Bacteria with immunostimulatory properties and compositions containing them

Abstract

To provide a composition containing bacteria that have immunostimulatory properties. [Solution] An immunostimulatory composition containing bacteria that bind to PNA.

Description

[Technical Field]

[0001] This disclosure relates to bacteria having immunostimulatory properties and compositions containing the same. [Background technology]

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

[0003] [Patent Document 1] Japanese Patent Publication No. 2017-201984 [Patent Document 2] Japanese Patent Publication No. 2014-217372 [Overview of the project] [Problems that the invention aims to solve]

[0004] While several species of bacteria possessing immunostimulatory capabilities have been reported, much remains unclear about the mechanisms by which these bacteria stimulate immunity and the common properties they share.

[0005] This disclosure aims to provide bacteria having immunostimulatory properties and compositions containing them. [Means for solving the problem]

[0006] The inventors have found that the binding affinity to a given lectin is one of the important parameters that determine the immunostimulatory ability of bacteria. In addition, the inventors have found that the sugar chain structure on the surface of bacterial cells is one of the important parameters that determine the immunostimulatory ability of bacteria.

[0007] This disclosure relates, for example, to the following: [1] Bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL. [2] The bacteria described in [1] that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB. [3] The bacterium described in [1] or [2], which binds to at least one lectin selected from the group consisting of PNA, MPL, and PHA-E. [4] Furthermore, a bacterium described in any one of [1] to [3] that does not bind to at least one lectin selected from the group consisting of F17AG, Gal1-S, VVA, AAA, UEA-I, EEL, LSL-N, MOA, SJA, Gal3, LEA, CGL2, Gal7-S, VFA, ACL, Orysata, Gal1 and PA-IL. [5] A bacterium described in any one of [1] to [4] that also satisfies any one of (a) to (c) below: (a) The ratio of the binding strength to PNA to the binding strength to PA-IL is 2.70 times or more; (b) The ratio of the binding force to MPL to the binding force to PA-IL is 0.90 or greater; (c) The ratio of the binding strength to PHA-E to the binding strength to PA-IL is 3.20 times or more. [6] Bacteria having at least one sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4 and NeuAcα2-6Gal / GalNAc on the surface of the bacterial cell. [7] The bacterium described in [6], having at least one sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), and (GlcNAcβ4Manα3)Manβ4 on the surface of the bacterial cell. [8] A bacterium belonging to one of the genera Lactococcus, Lactobacillus, Bifidobacterium, Pediococcus, Hendricksia, or Leuconostoc, as described in any one of [1] to [7]. A composition containing any one of the bacteria described in [9][1] to [8]. An immunostimulatory composition containing any one of the bacteria described in

[10] [1] to [8]. [10-1] Use of any one of the bacteria described in [1] to [8] for activating the target's immunity. [10-2] Any of the bacteria described in [1] to [8] for use in activating the target immune system. [10-3] Use of any one of the bacteria described in [1] to [8] in the manufacture of an immunostimulatory composition. An immunostimulatory method comprising administering a bacterium described in any one of [10-4][1] to [8] to a subject in need thereof. [10-5] Use of a composition containing any one of the bacteria described in [1] to [8] for activating the target's immunity. [10-6] A composition containing the bacteria described in any one of [1] to [8] for use in activating the immune system of a target. An immunostimulatory method comprising administering a composition containing the bacteria described in any one of [10-7][1] to [8] to a subject requiring it.

[11] A composition containing bacteria, which satisfies the following (A1): (A1) The ratio of bacterial cells that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, relative to the total number of bacterial cells contained in the composition, is 7% or more.

[12] The composition described in

[11] that satisfies (A2) below: (A2) The ratio of bacterial cells that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB, relative to the total number of bacterial cells contained in the composition, is 7% or more.

[13] The composition described in

[11] or

[12] that satisfies (A3) below: (A3) The ratio of the number of bacterial cells that bind to PNA contained in the composition to the total number of bacterial cells contained in the composition is 7% or more.

[14] A composition containing bacteria, which satisfies the following (B1): (B1) Bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, total cell number of 1.0 × 10⁶ 5 Contains one or more of these.

[15] The composition described in

[14] that satisfies (B2) below: (B2) Bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB, total number of cells: 1.0 × 10⁶ 5 Contains one or more of these.

[16] The composition described in

[14] or

[15] that satisfies (B3) below: (B3) Bacteria that bind to PNA, total number of cells: 1.0 × 10 5 Contains one or more of these.

[17] A composition containing bacteria, which satisfies the following (C1): (C1) The proportion of bacterial cells containing at least one sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Poly β(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on the surface of the bacterial cell is 7% or more of the total number of bacterial cells contained in the composition.

[18] The composition described in

[17] that satisfies (C2) below: (C2) The ratio of the number of bacterial cells containing the Galβ3GalNAc sugar chain structure on the surface of the bacterial cell to the total number of bacterial cells contained in the composition is 7% or more.

[19] A composition containing bacteria, which satisfies the following (D1): (D1)Bacteria having at least one sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on the surface of the bacterial cell, total number of cells: 1.0 × 10⁶ 5 Contains one or more of these.

[20] The composition described in

[19] that satisfies (D2) below: (D2) Bacteria possessing the glycan structure of (D2)Galβ3GalNAc on the cell surface, total number of cells: 1.0 × 10⁶ 5 Contains one or more of these.

[21] The composition according to any one of

[11] to

[20] , wherein the above-mentioned bacteria belong to one of the genera Lactococcus, Lactobacillus, Bifidobacterium, Pediococcus, Hendricksia, or Leuconostoc.

[22] An immunostimulatory composition, the composition according to any one of

[11] to

[21] . [22-1] Use of any one of the compositions described in

[11] to

[21] in activating the target immune system. [22-2] A composition according to any one of

[11] to

[21] for use in activating the immune system of a target. An immunostimulatory method comprising administering a composition described in any one of [22-3]

[11] to

[21] to a subject requiring it.

[23] A method for producing a composition, comprising at least one selected from the group consisting of (A1M), (B1M), (C1M), and (D1M): The ratio of bacterial cell counts that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, relative to the total number of bacterial cells contained in the (A1M) composition, shall be 7% or more. (B1M) Composition contains bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, totaling 1.0 × 10⁶ cells. 5 Contains more than one δ; The proportion of bacterial cells having a sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on their cell surface, relative to the total number of bacterial cells contained in the (C1M) composition, should be 7% or more. (D1M) Composition contains bacteria having a sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on the surface of the bacterial cell, totaling 1.0 × 10⁶ cells. 5 Contains at least one of these.

[24] The manufacturing method described in

[23] , including the following (A2M): Making the ratio of the number of bacteria cells that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB in the (A2M) composition to the total number of bacteria cells contained in the composition be 5% or more.

[25] The production method according to

[23] or

[24] , comprising the following (A3M): (A3M) Making the ratio of the number of bacteria cells that bind to PNA contained in the composition to the total number of bacteria cells contained in the composition be 7% or more.

[26] The production method according to any one of

[23] to

[25] , comprising the following (B2M): (B2M) Incorporating into the composition at least 1.0×10 5 bacteria cells or more that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB as the total number of cells.

[27] The production method according to any one of

[23] to

[26] , comprising the following (B3M): (B3M) Incorporating into the composition at least 1.0×10 5 bacteria cells or more that bind to PNA as the total number of cells.

[28] The production method according to any one of

[23] to

[27] , comprising the following (C2M): (C2M) Making the ratio of the number of bacteria cells having the sugar chain structure of Galβ3GalNAc on the cell surface contained in the composition to the total number of bacteria cells contained in the composition be 7% or more.

[29] The production method according to any one of

[23] to

[28] , comprising the following (D2M): (D2M) Incorporating into the composition at least 1.0×10 5 bacteria cells or more that have the sugar chain structure of Galβ3GalNAc on the cell surface as the total number of cells.

[30] A method for evaluating the immunostimulatory capacity of a composition containing bacteria, comprising at least one selected from the group consisting of (A1P), (B1P), (C1P), and (D1P): (A1P) To evaluate the ratio of bacterial cell counts that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, relative to the total number of bacterial cells contained in the composition; (B1P) To evaluate the number and / or concentration of bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL contained in the composition; (C1P) To evaluate the proportion of bacterial cells having at least one sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on the bacterial cell surface, relative to the total number of bacterial cells contained in the composition; Evaluate the number and / or concentration of bacteria in the (D1P) composition that have at least one sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Poly β(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on their cell surface.

[31] The method described in

[30] , including (A2P) below: (A2P) Evaluate the ratio of bacterial cell counts that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB, relative to the total number of bacterial cells contained in the composition.

[32] The method according to

[30] or

[31] , comprising at least one selected from the group consisting of (A3P) and (A4P): (A3P) Evaluate the ratio of the total number of bacterial cells that bind to at least one lectin selected from the group consisting of MPL and PNA contained in the above composition, relative to the total number of bacterial cells contained in the composition; (A4P) Evaluate the ratio of the total number of bacterial cells that bind to PHA-E contained in the composition to the total number of bacterial cells contained in the composition.

[33] The method described in any one of

[30] to

[32] , including the following (B2P): To evaluate the number and / or concentration of bacterial cells that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB contained in the (B2P) composition.

[34] The method according to any one of

[30] to

[33] , comprising at least one selected from the group consisting of (B3P) and (B4P): To evaluate the number and / or concentration of bacterial cells that bind to at least one lectin selected from the group consisting of MPL and PNA contained in the (B3P) composition; To evaluate the number of bacteria that bind to PHA-E contained in the (B4P) composition.

[35] The method according to any one of

[30] to

[34] , comprising at least one selected from the group consisting of (C2P) and (C3P): (C2P) To evaluate the ratio of the number of bacterial cells having the Galβ3GalNAc sugar chain structure contained in the composition to the total number of bacterial cells contained in the composition; To evaluate the ratio of the total number of bacterial cells having at least one sugar chain structure selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 on the bacterial cell surface to the total number of bacterial cells contained in the (C3P) composition.

[36] The method according to any one of

[30] to

[35] , comprising at least one selected from the group consisting of (D2P) and (D3P): (D2P) Evaluate the number and / or concentration of bacterial cells containing the Galβ3GalNAc sugar chain structure on the cell surface of the (D2P) composition. (D3P) Evaluate the number of bacteria containing the Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 sugar chain structure on the cell surface of the (D3P) composition.

[37] A method for evaluating the immunostimulatory capacity of bacteria, comprising at least one selected from the group consisting of (X1) and (Y1) below: (X1) Evaluate the binding affinity of bacteria to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL; (Y1) Evaluate the amount of at least one glycan structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc, which is present on the surface of the bacterial cell.

[38] A method for screening for immunostimulatory bacteria, comprising at least one selected from the following groups: (X1) and (Y1): (X1) Evaluate the binding affinity of bacteria to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL; (Y1) Evaluate the amount of at least one glycan structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc, which is present on the surface of the bacterial cell.

[39] The method described in

[37] or

[38] , including (X2) below: (X2) Evaluate the binding affinity of bacteria to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB.

[40] The method according to any one of

[37] to

[39] , comprising at least one selected from the group consisting of (X3) and (X4) below: (X3) Evaluate the binding affinity of bacteria to at least one lectin selected from the group consisting of MPL and PNA; (X4) Evaluate the binding affinity of bacteria to PHA.

[41] The method according to any one of

[37] to

[40] , comprising at least one selected from the group consisting of (Y2) and (Y3) below: (Y2) To evaluate the amount of Galβ3GalNAc sugar chain structure present on the surface of bacterial cells; (Y3) Evaluate the amount of at least one sugar chain structure selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 that the bacterial cell surface possesses. [Effects of the Invention]

[0008] This disclosure provides bacteria having immunostimulatory activity, indicated by their binding affinity to a predetermined lectin or the sugar chain structure on the bacterial cell surface, and compositions containing the same. Furthermore, this disclosure provides a method for producing compositions with high immunostimulatory activity, indicated by their binding affinity to a predetermined lectin or the sugar chain structure on the bacterial cell surface; a method for evaluating the quality of a bacterial composition, such as its immunostimulatory activity; a method for evaluating the immunostimulatory activity of bacteria; and a method for screening bacteria having immunostimulatory activity. [Modes for carrying out the invention]

[0009] The following describes embodiments for carrying out the present invention, but the present invention is not limited to the following embodiments.

[0010] In this disclosure, the phrase "at least one selected from the group consisting of" includes one element of that group or all combinations that can be formed by those elements, which could be, for example, one of the elements of that group, or any combination of two, three, four, five, six, seven, eight, nine or more elements of that group.

[0011] <Lectins> In this disclosure, the relationship between the abbreviation, full name, and recognized glycan structure of the lectins is as shown in Tables 1 and 2. These lectins are the lectins used in the lectin array RayBiotech Lectin Array 70 (RayBiotech, Inc., cat.GA-Lectin-70).

[0012] [Table 1] [Table 2]

[0013] <Immunostimulating bacteria> A bacterium according to one embodiment of this disclosure will be described. The bacterium according to one embodiment of this disclosure has immunostimulatory ability. Hereinafter, the bacterium having immunostimulatory ability according to this embodiment will also be referred to as "immunostimulatory bacteria".

[0014] In this disclosure, immunostimulatory ability (immune activation ability) means the ability to activate the innate immune system and / or the adaptive immune system, where the adaptive immune system is, for example, cellular immunity and / or humoral immunity. In one embodiment, immunostimulatory ability may be dendritic cell activation ability that activates the innate immune system of dendritic cells, and more specifically, plasmacytoid dendritic cell (pDC) activation ability. In one embodiment, immune activation may be an increase in the gene or protein expression level of cytokines or cell surface markers secreted from dendritic cells, preferably an increase in IFN-α production, and particularly preferably an increase in IFN-α production by pDCs. In vivo, pDCs are the main producers of IFN-α, and IFN-α is a type of cytokine that activates a comprehensive biological defense mechanism from the innate immune system to the adaptive immune system. Therefore, a substance that has the ability to promote IFN-α expression by pDCs can be said to be a substance that has dendritic cell activation ability and pDC activation ability, and can also be said to be a substance that has immunostimulatory ability. In one embodiment, the immunostimulatory ability may be the ability to be phagocytosed by dendritic cells, and more specifically, the ability to be phagocytosed by plasmacytoid dendritic cells. Therefore, for example, a substance phagocytosed by pDCs can be said to be a substance that has immunostimulatory ability.

[0015] For example, the immunostimulatory ability of bacteria can be evaluated using the amount of IFN-α expressed by pDCs after contact with the bacteria as an indicator. The amount of IFN-α expressed by pDCs is the amount of biomolecules that can serve as an indicator for evaluating the amount of IFN-α expressed by pDCs by bacteria. In one embodiment, the amount of IFN-α expressed may be the amount of IFN-α secreted extracellularly and / or the amount of IFN-α in pDCs, or the amount of mRNA encoding IFN-α in pDCs. In a preferred embodiment, the amount of IFN-α expressed may be measured by the ELISPOT method, which involves adsorbing extracellularly secreted IFN-α onto cell culture wells, or by measuring the amount of IFN-α secreted extracellularly and present in the culture supernatant. In these cases, the culture supernatant is the culture medium after contact between bacteria and pDCs in the contact step. The amount of IFN-α in the culture supernatant can be measured after the culture supernatant is collected, according to methods commonly used by those skilled in the art to quantify specific proteins. Furthermore, the amount of IFN-α in pDCs (e.g., in CAL-1 cells) can be measured by methods commonly used by those skilled in the art, such as intracellular cytokine staining using a flow cytometer, or by preparing a pDC lysate and then measuring the amount of IFN-α in the lysate according to methods commonly used by those skilled in the art to quantify specific proteins. In these cases, the amount of IFN-α may be measured by methods such as ELISA or Western blotting, and preferably by ELISA. For measuring the amount of IFN-α by ELISA, for example, the Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science) can be used. In addition, the amount of mRNA encoding IFN-α in pDCs (e.g., in CAL-1 cells) can be measured by preparing a pDC lysate using methods commonly used by those skilled in the art, and then measuring the amount of mRNA encoding IFN-α in the lysate according to methods commonly used by those skilled in the art to quantify mRNA. mRNA quantification may be performed by quantitative PCR methods such as real-time PCR (q-PCR), direct digital counting (e.g., nCounter®), or next-generation sequencing (NGS).

[0016] When evaluating the immunostimulatory capacity of bacteria using the amount of IFN-α expressed by pDCs after contact with the bacteria as an indicator, the indicator may be the amount of IFN-α in the culture supernatant measured according to the protocol shown below (hereinafter also referred to as the "standard measurement method for immunostimulatory capacity"). The immunostimulatory activity of bacteria is determined by the amount of IFN-α in the culture supernatant, measured according to the "Standard Measurement Method for Immunostimulatory Activity" shown below, which is 5 pg / mL or more, preferably 10 pg / mL or more, more preferably 20 pg / mL or more, more preferably 30 pg / mL or more, more preferably 50 pg / mL or more, more preferably 75 pg / mL or more, more preferably 100 pg / mL or more, more preferably 200 pg / mL or more, more preferably 300 pg / mL or more, more preferably 400 pg / mL or more, more preferably 500 pg / mL or more, more preferably 700 pg / mL or more, more preferably 1000 pg / mL or more, and most preferably 1600 pg / mL or more.

[0017] [Standard methods for measuring immunostimulatory capacity] 1. Using serum-free medium (RPMI-1640 medium (Sigma, R8758) supplemented with a final concentration of 1.0 volume% penicillin / streptomycin (Gibco, 15140-12)), CAL-1 cells were sampled in 5.0 × 10⁶ cells. 5 Prepare a cell suspension containing cells / mL, and seed 10 mL / well of the cell suspension into a 10 cm diameter culture dish and culture for 16 hours. 2. CAL-1 cells are collected by pipetting. 3. Place the recovered CAL-1 cells in serum-free medium in a 2.0 × 10⁶ layer. 5 Resuspend the cells at a concentration of cells / mL and seed each well of a 96-well microplate at a rate of 200 μL / well. 4. After culturing the seeded cells for 22 hours, collect the culture supernatant from each well and measure the IFN-α concentration in the culture supernatant. The measurement can be performed by, for example, the ELISA method, using a Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science), etc.

