Lactobacillus bacteria inducing production of interleukin

Abstract

Disclosed are: lactobacillus bacteria that satisfy at least one selected from the group consisting of the following (A) and (B); and a composition containing the lactobacillus bacteria. (A) In a culture supernatant obtained by inoculating a DMEM medium with RAW264.7 cells to achieve 5.0×105 cells / mL, adding lactobacillus bacteria to achieve a final concentration of 10 µg / mL, and culturing the same for 24 hours at 37°C and at a CO2 concentration of 5 vol%, the IL-10 concentration is 4500 pg / µL or more. (B) When the lactobacillus bacteria at a concentration of 10 µg / mL is brought into contact with Dectin-2 at a concentration of 10 µg / mL in a 15 mM HEPES buffer having a pH of 7.2 and containing 0.5 mM of CaCl2 and 150 mM of NaCl, the proportion of the number of the lactobacillus bacteria bonded to the Dectin-2 is 5% or more.

Description

Lactobacillus bacteria that induce interleukin production

[0001] This disclosure relates to Lactobacillus bacteria that induce interleukin production.

[0002] Lactobacillus bacteria that induce interleukin production are known. For example, Non-Patent Document 1 discloses that Lactobacillus paracasei KW3110 induces the production of interleukin-10 (IL-10) by macrophages through phagocytosis via binding to the lectin-like receptor Dectin-2. Furthermore, Lactobacillus bacteria that induce such interleukin production are expected to be used as active ingredients in functional foods or pharmaceuticals, and there are examples of such active ingredients being put into practical use. For example, Patent Document 1 discloses a composition containing lactic acid bacteria as an active ingredient for use in suppressing or improving eye fatigue. For example, Patent Document 2 discloses a food or beverage having anti-allergic function, characterized by the addition of Lactobacillus paracasei KW3110 or a mutant strain of Lactobacillus paracasei KW3110 having anti-allergic activity as an active ingredient.

[0003] Japanese Patent Publication No. 2018-043986 Japanese Patent Publication No. 2005-137357

[0004] Mia Yoshikawa et al., "Dectin-2 mediates phagocytosis of Lactobacillus paracasei KW3110 and IL-10 production by macrophages", Sci. Rep. 2021; 11(1): 17737.

[0005] Lactobacillus bacteria, which have a high ability to induce interleukin production, are expected to have high efficacy in suppressing or improving eye fatigue and providing anti-allergic effects when applied as active ingredients in functional foods or pharmaceuticals.

[0006] This disclosure aims to provide Lactobacillus bacteria that induce interleukin production. Furthermore, this disclosure aims to provide Lactobacillus bacteria with high interleukin production induction ability, and methods for producing Lactobacillus bacteria that can enhance interleukin production induction ability.

[0007] This disclosure relates, for example, to the following <X1> to <X20>. <X1> Lactobacillus species satisfying at least one selected from the group consisting of (A) and (B) below: (A) RAW264.7 cells in DMEM medium in 5.0 × 10 ５ After sowing to a cell / mL concentration, Lactobacillus bacteria were added to a final concentration of 10 μg / mL, and the mixture was heated at 37°C and CO2. ２ (B) The IL-10 concentration in the culture supernatant obtained by culturing at a concentration of 5% by volume for 24 hours is 4500 pg / μL or higher; (B) 10 μg / mL of Lactobacillus bacteria in 0.5 mM CaCl ２Furthermore, when contacted with 10 μg / mL of Dectin-2 in a pH 7.2 and 15 mM HEPES buffer containing 150 mM NaCl, the proportion of Lactobacillus bacteria bound to Dectin-2 is 5% or more. <X2> Lactobacillus bacteria according to <X1> that further satisfies (C) below: (C) Having a high mannose structure on the surface of the bacterial cell. <X3> Lactobacillus bacteria according to <X1> or <X2> that is Lactobacillus paracasei. <X4> Lactobacillus bacteria according to any one of <X1> to <X3> that is Lactobacillus paracasei KW3110 or a mutant thereof. <X5> A composition comprising Lactobacillus bacteria according to any one of <X1> to <X4>. A composition comprising a Lactobacillus bacterium described in any one of <X6> to <X4> as an active ingredient. <X7> The composition according to <X5> or <X6>, wherein the Lactobacillus bacterium is a dead bacterium. <X8> The composition according to any one of <X5> to <X7>, which is an immunomodulatory composition. <X9> The composition according to any one of <X5> to <X8>, which is an interleukin production induction composition. <X10> A method for regulating immunity in a subject, comprising administering a Lactobacillus bacterium described in any one of <X1> to <X4> or a composition according to any one of <X5> to <X7> to the subject. <X11> A method for inducing interleukin production in a subject, comprising administering a Lactobacillus bacterium described in any one of <X1> to <X4> or a composition according to any one of <X5> to <X7> to the subject. <X12> The method according to <X10> or <X11>, wherein the subject is a subject that requires it. <X13> Lactobacillus species according to any one of <X1> to <X4> or composition according to any one of <X5> to <X7> for use in immunomodulation. <X14> Lactobacillus species according to any one of <X1> to <X4> or composition according to any one of <X5> to <X7> for use in inducing interleukin production. <X15> Use of Lactobacillus species according to any one of <X1> to <X4> or composition according to any one of <X5> to <X7> in immunomodulation.<X16> Use of Lactobacillus bacteria described in any one of <X1> to <X4> or the composition described in any one of <X5> to <X7> in the induction of interleukin production. <X17> Use of Lactobacillus bacteria described in any one of <X1> to <X4> or the composition described in any one of <X5> to <X7> in the manufacture of a pharmaceutical or composition for regulating immunity. <X18> Use of Lactobacillus bacteria described in any one of <X1> to <X4> or the composition described in any one of <X5> to <X7> in the manufacture of a pharmaceutical or composition for inducing interleukin production. <X19> Use of Lactobacillus bacteria described in any one of <X1> to <X4> or the composition described in any one of <X5> to <X7> in the manufacture of a composition for activating immunity, improving immunity, relieving visual fatigue, or maintaining throat comfort. <X20> Use of Lactobacillus bacteria according to any one of <X1> to <X4> or the composition according to any one of <X5> to <X7> in the manufacture of a composition for activating immunity or improving immunity.

[0008] Furthermore, this disclosure also relates to, for example, the following <Y1> to <Y9>. <Y1> A method for producing Lactobacillus bacteria, comprising the step of culturing Lactobacillus bacteria in a medium maintained at a pH of 4.5 or higher and 7.0 or lower. <Y2> The method for producing Lactobacillus bacteria according to <Y1>, wherein the time for culturing the Lactobacillus bacteria in a medium maintained at a pH of 4.5 or higher and 7.0 or lower is 3 hours or more. <Y3> The method for producing Lactobacillus bacteria according to <Y1> or <Y2>, wherein during the cultivation of Lactobacillus bacteria, air is passed through the medium maintained at a pH of 4.5 or higher and 7.0 or lower at a pressure of 0.10 vvm or higher and 10.0 vvm or lower. <Y4> The method for producing Lactobacillus bacteria according to any one of <Y1> to <Y3>, further comprising killing the Lactobacillus bacteria after culturing them in a medium maintained at a pH of 4.5 or higher and 7.0 or lower. <Y5> A method for producing the above-mentioned Lactobacillus bacteria, which does not include the step of culturing the above-mentioned Lactobacillus bacteria in a medium with a pH of less than 4.5. <Y6> A method for producing the above-mentioned Lactobacillus bacteria, which is Lactobacillus paracasei. <Y7> A method for producing the above-mentioned Lactobacillus bacteria, which is Lactobacillus paracasei KW3110 or a mutant thereof. <Y8> A method for producing the above-mentioned Lactobacillus bacteria, which is higher in immunomodulatory function immediately after the culture step compared to immediately before the culture step. <Y9> A method for producing the above-mentioned Lactobacillus bacteria, which is higher in at least one selected from the group consisting of interleukin production induction ability, Dectin-2 binding ability, and macrophage phagocytosis ability, immediately after the culture step compared to immediately before the culture step.

[0009] Furthermore, this disclosure also relates to, for example, the following <Z1> to <Z8>. <Z1> A method for enhancing the immunomodulatory function of Lactobacillus bacteria, comprising the step of culturing Lactobacillus bacteria in a medium maintained at a pH of 4.5 or higher and 7.0 or lower. <Z2> The method according to <Z1>, wherein the time for culturing the Lactobacillus bacteria in a medium maintained at a pH of 4.5 or higher and 7.0 or lower is 3 hours or more. <Z3> The method according to <Z1> or <Z2>, wherein during the cultivation of the Lactobacillus bacteria, air is aerated into the medium maintained at a pH of 4.5 or higher and 7.0 or lower at a rate of 0.10 vvm or higher and 10.0 vvm or lower. <Z4> The method according to any one of <Z1> to <Z3>, further comprising killing the Lactobacillus bacteria after culturing them in a medium maintained at a pH of 4.5 or higher and 7.0 or lower. <Z5> The method according to any one of <Z1> to <Z4>, wherein the step of culturing the above-mentioned Lactobacillus species in a medium with a pH of less than 4.5 is not included. <Z6> The method according to any one of <Z1> to <Z5>, wherein the above-mentioned Lactobacillus species is Lactobacillus paracasei. <Z7> The method according to any one of <Z1> to <Z6>, wherein the above-mentioned Lactobacillus species is Lactobacillus paracasei KW3110 or a mutant thereof. <Z8> The method according to any one of <Z1> to <Z7>, wherein the above-mentioned immunomodulatory function is at least one selected from the group consisting of interleukin production induction ability, Dectin-2 binding ability and macrophage phagocytosis ability.

[0010] Furthermore, this disclosure also relates to a Lactobacillus bacterium described in any one of <X1> to <X5>, which is produced by the manufacturing method described in any one of <Y1> to <Y9>. This disclosure also relates to a composition described in any one of <X6> to <X8>, which contains a Lactobacillus bacterium produced by the manufacturing method described in any one of <Y1> to <Y9>. This disclosure also relates to a manufacturing method described in any one of <Y1> to <Y9>, which is, for example, a Lactobacillus bacterium produced by any one of <X1> to <X5>. Furthermore, the Lactobacillus bacterium related to <X6> to <X19> may also be a Lactobacillus bacterium produced by the manufacturing method described in any one of <Y1> to <Y9>.

[0011] This disclosure provides Lactobacillus bacteria that induce interleukin production and compositions containing the same. This disclosure also provides a method for producing Lactobacillus bacteria that induce interleukin production. Furthermore, this disclosure provides Lactobacillus bacteria with high interleukin production induction ability and compositions containing the same, as well as a method for producing Lactobacillus bacteria that enhance interleukin production induction ability.

[0012] Figure 1 shows the amount of interleukin-10 (IL-10) produced by RAW264.7 cells (pg / μL) when bacterial powder prepared by static culture (uncontrolled pH) or culture with controlled pH of 5.0, 5.5, 6.0, or 6.5 was added. Figure 2 shows the time change of the pH of the culture medium under the same static culture conditions as in Figure 1. Figure 3 shows the TII (OCU × μm) produced by RAW264.7 cells when bacterial powder prepared by static culture (uncontrolled pH) or culture with controlled pH of 5.0, 5.5, 6.0, or 6.5 was added. ２This is a diagram showing the (image) values. This is a diagram showing the percentage of bacteria bound to Dectin-2 among the bacteria prepared by static culture without pH control or culture with pH controlled to 5.5 in Example 3. This is a diagram showing the expression level of Clec4n mRNA in Example 4 when no bacteria were added (Control), when powder of bacteria prepared by culture with pH controlled to 5.5 was added, or when powder of bacteria prepared by static culture without pH control was added (Static). This is a diagram showing the expression level of Syk mRNA in Example 4 when no bacteria were added (Control), when powder of bacteria prepared by culture with pH controlled to 5.5 was added, or when powder of bacteria prepared by static culture without pH control was added (Static). This figure shows the expression levels of SLC15A3 mRNA in Example 4 when no bacteria were added (Control), when bacterial powder prepared by a pH-controlled culture was added, or when bacterial powder prepared by a static culture without pH control was added (Static). This figure shows the pH5.5-Static values ​​for the top 19 lectins that had the largest pH5.5-Static values ​​(the difference between the measured values ​​of bacteria prepared by a pH-controlled culture and the measured values ​​of bacteria prepared by a static culture without pH control) in Example 5.

[0013] The following describes the forms for implementing this disclosure, but this disclosure is not limited to the following embodiments.

[0014] 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 two, three, four, five, six, seven, eight, nine or more combinations of the elements of that group.

[0015] <Lectins> In this disclosure, the relationship between the abbreviation, formal name, and recognized sugar chain structure of 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).

[0016]

[0017] <IL-producing Lactobacillus> A Lactobacillus bacterium according to one embodiment of this disclosure will be described. The Lactobacillus bacterium according to one embodiment of this disclosure induces the production of interleukin (IL). Hereinafter, the Lactobacillus bacterium that induces IL production according to this embodiment will also be referred to as "IL-producing Lactobacillus". As will be explained below, the set of bacteria corresponding to IL-producing Lactobacillus may include the entire set of one biologically classified bacterial strain, but in one preferred embodiment, it may be a set that does not include the entire set of one biologically classified bacterial strain. For example, the set of bacteria corresponding to IL-producing Lactobacillus may be a set consisting of objects (live bacterial processed products) that maintain the morphology of bacteria, obtained by processing live bacteria of one or more predetermined bacterial strains, and such a set may be a subset of the entire set of one or more predetermined bacterial strains. That is, IL-producing Lactobacillus according to one embodiment may be a live bacterial processed product of Lactobacillus bacterium, or for example, a heat-treated product (heat-treated bacteria). The inventors have found that a product treated with live bacteria may have a higher IL production induction ability than bacterial cell powder prepared by a general method from the original bacterial strain.

[0018] IL-producing Lactobacillus are bacteria of the genus Lactobacillus. In this specification, Lactobacillus includes bacteria that were classified in the genus Lactobacillus before the reclassification of the genus. The genus Lactobacillus is not particularly limited, but for example, with the reclassification of the genus Lactobacillus, new genera have been added, such as Acetilactobacillus, Agrilactobacillus, Amylolactobacillus, Apilactobacillus, Bombilactobacillus, Companilactobacillus, Dellaglioa, Fructilactobacillus, Furfurilactobacillus, Holzapfelia, Lacticaseibacillus, and Lactiplantibacillus. This includes bacteria classified into genera such as s), Lapidilactobacillus, Latilactobacillus, Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolactobacillus, Paralactobacillus, Paucilactobacillus, Schleiferilactobacillus, and Secundilactobacillus.

[0019] Lactobacillus species are not particularly limited, but examples include Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus parakefiri, and Lactobacillus plantarum. Examples include *Lactobacillus plantarum* and *Lactobacillus pentosus*.

[0020] Specific examples of Lactobacillus species include, but are not limited to, 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, and Lactobacillus casei subspecies. Examples include Lactobacillus casei 327, Lactobacillus casei Shirota (Lactobacillus in the new classification), Lactobacillus bulgaricus OLL1073R-1, Lactobacillus bulgaricus 2038, Lactobacillus parakefilii (Lentilactobacillus parakefilii in the new classification) JCM8573, Lactobacillus plantarum (Lactiplantibacillus plantarum in the new classification) L-137, and Lactobacillus pentosa (Lactiplantibacillus pentosa in the new classification) ONRICb0240.

[0021] The IL-producing Lactobacillus may, in one embodiment, be Lactobacillus paracasei, in a preferred embodiment, be Lactobacillus paracasei KW3110 or a mutant thereof, and in a more preferred embodiment, be Lactobacillus paracasei KW3110.

[0022] Lactobacillus paracasei KW3110 can be obtained from the Japan Dairy Technology Association and is also 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)) as an international depositary authority under the Budapest Convention for the Deposit of Patent Organisms (Deposit Date: February 20, 2004). Furthermore, a mutant strain of Lactobacillus paracasei KW3110 has been deposited with the Patent Organism Depository Center as FERM BP-08635 (deposit date: February 20, 2004).

[0023] IL-producing Lactobacillus may be live or dead bacteria, and in one embodiment, may include dead bacteria, or may be dead bacteria. When IL-producing Lactobacillus is dead bacteria, IL-producing Lactobacillus is not particularly limited, but can be produced by killing live bacteria by heat treatment, pressurization treatment, high-pressure steam treatment, electromagnetic wave treatment, electron beam treatment, radiation treatment, ultraviolet treatment, alcohol treatment, electrolyzed water treatment, drug treatment or chemical treatment, etc., and then drying as necessary by freeze-drying, spray drying, drum drying, hot air drying or vacuum drying, etc. For example, IL-producing Lactobacillus according to one embodiment may be heat-treated bacteria.

