Lactic acid bacteria and immunostimulant

Abstract

The present invention provides an immunostimulant comprising Lactobacillus helveticus, wherein the Lactobacillus helveticus has three functions, which are dendritic cell-like cell activation, induction of IgA production, and induction of IL-12 production.

Description

Lactic acid bacteria and immunostimulants

[0001] The present invention relates to Lactobacillus lactic acid bacteria and immunostimulants.

[0002] Immunity plays a very important role in maintaining and improving health, and research into this field is being conducted in various fields. Immunity consists of innate immunity and adaptive immunity, which are responsible for the body's defense functions. With the increasing health consciousness, there is a strong desire for means to enhance these immune functions. One known means of enhancing such immune function is to ingest lactic acid bacteria. There are various types of lactic acid bacteria, but Lactobacillus lactic acid bacteria are industrially useful because they can be used in a wide variety of foods, such as the production of yogurt. Therefore, there is a strong desire to develop Lactobacillus lactic acid bacteria with high immunostimulatory activity.

[0003] Patent No. 6170190 Patent No. 6652331 Patent No. 6705628 Patent No. 6796299 Patent No. 5968655

[0004] An object of the present invention is to provide a novel immunostimulant using Lactobacillus lactic acid bacteria, which has high immunostimulant activity. In a preferred embodiment, an object of the present invention is to provide a novel Lactobacillus lactic acid bacterium that exhibits higher immunostimulant activity than the known lactic acid bacterium JCM5805 strain, and an immunostimulant using the Lactobacillus lactic acid bacterium.

[0005] Under these circumstances, the present inventors have investigated a wide variety of lactic acid bacteria from the perspective of immunostimulation, and as a result have found several lactic acid bacteria that exhibit high immunostimulatory activity from among the countless lactic acid bacteria that exist. Furthermore, they have found that some Lactobacillus helveticus, a lactic acid bacterium of the Lactobacillus genus, exhibit high activity. The present invention is based on this new finding. Accordingly, the present invention provides the following immunostimulators, lactic acid bacteria, etc.: Item 1. An immunostimulator containing Lactobacillus helveticus as an active ingredient, wherein the Lactobacillus helveticus has three functions: activating dendritic cell-like cells, inducing IgA production, and inducing IL-12 production.

[0006] Item 2. The immunostimulant according to Item 1, wherein the Lactobacillus helveticus is Lactobacillus helveticus GCL1815 or Lactobacillus helveticus GCJ5-4B.

[0007] Item 3. The immunostimulant according to Item 1 or 2, wherein the Lactobacillus helveticus satisfies at least one requirement selected from the group consisting of the following (1) to (3): (1) 8 x 10 5 1 × 10 cells / mL of human peripheral blood mononuclear cell solution was seeded in a well containing 250 μL of the solution. 7 (2) 1.25 x 10 cells / well of the lactic acid bacteria produce 100 ng / mL or more of IgA. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 (3) 1.25 x 10 cells / well of the lactic acid bacteria is added, the CD86 expression intensity is 1.5 times or more compared to a control without the addition of lactic acid bacteria. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 When the lactic acid bacteria were added at 100 cells / well, they produced 300 pg / mL or more of IL-12.

[0008] Item 4. The immunostimulant according to Item 3, wherein the Lactobacillus helveticus satisfies the three requirements (1) to (3).

[0009] Item 5. The immunostimulant according to any one of Items 1 to 4, wherein at least one of the three immunostimulatory functions of Lactobacillus helveticus, namely, activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production, is higher than that of Lactococcus lactis JCM5805 strain.

[0010] Item 6. The immunostimulant according to any one of Items 1 to 5, wherein the three immunostimulatory functions of Lactobacillus helveticus, namely, dendritic cell-like cell activation, IgA production induction, and IL-12 production induction, are higher than those of Lactococcus lactis JCM5805 strain.

[0011] Item 7. The immunostimulating agent according to any one of Items 1 to 6, wherein the immunostimulation is maintenance of immune function in healthy individuals.

[0012] Item 8. An oral composition for immunostimulation, comprising the immunostimulant according to any one of Items 1 to 7.

[0013] Item 9. The oral composition according to Item 8, which is a food or drink composition.

[0014] Item 10. A preventive or therapeutic agent for viral infections, comprising the immunostimulant according to any one of Items 1 to 7.

[0015] Item 11. A lactic acid bacterium belonging to the genus Lactobacillus, whose accession number at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) is NITE BP-03804.

[0016] Item 12. A lactic acid bacterium belonging to the genus Lactobacillus, whose accession number at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) is NITE BP-03805.

[0017] Item 13. A lactic acid bacterium for use in immunostimulation, the lactic acid bacterium being Lactobacillus helveticus, which has three functions of dendritic cell-like cell activation, induction of IgA production, and induction of IL-12 production.

[0018] Item 14. The lactic acid bacterium for use according to Item 13, which is Lactobacillus helveticus GCL1815 or Lactobacillus helveticus GCJ5-4B.

[0019] Item 15. The lactic acid bacterium for use according to Item 13 or 14, wherein the Lactobacillus helveticus satisfies at least one requirement selected from the group consisting of the following (1) to (3): (1) 8 x 10 5 1 × 10 cells / mL of human peripheral blood mononuclear cell solution was seeded in a well containing 250 μL of the solution. 7 (2) 1.25 x 10 cells / well of the lactic acid bacteria produce 100 ng / mL or more of IgA. 5200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 (3) 1.25 x 10 cells / well of the lactic acid bacteria is added, the CD86 expression intensity is 1.5 times or more compared to a control without the addition of lactic acid bacteria. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 When the lactic acid bacteria were added at 100 cells / well, they produced 300 pg / mL or more of IL-12.