[0018] CAL-1 cells are a human plasmacytoid dendritic cell (pDC) cancer cell line established from tumor cells in the peripheral blood of patients, and are deposited with the National Institute of Technology and Evaluation (NPMD, Japan) under depositary number FERM BP-10914.

[0019] The immunostimulatory ability of an immunostimulatory bacterium may be, for example, 0.01 times or more, 0.03 times or more, 0.05 times or more, 0.07 times or more, 0.09 times or more, 0.1 times or more, 0.3 times or more, 0.5 times or more, 0.7 times or more, 0.9 times or more, 1.0 times or more, greater than 1.0 times, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more, 1.7 times or more, or 2.0 times or more than the immunostimulatory ability of pDCs possessed by Lactococcus lactis subspecies lactis JCM5805. Furthermore, the immunostimulatory ability of immunostimulatory bacteria may be, for example, more than 1.0 times, 1.2 times or more, 1.5 times or more, 2.0 times or more, 3.0 times or more, 4.0 times or more, 5.0 times or more, 7.0 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 60 times or more, or 100 times or more of the immunostimulatory ability of pDCs possessed by Bifidobacterium pseudorhondronum JCM1205.

[0020] For example, the immunostimulatory ability of bacteria can be evaluated using the amount of phagocytosis of the bacteria by pDCs after contact with the bacteria as an indicator. The amount of immunostimulatory bacteria phagocytosed by pDCs can be evaluated, for example, by an evaluation method using fluorescent or fluorescently dye-labeled bacteria, as described later. In the above evaluation method, for example, immunostimulatory bacteria or control bacteria (Lactococcus lactis subspecies lactis JCM5805, Lactococcus lactis subspecies lactis ATCC15577, or Bifidobacterium pseudorhondronum JCM1205) are brought into contact with pDCs of the same cell number (or cell concentration) under the same conditions for a certain period of time, after which the bacteria are removed from the culture medium, and the fluorescence area per unit area of ​​the observation field of a fluorescence microscope (e.g., μm) in the pDCs is evaluated. 2 The amount of phagocytosis can be evaluated as the amount of / image). In this case, it is preferable that the amount of phagocytosis is the average value 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 the amount of phagocytosis by pDCs is evaluated using an evaluation method using fluorescent or fluorescently dye-labeled bacteria described later, the amount of phagocytosis by pDCs is 50 μm 2 / image or more, 100μm 2 / image or more, 200μm 2 / image or more, 400μm 2 / image or more, 500μm 2 / image or more, 600μm 2 / image or more, 700μm 2 / image or more, 800μm 2 / image or larger, or 900μm 2 It can be greater than or equal to / image. Here, "image" is 0.572mm. 2 It means the field of view.

[0021] Furthermore, the difference between the amount of phagocytosis by pDCs of immunostimulant bacteria and the amount of phagocytosis by pDCs of Lactococcus lactis subspecies lactis ATCC15577 is 1 μm. 2 / image or more, 20μm 2 / image or more, 50μm 2 / image or more, 70μm 2 / image or more, 100μm 2 / image or more, 140μm 2 / image or more, 200μm 2 / image or more, 300μm 2 / image or more, 400μm 2 / image or more, 500μm 2 / image or more, 600μm 2 / image or more, 700μm 2 / image or larger or 900μm 2 / image may be greater than or equal to / image. Furthermore, the immunostimulatory bacteria may be bacteria whose pDC phagocytosis amount is 0.01 times or more, 0.1 times or more, 0.3 times or more, 0.5 times or more, 0.7 times or more, 0.9 times or more, 1.0 times or more, greater than 1.0 times, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more, 1.7 times or more, or 2.0 times or more than the phagocytosis amount of Lactococcus lactis subspecies lactis JCM5805 pDC. Furthermore, the amount of immunostimulatory bacteria phagocytosed by pDCs may be more than 1.0 times, 1.2 times or more, 1.5 times or more, 2.0 times or more, 3.0 times or more, 4.0 times or more, 5.0 times or more, 7.0 times or more, 10 times or more, 15 times or more, 20 times or more, 30 times or more, 40 times or more, 60 times or more, or 100 times or more than the amount of immunostimulatory bacteria phagocytosed by pDCs of Lactococcus lactis subspecies lactis ATCC15577 and / or Bifidobacterium pseudorhondronum JCM1205.

[0022] In these cases, the amount of phagocytosis by pDCs is not particularly limited as long as it is an index value indicating the amount of phagocytosis by pDCs, and the evaluation of the amount of phagocytosis by pDCs can be done using methods that are commonly used by those skilled in the art to measure the amount of phagocytosis. For example, the amount of phagocytosis by pDCs may be the fluorescence intensity of pDCs measured after contact with bacteria labeled with a fluorescent or fluorescent dye. As a more detailed example, the amount of phagocytosis by pDCs can be evaluated using the fluorescence detected from pDCs when bacteria labeled with a fluorescent dye that becomes strongly fluorescent in the acidic environment of endosomes (e.g., pHrodo Red SE, Invitrogen, P36600) are in contact with pDCs for a sufficient time (e.g., 24 hours). As an indicator of fluorescence, one that is commonly used by those skilled in the art may be used. For example, one could use the area or the percentage of the field of view in a fluorescence image where the fluorescence intensity is greater than a predetermined value, the value obtained by multiplying the area value where the fluorescence intensity is greater than a predetermined 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 percentage of cells included in the gate when gating is performed on fluorescence intensity, or the fluorescence intensity of bulk cells contained in a cell suspension or microwells for analysis using a microwell plate reader. Furthermore, the amount of phagocytosis by pDCs may be evaluated using the expression level of a biomarker whose expression level fluctuates in accordance with the amount of phagocytosis by pDCs as an indicator. The values ​​described above can be treated as "amount of phagocytosis by pDCs".

[0023] In this embodiment, the amount of phagocytosis by pDCs may be the amount of phagocytosis evaluated using CAL-1 cells. As a detailed example, the amount of phagocytosis by pDCs may be the amount of phagocytosis evaluated by the method described below as "Evaluation method using bacteria labeled with a fluorescent dye." As a more detailed example, the amount of phagocytosis by pDCs may be the amount of phagocytosis evaluated by the method described in the examples.

[0024] [Evaluation method using bacteria labeled with fluorescent dyes] (Phasophagous experiment) Using a serum-free medium (for example, RPMI-1640 medium (Sigma, R8758) supplemented with a final concentration of 1.0 volume% penicillin / streptomycin (Gibco, 15140-12)), CAL-1 cells were sampled in a volume of 5.0 × 10⁶ cells. 5 Prepare a cell suspension containing cells / mL, seed 10 mL / well of the cell suspension into a 10 cm diameter culture dish, culture for 16 hours, and harvest by pipetting. Place the harvested CAL-1 cells in the same medium in a 2.0 × 10⁶ container. 5 The cells are resuspended at a concentration of cells / mL and seeded at 200 μL / well in each well of a 96-well microplate. Bacteria stained with pHrodo Red SE (Invitrogen, P36600) by the method described below are added at a final concentration of 10 μg / mL, and the fluorescence of pHrodo Red SE in each well is measured over time using the Incucyte® SX5 Live-Cell Analysis System (Sartorius).

[0025] (Bacterial staining with pHrodo Red SE) Dissolve pHrodo Red SE (Invitrogen, P36600) in DMSO to prepare a 10.2 mM solution. Weigh the bacterial powder into a 2 mL Eppendorf tube and add 0.1 M sodium bicarbonate adjusted to pH 9.0 to prepare a bacterial solution containing 20 mg / mL of bacteria. Transfer 95 μL of the bacterial solution to a new 2 mL Eppendorf tube and add 5 μL of the 10.2 mM pHrodo Red SE prepared as described above. After dispersing the bacteria by vortexing, cover with aluminum foil to protect from light and incubate at room temperature for 60 minutes. Then, add 750 μL of PBS, vortex, and centrifuge at 20,000 × g for 2 minutes at room temperature. Discard 800 μL of the supernatant, add 1.5 mL of PBS, and vortex to completely suspend the precipitate. Centrifuge again at 20,000 × g for 2 minutes at room temperature, and discard 1.5 mL of the supernatant. Add 140 μL of PBS to the suspension to prepare a 10 mg / mL bacterial suspension of the stained bacteria. Store in a light-shielded aluminum foil at 4°C until use. Dilute the stained bacteria 10-fold with PBS immediately before use to prepare a 1 mg / mL bacterial suspension. The bacterial suspension of stained bacteria should be used within 24 hours of preparation.

[0026] (Evaluation of phagocytic intake) The entire field of view (0.572 mm) to be evaluated was obtained from each well of the plate. 2 The amount of bacterial phagocytosis by CAL-1 cells is quantified using the total area (fluorescence detection area) where fluorescence of pHrodo Red SE is detected as an indicator. Using the Incucyte® SX5 Live-Cell Analysis System (Sartorius), one image (0.572 mm) is used depending on the number of images to be evaluated. 2 ) fluorescence detection area per unit (e.g., μm) 2The amount of phagocytosis can be evaluated by analyzing the image ( / image). At this time, even if the stained bacteria have not been phagocytosed, they will still fluoresce slightly, so to eliminate the effect of fluorescence not caused by phagocytosis, the Threshold value of the Orange Channel is set to 5.0. In addition, depending on the type of bacteria, the bacterial cells may form aggregates, causing fluorescence to exceed the Threshold value even if they have not been phagocytosed, so the Area of ​​the Filters in the Orange Channel is set to 20 to eliminate the effect of excessively large aggregates.

[0027] Immunostimulating bacteria are not limited to bacteria that possess immunostimulatory ability, but may be bacteria that are particularly useful to the human body. Immunostimulating bacteria may be, for example, Gram-positive bacteria or Gram-negative bacteria, and preferably Gram-positive bacteria. Specifically, immunostimulating bacteria may be lactic acid bacteria or acetic acid bacteria.

[0028] Lactic acid bacteria are bacteria that produce lactic acid as a metabolite. Examples of lactic acid bacteria include those of the genera Oenococcus, Bifidobacterium, Weissella, Tetragenococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Lactobacillus, and Hendrickxia (formerly classified as Bacillus). The lactic acid bacteria listed above and below may or may not be included.

[0029] In this specification, the term "Lactobacillus" includes bacteria that were classified under the genus Lactobacillus before the reclassification of the genus. For example, with the reclassification of the Lactobacillus genus, new genera have been added: Acetilactobacillus, Agrilactobacillus, Amylolactobacillus, Apilactobacillus, Bombilactobacillus, Companilactobacillus, Dellaglioa, Fructilactobacillus, Furfurilactobacillus, Holzapfelia, Lacticaseibacillus, Lactiplantibacillus, and Lapidilactobacillus. This includes bacteria classified under genera such as Lapidilactobacillus, Latilactobacillus, Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolactobacillus, Paralactobacillus, Paucilactobacillus, Schleiferilactobacillus, and Secundilactobacillus.

[0030] The genus Oenococcus is not particularly limited, but examples include Oenococcus oeni. A specific example of an Oenococcus is Oenococcus oeni JCM6125, but the genus Oenococcus may or may not include the bacteria listed above.

[0031] Examples of Bifidobacterium species include, but are not limited to, Bifidobacterium animalis subsp. lactis, Bifidobacterium pseudolongum, and Bifidobacterium longum subsp. infantis. Specific examples of Bifidobacterium species include Bifidobacterium animalis subspecies lactis JCM10602, Bifidobacterium animalis subspecies lactis BB-12, Bifidobacterium longum subspecies infantis JCM1222, Bifidobacterium longum subspecies infantis M-63, and Bifidobacterium longum subspecies Examples include Bifidobacterium longum BB536, Bifidobacterium longum subspecies longum N61, Bifidobacterium bifidum OLB6378, Bifidobacterium breve M-16V, and Bifidobacterium breve MCC1274, Bifidobacterium pseudolongum JCM1205, etc., but the Bifidobacterium species may or may not include the bacteria listed above.

[0032] The genus Weissella is not particularly limited, but examples include Weissella paramesenteroides and Weissella viridescens. Specific examples of Weissella include Weissella paramesenteroides JCM9890 and Weissella viridescens JCM1174, but the definition of Weissella may or may not include the bacteria listed above.

[0033] The genus Tetragenococcus is not particularly limited, but examples include Tetragenococcus halophilus. Specific examples of Tetragenococcus include Tetragenococcus halophilus NRIC0098 and Tetragenococcus halophilus No. 1, but the genus Tetragenococcus may or may not include the bacteria listed above.

[0034] Examples of Lactococcus species include Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, and Lactococcus plantarum.

[0035] Specific examples of Lactococcus species include Lactococcus lactis subspecies lactis JCM5805, Lactococcus lactis subspecies lactis NBRC12007, Lactococcus lactis subspecies lactis NRIC1150, Lactococcus lactis subspecies lactis JCM20101, Lactococcus lactis subspecies lactis JCM7638, Lactococcus lactis subspecies lactis ATCC7963, Lactococcus lactis subspecies lactis ATCC7962, Lactococcus lactis subspecies lactis ATCC29146, Lactococcus lactis subspecies lactis ATCC27861, and Lactococcus lactis subspecies lactis ATCC19435, Lactococcus lactis subspecies lactis ATCC15346, Lactococcus lactis subspecies lactis ATCC13675, Lactococcus lactis subspecies lactis ATCC12929, Lactococcus lactis subspecies lactis ATCC11955, Lactococcus lactis subspecies lactis ATCC11454, Lactococcus lactis subspecies lactis Examples include ATCC11007, Lactococcus garvieae NBRC100934, Lactococcus lactis subspecies cremoris JCM16167, Lactococcus lactis subspecies cremoris NBRC100676, Lactococcus lactis subspecies heldniae JCM1180, Lactococcus lactis subspecies heldniae JCM11040, and Lactococcus plantarum JCM11056, but the Lactococcus species may or may not include the bacteria listed above.

[0036] The genus Leuconostoc is not particularly limited, but examples include Leuconostoc carnosum and Leuconostoc lactis. Specific examples of Leuconostoc include Leuconostoc carnosum JCM9695 and Leuconostoc lactis NBRC12455, but the genus Leuconostoc may or may not include the bacteria listed above.

[0037] Examples of species belonging to the genus Pediococcus include, but are not limited to, 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 JCM8797, Pediococcus acidilactici K15, and Pediococcus damnosus JCM5886, but the term Pediococcus species may or may not include the bacteria listed above.

[0038] The genus Streptococcus is not particularly limited, but examples include Streptococcus thermophilus. Specific examples of Streptococcus species include Streptococcus thermophilus SBC8781, but the genus Streptococcus may or may not include the bacteria listed above.

[0039] Enterococcus species are not particularly limited, but examples include Enterococcus alcedinis and Enterococcus faecalis. Specific examples of Enterococcus species include Enterococcus faecalis EC-12, Enterococcus faecalis JCM5803T, Enterococcus faecalis JCM20307, ​​Enterococcus faecium JCM5804T, and Enterococcus faecium JCM8903, but the Enterococcus species may or may not include the bacteria listed above.

[0040] While not particularly limited to Lactobacillus species, examples include Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus rhamnosus, Lactobacillus gasseri, and Lactobacillus bulgaricus. Examples include Lactobacillus bulgaricus, Lactobacillus parakefiri, Lactobacillus plantarum, and Lactobacillus pentosus.

[0041] Specific examples of Lactobacillus species include Lactobacillus paracasei KW3110, Lactobacillus paracasei MCC1849, 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 subspecies casei 327, and Lactobacillus (in the new classification, Lactobacillus lactica). Examples include Lactobacillus casei Shirota, Lactobacillus bulgaricus OLL1073R-1, Lactobacillus bulgaricus 2038, Lactobacillus parakephyri (Lentilactobacillus parakephyri in the new classification) JCM8573, Lactobacillus plantarum (Lactipruntilabacillus plantarum in the new classification) L-137, and Lactobacillus pentosus (Lactipruntilabacillus pentosus in the new classification) ONRICb0240, but the genus Lactobacillus may or may not include the bacteria listed above.

[0042] The genus Hendrickxia is not particularly limited, but examples include Hendrickxia coagulans (also known as Bacillus coagulans). Specific examples of Hendrickxia include strains SANK70258 and BC99 of Hendrickxia coagulans, but the definition of Hendrickxia may or may not include the bacteria listed above.

[0043] The acetic acid bacteria are not particularly limited, but examples include bacteria of the genus Gluconacetobacter, Acetobacter, and Gluconobacter, preferably Gluconacetobacter, more preferably Gluconacetobacter hanzenii, and even more preferably Gluconacetobacter hanzenii GK-1. The acetic acid bacteria may or may not include the lactic acid bacteria listed above and below.

[0044] In addition to the above, immunostimulatory bacteria may include bacteria of the genera Akkermansia, Bacteroides, Blautia, Clostridium, Collinsella, Faecalibacterium, Faecalicatena, Lacrimispora, Paeniclostridium, Parabacteroides, or Roseburia. Immunostimulatory bacteria may or may not include these bacteria.

[0045] A specific example of a fungus belonging to the genus Akkermansia is Akkermansia muciniphila JCM30893, but the genus Akkermansia may or may not include the bacteria listed above.

[0046] Specific examples of Bacteroides species include, for example, Bacteroides caccae JCM9498T, Bacteroides fragilis JCM11019T, Bacteroides fragilis JCM11017, Bacteroides fragilis JCM17586, Bacteroides fragilis JCM17587, Bacteroides ovatus JCM5824T, Bacteroides setaiotaomicron ATCC29148T, Bacteroides setaiotaomicron ATCC29741, Bacteroides setaiotaomicron ATCC12290, Bacteroides uniformis JCM5828T, Bacteroides uniformis JCM13286, and Bacteroides uniformis Examples include JCM13287 and Bacteroides uniformis JCM13288, but the genus Bacteroides may or may not include the bacteria listed above.