[0024] <IL-10 Production Inducing Lactobacillus> In one embodiment, IL-10 production induction lactobacillus may be a Lactobacillus species that induces (or promotes) the production of interleukin-10 (hereinafter also referred to as "IL-10") by macrophages. IL-10 is a type of cytokine involved in the immune system and has the function of regulating immune responses and inflammatory responses. Hereinafter, the Lactobacillus species that induces IL-10 production by macrophages in this embodiment will also be referred to as "IL-10 production induction lactobacillus".

[0025] The IL-10 production-inducing Lactobacillus according to this disclosure may be a Lactobacillus species in which, when in contact with a macrophage, the amount of IL-10 expressed by the macrophage (IL-10 expression level) is above a predetermined lower limit. The macrophages used for evaluation are not particularly limited, but may be, for example, cultured cells of a macrophage line or primary cultured macrophages. In one embodiment, they may be cultured cells of a macrophage line, and these cultured cells may be mammalian cells. In a preferred embodiment, the macrophages used for evaluation may be RAW264.7 cells. RAW264.7 cells are a macrophage-like cell line established from mouse ascites tumors.

[0026] The expression level of IL-10 by macrophages is the amount of biomolecules that can serve as an indicator for evaluating the amount of IL-10 expressed by macrophages that have come into contact with Lactobacillus bacteria. In one embodiment, the expression level of IL-10 may be the amount of IL-10 protein secreted extracellularly and / or the amount of IL-10 protein in macrophages, or the amount of mRNA encoding IL-10 in macrophages. In a preferred embodiment, the expression level of IL-10 may be the amount of IL-10 protein adsorbed in the ELISPOT method, which measures the amount of IL-10 protein adsorbed onto cell culture wells after the extracellularly secreted IL-10 protein is adsorbed, or the amount of IL-10 protein secreted extracellularly and present in the culture supernatant. In these cases, the culture supernatant is the culture medium after contact between Lactobacillus bacteria and macrophages. The amount of IL-10 protein in the culture supernatant can be measured after the culture supernatant is collected, according to a method commonly used by those skilled in the art to quantify a specific protein. Furthermore, the amount of IL-10 in macrophages can be measured by methods commonly used by those skilled in the art, but is not particularly limited. For example, it can be measured by an intracellular cytokine staining method using a flow cytometer, or by preparing a macrophage lysate and then measuring the amount of IL-10 protein in the lysate using methods commonly used by those skilled in the art to quantify specific proteins. In these cases, the measurement of the amount of IL-10 protein is not particularly limited, but may be performed by, for example, the ELISA method or the Western blotting method, and may be performed by the ELISA method. For measuring the amount of IL-10 protein by the ELISA method, there are no particular limitations, but for example, the BD OptEIA Mouse IL-10 ELISA Set (BD Biosciences) can be used. Furthermore, the amount of IL-10 encoding mRNA in macrophages can be measured after preparing macrophage lysates by a method commonly used by those skilled in the art, by measuring the amount of IL-10 encoding mRNA in the lysates according to a method commonly used by those skilled in the art to quantify mRNA.The quantification of mRNA is not particularly limited, but may be performed by quantitative PCR methods such as real-time PCR (q-PCR), direct digital counting (e.g., nCounter®), or by methods using a next-generation sequencer (NGS).

[0027] In one preferred embodiment, the IL-10-inducing Lactobacillus may be a Lactobacillus species whose IL-10 production, as evaluated according to the macrophage-mediated IL-10 induction ability evaluation protocol (hereinafter also referred to as the "standard evaluation protocol for IL-10 production induction ability"), is equal to or greater than a predetermined lower limit.

[0028] [Standard Evaluation Protocol for IL-10 Production Induction Ability] As the culture medium for RAW264.7 cells, use DMEM (Dulbecco's modified Eagle medium, Thermo Fisher Scientific, catalog number: 11965-092) mixed with 10% by volume of fetal bovine serum and antibiotics. Place 5.0 × 10⁶ RAW264.7 cells in the medium. ５ Seeds are sown at a concentration of 10 μg / ml. Lactobacillus bacteria are then added to the seeds at 37°C and CO2. ２ Incubate at a concentration of 5% by volume for 24 hours. Collect the culture supernatant after incubation and measure the IL-10 protein concentration in the culture supernatant.

[0029] In the above "Standard Evaluation Protocol for IL-10 Production Induction Potential," the antibiotics are not particularly limited, but may be, for example, 100 U / mL penicillin and 100 μg / mL streptomycin. In the above "Standard Evaluation Protocol for IL-10 Production Induction Potential," the culture of Lactobacillus bacteria may be carried out, for example, in an environment with 100% humidity. In the above "Standard Evaluation Protocol for IL-10 Production Induction Potential," the measurement of IL-10 protein concentration may be carried out, for example, according to the ELISA method.

[0030] Thus, in one preferred embodiment, IL-10 production-inducing Lactobacillus cells are raised in DMEM medium in 5.0 × 10⁶ RAW264.7 cells. ５After sowing to a cell / mL concentration, Lactobacillus bacteria were added to a final concentration of 10 μg / mL, and the mixture was heated at 37°C and CO2. ２ The Lactobacillus bacteria may be those whose IL-10 concentration in the culture supernatant obtained by culturing at a concentration of 5% by volume for 24 hours is above a predetermined lower limit, and the DMEM medium may contain 10% by volume of fetal bovine serum.

[0031] In these cases, the amount of IL-10 produced is not particularly limited, based on the mass of the IL-10 protein produced, but may be, for example, 4,500 mg or more, 5,000 mg or more, 7,000 mg or more, 9,000 mg or more, 10.00 mg or more, 11.00 mg or more, or 12.00 mg or more. In these cases, the amount of IL-10 produced is, for example, 50.00 mg or less or 20.00 mg or less, based on the mass of the IL-10 protein produced. These lower and upper limits can be combined arbitrarily.

[0032] Furthermore, the amount of IL-10 produced in these cases is not particularly limited, based on the IL-10 protein concentration in the culture supernatant, but may be, for example, 4500 pg / μL or more, 5000 pg / μL or more, 7000 pg / μL or more, 9000 pg / μL or more, 10000 pg / μL or more, 11000 pg / μL or more, or 12000 pg / μL or more. In these cases, the amount of IL-10 produced is not particularly limited, based on the mass of IL-10 protein in the culture supernatant, but may be, for example, 50000 pg / μL or less or 20000 pg / μL or less. These lower and upper limits can be combined arbitrarily.

[0033] <Dectin-2 Binding Lactobacillus> In one embodiment, the IL-producing lactobacillus may be a Lactobacillus species that binds to Dectin-2. Hereinafter, the IL-producing lactobacillus according to this embodiment will also be referred to as "Dectin-2 binding lactobacillus".

[0034] Dectin-2 is a type of lectin-like receptor expressed on the surface of macrophages. The gene symbol for the gene encoding Dectin-2 is Clec4n (C-type lectin domain family 4, member n). Lactobacillus bacteria are known to induce IL-10 production by macrophages by phagocytosis via binding to Dectin-2 present on the surface of macrophages (Non-Patent Literature 1). The present inventors have found that Lactobacillus bacteria with high interleukin production induction ability also have high binding affinity to Dectin-2, and have clarified that binding affinity to Dectin-2 is one of the important parameters that determine the interleukin production induction ability in Lactobacillus bacteria.

[0035] [Dectin-2-binding Lactobacillus evaluated by binding assay] In one embodiment, a Dectin-2-binding Lactobacillus may be a Lactobacillus species confirmed to bind to Dectin-2 in a predetermined proportion or number of bacteria or more by evaluation using an in vitro method (binding assay) that allows evaluation of the binding of Lactobacillus species to Dectin-2 based on a detection index. The binding assay may be a cell-free in vitro method. The Dectin-2 used in the binding assay may be Dectin-2 present in a free state in an aqueous medium, Dectin-2 expressed in a cell line, or Dectin-2 expressed on an artificial lipid bilayer. In a preferred embodiment, it may be Dectin-2 present in a free state in an aqueous medium, and these Dectin-2 may be purified. Such Dectin-2 may be a commercially available protein, or a protein that has been isolated and purified after being expressed in host cells using a vector or the like. Such Dectin-2 may be a chimeric protein tagged with an isolation tag such as FlagTag.

[0036] A binding assay according to one aspect may be an assay in which Lactobacillus bacteria at a predetermined concentration are contacted with Dectin-2 at a predetermined concentration in a solution, and Lactobacillus bacteria in which the ratio of the number of Lactobacillus bacteria bound to Dectin-2 in the binding assay is equal to or higher than a predetermined lower limit value may be determined as Dectin-2-binding Lactobacillus bacteria.

[0037] A binding assay according to a preferred aspect may be an assay in which 10 μg / mL of Lactobacillus bacteria are contacted with 10 μg / mL of Dectin-2 in a pH 7.2 and 15 mM HEPES buffer containing 0.5 mM CaCl ２ and 150 mM NaCl, and Lactobacillus bacteria in which the ratio of the number of Lactobacillus bacteria bound to Dectin-2 in the binding assay is equal to or higher than a predetermined lower limit value may be determined as Dectin-2-binding Lactobacillus bacteria.

[0038] In these binding assays, the HEPES buffer is not particularly limited. For example, 27.7 mg of CaCl ２ , 4.383 g of NaCl, and 1.787 g of HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) are dissolved in 400 mL of pure water (for example, ultrapure water purified by an ultrapure water production apparatus milliQ (registered trademark, manufactured by Merck Millipore)), adjusted to pH 7.2 using NaOH, and then pure water is added until the volume reaches 500 mL, and the resulting solution may be filtered through a 0.22-μm filter.

[0039] In these binding assays, the environment in which Dectin-2 and Lactobacillus bacteria are contacted may be an environment commonly employed in experiments using mammalian proteins and is not particularly limited. For example, it may be an environment of 15°C or higher and 40°C or lower, or an environment at room temperature of 15°C or higher and 35°C or lower and in air, and it may further be an environment shielded from light or the like.

[0040] In these binding assays, the time for contacting Dectin-2 with Lactobacillus bacteria is limited to a time at which the binding of Dectin-2 to Lactobacillus bacteria can be detected. In one embodiment, this time may be the time at which the binding of Dectin-2 to Lactobacillus bacteria reaches equilibrium. The time for contacting Dectin-2 with Lactobacillus bacteria is not particularly limited, but may be, for example, 5 minutes to 24 hours, or 1 hour.

[0041] In these binding assays, the proportion of Lactobacillus bacteria bound to Dectin-2, which indicates that a Lactobacillus bacterium is a Dectin-2-binding Lactobacillus, is not particularly limited, but may be, for example, 5% or more, and may be 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, or 14% or more. Furthermore, the proportion of Lactobacillus bacteria bound to Dectin-2 obtained in these binding assays in Dectin-2-binding Lactobacillus is not particularly limited, but may be, for example, 50% or less, and may be 30% or less or 20% or less. These lower and upper limits can be combined arbitrarily, and the percentage of Lactobacillus bacteria bound to Dectin-2 obtained in these binding assays in Dectin-2-binding Lactobacillus can be, for example, 6% to 50%, 6% to 30%, 6% to 20%, 7% to 50%, 7% to 30%, 7% to 20%, 8% to 50%, 8% to 30%, 8% to 20%, and 9%. It may be 50% or less, 9% to 30%, 9% to 20%, 10% to 50%, 10% to 30%, 10% to 20%, 11% to 50%, 11% to 30%, 11% to 20%, 12% to 50%, 12% to 30%, 12% to 20%, 13% to 50%, 13% to 30%, 13% to 20%, 14% to 50%, 14% to 30%, or 14% to 20%.

[0042] [Dectin-2-binding Lactobacillus evaluated using IL-10 expression levels by macrophages as an indicator] In one embodiment, a Dectin-2-binding Lactobacillus may be a Lactobacillus species whose ability to induce IL-10 production in macrophages is at a predetermined multiplier or higher than the ability to induce IL-10 production in macrophages in which Dectin-2 expression is suppressed or the glycosylation ability of Dectin-2 is suppressed. In this case, the IL-10 production induction ability may be indicated by the amount of IL-10 expressed by macrophages that have come into contact with the Lactobacillus species, as explained in the section on IL-10 production-inducing Lactobacillus, and is not particularly limited, but for example, a 3x IL-10 production induction ability may mean that the amount of IL-10 expressed by the macrophages is 3x. Hereinafter, macrophages used to evaluate Dectin-2-binding Lactobacillus will also be referred to as "interest macrophages". Furthermore, in the following, macrophages in which Dectin-2 expression is suppressed or the glycosylation ability of Dectin-2 is suppressed will also be referred to as "control macrophages."

[0043] Regarding the control macrophages, macrophages in which Dectin-2 expression is suppressed are not particularly limited, but may be, for example, macrophages in which Dectin-2 has been knocked down or knocked out. Dectin-2 knockdown is not particularly limited, but can be carried out in a conventional manner using, for example, antisense nucleic acids, siRNA, or shRNA targeting Dectin-2 mRNA. Dectin-2 knockout is not particularly limited, but can be carried out in a conventional manner using, for example, gene editing technologies such as the CRISPR / Cas9 system, TALEN (Transscription Activator-Like Effector Nucleases) system, or ZFN (Zinc Finger Nucleases), or homologous recombination technology.

[0044] Furthermore, regarding the control macrophages, the macrophages in which the glycosylation ability of Dectin-2 is suppressed are not particularly limited, but may be, for example, macrophages in which Dectin-2 is blocked with a Dectin-2 binding peptide and / or an anti-Dectin-2 antibody, or macrophages in which Dectin-2 is competitively inhibited with a ligand. As the Dectin-2 binding peptide, a commercially available product may be used, and Dectin 2 Blocking Peptide (MybioSource, MBS829738) is an example. As the anti-Dectin-2 antibody, a commercially available product may be used, and for example, Anti-Dectin 2 Antibody (MybioSource, MBS8219862) may be used. The ligand for Dectin-2 may be, for example, a sugar chain containing a high mannose structure, as described later, and a glycoprotein containing the same.

[0045] The control macrophages may have a ratio of Dectin-2 present on their cell surface, or a ratio of Dectin-2 with glycosylation ability, relative to the interest macrophages, which is not particularly limited, but may be, for example, 80% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5.0% or less, 2.0% or less, 1.0% or less, or 0%.

[0046] In one embodiment, the control macrophage may be a macrophage in which Dectin-2 is blocked by the Dectin-2 binding peptide and / or anti-Dectin-2 antibody, prepared by contacting one macrophage cell with a total amount of Dectin-2 binding peptide and / or anti-Dectin-2 antibody in a ratio of 0.0010 ng to 10 ng. In this embodiment, the contact of the macrophage with the Dectin-2 binding peptide and / or anti-Dectin-2 antibody is not particularly limited, but for example, 5.0 × 10 ５This procedure may be performed on macrophages seeded at a concentration of Cells / mL, 24 hours after seeding. In this embodiment, the final concentrations of the Dectin-2 binding peptide and the anti-Dectin-2 antibody are not particularly limited, but may be, for example, 100 μg / mL or 10 μg / mL, respectively. In this embodiment, the time for contacting macrophages with the Dectin-2 binding peptide and / or anti-Dectin-2 antibody may be any time for which Dectin-2 is blocked, and is not particularly limited, but may be, for example, 10 minutes to 72 hours, or 24 hours as an example. In this embodiment, the environment for contacting macrophages with the Dectin-2 binding peptide and / or anti-Dectin-2 antibody may be any environment for which Dectin-2 is blocked, for example, 0°C to 40°C, or 4°C as an example.