[0020] Item 16. The lactic acid bacterium for use according to Item 15, wherein the Lactobacillus helveticus satisfies the three requirements (1) to (3) above.

[0021] Item 17. The lactic acid bacterium for use according to any one of Items 13 to 16, wherein at least one of the three immunostimulatory functions of Lactobacillus helveticus, namely, activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production, is higher than that of Lactococcus lactis JCM5805 strain.

[0022] Item 18. The lactic acid bacterium for use according to any one of Items 13 to 17, wherein the three immunostimulatory functions of the Lactobacillus helveticus, namely, dendritic cell-like cell activation, IgA production induction, and IL-12 production induction, are higher than those of the Lactococcus lactis JCM5805 strain.

[0023] Item 19. The lactic acid bacterium for use according to any one of Items 13 to 18, wherein the immunostimulation is maintenance of immune function in healthy individuals.

[0024] Item 20. A composition for immunostimulation comprising Lactobacillus helveticus as an active ingredient, wherein the Lactobacillus helveticus has three functions: activating dendritic cell-like cells, inducing IgA production, and inducing IL-12 production.

[0025] Item 21. The composition according to Item 20, wherein the Lactobacillus helveticus is Lactobacillus helveticus GCL1815 or Lactobacillus helveticus GCJ5-4B.

[0026] Item 22. The composition according to Item 20 or 21, wherein the Lactobacillus helveticus satisfies at least one requirement selected from the group consisting of the following (1) to (3): (1) 8 x 10 5 1 × 10 cells / mL of human peripheral blood mononuclear cell solution was seeded in a well containing 250 μL of the solution. 7 (2) 1.25 x 10 cells / well of the lactic acid bacteria produce 100 ng / mL or more of IgA. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 (3) 1.25 x 10 cells / well of the lactic acid bacteria is added, the CD86 expression intensity is 1.5 times or more compared to a control without the addition of lactic acid bacteria. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 When the lactic acid bacteria were added at 100 cells / well, they produced 300 pg / mL or more of IL-12.

[0027] Item 23. The composition according to Item 22, wherein the Lactobacillus helveticus satisfies the three requirements (1) to (3).

[0028] Item 24. The composition according to any one of Items 20 to 23, wherein at least one of the three immunostimulatory functions of Lactobacillus helveticus, namely, activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production, is higher than that of Lactococcus lactis JCM5805 strain.

[0029] Item 25. The composition according to any one of Items 20 to 24, wherein the three immunostimulatory functions of Lactobacillus helveticus, namely, dendritic cell-like cell activation, IgA production induction, and IL-12 production induction, are higher than those of Lactococcus lactis JCM5805 strain.

[0030] Item 26. The composition according to any one of Items 20 to 25, wherein the immunostimulation is maintenance of immune function in healthy individuals. Item 27. A lactic acid bacterium that is Lactobacillus helveticus, which has three functions of activating dendritic cell-like cells, inducing IgA production, and inducing IL-12 production.

[0031] Item 28. The lactic acid bacterium according to Item 27, which is Lactobacillus helveticus GCL1815 or Lactobacillus helveticus GCJ5-4B.

[0032] Item 29. The lactic acid bacterium according to Item 27 or 28, wherein the Lactobacillus helveticus satisfies at least one requirement selected from the group consisting of the following (1) to (3): (1) 8 x 10 5 1 × 10 cells / mL of human peripheral blood mononuclear cell solution was seeded in a well containing 250 μL of the solution. 7 (2) 1.25 x 10 cells / well of the lactic acid bacteria produce 100 ng / mL or more of IgA. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 (3) 1.25 x 10 cells / well of the lactic acid bacteria is added, the CD86 expression intensity is 1.5 times or more compared to a control without the addition of lactic acid bacteria. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 When the lactic acid bacteria were added at 100 cells / well, they produced 300 pg / mL or more of IL-12.

[0033] Item 30. The lactic acid bacterium according to Item 29, wherein the Lactobacillus helveticus satisfies the three requirements (1) to (3).

[0034] Item 31. The lactic acid bacterium for use according to any one of Items 13 to 16, wherein at least one of the three immunostimulatory functions of Lactobacillus helveticus, namely, activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production, is higher than that of Lactococcus lactis JCM5805 strain.

[0035] Item 32. The lactic acid bacterium according to any one of Items 27 to 31, wherein the three immunostimulatory functions of the Lactobacillus helveticus, namely, dendritic cell-like cell activation, IgA production induction, and IL-12 production induction, are higher than those of the Lactococcus lactis JCM5805 strain.

[0036] Item 33. An oral composition for maintaining immune function in healthy individuals, comprising Lactobacillus helveticus as an active ingredient, wherein the Lactobacillus helveticus has three functions: activating dendritic cell-like cells, inducing IgA production, and inducing IL-12 production.

[0037] Item 34. Use of Lactobacillus helveticus for producing an immunostimulant, wherein the Lactobacillus helveticus has three functions: dendritic cell-like cell activation, induction of IgA production, and induction of IL-12 production.

[0038] Item 35. A method for immunostimulation, comprising administering an effective amount of Lactobacillus helveticus to a subject in need thereof, wherein the Lactobacillus helveticus has the three functions of dendritic cell-like cell activation, IgA production induction, and IL-12 production induction. Item 36. A method for preventing or treating a viral infection, comprising administering an effective amount of Lactobacillus helveticus to a subject in need thereof, wherein the Lactobacillus helveticus has the three functions of dendritic cell-like cell activation, IgA production induction, and IL-12 production induction. Item 37. The use according to Item 34 or the method according to Item 35 or 36, wherein the Lactobacillus helveticus is Lactobacillus helveticus GCL1815 or Lactobacillus helveticus GCJ5-4B.

