with the same concentration of huIgfc as BDCA2 reached approximately 0.1%. The proportion of PE+ population in the FITC+ population was calculated as the proportion of lactic acid bacteria binding to BDCA2.

[0417] Figure 11 This is a graph showing the relative values ​​of LC-Plasma bound to BDCA2 at a concentration of 1 μg / mL, under conditions where the concentration of BDCA2 evaluated in the BDCA2 binding assay is 1, 5, 10, 20, or 50 μg / mL relative to LC-Plasma 10 μg / mL. Figure 11The proportion of LC-Plasma bound to BDCA2 increased in a BDCA2 concentration-dependent manner. This result indicates that the BDCA2 binding assay can be used to evaluate the binding capacity of bacteria to BDCA2.

[0418] <Example 6: pDC phagocytosis of bacteria, phagocytosis via binding to BDCA2, and binding ability to BDCA2>

[0419] For the lactic acid bacteria listed in Tables 3 and 4, the amount of pDC phagocytosis and the amount of pDC phagocytosis via binding to BDCA2 were evaluated. For the lactic acid bacteria listed in Table 5, the binding ability to BDCA2 was evaluated. The amount of pDC phagocytosis was evaluated using the same method as in Example 2, and is shown in Table 3 as (A-1) the fluorescence detection area 22 hours after the addition of lactic acid bacteria. Additionally, the values ​​obtained by subtracting the fluorescence detection area 22 hours after the addition of *Lactococcus lactis* subsp. *lactococcus* ATCC15577 from these fluorescence detection areas (A-2) and (A-3) the values ​​obtained by dividing these fluorescence detection areas by the fluorescence detection area 22 hours after the addition of *Lactococcus lactis* subsp. *lactococcus* ATCC15577 (ratios) are shown in Table 4. The amount of pDC phagocytosis via binding to BDCA2 was evaluated using 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 *Lactococcus lactis* subsp. *lactococcus* 15577 from these phagocytosed amounts is shown in Table 4 as (B-2), and the value (ratio) obtained by dividing these phagocytosed amounts by the amount of *Lactococcus lactis* subsp. *lactococcus* 15577 as (B-3) is shown in Table 4. The binding ability with BDCA2 was evaluated using the same BDCA2 binding assay as in Example 5. Specifically, the experiment was conducted using BDCA2 or recombinant human IgG1 Fc protein (R&D Systems, 110-HG) at a final concentration of 10 μg / mL, with the proportion of lactic acid bacteria binding to BDCA2 as an indicator. In Table 5, the proportions bound to BDCA2 are shown as (C-1), the values ​​obtained by subtracting the proportion of *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 from these proportions are shown as (C-2), and the values ​​(ratios) obtained by dividing these proportions by the proportion of *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 are shown as (C-3). It should be noted that these determinations were performed over 3 or 4 days; therefore, the relative values ​​shown as (A-2), (A-3), (B-2), (B-3), (C-2), and (C-3) relative to *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 (the results of (A-1), (B-1), or (C-1)) are obtained by using the values ​​obtained from testing *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 on each determination day as the basis for subtraction or division. In addition, (A-2) the value obtained by subtracting the fluorescence detection area of ​​Lactococcus lactis subsp. 15577 22 hours after adding each lactic acid bacteria from the fluorescence detection area after adding each lactic acid bacteria, and (B-2) the value obtained by subtracting the amount of Lactococcus lactis subsp. 15577 22 hours after adding each lactic acid bacteria from the amount of phagocytosis of each lactic acid bacteria via binding to BDCA2 from the amount of phagocytosis of Lactococcus lactis subsp. 15577 via binding to BDCA2 from the amount of phagocytosis of each lactic acid bacteria via binding to BDCA2, are shown as 0 if the value is less than 0.

[0420] [Table 3]

[0421] [Table 4]

[0422] [Table 5]

[0423] Lactic acid bacteria with higher pDC phagocytosis, pDC phagocytosis via BDCA2 binding, and / or BDCA2 binding capacity than *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 can be considered to have immune-activating capacity. Therefore, based on the results in Tables 3–5, lactic acid bacteria with higher pDC phagocytosis, pDC phagocytosis via BDCA2 binding, and / or BDCA2 binding capacity than *Lactococcus lactis* subsp. *lactococcus* ATCC 15577 can be considered to have immune-activating capacity.