[0047] Dectin-2-binding Lactobacillus may have an IL-10 production induction ability in interest macrophages that is equal to or greater than a predetermined value of its IL-10 production induction ability in control macrophages. This predetermined value is not particularly limited, but may be, for example, 1.05 times, 1.10 times, 1.20 times, 1.30 times, 1.40 times, 1.50 times, 1.60 times, 1.70 times, 1.80 times, 1.90 times, 2.00 times, 3.00 times, 4.00 times, 5.00 times, 7.00 times, 10.0 times, 20.0 times, 30.0 times, 40.0 times, or 50.0 times. Dectin-2-binding lactobacillus may have an IL-10 production-inducing ability in interest macrophages that is less than or equal to a predetermined value of its IL-10 production-inducing ability in control macrophages. This predetermined value is not particularly limited, but may be 10,000 times, 1,000 times, 100 times, 30.0 times, 10.0 times, or 6.00 times. These lower and upper limits can be combined arbitrarily, and Dectin-2-binding Lactobacillus has an IL-10 production induction ability in interest macrophages that is, for example, 1.05 to 6.00 times, 1.05 to 10.0 times, 1.05 to 30.0 times, 1.05 to 100 times, 1.05 to 1000 times, 1.05 to 10000 times, 1.10 to 6.00 times, 1.10 to 10.0 times, 1.10 to 30.0 times, 1.10 to 100 times, 1.10 to 10000 times, 1.20 to 6.00 times, and 1.20 to 10.0 times. , 1.20 times or more and 30.0 times or less, 1.20 times or more and 100 times or less, 1.20 times or more and 1000 times or less, 1.20 times or more and 10,000 times or less, 1.30 times or more and 6.00 times or less, 1. 30 times or more and 10.0 times or less, 1.30 times or more and 30.0 times or less, 1.30 times or more and 100 times or less, 1.30 times or more and 1000 times or less, 1.30 times or more and 10,000 times or less, 1.40 times or more 6.00 times or less, 1.40 times or more and 10.0 times or less, 1.40 times or more and 30.0 times or less, 1.40 times or more and 100 times or less, 1.40 times or more and 1000 times or less, 1.40 times or more and 1000 times or less 0 times or less, 1.50 times or more and 6.00 times or less, 1.50 times or more and 10.0 times or less, 1.50 times or more and 30.0 times or less, 1.50 times or more and 100 times or less, 1.50 times or more and 1000 times or less,1.50 times to 10,000 times, 1.60 times to 6.00 times, 1.60 times to 10.0 times, 1.60 times to 30.0 times, 1.60 times to 100 times, 1.60 times to 1000 times, 1.60 times to 10,000 times, 1.70 times to 6.00 times, 1.70 times to 10.0 times, 1.70 times to 30.0 times, 1.70 times to 100 times, 1.70 times to 1000 times, 1.70 times to 10,000 times, 1.80 times to 6.00 times, 1.80 times to 10.0 times, 1.80 times to 30.0 times, 1.80 times and below Above 100 times, 1.80 times to below 1000 times, 1.80 times to below 10000 times, 1.90 times to below 6.00 times, 1.90 times to below 10.0 times, 1.90 times to below 30.0 times, 1.90 times to below 100 times, 1.90 times to below 1000 times, 1.90 times to below 10000 times, 2.00 times to below 6.00 times, 2.00 times to below 10.0 times, 2.00 times to below 30.0 times, 2.00 times to below 100 times, 2.00 times to below 1000 times, 2.00 times to below 10000 times, 3.00 times to below 6.00 times, 3.00 times to below 10.0 times 3.00 times to 30.0 times, 3.00 times to 100 times, 3.00 times to 1000 times, 3.00 times to 10000 times, 4.00 times to 6.00 times, 4.00 times to 10.0 times, 4.00 times to 30.0 times, 4.00 times to 100 times, 4.00 times to 1000 times, 4.00 times to 10000 times, 5.00 times to 6.00 times, 5.00 times to 10.0 times, 5.00 times to 30.0 times, 5.00 times to 100 times, 5.00 times to 1000 times, 5.00 times to 10000 times, 6.00 times and below Above 10.0 times but below, 6.00 times but below 30.0 times, 6.00 times but below 100 times, 6.00 times but below 1000 times, 6.00 times but below 10000 times, 7.00 times but below 10.0 times, 7.00 times but below 30.0 times, 7.00 times but below 100 times, 7.00 times but below 1000 times, 7.00 times but below 10000 times, 10.0 times but below 30.0 times, 10.0 times but below 1000 times, 10.0 times but below 10000 times, 20.0 times but below 30.0 times, 20.0 times but below 100 times, 20.0 times but below 1000 times.It may be 20.0 times or more and 10,000 times or less, 30.0 times or more and 100 times or less, 30.0 times or more and 1,000 times or less, 30.0 times or more and 10,000 times or less, 40.0 times or more and 1000 times or less, 40.0 times or more and 10,000 times or less, 50.0 times or more and 100 times or less, or 50.0 times or more and 10,000 times or less.

[0048] [Dectin-2-binding Lactobacillus evaluated using macrophage phagocytosis as an indicator] In one embodiment, Dectin-2-binding Lactobacillus may be a Lactobacillus species in which the efficiency of phagocytosis by macrophages is at a predetermined ratio or higher compared to the efficiency of phagocytosis by macrophages in which Dectin-2 expression is suppressed or the glycosylation ability of Dectin-2 is suppressed. In other words, in one embodiment, Dectin-2-binding Lactobacillus may be a Lactobacillus species in which phagocytosis by interest macrophages is at a predetermined ratio or higher compared to the efficiency of phagocytosis by control macrophages. Here, interest macrophages and control macrophages are the same as those described in the above-mentioned "Dectin-2-binding Lactobacillus evaluated using macrophage IL-10 expression level as an indicator".

[0049] In this embodiment, the efficiency of bacterial phagocytosis by macrophages can be evaluated based on a parameter (phagocytosis parameter) that indicates the amount of bacteria phagocytosed by macrophages after contact between bacteria and macrophages for a predetermined time. While not particularly limited, for example, a threefold increase in the efficiency of bacterial phagocytosis by macrophages may mean that the phagocytosis parameter is threefold. The time for contact between bacteria and macrophages is not particularly limited, but may be, for example, 10 minutes or more and 168 hours or less, and may be 24 hours as an example.

[0050] The parameter for phagocytosis is not particularly limited, but may be, for example, the area or volume in macrophages where fluorescence is detected after contact with bacteria labeled with a fluorescent dye or fluorescent dye, or the total amount of fluorescence in macrophages. Here, the total amount of fluorescence means the product of the fluorescence intensity per unit area or unit volume in which fluorescence is detected and the area or volume in which fluorescence is detected. In one embodiment, the parameter for phagocytosis may be the area or total amount of fluorescence in which fluorescence is detected, obtained by the "Standard Evaluation Method Using Bacteria Labeled with a Fluorescent Dye" shown below. Furthermore, a fluorescent dye that can be suitably used for such evaluation may be a fluorescent dye that exhibits strong fluorescence in the acidic environment of endosomes (for example, pHrodo Red SE, Invitrogen, P36600).

[0051] [Standard evaluation method using bacteria labeled with fluorescent dyes] (Phagocytosis experiment) RAW264.7 cells were placed in the culture medium in a quantity of 5.0 × 10⁶ ５ Seeds are seeded into each well at a density of cells / mL. Bacteria stained with pHo Red SE according to the method described in "Bacterial Staining with pHo Red SE" below are added to a concentration of 10 μg / mL and incubated at 37°C for 24 hours. After incubation, the fluorescence of pHo Red SE in each well is measured.

[0052] (Bacterial staining with pHo Red SE) As a culture medium for RAW264.7 cells, use DMEM mixed with 10% by volume fetal bovine serum and antibiotics. Dissolve pHo Red SE (Invitrogen, P36600) in DMSO to prepare a 10.2 mM solution. Add 0.1 M sodium bicarbonate adjusted to pH 9.0 to Lactobacillus bacteria to prepare a 20 mg / mL bacterial solution. Mix 19 volumes of the bacterial solution with 1 volume of the 10.2 mM pHo Red SE solution and incubate at room temperature for 60 minutes in the dark. After staining, classify and wash the bacteria from the solution and use a 1 mg / mL bacterial suspension.

[0053] In one embodiment of the "Standard Evaluation Method Using Bacteria Labeled with Fluorescent Dyes" described above, the measurement of fluorescence is not particularly limited, but for example, it can be measured using the Incucyte® SX5 Live-Cell Analysis System (Sartorius), for example, with a field of view area of ​​0.572 mm² per image. ２ It may also be measured as follows. In this case, fluorescence intensity analysis using the Incucyte® SX5 Live-Cell Analysis System (Sartorius) is not particularly limited, but may be performed as described in the following [Incucyte fluorescence intensity analysis conditions].

[0054] (Incucyte Fluorescence Intensity Analysis Conditions) In Incucyte fluorescence intensity analysis, two channels, a "Phase channel" and an "Orange channel," are set. The Phase channel detects macrophages based on phase difference. The Orange channel detects fluorescence of Hydro Red SE. Detailed detection conditions are as shown in the Scan Settings and Analysis Settings below. Scan Settings Scan Type: Adherent Cell-by-Cell Vessel Type: 24-well Corning Image Channels: Phase, Orange (Acquisition Time: 400 ms) Objective: 20× Images per Well: 9 Analysis Settings Analysis Type: Basic Analyzer Phase Channel Segmentation: AI Confluence Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2): 20,000 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 ): Not set - Eccentricity: Not set - Mean Intensity: Not set - Mean Intensity: Not set

[0055] Dectin-2-binding Lactobacillus may be phagocytosed by interest macrophages at a rate equal to or greater than a predetermined value of the phagocytosed by control macrophages. This predetermined value is not particularly limited, but may be, for example, 1.05 times, 1.10 times, 1.20 times, 1.30 times, 1.40 times, 1.50 times, 1.60 times, 1.70 times, 1.80 times, 1.90 times, 2.00 times, 3.00 times, 4.00 times, 5.00 times, 7.00 times, 10.0 times, 20.0 times, 30.0 times, 40.0 times, or 50.0 times. Dectin-2-binding lactobacillus may be phagocytosed by interest macrophages at an efficiency of less than or equal to a predetermined value of phagocytosis by control macrophages. This predetermined value is not particularly limited, but may be 10,000 times, 1,000 times, 100 times, 30.0 times, 10.0 times, or 6.00 times. These lower and upper limits can be arbitrarily combined, and Dectin-2-binding lactobacillus may be phagocytosed by interest macrophages at an efficiency of less than or equal to a predetermined value of phagocytosis by control macrophages, for example, 1.05 times or more and 6.00 times or less, 1.05 times or more and 10.0 times or less, 1.05 times or more and 30.0 times or less, 1.05 times or more and 100 times or less, 1.05 times or more and 100 times or less. 0 times or less, 1.05 times or more and 10,000 times or less, 1.10 times or more and 6.00 times or less, 1.10 times or more and 10.0 times or less, 1.10 times or more and 30.0 times or less, 1.10 times or more and 100 times or less, 1 .10 times or more and 1000 times or less, 1.10 times or more and 10,000 times or less, 1.20 times or more and 6.00 times or less, 1.20 times or more and 30.0 times or less, 1.20 times or more 100 times or less, 1.20 times or more and 1000 times or less, 1.20 times or more and 10,000 times or less, 1.30 times or more and 6.00 times or less, 1.30 times or more and 10.0 times or less, 1.30 times or more and 30.0 times Below, 1.30 times or more and 100 times or less, 1.30 times or more and 1000 times or less, 1.30 times or more and 10,000 times or less, 1.40 times or more and 6.00 times or less, 1.40 times or more and 10.0 times or less, 1.4 0 times to 30.0 times, 1.40 times to 100 times, 1.40 times to 1000 times, 1.40 times to 10,000 times, 1.50 times to 6.00 times, 1.50 times to 10 .0 times or less, 1.50 times or more and 30.0 times or less, 1.50 times or more and 100 times or less, 1.50 times or more and 1000 times or less, 1.60 times or more and 6.00 times or less,1.60 times to less than 10.0 times, 1.60 times to less than 30.0 times, 1.60 times to less than 100 times, 1.60 times to less than 1000 times, 1.60 times to less than 10000 times, 1.70 times to less than 6.00 times, 1.70 times to less than 10.0 times, 1.70 times to less than 30.0 times, 1.70 times to less than 100 times, 1.70 times to less than 1000 times, 1.70 times to less than 10000 times, 1.80 times to less than 6.00 times, 1.80 times to less than 10.0 times, 1.80 times to less than 30.0 times, 1.80 times to less than 100 times, 1.80 times to less than 1000 times, 1.80 times to more than 1... Below 0000 times, 1.90 times to 6.00 times, 1.90 times to 10.0 times, 1.90 times to 30.0 times, 1.90 times to 100 times, 1.90 times to 1000 times, 1.90 times to 10000 times, 2.00 times to 6.00 times, 2.00 times to 10.0 times, 2.00 times to 30.0 times, 2.00 times to 1000 times, 2.00 times to 10000 times, 3.00 times to 6.00 times, 3.00 times to 10.0 times, 3.00 times to 30.0 times, 3.00 times to 1000 times. 3.00 to 1000 times, 3.00 to 10000 times, 4.00 to 6.00 times, 4.00 to 10.0 times, 4.00 to 30.0 times, 4.00 to 100 times, 4.00 to 1000 times, 4.00 to 10000 times, 4.00 to 10000 times, 5.00 to 6.00 times, 5.00 to 10.0 times, 5.00 to 30.0 times, 5.00 to 100 times, 5.00 to 1000 times, 5.00 to 10000 times, 6.00 to 10.0 times, 6.00 to 30.0 times, 6.00 times and below Above 100 times, 6.00 times to 1000 times, 6.00 times to 10000 times, 7.00 times to 10.0 times, 7.00 times to 30.0 times, 7.00 times to 100 times, 7.00 times to 1000 times, 7.00 times to 10000 times, 10.0 times to 30.0 times, 10.0 times to 100 times, 10.0 times to 1000 times, 10.0 times to 10000 times, 20.0 times to 30.0 times, 20.0 times to 100 times, 20.0 times to 1000 times, 20.0 times to 10000 times, 30.0 times to 100 times.It may be 30.0 times or more and 1000 times or less, 30.0 times or more and 10000 times or less, 40.0 times or more and 100 times or less, 40.0 times or more and 10000 times or less, 50.0 times or more and 1000 times or less, or 50.0 times or more and 10000 times or less.

[0056] <ORYSATA-binding Lactobacillus> In one embodiment, IL-producing Lactobacillus may be a Lactobacillus bacterium that binds to the lectin ORYSATA. Hereinafter, the Lactobacillus bacterium according to this embodiment will also be referred to as "ORYSATA-binding Lactobacillus". ORYSATA (Oryza passive lectin) is a lectin derived from rice that recognizes a high mannose structure. The inventors have found that Lactobacillus bacteria with high interleukin production induction ability have high binding affinity to ORYSATA, and have clarified that binding affinity to ORYSATA is one of the important parameters that determine the interleukin production induction ability in Lactobacillus bacteria.

[0057] The binding of bacteria to lectins is not particularly limited, but can be evaluated based on, for example, the presence, proportion, or amount of bacteria captured on a plate or carrier after contact with a plate or carrier on which lectins are supported, or the presence, proportion, or amount of complexes formed after contact between lectins and bacteria. In this case, the amount of bacteria and the amount of lectin are set under conditions where, when the amount of one is fixed, the proportion and amount of complexes increase or decrease linearly with respect to the amount of the other. The presence, proportion, or amount of bacteria captured on a plate or carrier after contact with a plate or carrier on which lectins are supported is not particularly limited, but for example, if the bacteria are fluorescently labeled beforehand, the fluorescence intensity on the plate or carrier measured with a microwell plate reader or flow cytometer can be used as an indicator, or the luminescence intensity in ELISA can be used as an indicator. The presence, proportion, or amount of complexes formed after contact between lectins and bacteria is not particularly limited. For example, if both are fluorescently labeled beforehand, the number of particles in which the fluorescence of both is detected simultaneously, as measured by a flow cytometer, can be used as an indicator for evaluation. Alternatively, if both are labeled beforehand with donor or acceptor beads or molecules that induce energy transfer, the intensity of luminescence or fluorescence originating from the acceptor, generated by donor excitation, can be used as an indicator for evaluation. Hereinafter, such values ​​that can be used as indicators of the binding affinity between bacteria and lectins will also be referred to as "binding indicator signal values."

[0058] In this disclosure, for example, when evaluated by the evaluation method described above, bacteria whose binding index signal value satisfies at least one of the following (p) to (r) can be confirmed to be bacteria that bind to a certain lectin. In one embodiment, bacteria whose binding index signal value satisfies (q) can be confirmed to be bacteria that bind to a certain lectin. Hereinafter, the bacteria to be evaluated will also be referred to as "target bacteria" (Bacteria of interest). (p) The binding index signal value obtained by measurement using the target bacteria is at least a predetermined multiplier compared to the binding index signal value obtained by measurement using control bacteria (negative control bacteria) with low interleukin production induction ability. (q) The binding index signal value obtained by measurement using the target bacteria and 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 bacteria and lectin. (r) The binding index signal value obtained by measurement using the target bacteria and lectin is at least a predetermined multiplier compared to the binding index signal value measured in the absence of lectin and target bacteria.

[0059] In one embodiment of (p) above, the negative control bacteria may be, for example, bacteria whose measured amount of IL-10 protein production according to the above-described standard evaluation protocol for IL-10 production ability is less than 4500 pg / μL, 4000 pg / μL or less, 3000 pg / μL or less, 2000 pg / μL or less, 1000 pg / μL or less, 500 pg / μL or less, or 200 pg / μL or less, and as an example, bacteria whose measured amount of IL-10 protein production according to the above-described standard evaluation protocol for IL-10 production ability is 1000 pg / μL or less. In one embodiment of (p) above, the negative control bacteria may be, for example, macrophages prepared by static culture without pH control.

[0060] The predetermined magnification in one embodiment of (p) above is not particularly limited, but 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, or 1.65x.