[0047] Specific examples of Blautia species include Blautia acetategigens JCM34803T, Blautia ammonialitica JCM34802T, Blautia algi JCM31394T, Blautia caecimlis JCM34498T, Blautia coccoides JCM1395T, Blautia fexis JCM17205T, Blautia glucellacea JCM17039T, Blautia hansenii JCM14655, Blautia hansenii JCM35484, Blautia hominis JCM32276T, Blautia hydrogenotropica JCM31266, Blautia liqualis JCM34225T, Blautia luti JCM17040T, and Blautia obeum. Examples include JCM31340, Brautia producta JCM1471T, Brautia pseudococcoides JCM35243T, Brautia cinckii JCM14657T, Brautia wexlerae JCM31267, and Brautia wexlerae JCM35486, but the genus Brautia may or may not include the bacteria listed above.

[0048] Specific examples of Clostridium species include Clostridium butyricum JCM NT, Clostridium nexile JCM31500T, and Clostridium cymbiosum JCM1297T. ​​However, the term Clostridium may or may not include the bacteria listed above.

[0049] Examples of bacteria belonging to the genus Collinsella include Collinsella aerofasciens JCM10188T, Collinsella intestinalis JCM10643T, Collinsella stercolis JCM10641T, and Collinsella tanakaei JCM16071T. However, the term Collinsella may or may not include the bacteria listed above.

[0050] Specific examples of bacteria belonging to the genus Faecalibacterium include, for example, Faecalibacterium hattleyi JCM39210, Faecalibacterium longum JCM39208, Faecalibacterium prausnizzi JCM31915, Faecalibacterium prausnizzi JCM39207, and Faecalibacterium prausnizzi JCM39209. However, the genus Faecalibacterium may or may not include the bacteria listed above.

[0051] A specific example of a bacterium belonging to the genus Faecalicatena is Faecalicatena oroticum JCM1429T, but the genus Faecalicatena may or may not include the bacteria listed above.

[0052] Specific examples of bacteria belonging to the genus Lacrimispora include, for example, Lacrimispora celerecrescent JCM15734T, Lacrimispora sphenoides JCM1415T, and Lacrimispora xylanoritica JCM15735T. However, the genus Lacrimispora may or may not include the bacteria listed above.

[0053] A specific example of a Paeniclostridium species is Paeniclostridium sorderii JCM3814T, but the Paeniclostridium species may or may not include the bacteria listed above.

[0054] An example of a bacterium belonging to the genus Parabacteroides is Parabacteroides meldae JCM9497T, but the term Parabacteroides may or may not include the bacteria listed above.

[0055] Specific examples of bacteria belonging to the genus Roseburia include, for example, Roseburia hominis JCM17582, Roseburia intestinalis JCM17583, and Roseburia inulinovorans JCM17584. However, the genus Roseburia may or may not include the bacteria listed above.

[0056] In one preferred embodiment, the immunostimulatory bacteria may be bacteria belonging to any one of the genera Lactococcus, Lactobacillus, Bifidobacterium, Pediococcus, Hendricksia, or Leuconostoc. Among these, the immunostimulatory bacteria are particularly preferably Lactococcus species.

[0057] The bacterial strains listed above can be obtained from public depositary institutions, etc. For example, the JCM strain can be obtained from the Microbial Materials Development Laboratory, BioResource Center, RIKEN (3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture), the NBRC strain from the Biological Genetics Division, National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu, Chiba Prefecture), the NRIC strain from the Strain Preservation Room, Tokyo University of Agriculture (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo), and the ATCC strain from the American Type Culture Collection (USA).

[0058] Furthermore, Lactobacillus paracasei KW3110 is deposited as FERM BP-08634 at the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (AIST) (1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566, Central No. 6) (currently, the Patent Organism Depository Center of the Biotechnology Center of the National Institute of Technology and Evaluation (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818, Japan)) as the international depositary authority under the Budapest Convention for the Deposit of Patent Microorganisms (Deposit date: February 20, 2004). In addition, a derivative strain of Lactobacillus paracasei KW3110 is deposited at the same Patent Organism Depository Center as FERM BP-08635 (Deposit date: February 20, 2004).

[0059] As mentioned above, Lactococcus lactis subspecies lactis JCM5805 can be obtained from the Microbial Materials Development Laboratory at the RIKEN BioResource Center. However, in this invention, the same strain of Lactococcus lactis subspecies lactis JCM5805 stored at institutions other than the RIKEN BioResource Center Microbial Materials Development Laboratory can be used. Specifically, the same strain of Lactococcus lactis subspecies lactis JCM5805 can be obtained from the National Institute of Technology and Evaluation (NITE) Biological Genetics Division (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture), Tokyo University of Agriculture Strain Preservation Room (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo), and the American Type Culture Collection (USA), among others. Lactococcus lactis subspecies lactis JCM5805 is deposited in the American type culture collection as Lactococcus lactis subspecies lactis ATCC9936 and Lactococcus lactis subspecies lactis ATCC19435.

[0060] The immunostimulatory bacterium may be a live bacterium or a dead bacterium, and in one aspect, it may be a dead bacterium. When the immunostimulatory bacterium is a dead bacterium, the immunostimulatory bacterium is obtained by, for example, sterilizing live bacteria by heat treatment, pressure treatment, high-pressure steam treatment, electromagnetic wave treatment, electron beam treatment, radiation treatment, ultraviolet treatment, alcohol treatment, or electrolyzed water treatment, etc., and then drying by freeze-drying, spray drying, drum drying, hot air drying, or vacuum drying, etc. as necessary.

[0061] <Pos-lectin-binding bacterium> In one aspect, the immunostimulatory bacterium may be a bacterium that binds to a predetermined lectin. When the immunostimulatory bacterium is a bacterium that binds to a predetermined lectin, the immunostimulatory ability in the bacterium is likely to be enhanced. Hereinafter, a lectin whose binding ability shows a positive correlation with the immunostimulatory ability of the bacterium is also referred to as "Pos-lectin". Further, hereinafter, a bacterium that binds to Pos-lectin is also referred to as "Pos-lectin-binding bacterium".

[0062] In this aspect, the Pos-lectin may be, for example, at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, and may be a combination of at least two, three, four, five or more arbitrary lectins selected from this group. The inventors have found that there is a positive correlation between the binding ability to these lectins and the promoting activity of IFN-α production by pDC in bacteria. Therefore, when the Pos-lectin is as described above, the immunostimulatory ability in the Pos-lectin-binding bacterium is likely to be enhanced.

[0063] In a preferred embodiment, the Pos-lectin may be at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB, or a combination of at least two, three, four, five, or more lectins selected from this group. The inventors have found a significant positive correlation in bacteria between the binding affinity to these lectins and the activity of promoting IFN-α production by pDCs. Therefore, when the Pos-lectin is as described above, the immunostimulatory activity in Pos-lectin-binding bacteria tends to be higher.

[0064] In a more preferred embodiment, the Pos-lectin may be at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, and LcH, or a combination of at least two, three, four, five, or more lectins selected from this group. The inventors have found a significant positive correlation in bacteria between the binding affinity to these lectins and the activity of promoting IFN-α production by pDCs. Therefore, when the Pos-lectin is as described above, the immunostimulatory activity in Pos-lectin-binding bacteria tends to be even higher.

[0065] It is known that certain types of bacteria can promote cytokine production by dendritic cells by being phagocytosed by them. In fact, according to evaluations conducted by the inventors in the present invention, bacteria that are more easily phagocytosed by pDCs tend to have a higher ability to promote IFN-α production. Therefore, although we do not wish to be bound by any theory, it is thought that Pos-lectin-binding bacteria can promote IFN-α production by pDCs by binding to proteins (e.g., lectins or lectin-like receptors) that recognize the same or similar sugar chain structure as Pos-lectin expressed on the surface of pDCs, and then being phagocytosed by pDCs.

[0066] For example, among the Pos-lectins that showed a particularly significant positive correlation, PNA, MPL, and PHA-E are lectins that recognize glycan structures with galactose at their termini. Therefore, it is thought that bacteria that can bind to such Pos-lectins can promote IFN-α production by pDCs by binding to lectins or lectin-like receptors (e.g., BDCA2 (Blood Dendritic Cell Antigen 2)) that recognize glycan structures with galactose at their termini, expressed on the surface of pDCs, and then being phagocytosed by the pDCs. Thus, in another, more preferred embodiment, the Pos-lectin may be at least one lectin selected from the group consisting of PNA, MPL, and PHA-E.

[0067] For example, among the Pos-lectins that showed a particularly significant positive correlation, HHA, ASA, NPA, GNA, and LcH are lectins that recognize glycan structures with mannose at their terminus. Therefore, it is thought that bacteria that can bind to such Pos-lectins can promote IFN-α production by pDCs by binding to lectins or lectin-like receptors (e.g., DC-SIGN, Mincle, Dectin-2) that recognize glycan structures with mannose at their terminus and are expressed on the surface of pDCs, and then being phagocytosed by the pDCs. Thus, in another, more preferred embodiment, the Pos-lectin may be at least one lectin selected from the group consisting of HHA, ASA, NPA, GNA, and LcH.

[0068] For example, among the Pos-lectins that showed a particularly significant positive correlation, RS-Fuc is a lectin that recognizes a glycan structure with fucose at its terminus. Therefore, it is thought that bacteria that can bind to such Pos-lectins can promote IFN-α production by pDCs by binding to lectins or lectin-like receptors (e.g., DC-SIGN) that recognize a glycan structure with fucose at its terminus, expressed on the surface of pDCs, and being phagocytosed by the pDCs. Thus, in another, more preferred embodiment, the Pos-lectin may be RS-Fuc.

[0069] The binding of bacteria to Pos-lectin can be evaluated, for example, based on the presence, percentage, and amount of bacteria captured on a plate or carrier after contact with the plate or carrier on which Pos-lectin is supported, or the presence, percentage, and amount of complexes formed after contact between Pos-lectin and bacteria. In this case, the amount of bacteria and the amount of Pos-lectin are set under conditions where, when one amount is fixed, the percentage and amount of bacterial-lectin complexes formed increase or decrease linearly relative to the other amount. The presence, percentage, and amount of bacteria captured on a plate or carrier after contact with the plate or carrier on which Pos-lectin is supported can be evaluated, for example, by measuring the fluorescence intensity on the plate or carrier using a microwell plate reader or flow cytometer if the bacteria have been fluorescently labeled beforehand, or by using the luminescence intensity in the ELISA method as an indicator. The presence, proportion, and amount of complexes formed after contact between Pos-lectin and bacteria can be evaluated using, for example, the number of particles in which the fluorescence of both is detected simultaneously, measured by a flow cytometer, if both are pre-labeled with fluorescence. Alternatively, if both are pre-labeled with donor and acceptor beads or molecules that induce energy transfer, the intensity of the luminescence or fluorescence originating from the acceptor, generated by the excitation of the donor, can be used as an indicator. Hereafter, such values ​​that can be used as indicators of the binding affinity between bacteria and Pos-lectin will also be referred to as "binding indicator signal values."

[0070] In this disclosure, for example, when evaluated by the evaluation method described above, bacteria whose binding indicator signal value satisfies at least one of the following (p) to (r) can be evaluated as bacteria that bind to a certain Pos-lectin. In one embodiment, bacteria whose binding indicator signal value satisfies (p) may be evaluated as bacteria that bind to a certain Pos-lectin. Hereafter, the bacteria to be evaluated will also be referred to as "bacteria of interest". (p) The binding index signal value obtained by measurement using the target bacteria and Pos-lectin is at or above a predetermined ratio compared to the binding index signal value obtained by measurement using Pos-lectin with control bacteria (negative control bacteria) that have low immunostimulatory activity. (q) The binding index signal value obtained by measurement using the target bacterium and Pos-lectin is at least a predetermined multiplier compared to the binding index signal value measured using a pair of molecules known to bind to each other (binding pair) instead of the target bacterium and Pos-lectin. (r) The binding index signal value obtained by measurement using the target bacterium and Pos-lectin is at or above a predetermined ratio compared to the binding index signal value measured in the absence of Pos-lectin and the target bacterium.

[0071] In one embodiment of (p) above, the negative control bacteria may be, for example, bacteria whose IFN-α production is measured by the standard method for measuring immunostimulatory activity described above at 100 pg / mL or less, 30 pg / mL or less, 10 pg / mL or less, or 5 pg / mL or less, and as an example, bacteria whose IFN-α production is measured by the standard method for measuring immunostimulatory activity described above at 10 pg / mL or less. In one embodiment of (p) above, the negative control bacteria may be, for example, Bifidobacterium reuteri JCM17295, Bifidobacterium pseudorhongum JCM1205, or Lactococcus lactis subspecies lactis ATCC15577. In a preferred embodiment of (p) above, the negative control bacteria may be Bifidobacterium pseudorhongum JCM1205.

[0072] The predetermined magnification in one embodiment of (p) above may be, for example, 1.01x, 1.05x, 1.10x, 1.15x, 1.20x, 1.25x, 1.30x, 1.35x, 1.40x, 1.45x, 1.50x, 1.55x, 1.60x, 1.65x, 1.70x, 1.75x, 1.80x, 1.85x, 1.90x, 1.95x, 2.00x, 2.20x, 2.40x, 2.60x, 2.80x, 3.00x, 3.50x, 4.00x, 4.50x, or 5.00x. These magnifications are appropriately set according to the type of negative control bacteria and Pos-lectin. In one preferred embodiment of (p) above, the predetermined magnification may be 1.05x.

[0073] In one embodiment of (q) above, the binding pair may be a pair of molecules known to bind to each other, for example, a pair of a protein and a molecule or antibody that binds to it, or a pair of a primary antibody and a secondary antibody, and in one embodiment, it may be an affinity tag protein and its ligand. Examples of binding pairs include a pair of biotin and streptavidin, a pair of His tag and a His tag protein, and a pair of FLAG tag and a FLAG tag protein.

[0074] In one preferred embodiment of (q) above, the binding index signal value may be the binding index signal value obtained according to the standard lectin array protocol described below, and the binding index signal value measured using binding pairs (i.e., the binding index signal value that serves as a positive control) may be the binding index signal value obtained as POS1 according to the kit used. The above POS1 in the standard lectin array protocol is the result obtained when, as a positive control, Cy3-Streptavidin is added instead of bacteria to wells on which biotin-labeled IgG is immobilized instead of lectin at different concentrations.

[0075] The predetermined magnification in one aspect of (q) above may be, for example, 0.0001x, 0.0003x, 0.0005x, 0.00075x, 0.001x, 0.0015x, 0.002x, 0.0025x, 0.003x, 0.004x, 0.005x, 0.006x, 0.007x, 0.008x, 0.009x, 0.01x, 0.03x, 0.1x, or 0.3x. These magnifications are set appropriately according to the type of binding pair and Pos-lectin.

[0076] The predetermined magnification in one aspect of (r) above may be, for example, 2x, 4x, 8x, 16x, 32x, 64x, 128x, 256x, 512x, or 1024x. These magnifications are set appropriately according to the type of Pos-lectin.

[0077] In one preferred embodiment of (p) to (r) above, the binding indicator signal value may be, for example, the fluorescence intensity in the plate or carrier measured by a microwell plate reader or flow cytometer after the bacteria have been fluorescently labeled in advance and the bacteria have been brought into contact with the plate or carrier on which the lectin is supported. For example, in one preferred embodiment of (p) to (r) above, the binding indicator signal value may be the signal value obtained by the protocol shown below (standard lectin array protocol).

[0078] [Standard Lectin Array Protocol] (Array and labeling kit) The array used is the RayBiotech Lectin Array 70 (RayBiotech, Inc., cat.GA-Lectin-70). The labeling solvent used is 1× Labeling Reagent. This is prepared by adding 100 μL of 1× PBS (pH 8.0) to a Labeling Reagent tube. Cy3 equivalent dye-conjugated streptavidin is used. This is prepared by adding 1400 μL of Sample Diluent (reagent included in the kit) to a streptavidin tube. (Sample preparation) 1. Suspend the bacteria in 500 μL of PBS to a concentration of 1 mg / mL, and then expose the suspension to ultrasound (sonication) in a 1.5 mL Eppendorf tube. 200 μL of the sonicated mixture obtained in 2.1 is taken and dialysis is performed in 1×PBS (pH 8.0) at 4°C for 20 hours. The regenerated cellulose membrane provided by the Dialysis Vials included in the kit is used as the dialysis membrane. Add 100 μL of 1×Labeling Reagent to approximately 200 μL of the post-dialysis solution obtained in 3.2, incubate at room temperature for 60 minutes, and then add 3 μL of Stop Solution (reagent included in the kit). The solution labeled with 4.3 is dialyzed in 1×PBS (pH 8.0) at 4°C for 20 hours. The dialysis solution obtained in 5.4 is centrifuged at 4°C and 1000 rpm for 5 minutes, and the supernatant is collected. This supernatant is used as a labeled sample for the subsequent hybridization. (Hybridization) 1. Apply 100 μL of sample diluent to the array, block it by incubating at room temperature for 30 minutes, and then remove the sample diluent by decanting. 2. Next, apply 150 μL of the solution, which is obtained by adding 50 μL of Sample Diluent to 100 μL of the labeled sample prepared above, to the array. After application, incubate the array at 4°C for 17 hours. 3. Clean the array according to the protocol provided with the kit. Apply 4.80 μL of Cy3 equivalent dye-conjugated streptavidin to the washed array and incubate at room temperature for 1 hour. 5. Clean the array according to the protocol provided with the kit. 6. Centrifuge the washed array at room temperature at 1,000 rpm for 3 minutes. (Array scanning and data analysis) 1. Scan the cleaned array using an array scanner (e.g., GenePix® 4100A (Molecular Devices, LLC)). 2. Using analysis software (e.g., GenePix Pro 7 Software (Molecular Devices, LLC)), the fluorescence intensity value at each spot is quantified from the obtained image data, and the signal value is calculated.

[0079] As described above, the Pos-lectin-binding bacteria according to one preferred embodiment may be bacteria whose signal value obtained by the standard lectin array protocol is 1.05 times or more than the signal value similarly measured for Bifidobacterium pseudorhondronum JCM1205.