[0039] Item 38. The use according to Item 34, the method according to Item 35 or 36, or the use or method according to Item 37, wherein the Lactobacillus helveticus satisfies at least one requirement selected from the group consisting of the following (1) to (3): (1) 8 x 10 51 × 10 cells / mL of human peripheral blood mononuclear cell solution was seeded in a well containing 250 μL of the solution. 7 (2) 1.25 x 10 cells / well of the lactic acid bacteria produce 100 ng / mL or more of IgA. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 (3) 1.25 x 10 cells / well of the lactic acid bacteria is added, the CD86 expression intensity is 1.5 times or more compared to a control without the addition of lactic acid bacteria. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 When the lactic acid bacteria were added at 100 cells / well, they produced 300 pg / mL or more of IL-12.

[0040] Item 39. The use or method according to Item 38, wherein the Lactobacillus helveticus satisfies the three requirements (1) to (3).

[0041] Item 40. The use according to Item 34, the method according to Item 35 or 36, or the use or method according to any one of Items 37 to 39, wherein at least one of the three immunostimulatory functions of Lactobacillus helveticus, namely, dendritic cell-like cell activation, induction of IgA production, and induction of IL-12 production, is higher than that of Lactococcus lactis JCM5805 strain.

[0042] Item 41. The use according to Item 34, the method according to Item 35 or 36, or the use or method according to any one of Items 37 to 40, wherein the three immunostimulatory functions of the Lactobacillus helveticus, namely, dendritic cell-like cell activation, IgA production induction, and IL-12 production induction, are higher than those of the Lactococcus lactis JCM5805 strain.

[0043] Item 42. The use according to Item 34, the method according to Item 35, or the use or method according to any one of Items 37 to 41, wherein the immunostimulation is maintenance of immune function in healthy individuals.

[0044] Item 43. The method according to any one of Items 35 to 42, wherein the Lactobacillus helveticus is administered orally.

[0045] Item 44. The use according to any one of Items 34 to 42, wherein the immunostimulant is an oral composition.

[0046] Item 45. The method according to any one of Items 35 to 43, wherein the Lactobacillus helveticus is administered as a food or drink.

[0047] Item 46. The use according to any one of Items 34 to 42 and 44, wherein the immunostimulant is a composition for food or drink.

[0048] Item 47. The use according to any one of Items 34 to 42, 44 and 46, wherein the immunostimulant is a prophylactic or therapeutic agent for viral infections.

[0049] According to the present invention, it is possible to provide a novel immunostimulating agent using Lactobacillus lactic acid bacteria, which has high immunostimulating activity.

[0050] The results of evaluating the IgA production ability of Preparation Examples 1 and 2 are shown below. Abbreviations in the figure are as follows: je1: Lactobacillus jensenii 1, fe6: Limosilactobacillus fermentum 6, fe20: Limosilactobacillus fermentum 20, cr2: Lactobacillus crispatus 2, pe5: Lactiplantibacillus pentosus 5, la23: Lactococcus lactis 23, la24: Lactococcus lactis 24, he11: Lactobacillus helveticus GCJ5-4B (accession number NITE BP-03805), he12: Lactobacillus helveticus GCL1815 (accession number NITE BP-03804), ga8: Lactobacillus gasseri 8, la44: Lactococcus lactis 44, pe6: Lactiplantibacillus pentosus 6, cr11: Lactobacillus crispatus 11, la15: Lactococcus lactis 15. The results of evaluating the activation of dendritic cell-like cells in Preparation Examples 1 and 2 are shown. The results of evaluating the activation of NK cells in Preparation Examples 1 and 2 are shown.

[0051] Immunostimulant The present invention provides an immunostimulant containing Lactobacillus helveticus as an active ingredient, wherein the Lactobacillus helveticus has three functions: activating dendritic cell-like cells, inducing IgA production, and inducing IL-12 production.

[0052] Examples of Lactobacillus helveticus include Lactobacillus helveticus GCL1815 (also referred to as "Lactobacillus helveticus GCD8-9D"; the same applies below), Lactobacillus helveticus GCJ5-4B, and the like, with Lactobacillus helveticus GCL1815 and Lactobacillus helveticus GCJ5-4B being preferred. In a preferred embodiment, the Lactobacillus helveticus is a strain deposited on January 20, 2023 at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Room 122 (Postal Code 292-0818) and having the accession number NITE BP-03804 or NITE BP-03805).

[0053] In the present invention, Lactobacillus helveticus, the active ingredient of the immunostimulant, has all three functions: dendritic cell-like cell activation, IgA production induction, and IL-12 production induction. Specifically, Lactobacillus helveticus, the active ingredient of the immunostimulant, first activates dendritic cell-like cells. Dendritic cells play a major role in immune function. More specifically, for example, dendritic cells, along with macrophages and NK cells, play an important role in innate immunity, and when foreign substances such as bacteria and viruses enter the body, they first function to eliminate them. In addition, dendritic cells transmit information about the invading foreign substance to T cells and B cells by antigen presentation, and also play an important role in adaptive immunity, such as promoting the production of antibodies that act specifically against foreign substances and activating cytotoxic T cells.