[0061] In one aspect of (q) above, the binding pair may be a pair of molecules known to bind to each other, and is not particularly limited, but may be, 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 aspect, it may be a pair of an affinity tag protein and its ligand. Examples of binding pairs, though not particularly limited, 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.

[0062] 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 later, 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 POS1 in the standard lectin array protocol is the result obtained when, as a positive control, biotin-labeled IgG is immobilized at different concentrations instead of lectins in wells, and Cy3-Streptavidin is added instead of bacteria for detection.

[0063] The predetermined magnification in one aspect of (q) above is not particularly limited, but may be, for example, 0.0001x, 0.010x, 0.012x, 0.013x, 0.014x, 0.015x, 0.016x, 0.017x, or 0.018x. In one aspect of (p) above, the predetermined magnification may be 0.012x. These magnifications are set appropriately according to the type of binding pair or lectin.

[0064] The predetermined magnification in one aspect of (r) above is not particularly limited, but may be, for example, 34.0x, 39.0x, 44.0x, 49.0x, 54.0x, or 59.0x.

[0065] In one embodiment of (p) to (r) above, the binding indicator signal value is not particularly limited, but 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).

[0066] [Standard Lectin Array Protocol] (Array and Labeling Kit) The array used is 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. For Cy3 equivalent dye-conjugated streptavidin, use Cy3 equivalent dye-conjugated. 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 expose it to ultrasound (sonication) in a 1.5 mL Eppendorf tube. 2. Take 200 μL of the mixture obtained after sonication in step 1, and perform dialysis in 1 × PBS (pH 8.0) at 4°C for 20 hours. As the dialysis membrane, use the regenerated cellulose membrane provided by the Dialysis Vials included in the kit. 3. Add 100 μL of 1 × Labeling Reagent to approximately 200 μL of the dialysis solution obtained in step 2, incubate at room temperature for 60 minutes, and then add 3 μL of Stop Solution (reagent included in the kit). 4.3 The labeled solution is dialyzed in 1×PBS (pH 8.0) at 4°C for 20 hours. 5.4 The dialyzed solution is centrifuged at 4°C and 1,000 rpm for 5 minutes, and the supernatant is collected. This supernatant is used as the labeled sample for the subsequent hybridization. (Hybridization) 1. Apply 100 μL of Sample Diluent to the array, incubate at room temperature for 30 minutes, and then remove the Sample Diluent by decanting. 2. Next, apply 150 μL of a solution prepared 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. Wash the array according to the protocol provided in the kit.4. Apply 80 μL of Cy3 equivalent dye-conjugated streptavidin to the washed array and incubate at room temperature for 1 hour. 5. Wash the array according to the protocol provided in 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 washed array using an array scanner (e.g., GenePix® 4100A (Molecular Devices, LLC)). 2. Quantify the fluorescence intensity value at each spot from the obtained image using analysis software (e.g., GenePix Pro 7 Software (Molecular Devices, LLC)) and calculate the signal value.

[0067] <High Mannose Structure-Containing Bacteria> In one embodiment, the IL-producing Lactobacillus may be a Lactobacillus bacterium having a high mannose structure on its cell surface. Hereinafter, the Lactobacillus bacterium according to this embodiment will also be referred to as "high mannose structure-containing bacteria." The high mannose structure is a sugar chain structure recognized by ORYSATA. The inventors have shown that the ability to bind to the high mannose structure is one of the important parameters that determine the interleukin production induction ability in Lactobacillus bacteria. Therefore, high mannose structure-containing bacteria may have the ability to induce interleukin production, similar to ORYSATA-binding bacteria.

[0068] In this disclosure, if a bacterium has a 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 a glycan, but in one embodiment, a bacterium having a 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.

[0069] While there are no particular limitations on whether bacteria possess a certain sugar chain structure on their cell surface, it can be confirmed, for example, by following a method similar to the evaluation method for the binding of bacteria to ORYSATA as described for ORYSATA-binding bacteria. Bacteria confirmed to be ORYSATA-binding bacteria can then be confirmed as bacteria possessing a high mannose structure on their cell surface.

[0070] The presence of a certain sugar chain structure on the surface of a bacterium can be confirmed, in addition to the above, by mass spectrometry such as LC-MS, LC-MS / MS, MALDI-TOF MS, or FAB-MS of 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.

[0071] The high mannose structure to which ORYSATA binds is not particularly limited, but may be, for example, a structure consisting of 3 to 15 consecutive monosaccharides, with 50% or more of the constituent monosaccharides being mannose. Preferably, it is an N-linked double-chain complex glycan structure that extends from N-acetylglucosamine (GlcNAc) and is located at the end or inside the glycan. The high mannose structure is not particularly limited, but is disclosed in non-patent literature such as Masamichi Nagae et al., "Distinct roles for each N-glycan branch interacting with mannose-binding type Jacalin-related lectins Orysata and Calsepa", Glycobiology 27(12), 1120-1133 (2017). The double-chain complex glycan structure is a glycan structure in which the glycan branches into two at the mannose directly connected to N-acetylglucosamine, which is the base of the extension.

[0072] In one embodiment, the number of monosaccharides constituting the high mannose structure may be 3 or more monosaccharides, 5 or more monosaccharides, 7 or more monosaccharides, or 9 or more monosaccharides, and may be 15 or less monosaccharides, 13 or less monosaccharides, 11 or less monosaccharides, or 9 or less monosaccharides. For example, in one embodiment, the number of monosaccharides constituting the high mannose structure may be 3 or more monosaccharides and 15 or less monosaccharides, 3 or more monosaccharides and 13 or less monosaccharides, 3 or more monosaccharides and 11 or less monosaccharides, 3 or more monosaccharides and 9 or less monosaccharides, 5 or more monosaccharides and 15 or less monosaccharides, 5 or more monosaccharides and 13 or less monosaccharides, 5 or more monosaccharides and 11 or less monosaccharides, 5 or more monosaccharides and 9 or less monosaccharides, 7 or more monosaccharides and 15 or less monosaccharides, 7 or more monosaccharides and 13 or less monosaccharides, 7 or more monosaccharides and 11 or less monosaccharides, 7 or more monosaccharides and 9 or less monosaccharides, 9 or more monosaccharides and 15 or less monosaccharides, 9 or more monosaccharides and 13 or less monosaccharides, 9 or more monosaccharides and 11 or less monosaccharides, or 9 monosaccharides. In one preferred embodiment, the number of monosaccharides constituting the high mannose structure may be 3 to 9 monosaccharides.

[0073] In one embodiment, the proportion of mannose molecules in the monosaccharides constituting the high mannose structure may be 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, and may be 100% or less. In one embodiment, the high mannose structure may be a sugar chain structure composed of mannose and N-acetylglucosamine.

[0074] As described above, the High mannose structure according to one preferred embodiment is an N-linked double-chain complex glycan structure that is present at the terminal or internal part of the glycan and is extended from N-acetylglucosamine (GlcNAc), and may be a glycan structure composed only of 3 to 9 mannose molecules.

[0075] <Method for producing IL-10-inducing Lactobacillus> IL-10-inducing Lactobacillus is not particularly limited, but can be produced, for example, by following the method described in "Method for producing Lactobacillus bacteria" below. That is, IL-10-inducing Lactobacillus can be the "target bacteria" described later in "Method for producing Lactobacillus bacteria".

[0076] IL-10 production-inducing Lactobacillus is not particularly limited, but can be produced, for example, by increasing the amount of sugar chains containing a high-mannose structure on the surface of commercially available Lactobacillus bacteria. The method for increasing the amount of sugar chains containing a high-mannose structure on the surface of bacterial cells is not particularly limited, but can be carried out, for example, by selecting a preferred bacterium from a group of bacteria obtained by breeding including spontaneous mutations or accidental mutations, by overexpressing a protein that forms glycoproteins that readily have a high-mannose structure, or by culturing bacteria in a high-mannose medium.

[0077] <Composition> Next, a composition according to one embodiment of the present disclosure will be described. The composition according to this embodiment contains the IL-producing Lactobacillus according to one embodiment described so far. In one embodiment, the composition according to this embodiment contains the IL-producing Lactobacillus described so far as an active ingredient. The composition according to this embodiment may contain one or more types of IL-producing Lactobacillus. The Lactobacillus 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 Lactobacillus bacteria contained in the composition according to this embodiment may be dead bacteria.

[0078] The composition according to this embodiment may be an immunomodulatory composition, an immunosuppressive composition, a cytokine production induction composition, an interleukin production induction composition, or an interleukin-10 production induction composition. Furthermore, as described in Patent Document 1, etc., since interleukins are involved in the suppression or improvement of eye fatigue, the composition according to this embodiment may be a composition used for the suppression or improvement of eye fatigue. Furthermore, as described in Patent Document 2, etc., since interleukins are involved in anti-allergic function, the composition according to this embodiment may be an immunosuppressive composition, an inflammation suppression composition (for example, a composition for suppressing inflammation caused by inflammasome activation), or an anti-allergic composition.

[0079] Furthermore, as described in Non-Patent Literature 1, it is known that in macrophages that phagocytose Lactobacillus bacteria via binding to Dectin-2, the expression of Syk (Spleen tyrosine kinase), a tyrosine kinase that promotes the activation of intracellular signals, increases first. Therefore, the composition according to this embodiment may also be a composition for promoting Syk expression.

[0080] Furthermore, as described in Non-Patent Literature 1, it is known that in macrophages that phagocytose Lactobacillus bacteria via binding to Dectin-2, the Lactobacillus bacteria are first trafficked to endosomal lysosomes for degradation, then the degradation products are translocated from the lysosomes to the cytoplasm via PHT2 (Peptide / Histidine Transporter 2), and finally the translocated degradation products activate NOD2 (Nucleotide-binding Oligomerization Domain 2), thereby activating the subsequent IL-10 production signal. Therefore, the composition according to this embodiment may also be a composition for promoting PHT2 expression.

[0081] The composition according to this embodiment may be a food composition, a pharmaceutical composition, a quasi-drug, bacterial powder (such as a powder obtained by drying bacterial cells or a powder containing the same), an additive, or animal feed, and preferably a food composition or bacterial powder.

[0082] In the composition according to this embodiment, the content of IL-producing lactobacillus is not particularly limited and should be an amount that satisfies the effective amount for inducing interleukin production. Therefore, the content of IL-producing lactobacillus 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 IL-producing lactobacillus 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, or 3.0% by mass or more. The upper limit may be 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 also 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 and upper limits can be combined in any way and are not particularly limited. For example, with respect to the total dry weight of the composition according to this embodiment, the dry weight of the IL-producing Lactobacillus according to this embodiment is 0.0001% by mass or more and 15% by mass or less, 0.0001% by mass or more and 20% by mass or less, 0.0001% by mass or more and 25% by mass or less, 0.0001% by mass or more and 30% by mass or less, 0.0001% by mass or more and 50% by mass or less, 0.0001% by mass or more and 60% by mass or less, 0.0001% by mass or more and 70% by mass or less, 0.0001% by mass or more and 80% by mass or less, 0.0001% by mass or more and 90% by mass or less, 0.0001 Mass% or more and 95 mass% or less, 0.0001 mass% or more and 100 mass% or less, 0.001 mass% or more and 15 mass% or less, 0.001 mass% or more and 20 mass% or less, 0.001 mass% or more and 25 mass% or less, 0.001 mass% or more and 30 mass% or less, 0.001 mass% or more and 5 0 mass% or less, 0.001 mass% or more and 60 mass% or less, 0.001 mass% or more and 70 mass% or less, 0.001 mass% or more and 80 mass% or less, 0.001 mass% or more and 90 mass% or less, 0.001 mass% or more and 95 mass% or less, 0.001 mass% or more and 100 mass% or less,0.005% to 15% by mass, 0.005% to 20% by mass, 0.005% to 25% by mass, 0.005% to 30% by mass, 0.005% to 50% by mass, 0.005% to 60% by mass, 0.005% to 70% by mass, 0.005% to 80% by mass, 0.005% to 90% by mass, 0.005% to 95% by mass, 0.005% to 100% by mass, 0.01% to 15% by mass, 0.01% to 20% by mass, 0.01% to 25% by mass Below %, 0.01% to 30% by mass, 0.01% to 50% by mass, 0.01% to 60% by mass, 0.01% to 70% by mass, 0.01% to 80% by mass, 0.01% to 90% by mass, 0.01% to 95% by mass, 0.01% to 100% by mass, 0.02% to 15% by mass, 0.02% to 20% by mass, 0.02% to 25% by mass, 0.02% to 30% by mass, 0.02% to 50% by mass, 0.02% to 60% by mass, 0.0 2% to 70% by mass, 0.02% to 80% by mass, 0.02% to 90% by mass, 0.02% to 95% by mass, 0.02% to 100% by mass, 0.05% to 15% by mass, 0.05% to 20% by mass, 0.05% to 25% by mass, 0.05% to 30% by mass, 0.05% to 50% by mass, 0.05% to 60% by mass, 0.05% to 70% by mass, 0.05% to 80% by mass, 0.05% to 90% by mass, 0.05% to 90% by mass, 0.05% to 90% by mass. Less than 5% by mass, 0.05% to 100% by mass, 0.10% to 15% by mass, 0.10% to 20% by mass, 0.10% to 25% by mass, 0.10% to 30% by mass, 0.10% to 50% by mass, 0.10% to 60% by mass, 0.10% to 70% by mass, 0.10% to 80% by mass, 0.10% to 90% by mass, 0.10% to 95% by mass, 0.10% to 100% by mass, 0.30% to 15% by mass, 0.30% to 20% by mass.0.30% to 25% by mass, 0.30% to 30% by mass, 0.30% to 50% by mass, 0.30% to 60% by mass, 0.30% to 70% by mass, 0.30% to 80% by mass, 0.30% to 90% by mass, 0.30% to 95% by mass, 0.30% to 100% by mass, 1.0% to 15% by mass, 1.0% to 20% by mass, 1.0% to 25% by mass, 1.0% to 30% by mass, 1.0% to 50% by mass, 1.0% to 60% by mass Below, 1.0% to 70% of mass, 1.0% to 80% of mass, 1.0% to 90% of mass, 1.0% to 95% of mass, 1.0% to 100% of mass, 1.5% to 15% of mass, 1.5% to 20% of mass, 1.5% to 25% of mass, 1.5% to 30% of mass, 1.5% to 50% of mass, 1.5% to 60% of mass, 1.5% to 70% of mass, 1.5% to 80% of mass, 1.5% to 90% of mass, 1.5% to 95% of mass, 1.5% of mass... Quantity % ≥ 100% ≤ mass %, 2.0% ≥ 15% ≤ mass %, 2.0% ≥ 20% ≤ mass %, 2.0% ≥ 25% ≤ mass %, 2.0% ≥ 30% ≤ mass %, 2.0% ≥ 50% ≤ mass %, 2.0% ≥ 60% ≤ mass %, 2.0% ≥ 70% ≤ mass %, 2.0% ≥ 80% ≤ mass %, 2.0% ≥ 90% ≤ mass %, 2.0% ≥ 95% ≤ mass %, 2.0% ≥ 100% ≤ mass %, 2.5% ≥ 15% ≤ mass %, 2.5% ≥ 20% ≤ mass %, 2.5% ≥ 25% ≤ mass %, 2.5% ≥ 3 Less than 0% by mass, 2.5% to 50% by mass, 2.5% to 60% by mass, 2.5% to 70% by mass, 2.5% to 80% by mass, 2.5% to 90% by mass, 2.5% to 95% by mass, 2.5% to 100% by mass, 3.0% to 15% by mass, 3.0% to 20% by mass, 3.0% to 25% by mass, 3.0% to 30% by mass, 3.0% to 50% by mass, 3.0% to 60% by mass, 3.0% to 70% by mass, 3.0% to 80% by mass.3.0% to 90% of mass, 3.0% to 95% of mass, 3.0% to 100% of mass, 5.0% to 15% of mass, 5.0% to 20% of mass, 5.0% to 25% of mass, 5.0% to 30% of mass, 5.0% to 50% of mass, 5.0% to 60% of mass, 5.0% to 70% of mass, 5.0% to 80% of mass, 5.0% of mass... 5.0% to 90% of mass, 5.0% to 95% of mass, 5.0% to 100% of mass, 7.0% to 15% of mass, 7.0% to 20% of mass, 7.0% to 25% of mass, 7.0% to 30% of mass, 7.0% to 50% of mass, 7.0% to 60% of mass, 7.0% to 70% of mass, 7.0% to 80% of mass, 7.0% to 90% of mass. Below 100% of mass, 7.0% to 95% of mass, 7.0% to 100% of mass, 10.0% to 15% of mass, 10.0% to 20% of mass, 10.0% to 25% of mass, 10.0% to 30% of mass, 10.0% to 50% of mass, 10.0% to 60% of mass, 10.0% to 70% of mass, 10.0% to 80% of mass, 10.0% of mass Above 90% quality percentage and below, 10.0% quality percentage and above 95% quality percentage and below, 10.0% quality percentage and above 100% quality percentage and below, 80.0% quality percentage and above 90% quality percentage and below, 80.0% quality percentage and above 95% quality percentage and below, 80.0% quality percentage and above 100% quality percentage and below, 90.0% quality percentage and above 95% quality percentage and below, 90.0% quality percentage and above 100% quality percentage and below. Also, 95.0% quality percentage and above 100% quality percentage and below. (The rest of the text appears to be a series of seemingly unrelated characters and phrases, possibly from different sources.)