[0080] Furthermore, as explained above, the inventors have found that in bacteria, the greater the binding affinity to Pos-lectin, the more likely the immunostimulatory ability is to be. Therefore, another embodiment of the present disclosure may be a method for evaluating the immunostimulatory ability of bacteria, which includes evaluating the binding affinity to Pos-lectin in bacteria. Yet another embodiment of the present disclosure may be a method for screening bacteria having immunostimulatory ability, which includes evaluating the binding affinity to Pos-lectin in bacteria. In these methods, the method for evaluating the binding affinity to Pos-lectin is as described above, and in these methods, bacteria that are determined to be Pos-lectin-binding bacteria in the above evaluation method can be determined to be bacteria with high immunostimulatory ability or bacteria having immunostimulatory ability, and bacteria with a greater binding affinity to Pos-lectin can be evaluated as having higher immunostimulatory ability.

[0081] In addition, in the above-mentioned "method for evaluating the immunostimulatory ability of bacteria" and "method for screening bacteria having immunostimulatory ability", as a preferred embodiment, Pos-lectin may be at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB, and may be a combination of at least two, three, four, five or more arbitrary lectins selected from this group. As a more preferred embodiment, Pos-lectin may be at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, and LcH, and may be a combination of at least two, three, four, five or more arbitrary lectins selected from this group.

[0082] <Bacteria non-binding to Neg-lectin> In one embodiment, the immunostimulatory bacteria may be bacteria that do not bind to a predetermined lectin. When the immunostimulatory bacteria are bacteria that do not bind to a predetermined lectin, the immunostimulatory ability in the bacteria is likely to be high. Hereinafter, a lectin whose binding ability shows a negative correlation with the immunostimulatory ability of bacteria is also referred to as "Neg-lectin". Further, hereinafter, bacteria that do not bind to Neg-lectin are also referred to as "bacteria non-binding to Neg-lectin".

[0083] In this embodiment, the Neg-lectin may be at least one lectin selected from the group consisting of, for example, F17AG, Gal1-S, VVA, AAA, UEA-I, EEL, LSL-N, MOA, SJA, Gal3, LEA, CGL2, Gal7-S, VFA, ACL, Orysata, Gal1, and PA-IL, or it may be any combination of at least two, three, four, five, or more lectins selected from this group. The inventors have found that in bacteria, there is a negative correlation between the binding affinity to these lectins and the activity of promoting IFN-α production by pDCs. Therefore, when the Neg-lectin is as described above, the immunostimulatory activity in Neg-lectin-unbound bacteria tends to be increased.

[0084] In a preferred embodiment, the Neg-lectin may be PA-IL. The inventors have found a relatively strong negative correlation between the binding affinity to PA-IL and the activity of promoting IFN-α production by pDCs in bacteria. Therefore, when the Neg-lectin is PA-IL, the immunostimulatory activity in Neg-lectin-unbound bacteria tends to be higher.

[0085] The non-binding of bacteria to Neg-lectin can be evaluated, for example, based on the presence, proportion, and amount of bacteria captured on a plate or carrier after contact with a plate or carrier carrying Neg-lectin, or the presence, proportion, and amount of complexes formed after contact between Neg-lectin and bacteria. In this case, the amount of bacteria and the amount of Neg-lectin are set under conditions where, when one amount is fixed, the proportion and amount of complexes increase or decrease linearly relative to the other amount. These can be evaluated using the "binding indicator signal value," similar to the explanation for Pos-lectin-binding bacteria.

[0086] In this disclosure, for example, when evaluated by the evaluation method described above, bacteria whose binding indicator signal value satisfies at least one of the following (s) to (u) can be evaluated as bacteria that do not bind to a certain Neg-lectin. In one embodiment, bacteria whose binding indicator signal value satisfies (s) can be evaluated as bacteria that do not bind to a certain Neg-lectin. (s) The binding index signal value obtained by measurement using the target bacteria and Neg-lectin is less than or equal to a predetermined ratio compared to the binding index signal value obtained by measurement using the negative control bacteria and Neg-lectin. (t) The binding index signal value obtained by measurement using the target bacterium and Neg-lectin is less than or equal to a predetermined multiplier of the binding index signal value measured using a binding pair instead of the target bacterium and Neg-lectin. (u) The binding index signal value obtained by measurement using the target bacterium and Neg-lectin is less than or equal to a predetermined ratio compared to the binding index signal value measured in the absence of Neg-lectin and the target bacterium.

[0087] The negative control bacteria in one aspect of (s) above are the same as those described in (p) relating to Pos-lectin-binding bacteria. The predetermined magnification in one aspect of (s) above may be, for example, 0.99x, 0.95x, 0.90x, 0.85x, 0.80x, 0.75x, 0.70x, 0.65x, 0.60x, 0.55x, 0.50x, 0.45x, 0.40x, 0.35x, 0.30x, 0.25x, 0.20x, 0.15x, 0.10x, or 0.05x. These magnifications are set appropriately according to the negative control bacteria and the type of Neg-lectin. In a preferred aspect of (s) above, the predetermined magnification may be 0.80x.

[0088] The binding pair in one aspect of (t) above is the same as that described in (q) relating to Pos-lectin-binding bacteria. The predetermined magnification in one aspect of (t) above may be, for example, 0.3x, 0.1x, 0.03x, 0.01x, 0.003x, 0.001x, 0.0003x, 0.0001x, or 0.00003x. These magnifications are set appropriately according to the binding pair and the type of Neg-lectin.

[0089] The predetermined magnification in one aspect of (u) above may be, for example, 1024x, 512x, 256x, 128x, 64x, 32x, 16x, 8x, 4x, or 2x. These magnifications are set appropriately according to the type of Neg-lectin.

[0090] In one preferred embodiment of (s) to (u) above, the binding indicator signal value may be, for example, the fluorescence intensity in the plate or carrier measured by a microwell plate reader or flow cytometer after the bacteria have been fluorescently labeled in advance and the bacteria have been brought into contact with the plate or carrier on which the lectin is supported. For example, in one preferred embodiment of (s) to (u) above, the binding indicator signal value may be the signal value obtained by the standard lectin array protocol described above.

[0091] Also, as described above, the inventors have found that in bacteria, the lower the binding ability to Neg-lectin, the higher the immunostimulatory ability tends to be. Therefore, another embodiment of the present disclosure may be a method for evaluating the immunostimulatory ability of bacteria, which includes evaluating the binding ability of the bacteria to Neg-lectin. Further, still another embodiment of the present disclosure may be a method for screening bacteria having immunostimulatory ability, which includes evaluating the binding ability of the bacteria to Neg-lectin. In these methods, the method for evaluating the binding ability to Neg-lectin is as described above. Also, in these methods, bacteria determined to correspond to Neg-lectin non-binding bacteria in the above-described evaluation method can be determined to be bacteria with high immunostimulatory ability or bacteria having immunostimulatory ability, and it can be evaluated that bacteria with a lower binding ability to Neg-lectin have higher immunostimulatory ability.

[0092] In addition, in the above-mentioned "method for evaluating the immunostimulatory ability of bacteria" and "method for screening bacteria having immunostimulatory ability", as a preferred aspect, Neg-lectin may be at least one lectin selected from the group consisting of F17AG, Gal1-S, VVA, AAA, UEA-I, EEL, LSL-N, MOA, SJA, Gal3, LEA, CGL2, Gal7-S, VFA, ACL, Orysata, Gal1, and PA-IL, and may be at least two, three, four, five or more lectins selected from this group. As a more preferred aspect, Neg-lectin may be PA-IL.

[0093] <Evaluation of being Pos-lectin binding bacteria and Neg-lectin non-binding bacteria> In one embodiment, whether a bacterium is a Pos-lectin binding bacterium and a Neg-lectin non-binding bacterium can be evaluated based on the ratio of its binding affinity to Pos-lectin to its binding affinity to Neg-lectin. For example, if this ratio is greater than or equal to a predetermined ratio, the bacterium can be evaluated as a Pos-lectin binding bacterium and a Neg-lectin non-binding bacterium. Here, the binding affinity of the bacterium to lectin may, in one embodiment, be a binding index signal value obtained from one of the binding affinity evaluation methods described above, and in a preferred embodiment, it may be a binding index signal value measured by the standard lectin array protocol described above.

[0094] In one embodiment, bacteria whose binding affinity to Pos-lectin is greater than or equal to a predetermined ratio to their binding affinity to PA-IL may be evaluated as Pos-lectin-binding bacteria and Neg-lectin-non-binding bacteria. More specifically, regarding the binding affinity to Pos-lectin relative to its binding affinity to PA-IL, bacteria whose combination of "type of Pos-lectin; ratio of binding indicator signals" satisfies at least one of the following conditions can be evaluated as Pos-lectin-binding bacteria and Neg-lectin-non-binding bacteria: PNA; 2.70x or more, MPL; 0.90 times or more, PHA-E; 3.20 times or more HHA; 1.2 times or more ASA; 2.0 times or more, NPA; 0.80 times or more, UEA-II; 0.60 times or more GNA; 3.30 times or more RS-Fuc;2.30 times or more, PHA-P;1.50 times or more, BC2L-A;1.20 times or more, AAL; 2.0 times or more, LcH; 1.0 times or more, PSA; 1.50 times or more, PHA-L; 1.30 times or more, STL; 2.0 times or more, GRFT; 3.60 times or more, SAMB; 0.80 times or higher.

[0095] In a preferred embodiment, the immunostimulatory bacterium may be a bacterium that satisfies any one of the following, and may also be a bacterium that satisfies any one of (a) to (c) below and satisfies the requirements of the above-mentioned Pos-lectin-binding bacterium and / or Neg-lectin non-binding bacterium: (a) The ratio of the binding force with PNA to the binding force with PA-IL is 2.70 times or more; (b) The ratio of the binding force with MPL to the binding force with PA-IL is 0.90 times or more; (c) The ratio of the binding force with PHA-E to the binding force with PA-IL is 3.20 times or more.

[0096] Here, the binding force between the immunostimulatory bacterium and PA-IL, and the binding force between the immunostimulatory bacterium and each Pos-lectin can be measured or evaluated by, for example, the following method. After contacting 1 mg / mL of the biotin-labeled bacterium with a carrier carrying a certain amount of lectin, the bacterium bound to the lectin is fluorescently labeled, and the binding force with the lectin is calculated by measuring the fluorescence intensity on the carrier. For the fluorescent label, for example, a fluorescent dye equivalent to Cy3 may be used, and the lectin may be one type or multiple types. For measuring the fluorescence intensity, an optical device such as a microplate reader, a flow cytometer or an array scanner can be used.

[0097] As an example, after suspending and ultrasonicating the bacterium in PBS, dialysis and biotin labeling are performed, and the supernatant obtained by centrifugation after dialysis again is used as a labeled sample. The labeled sample is hybridized on a lectin array, washed, fluorescently labeled streptavidin is added and reacted, the fluorescence intensity is measured with an array scanner after re-washing, and the signal value for each spot is calculated by analysis software. At this time, when either the amount of the bacterium or the amount of PA-IL and Pos-lectin is fixed, the ratio and amount of the complex formed between the bacterium and the lectin increase or decrease linearly with respect to the amount of the other. The conditions are set.

[0098] <Bacterium containing Pos-sugar chain structure> Immunostimulating bacteria may, in one embodiment, be bacteria that have a predetermined sugar chain structure on their cell surface. When immunostimulating bacteria are bacteria that have a predetermined sugar chain structure on their cell surface, their immunostimulatory ability tends to be enhanced. Hereafter, such a sugar chain structure that, when present on the cell surface, tends to enhance the immunostimulatory ability of bacteria will also be referred to as the "Pos-sugar chain structure." Furthermore, hereafter, bacteria that have a Pos-sugar chain structure on their cell surface will also be referred to as "Pos-sugar chain structure-containing bacteria."

[0099] In this embodiment, the Pos-glycan structure may be at least one glycan structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc, or at least two, three, four, five, or more glycan structures selected from this group. The glycan structures included in the above group are glycans recognized by Pos-lectins in bacteria, where there is a significant positive correlation between binding affinity and activity in promoting IFN-α production by pDCs. Therefore, although we do not wish to be bound by any theory, if the Pos-glycan structure is as described above, it is thought that it may promote IFN-α production by pDCs by binding to a protein (e.g., lectin or lectin-like receptor) that recognizes the same glycan structure as the Pos-lectin recognition structure expressed on the surface of pDCs, and then being phagocytosed by the pDC.

[0100] For example, among the Pos-glycan structures, Galβ3GalNAc is a glycan structure that has galactose at its terminus and is recognized by the Pos-lectins MPL and PNA. Also, among the Pos-glycan structures, Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 are glycan structures that have galactose at their terminus and are recognized by the Pos-lectin PHA-E. Here, PNA, MPL, and PHA-E are Pos-lectins in bacteria that show a significant positive correlation between their binding affinity and their activity in promoting IFN-α production by pDCs. Therefore, it is thought that bacteria having such Pos-glycan structures on their cell surface can promote IFN-α production by pDCs by binding to lectins or lectin-like receptors (e.g., BDCA2) that recognize glycan structures with galactose at their terminus, expressed on the surface of pDCs, and being phagocytosed by pDCs. Thus, in one preferred embodiment, the Pos-glycan structure may be at least one glycan structure selected from the group consisting of Galβ3GalNAc and Galβ4GlcNAcβ2Manα6(GlcNAcβ4), and (GlcNAcβ4Manα3)Manβ4. Furthermore, the immunostimulatory bacteria may be, for example, bacteria that bind to BDCA2, bacteria that bind to BDCA2 via the Pos-glycan structure, or bacteria that bind to BDCA2 via the Pos-glycan structure and are phagocytosed by pDCs.

[0101] In this disclosure, if a bacterium has a certain glycan structure on its cell surface, it means that the glycan structure is present on the bacterial surface (e.g., the surface of the cell wall) in a manner that it can be in contact with other proteins or cells. The glycan structure may be located at the end or within the glycan, but in one embodiment, a bacterium having a certain glycan structure on its cell surface may have that glycan structure at the end of a glycan forming the cell wall. In this embodiment, if a glycan structure is located at the end of a glycan, it means that any end (terminal sugar) of the glycan structure is located at the end of the glycan. Also in this embodiment, for example, in a Pos-glycan structure, the sugar listed first in its notation (e.g., galactose in Galβ3GalNAc) may be located at the end of a glycan forming the cell wall.

[0102] The presence of a certain sugar chain structure on the cell surface of a bacterium can be evaluated, for example, by a method similar to the method for evaluating bacterial-lectin binding described above for Pos-lectin-binding bacteria. Specifically, based on known information about the sugar chain structures recognized by each lectin, a bacterium evaluated to bind to a certain lectin can be evaluated as a bacterium that possesses the sugar chain structure recognized by that lectin on its cell surface. That is, for example, a bacterium that possesses the sugar chain structure recognized by a certain lectin on its cell surface may be a bacterium in which the binding index signal value measured according to the method for evaluating bacterial-lectin binding described above in a test using the lectin and the bacterium satisfies at least one of (p) to (r) above, and in one embodiment, the binding index signal value may be a bacterium that satisfies (p), and in a preferred embodiment, the signal value obtained by the standard lectin array protocol may be 1.05 times or more than the signal value similarly measured for Bifidobacterium pseudorhondronum JCM1205.

[0103] In addition to the above, the presence of a certain sugar chain structure on the surface of a bacterium can also be evaluated by mass spectrometry such as LC-MS, LC-MS / MS, MALDI-TOF MS, or FAB-MS on sugar chains or fragments extracted from bacteria, nuclear magnetic resonance (NMR) analysis of bacteria or sugar chains extracted therefrom, chromatography of sugar chains or fragments extracted from bacteria, or fragment analysis of bacteria or sugar chains extracted therefrom using glycoseptic enzymes.

[0104] Furthermore, as described above, the present inventors have found that bacteria tend to have higher immunostimulatory ability when they have a large amount of Pos-glycan structures on their cell surface. Therefore, another embodiment of the present disclosure may be a method for evaluating the immunostimulatory ability of bacteria, which includes evaluating the amount of Pos-glycan structures that bacteria have on their cell surface. Yet another embodiment of the present disclosure may be a method for screening bacteria that have immunostimulatory ability, which includes evaluating the amount of Pos-glycan structures that bacteria have on their cell surface. In these methods, the evaluation of the amount of Pos-glycan structures may be performed using lectin binding affinity as an indicator, obtained by a method similar to the evaluation method for bacterial-lectin binding described above for Pos-lectin-binding bacteria. Furthermore, in these methods, bacteria that are judged to have Pos-glycan structures on their cell surface by the above evaluation method can be judged to be bacteria with high immunostimulatory ability or bacteria that have immunostimulatory ability, and bacteria with a larger amount of Pos-glycan structures on their cell surface can be evaluated as having higher immunostimulatory ability.

[0105] <Composition> Next, a composition according to one embodiment of the present disclosure will be described. The composition according to this embodiment contains the immunostimulating bacteria according to the above-described embodiment. In one embodiment, the composition according to this embodiment contains the immunostimulating bacteria described above. The composition according to this embodiment particularly contains the immunostimulating bacteria described above as an active ingredient. The composition according to this embodiment may contain one or more types of immunostimulating bacteria. The bacteria contained in the composition according to this embodiment may be live or dead, may contain dead bacteria, or may be a mixture of live and dead bacteria. In one embodiment, the bacteria contained in the composition according to this embodiment may consist of dead bacteria.

[0106] The composition according to this embodiment may be an immunostimulatory composition (immune activation composition), a dendritic cell activation composition, a plasmacytoid dendritic cell activation composition (pDC activation composition), or an IFN-α production promotion composition.

[0107] <Composition Containing Pos-Lectin-Binding Bacteria> In one aspect, the composition according to this embodiment contains Pos-lectin-binding bacteria. That is, for example, in one aspect, the composition according to this embodiment may contain bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II , ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL. In a preferred aspect, it may contain bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB. In a more preferred aspect, it may contain bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, and LcH, or at least one lectin selected from the group consisting of PNA, MPL, and PHA-E.

[0108] In one aspect, in the composition according to this embodiment, the ratio of the number of cells of the Pos-lectin-binding bacteria contained in the composition to the total number of cells of the bacteria contained in the composition may be not less than a predetermined lower limit. Also, in a preferred aspect, the composition according to this embodiment may satisfy at least one selected from the following group: The ratio of the number of bacterial cells that bind to at least one lectin selected from the group consisting of MPL and PNA, contained in the composition, to the total number of bacterial cells contained in the composition, is above a predetermined lower limit; The ratio of the number of bacterial cells that bind to PHA-E contained in the composition to the total number of bacterial cells contained in the composition is above a predetermined lower limit.

