Lactic acid bacteria and immunostimulants

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

[Technical Field]

[0001] This invention relates to Lactobacillus lactic acid bacteria and immunostimulants. [Background technology]

[0002] Immunity plays a crucial role in maintaining and improving health, and research into this area is being conducted in various fields. Immunity consists of innate and adaptive immunity, both of which are responsible for the body's defense mechanisms. With increasing health consciousness, there is a strong desire for ways to enhance these immune functions. One known method for enhancing immune function is the intake of lactic acid bacteria. While there are various types of lactic acid bacteria, Lactobacillus species are industrially useful because they can be used in a wide variety of foods, such as yogurt production. Therefore, there is a strong demand for the development of Lactobacillus species with high immunostimulatory activity. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Patent No. 6170190 [Patent Document 2] Patent No. 6652331 [Patent Document 3] Patent No. 6705628 [Patent Document 4] Patent No. 6796299 [Patent Document 5] Patent No. 5968655 [Overview of the project] [Problems that the invention aims to solve]

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

Means for Solving the Problems

[0005] Under such circumstances, the present inventors conducted studies on various lactic acid bacteria from the perspective of immunostimulation. As a result, they found multiple lactic acid bacteria that exhibit high immunostimulatory activity among the innumerable lactic acid bacteria. Moreover, they found that there are highly active ones among Lactobacillus helveticus, which is a lactic acid bacterium of the genus Lactobacillus. The present invention is based on such new findings. Therefore, the present invention provides the following immunostimulants, lactic acid bacteria, etc.: Item 1. An immunostimulant containing Lactobacillus helveticus as an active ingredient, wherein the Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of 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) When 1×10 5 cells / well of this lactic acid bacterium is added to a well seeded with 250 μL of human-derived peripheral blood mononuclear cell fluid at a concentration of 8×10 7 cells / mL, it produces 100 ng / mL or more of IgA. (2) When 4×10 5 cells / well of this lactic acid bacterium is added to a well seeded with 200 μL of human-derived dendritic cell-like cells at a concentration of 1.25×10 6The expression intensity of CD86 when the lactic acid bacteria are added to the cells / wells is 1.5 times or more than that of the control without the addition of lactic acid bacteria. (3) 200 μL of human-derived dendritic cell-like cells at a concentration of 1.25×10 5 cells / mL were seeded, and when 4×10 6 cells / well of the lactic acid bacteria are added, it produces 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 of the above (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 dendritic cell-like cell activation, IgA production induction, and IL-12 production induction possessed by the Lactobacillus helveticus 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 dendritic cell-like cell activation, IgA production induction, and IL-12 production induction possessed by the Lactobacillus helveticus are higher than that of Lactococcus lactis JCM5805 strain.

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

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

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

[0014] Item 10. A preventive or therapeutic agent for viral infectious diseases, containing the immunostimulant according to any one of items 1 to 7.

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

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

[0017] Item 13. A lactic acid bacterium for use in immunostimulation, which is Lactobacillus helveticus having 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) When adding 1×10 5 cells / well of the lactic acid bacterium to a well seeded with 250 μL of human-derived peripheral blood mononuclear cell suspension at a concentration of 8×10 7 cells / mL, it produces 100 ng / mL or more of IgA. (2) When adding 4×10 5 cells / well of the lactic acid bacterium to a well seeded with 200 μL of human-derived dendritic cell-like cells at a concentration of 1.25×10 6 cells / mL, the expression intensity of CD86 is 1.5 times or more compared to the control without adding the lactic acid bacterium. (3) When adding 4×10 5 cells / well of the lactic acid bacterium to a well seeded with 200 μL of human-derived dendritic cell-like cells at a concentration of 1.25×10 6 cells / mL, it produces 300 pg / mL or more of IL-12.

[0020] Item 16. Lactobacillus helveticus for use as described in Item 15, wherein the Lactobacillus helveticus satisfies the three requirements (1) to (3) above.