[0054] Thus, dendritic cells are involved in both innate and adaptive immunity and have a significant impact on the overall immune system. There are various types of dendritic cells, but they can be broadly classified into plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs). pDCs exhibit growth inhibitory activity against viruses and are major producers of type I interferon, which also contributes to the activation of NK cells, but they have weak antigen-presenting ability. On the other hand, cDCs not only produce IL-12, which activates NK cells, but also have strong antigen-presenting ability, thus playing an important role in inducing adaptive immunity. IgA, a type of adaptive immunity, has low specificity compared to other immunoglobulins and acts on many types of bacteria and viruses, and is therefore thought to contribute to the prevention of a wide range of infectious diseases. cDCs, which activate both innate and adaptive immunity, are one of the cells that play a central role in preventing infectious diseases. In the present invention, "dendritic cell-like cells" is a general term for dendritic cells collected from a living body and cells with the function of cDCs induced from stem cells such as monocytes, iPS cells, and ES cells. By measuring the degree of activation of dendritic cell-like cells, the effect of lactic acid bacteria on dendritic cells in the living body can be evaluated.

[0055] Specifically, dendritic cell-like cell activation by lactic acid bacteria is, for example, 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6The CD86 expression intensity can be evaluated by adding lactic acid bacteria at 10 ...

[0056] Furthermore, the induction of IgA production by lactic acid bacteria is, for example, 8 × 10 5 1 × 10 cells / mL of human peripheral blood mononuclear cell solution was seeded in a well containing 250 μL of the solution. 7 The lactic acid bacteria can be evaluated by adding 100 ng / mL of lactic acid bacteria per well and measuring the IgA produced. More specifically, the measurement can be carried out according to the method described in the Examples of the present application. In the present invention, among Lactobacillus helveticus, those that can detect IgA of 100 ng / mL or more, more preferably 150 ng / mL or more when measured by the above method are preferred. In addition, in the present invention, among Lactobacillus helveticus, those that can detect IgA of 100 ng / mL or more, more preferably 150 ng / mL or more when measured by the above method are preferred, and the IgA production amount measured by the above method is, for example, 105% or more, preferably 110% or more, preferably 120% or more, preferably 140% or more, preferably 170% or more (the ratio of [IgA production amount (ng / mL) in the group containing the active ingredient of the present invention] / [IgA production amount (ng / mL) in the control] compared with the control without lactic acid bacteria.

[0057] The induction of IL-12 production by lactic acid bacteria is, for example, 1.25 × 10 5200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 The IL-12 production can be evaluated by adding the lactic acid bacteria at 1000 cells / well and measuring the IL-12 produced. More specifically, the measurement can be performed according to the method described in the Examples of the present application. In the present invention, Lactobacillus helveticus is preferred, which has an IL-12 production of preferably 300 pg / mL or more, more preferably 600 pg / mL or more, and more preferably 900 pg / mL or more when measured by the above method.

[0058] Furthermore, in the present invention, Lactobacillus helveticus strains are preferred which, when measured by the above-mentioned method, produce IL-12 that is, for example, preferably 110% or more, more preferably 180% or more, even more preferably 290% or more, still more preferably 500% or more, and particularly preferably 1000% or more, compared to a control to which no lactic acid bacteria have been added (as a ratio of [IL-12 production (pg / mL) in the group to which lactic acid bacteria containing the active ingredient of the present invention have been added] / [IL-12 production (pg / mL) in the control]).

[0059] In a more preferred embodiment, it is preferable to use a Lactobacillus helveticus strain that exhibits at least one (preferably at least two, more preferably all three) immunostimulatory function selected from the group consisting of dendritic cell-like cell activation, IgA production induction, and IL-12 production induction, which is stronger than that of Lactococcus lactis JCM5805 strain. In this embodiment, the methods for measuring dendritic cell-like cell activation, IgA production induction, and IL-12 production induction are the same as those described above. The above functions of the JCM5805 strain lactic acid bacteria can be measured by performing the same procedures as described above, except that the JCM5805 strain lactic acid bacteria is used instead of the Lactobacillus helveticus, which is the active ingredient of the immunostimulant of the present invention.

[0060] In the present invention, the Lactobacillus helveticus may be killed (heat-killed or the like) or live. In the present invention, the Lactobacillus helveticus may be used alone or in combination of two or more.

[0061] In the present invention, the lactic acid bacteria are used as an active ingredient of an immunostimulant. Examples of "immunostimulation" uses include the activation of dendritic cell-like cells, induction of IgA production, induction of IL-12 production, etc. Furthermore, in the present invention, the term "immunostimulation" encompasses not only improving the immune function of people with weakened immune functions, but also maintaining the immune function of healthy people.

[0062] In the present invention, Lactobacillus helveticus, the active ingredient of the present invention, may be used as an immunostimulant itself, or may be used as a composition combining the lactic acid bacteria powder with various pharmaceutically acceptable carriers that can be added to foods (e.g., isotonicity agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, excipients, binders, etc.). In this embodiment, the content of Lactobacillus helveticus in the immunostimulant composition is not limited, and can be appropriately set within the range of, for example, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, 99% by mass or more, etc. The number of Lactobacillus helveticus bacteria in the Lactobacillus helveticus bacterial powder used in the immunostimulant is not limited, but can be appropriately set within the range of, for example, 100 million to 100 trillion cells / g, preferably 10 billion to 10 trillion cells / g, more preferably 50 billion to 1 trillion cells / g, even more preferably 100 billion cells / g or more, and particularly preferably 300 billion cells / g or more.

[0063] Examples of the isotonicity agent include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonicity agents can be used alone or in combination of two or more.

[0064] Examples of the chelating agent include edetate salts such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or a salt thereof, sodium hexametaphosphate, citric acid, etc. These chelating agents may be used alone or in combination of two or more.