[0109] In these cases, the above percentages may be, for example, 0.1% or more, 0.25% or more, 0.5% or more, 1% or more, 2.5% or more, 5% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 23% or more, 26% or more, 29% or more, 34% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more, and in one embodiment, it may be 5% or more.

[0110] In these cases, if the composition contains two or more types of Pos-lectin-binding bacteria, the proportion of Pos-lectin-binding bacteria is calculated based on the total number of cells of each type of Pos-lectin-binding bacteria.

[0111] In one embodiment, the composition according to this embodiment contains a number of Pos-lectin-binding bacteria in total cell counts equal to or greater than a predetermined lower limit. In another preferred embodiment, the composition according to this embodiment may satisfy at least one selected from the group consisting of the following: The product contains bacteria that bind to at least one lectin selected from the group consisting of PNA and MPL, in a total number of cells above a predetermined lower limit; It contains bacteria that bind to PHA-E in a number that is above a predetermined lower limit in terms of total cell count.

[0112] In these cases, the number of cells is, for example, 1.0 × 10⁻⁶. 4 pcs or more, 5.0×10 4 The above is 1.0 × 10 5or more than, 3.0×10 5 or more than, 1.0×10 6 or more than, 3.0×10 6 or more than, 1.0×10 7 or more than, 3.0×10 7 or more than, 1.0×10 8 or more than, 3.0×10 8 or more than, 5.0×10 8 or more than, 1.0×10 9 or more than, 5.0×10 9 or more than, 1.0×10 10 or more than, 5.0×10 10 or more than, 1.0×10 11 or more than or 1.0×10 12 or more than, and in one embodiment 1.0×10 5 or more than may also be possible.

[0113] In addition, in these cases, when the composition contains two or more types of Pos-lectin-binding bacteria, the total cell number of the Pos-lectin-binding bacteria is defined based on the sum of the cell numbers of each type of Pos-lectin-binding bacteria.

[0114] <The composition containing Neg-lectin non-binding bacteria> The composition according to this embodiment, in one aspect, contains Neg-lectin non-binding bacteria. That is, for example, the composition according to this embodiment, in one aspect, may contain bacteria that do not bind to at least one lectin selected from the group consisting of F17AG, Gal1-S, VVA, AAA, UEA-I, EEL, LSL-N, MOA, SJA, Gal3, LEA, CGL2, Gal7-S, VFA, ACL, Orysata, Gal1, and PA-IL, and in a preferred aspect, may contain bacteria that do not bind to PA-IL.

[0115] In one embodiment, the composition according to this embodiment may have a ratio of the number of Neg-lectin-unbound bacteria contained in the composition to the total number of bacteria contained in the composition that is above a predetermined lower limit. In another preferred embodiment, the composition according to this embodiment may have a ratio of the number of bacteria that do not bind to PA-IL contained in the composition to the total number of bacteria contained in the composition that is above a predetermined lower limit.

[0116] In these cases, the above percentages may be, for example, 0.1% or more, 0.25% or more, 0.5% or more, 1% or more, 2.5% or more, 5% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 23% or more, 26% or more, 29% or more, 34% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more, and in one embodiment, it may be 5% or more.

[0117] In these cases, if the composition contains two or more types of Neg-lectin-unbound bacteria, the proportion of Neg-lectin-unbound bacteria is calculated based on the total number of cells of each type of Neg-lectin-unbound bacteria.

[0118] In one embodiment, the composition according to this embodiment may contain a number of Neg-lectin-unbound bacteria in a total number that is above a predetermined lower limit. In another preferred embodiment, the composition according to this embodiment may contain a number of PA-IL-binding bacteria in a total number that is above a predetermined lower limit.

[0119] In these cases, the number of cells is, for example, 1.0 × 10⁻⁶. 4 pcs or more, 5.0×10 4 The above is 1.0 × 10 5 pcs or more, 3.0×10 5 pcs or more, 1.0×10 6 pcs or more, 3.0×10 6 pcs or more, 1.0×10 7 pcs or more, 3.0×107 pcs or more, 1.0×10 8 pcs or more, 3.0×10 8 pcs or more, 5.0×10 8 pcs or more, 1.0×10 9 pcs or more, 5.0×10 9 pcs or more, 1.0×10 10 pcs or more, 5.0×10 10 pcs or more, 1.0×10 11 One or more or 1.0 × 10 12 There may be more than one, and in one embodiment, 1.0 × 10 5 There may be more than one.

[0120] In these cases, if the composition contains two or more types of Neg-lectin-unbound bacteria, the total number of Neg-lectin-unbound bacteria is determined based on the sum of the number of cells of each type of Neg-lectin-unbound bacteria.

[0121] <Method for evaluating the proportion and total cell number of Pos-lectin-binding bacteria and Neg-lectin-non-binding bacteria in a composition> The proportion and total number of Pos-lectin-binding bacteria and Neg-lectin-unbinding bacteria in the composition can be evaluated, for example, by flow cytometry.

[0122] For example, the proportion of Pos-lectin-binding bacteria in a composition can be determined by fluorescently labeling all bacteria and Pos-lectins in the composition, allowing for their detection and differentiation. After contacting them for a certain period of time (e.g., 1 hour), evaluation is performed using flow cytometry. The proportion of particles in which fluorescence derived from the fluorescent dye labeled for Pos-lectins is detected among the particles (i.e., bacteria) in which fluorescence derived from the fluorescent dye labeled for all bacteria is detected represents the proportion of Pos-lectin-binding bacteria in the composition. Furthermore, the total number of Pos-lectin-binding bacteria in the composition can be determined by contacting fluorescently labeled bacteria with Pos-lectin using the same method as described above, and then evaluating the results by flow cytometry. The number of particles in which both the fluorescence of the fluorescent dye labeling the bacteria and the fluorescent dye labeling the Pos-lectin are detected represents the number of Pos-lectin-binding bacteria in the composition. This evaluation may be performed using only a portion of the composition, not the entire amount. When evaluating by flow cytometry, a certain volume of bead reagent (counting beads) that is distinguishable from bacteria and Pos-lectin and has a known number of particles per a certain volume may be added. The number of counting beads detected can then be used to calculate the total amount and the number per volume or mass of the composition.

[0123] For example, the proportion of Neg-lectin-unbound bacteria in a composition can be determined by fluorescently labeling all bacteria and Neg-lectin in the composition so that they can be detected separately, contacting them for a certain period of time (e.g., 1 hour), and then evaluating them using flow cytometry. The proportion of particles in which fluorescence from the Neg-lectin-labeled fluorescent dye is not detected among particles (i.e., bacteria) in which fluorescence from the Neg-lectin-labeled fluorescent dye is detected represents the proportion of Neg-lectin-unbound bacteria in the composition. Furthermore, the total number of Neg-lectin-unbound bacteria in the composition can be determined by contacting fluorescently labeled bacteria with Neg-lectin using the same method as described above, and then evaluating the results by flow cytometry. The number of particles in which the fluorescent dye labeling the bacteria is detected, but the fluorescence of the fluorescent dye labeling the Neg-lectin is not detected, represents the number of Neg-lectin-unbound bacteria in the composition. This evaluation may be performed using only a portion of the composition, not the entire amount. When evaluating by flow cytometry, a certain volume of bead reagent (counting beads) that is distinguishable from bacteria and Neg-lectin and has a known number of particles per a certain volume may be added. The number of particles of the detected counting beads can then be used to calculate the total amount and the number per volume or mass contained in the composition.

[0124] In these cases, all bacteria contained in the composition can be fluorescently labeled using fluorescent dyes that can stain both living and dead cells, such as FITC (Fluoresceinisothiocyanate isomer-I), the Hoechst® series, and SYTO9. Other fluorescent dyes besides those exemplified above can also be used. When using dead bacteria, DAPI (4',6-diamidino-2-phenylindole) can also be used. Furthermore, bacteria that bind to a certain lectin contained in the composition can be fluorescently labeled, for example, by contacting the composition with the fluorescently labeled lectin.

[0125] For example, the ratio and number of Pos-lectin-binding bacteria and Neg-lectin-non-binding bacteria in the composition may be evaluated according to the flow cytometry method whose detailed protocol is shown below. [Evaluation of Pos-lectin-binding bacteria and Neg-lectin-non-binding bacteria by flow cytometry method] 1. Add 1 mL of ultrapure water to a fluorescently labeled PNA lectin (for example, Lectin PNA From Arachis hypogaea (peanut), Alexa Fluor® 488 Conjugate (Invitrogen, L21409)). Dilute it 100-fold with a buffer (for example, PBS) to prepare a lectin solution. 2. Suspend the bacteria in a buffer (for example, PBS) at 1 mg / mL, and add a solution of a fluorescent dye (for example, DAPI solution (Dojindo Chemical Laboratories, BS04)) thereto (for example, 1 μL of DAPI solution per 1 mg of bacteria). Then, allow the mixture to stand at room temperature (for example, for 1 hour) to label the bacteria with the fluorescent dye. Wash the bacteria with a buffer as needed (for example, 20,000 g × 1 min, twice with 1 mL of PBS). 3. Apply ultrasonic waves (sonication) to the mixture to disperse the bacteria throughout the mixture. Sonication can be performed, for example, by contacting the mixture with a water bath that generates ultrasonic waves, or by immersing the probe of an ultrasonic generator (for example, Handy Sonic (UR-21P)) into the mixture. 4. Add the lectin solution prepared in 1. to each well of a microplate to a final concentration of 100 μg / mL, and add the fluorescently labeled bacterial solution prepared in 3. thereto at a final concentration of bacteria of 10 μg / mL. After incubating for 1 hour under light-shielded conditions, collect the bacteria and wash them as needed for measurement with a flow cytometer.

[0126] [Composition containing bacteria containing Pos-glycan structure] In one embodiment, the composition according to this embodiment contains bacteria containing a Pos-glycan structure. That is, for example, the composition according to this embodiment may contain bacteria having at least one glycan structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Poly β(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4 and NeuAcα2-6Gal / GalNAc on the surface of the bacterial cell. In a preferred embodiment, it may contain bacteria having at least one glycan structure selected from the group consisting of Galβ3GalNAc and Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4 on the surface of the bacterial cell.

[0127] In one embodiment, the composition according to this embodiment may have a ratio of the number of Pos-glycan structure-containing bacteria cells to the total number of bacteria cells contained in the composition that is above a predetermined lower limit. Furthermore, in one preferred embodiment, the composition according to this embodiment may satisfy at least one selected from the group consisting of the following: The ratio of the number of bacterial cells containing the Galβ3GalNAc sugar chain structure on the cell surface to the total number of bacterial cells contained in the composition is above a predetermined lower limit; The ratio of the number of bacterial cells containing Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4, to the total number of bacterial cells contained in the composition, is above a predetermined lower limit.

[0128] In these cases, the above percentages may be, for example, 0.1% or more, 0.25% or more, 0.5% or more, 1% or more, 2.5% or more, 5% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 23% or more, 26% or more, 29% or more, 34% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more, and in one embodiment, it may be 5% or more.

[0129] In these cases, if the composition contains two or more types of Pos-glycan structure-containing bacteria, the proportion of Pos-glycan structure-containing bacteria is calculated based on the total number of cells of each type of Pos-glycan structure-containing bacteria.

[0130] In one embodiment, the composition according to this embodiment may contain a number of Pos-glycan structure-containing bacteria in total cell counts above a predetermined lower limit. Furthermore, in one preferred embodiment, the composition according to this embodiment may satisfy at least one selected from the following group: The product contains bacteria having the Galβ3GalNAc sugar chain structure on their cell surface, in a total number exceeding a predetermined lower limit; The product contains a number of bacteria, in total, that are above a predetermined lower limit, that have at least one sugar chain structure selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 on their cell surface.

[0131] In these cases, the number of cells is, for example, 1.0 × 10⁻⁶. 4 pcs or more, 5.0×10 4 The above is 1.0 × 10 5 pcs or more, 3.0×10 5 pcs or more, 1.0×10 6 pcs or more, 3.0×10 6 pcs or more, 1.0×10 7 pcs or more, 3.0×10 7 pcs or more, 1.0×10 8 pcs or more, 3.0×108 or more than 5.0×10 8 or more than 1.0×10 9 or more than 5.0×10 9 or more than 1.0×10 10 or more than 1.0×10 11 or more than 1.0×10 12 or more than 1.0×10 5 or more than 1.0×10

[0132] In these cases, when the composition contains two or more types of bacteria containing a Pos-glycan structure, the total cell number of the bacteria containing a Pos-glycan structure is defined based on the sum of the cell numbers of the bacteria containing a Pos-glycan structure of each type.

[0133] The content ratio and total cell number of the bacteria containing a Pos-glycan structure in the composition can be evaluated according to the same method as the evaluation of the content ratio and total cell number of the Pos-lectin-binding bacteria described in "Evaluation Method for the Ratio and Total Cell Number of Pos-Lectin-Binding Bacteria and Neg-Lectin-Non-Binding Bacteria in a Composition". Specifically, based on the known information on the sugar chain structure recognized by each lectin, the content ratio and total cell number of the bacteria evaluated to bind to a certain lectin in the composition can be evaluated as the content ratio and total cell number of the bacteria having the sugar chain structure recognized by that lectin on the cell surface in the composition.

[0134] <Form of the composition> The composition according to this embodiment may be a food composition, a pharmaceutical composition, a quasi-drug, a bacterial powder (powder obtained by drying bacterial cells or powder containing the same), an additive, or a feed, and preferably may be a food composition or a bacterial powder.

[0135] In the composition according to this embodiment, the content of bacteria is not particularly limited as long as it is an amount that satisfies the effective amount of immune activation, and therefore, it may vary depending on the form of the composition according to this embodiment. For example, with respect to the total dry weight of the composition according to this embodiment, the dry weight of the bacteria according to 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, and may be 100% by mass or less, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, or 15% by mass or less. These lower limit values and upper limit values can be arbitrarily combined respectively. For example, with respect to the total dry weight of the composition according to this embodiment, the dry weight of the bacteria according to this embodiment may be 0.0001% by mass to 100% by mass, 0.001% by mass to 95% by mass, 0.01% by mass to 90% by mass, or 0.10% by mass to 80% by mass.

[0136] When the composition according to this embodiment is in a liquid state, the number of cells of the bacteria according to this embodiment is 1.0×10 3 cells / mL or more, 1.0×10 4 cells / mL or more, 1.0×10 5 cells / mL or more, 1.0×10 6 cells / mL or more, 1.0×10 7 cells / mL or more, or 4.0×10 7 cells / mL or more, and may be 1.0×10 11 cells / mL or less, 1.0×10 10 cells / mL or less, 3.0×10 9 cells / mL or less, or 1.0×10 9 cells / mL or less. These lower limit values and upper limit values can be arbitrarily combined respectively. For example, the number of cells of the bacteria according to this embodiment is 1.0×103 cells / mL~1.0×10 11 cells / mL, 1.0×10 5 cells / mL~1.0×10 10 cells / mL, 1.0×10 6 cells / mL~3.0×10 9 cells / mL or 1.0 × 10⁶ 7 cells / mL~1.0×10 9 The concentration may be cells / mL. In this case, the daily intake of the liquid immunostimulatory composition may be 10mL to 1,000mL, 30mL to 800mL, 50mL to 500mL, or 100mL to 250mL.

[0137] In the composition according to this embodiment, the number of bacteria per unit package according to this embodiment is 1.0 × 10 4 pcs or more, 5.0×10 4 The above is 1.0 × 10 5 pcs or more, 3.0×10 5 pcs or more, 1.0×10 6 pcs or more, 3.0×10 6 pcs or more, 1.0×10 7 pcs or more, 3.0×10 7 pcs or more, 1.0×10 8 pcs or more, 3.0×10 8 pcs or more, 5.0×10 8 One or more or 1.0 × 10 9 pcs or more, 5.0×10 9 pcs or more, 1.0×10 10 pcs or more, 5.0×10 10 pcs or more, 1.0×10 11 There may be more than 1.0 × 10 14 pcs or less, 1.0×10 13 Less than or equal to 1.0 × 10 12 The number may be less than or equal to 10. Furthermore, these upper and lower limits can be combined arbitrarily. For example, in the composition according to this embodiment, the number of bacteria per unit package according to this embodiment is 1.0 × 10. 4 pcs or more, 5.0×10 4 The above is 1.0 × 10 5 pcs or more 1.0×10 14 pcs or less, 1.0×10 6 pcs or more 1.0×1013 less than 1.0×10 7 more than 1.0×10 12 less than 1.0×10 8 more than 1.0×10 12 less than 3.0×10 8 more than 1.0×10 12 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, or more than 1.0×10 9 more than 1.0×10 12 may be less than.

[0138] From the viewpoint of reducing the intake burden, the composition according to the present embodiment is preferably used for oral intake. When used for oral intake, bacteria or a composition containing bacteria preferably has high resistance to gastric juice, intestinal juice, etc., and for example, preferably has strong acid resistance. The bacteria are not particularly limited, and either live bacteria or dead bacteria can be used, but from the viewpoints of immunostimulating effect, stability, production efficiency, etc., dead bacteria are preferred, and heat-killed bacteria are more preferred.

[0139] The composition according to this embodiment can be administered orally to humans and non-human mammals, and a typical form of administration is a food composition. The provided food composition is a food composition containing the bacteria according to this embodiment in an effective amount. Here, "contained in an effective amount" means that when the amount normally consumed in each food composition is ingested, the immunostimulating bacteria according to the above embodiment are ingested to the extent that the effects such as immune activation are exerted. Furthermore, the term "food composition" is used to include health foods, functional foods, nutritional supplements, health functional foods (e.g., Foods for Specified Health Uses, Nutritional Functional Foods, Foods with Function Claims), foods for special dietary uses (e.g., foods for infants, foods for pregnant and lactating women, foods for the sick), and supplements. It goes without saying that when the immunostimulating bacteria according to the above embodiment are administered to mammals other than humans, the food referred to in this invention is used as feed.