[0083] In the case of the liquid liquid composition in the form of this application form, the composition of this application form contains the するIL inducing ラクトバチルスのnumberは, 1.0 × 10 ３ Cells / mL or higher, 1.0 × 10⁻⁶ ４ Cells / mL or higher, 1.0 × 10⁻⁶ ５ Cells / mL or higher, 1.0 × 10⁻⁶ ６ Cells / mL or higher, 1.0 × 10⁻⁶ ７ Cells / mL or more and 4.0 × 10 ７ cells / mL or more, 1.0×10 １１Cells / mL or less, 1.0 x 10 １０ Cells / mL or less, 3.0 x 10 ９ Cells / mL or less or 1.0 × 10 ９ The number of cells / mL or less may also be less. These lower and upper limits can be combined in any way and are not particularly limited. For example, the number of IL-producing Lactobacillus contained in the composition according to this embodiment is 1.0 × 10 ３ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ３ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ３ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ３ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 1.0 x 10 ４ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ４ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ４ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ４ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 1.0 x 10 ５ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ５ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ５ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ５ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 1.0 x 10 ６ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ６ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ６ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ６Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 1.0 x 10 ７ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 1.0 x 10 ７ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 1.0 x 10 ７ Cells / mL or more 3.0 x 10 ９ Cells / mL or less, 1.0 x 10 ７ Cells / mL or more 1.0 x 10 ９ Cells / mL or less, 4.0 x 10 ７ Cells / mL or more 1.0 x 10 １１ Cells / mL or less, 4.0 x 10 ７ Cells / mL or more 1.0 x 10 １０ Cells / mL or less, 4.0 x 10 ７ Cells / mL or more 3.0 x 10 ９ Cells / mL or less or 4.0 × 10 ７ Cells / mL or more 1.0 x 10 ９ The amount may be cells / mL. In this case, the daily intake of the liquid composition may be 10 mL to 1,000 mL, 10 mL to 800 mL, 10 mL to 500 mL, 10 mL to 250 mL, 30 mL to 1,000 mL, 30 mL to 800 mL, 30 mL to 500 mL, 30 mL to 250 mL, 50 mL to 1,000 mL, 50 mL to 800 mL, 50 mL to 500 mL, 50 mL to 250 mL, 100 mL to 1,000 mL, 100 mL to 800 mL, 100 mL to 500 mL, or 100 to 250 mL.

[0084] In the composition according to this embodiment, the number of IL-producing Lactobacillus bacteria per unit package 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 ８or more, 1.0×10 ９ or more, 5.0×10 ９ or more, 1.0×10 １０ or more or 5.0×10 １０ or more and may be 1.0×10 １４ or less, 1.0×10 １３ or less, 1.0×10 １２ or 1.0×10 １１ or less. Also, these upper and lower limits can be arbitrarily combined and are not particularly limited. For example, in the composition according to this embodiment, the number of IL-producing induced Lactobacillus bacteria per unit package is 1.0×10 ５ or more and 1.0×10 １１ or less, 1.0×10 ５ or more and 1.0×10 １２ or less, 1.0×10 ５ or more and 1.0×10 １３ or less, 1.0×10 ５ or more and 1.0×10 １４ or less, 3.0×10 ５ or more and 1.0×10 １１ or less, 3.0×10 ５ or more and 1.0×10 １２ or less, 3.0×10 ５ or more and 1.0×10 １３ or less, 3.0×10 ５ or more and 1.0×10 １４ or less, 1.0×10 ６ or more and 1.0×10 １１ or less, 1.0×10 ６ or more and 1.0×10 １２ or less, 1.0×10 ６ or more and 1.0×10 １３ or less, 1.0×10 ６ or more and 1.0×10 １４ or less, 3.0×10 ６ or more and 1.0×10 １１ or less, 3.0×10 ６ or more and 1.0×10 １２ or less, 3.0×10 ６ or more and 1.0×10 １３ or less, 3.0×10 ６ or more and 1.0×10１４ Less than 1.0 × 10 ７ More than 1.0 × 10 １１ Less than 1.0 × 10 ７ More than 1.0 × 10 １２ Less than 1.0 × 10 ７ More than 1.0 × 10 １３ Less than 1.0 × 10 ７ More than 1.0 × 10 １４ Less than 3.0 × 10 ７ More than 1.0 × 10 １１ Less than 3.0 × 10 ７ More than 1.0 × 10 １２ Less than 3.0 × 10 ７ More than 1.0 × 10 １３ Less than 3.0 × 10 ７ More than 1.0 × 10 １４ Less than 1.0 × 10 ８ More than 1.0 × 10 １１ Less than 1.0 × 10 ８ More than 1.0 × 10 １２ Less than 1.0 × 10 ８ More than 1.0 × 10 １３ Less than 1.0 × 10 ８ More than 1.0 × 10 １４ Less than 3.0 × 10 ８ More than 1.0 × 10 １１ Less than 3.0 × 10 ８ More than 1.0 × 10 １２ Less than 3.0 × 10 ８ More than 1.0 × 10 １３ Less than 3.0 × 10 ８ More than 1.0 × 10 １４ Less than 5.0 × 10 ８ More than 1.0 × 10 １１ Less than 5.0 × 10 ８ More than 1.0 × 10 １２ Less than 5.0 × 10 ８ More than 1.0 × 10 １３ Less than 5.0 × 10 ８ More than 1.0 × 10 １４ Less than 1.0 × 10 ９ More than 1.0 × 10 １１ Less than 1.0 × 10 ９ More than 1.0 × 10 １２Less than or equal to 1.0×10 ９ pcs or more 1.0×10 １３ Less than or equal to 1.0×10 ９ pcs or more 1.0×10 １４ Less than or equal to 5.0×10 ９ pcs or more 1.0×10 １１ Less than or equal to 5.0×10 ９ pcs or more 1.0×10 １２ Less than or equal to 5.0×10 ９ pcs or more 1.0×10 １３ Less than or equal to 5.0×10 ９ pcs or more 1.0×10 １４ Less than or equal to 1.0×10 １０ pcs or more 1.0×10 １１ Less than or equal to 1.0×10 １０ pcs or more 1.0×10 １２ Less than or equal to 1.0×10 １０ pcs or more 1.0×10 １３ Less than or equal to 1.0×10 １０ pcs or more 1.0×10 １４ Less than or equal to 5.0×10 １０ pcs or more 1.0×10 １１ Less than or equal to 5.0×10 １０ pcs or more 1.0×10 １２ Less than or equal to 5.0×10 １０ pcs or more 1.0×10 １３ 1 or less or 5.0 x 10 １０ pcs or more 1.0×10 １４ It may be one or fewer.

[0085] The composition according to this embodiment is preferably used for oral administration from the viewpoint of reducing the burden of intake. When used for oral administration, the IL-producing Lactobacillus or the composition containing it may have high resistance to gastric juice and intestinal juice, etc., and is not particularly limited, but for example, it is preferable to have strong acid resistance. The IL-producing Lactobacillus is not particularly limited, and either live or dead bacteria can be used, but dead bacteria are preferred from the viewpoint of interleukin production induction effect, stability and manufacturing efficiency, and heat-killed dead bacteria (heat-treated bacteria) are more preferred.

[0086] The compositions according to this embodiment can be administered orally to humans and non-human mammals, with food compositions being a typical form of administration. The provided food composition contains an effective amount of IL-producing Lactobacillus. Here, "contained in an effective amount" means that when the amount normally consumed in each food composition is taken, the active ingredient of the present invention is taken in such an amount that the effects of inducing interleukin production, etc., are exerted. 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 active ingredient of the present invention is administered to mammals other than humans, the food referred to in this invention is used as feed.

[0087] The composition according to this embodiment has effects such as inducing interleukin production, and can therefore be provided in foods consumed on a daily basis. 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. The unit package form per serving is not particularly limited, but examples include forms that specify a fixed amount in a pack, packaging, can, or bottle. In order to better exert the various effects of the composition according to this embodiment, the amount to be consumed per serving may be determined according to the daily intake amount of the active ingredient of the present invention, 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.

[0088] 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) intakes. Therefore, the unit packaging form of the composition according to this embodiment can contain the active ingredient of the present invention in the amount of the daily intake amount described later, or it can contain the active ingredient of the present invention in an amount of one-half to one-sixth of the amount of the daily intake amount described later. For the convenience of intake, it is preferable to provide the composition according to this embodiment in a unit packaging form per serving (i.e., a unit packaging form per day) in which the intake amount per serving is the effective daily intake amount.

[0089] The composition according to this embodiment can be administered to subjects who require the induction of interleukin production. Subjects who require the induction of interleukin production are not particularly limited, but examples include subjects infected with a virus, subjects with a cold, subjects aged 65 or older, and subjects who are routinely exposed to blue light.

[0090] The form of the "food composition" in the composition according to this embodiment is not particularly limited, but 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 kneading the active ingredient of the composition according to this embodiment with excipients, binders, etc., and then compressing them; granules produced by granulating the active ingredient with excipients, binders, etc.; orally disintegrating tablets; and capsules containing the active ingredient 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. The latter unit packaging is not particularly limited, but it is preferable, for example, to display the per-serving or per-day intake amount so that the consumer can effectively ingest the active ingredient of the present invention according to that indication.

[0091] Examples of food compositions provided 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 the function of inducing interleukin production. Such food compositions are not particularly limited, but for example, they can be labeled as follows. - "Reduces eye fatigue for those experiencing eye strain" - "For those experiencing eye strain" - "Reduces eye strain caused by computers, etc." - "Reduces eye fatigue" - "Protects eyes from light stimulation such as blue light" - "Protects eyes exposed to light stimulation and improves eye condition" - "Maintains, improves, supports, keeps normal visual function from light stimulation such as blue light, helps maintain visual function, reduces eye function decline" - "Relieves, reduces, and prevents eye fatigue from light stimulation such as blue light" - "Relieves, alleviates, reduces eye strain caused by light stimulation such as blue light, makes eyes less prone to fatigue, and reduces blurred vision" - "Relieves, reduces, and prevents shoulder and lower back stiffness and pain caused by eye strain from light stimulation such as blue light" - "Maintains, improves, supports, maintains, and keeps normal eye moisture from light stimulation such as blue light" - "Relieves, reduces, and prevents dry eyes from light stimulation such as blue light" - "Relieves dry eyes caused by light stimulation such as blue light, maintains, improves, supports, and keeps eye function normal" - "Maintains, improves, supports, and keeps normal focusing ability which is reduced by light stimulation such as blue light" - "Maintains, improves, supports, and keeps normal contrast sensitivity which is reduced by light stimulation such as blue light" - "Prevents smartphone-induced presbyopia" - "Fights smartphone-induced presbyopia" - "Improves eye condition which is reduced by light stimulation such as blue light" - "For those with hay fever symptoms" - "Improves the quality of life for those with hay fever symptoms" - "Relieves eye and nasal discomfort caused by pollen, dust, house dust, etc." - "Reduces interference with outdoor activities caused by pollen, dust, house dust, etc." - "For those concerned about gout" - "For those with high uric acid levels" - "For those concerned about joint pain" - "For those who need joint care"

[0092] The food composition provided as a composition according to this embodiment is not particularly limited as long as it contains an active ingredient, but examples 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, fresh cream made from dairy products or other oil and fat raw materials, etc. Dairy products such as 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 cream such as jelly or pudding; various confectionery such as snack foods; alcoholic beverages 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 using eggs, processed foods of seafood or livestock (including offal such as liver) (including delicacies), processed foods such as soups such as miso soup, seasonings such as miso, soy sauce, furikake, and other seasonings, or liquid foods such as concentrated liquid foods.

[0093] Tea beverages include, but are not particularly limited to, fermented teas, semi-fermented teas, and unfermented teas. Examples include 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 (e.g., mint tea, jasmine tea).

[0094] The fruits used in fruit juice beverages and beverages containing both fruit and vegetable juices are not particularly limited, but examples include apples, oranges, grapes, bananas, pears, peaches, mangoes, acai, blueberries, and plums. Similarly, the vegetables used in vegetable juice beverages and beverages containing both fruit and vegetable juices are not particularly limited, but examples include tomatoes, carrots, celery, pumpkins, cucumbers, and watermelons.

[0095] 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.

[0096] 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.

[0097] When the composition according to this embodiment is used as a pharmaceutical composition or quasi-drug, the target diseases are not particularly limited, but include, for example, inflammation caused by inflammasome activation and inflammation caused by autoinflammatory syndromes (syndromes that do not conform to immune diseases such as autoimmunity and allergies). Autoinflammatory syndromes in a narrow sense include familial Mediterranean fever (FMF), TNF receptor-associated periodic syndromes (TRAPS), hyper-IgD syndrome / mevalonate kinase deficiency (MKD), cryopyrin-associated periodic fever syndrome (CAPS), Blau syndrome / juvenile-onset sarcoidosis, suppurative aseptic arthritis, gangrenous urea / acne syndrome (PAPA group), Majeed syndrome, NLRP12-associated periodic fever syndrome (NAPS12), IL-1 receptor antagonist deficiency, CARD14 deficiency, phospholipase Cγ2-associated antibody deficiency, immune disorders (PLAID), and HOIL-1 deficiency. Furthermore, autoinflammatory syndromes in a broad sense include periodic fever-aphthous stomatitis-pharyngitis-cervical lymphadenitis syndrome (PFAPA), systemic juvenile idiopathic arthritis, adult-onset Still's disease, Crohn's disease, crystal deposition-induced arthropathy (including gout and pseudogout), asbestosis / silicosis, Schnitzler syndrome, and type 2 diabetes (Hiroaki Ida, Journal of the Japanese Society of Internal Medicine, Vol. 104, No. 9, pp. 1964-1973 (2015) and Medicina, 41(12):340-345 (2004)).

[0098] The composition according to this embodiment may be a bacterial cell powder of IL-producing Lactobacillus (a powder obtained by drying bacterial cells or a powder containing the same). Such a bacterial cell powder is not particularly limited, but can be obtained, for example, by culturing, sterilizing, and drying bacteria as appropriate.

[0099] The bacterial cell powder of this embodiment may contain, in addition to IL-producing Lactobacillus and components derived from the culture medium used during bacterial culture, components that are acceptable as food, pharmaceuticals or feed, and are not particularly limited, but 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, colorants, preservatives, sustained-release regulators, surfactants, and solubilizers.

[0100] 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.

[0101] 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 a functional food having an interleukin production-inducing effect.

[0102] The amount of 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 which it is combined. The daily intake of the composition according to this embodiment for an adult is not particularly limited, but can be specified, for example, by the number of IL-producing Lactobacillus bacteria, which is the active ingredient, with a lower limit of 1 × 10⁻⁶ ８ pieces, 1×10 ９ individual or 1 x 10 １０ It can be one, and the upper limit is 1 x 10 １４ pieces, 1×10 １３ individual or 1 x 10 １２ It can be set to 1 x 10. These upper and lower limits can be combined arbitrarily, and the range of intake is not particularly limited, but for example, 1 x 10 ８ More than 1×10 pieces １４ Less than or equal to 1 x 10 ８ More than 1×10 pieces １３ Less than or equal to 1 x 10 ８More than 1×10 pieces １２ Less than or equal to 1 x 10 ９ More than 1×10 pieces １４ Less than or equal to 1 x 10 ９ More than 1×10 pieces １３ Less than or equal to 1 x 10 ９ More than 1×10 pieces １２ Less than or equal to 1 x 10 １０ More than 1×10 pieces １４ Less than or equal to 1 x 10 １０ More than 1×10 pieces １３ or less or 1 x 10 １０ More than 1×10 pieces １２ The number of IL-producing Lactobacillus bacteria can be measured using known microscopes, flow cytometers, or rapid non-culture microorganism testing devices (e.g., ELESTA PixeeMo (AFI Technology Co., Ltd.)), but measurement by microscope is preferred from the viewpoint of high versatility.