[0065] The stabilizer may, for example, be sodium hydrogen sulfite.

[0066] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol. These pH adjusters can be used alone or in combination of two or more.

[0067] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used alone or in combination of two or more.

[0068] Examples of antioxidants include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, etc. These antioxidants may be used alone or in combination of two or more.

[0069] Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc. These solubilizing agents can be used alone or in combination of two or more.

[0070] Examples of thickeners include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. These thickeners may be used alone or in combination of two or more.

[0071] Examples of excipients include lactose, corn starch, L-cysteine, trehalose, maltitol, sorbitol, etc. These excipients may be used alone or in combination of two or more.

[0072] Examples of binders include crystalline cellulose, starch, sucrose, hydroxypropyl cellulose, gelatin, powdered gum arabic, polyvinylpyrrolidone, pullulan, dextrin, cyclodextrin, methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, polyethylene glycol, etc. These binders can be used alone or in combination of two or more.

[0073] In addition, the composition may further contain a substance known to have immunostimulating function in addition to the Lactobacillus helveticus. Examples of substances known to have immunostimulating function include vitamin C, vitamin A, zinc, etc. These substances can be used alone or in combination of two or more.

[0074] Ingestion of the immunostimulant of the present invention by a subject (preferably a mammal such as a human) results in an immunostimulating effect. The intake amount of the immunostimulant of the present invention is not limited, but the daily intake of the active ingredient, Lactobacillus helveticus, can be appropriately set within a range of, for example, 10 million to 10 trillion cells, preferably 100 million to 1 trillion cells, more preferably 1 billion to 100 billion cells, even more preferably 5 billion to 30 billion cells, and particularly preferably 10 billion to 20 billion cells. The intake amount of the immunostimulant of the present invention based on the weight of Lactobacillus helveticus is also not limited, but the daily intake of the active ingredient, Lactobacillus helveticus, can be appropriately set within a range of, for example, 1 mg to 10 g, preferably 10 mg to 1000 mg, more preferably 40 mg to 300 mg, and even more preferably 200 mg or more.

[0075] Oral Composition In another embodiment, the present invention provides an oral composition containing the immunostimulant.Accordingly, the present invention provides an oral composition containing the Lactobacillus helveticus.The oral composition includes food and drink compositions, pharmaceutical compositions, etc.In the present invention, the food and drink compositions also include health functional foods (nutrient functional foods, specified health foods, functional food) and the like.

[0076] Examples of food and beverage compositions include beverages such as vegetable juice drinks, fruit juice drinks, mixed vegetable and fruit juice drinks, fermented milk drinks, and almond-containing drinks; ice creams, frozen desserts, almond-containing foods, biscuits (cream sandwich biscuits, etc.), chocolates (including semi-chocolate), and fermented milk foods (yogurt, cheese). Examples of ice creams include lacto ice cream (lacto ice cream containing fermented milk, etc.). Examples of cream sandwich biscuits include cream blended with Lactobacillus helveticus. Examples of semi-chocolate include corn-containing semi-chocolate. Among these foods and beverages, dairy products and foods containing dairy products are preferred in the present invention. Examples of dairy products include fermented milk, ice creams, and milk drinks. Examples of foods and beverages containing dairy products include cream sandwich biscuits and milk chocolate. Examples of frozen desserts include milk-containing frozen desserts (including fermented milk-containing frozen desserts). The food and beverage compositions of the present invention also include supplements. Examples of supplements include sports supplements (amino acid-containing sports supplements, etc.). Examples of amino acid-containing sports supplements include glutamine-containing sports supplements.

[0077] In the embodiment of the oral composition, the details of the active ingredient Lactobacillus helveticus, its intake amount, etc. are the same as those described above in the description of the immunostimulant. In this embodiment, the content of Lactobacillus helveticus in the oral composition is not particularly limited, but can be appropriately set within the range of, for example, 0.00001 to 99% by mass, preferably 0.0001 to 50% by mass, more preferably 0.001 to 10% by mass, even more preferably 0.01 to 1% by mass, and particularly preferably 0.1% by mass or more. The oral composition of the present invention can be used for immunostimulation purposes.

[0078] Preventive or therapeutic agent for viral infections Administration of a specific Lactobacillus helveticus, the active ingredient of the present invention, can produce an immunostimulating effect. Therefore, the immunostimulating effect of the present invention can be expected to prevent or treat viral infections. Therefore, the present invention provides a preventive or therapeutic agent for viral infections containing the immunostimulant. The present invention also provides a preventive or therapeutic agent for viral infections containing the above-mentioned Lactobacillus helveticus. Target viruses include, but are not limited to, influenza virus, coronavirus, adenovirus, respiratory syncytial virus, human metapneumovirus, rhinovirus, etc. Details of the active ingredient Lactobacillus helveticus in embodiments of the preventive or therapeutic agent for viral infections, its intake amount, etc. are the same as those described above in the description of the immunostimulant and oral composition.

[0079] According to the present invention, the above-mentioned Lactobacillus helveticus can bring about immunostimulating effect, and prevent or treat viral infections.In addition, the above-mentioned Lactobacillus helveticus is novel.Therefore, the present invention also provides the above-mentioned Lactobacillus helveticus itself.The method of using Lactobacillus helveticus in this embodiment is the same as that described above in the description of immunostimulant, oral composition and prevent or treat viral infections.