[0140] The composition according to this embodiment has effects such as immune activation, and can therefore be provided as an ingredient in foods consumed daily. In this case, the composition according to this embodiment can be provided in a unit package form in which the amount to be consumed per serving is predetermined. Examples of unit package forms per serving include packs, packaging, cans, or bottles that specify a fixed amount. In order to better exert the various effects of the composition according to this embodiment, the amount to be consumed per serving can be determined according to the daily intake amount of the immune-activating bacteria according to the above-described embodiment, which will be described later. The food according to this embodiment may be provided with information regarding the amount to be consumed displayed on the packaging, or together with a document containing such information.

[0141] The predetermined intake amount per serving in the unit packaging form may be the effective daily intake amount, or it may be the effective daily intake amount divided into two or more (preferably two to six) doses. Therefore, the unit packaging form of the composition according to this embodiment can contain the immunostimulating bacteria according to the above-described embodiment in the daily intake amount described later, or it can contain the immunostimulating bacteria according to the above-described embodiment in an amount of one-half to one-sixth of the daily intake amount described later. For convenience of intake, it is preferable to provide the composition according to this embodiment in a unit packaging form per serving (i.e., a daily unit packaging form) in which the intake amount per serving is the effective daily intake amount.

[0142] The composition according to this embodiment can be administered to subjects requiring immunostimulation. Subjects requiring immunostimulation are not particularly limited, but examples include subjects infected with a virus, subjects who wish to prevent viral infection, subjects with a cold, subjects who wish to prevent a cold, and subjects aged 65 or older.

[0143] The form of the "food composition" in this embodiment is not particularly limited and may be, for example, in the form of a beverage, a semi-liquid or gel-like form, a solid or powder-like form. Examples of "supplements" include tablets produced by mixing the active ingredient of the composition according to this embodiment with excipients, binders, etc., and then compressing them into tablets; granules produced by granulating the immunostimulating bacteria according to the above embodiment with excipients, binders, etc.; orally disintegrating tablets; and capsules containing the immunostimulating bacteria according to the above embodiment enclosed in capsules, etc. When providing as a supplement, in addition to the above-mentioned per-serving or per-day unit packaging, it is also preferable to provide it in per-week, per-two-week, per-month, or per-two-month unit packaging. In the latter unit packaging, it is preferable to display, for example, the amount to be consumed per serving or per day, so that the consumer can effectively ingest the immunostimulating bacteria according to the above embodiment according to that display.

[0144] Examples of food compositions provided as compositions according to this embodiment include health foods, functional foods, nutritional compositions, nutritional supplements, supplements, health foods, foods for specified health uses, foods with nutritional function claims, or foods with functional claims, all of which have immunostimulatory properties. Such food compositions can be labeled with claims such as: helping to maintain the immune function of healthy people or supporting the maintenance of the immune function of healthy people (immune care); for people concerned about a decline in immune function; suppressing a decline in immune function; preventing a decline in immune function; for people concerned about sunburn; for people concerned about skin damage in daily life; for people concerned about dry skin; for people concerned about skin flushing; for people concerned about skin erythema; for people concerned about redness of the skin; for people concerned about facial redness; for people concerned about rough hands; helping to improve immunity; helping to improve skin moisture conditions, etc.

[0145] The food composition provided as a composition according to this embodiment is not particularly limited as long as it contains the immunostimulating bacteria according to the above-described embodiment, but for example, it may include soft drinks, carbonated drinks, fruit juice drinks, vegetable juice drinks, fruit and vegetable juice drinks, milk and other dairy products, soy milk, dairy beverages, drinkable yogurt, drinkable or stick-type jelly, coffee, cocoa, tea beverages, nutritional drinks, energy drinks, sports drinks, mineral water (including both sparkling and non-sparkling), near water, non-alcoholic beverages such as non-alcoholic beer-flavored beverages; carbohydrate-containing foods and beverages such as rice dishes, noodles, bread or pasta; cheeses, hard or soft yogurt, dairy products and other oil and fat raw materials. Examples of prohibited items include dairy products such as fresh cream and ice cream; Western-style confectionery such as cookies, cakes, and chocolates; Japanese-style confectionery such as manju or yokan; tablet candies such as ramune (refreshing candies); candies, gums, gummies, frozen desserts and ice creams such as jelly or pudding; and various other confectionery products such as whiskey, bourbon, spirits, liqueurs, wine, fruit wine, sake, Chinese liquor, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, sparkling wine, other miscellaneous alcoholic beverages, and chuhai; processed foods such as processed egg products, processed seafood or meat products (including offal such as liver) (including delicacies), processed soups such as miso soup, seasonings such as miso, soy sauce, furikake, and other seasonings, or liquid foods such as high-calorie liquid foods.

[0146] Tea beverages include fermented teas, semi-fermented teas, and unfermented teas, such as black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, hojicha, oolong tea, turmeric tea, pu-erh tea, rooibos tea, rose tea, chrysanthemum tea, ginkgo leaf tea, and herbal teas (for example, mint tea, jasmine tea).

[0147] Fruits used in fruit juice beverages and beverages containing both fruit and vegetable juices include, for example, apples, oranges, grapes, bananas, pears, peaches, mangoes, acai, blueberries, and plums. Vegetables used in vegetable juice beverages and beverages containing both fruit and vegetable juices include, for example, tomatoes, carrots, celery, pumpkins, cucumbers, and watermelons.

[0148] When providing the composition according to this embodiment as animal feed, it can be carried out in accordance with the above-described description of food products.

[0149] When the composition according to this embodiment is provided as a pharmaceutical composition or quasi-drug, it can be formulated as an oral or parenteral preparation. Examples of oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, liquids, jellies, emulsions, and suspensions. Examples of parenteral preparations include injectable preparations suitable for local administration (including intradermal, subcutaneous, intramuscular, and intravenous injections), inhalants (e.g., inhaled aerosols, inhaled powders, inhaled solutions), nasal drops (e.g., nasal powders, nasal solutions), ointments, creams, gels, suppositories, patches, and compresses. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly practiced in the art. Examples of pharmaceutically acceptable carriers include excipients, binders, diluents, additives, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives.

[0150] When the composition according to this embodiment is used as a pharmaceutical composition or quasi-drug, the target diseases include, for example, cancers such as renal cancer, multiple myeloma, chronic myeloid leukemia, hairy cell leukemia, glioblastoma, medulloblastoma, astrocytoma, malignant melanoma, mycosis fungoides, and adult T-cell leukemia, which are already known to be indications for Type I IFN; viral infections such as subacute sclerosing panencephalitis, HTLV-1 myelopathy, hepatitis B, and hepatitis C; bacterial infections such as Chlamydia (sexually transmitted disease), Mycobacterium (tuberculosis), Listeria (sepsis, etc.), Staphylococcus (food poisoning), and Helicobacter (gastritis); and autoimmune diseases such as multiple sclerosis. The composition according to this embodiment can be used for the prevention or treatment of the above diseases. Furthermore, since the activity of Type I IFN in the composition according to this embodiment is known to be the inhibitory function of differentiation from osteoblasts to osteoclasts, it can also be used for the prevention or treatment of osteoporosis, etc.

[0151] The composition according to this embodiment can be used as a vaccine by using genetic engineering techniques to express or secrete antigens corresponding to specific diseases within or on the surface of bacteria that are the active ingredient. In particular, since the cell wall of bacteria protects antigens from stomach acid, strains expressing heterologous antigens within or on the surface of bacteria are suitable as hosts for oral vaccines.

[0152] The composition according to this embodiment may be a bacterial cell powder according to this embodiment (a powder obtained by drying bacterial cells or a powder containing the same). Such a bacterial cell powder can be obtained, for example, by culturing, sterilizing, and drying bacteria according to a conventional manufacturing method.

[0153] The bacterial powder of this embodiment may contain, in addition to the bacteria and culture medium components according to this embodiment, components that are acceptable as food, pharmaceuticals or animal feed, and may further contain at least one 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, preservatives, sustained-release regulators, surfactants, and solubilizers.

[0154] The bacterial cell powder of this embodiment can be used as a raw material for the food composition, pharmaceutical composition, or animal feed of this embodiment.

[0155] When the composition according to this embodiment is provided as an additive, it can be implemented in accordance with the above-described descriptions relating to food compositions, animal feed, quasi-drugs, pharmaceutical compositions, or bacterial powders. When the composition according to this embodiment is provided as a food additive, it can be used as a functional ingredient in functional foods having an immunostimulatory effect.

[0156] The composition according to this embodiment can be determined depending on the recipient's sex, age and weight, symptoms, time of ingestion, dosage form, route of ingestion, and the materials or drugs to be combined with it. The daily intake of the composition according to this embodiment for adults can be specified, for example, by the number of bacteria that are the active ingredient, and its lower limit is 1.0 × 10⁻⁶. 4 pieces, 5.0×10 4 pieces, 1.0×10 5 pieces, 3.0×10 5 pieces, 1.0×10 6 pieces, 3.0×10 6 pieces, 1.0×10 7 pieces, 3.0×10 7 pieces, 1×10 8 pieces, 3.0×10 8 pieces, 5.0×10 8 pieces, 1.0×10 9 pieces, 5.0×10 9 pieces, 1.0×10 10 pieces, 5.0×10 10pieces, or 1.0 × 10 11 It can be one, and its upper limit is 1.0 × 10 14 pieces, 1.0×10 13 pieces, 1.0×10 12 It can be set to one. These upper and lower limits can be combined arbitrarily, and the above intake range is, for example, 1.0 × 10 5 pcs or more 1.0×10 14 pcs or less, 1.0×10 6 pcs or more 1.0×10 13 pcs or less, 1.0×10 7 pcs or more 1.0×10 12 pcs or less, 1.0×10 8 pcs or more 1.0×10 12 pcs or less, 3.0×10 8 pcs or more 1.0×10 12 pcs or less, 3.0×10 8 pcs or more 1.0×10 12 Less than or equal to 5.0×10 8 pcs or more 1.0×10 14 Less than or equal to 5.0×10 8 pcs or more 1.0×10 13 Less than or equal to 5.0×10 8 pcs or more 1.0×10 12 pcs or less, 1.0×10 9 pcs or more 1.0×10 14 pcs or less, 1.0×10 9 pcs or more 1.0×10 13 1 or fewer, or 1.0 × 10 9 pcs or more 1.0×10 12 The number of bacteria can be reduced to less than or equal to 10. The bacterial count can be measured using known microscopes, flow cytometers, or non-culture microbial rapid testing devices (e.g., ELESTA PixeeMo (AFI Technology Co., Ltd.)), but measurement by microscope is preferred from the standpoint of high versatility.

[0157] The daily intake of the composition according to this embodiment for adults can also be determined by the dry cell mass of the active ingredient, bacteria, and its lower limit is 2.5 × 10⁻⁶. -4 mg, 2.5 × 10 -3 mg, 2.5 × 10 -2 mg, 2.5 × 10-1 It can be 1 mg or 2.5 mg, and its upper limit is 2.5×10 4 mg, 2.5×10 3 mg, 2.5×10 2 mg. These upper and lower limits can be arbitrarily combined respectively, and the range of the above intake amounts can be, for example, 2.5×10 -2 mg to 2.5×10 4 mg, 2.5×10 -1 mg to 2.5×10 3 mg, 2.5 mg to 2.5×10 2 mg.

[0158] The intake amount, the following intake timing and the intake period of the composition according to this embodiment are applicable both when the composition according to this embodiment is used for non-therapeutic purposes and therapeutic purposes. In the case of therapeutic purposes, the intake can be read as administration.

[0159] It is preferable to continue the intake of the composition according to this embodiment within the period when an immune activation effect is expected. From the viewpoint of better exerting the immune activation effect, the intake period of the composition according to this embodiment can be, for example, the above daily dose intake for 1 week or more, 2 weeks or more, 3 weeks or more, preferably 1 month or more (4 weeks or more). The intake interval of the composition according to this embodiment can be taking the above daily dose once every 3 days, once every 2 days or once a day, preferably once a day.

[0160] The composition according to this embodiment may also be taken starting before an event or time when an immune-boosting effect is expected. Examples of events when an immune-boosting effect is expected include activities that may lead to viral infection (e.g., participation in events with a high risk of viral infection, travel to areas with outbreaks), and examples of times when an immune-boosting effect is expected include the peak season for viral infections. Examples of timing for taking the composition before an event when an immune-boosting effect is expected include 1 day or more before, 3 days or more before, 1 week or more before, 2 weeks or more before, 3 weeks or more before, 1 month or more before (4 weeks or more before), or 2 months or more before (8 weeks or more before). Furthermore, although not particularly limited, it may be taken continuously with intervals between intakes from the start of intake until the event when an immune-boosting effect is expected. The composition according to this embodiment may also be taken starting after an event or time when an immune-boosting effect is expected. Examples of timing for taking the composition after an event when an immune-boosting effect is expected include 1 day or more after, 3 days or more after, 1 week or more after, or 2 weeks or more after. Furthermore, although not particularly limited, when ingested after an event in which an immune-boosting effect is expected, it may be possible to continue ingesting the composition with intervals between doses. In the present invention, it is particularly preferable to start ingesting the composition according to this embodiment before an event in which an immune-boosting effect is expected, and to continue ingesting it until after the event.

[0161] <Method for producing the composition> Next, a method for producing a composition according to one embodiment of this disclosure will be described. The composition produced by the production method of this embodiment is the composition according to the embodiment described above. That is, the composition produced contains immunostimulatory bacteria and may be, for example, a food composition, a pharmaceutical composition, a quasi-drug, bacterial powder, an additive, or a feed, and preferably a food composition or bacterial powder.

[0162] The manufacturing method of this embodiment includes at least one step selected from the following group (an immunostimulatory bacterial quantity adjustment step): (XM) The ratio of the number of immunostimulatory bacteria cells contained in the composition to the total number of bacteria cells contained in the composition shall be above a predetermined lower limit; The (YM) composition contains immunostimulatory bacteria in a number that is above a predetermined lower limit in terms of total cell count.

[0163] The immunostimulatory bacterial load adjustment step may, in one embodiment, include at least one selected from the group consisting of (AM), (BM), (CM), and (DM): (AM) The ratio of the number of Pos-lectin-binding bacteria contained in the composition to the total number of bacteria contained in the composition shall be above a predetermined lower limit; (BM) Composition contains Pos-lectin-binding bacteria in a number that is above a predetermined lower limit in terms of total cell count; (CM) The ratio of the number of Pos-glycan structure-containing bacteria contained in the composition to the total number of bacteria contained in the composition shall be above a predetermined lower limit; (DM) The composition contains Pos-glycan structure-containing bacteria in a number that is above a predetermined lower limit in terms of total cell count.

[0164] In one embodiment, the immunostimulatory bacterial load adjustment step may include at least one selected from the group consisting of (A1M), (B1M), (C1M), and (D1M): The ratio of bacterial cell counts that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, relative to the total number of bacterial cells contained in the (A1M) composition, shall be 7% or more. (B1M) Composition contains bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, totaling 1.0 × 10⁶ cells. 5 Contains more than one δ; The proportion of bacterial cells having a sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on their cell surface, relative to the total number of bacterial cells contained in the (C1M) composition, should be 7% or more. (D1M) Composition contains bacteria having a sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on the surface of the bacterial cell, totaling 1.0 × 10⁶ cells. 5 Contains at least one of these.

[0165] The above (AM) may, in one embodiment, include (A2): The ratio of bacterial cells that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB, relative to the total number of bacterial cells contained in the (A2M) composition, should be 7% or more.

[0166] In a preferred embodiment, the above (AM) may include at least one selected from the group consisting of (A3M) and (A4M): (A3M) The ratio of the number of bacterial cells that bind to at least one lectin selected from the group consisting of MPL and PNA, contained in the composition, to the total number of bacterial cells contained in the composition, must be above a predetermined lower limit; (A4M) The ratio of the number of bacterial cells that bind to PHA-E contained in the composition to the total number of bacterial cells contained in the composition shall be above a predetermined lower limit.

[0167] The above (BM) may, in one embodiment, include (B2M): (B2M) Composition contains bacteria that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB, totaling 1.0 × 10⁶ cells. 5 Contains at least one of these.

[0168] In a preferred embodiment, the above (BM) may include at least one selected from the group consisting of (B3M) and (B4M): The (B3M) composition contains bacteria that bind to at least one lectin selected from the group consisting of MPL and PNA, in a total number of cells above a predetermined lower limit; The (B4M) composition contains bacteria that bind to PHA-E in a number that is above a predetermined lower limit in terms of total cell count.

[0169] In a preferred embodiment, the above (CM) may include at least one selected from the group consisting of (C2M) and (C3M): (C2M) The ratio of the number of bacterial cells containing the Galβ3GalNAc sugar chain structure on the cell surface to the total number of bacterial cells contained in the composition shall be above a predetermined lower limit; (C3M) The ratio of the number of bacterial cells containing Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4, which are selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4, to the total number of bacterial cells contained in the composition is to be above a predetermined lower limit.

[0170] In a preferred embodiment, the above (DM) may include at least one selected from the group consisting of (D2M) and (D3M): The (D2M) composition contains bacteria having a Galβ3GalNAc sugar chain structure on its cell surface, in a total number of cells above a predetermined lower limit; The (D3M) composition contains bacteria having at least one sugar chain structure selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 on its cell surface, in a total number of cells equal to or greater than a predetermined lower limit.

[0171] In one embodiment, the above percentages in (XM), (AM), (CM), (A1M), (A2M), (A3M), (A4M), (C2M), and (C3M) may be, for example, 0.1% or more, 0.25% or more, 0.5% or more, 1% or more, 2.5% or more, 5% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 23% or more, 26% or more, 29% or more, 34% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more.

[0172] In (XM), (AM), (CM), (A1M), (A2M), (A3M), (A4M), (C2M), and (C3M), if the composition contains two or more types of immunostimulant bacteria, the proportion of immunostimulant bacteria is calculated based on the total number of cells of each type of immunostimulant bacteria.