[0424] <Example 7: The Importance of Phagocytosis in the Generation of IFN-α in pDC>

[0425] For the CAL-1 cell suspensions in each well inoculated in the same manner as in Example 1, in the group with added pinocembrin D (denoted as CytD in the chart), 400 μM pinocembrin D (Fujifilm Wako, 037-17561) dissolved in 4 v / v dimethyl sulfoxide (DMSO) was added at final concentrations of 10 μM and 0.1 v / v for DMSO, and incubated for 30 minutes. As a control, the DMSO group was added only at a final concentration of 0.1 v / v for 4 v / v, and incubated for 30 minutes as well. After incubation, the lactic acid bacteria listed in Table 1, stained with pHrodo Red SE (Invitrogen, P36600) using the above-described method of [bacterial staining using pHrodo Red SE], were added to each well at a final concentration of 10 μg / mL and cultured for 24 hours. Phagocytic activity and IFN-α concentration in each well were measured using the same method as in Example 1.

[0426] In addition, to confirm that DMSO and pinocembrin D are non-cytotoxic, pinocembrin D or DMSO, along with an equal volume of PBS as a control, were added as described above, and the cells were incubated for 30 minutes followed by 24 hours. The number of viable cells was determined using a cell counting kit-8 (Dongren Chemical, CK04) to evaluate cytotoxicity at this point.

[0427] Cell Count Kit-8 is a kit for determining cell counts in cell proliferation or chemical sensitivity assays. By employing a novel tetrazolium salt, WST-8, which generates highly sensitive, water-soluble formazan as a chromogenic substrate, it enables highly sensitive determination compared to previous cell counting kits. WST-8 is reduced by intracellular dehydrogenases to generate water-soluble formazan. The number of viable cells can be easily measured by directly measuring the absorbance of this formazan at 450 nm. The cell count is linearly proportional to the amount of formazan generated. Cytotoxicity can be evaluated by observing changes in the number of viable cells.

[0428] In addition, as detailed in Example 1, pinocembrin D is an inhibitor of actin polymerization, and it is known that phagocytosis is inhibited in cells with added pinocembrin D.

[0429] Figure 12 This is a graph showing the results obtained after adding DMSO or pine cytosine D+DMSO (denoted as CytD in the graph) and measuring the number of viable cells 24 hours later using a cell counting kit-8. Figure 13 This shows the total fluorescence intensity (total integral intensity, TII, OCU×μm) of the six tested lactic acid bacteria 24 hours after addition, representing indicators of phagocytic activity. 2 The image is a diagram (image). Figure 14 This is a graph showing the IFN-α concentration in the supernatant 24 hours after adding the six tested lactic acid bacteria. Based on... Figure 12 The results showed that the number of viable cells did not change compared to the group receiving PBS, with the addition of DMSO or pinocembrin D indicating that the inhibitory agents used in this experiment did not produce cytotoxicity. Figure 13 As a result, treatment with pinocembrin D completely inhibited the phagocytic activity of various lactic acid bacteria. At this point, according to... Figure 14 As a result, IFN-α production was completely eliminated by inhibiting phagocytic activity. This result indicates that phagocytosis is essential for the immune activation of pDCs in lactic acid bacteria, and considering the results of Example 1, it shows that phagocytosis prescribes the immune activation of pDCs.

[0430] <Example 8: Phagocytosis rate determination using FACS>

[0431] 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 recovered using FACS buffer and stained with 7-AAD (manufacturer: BD biosciences, BD LSRFortessa). After washing the cells with FACS buffer, the washed cells were subjected to flow cytometry (FACS) using a BD biosciences, BD LSRFortessa (registered trademark) X-20 flow cytometer. The IFN-α content of the supernatant upon cell recovery was evaluated in the same manner as in Example 1.