[0080] Preparation of Killed Lactic Acid Bacteria Powder Preparation Example 1: A lactic acid bacteria strain owned by the company was statically cultured in MRS medium (Merck Millipore) at 30°C or 37°C for 48 hours. After culture, the cells were collected by centrifugation at 8,000 × G for 10 minutes, washed three times with endotoxin-free saline (hereinafter referred to as saline), and then sterilized by autoclaving at 121°C for 15 minutes. The cells were then freeze-dried, and the concentration was adjusted to 10 mg / mL with saline to obtain a lactic acid bacteria suspension.

[0081] Preparation Example 2 Heat-killed cells of Lactococcus lactis JCM5805 strain (hereinafter referred to as JCM5805 strain) were prepared in the same manner as in Preparation Example 1. The cells were then freeze-dried, and the concentration was adjusted to 10 mg / mL with physiological saline to obtain a lactic acid bacteria suspension. The "JCM" strain indicates a strain provided by the Institute of Physical and Chemical Research.

[0082] Screening by IgA Production Inducibility Preparation of Peripheral Blood Mononuclear Cell Medium (hereinafter referred to as PBMC medium) PBMC medium was prepared by adding RPMI 1640 (Thermo Fisher Scientific) to RPMI 1640 so that the concentrations of inactivated fetal bovine serum (Thermo Fisher Scientific) (hereinafter referred to as FBS) were 10% by mass, MEM vitamin solution (Thermo Fisher Scientific) were 1% by mass, MEM non-essential amino acid solution (Thermo Fisher Scientific) were 1% by mass, penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd.) were 1% by mass, sodium pyruvate solution (Thermo Fisher Scientific) were 1% by mass, and 2-mercaptoethanol (Thermo Fisher Scientific) were 0.1% by mass.

[0083] Preparation of human peripheral blood mononuclear cells (PBMCs): Frozen human peripheral blood mononuclear cells (Astarte Biologics) derived from normal donors (hereinafter referred to as PBMCs) were quickly thawed in a 37°C water bath and added to a 50 mL centrifuge tube containing 10 mL of RPMI 1640 medium containing 10% FBS. The tube was then gently mixed by inversion. The tube was centrifuged at 200 × G for 5 minutes to pellet the PBMCs. The supernatant was removed using an aspirator, and the pelleted PBMCs were resuspended in 10 mL of PBMC medium to obtain a PBMC suspension.

[0084] PBMC seeding, lactobacillus addition, and culture: 20 μL of the PBMC suspension and 2 μL of acridione orange (Logos Biosystems) were added to a 1.5 mL microtube and mixed well. The cell concentration was then calculated using a cell counter (Logos Biosystems, L20001). The PBMC suspension was diluted in PBMC medium until the cell concentration reached 8 × 10 5 The cells were diluted to 1000 cells / mL and seeded in 250 μL aliquots onto a 96-well plate (TPP). The lactic acid bacteria suspension from Preparation Example 1 was then added to each well of the 96-well plate at 50 μg / mL. A control well was also prepared without lactic acid bacteria. The cells were then statically cultured for 120 hours in a CO2 incubator at 37°C and 5% CO2.

[0085] IgA Measurement: After culturing PBMCs for 120 hours, the 96-well plate was removed and 250 μL of the culture medium was dispensed into 8-tube tubes. PBMCs were precipitated by centrifugation at 200 × g for 10 minutes, and 220 μL of the culture supernatant was dispensed into a new 8-tube tube. After further centrifugation at 1,500 × g for 10 minutes, the culture supernatant was subjected to IgA quantification by ELISA. IgA quantification was performed using the Abcam Human IgA ELISA Kit, and IgA concentrations were measured according to the kit's protocol booklet. To confirm experimental reproducibility, the procedure from PBMC seeding to IgA measurement was performed twice.

[0086] Test results Lactic acid bacteria were selected that had an IgA concentration more than double that of the control culture medium in which PBMCs were cultured without the addition of lactic acid bacteria prepared in Preparation Example 1. Screening was performed under the above conditions, and 70 types of lactic acid bacteria were selected as lactic acid bacteria capable of inducing IgA production. A list is shown in Table 1. The IgA production rate was calculated using the following formula: IgA production rate = IgA concentration in PBMC culture medium (with lactic acid bacteria added) / IgA concentration in PBMC culture medium (without lactic acid bacteria added)

[0087] Of these, 14 types of lactic acid bacteria were selected from the viewpoints of different bacterial species and ease of cultivation, and were used in the following tests. The selected lactic acid bacteria are marked with an * in Table 1.

[0088] Evaluation of IgA Production Induction Isolation of Peripheral Blood Mononuclear Cells from Human Peripheral Blood Twenty milliliters of blood was collected from 12 healthy adults and placed in blood collection tubes containing an anticoagulant (heparin sodium). Lymphoprep® tubes (AXS) were centrifuged at 400 × g for 1 minute to remove the Lymphoprep® from the tubes before use. Blood and saline were mixed 1:1 in a 50-mL centrifuge tube, and 20–30 mL of the mixture was transferred to a Lymphoprep® tube and centrifuged at 800 × g for 20 minutes at room temperature. The resulting layer of peripheral blood mononuclear cells was then collected using a Pasteur pipette and transferred to a new 50-mL centrifuge tube. 20 mL of saline was added to the 50-mL centrifuge tube containing the collected peripheral blood mononuclear cells, and the cells were pelleted by centrifugation at 250 × g for 10 minutes. The supernatant was removed using an aspirator, and 1 mL of PBMC medium was added to the pelleted peripheral blood mononuclear cells to suspend them, thereby obtaining a peripheral blood mononuclear cell solution.