[0173] In one embodiment, the number of cells in (YM), (BM), (DM), (B1M), (B2M), (B3M), (B4M), (D2M), and (D3M) is 1.0 × 10⁶. 4 There are more than 5.0 × 10 4 The above is 1.0 × 10 5 pcs or more, 3.0×10 5 pcs or more, 1.0×10 6 pcs or more, 3.0×10 6pcs or more, 1.0×10 7 pcs or more, 3.0×10 7 pcs or more, 1.0×10 8 pcs or more, 3.0×10 8 pcs or more, 5.0×10 8 pcs or more, 1.0×10 9 pcs or more, 5.0×10 9 pcs or more, 1.0×10 10 pcs or more, 1.0×10 11 One or more or 1.0 × 10 12 There may be more than one.

[0174] In (YM), (BM), (DM), (B1M), (B2M), (B3M), (B4M), (D2M), and (D3M), if the composition contains two or more immunostimulatory bacteria, the total number of immunostimulatory bacteria is determined based on the sum of the number of cells of each type of immunostimulatory bacteria.

[0175] The immunostimulatory bacteria quantity adjustment step can be carried out by adding immunostimulatory bacteria to the mixture in the composition manufacturing process. The manufacturing method of this embodiment can be implemented by further performing the immunostimulatory bacteria quantity adjustment step in a general manufacturing method of a composition, depending on the form of the composition to be manufactured. The immunostimulatory bacteria quantity adjustment step may be performed simultaneously with the steps originally included in the general manufacturing method of the composition, or it may be performed independently of the steps originally included.

[0176] <Method for evaluating the quality of the composition> Next, a method for evaluating the quality of a composition according to one embodiment of this disclosure will be described. The composition to be evaluated by the evaluation method of this embodiment is, for example, the composition according to the embodiment described above. That is, the composition to be evaluated by the evaluation method of this embodiment may be a composition containing bacteria, and may be, for example, a food composition, a pharmaceutical composition, a quasi-drug, bacterial powder, an additive, or a feed, and in one embodiment it may be a food composition or bacterial powder. Here, quality includes "immunostimulatory ability".

[0177] The evaluation method of this embodiment includes at least one step selected from the following group (an immunostimulatory bacterial quantity evaluation step): (XP) Evaluate the ratio of the number of immunostimulant bacteria cells contained in the composition to the total number of bacteria cells contained in the composition (for example, measure the ratio of immunostimulant bacteria cells contained in the composition, and if the ratio is equal to or greater than a predetermined value (or reference value), evaluate that the composition has immunostimulant activity); (YP) Evaluate the number and / or concentration of immunostimulatory bacteria contained in the composition (for example, if the number and / or concentration of immunostimulatory bacteria is equal to or greater than a predetermined value (or reference value), evaluate that the composition has immunostimulatory ability).

[0178] The immunostimulatory bacterial load assessment step may, in one embodiment, include at least one selected from the group consisting of (AP), (BP), (CP), and (DP): (AP) Evaluate the ratio of the number of Pos-lectin-binding bacteria cells contained in the composition to the total number of bacterial cells contained in the composition; To evaluate the number and / or concentration of Pos-lectin-binding bacteria contained in the (BP) composition; (CP) To evaluate the ratio of the number of Pos-glycan structure-containing bacteria in the composition to the total number of bacterial cells in the composition; To evaluate the number and / or concentration of Pos-glycan structure-containing bacteria contained in the (DP) composition. Here, if the above proportion or the number of bacterial cells and / or concentration is equal to or greater than a predetermined value (or reference value), the composition can be evaluated as having immunostimulatory ability.

[0179] The immunostimulatory bacterial load assessment step may, in one embodiment, include at least one selected from the group consisting of (A1P), (B1P), (C1P), and (D1P): (A1P) To evaluate the ratio of bacterial cell counts that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL, relative to the total number of bacterial cells contained in the composition; (B1P) To evaluate the number and / or concentration of bacterial cells that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, SAMB, Calsepa, Lentil, DiscoidinII, Lotus, BanLec, SNA-II, SNA-I, BC2LCN, GS-I, SBA, WGA, PALA, DBA, MAA, Jacalin, Gal2, GS-II, ECA, BPA, WFA, Con A, PSL1a, UDA, Gal9, LBA, DSA, Malectin, PA-IIL, PPL, ACG, DiscoidinI, Gal3C-S, CNL, and PTL contained in the composition; (C1P) To evaluate the proportion of bacterial cells having at least one sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on the bacterial cell surface, relative to the total number of bacterial cells contained in the composition; To evaluate the cell number and / or concentration of bacteria having at least one sugar chain structure selected from the group consisting of Galβ3GalNAc, Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man, Polyβ(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4, and NeuAcα2-6Gal / GalNAc on the surface of the bacterial cell. Here, if the above proportion or the number of bacterial cells is equal to or greater than a predetermined value (or reference value), the composition can be evaluated as having immunostimulatory activity.

[0180] The above (AP) may, in one embodiment, include (A2P): (A2P) Evaluate the ratio of bacterial cell counts that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB, relative to the total number of bacterial cells contained in the composition.

[0181] In a preferred embodiment, the above (AP) may include at least one selected from the group consisting of (A3P) and (A4P): (A3P) To evaluate the ratio of bacterial cell numbers that bind to at least one lectin selected from the group consisting of MPL and PNA contained in the composition, relative to the total number of bacterial cells contained in the composition; (A4P) Evaluate the ratio of the number of bacterial cells that bind to PHA-E contained in the composition to the total number of bacterial cells contained in the composition.

[0182] The above (BP) may, in one embodiment, include (B2P): To evaluate the number and / or concentration of bacterial cells that bind to at least one lectin selected from the group consisting of PNA, MPL, PHA-E, HHA, ASA, NPA, UEA-II, PHA-E, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB contained in the (B2P) composition. In a preferred embodiment, the above (BP) may include at least one selected from the group consisting of (B3P) and (B4P): To evaluate the number of bacterial cells that bind to at least one lectin selected from the group consisting of MPL and PNA contained in the (B3P) composition; To evaluate the number of bacterial cells that bind to PHA-E contained in the (B4P) composition.

[0183] In a preferred embodiment, the above (CP) may include at least one selected from the group consisting of (C2P) and (C3P): (C2P) To evaluate the ratio of the number of bacterial cells having the Galβ3GalNAc sugar chain structure contained in the composition to the total number of bacterial cells contained in the composition; To evaluate the ratio of bacterial cells having at least one sugar chain structure selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 on the bacterial cell surface to the total number of bacterial cells contained in the (C3P) composition.

[0184] In a preferred embodiment, the above (DP) may include at least one selected from the group consisting of (D2P) and (D3P): To evaluate the number and / or concentration of bacterial cells having the Galβ3GalNAc sugar chain structure on their surface, contained in the (D2P) composition; To evaluate the number and / or concentration of bacterial cells having at least one sugar chain structure selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 on the surface of the bacterial cell, contained in the (D3P) composition.

[0185] In one embodiment, the above percentages for (XP), (AP), (CP), (A1P), (A2P), (A3P), (A4P), (C2P), and (C3P) may be 0.1% or more, 0.25% or more, 0.5% or more, 1% or more, 2.5% or more, 5% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 23% or more, 26% or more, 29% or more, 34% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more. When the above percentages are the above values, the composition can be evaluated as having immunostimulatory activity.

[0186] In (XP), (AP), (CP), (A1P), (A2P), (A3P), (A4P), (C2P), and (C3P), if the composition contains two or more types of immunostimulant bacteria, the proportion of immunostimulant bacteria is calculated based on the total number of cells of each type of immunostimulant bacteria.

[0187] In one embodiment, the number of cells in (YP), (BP), (DP), (B1P), (B2P), (B3P), (B4P), (D2P), and (D3P) is 1.0 × 10⁶. 4 There are more than 5.0 × 10 4 The above is 1.0 × 10 5 pcs or more, 3.0×10 5 pcs or more, 1.0×10 6pcs or more, 3.0×10 6 pcs or more, 1.0×10 7 pcs or more, 3.0×10 7 pcs or more, 1.0×10 8 pcs or more, 3.0×10 8 pcs or more, 5.0×10 8 pcs or more, 1.0×10 9 pcs or more, 5.0×10 9 pcs or more, 1.0×10 10 pcs or more, 1.0×10 11 One or more or 1.0 × 10 12 The number of cells may be greater than or equal to the above value. If the number of cells is as described above, the composition can be evaluated as having immunostimulatory activity.

[0188] The above concentrations in (YP), (BP), (DP), (B1P), (B2P), (B3P), (B4P), (D2P), and (D3P) are 1.0 × 10 in one embodiment when the composition is a liquid. 4 The concentration is 5.0 × 10⁻¹⁰ or higher per mL. 4 pcs / mL or more, 1.0×10 5 pcs / mL or more, 3.0×10 5 pcs / mL or more, 1.0×10 6 pcs / mL or more, 3.0×10 6 pcs / mL or more, 1.0×10 7 pcs / mL or more, 3.0×10 7 pcs / mL or more, 1.0×10 8 pcs / mL or more, 3.0×10 8 pcs / mL or more, 5.0×10 8 pcs / mL or more, 1.0×10 9 pcs / mL or more, 5.0×10 9 pcs / mL or more, 1.0×10 10 pcs / mL or more, 1.0×10 11 cells / mL or more, or 1.0 × 10 12 The concentration may be 1 / mL or higher. When the above concentration is at the above value, the composition can be evaluated as having immunostimulatory activity.

[0189] The above concentrations in (YP), (BP), (DP), (B1P), (B2P), (B3P), (B4P), (D2P), and (D3P) are, in one embodiment when the composition is solid, 1.0 × 10 4 It is 5.0 × 10 or more pieces / g 4 pcs / g or more, 1.0×10 5 pcs / g or more, 3.0×10 5 pcs / g or more, 1.0×10 6 pcs / g or more, 3.0×10 6 pcs / g or more, 1.0×10 7 pcs / g or more, 3.0×10 7 pcs / g or more, 1.0×10 8 pcs / g or more, 3.0×10 8 pcs / g or more, 5.0×10 8 pcs / g or more, 1.0×10 9 pcs / g or more, 5.0×10 9 pcs / g or more, 1.0×10 10 pcs / g or more, 1.0×10 11 pieces / g or more or 1.0 x 10 12 The concentration may be greater than or equal to the number of particles / g. When the above concentration is at the above value, the composition can be evaluated as having immunostimulatory activity.

[0190] The above concentrations in (YP), (BP), (DP), (B1P), (B2P), (B3P), (B4P), (D2P), and (D3P) are 0.1 μg / mL or higher in one embodiment when the composition is a liquid, and may be 0.5 μg / mL or higher, 0.7 μg / mL or higher, 0.9 μg / mL or higher, 1 μg / mL or higher, 2 μg / mL or higher, 3 μg / mL or higher, 5 μg / mL or higher, 7 μg / mL or higher, 10 μg / mL or higher, 15 μg / mL or higher, 20 μg / mL or higher, or 25 μg / mL or higher. When the above concentrations are as specified above, the composition can be evaluated as having immunostimulatory activity. Here, the mass of the bacterial cells is the mass of the dried bacterial cells.

[0191] The above concentrations in (YP), (BP), (DP), (B1P), (B2P), (B3P), (B4P), (D2P), and (D3P) are 0.1 μg / g or higher in one embodiment when the composition is solid, and may be 0.5 μg / g or higher, 0.7 μg / g or higher, 0.9 μg / g or higher, 1 μg / g or higher, 2 μg / g or higher, 3 μg / g or higher, 5 μg / g or higher, 7 μg / g or higher, 10 μg / g or higher, 15 μg / g or higher, 20 μg / g or higher, or 25 μg / g or higher. When the above concentrations are as described above, the composition can be evaluated as having immunostimulatory activity. Here, the mass of the bacterial cells is the mass of the dried bacterial cells.

[0192] In (YP), (BP), (DP), (B1P), (B2P), (B3P), (B4P), (D2P), and (D3P), if the composition contains two or more types of immunostimulatory bacteria, the total number of immunostimulatory bacteria is evaluated based on the sum of the number of cells of each type of immunostimulatory bacteria.

[0193] In these evaluation methods, the number, concentration, and proportion of immunostimulatory bacteria can be evaluated, for example, by the same method as described in the method for evaluating the proportion and total number of Pos-lectin-binding bacteria and Neg-lectin-non-binding bacteria in the above-described composition, and in one embodiment, they may be evaluated by flow cytometry.

[0194] The evaluation of the quality of the composition according to this embodiment may be any evaluation that can provide information about factors affecting the immunostimulatory ability of the composition. For example, the evaluation of the quality of the composition according to this embodiment may be a test to determine whether the immunostimulatory composition contains an amount of immunostimulatory bacteria that can suitably exhibit immunostimulatory ability, an indirect evaluation of the immunostimulatory activity in the composition, an indirect evaluation of the purity or quality of the bacteria used as raw materials for the composition, an evaluation of the manufacturing conditions of the composition, or an evaluation of the storage conditions of the composition. In one embodiment, the evaluation of the quality of the composition may be a test to determine whether the immunostimulatory food composition or pharmaceutical composition contains an amount of immunostimulatory bacteria that can suitably exhibit immunostimulatory ability.

[0195] The evaluation method of this embodiment may be carried out at any stage from the start of the composition's manufacture until the composition is used. For example, it may be carried out by measuring the proportion of immunostimulant bacteria cells or the number and / or concentration of immunostimulant bacteria cells in an intermediate mixture during the composition's manufacture process. In this case, the quality may be evaluated using whether the final proportion or number, theoretically estimated from the proportion or number in the intermediate mixture based on the dilution ratio, etc., is above a predetermined lower limit. Alternatively, for example, it may be carried out by measuring the proportion of immunostimulant bacteria cells or the number and / or concentration of immunostimulant bacteria cells in the composition during the period from after the composition's manufacture until its use, such as during storage, distribution, or immediately before use. [Examples]

[0196] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0197] In the following examples, CAL-1 cells (deposit number FERM BP-10914) obtained from Nagasaki University were used as the sample (hereinafter referred to as "the sample"). (All rights to the sample belong to Nagasaki University, and permission to use the sample was obtained from Nagasaki University.) Cell culture was carried out under conditions of 37°C and 5% CO2. The results are shown as mean ± standard deviation. For the maintenance culture of CAL-1 cells, glutamine-free RPMI-1640 medium (Gibco, 21870076) was used, supplemented with 1.0 vol% GlutaMax (Gibco, 35050-061), 1.0 vol% MEM Non-Essential Amino Acids Solution (100X) (Gibco, 11140050, NEAA), 1.0 vol% penicillin / streptomycin (Gibco, 15140-12), and 10% FBS (Sigma) (hereinafter also referred to as "serum-containing medium"). For the tests using CAL-1 cells, the medium used was the serum-containing medium from which NEAA and FBS had been removed (hereinafter also referred to as "serum-free medium").

[0198] Table 3 shows the lactic acid bacteria strains used in the following examples. Table 4 shows the culture methods indicated as conditions A to C in Table 3. In the examples, with the exception of Lactobacillus rhamnosus CRL1505, the lactic acid bacteria were cultured according to the culture methods described in Table 4 in accordance with the correspondence between A to C shown in Table 3, and then used as bacterial powder (dried bacterial cells or powders containing them, etc.) prepared as follows: Bacteria cultured in MRS medium (MRS BROTH, CODE: CM0359, Oxoid Co., Ltd.) were subjected to centrifugation (5000 rpm) of the culture solution, and the supernatant was removed to obtain a bacterial suspension. Subsequently, the obtained bacterial suspension and phosphate-buffered saline (Takara Bio Co., Ltd.) were mixed in a ratio of 1:19 and washed twice by centrifugation (5000 rpm). The lactic acid bacteria in the bacterial suspension were killed by heat treatment after washing. The heat treatment involved raising the temperature from room temperature to 80°C over 30 minutes, holding it at 80°C for 30 minutes, and then cooling it back down to room temperature over 30 minutes. After cooling, the sample was freeze-dried to obtain bacterial powder.

[0199] [Table 3]

[0200] [Table 4]

[0201] For Lactobacillus rhamnosus CRL1505, the bacterial powder prepared as follows was used. [Method for preparing bacterial powder of Lactobacillus rhamnosus CRL1505] A supplement containing Lactobacillus rhamnosus CRL1505, FLORASSIST Probiotic Immune & Nasal Defense (Life Extension, #02208), was dissolved in 10 mL of MRS medium to prepare a solution containing Lactobacillus rhamnosus CRL1505. This solution was spread onto MRS agar plates, and streaking culture was performed to obtain single colonies of Lactobacillus rhamnosus CRL1505. The Lactobacillus rhamnosus CRL1505 colonies were added to MRS medium, and 700 μL of the culture solution, incubated at 37°C for 24 hours, was mixed with 300 μL of 80% glycerol to prepare a glycerol stock of Lactobacillus rhamnosus CRL1505, which was stored at -80°C.

[0202] 100 μL of Lactobacillus rhamnosus CRL1505 glycerol stock was added to 100 mL of MRS medium and incubated at 37°C for 24 hours. The culture solution was collected, centrifuged twice, and washed with ultrapure water. It was then suspended in 20 mL of ultrapure water, sterilized at 80°C for 30 minutes, and freeze-dried. This was used as the bacterial powder of Lactobacillus rhamnosus CRL1505 in the following examples.

[0203] <Example 1: Relationship between bacterial lectin binding ability and immunostimulatory activity> We measured the lectin-binding activity and immunostimulatory capacity of various bacteria and investigated their correlation. In addition, we evaluated the amount of phagocytosis by pDCs (pterogenic dendritic cells) for various bacteria.

[0204] [Measurement of lectin binding] Lectin binding activity was measured using a lectin array. The following arrays and labeling kits were used.

[0205] The array used was the RayBiotech Lectin Array 70 (RayBiotech, Inc., cat.GA-Lectin-70). The labeling solvent used was 1× Labeling Reagent. This was prepared by adding 100 μL of 1× PBS (pH 8.0) to a Labeling Reagent tube. 1× Labeling Reagent is a reagent used to label bacterial and other samples with biotin.

[0206] Cy3 equivalent dye-conjugated streptavidin was used. This was prepared by adding 1400 μL of Sample Diluent (reagent included in the kit) to a streptavidin tube.