[0432] The analysis of FACS results was performed using the following steps. First, the population containing CAL-1 cells was gated using FSC / SSC. Then, using the wavelength corresponding to SSC / 7-AAD, only the population unstained by 7-AAD was extracted (7-AAD- population: the population of live CAL-1 cells). For this 7-AAD- population, the proportion of the population that detected fluorescence corresponding to the wavelength corresponding to PE (i.e., fluorescence from pHrodo Red SE labeled with lactate) was extracted using the wavelength corresponding to PE (PE+ population). The proportion of the PE+ population relative to the 7-AAD- population was taken as the phagocytosis rate. It should be noted that the threshold for the PE+ population was set to the lowest value of the population with the stronger PE intensity among the two populations divided when plotting PE / SSC.

[0433] Table 6 summarizes the phagocytic rate and IFN-α assay results for various lactic acid bacteria. Based on the results in Table 6, a positive correlation was confirmed between the phagocytic rate obtained by flow cytometry and IFN-α. A positive correlation was observed between the phagocytic behavior of each lactic acid bacteria and its immune activation capacity (R0). 2 =0.69). This result indicates that the amount of bacteria phagocytosed determines their immune activation capacity.

[0434] [Table 6]

[0435] <Example 9: Obtaining Lactic Acid Strains with High Phagocytosis Rate>

[0436] In this embodiment, LC-Plasma was cultured using two different culture media to evaluate phagocytosis rate and IFN-α. One type of LC-Plasma cultured using the two different media was cultured according to Table 1, B. The other type was cultured as follows: Bacterial cells were suspended in MRS medium (product name: Difco Lactobacilli MRS Broth, manufacturer: Becton, Dickinson and Company, hereinafter the same) and inoculated onto MRS agar. After culturing at 30°C for 48 hours, colonies were picked and inoculated into 10 mL of M17 medium (product name: M17 Broth, manufacturer: Becton, Dickinson and Company) supplemented with 1% glucose (product name: D(+)-Glucose, manufacturer: Fujifilm and Koden Chemicals) at a final concentration of 1%, and cultured at 30°C for 24 hours. 1 mL of the cultured bacterial suspension was then inoculated into 100 mL of M17 medium and cultured at 30°C for 24 hours.

[0437] For LC-Plasma cultured in two different culture media, the phagocytosis rate and IFN-α were evaluated in the same manner as in Example 8.

[0438] Table 7 summarizes the phagocytic rate and IFN-α assay results of the strains. According to the results in Table 7, LC-Plasma cultured in M17 medium supplemented with 1% glucose had a higher phagocytic rate than LC-Plasma cultured in MRS medium, indicating that lactic acid bacteria with enhanced immune activation capabilities can be obtained.

[0439] [Table 7]

Claims

1. An immune-activating composition comprising bacteria that bind to blood dendritic cell antigen 2 (BDCA2) as an active ingredient.

2. The composition according to claim 1, wherein, The bacteria described above have a greater binding capacity to BDCA2 than Lactococcus lactis subsp. lactis ATCC15577.

3. The composition according to claim 2, wherein, The binding capacity with BDCA2 is evaluated based on the proportion of bacteria that bind to BDCA2 as assessed in the binding assay.

4. An immune-activating composition comprising bacteria as an active ingredient, said bacteria being phagocytosed by plasmacytoid dendritic cells (pDCs), and said bacteria satisfying the following requirement X: [Requirement X] Make the final concentration 2.0 × 10⁻⁶ 5 After contacting pDCs at a final concentration of 10 μg / mL with serum-free culture medium for 24 hours, the number of pDCs that engulfed the bacteria was greater than 2.5% of the total number of pDCs that had been contacted. The pDC is a pDC that has been pre-cultured in a serum-free medium.

5. An immune-activating composition comprising bacteria as an active ingredient, said bacteria being phagocytosed by plasmacytoid dendritic cells (pDCs), wherein, The amount of bacteria phagocytosed by pDC is more than 2.0 times that of Lactococcus lactis subsp. lactis ATCC15577.