[0089] Seeding of peripheral blood mononuclear cells, addition of lactobacillus, and culture: 90 μL of PBMC medium and 10 μL of peripheral blood mononuclear cell solution were measured into a 1.5 mL microtube and mixed well. 20 μL was then aliquoted into a new 1.5 mL microtube, and 2 μL of acridione orange (Logos Biosystems) was added and mixed well. The cell concentration was then calculated using a cell counter (Logos Biosystems, L20001). The peripheral blood mononuclear cell solution was diluted with PBMC medium to a concentration of 8 × 10 5 The suspension was diluted to a concentration of 1 × 10 cells / mL and 250 μL of each was seeded onto a 96-well plate (TPP). 7As a positive control, lipopolysaccharide (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as LPS) was added to the wells of a 96-well plate at a final concentration of 10 ng / mL. A control well was also prepared without Lactobacillus. Static culture was performed for 120 hours in a CO2 incubator at 37°C and 5% CO2.

[0090] IgA Measurement: After culturing peripheral blood mononuclear cells for 120 hours, the 96-well plate was removed and 250 μL of the culture medium was dispensed into 8-tube tubes. The cells were centrifuged at 200 × G for 10 minutes to precipitate the peripheral blood mononuclear cells, and 220 μL of the culture supernatant was dispensed into new 8-tube tubes. After further centrifugation at 1,500 × G for 10 minutes, the culture supernatant was subjected to IgA quantification by ELISA. IgA quantification was performed using the Abcam Human IgA ELISA Kit, and IgA concentrations were measured according to the kit's protocol booklet.

[0091] Evaluation of dendritic cell-like cell activation Preparation of differentiation medium Mylc dedicated medium B (manufactured by Mycan Technologies Inc.) and Mylc dedicated medium supplement B (manufactured by Mycan Technologies Inc.) were mixed at a ratio of 200:1 to obtain a differentiation medium.

[0092] Preparation of Lactic Acid Bacteria Suspension (1 mg / mL) The lactic acid bacteria suspensions obtained in Preparation Examples 1 and 2 were diluted 10-fold with physiological saline to obtain a lactic acid bacteria suspension (1 mg / mL). The bacterial count of the lactic acid bacteria suspension (1 mg / mL) was measured by the bleed method, and the bacterial count was 4 × 10 7 The cells were diluted with differentiation medium to give cells / mL.

[0093] Dendritic cell seeding, lactic acid bacteria addition, and culture. Immortalized dendritic cells, aMylc-2-A differentiated cells (hereafter referred to as dendritic cells), purchased from Mycan Technologies Inc. and induced to differentiate for three days were collected. Next, the cells were centrifuged at 300 × G for 5 minutes to precipitate the dendritic cells, the supernatant was removed with an aspirator, and 2 mL of differentiation medium was added to the pelleted dendritic cells. After calculating the cell concentration using a cell counter, the dendritic cells were diluted to 2.5 × 10 in differentiation medium. 5 The suspension was diluted to a concentration of 100 cells / mL and 100 μL of the diluted suspension was seeded into a Nunclon Sphera 96-well round-bottom plate (Thermo Fisher Scientific). 7 100 μL of lactic acid bacteria (100 μL / mL) was added to each well, and 100 μL of LPS was added to each well as a positive control to a final concentration of 1 ng / mL. A control well was also prepared without the addition of lactic acid bacteria. The cells were cultured statically in a CO2 incubator at 37°C with 5% CO2 for 24 hours.

[0094] Recovery of dendritic cells and culture supernatant. The 96-well plate in which dendritic cells had been cultured for 24 hours was removed, and 200 μL of the culture medium was dispensed into an 8-tube tube. The dendritic cells were precipitated by centrifugation at 500 × G for 10 minutes, and 140 μL of the culture supernatant was dispensed into a new 8-tube tube. The culture supernatant remaining in the 8-tube tube was removed using an aspirator, yielding a dendritic cell precipitate.

[0095] Preparation of reagent for measuring cell surface marker (CD86) 1. FcR blocking solution An FcR blocking solution was obtained by mixing eBioscience (registered trademark) Flow Cytometry Staining Buffer (manufactured by Invitrogen) (hereinafter referred to as staining buffer) and an Fc receptor binding inhibitor (manufactured by eBioscience) at a ratio of 19:1.

[0096] 2. Antibody solution The staining buffer and PE anti-human CD86 antibody were mixed at a ratio of 49:1 to obtain an antibody solution.

[0097] Measurement of cell surface marker (CD86). Dendritic cell pellets were suspended in 200 μL of staining buffer. The cells were centrifuged at 500 × g for 5 minutes, the supernatant removed using an aspirator, and 20 μL of FcR blocking solution was added to suspend the cells. The cells were then allowed to stand on ice for 20 minutes. 20 μL of antibody solution was added and the cells were suspended. The cells were then allowed to stand on ice for 30 minutes. After the suspension, 160 μL of staining buffer was added to suspend the cells. The cells were centrifuged at 500 × g for 5 minutes, and the supernatant was removed using an aspirator. Next, 200 μL of staining buffer was added to suspend the cells. The cells were centrifuged at 500 × g for 5 minutes, and the supernatant was removed using an aspirator. The cell surface marker (CD86) expression intensity was measured using a flow cytometer (Sony Corporation, SA3800) (laser wavelength: 488 nm) for the solution after resuspending the cells in 200 μL of staining buffer.

[0098] Evaluation of NK cell activation IL-12 was measured using the culture supernatant obtained by the method described above in "Collection of dendritic cells and culture supernatant" to evaluate NK cell activation. IL-12 was measured using the Human IL-12 p70 ELISA Kit (Abcam) and the IL-12 concentration was measured according to the protocol booklet included with the kit.