[0021] Item 17. A 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—dendritic cell-like cell activation, IgA production induction, and IL-12 production induction—is higher than that of Lactococcus lactis JCM5805 strain.

[0022] Item 18. Lactobacillus helveticus having three immunostimulatory functions, namely dendritic cell-like cell activation, IgA production induction, and IL-12 production induction, which are higher than those of Lactococcus lactis JCM5805 strain, for use as described in any one of items 13 to 17.

[0023] Item 19. Lactobacillus for use as described in any one of items 13 to 18, wherein the immunostimulation is the maintenance of the immune function of a healthy person.

[0024] Item 20. A composition for immunostimulation comprising Lactobacillus helveticus as an active ingredient, wherein Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of 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 (1) to (3) below. (1) 8 × 10 51 × 10⁶ wells were seeded with 250 μL of human peripheral blood mononuclear cell solution at a concentration of cells / mL. 7 When the lactic acid bacteria are added in a cell / well quantity, they produce IgA at a concentration of 100 ng / mL or higher. (2) 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6 The CD86 expression level in cells / wells when the lactic acid bacteria are added is 1.5 times or more compared to the control without the lactic acid bacteria. (3) 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6 When the lactic acid bacteria in cells / well are added, they produce IL-12 at a concentration of 300 pg / mL or higher.

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

[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—dendritic cell-like cell activation, IgA production induction, and IL-12 production induction—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—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 the maintenance of the immune function of a healthy person. Item 27. A lactic acid bacterium that is Lactobacillus helveticus, wherein the lactic acid bacterium has three functions: dendritic cell-like cell activation, IgA production induction, and IL-12 production induction.

[0031] Item 28. Lactobacillus helveticus GCL1815, or Lactobacillus helveticus GCJ5-4B, as described in item 27.

[0032] Item 29. The lactic acid bacteria according to item 27 or 28, wherein the Lactobacillus helveticus satisfies at least one requirement selected from the group consisting of (1) to (3) below. (1) 8 × 10 5 1 × 10⁶ wells were seeded with 250 μL of human peripheral blood mononuclear cell solution at a concentration of cells / mL. 7 When the lactic acid bacteria are added in a cell / well quantity, they produce IgA at a concentration of 100 ng / mL or higher. (2) 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6 The CD86 expression level in cells / wells when the lactic acid bacteria are added is 1.5 times or more compared to the control without the lactic acid bacteria. (3) 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6 When the lactic acid bacteria in cells / well are added, they produce IL-12 at a concentration of 300 pg / mL or higher.

[0033] Item 30. The lactic acid bacteria described in Item 29, wherein Lactobacillus helveticus satisfies the three requirements (1) to (3) above.

[0034] Item 31. A 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—dendritic cell-like cell activation, IgA production induction, and IL-12 production induction—is higher than that of Lactococcus lactis JCM5805 strain.

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

[0036] Item 33. An oral composition for maintaining the immune function of a healthy person, comprising Lactobacillus helveticus as an active ingredient, wherein Lactobacillus helveticus has three functions: dendritic cell-like cell activation, IgA production induction, and IL-12 production induction.

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

[0038] Item 35. An immunostimulatory method comprising administering an effective amount of Lactobacillus helveticus to a subject in need thereof, wherein Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production. 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 Lactobacillus helveticus has three functions: dendritic cell-like cell activation, induction of IgA production, and induction of IL-12 production. 37. The use described in 34 or the method described in 35 or 36, wherein the Lactobacillus helveticus is Lactobacillus helveticus GCL1815 or Lactobacillus helveticus GCJ5-4B.