6. A composition comprising Lactococcus lactis subsp. lactis JCM5805 as a bacterium, wherein said Lactococcus lactis subsp. lactis JCM 5805 satisfies the following requirement Y: [Requirement Y] Make the final concentration 2.0 × 10⁻⁶ 5 After 24 hours of contacting pDCs of *Lactococcus lactis* subsp. *lactococcus* JCM 5805 at a final concentration of 10 μg / mL with serum-free culture medium, the number of pDCs that engulfed *Lactococcus lactis* subsp. *lactococcus* JCM 5805 was greater than 15.0% of the total number of pDCs that had been contacted. The pDC is a pDC that has been pre-cultured in a serum-free medium.

7. The composition according to claim 6, wherein, The composition is an immune-activating composition.

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

9. The composition according to any one of claims 1 to 5, wherein, The bacteria are selected from one or more of the following groups: Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Tetracoccus, Staphylococcus, Weissella, Bifidobacterium, and Lactobacillus.

10. The composition according to any one of claims 1 to 5, wherein, The bacteria in question is Lactococcus lactis subsp. lactis.

11. The composition according to any one of claims 1 to 5, wherein, The bacteria selected are *Lactococcus plantarum* JCM 11056, *Lactococcus curvatus* JCM 1096, *Lactococcus murinus* JCM 1717, *Lactococcus parakefiri* JCM 8573, *Lactococcus paraplantarum* JCM 12533, *Leuconostoc carnosum* JCM 9695, *Streptococcus salivarius* JCM 5707, *Enterococcus lactis* JCM 30200, *Oenococcus oeni* JCM 6125, and *Weissella paramesenteroides* JCM. 9890, *Weissella viridescens* JCM 1174, *Bifidobacterium animalis subsp. animalis* JCM 1190, *Bifidobacterium animalis subsp. lactis* JCM 10602, *Bifidobacterium bifidum* JCM 1255, *Bifidobacterium longum subsp. Infantis* JCM 1222, *Bifidobacterium pseudolongum* JCM 1205, *Lactococcus lactis* subsp. lactis ATCC 7963, *Lactococcus lactis* subsp. lactis ATCC 7962, *Lactococcus lactis* subsp. *lactis* ATCC 15346, *Lactococcus lactis* subsp. *lactis* ATCC 13675, *Lactococcus lactis* subsp. *lactis* ATCC 12929, *Lactococcus lactis* subsp. *lactis*One or more bacteria belonging to the group consisting of *Lactococcus lactis* ATCC 11955, *Lactococcus lactis* subsp. *lactis* ATCC 11454, *Lactococcus lactis* subsp. *lactis* ATCC 11007, and *Lactobacillus rhamnosus* CRL 1505.

12. The composition according to any one of claims 1 to 7, wherein, The bacteria include dead bacteria.

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

14. The composition according to claim 13, wherein, The pDC's phagocytosis of the bacteria includes phagocytosis via binding to BDCA2 and phagocytosis without binding to BDCA2, and the amount of bacteria phagocytosed via binding to BDCA2 is more than 2.0 times the amount of Lactococcus lactis subsp. lactis ATCC15577 phagocytosed via binding to BDCA2.

15. The composition according to claim 14, wherein, The amount of phagocytosis without binding to BDCA2 is the amount of bacteria phagocytosed by the pDC when the BDCA2 of the pDC cannot bind to the ligand.

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

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

18. The composition according to claim 17, wherein, The bacterial count of each unit package of the food composition, pharmaceutical composition, or feed is 1 × 10⁻⁶. 8 More than one.

19. A method for screening bacteria with immune-activating capabilities, comprising the step of screening said bacteria with immune-activating capabilities by using binding to blood dendritic cell antigen 2 (BDCA2) as an indicator.

20. The method according to claim 19, wherein, The screening step is performed by measuring the binding of candidate bacteria to BDCA2.

21. A method for enhancing the immune activation capacity and / or the ability of bacteria to be phagocytosed by plasmacytoid dendritic cells (pDCs), comprising culturing bacteria in M17 medium supplemented with glucose at a final concentration of 0.1 to 10% by mass.

22. A method for manufacturing an immune-activating composition containing Lactococcus lactis subsp. lactis JCM 5805 as an active ingredient, comprising the step of culturing Lactococcus lactis subsp. lactis JCM 5805 in M17 medium supplemented with glucose at a final concentration of 0.1 to 10% by mass.

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