[0103] The daily intake of the composition according to this embodiment for adults is not particularly limited, but can be determined, for example, by the dry cell mass of the active ingredient, IL-producing Lactobacillus, with a lower limit of 2.5 × 10⁻⁶. －２ mg, 2.5 x 10 －１ It can be mg or 2.5 mg, with an upper limit of 2.5 × 10 ４ mg, 2.5 x 10 ３ mg or 2.5 x 10 ２ It can be expressed as mg. These upper and lower limits can be combined arbitrarily, and the above range of intake is not particularly limited, but for example, 2.5 × 10 －２ mg or more 2.5×10 ４ mg or less, 2.5×10 －２ mg or more 2.5×10 ３ mg or less, 2.5×10 －２ mg or more 2.5×10 ２ mg or less, 2.5×10 －１ mg or more 2.5×10 ４ mg or less, 2.5×10 －１ mg or more 2.5×10 ３ mg or less, 2.5×10 －１ mg or more 2.5×10 ２mg or less, 2.5 mg or more 2.5 x 10 ４ mg or less, 2.5 mg or more 2.5 x 10 ３ mg or less or 2.5 mg or more, 2.5 x 10 ２ It can be reduced to mg or less.

[0104] The amount of the composition to be ingested according to this embodiment, as well as the timing and duration of ingestion described below, apply whether the composition according to this embodiment is used for non-therapeutic or therapeutic purposes. In the case of therapeutic purposes, ingestion can be replaced with administration.

[0105] The composition according to this embodiment is preferably taken continuously for the period during which the interleukin production-inducing effect is expected. The period of intake of the active ingredient of the present invention is not particularly limited from the viewpoint of better exhibiting the interleukin production-inducing effect, but for example, the intake of the above daily amount can be for one week or more, two weeks or more, three weeks or more, preferably one month or more (four weeks or more). The intake interval of the active ingredient of the present invention is not particularly limited, but can be once every three days, once every two days, or once a day, but for example, once a day.

[0106] The composition according to this embodiment may also be taken before an event or time in which an interleukin production-inducing effect is expected. Examples of events in which an interleukin production-inducing 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 an epidemic area), and examples of times in which an interleukin production-inducing effect is expected include the peak season for viral infections. The timing of taking the composition before an event in which an interleukin production-inducing effect is expected is not particularly limited, but examples include one day or more before, three days or more before, one week or more before, two weeks or more before, three weeks or more before, one month or more before (four weeks or more before), or two months or more before (eight weeks or more before). Furthermore, although not particularly limited, the composition may be taken continuously with intervals between intakes from the start of intake until the event in which an interleukin production-inducing effect is expected. The active ingredient of the present invention may also be taken after an event or time in which an interleukin production-inducing effect is expected. The timing of intake after an event in which an interleukin production-inducing effect is expected is not particularly limited, but examples include one day or more later, three days or more later, one week or more later, or two weeks or more later. Also, although not particularly limited, when taking the active ingredient after an event in which an interleukin production-inducing effect is expected, it may be possible to take it continuously with intervals between intakes. In the present invention, it is particularly preferable to start taking the active ingredient of the present invention before an event in which an interleukin production-inducing effect is expected, and to continue taking it after the event.

[0107] <Method for Producing Lactobacillus Bacteria> Next, a method for producing Lactobacillus bacteria according to one embodiment of this disclosure will be described. In this embodiment, although not particularly limited, IL-producing Lactobacillus can be produced, for example. Hereinafter, Lactobacillus bacteria that serve as raw materials for the method of production of this embodiment (i.e., Lactobacillus bacteria before the culture step described later) will also be referred to as "raw material bacteria," and Lactobacillus bacteria that are ultimately produced by the method of production of this embodiment will also be referred to as "target bacteria."

[0108] The biological classification of the raw material bacteria is the same as that described for IL-producing Lactobacillus. That is, the raw material bacteria may be Lactobacillus species as described in the section on "IL-producing Lactobacillus," and in one embodiment it may be Lactobacillus paracasei, in a preferred embodiment it may be Lactobacillus paracasei KW3110 or a mutant thereof, and in a more preferred embodiment it may be Lactobacillus paracasei KW3110.

[0109] The manufacturing method of this embodiment includes a step of culturing Lactobacillus bacteria in a medium maintained at a pH of 4.5 to 7.0 (cultivation step). The inventors have found that culturing Lactobacillus bacteria in a medium maintained at a pH of 4.5 to 7.0 tends to increase the ability to induce interleukin production in bacteria compared to culturing them without maintaining a pH of 4.5 to 7.0. In addition to maintaining a specific pH, "maintaining" the pH in the culturing step also includes controlling the pH so that it remains within a predetermined range, that is, controlling it so that the pH does not fall below the lower limit or exceed the upper limit.

[0110] The culture medium used in the culture process may be any medium that a person skilled in the art might use for culturing bacteria, and in one embodiment, it may be MRS medium.

[0111] The pH at which the culture medium is maintained during the culture process is not particularly limited, but may be, for example, 4.5 or higher, 4.8 or higher, 5.0 or higher, 5.3 or higher, 5.5 or higher, or 5.8 or higher, and may also be 7.0 or lower, 6.7 or lower, 6.5 or lower, or 6.2 or lower. The pH in the culture process is not particularly limited, but may be, for example, 4.5 to 7.0, 4.5 to 6.7, 4.5 to 6.5, 4.5 to 6.2, 4.8 to 7.0, 4.8 to 6.7, 4.8 to 6.5, 4.8 to 6.2, 5.0 to 7.0, 5.0 to 6.7, 5.0 to 6.5, 5.0 to 6.2, 5.3 to 7.0, 5.3 to 6.7, 5.3 to 6.5, 5.3 to 6.2, 5.5 to 7.0, 5.5 to 6.7, 5.5 to 6.5, 5.5 to 6.2, 5.8 to 7.0, 5.8 to 6.7, 5.8 to 6.5, or 5.8 to 6.2. Among these, from the viewpoint of easily increasing the ability to induce interleukin production, the pH in the culture step according to one embodiment may be 5.0 or more and 6.5 or less, and the pH in the culture step according to a preferred embodiment may be 5.5 or more and 6.5 or less. Maintaining the pH in the culture step is not particularly limited, but for example, it can be done by measuring the pH over time in the culture step, adding the necessary amount of base (e.g., sodium hydroxide) to the culture medium when the pH decreases, and adding the necessary amount of acid (e.g., phosphoric acid) to the culture medium when the pH increases.

[0112] The culture time in the culture process is not particularly limited, but may be, for example, 1 hour or more, 3 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, 16 hours or more, or 18 hours or more, and may be 336 hours or less, 168 hours or less, 72 hours or less, or 48 hours or less. The following are possible: 10 hours to 72 hours, 10 hours to 48 hours, 12 hours to 336 hours, 12 hours to 168 hours, 12 hours to 72 hours, 12 hours to 48 hours, 14 hours to 336 hours, 14 hours to 168 hours, 14 hours to 72 hours, 14 hours to 48 hours, 16 hours to 336 hours, 16 hours to 168 hours, 16 hours to 72 hours, 16 hours to 48 hours, 18 hours to 336 hours, 18 hours to 168 hours, 18 hours to 72 hours, or 18 hours to 48 hours. In one embodiment, the culture time in the culture step may be 3 hours or more. If the maintenance of pH in the culture step is achieved by controlling the pH to stay within a predetermined range, the culture time in the culture step refers to the time during which the pH stays within that predetermined range.

[0113] The culture temperature in the culture process is not particularly limited, but may be, for example, 20°C or higher, 22°C or higher, 25°C or higher, or 30°C or higher, and may be 45°C or lower, 40°C or lower, 38°C or lower, 35°C or lower, or 33°C or lower, and as an example, it may be 32°C.

[0114] In the culture process, air may be aerated into the culture medium. The air aerated is any gas containing 10% to 30% by volume of oxygen. In one embodiment, it may be air from the atmosphere or a gas whose oxygen concentration has been adjusted to a range of 10% to 30% by volume. In a preferred embodiment, it may be air from the atmosphere. The amount of air aerated in the culture process is not particularly limited, but may be, for example, 0.10 vvm to 10.0 vvm or 0.30 vvm to 3.0 vvm.

[0115] The concentration of the raw material bacteria at the start of the culture process is not particularly limited, based on the total volume of the culture medium, but may be, for example, 0.0010% by volume or more, 0.010% by volume or more, or 0.030% by volume or more, and may be 10.0% by volume or less, 1.0% by volume or less, or 0.30% by volume or less. These upper and lower limits can be combined arbitrarily, and the concentration of raw material bacteria at the start of the culture process may be, for example, 0.0010% by volume or more and 10.0% by volume or less, 0.0010% by volume or more and 1.0% by volume or less, 0.0010% by volume or more and 0.30% by volume or less, 0.010% by volume or more and 10.0% by volume or less, 0.010% by volume or more and 1.0% by volume or less, 0.010% by volume or more and 0.30% by volume or less, 0.030% by volume or more and 10.0% by volume or less, or 0.030% by volume or more and 0.30% by volume or less, based on the total amount of culture medium.

[0116] The manufacturing method of this embodiment may include a step of inactivating the cultured Lactobacillus bacteria after the culture step (inactivation step). When the manufacturing method of this embodiment includes an inactivation step, the Lactobacillus bacteria are immobilized in a state where their interleukin production induction ability is enhanced during the culture step, thus making it easier to increase the interleukin production induction ability of the target bacteria.

[0117] The method for killing Lactobacillus bacteria in the sterilization process is not particularly limited, as long as it can kill Lactobacillus bacteria, but may include, for example, heat treatment, ultraviolet irradiation, pressurization, high-pressure steam treatment, electromagnetic wave treatment, electron beam treatment, radiation treatment (e.g., irradiation with radiation such as gamma rays), ultraviolet treatment, alcohol treatment, electrolyzed water treatment, drug treatment (e.g., treatment with drugs such as antibiotics), or chemical treatment (e.g., treatment with chemical substances such as formalin). From the viewpoint of easily increasing the ability of the target bacteria to induce interleukin production, in one preferred embodiment, the method for killing Lactobacillus bacteria in the sterilization process may be heat treatment. The heat treatment is not particularly limited, but can be carried out by a heating device with a temperature setting, such as a plate sterilizer, a tubular sterilizer, a direct heating sterilizer, a jacketed tank, and an autoclave.

[0118] When the method of sterilization is heat treatment, the temperature in the heat treatment is not particularly limited, but may be, for example, 60°C or higher, 65°C or higher, 70°C or higher, or 75°C or higher, and may be less than 100°C, 95°C or lower, 90°C or lower, or 85°C or lower. When the method of sterilization is heat treatment, the temperature in the heat treatment is not particularly limited, but may be, for example, 60°C or higher but less than 100°C, 60°C or higher but less than 95°C, 60°C or higher but less than 90°C, 60°C or higher but less than 85°C, 65°C or higher but less than 100°C, 65°C or higher but less than 95°C, 65°C or higher but less than 90°C, 65°C or higher but less than 85°C, 70°C or higher but less than 100°C, 70°C or higher but less than 95°C, 70°C or higher but less than 90°C, 70°C or higher but less than 85°C, 75°C or higher but less than 100°C, 75°C or higher but less than 95°C, 75°C or higher but less than 90°C, or 75°C or higher but less than 85°C.

[0119] If the method of sterilization is heat treatment, the heating time in the heat treatment is not particularly limited, but may be, for example, 1 minute or more, 4 minutes or more, or 10 minutes or more, and may be 6 hours or less, 1 hour or less, or 30 minutes or less. If the method of sterilization is heat treatment, the heating time in the heat treatment is not particularly limited, but may be, for example, 1 minute or more and 6 hours or less, 1 minute or more and 1 hour or less, 1 minute or more and 30 minutes or less, 4 minutes or more and 6 hours or less, 4 minutes or more and 1 hour or less, 4 minutes or more and 30 minutes or less, 10 minutes or more and 6 hours or less, 10 minutes or more and 1 hour or less, or 10 minutes or more and 30 minutes or less.

[0120] If the method of sterilization is heat treatment, the heat treatment may be carried out in the form of a suspension or a powder, and in one embodiment, it may be carried out in the form of a suspension. When the heat treatment is carried out in the form of a suspension, the solvent is not particularly limited, but may be, for example, pure water.

[0121] The manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH below the lower limit of the pH range in which the culture medium is maintained during the culture process (low pH culture step). That is, for example, if the pH of the medium is maintained at 4.5 or higher during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH of less than 4.5. Also, for example, if the pH of the medium is maintained at 4.8 or higher during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH of less than 4.8. Also, for example, if the pH of the medium is maintained at 5.0 or higher during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH of less than 5.0. Also, for example, if the pH of the medium is maintained at 5.3 or higher during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH of less than 5.3. Furthermore, for example, if the pH of the culture medium is maintained at 5.5 or higher during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH of less than 5.5. Also, for example, if the pH of the culture medium is maintained at 5.8 or higher during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH of less than 5.8. If the manufacturing method of this embodiment does not include a low-pH culture step, the interleukin production induction ability of the target bacteria tends to be higher. The fact that the manufacturing method of this embodiment does not include a low pH culture step means that the time spent culturing in a medium with a pH below the lower limit of the pH range in which the medium is maintained during the culture step is not particularly limited, but may be, for example, less than 16 hours, less than 14 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 1 minute, or 0 minutes, and in one embodiment, such a time may mean 0 minutes.Furthermore, the statement that the manufacturing method of this embodiment does not include a low-pH culture step means that the ratio of the time spent culturing in a medium with a pH below the lower limit of the pH range in which the medium is maintained during the culture step, relative to the total culture time in the culture step, is not particularly limited, but may be, for example, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, or 0%.

[0122] The manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH exceeding the upper limit of the pH range in which the medium is maintained during the culture process (high pH culture step). That is, for example, if the pH of the medium is maintained at 7.0 or below during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH greater than 7.0. Also, for example, if the pH of the medium is maintained at 6.7 or below during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH greater than 6.7. Also, for example, if the pH of the medium is maintained at 6.5 or below during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH greater than 6.5. Also, for example, if the pH of the medium is maintained at 6.2 or below during the culture process, the manufacturing method according to one aspect of this embodiment does not need to include a step of culturing Lactobacillus bacteria in a medium with a pH greater than 6.2. If the manufacturing method of this embodiment does not include a high pH culture step, the ability to induce interleukin production of the target bacteria tends to be higher. The absence of a high pH culture step in the manufacturing method of this embodiment means that the time spent culturing in a medium with a pH exceeding the upper limit of the pH range in which the medium is maintained in the culture step is not particularly limited, but may be, for example, less than 16 hours, less than 14 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 1 minute, or 0 minutes, and in one embodiment, it may mean that such a time is 0 minutes. Furthermore, the absence of a high pH culture step in the manufacturing method of this embodiment means that the ratio of the time spent culturing in a medium with a pH exceeding the upper limit of the pH range in which the medium is maintained in the culture step to the total culture time in the culture step is not particularly limited, but may be, for example, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, or 0%.

[0123] A manufacturing method according to one aspect of this embodiment may include a washing step (washing step) for Lactobacillus bacteria after the culture step and before the inactivation step. The washing method in the washing step is not particularly limited, but in a preferred embodiment, it may be washing by centrifugation (centrifugal washing). In centrifugal washing, the culture solution after the culture step is centrifuged to precipitate the bacteria, then a portion or all of the supernatant is removed, and instead, a washing solution may be added in the same amount as the removed supernatant. Subsequently, the bacteria are washed by repeating centrifugation, removal of a portion or all of the supernatant, and addition of the washing solution. The washing solution may be one that can be normally used to wash bacteria, and is not particularly limited, but may be, for example, pure water or a buffer such as phosphate buffer. The number of washings in the washing step (i.e., the number of centrifugations) is not particularly limited, but may be, for example, 1, 2, 3, 4, or 5 times. The centrifugal separation speed may be any of the conditions typically used to precipitate bacteria, and is not particularly limited, but may be, for example, 1,000 to 10,000 rpm, or 5,000 rpm as an example.

[0124] A manufacturing method according to one aspect of this embodiment may include a step of drying the Lactobacillus bacteria (drying step) after the sterilization step. The drying treatment in the drying step is not particularly limited, but may be, for example, spray drying or freeze-drying. Spray drying may be performed using a spray dryer used for spray drying bacteria.

[0125] As described above, the manufacturing method according to one aspect of this embodiment is not particularly limited, but for example, it may include a culture step and a sterilization step in this order, or for example, a culture step, a sterilization step and a drying step in this order, or for example, a culture step, a washing step, a sterilization step and a drying step in this order.

[0126] In one aspect of this embodiment, the immunomodulatory function of Lactobacillus bacteria may be enhanced. In one aspect of this embodiment, the immunosuppressive function of Lactobacillus bacteria may be enhanced, specifically, the immunomodulatory function of Lactobacillus bacteria immediately after the culture step may be higher than that immediately before the culture step. In one aspect of this embodiment, the cytokine production induction ability of Lactobacillus bacteria may be enhanced. In one aspect of this embodiment, the interleukin production induction ability of Lactobacillus bacteria may be enhanced. That is, the target bacteria may have a higher interleukin production induction ability than the raw material bacteria. For example, in the production method of this embodiment, the raw material bacteria may not be IL-producing Lactobacillus, and the target bacteria may be IL-producing Lactobacillus. Also, as described in Patent Document 1, etc., interleukins are involved in the suppression or improvement of eye fatigue, so in one aspect of this embodiment, the eye fatigue suppression or improvement function of Lactobacillus bacteria may be enhanced. Furthermore, as described in Patent Document 2 and other documents, interleukins are involved in anti-allergic function. Therefore, in the manufacturing method according to one aspect of this embodiment, the inhibitory function of Lactobacillus bacteria on inflammation (for example, inflammation caused by activation of the inflammasome) may be enhanced, and the anti-allergic function of Lactobacillus bacteria may be enhanced.