[0099] Evaluation of IgA Production Induction The results of evaluating the IgA production ability of Preparation Examples 1 and 2 are shown in Figure 1. As shown in Figure 1, compared to the control without added lactic acid bacteria, the ability to induce IgA production in peripheral blood mononuclear cells was significantly increased by adding and culturing the lactic acid bacteria of Preparation Example 1, with the exception of cr2, la44, cr11, and la15. Furthermore, compared to the addition of the JCM5805 strain of Preparation Example 2, the ability to induce IgA production in peripheral blood mononuclear cells was significantly increased by adding and culturing the lactic acid bacteria of Preparation Example 1, with the exception of cr2, la44, cr11, and la15. In animals, including humans, ingesting these lactic acid bacteria is expected to enhance IgA production and prevent the invasion of viruses and bacteria into the body, thereby improving infection defense capabilities.

[0100] Evaluation of Dendritic Cell-Like Cell Activation The results of evaluating dendritic cell-like cell activation in Preparation Examples 1 and 2 are shown in Figure 2. Dendritic cell activation was evaluated by measuring the expression intensity of a cell surface marker (CD86). As shown in Figure 2, compared to the control without lactic acid bacteria, culturing with the addition of lactic acid bacteria from Preparation Example 1 significantly activated dendritic cells, with the exception of pe6. Furthermore, compared to the addition of the JCM5805 strain from Preparation Example 2, culturing with the addition of lactic acid bacteria from Preparation Example 1 significantly activated dendritic cells, with the exception of je1, fe20, pe5, la23, la24, la44, pe6, and la15. In animals, including humans, ingesting these lactic acid bacteria is expected to activate dendritic cells and thereby stimulate the overall immune system.

[0101] Evaluation of NK Cell Activation The results of evaluating NK cell activation in Preparation Examples 1 and 2 are shown in Figure 3. NK cell activation was evaluated by measuring IL-12 concentration. As shown in Figure 3, compared to the control without lactic acid bacteria, culturing with the addition of lactic acid bacteria from Preparation Example 1 significantly activated NK cells, except for Ia44. He11 and He12 in particular significantly activated NK cells. Furthermore, culturing with the addition of lactic acid bacteria from Preparation Example 1 significantly activated NK cells compared to the addition of the JCM5805 strain from Preparation Example 2. In animals, including humans, ingesting these lactic acid bacteria activates NK cells, which is expected to enhance their ability to attack bacterial and virally infected cells and prevent the spread of infection, thereby enhancing their ability to protect against infection.

[0102] As shown in the above tests, he11 and he12 exhibited all three functions: dendritic cell-like cell activation, IgA production induction, and IL-12 production induction. Specifically, he11 and he12 exhibited all of the following functions (1) to (3): (1) 8 x 10 5 1 × 10 cells / mL of human peripheral blood mononuclear cell solution was seeded in a well containing 250 μL of the solution. 7 (2) 1.25 x 10 cells / well of the lactic acid bacteria produce 100 ng / mL or more of IgA. 5200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 (3) 1.25 x 10 cells / well of the lactic acid bacteria is added, the CD86 expression intensity is 1.5 times or more compared to a control without the addition of lactic acid bacteria. 5 200 μL of human-derived dendritic cell-like cells at a concentration of 4 × 10 cells / mL were seeded in a well. 6 When the lactic acid bacteria were added at 1000 μg / well, they produced 300 pg / mL or more of IL-12. Furthermore, he11 and he12 were superior to the JCM5805 strain in all three immunostimulatory functions: dendritic cell-like cell activation, IgA production induction, and IL-12 production induction.

[0103] Formulation Example 1: Ice cream containing killed lactic acid bacteria powder was obtained using the ingredients shown in Table 2. Specifically, egg yolk and sugar were first placed in a bowl and mixed thoroughly. Fresh cream, milk, and killed lactic acid bacteria powder were placed in a separate pot and heated. When the pot began to bubble, the pot was removed from the heat and the egg yolk and sugar were gradually added to the bowl while mixing. The resulting mixture was placed in a metal container to cool, then covered and cooled to -20°C. After 3 hours, the mixture was stirred, and then stirred four times every 30 minutes to obtain ice cream containing killed lactic acid bacteria powder.

[0104]

[0105] Formulation Example 2 Using the ingredients shown in Table 3, yogurt containing killed lactic acid bacteria powder was obtained.

[0106]

[0107] Formulation Example 3: Chocolate containing killed lactic acid bacteria powder was obtained using the ingredients shown in Table 4. Specifically, the chocolate was melted in a hot water bath at about 50°C, the killed lactic acid bacteria powder was added and mixed well, the mixture was placed in a mold, and the mixture was left to stand at room temperature to solidify, thereby obtaining a chocolate containing killed lactic acid bacteria powder.

[0108]

Claims

1. An immunostimulant containing Lactobacillus helveticus GCL1815 or Lactobacillus helveticus GCJ5-4B as an active ingredient.

2. The immunostimulator according to claim 1, wherein the immunostimulation is maintenance of immune function in healthy individuals.

3. An oral composition for immunostimulation, comprising the immunostimulant according to claim 1 or 2.

4. The oral composition according to claim 3, which is a food or beverage composition.

5. A preventive or therapeutic agent for viral infections, comprising the immunostimulant according to claim 1 or 2.

6. A lactic acid bacterium belonging to the genus Lactobacillus, whose accession number at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) is NITE BP-03804.

7. A lactic acid bacterium belonging to the genus Lactobacillus, whose accession number at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) is NITE BP-03805.