[0039] 38. The use described in 34, the method described in 35 or 36, or the use or method described in 37, wherein Lactobacillus helveticus satisfies at least one requirement selected from the group consisting of (1) to (3) below. (1) 8 × 10 5 1 × 10⁶ wells were seeded with 250 μL of human peripheral blood mononuclear cell solution at a concentration of cells / mL. 7 When the lactic acid bacteria are added in a cell / well quantity, they produce IgA at a concentration of 100 ng / mL or higher. (2) 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6 The CD86 expression level in cells / wells when the lactic acid bacteria are added is 1.5 times or more compared to the control without the lactic acid bacteria. (3) 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6 When the lactic acid bacteria in cells / well are added, they produce IL-12 at a concentration of 300 pg / mL or higher.

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

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

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

[0043] Item 42. The use described in item 34, the method described in item 35, or any one of items 37 to 41, wherein the immunostimulation is the maintenance of the immune function of a healthy person.

[0044] Item 43. The method according to any one of items 35 to 42, wherein 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 Lactobacillus helveticus is administered as food or drink.

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

[0048] Item 47. The use described in any one of items 34-42, 44, and 46, wherein the immunostimulant is a prophylactic or therapeutic agent for viral infections. [Effects of the Invention]

[0049] According to the present invention, it is possible to provide a novel immunostimulant using Lactobacillus lactic acid bacteria that has high immunostimulation. [Brief explanation of the drawing]

[0050] [Figure 1]The results of evaluating the IgA production capacity of Preparation Example 1 and Preparation Example 2 are shown. 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 [Figure 2] The results of evaluating the activation of dendritic cell-like cells in Preparation Example 1 and Preparation Example 2 are shown. [Figure 3] The results of evaluating NK cell activation in Preparation Example 1 and Preparation Example 2 are shown. [Modes for carrying out the invention]

[0051] Immunostimulants The present invention provides an immunostimulant comprising Lactobacillus helveticus as an active ingredient, wherein Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0052] Examples of Lactobacillus helveticus include Lactobacillus helveticus GCL1815 (including those denoted as "Lactobacillus helveticus GCD8-9D"; the same applies hereinafter), Lactobacillus helveticus GCJ5-4B, etc., with Lactobacillus helveticus GCL1815 and Lactobacillus helveticus GCJ5-4B being preferred. In a preferred embodiment, examples of Lactobacillus helveticus include strains deposited on January 20, 2023, at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (postal code 292-0818), with accession numbers NITE BP-03804 or NITE BP-03805).

[0053] In this invention, Lactobacillus helveticus, the active ingredient of the immunostimulant, possesses all three functions: dendritic cell-like cell activation, IgA production induction, and IL-12 production induction. Specifically, Lactobacillus helveticus, the active ingredient in immunostimulants, 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 invade the body, they are the first to eliminate them. Furthermore, dendritic cells transmit information about invading foreign substances to T cells and B cells through antigen presentation, and play an important role in adaptive immunity by 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 inhibitory activity against viral replication and are the main producers of type I interferon, which also contributes to the activation of NK cells, but their antigen-presenting ability is weak. On the other hand, cDCs produce IL-12, which activates NK cells, and also have strong antigen-presenting ability, thus playing an important role in inducing adaptive immunity. IgA, a type of adaptive immune marker, has low specificity compared to other immunoglobulins and acts on many types of bacteria and viruses, so it is 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 this invention, "dendritic cell-like cells" is a general term for dendritic cells collected from living organisms 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 vivo can be evaluated.

[0055] Specifically, dendritic cell-like cell activation by lactic acid bacteria is, for example, 1.25 × 10⁻⁶ 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6The expression intensity of CD86 can be evaluated by adding lactic acid bacteria cells / well and measuring the CD86 expression intensity. More specifically, the above measurement can be performed according to the method described in the examples of this application. In the present invention, it is preferable to select Lactobacillus helveticus such that the CD86 expression intensity measured by the above method is, for example, 105% or more, preferably 130% or more, more preferably 145% or more, even more preferably 150% or more, still more preferably 180% or more, and particularly preferably 200% or more, compared to a control without the addition of lactic acid bacteria (as the ratio of [CD86 expression intensity in the group with lactic acid bacteria containing the active ingredient of the present invention] / [CD86 expression intensity in the control]). The dendritic cell-like cells used for the above measurement are not limited, but typically, immortalized dendritic cells obtained by differentiating immature dendritic cells (Mylc cells) used in the examples of this application can be used.