[0127] In one aspect of this embodiment, the interleukin-10 production-inducing ability of Lactobacillus bacteria may be enhanced. That is, the target bacteria may have a higher interleukin-10 production-inducing ability than the raw material bacteria. For example, in the production method of this embodiment, the raw material bacteria may not be Lactobacillus that induces IL-10 production, and the target bacteria may be Lactobacillus that induces IL-10 production. Furthermore, for example, in the manufacturing method of this embodiment, the IL-10 production induction ability of the target bacteria (Lactobacillus bacteria produced) may be, but is not limited to, 1.05 times or more, 1.10 times or more, 1.20 times or more, 1.30 times or more, 1.40 times or more, 1.50 times or more, 1.60 times or more, 1.70 times or more, 1.80 times or more, 1.90 times or more, 2.00 times or more, 3.00 times or more, 4.00 times or more, 5.00 times or more, 7.00 times or more, 10.0 times or more, 20.0 times or more, 30.0 times or more, 40.0 times or more, or 50.0 times or more, and may also be 10,000 times or less, 1,000 times or less, 100 times or less, 30.0 times or less, 10.0 times or less, or 6.00 times or less. In this case, the IL-10 production induction ability may be evaluated according to the method described in the section on "IL-10 production induction Lactobacillus." Furthermore, since interleukin-10 is known to be involved in the regulation of immunity, the manufacturing method according to one aspect of this embodiment may enhance the immunomodulatory function of Lactobacillus bacteria. For example, the immunomodulatory function of Lactobacillus bacteria immediately after the culture step may be higher than that immediately before the culture step.

[0128] In one embodiment of this manufacturing method, the ability of Lactobacillus bacteria to bind to Dectin-2 may be enhanced. That is, the target bacteria may have a higher ability to bind to Dectin-2 than the raw material bacteria. For example, in the manufacturing method of this embodiment, the raw material bacteria may not be Dectin-2 binding Lactobacillus, and the target bacteria may be Dectin-2 binding Lactobacillus. Furthermore, for example, in the manufacturing method of this embodiment, the binding ability of the target bacteria (Lactobacillus bacteria produced) to Dectin-2 is not particularly limited to, but may be, for example, 1.05 times or more, 1.10 times or more, 1.20 times or more, 1.30 times or more, 1.40 times or more, 1.50 times or more, 1.60 times or more, 1.70 times or more, 1.80 times or more, 1.80 times or more, 1.90 times or more, 2.00 times or more, or 3.00 times or more, and may also be 100 times or less, 30.0 times or less, 10.0 times or less, or 6.00 times or less. In this case, the binding ability to Dectin-2 may be the binding parameter obtained according to the binding assay described in the section on "Dectin-2-binding Lactobacillus," and may be, for example, the percentage of Lactobacillus bacteria that have been bound to Dectin-2, obtained in this manner.

[0129] In one embodiment of the manufacturing method, the phagocytic ability of Lactobacillus bacteria by macrophages may be enhanced. That is, the target bacteria may have a higher phagocytic ability by macrophages than the raw material bacteria. For example, in the manufacturing method of this embodiment, the raw material bacteria may not be "Dectin-2 binding Lactobacillus evaluated using the amount of macrophage phagocytosis as an indicator," and the target bacteria may be "Dectin-2 binding Lactobacillus evaluated using the amount of macrophage phagocytosis as an indicator." Also, for example, in the manufacturing method of this embodiment, the phagocytic ability of the target bacteria (the Lactobacillus bacteria produced) by macrophages may be, without particular limitation, 1.05 times or more, 1.10 times or more, 1.20 times or more, 1.30 times or more, 1.40 times or more, 1.50 times or more, 1.60 times or more, 1.7 times or more than the phagocytic ability of the raw material bacteria (for example, Lactobacillus bacteria used in the culture process). It may be 0 times or more, 1.80 times or more, 1.80 times or more, 1.90 times or more, 2.00 times or more, 3.00 times or more, 4.00 times or more, 5.00 times or more, 7.00 times or more, 10.0 times or more, 20.0 times or more, 30.0 times or more, 40.0 times or more, or 50.0 times or more, and may be 10,000 times or less, 1,000 times or less, 100 times or less, 30.0 times or less, 10.0 times or less, or 6.00 times or less. In this case, the phagocytic capacity by macrophages is not particularly limited, but for example, the parameter of macrophage phagocytosis amount, as explained in the section "Dectin-2 binding Lactobacillus evaluated using macrophage phagocytosis amount as an indicator," may be used as an indicator.

[0130] In one embodiment of this manufacturing method, the ability of Lactobacillus bacteria to promote Syk expression may be enhanced. That is, the target bacteria may have a higher ability to promote Syk expression than the raw material bacteria. For example, in the manufacturing method of this embodiment, the Syk expression promoting ability of the target bacteria (Lactobacillus bacteria produced) may be, but is not particularly limited, 1.70 times or more, 1.80 times or more, 1.80 times or more, 1.90 times or more, 2.00 times or more, 2.10 times or more, 2.20 times or more, 2.30 times or more, 2.40 times or more, 2.50 times or more, 2.60 times or more, 2.70 times or more, 2.80 times or more, 2.90 times or more, 3.00 times or more, 3.10 times or more, or 3.20 times or more, and may also be 100 times or less, 30.0 times or less, 10.0 times or less, 6.00 times or less, or 4.00 times or less. The ability to promote Syk expression is not particularly limited, but for example, the expression level of Syk or the mRNA encoding it in macrophages that have been exposed to Lactobacillus species according to the evaluation method for IL-10 production induction described in the section on "IL-10 production-inducing Lactobacillus" may be used as an indicator.

[0131] In one embodiment of this manufacturing method, the PHT2 expression-promoting ability of Lactobacillus bacteria may be enhanced. That is, the target bacteria may have a higher PHT2 expression-promoting ability than the raw material bacteria. For example, in the manufacturing method of this embodiment, the PHT2 expression-promoting ability of the target bacteria (the Lactobacillus bacteria produced) may be, but is not particularly limited, 8.00 times or more, 9.00 times or more, 10.0 times or more, 11.0 times or more, 12.0 times or more, 13.0 times or more, 14.0 times or more, 15.0 times or more, 16.0 times or more, 17.0 times or more, 18.0 times or more, or 19.0 times or more of the PHT2 expression-promoting ability of the raw material bacteria (for example, Lactobacillus bacteria used in the culture step), and may be 1000 times or less, 100 times or less, 60.0 times or less, or 30.0 times or less. The PHT2 expression-promoting ability is not particularly limited, but for example, the expression level of PHT2 or the expression level of SLC15A3 mRNA, which is the mRNA encoding PHT2, in macrophages that have been exposed to Lactobacillus species according to the evaluation method for IL-10 production induction ability described in the section on "IL-10 production-inducing Lactobacillus" may be used as an indicator.

[0132] In one embodiment of this manufacturing method, the Dectin-2 expression promoting ability of Lactobacillus bacteria may be enhanced. That is, the target bacteria may have a higher Dectin-2 expression promoting ability than the raw material bacteria. For example, in the manufacturing method of this embodiment, the Dectin-2 expression promoting ability of the target bacteria (the Lactobacillus bacteria produced) may be, but is not particularly limited, 1.10 times or more, 1.20 times or more, 1.30 times or more, 2.00 times or more, 2.50 times or more, 3.00 times or more, or 4.00 times or more of the Dectin-2 expression promoting ability of the raw material bacteria (for example, Lactobacillus bacteria used in the culture step), and may also be 1000 times or less, 100 times or less, 10.0 times or less, or 5.00 times or less. The Dectin-2 expression-promoting ability is not particularly limited, but for example, the expression level of Dectin-2 or the expression level of Clec4n mRNA, which is the mRNA encoding Dectin-2, in macrophages that have been exposed to Lactobacillus species according to the evaluation method for IL-10 production induction ability described in the section on "IL-10 production-inducing Lactobacillus" may be used as an indicator.

[0133] Furthermore, another embodiment of the present disclosure may include a method for enhancing the immunomodulatory function of Lactobacillus bacteria, including a culture step; a method for enhancing the immunosuppressive function of Lactobacillus bacteria, including a culture step; a method for enhancing the cytokine production induction ability of Lactobacillus bacteria, including a culture step; a method for enhancing the interleukin production induction ability of Lactobacillus bacteria, including a culture step; a method for enhancing the interleukin-10 production induction ability of Lactobacillus bacteria, including a culture step; a method for enhancing the Dectin-2 binding ability of Lactobacillus bacteria, including a culture step; a method for enhancing the phagocytic ability of Lactobacillus bacteria by macrophages, including a culture step; a method for enhancing the Syk expression promotion ability of Lactobacillus bacteria, including a culture step; and a method for enhancing the PHT2 expression promotion ability of Lactobacillus bacteria, including a culture step.

[0134] <Use of Lactobacillus species or compositions containing the same> Other embodiments of the present disclosure relate to the use of IL-producing Lactobacillus species or compositions containing the same in the modulation of immunity or induction of interleukin production. Detailed aspects of the IL-producing Lactobacillus species, compositions and their uses according to the present embodiment are as described above for Lactobacillus species and compositions according to one embodiment of this embodiment.

[0135] The use in this embodiment may be non-therapeutic and / or therapeutic, for example, non-therapeutic.

[0136] In this disclosure, “therapeutic” use means use in the course of treating the body of a human or animal. Therapeutic use in this disclosure may be, for example, use for the purpose of or in conjunction with a medical practice. Therapeutic use in this disclosure may involve, for example, a medical professional administering the substance to a human or animal, or instructing a human or animal to administer the substance. Therapeutic use in this disclosure may be, for example, use for therapeutic or preventive purposes, use for therapeutic purposes, or use for preventive purposes involving the ingestion of a food composition, or use for therapeutic purposes.

[0137] In this disclosure, “non-therapeutic” use means any use of the substance that does not constitute a therapeutic use. Non-therapeutic use as relating to this disclosure may, for example, be use that is not intended for and / or does not involve a medical procedure. Non-therapeutic use as relating to this disclosure does not, for example, involve a medical professional administering the substance to a human or animal, and / or instructing a human or animal to administer the substance. Non-therapeutic use as relating to this disclosure may, for example, be use for preventive or health-promoting purposes, and may be use for preventive or health-promoting purposes that involves the administration of a pharmaceutical composition or quasi-drug.

[0138] <Lactobacillus species or compositions containing the same for use> Other embodiments of the present disclosure relate to IL-producing Lactobacillus species or compositions containing the same for use in immunomodulation or induction of interleukin production. Detailed aspects of the IL-producing Lactobacillus species, compositions and their uses according to the present embodiment are as described above for Lactobacillus species and compositions according to one embodiment of this embodiment. The uses in this embodiment may be non-therapeutic and / or therapeutic, for example, therapeutic.

[0139] <Use of Lactobacillus in the Preparation of Compositions> Further embodiments of this disclosure relate to the use of IL-producing Lactobacillus or compositions containing them in the preparation of compositions or pharmaceuticals for regulating immunity or inducing interleukin production. Detailed aspects of the IL-producing Lactobacillus, compositions and their uses according to this embodiment are as described above for Lactobacillus and compositions according to one embodiment of this embodiment. Such compositions and pharmaceuticals are useful for regulating immunity, for example, for immunostimulation or immune enhancement.

[0140] Further embodiments of this disclosure relate to the use of IL-inducing Lactobacillus or compositions containing the same in the manufacture of compositions for activating immunity, improving immunity, relieving visual fatigue, or maintaining throat comfort. Further embodiments of this disclosure also relate to the use of IL-inducing Lactobacillus or compositions containing the same in the manufacture of compositions for activating immunity or improving immunity. Compositions according to these embodiments are, for example, useful for activating or improving immunity. Further embodiments of this disclosure also relate to the use of IL-inducing Lactobacillus or compositions containing the same in the manufacture of compositions useful for activating immunity or improving immunity. Detailed aspects of the IL-inducing Lactobacillus, compositions and their uses according to these embodiments are as described above for Lactobacillus species and compositions according to one embodiment of this embodiment.

[0141] The present disclosure will be described in more detail below using examples, but the present disclosure should not be construed as being limited to these examples. In these examples, the results shown as bar graphs with error ranges are expressed as mean ± standard deviation (Mean ± S.D).

[0142] In Example 1 below, unless otherwise specified, Lactobacillus paracasei KW3110 obtained from the Japan Dairy Technology Association was used as the Lactobacillus species. Hereafter, Lactobacillus paracasei KW3110 will also be referred to as "KW".

[0143] In the following examples, RAW264.7 cells (purchased from American Type Culture Collection) were used as the macrophage cell line. The culture medium for RAW264.7 cells was DMEM (Dulbecco's modified Eagle medium, Thermo Fisher Scientific, catalog number: 11965-092) mixed with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Hereafter, when the culture medium used for culturing RAW264.7 cells is simply referred to as "culture medium," it means the medium with the above composition.

[0144] <Example 1: Evaluation of interleukin production induction ability by Lactobacillus bacteria prepared under various conditions> The ability of Lactobacillus bacteria prepared by static culture without pH control and Lactobacillus bacteria prepared by culture in a pH-controlled environment to induce interleukin production by macrophages was evaluated.

[0145] [Preparation of bacterial powder by static culture] In a 150 ml storage bottle (CORNING), 100 ml of MRS medium (MRS BROTH, CM0359, Oxoid) was inoculated (seeded) with KW at a concentration of 0.1% (v / v), and cultured in an incubator (Tokyo Rikakikai Co., Ltd.) at 32°C for 24 hours (static culture). The culture solution after static culture was concentrated by centrifugation (5000 rpm), and the supernatant was removed to obtain a bacterial suspension. Subsequently, the obtained bacterial suspension and milliQ were mixed in a ratio of 1:19, and washed twice by centrifugation (5000 rpm). The washed KW was suspended in 20 ml of milliQ and heated in an autoclave at 80°C for 15 minutes to obtain a heat-treated bacterial sample. The bacterial sample was dried by freeze-drying to obtain bacterial powder.

[0146] [Preparation of bacterial powder by culturing in a pH-controlled environment] 80 ml of MRS medium was added to a Junior 8 culture device (Biot), and KW was inoculated (seeded) to a concentration of 0.1% (v / v). The inoculated KW was cultured for 21 hours under controlled conditions: pH of 5.0, 5.5, 6.0, or 6.5, temperature of 32°C, stirring speed of 100 rpm, and aeration rate of 1.0 vvm. NaOH was added as needed to maintain the pH. Air was used for aeration. The culture solution after culturing was centrifuged (5000 rpm), and the supernatant was removed to obtain a bacterial suspension. Subsequently, the obtained bacterial suspension was mixed with milliQ in a ratio of 1:19, and washed twice by centrifugation (5000 rpm). The washed KW was suspended in 20 mL of milliQ and heated in an autoclave at 80°C for 15 minutes to obtain a heat-treated bacterial sample. The bacterial sample was dried by freeze-drying to obtain bacterial powder.

[0147] [Evaluation of the ability to induce interleukin production in macrophages] RAW264.7 cells were placed in culture medium in a quantity of 5.0 × 10⁶ cells. ５ A concentration of cells / mL and 1 mL per well (i.e., 5.0 × 10⁻¹⁶). ５ The bacteria were seeded in a 24-well plate at a concentration of (1 cell / well). The bacterial powder prepared above was then added to the plate to a concentration of 10 μg / ml, and the plate was heated at 37°C and CO2. ２The cells were incubated at a concentration of 5 vol% and 100% humidity for 24 hours. The culture supernatant was collected from each well, and the IL-10 concentration in the culture supernatant was measured by ELISA using a BD OptEIA Mouse IL-10 ELISA Set (BD Bioscience).

[0148] Figure 1 shows the amount of interleukin-10 (IL-10) produced by RAW264.7 cells (pg / μL) when bacterial powder prepared by static culture or culture controlled at pH 5.0, pH 5.5, pH 6.0, or pH 6.5 was added. According to Figure 1, Lactobacillus bacteria cultured at pH 5.0, 5.5, 6.0, or 6.5 showed a higher ability to induce interleukin production compared to those cultured statically without pH control.