[0207] The samples used for lectin array measurements were prepared according to the protocol described below. 1. First, bacteria were suspended in 500 μL of PBS at a concentration of 1 mg / mL in a 1.5 mL Eppendorf tube and exposed to sonication using a water bath. Sonication was performed using a US CLEANER (ASONE) at high mode for 30 seconds at room temperature. 200 μL of the sonicated mixture obtained in 2.1 was taken and dialysis was performed in 1×PBS (pH 8.0) at 4°C for 20 hours. Dialysis was performed using the Dialysis Vials provided in the kit. The Dialysis Vials are equipped with a regenerated cellulose membrane as the dialysis membrane. 3.2 The post-dialysis solution was mixed with 1× Labeling Reagent and incubated at room temperature for 60 minutes, after which Stop Solution (reagent included in the kit) was added. The solution labeled with 4.3 was dialyzed in 1×PBS (pH 8.0) at 4°C for 20 hours. The dialysis solution obtained in 5.4 was centrifuged at 4°C and 1000 rpm for 5 minutes, and the supernatant was collected. This supernatant was used as a labeled sample for the subsequent hybridization.

[0208] Hybridization of bacteria in the sample with lectins on the array was performed according to the protocol described below. 1. 100 μL of sample diluent was applied to the array and incubated at room temperature for 30 minutes. 2. Next, 150 μL of a solution prepared by adding 50 μL of Sample Diluent to 100 μL of the labeled sample prepared above was applied to the array. After application, the array was incubated at 4°C for 17 hours. 3. The array was cleaned according to the protocol provided with the kit. 4.80 μL of Cy3 equivalent dye-conjugated streptavidin was applied to the washed array and incubated at room temperature for 1 hour. 5. The array was cleaned according to the protocol provided with the kit. 6. The washed array was centrifuged at room temperature at 1000 rpm for 3 minutes.

[0209] Array scanning and data analysis were performed according to the protocol described below. 1. The array was scanned after cleaning using the GenePix® 4100A (Molecular Devices, LLC) array scanner. 2. Using the analysis software GenePix Pro 7 Software (Molecular Devices, LLC), the fluorescence intensity values ​​at each spot were quantified from the obtained image data (TIFF images, 16-bit format), and the signal values ​​were calculated.

[0210] Lectin arrays are a technique for detecting the binding of molecules with specific sugar chain structures. Lectins are proteins that specifically bind to sugar chains. By binding a sample, such as lactic acid bacteria, labeled (e.g., biotin-labeled) onto a substrate immobilized with lectins, and then labeling the labeled lactic acid bacteria that remain bound after washing (e.g., streptavidin conjugated with a fluorescent dye in the case of biotin labels) and measuring the fluorescence intensity, the amount of a specific sugar chain structure can be evaluated. Strong binding to a specific lectin in a lectin array indicates that the target has a large amount of the sugar chain structure recognized by that lectin, while weak binding suggests that the amount of that sugar chain structure is small.

[0211] [Bacterial staining with pHrodo Red SE] To evaluate the phagocytic capacity of each bacterium by CAL-1 cells, the bacteria were stained with the fluorescent dye pHrodo Red SE. pHrodo Red SE (Invitrogen, P36600) was dissolved in DMSO to prepare a 10.2 mM solution. Bacterial powder was weighed into a 2 mL Eppendorf tube, and a 20 mg / mL bacterial solution was prepared by adding 0.1 M sodium bicarbonate adjusted to pH 9.0. Then, 95 μL of this solution was transferred to a new 2 mL Eppendorf tube, and 5 μL of the prepared 10.2 mM pHrodo Red SE was added. After dispersing by vortexing, the solution was covered with aluminum foil to protect it from light and incubated at room temperature for 60 minutes. Subsequently, 750 μL of PBS (Takara Bio Inc.) was added, vortexed, and the solution was centrifuged at 20,000 g for 2 minutes at room temperature. After discarding 800 μL of the supernatant, 1.5 mL of PBS was added, and the precipitate was completely suspended by vortexing. The mixture was centrifuged again at room temperature at 20,000 g for 2 minutes, and 1.5 mL of the supernatant was discarded. Finally, 140 μL of PBS was added and the mixture was suspended to prepare a bacterial suspension containing 10 mg / mL of stained bacteria. The stained bacterial suspension was stored at 4°C, protected from light by aluminum foil, until use. Immediately before use, it was diluted 10-fold with PBS to produce a bacterial suspension containing 1 mg / mL of bacteria. The stained bacterial suspension was used within 24 hours of preparation.

[0212] [Evaluation of phagocytic capacity and measurement of immunostimulatory activity] Phagocytosis and immunostimulatory activity were measured according to the protocol described below. Using serum-free medium, CAL-1 cells were divided into 5.0 × 10⁶ cells. 5 A cell suspension containing cells / mL was prepared, and the cell suspension was seeded at 10 mL / well in a 10 cm diameter culture dish and cultured for 16 hours. CAL-1 cells were harvested by pipetting. The harvested CAL-1 cells were placed in serum-free medium in a 2.0 × 10⁶ layer. 5 The cells were resuspended at a concentration of cells / mL and seeded at 200 μL / well in each well of a 96-well microplate. To each seeded well, the respective bacteria, stained with pHrodo Red SE (Invitrogen, P36600) according to the method described above [Staining bacteria with pHrodo Red SE], were added to a final concentration of 10 μg / mL. Subsequently, the fluorescence of pHrodo Red SE in each well was measured over time using the Incucyte® SX5 Live-Cell Analysis System (Sartorius). After 22 hours of incubation, the culture supernatant was collected from each well, and the IFN-α concentration in the culture supernatant was measured by ELISA using the Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science).

[0213] pHrodo Red SE is a staining reagent whose fluorescence intensity is enhanced in low pH environments. When bacteria stained with pHrodo Red SE are phagocytosed by CAL-1 cells, the bacteria enter endosomes, creating a low pH environment which increases the fluorescence intensity of pHrodo Red SE. Therefore, the amount of bacteria phagocytosed by CAL-1 cells can be quantified using the total area in the field of view where pHrodo Red SE fluorescence is detected (fluorescence detection area). The fluorescence intensity analysis conditions for the Incucyte® SX5 Live-Cell Analysis System (Sartorius) were as described in [Incucyte Fluorescence Intensity Analysis Conditions 1] below. The field of view area per image was 0.572 mm². 2In addition to the fluorescence detection area, the total amount of fluorescence, which corresponds to the sum of the brightness of each pixel within the field of view, was also calculated.

[0214] [Incucyte fluorescence intensity analysis conditions 1] In the incucyte fluorescence intensity analysis, two channels were set up: a "Phase channel" and an "Orange channel." The Phase channel detected CAL-1 cells based on phase difference. The Orange channel detected the fluorescence of pHrodo Red SE. The detailed detection conditions are as shown in the Scan Settings and Analysis Settings below. Scan Configuration Scan Type:Non-Adherent Cell-by-Cell Vessel Type: 96-well Corning Image Channels:Phase, Orange (Acquisition Time: 400 ms) Objective: 20× Images per Well:9 Calculation Settings Analysis Type: Basic Analyzer Phase Channel Segmentation: AI Confluence Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): 60,000 or more - Eccentricity: Not set Orange Channel Segmentation: Surface Fit - Threshold (OCU): 5.000 - Edge Split: On - Edge Sensitivity: 0 Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): No settings - Eccentricity: Not set - Mean Intensity: Not set - Mean Intensity: Not set

[0215] [result] Tables 5-13 show the fluorescence detection area (Total_area, μm2 / Image), total fluorescence intensity (OCU×μm2 / Image), IFN-α concentration in the culture supernatant (pg / mL) 22 hours after the start of culture, and the fluorescence signal value, which is an indicator of binding affinity to each lectin, for each bacterium 22 hours after the start of culture. In Table 5, POS1 and POS2 show the results when Cy3-Streptavidin was added to wells where biotin-labeled IgG was immobilized at different concentrations instead of lectin, as a positive control, without adding the biotin-labeled sample. In Table 5, NEG shows the results when PBS was added instead of the labeled sample to wells where lectin was not immobilized, as a negative control. Note that "Strain" in Tables 5-13 refers to the abbreviation in Table 3.

[0216] Tables 14 and 15 show the results of calculating the correlation between the signal value of the lectin array in bacteria and the IFN-α concentration in the culture supernatant 22 hours after the start of culture, for each lectin, based on the results in Tables 5-13. The correlation coefficient and P-value are the correlation coefficient and P-value in Pearson correlation. According to these results, of the 70 lectins used in the lectin array, 52 lectins showed a positive correlation between lectin binding in bacteria and immunostimulatory activity, while 18 lectins showed a negative correlation between lectin binding in bacteria and immunostimulatory activity. Furthermore, among the lectins that showed a positive correlation, 18 lectins showed a significant positive correlation (p<0.05) between lectin binding in bacteria and immunostimulatory activity, and among those, 13 lectins showed a prominent positive correlation (p<0.01) between lectin binding in bacteria and immunostimulatory activity. The results, which showed a strong positive correlation with many lectins, revealed that lectin binding ability is one of the factors that determine the ability of bacteria to promote IFN-α production in pDCs. At the same time, it was found that bacteria that showed high binding ability to specific lectins possessed high immunostimulatory activity. Furthermore, since lectins recognize and bind to the sugar chain structure on the surface of bacterial cells, it was strongly suggested that bacteria that have the sugar chain structure recognized by lectins that showed a significant positive correlation between lectin binding ability and immunostimulatory activity possess high immunostimulatory activity.

[0217] Furthermore, a more detailed examination of the lectins that showed particularly strong positive correlations revealed that among the seven lectins with particularly small p-values, three lectins—PNA, MPL, and PHA-E—recognized glycan structures with galactose at their terminus. Focusing on these three lectins, multiple regression analysis was performed, showing that the binding ability of these three lectins—PNA, MPL, and PHA-E—to bacteria was very strongly positively correlated with IFN-α production (adjusted R). 2(Value = 0.79, P < 0.001). PNA and MPL are lectins that recognize Galβ3GalNAc, and PHA-E is a lectin that recognizes Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0218] <Example 2: Evaluation of the binding ratio of specific lectins in various bacteria> We measured the binding ratio of various bacteria to specific lectins and confirmed that the binding ratio to specific lectins correlated with the immunostimulatory activity measured in Example 1.

[0219] [Measurement of binding ratio with lectins] 1. Staining of bacteria For each of the ATCC15577, JCM5805, ATCC7962, ATCC11955, and JCM9695 strains, the bacteria were suspended in 300 μL of 0.1 M NaHCO3 (pH 9) to a concentration of 2 mg / mL. Then, 300 μL of 0.1 M NaHCO3 (pH 9) was added to adjust the FITC concentration to 0.2 mg / mL. The samples were allowed to stand at room temperature and protected from light for 60 minutes, after which they were washed twice with PBS (20,000 g × 1 min, 1 mL PBS). Subsequently, the samples were suspended in PBS to a concentration of 2 mg / mL.

[0220] 2. Binding test with PNA lectin Arachis hypogaea Lectin (PNA)-Cy3 (bioWORLD, 21761088-1) was prepared in Milli-Q to a concentration of 1 mg / mL. Then, it was diluted to 0.1 mg / mL with a solution of PBS with CaCl2 added to a final concentration of 0.5 mM (hereafter referred to as PBS+). Next, 100 μL of the above solution was added to a low-adsorption 96-well plate. Then, 5 μL of bacteria diluted to 0.2 mg / mL with PBS+ was added to each well. That is, 10 μg of PNA lectin was added for every 1 μg of bacteria. The plate was protected from light with aluminum foil and incubated at room temperature for 1 hour. Afterward, the wells were centrifuged twice with PBS+, and finally, 200 μL of PBS+ was added. The solutions in each well were subjected to flow cytometry (FACS) using a flow cytometer (BD biosciences, BD LSRFortessa® X-20 flow cytometer).

[0221] 3. Analysis using FACS The FACS results were analyzed using the following procedure. First, the population containing bacteria was gated with FSC / SSC. Next, single cells were gated with FSC-H and FSC-A. Further gates were applied with wavelengths corresponding to SSC / FITC, and only the population stained with FITC (FITC+ population) was extracted. From this FITC+ population, gates were applied with wavelengths corresponding to SSC / PE, and the proportion of the population that fluoresced with wavelengths corresponding to PE (PE+ population) was extracted. The threshold for the PE+ population was set so that the PE+ population was approximately 0.1% when only bacteria were subjected to FACS. The proportion of the PE+ population within the FITC+ population was calculated as the proportion of bacteria bound to PNA lectin.

[0222] [result] Table 16 shows the immunostimulatory activity (IFN-α production) measured in Example 1 for various bacteria, and the percentage of bacteria bound to PNA lectin relative to the total number of bacterial cells, as measured in Example 2. [Table 16]

[0223] The results above indicate that bacteria exhibiting a high binding rate to PNA lectin possess high immunostimulatory activity. Furthermore, since lectins recognize and bind to the sugar chain structure on the bacterial cell surface, it is strongly suggested that bacteria possessing the sugar chain structure recognized by lectins, which showed a significant positive correlation between lectin binding rate and immunostimulatory activity, also possess high immunostimulatory activity.

Claims

1. An immunostimulatory composition containing bacteria that bind to PNA.

2. The composition according to claim 1, wherein the bacteria further bind to at least one lectin selected from the group consisting of MPL, PHA-E, HHA, ASA, NPA, UEA-II, GNA, RS-Fuc, PHA-P, BC2L-A, AAL, LcH, PSA, PHA-L, STL, GRFT, and SAMB.

3. The composition according to claim 1, wherein the bacteria further bind to at least one lectin selected from the group consisting of MPL and PHA-E.

4. The composition according to claim 1, wherein the bacteria further do not bind to at least one lectin selected from the group consisting of F17AG, Gal1-S, VVA, AAA, UEA-I, EEL, LSL-N, MOA, SJA, Gal3, LEA, CGL2, Gal7-S, VFA, ACL, Orysata, Gal1, and PA-IL.

5. The composition according to claim 1, wherein the bacteria further satisfy (a): (a) The ratio of the binding strength to PNA to the binding strength to PA-IL is 2.70 times or more.

6. An immunostimulatory composition containing bacteria having a Galβ3GalNAc sugar chain structure on the surface of the bacterial cell.

7. The composition according to claim 6, wherein the bacterium further has at least one sugar chain structure selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4), (GlcNAcβ4Manα3)Manβ4, Man,Poly β(1,4)GlcNAc, Fucose, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3, Fucα6GlcNAc, αGlc, (GlcNAc)2-4 and NeuAcα2-6Gal / GalNAc on the surface of the bacterial cell.

8. The composition according to claim 6, wherein the bacterium further has at least one sugar chain structure selected from the group consisting of Galβ4GlcNAcβ2Manα6(GlcNAcβ4) and (GlcNAcβ4Manα3)Manβ4 on the surface of the bacterial cell.

9. An immunostimulatory composition that satisfies the following (A3): (A3) The ratio of the number of bacterial cells that bind to PNA contained in the composition to the total number of bacterial cells contained in the composition is 7% or more.

10. An immunostimulatory composition that satisfies the following (B3): (B3) Bacteria that bind to PNA, total number of cells: 1.0 × 10 ５ Contains one or more of these.

11. An immunostimulatory composition that satisfies the following (C2): (C2) The ratio of the number of bacterial cells containing the Galβ3GalNAc sugar chain structure on the surface of the bacterial cell to the total number of bacterial cells contained in the composition is 7% or more.

12. An immunostimulatory composition that satisfies the following (D2): (D2) Bacteria having the sugar chain structure of Galβ3GalNAc on the surface of the bacterial cell, total number of cells: 1.0 × 10 ５ Contains one or more of these.

13. The composition according to any one of claims 1 to 12, wherein the bacteria is a bacterium belonging to one of the genera Lactococcus, Lactobacillus, Bifidobacterium, Pediococcus, Hendricksia, or Leuconostoc.

14. The composition according to any one of claims 1 to 12, wherein the composition is for activating dendritic cells.

15. The composition according to any one of claims 1 to 12, wherein the composition is for promoting IFN-α production.

16. A method for producing an immunostimulatory composition, comprising at least one selected from the group consisting of (A1M), (B1M), (C1M), and (D1M): (A1M) The ratio of the number of bacterial cells that bind to the PNA contained in the composition to the total number of bacterial cells contained in the composition shall be 7% or more; (B1M) Composition contains bacteria that bind to PNA, totaling 1.0 × 10⁶ cells. ５ To contain one or more of these; (C1M) The ratio of the number of bacterial cells containing the Galβ3GalNAc sugar chain structure on the cell surface to the total number of bacterial cells contained in the composition shall be 7% or more; (D1M) Composition contains bacteria having a Galβ3GalNAc sugar chain structure on the surface of the bacterial cells, totaling 1.0 × 10⁶ cells. ５ Contains at least one of these.

17. A method for evaluating the immunostimulatory capacity of a composition containing bacteria, comprising at least one selected from the group consisting of (A1P), (B1P), (C1P), and (D1P): (A1P) To evaluate the ratio of the number of bacterial cells that bind to the PNA contained in the composition to the total number of bacterial cells contained in the composition; (B1P) To evaluate the number and / or concentration of bacterial cells that bind to PNA contained in the composition; (C1P) To evaluate the ratio of the number of bacterial cells having the Galβ3GalNAc sugar chain structure contained in the composition to the total number of bacterial cells contained in the composition; To evaluate the number and / or concentration of bacterial cells having the Galβ3GalNAc sugar chain structure on their surface, contained in the (D1P) composition.

18. A method for evaluating the immunostimulatory capacity of bacteria, comprising at least one selected from the group consisting of (X1) and (Y1) below: (X1) To evaluate the binding affinity of bacteria to PNA; (Y1) Evaluate the amount of Galβ3GalNAc sugar chain structure present on the surface of the bacterial cell.

19. A method for screening for immunostimulatory bacteria, comprising at least one selected from the following groups (X1) and (Y1): (X1) To evaluate the binding affinity of bacteria to PNA; (Y1) Evaluate the amount of Galβ3GalNAc sugar chain structure present on the surface of the bacterial cell.

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