[0056] Furthermore, the induction of IgA production by lactic acid bacteria is, for example, 8 × 10 5 1 × 10⁶ wells were seeded with 250 μL of human peripheral blood mononuclear cell solution at a concentration of cells / mL. 7 The lactic acid bacteria can be evaluated by adding cells / wells of the lactic acid bacteria and measuring the amount of IgA produced. More specifically, the above measurement can be performed according to the method described in the examples of this application. In the present invention, it is preferable that Lactobacillus helveticus is such that when measured by the above method, an amount of IgA of 100 ng / mL or more, more preferably 150 ng / mL or more, is detected. Furthermore, in the present invention, it is preferable that Lactobacillus helveticus is such that when measured by the above method, the amount of IgA produced is such that, compared to a control without the addition of lactic acid bacteria (as the ratio of [IgA production in the group with the active ingredient of the present invention and lactic acid bacteria (ng / mL)] / [IgA production in the control (ng / mL)], for example, 105% or more, preferably 110% or more, preferably 120% or more, preferably 140% or more, and preferably 170% or more).

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

[0058] Furthermore, in the present invention, among Lactobacillus helveticus, it is preferable that the amount of IL-12 produced when measured by the above method is, compared to a control without the addition of lactic acid bacteria (in the ratio of [IL-12 production in the group with the active ingredient of the present invention and lactic acid bacteria (pg / mL)] / [IL-12 production in the control (pg / mL)]), 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.

[0059] In a more preferred embodiment, it is preferable to use a Lactobacillus helveticus strain that exhibits a higher level of immunostimulatory activity than Lactococcus lactis JCM5805 strain in at least one (preferably at least two, more preferably all three) immunostimulatory activity selected from the group consisting of dendritic cell-like cell activation, IgA production induction, and IL-12 production induction. The method for measuring dendritic cell-like cell activation, IgA production induction, and IL-12 production induction in this embodiment is the same as described above. The above-mentioned functions of Lactococcus lactis JCM5805 strain can be measured by performing the same procedure as described above, except that Lactobacillus helveticus, the active ingredient of the immunostimulator of the present invention, is used instead of Lactobacillus helveticus.

[0060] In the present invention, the Lactobacillus helveticus can be either dead cells (such as heat-killed cells) or live cells. In the present invention, the Lactobacillus helveticus can be used alone or in combination of two or more types.

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

[0062] In the present invention, Lactobacillus helveticus itself, which is the active ingredient of the present invention, may be used as an immunostimulant, or it may be used as a composition in which the lactic acid bacteria powder is combined with various carriers that are pharmaceutically acceptable or can be added to food (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, viscosity enhancers, excipients, binders, etc.). In this embodiment, the content of Lactobacillus helveticus in the immunostimulant composition is not limited, but can be appropriately set in 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 powder used in immunostimulants is not limited, but can be appropriately set within a range such as 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 isotonic agents 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 isotonic agents can be used individually or in combination of two or more.

[0064] Examples of chelating agents include edetates such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate, and citric acid. These chelating agents can be used individually or in combination of two or more.

[0065] Examples of stabilizers include sodium bisulfite.

[0066] Examples of pH adjusting agents 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 bicarbonates 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 adjusting agents can be used individually 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, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, quaternary ammonium salts such as cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used individually or in combination of two or more.

[0068] Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherols. These antioxidants can be used individually or in combination of two or more.

[0069] Examples of solubilizers include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, and D-mannitol. These solubilizers can be used individually or in combination of two or more.

[0070] Examples of viscosity-reducing agents include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, chondroitin sulfate sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol. These viscosity-reducing agents can be used individually or in combination of two or more.