[0149] Figure 2 shows the change in pH of the culture medium over time under the same static culture conditions as in Figure 1. The change in pH of the culture medium over time was measured using the "Procedure for Measuring Change in pH Over Time" described below. According to the results in Figure 2, under static culture conditions, the pH of the culture medium decreased as the culture time progressed. [Procedure for Measuring Change in pH Over Time] 80 ml of MRS medium was added to a culture device (Junior 8, Biot Inc.), and inoculated (seeded) to a KW of 0.1% (v / v). The change in pH over time was measured using the culture device (using a pH sensor that had been calibrated before use) when the culture was performed with the pH control, stirring, and aeration control mechanisms stationary and the temperature controlled at 32°C.

[0150] <Example 2: Evaluation of the phagocytic potential of Lactobacillus bacteria prepared under various conditions> The amount of phagocytosis by RAW264.7 cells of various bacteria prepared in the same manner as in Example 1 was evaluated. RAW264.7 cells were placed in the culture medium at a rate of 5.0 × 10⁶ ５ At a concentration of 1 cell / mL, 1 mL per well (i.e., 5.0 × 10⁻¹⁶ cells / mL) ５The bacteria were seeded in a 24-well plate at a concentration of (10 cells / well). Powders of each bacterium, prepared in the same manner as in Example 1, were stained with pHo Red SE according to the method described in [Bacterial Staining with pHo Red SE] below, and added to the plate at a concentration of 10 μg / ml. The plates were then incubated at 37°C for 1 day. Subsequently, the fluorescence of pHo Red SE in each well was measured over time up to 24 hours using an Incucyte® SX5 Live-Cell Analysis System (Sartorius). The fluorescence intensity analysis conditions for the Incucyte® SX5 Live-Cell Analysis System (Sartorius) were as described in [Incucyte Fluorescence Intensity Analysis Conditions] below.

[0151] [Bacterial staining with pHo Red SE] pHo 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. 95 μL of the bacterial solution was transferred to a new 2 mL Eppendorf tube, and 5 μL of 10.2 mM pHo Red SE was added. The mixture was stirred using a vortex mixer to disperse the bacteria, then covered with aluminum foil to protect from light and incubated at room temperature for 60 minutes. Subsequently, 750 μL of PBS was added, the mixture was stirred using a vortex mixer, and then 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 stirring with a vortex mixer. The mixture was centrifuged again at room temperature at 20,000 × g for 2 minutes, and 1.5 mL of the supernatant was discarded. 140 μL of PBS was added and the mixture was suspended to prepare a 10 mg / mL bacterial suspension of the stained bacteria. This suspension was stored at 4°C, protected from light by aluminum foil, until use. The stained bacteria were diluted 10-fold with PBS immediately before use to prepare a 1 mg / mL bacterial suspension. The bacterial suspension of the stained bacteria was used within 24 hours of preparation.

[0152] pHo Red SE is a staining reagent whose fluorescence intensity is enhanced in low pH environments. When bacteria stained with pHo Red SE are phagocytosed by RAW264.7 cells, the bacteria enter endosomes, creating a low pH environment that increases the fluorescence intensity of pHo Red SE. Therefore, the Total Integrated Intensity (OCU × μm) is calculated by multiplying the total area (fluorescence detection area) where pHo Red SE fluorescence above the threshold is detected by the fluorescence intensity (OCU) of that area. ２ The image (hereinafter referred to as TII) can be used to quantify the amount of lactic acid bacteria phagocytosed by RAW264.7 cells. The field of view per image is 0.572 mm². ２ That was the case.

[0153] [Incucyte Fluorescence Intensity Analysis Conditions] In the Incucyte fluorescence intensity analysis, two channels, a "Phase channel" and an "Orange channel," were set. The Phase channel detected RAW264.7 cells based on phase contrast. The Orange channel detected the fluorescence of Hydro Red SE. The detailed detection conditions are as shown in the Scan Settings and Analysis Settings below. Scan Settings Scan Type: Adherent Cell-by-Cell Vessel Type: 24-well Corning Image Channels: Phase, Orange (Acquisition Time: 400 ms) Objective: 20× Images per Well: 9 Analysis Settings Analysis Type: Basic Analyzer Phase Channel Segmentation: AI Confluence Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2): 20,000 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 ): Not set - Eccentricity: Not set - Mean Intensity: Not set - Mean Intensity: Not set

[0154] Figure 3 shows the TII (OCU × μm) in RAW264.7 cells when bacterial powder prepared by static culture (KW Static) or controlled culture at pH 5.0, pH 5.5, pH 6.0, or pH 6.5 is added. ２ This is a diagram (image). According to Figure 3, Lactobacillus bacteria cultured with pH controlled to 5.0, 5.5, 6.0, or 6.5 showed a greater amount of phagocytosis by macrophages compared to those cultured statically without pH control.

[0155] <Example 3: Evaluation of the binding affinity of Lactobacillus bacteria prepared under various conditions to Dectin-2> Certain Lactobacillus bacteria are known to induce IL-10 production by macrophages by being phagocytosed via binding to Dectin-2, a lectin receptor present on the surface of macrophages (Non-Patent Literature 1). Therefore, we investigated whether there was a difference in the binding affinity to Dectin-2 between Lactobacillus bacteria prepared by static culture without pH control and Lactobacillus bacteria prepared by culture in a pH-controlled environment.

[0156] To distinguish between impurities and bacteria, powders were prepared by FITC staining of each bacterium, which was prepared by static culture or pH 5.5 controlled culture according to a standard method, similar to Example 1. 0.5 mM CaCl ２ 100 μL each of HEPES buffer (hereinafter simply referred to as "buffer") with pH 7.2 and 15 mM containing 150 mM NaCl was added to low-adsorption tubes. Recombinant mouse Dectin-2 alpha. CF (RSD, 1525-DC-050; a protein in which the N-terminus of recombinant mouse Dectin-2 is modified with a His tag; hereinafter simply referred to as "Dectin-2") or 10×His-tag peptide (abcam) was added to these tubes to a final concentration of 10 μg / mL. Then, FITC-stained bacterial powder was added to these tubes to a final concentration of 10 μg / mL, and the tubes were incubated at room temperature for 1 hour under conditions protected from light by aluminum foil. After adding 150 μL of buffer to each of the tubes, they were centrifuged at 400 g for 5 minutes at room temperature, and the supernatant was discarded. 100 μL of a 20-fold dilution of PE-His-tag detection antibody in buffer (hereinafter also referred to as "secondary antibody diluent") was added to each tube, and incubated at room temperature for 15 minutes. The solutions in each of the tubes were subjected to flow cytometry (FACS) using a flow cytometer (BD biosciences, BD LSRFortessa® X-20 flow cytometer).

[0157] The FACS results were analyzed using the following procedure. First, the bacterial population was gated using FSC / SSC. Next, the population was gated with the wavelength corresponding to SSC / FITC, and the population stained with FITC was considered to be bacteria and extracted. For that bacterial population, the population was gated with the wavelength corresponding to SSC / PE, and the proportion of the population that fluoresced with the wavelength corresponding to PE (PE+ population) was extracted. The threshold for the PE+ population was set so that the PE+ population was approximately 0.1% in the group to which the same concentration of His-tag as each Dectin-2 was added. The proportion of the PE+ population within the bacterial population was calculated as the proportion of bacteria bound to each Dectin-2.

[0158] Figure 4 shows the percentage of bacteria bound to Dectin-2 among bacteria prepared by static culture or culture controlled at pH 5.5. According to these results, the percentage of bacteria bound to Dectin-2 was higher in Lactobacillus bacteria cultured at pH 5.5 compared to those cultured statically without pH control. Therefore, it was revealed that Lactobacillus bacteria cultured at pH within a predetermined range have high Dectin-2 binding ability. Furthermore, it was demonstrated that a higher rate of Dectin-2 binding in bacteria facilitates the induction of IL-10 production by macrophages.

[0159] <Example 4: Investigation of the effects of Lactobacillus bacteria prepared under various conditions on intracellular signaling in macrophages> It is known that in macrophages that phagocytose certain Lactobacillus bacteria via binding to Dectin-2, the expression of Syk (Spleen tyrosine kinase), a tyrosine kinase that promotes the activation of intracellular signaling, increases first (Non-Patent Literature 1). Furthermore, it is known that in macrophages that phagocytose certain Lactobacillus species via binding to Dectin-2, the Lactobacillus species are first trafficked to endosomal lysosomes for degradation, then the degradation products are translocated from the lysosomes to the cytoplasm via PHT2 (Peptide / Histidine Transporter 2), and finally the translocated degradation products activate NOD2 (Nucleotide-binding Oligomerization Domain 2), thereby activating the subsequent IL-10 production signal (Non-Patent Literature 1). Therefore, we evaluated the expression levels of the genes encoding Syk and PHT2, which are proteins that constitute these pathways, in macrophages that phagocytosed Lactobacillus species. The gene symbol for the gene encoding PHT2 is SLC15A3 (Solute Carrier Family 15 Member 3). Additionally, the expression level of the gene encoding Dectin-2, Clec4n (C-type lectin domain family 4, member n), was also evaluated.

[0160] In Example 2, after evaluating the phagocytic levels of each bacterium, total RNA was extracted from RAW264.7 cells using the RNeasy mini kit (QIAGEN) according to the protocol specified by the kit. Next, cDNA was synthesized using the extracted RNA as a template with the iScript cDNA Synthesis Kit (Bio-Rad), and qPCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio). The instrument used for qPCR was the LightCycle 480 system II (Roche). The mRNA expression levels for each condition were calculated as relative values ​​using the ΔΔCt method, with RAW264.7 cells without bacterial addition (control) set to 1.

[0161] Figure 5 shows the expression levels of Clec4n mRNA when no bacteria are added (Control), when bacterial powder prepared by a culture controlled at pH 5.5 is added, or when bacterial powder prepared by static culture is added (Static). Figure 6 shows the expression levels of Syk mRNA when no bacteria are added (Control), when bacterial powder prepared by a culture controlled at pH 5.5 is added, or when bacterial powder prepared by static culture is added (Static). Figure 7 shows the expression levels of SLC15A3 mRNA when no bacteria are added (Control), when bacterial powder prepared by a culture controlled at pH 5.5 is added, or when bacterial powder prepared by static culture is added (Static).

[0162] As shown in Figure 5, the expression level of Clec4n mRNA, the gene encoding Dectin-2, was higher when bacterial powder cultured at a controlled pH of 5.5 was added compared to when bacterial powder cultured at a controlled pH of 5.5 was added. From this result, it was considered that a positive feedback loop occurred in which the expression level of Dectin-2 increased when bacterial powder cultured at a controlled pH of 5.5 was added compared to when bacterial powder cultured at a controlled pH of 5.5 was added, resulting in a greater increase in KW recognition ability.

[0163] As shown in Figures 6 and 7, the expression levels of Syk mRNA and SLC15A3 mRNA were higher when bacterial powder cultured at a controlled pH of 5.5 was added compared to when bacterial powder cultured at a controlled pH of 5.5 was added. These results indicate that intracellular signals related to immunity are more enhanced by Dectin-2-mediated phagocytosis when bacterial powder cultured at a controlled pH of 5.5 is added compared to when bacterial powder cultured at a controlled pH of 5.5 is added compared to when bacterial powder cultured at a controlled pH of 5.5.

[0164] <Example 5: Evaluation of lectin binding ability of Lactobacillus bacteria prepared under various conditions> The binding ability of Lactobacillus bacteria prepared under various conditions to various lectins was evaluated.

[0165] [Measurement of Lectin Binding] Lectin binding was measured using a lectin array. The following arrays and labeling kits were used. The array used was 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. For Cy3 equivalent dye-conjugated streptavidin, Cy3 equivalent dye-conjugated was used. This was prepared by adding 1400 μL of Sample Diluent (reagent included in the kit) to streptavidin tube.

[0166] The samples used for measurement using the lectin array were prepared according to the protocol shown below. 1. First, bacteria were suspended in PBS at a concentration of 1 mg / mL in 500 μL of PBS in a 1.5 mL Eppendorf tube and exposed to water bath sonication. Sonication was performed at room temperature for 30 seconds on mode: High using a US CLEANER (ASONE). 2. 200 μL of the mixture obtained after sonication in step 1 was taken and dialysis was performed in 1 × PBS (pH 8.0) at 4°C for 20 hours. For dialysis, the Dialesis Vials included in the kit were used. The Dialesis Vials are equipped with a regenerated cellulose membrane as the dialysis membrane. 3.2 The post-dialysis solution obtained in step 3.2 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. 4.3 The labeled solution was dialyzed in 1× PBS (pH 8.0) at 4°C for 20 hours. 5.4 The post-dialysis solution obtained in step 5.4 was centrifuged at 4°C and 1000 rpm for 5 minutes, and the supernatant was collected. This supernatant was used as the labeled sample for the subsequent hybridization.

[0167] Hybridization of bacteria in the sample with lectins on the array was performed according to the following protocol: 1. 100 μL of Sample Diluent was applied to the array and incubated at room temperature for 30 minutes. 2. Subsequently, 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 washed according to the protocol provided in 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 washed according to the protocol provided in the kit. 6. The washed array was centrifuged at room temperature at 1000 rpm for 3 minutes.

[0168] Array scanning and data analysis were performed according to the following protocol: 1. The washed array was scanned using the GenePix® 4100A array scanner (Molecular Devices, LLC). 2. The fluorescence intensity values ​​at each spot were quantified and signal values ​​were calculated from the obtained image data (TIFF image, 16-bit format) using the GenePix Pro 7 Software (Molecular Devices, LLC).

[0169] 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 (for example, by contacting them with streptavidin conjugated with a fluorescent dye in the case of biotin labels), the amount of a specific sugar chain structure can be evaluated by measuring its fluorescence intensity. Strong binding to a specific lectin in a lectin array indicates that there is a large amount of the sugar chain structure recognized by that lectin in the target, while weak binding suggests that there is a small amount of that sugar chain structure.

[0170] Tables 3-14 show the lectin array results for bacteria prepared by culture at a controlled pH of 5.5 and bacteria prepared by static culture (Static). In addition to the measured values, these tables also show the value obtained by subtracting the measured values ​​of bacteria prepared by static culture from the measured values ​​of bacteria prepared by culture at a controlled pH of 5.5 (pH5.5-Static), and the value obtained by dividing the measured values ​​of bacteria prepared by culture at a controlled pH of 5.5 by the measured values ​​of bacteria prepared by static culture (pH5.5 / Static). In the tables, POS1 and POS2 show the results when, as positive controls, biotin-labeled IgG was immobilized in wells instead of lectin, and Cy3-Streptavidin was added instead of the labeled sample. In the tables, NEG shows the results when, as a negative control, PBS was added instead of the labeled sample in wells without immobilized lectin. Figure 8 shows the pH 5.5-Static values ​​for the top 19 lectins with the largest pH 5.5-Static values ​​(the difference between the measured values ​​of bacteria prepared by static culture and those prepared by culture under pH control). These results indicate that when powder from bacteria cultured under pH control was added, the binding affinity to multiple lectins was higher than when powder from bacteria cultured under static culture without pH control was added. The difference was particularly significant in the binding affinity to ORYSATA. Therefore, considering that bacteria cultured under pH control showed higher interleukin production induction ability in macrophages, it is suggested that Lactobacillus species exhibiting high binding affinity to ORYSATA also exhibit high interleukin production induction ability. Furthermore, it is suggested that Lactobacillus species possessing the high-mannose structure, a sugar chain structure recognized by ORYSATA, on their cell surface exhibit high interleukin production induction ability.

[0171]

Claims

Lactobacillus bacteria that satisfy at least one of the following groups (A) and (B): (A) RAW264.7 cells in DMEM medium, 5.0 × 10 ５ After sowing to a cell / mL concentration, Lactobacillus bacteria were added to a final concentration of 10 μg / mL, and the mixture was heated at 37°C and CO2. ２ The IL-10 concentration in the culture supernatant obtained after incubation at a concentration of 5% by volume for 24 hours is 4500 pg / μL or higher; (B) Lactobacillus bacteria at 10 μg / mL, in 0.5 mM CaCl ２ Furthermore, when 10 μg / mL of Dectin-2 is brought into contact with 15 mM HEPES buffer at pH 7.2 containing 150 mM NaCl, the proportion of Lactobacillus bacteria bound to Dectin-2 is 5% or more. 　 A Lactobacillus bacterium according to claim 1, further satisfying (C) below: (C) Has a high mannose structure on the surface of the bacterial cell. 　 The Lactobacillus species described in claim 1, which is Lactobacillus paracasei. 　 A Lactobacillus bacterium according to claim 1, which is Lactobacillus paracasei KW3110 or a mutant thereof. 　 A composition comprising a Lactobacillus bacterium according to any one of claims 1 to 4. 　 The composition according to claim 5, wherein the Lactobacillus bacteria are dead bacteria. 　 The composition according to claim 5, which is an immunomodulatory composition. 　 The composition according to claim 5, which is a composition for inducing interleukin production.

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