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

[0072] Examples of binders include crystalline cellulose, starch, sucrose, hydroxypropylcellulose, gelatin, acacia powder, polyvinylpyrrolidone, pullulan, dextrin, cyclodextrin, methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyethylene glycol, and the like. These binders can be used individually or in combination of two or more.

[0073] Furthermore, the above composition may further contain substances known to have immunostimulatory functions in addition to Lactobacillus helveticus. Examples of substances known to have immunostimulatory functions include vitamin C, vitamin A, and zinc. These substances can be used individually or in combination of two or more.

[0074] The immunostimulant of the present invention provides an immunostimulatory effect when ingested by a target (preferably a mammal such as a human). The amount of the immunostimulant of the present invention to be ingested is not limited, but the daily intake of the active ingredient, Lactobacillus helveticus, can be appropriately set in the 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 amount of the immunostimulant of the present invention to be ingested 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 in the 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 Lactobacillus helveticus. Oral compositions include food and beverage compositions, pharmaceutical compositions, etc. Food and beverage compositions in the present invention also include health functional foods (nutrient functional foods, foods for specified health uses, foods with functional claims), etc.

[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; and foods such as ice cream, frozen desserts, almond-containing foods, biscuits (cream sandwich biscuits, etc.), chocolates (including semi-chocolate, etc.), and fermented milk foods (yogurt, cheese, etc.). As for ice cream, lacto ice (lacto ice containing fermented milk, etc.) is preferred. As for cream sandwich biscuits, those in which Lactobacillus helveticus is incorporated into the cream are preferred. As for semi-chocolate, corn-containing semi-chocolate, etc. In the present invention, among these food and beverages, dairy products and food and beverages containing dairy products are preferred. As for dairy products, fermented milk, ice cream, and dairy beverages are preferred. As for food and beverages containing dairy products, cream sandwich biscuits and milk chocolate are preferred. As for frozen desserts, milk-containing frozen desserts (including frozen desserts containing fermented milk, etc.) are preferred. The food and beverage compositions in the present invention also include supplements, etc. As for supplements, sports supplements (sports supplements containing amino acids, etc.) are preferred. Examples of sports supplements containing amino acids include glutamine-containing sports supplements.

[0077] Details of the active ingredient Lactobacillus helveticus in the embodiment of the oral composition, its intake amount, etc., are the same as described above in the description of the immunostimulant. In this embodiment, the Lactobacillus helveticus content in the oral composition is not particularly limited, but can be appropriately set in 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 immunostimulatory purposes.

[0078] Preventive or therapeutic agents for viral infections An immunostimulatory effect can be induced by administering a specific Lactobacillus helveticus, which is the active ingredient of the present invention. Therefore, the immunostimulatory effect provided by the present invention can be expected to prevent or treat viral infections. Accordingly, the present invention provides a preventive or therapeutic agent for viral infections containing the aforementioned immunostimulator. The present invention also provides a preventive or therapeutic agent for viral infections containing the aforementioned Lactobacillus helveticus. The target viruses are not limited to, but examples include influenza virus, coronavirus, adenovirus, RSV, human metapneumovirus, rhinovirus, etc. Details of the active ingredient Lactobacillus helveticus in embodiments of the preventive or therapeutic agent for viral infections, its intake, etc., are the same as described above in the description of the immunostimulator and oral composition.

[0079] Lactobacillus helveticus According to the present invention, the aforementioned Lactobacillus helveticus can provide immunostimulatory effects, as well as preventive or therapeutic effects against viral infections. Furthermore, the aforementioned Lactobacillus helveticus is novel. Accordingly, the present invention also provides Lactobacillus helveticus itself. The method of using Lactobacillus helveticus in this embodiment is the same as described above in the descriptions of immunostimulants, oral compositions, and agents for the prevention or treatment of viral infections. [Examples]

[0080] Preparation of lactic acid bacteria dead bacteria powder Preparation Example 1 Our proprietary lactic acid bacteria strains were cultured statically for 48 hours at 30°C or 37°C using MRS medium (Merck Millipore). After culturing, the cells were collected by centrifugation at 8,000×G for 10 minutes, washed three times with endotoxin-free physiological saline (hereinafter referred to as physiological saline), and then sterilized by autoclaving at 121°C for 15 minutes. Subsequently, the bacterial cells were freeze-dried, and a lactic acid bacteria suspension was obtained by adjusting the concentration with physiological saline to 10 mg / mL.

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

[0082] Screening based on IgA production induction ability 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 (Thermo Fisher Scientific) to 10% by mass of inactivated fetal bovine serum (Thermo Fisher Scientific) (hereinafter referred to as FBS), 1% by mass of MEM vitamin solution (Thermo Fisher Scientific), 1% by mass of MEM non-essential amino acid solution (Thermo Fisher Scientific), 1% by mass of penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd.), 1% by mass of sodium pyruvate solution (Thermo Fisher Scientific), and 0.1% by mass of 2-mercaptoethanol (Thermo Fisher Scientific).

[0083] Preparation of human peripheral blood mononuclear cells (PBMCs) Frozen human peripheral blood mononuclear cells (PBMCs) derived from normal donors (Astarte Biologics) were rapidly thawed in a 37°C water bath and added to a 50 mL centrifuge tube containing 10% FBS-containing RPMI1640 medium. The tubes were then slowly inverted and mixed. The PBMCs were precipitated by centrifugation at 200×G for 5 minutes, the supernatant was removed with an aspirator, and 10 mL of PBMC medium was added to the pelletized PBMCs to resuspend them and obtain a PBMC suspension.

[0084] Seeding of PBMC, addition of lactic acid bacteria, and cultivation. 20 μL of PBMC suspension and 2 μL of Acridion Orange (Logos Biosystems) were added to a 1.5 mL microcentrifuge tube and mixed thoroughly. Then, the cell concentration was calculated using a cell counter (Logos Biosystems, L20001). The PBMC suspension was then mixed in PBMC medium, and the cell concentration was 8 × 10⁶. 5 The lactic acid bacteria suspension was diluted to a concentration of cells / mL and seeded in 250 μL portions in a 96-well plate (TPP). Furthermore, 50 μg / mL of the lactic acid bacteria suspension from Preparation Example 1 was added to each well of the 96-well plate. A control well without added lactic acid bacteria was also prepared. The samples were incubated statically for 120 hours in a CO2 incubator at 37°C and 5% CO2.

[0085] IgA measurement A 96-well plate in which PBMCs had been cultured for 120 hours was removed, and 250 μL of the culture medium was transferred to an 8-strap microtube. The PBMCs were precipitated by centrifugation at 200×G for 10 minutes, and 220 μL of the culture supernatant was transferred to a new 8-strap microtube. The supernatant was further centrifuged at 1500×G for 10 minutes, and the culture supernatant was subjected to IgA quantification by ELISA. IgA quantification was performed using the Abcam Human IgA ELISA Kit, and the IgA concentration was measured according to the instructions in the kit's Protocol Booklet. To confirm the reproducibility of the experiment, the procedure from PBMC seeding to IgA measurement was performed twice.

[0086] Test results Lactic acid bacteria were selected from a culture medium in which PBMCs were cultured without the addition of lactic acid bacteria (control), and then the lactic acid bacteria prepared in Preparation Example 1 were added to the culture medium in which PBMCs were cultured, resulting in a doubling or more of the IgA concentration in the culture medium. Based on the screening conducted under the above conditions, 70 types of lactic acid bacteria were selected as having the ability to induce 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] [Table 1] TIFF0007835954000002.tif127170 Of these, 14 types of lactic acid bacteria were selected based on factors such as different bacterial species and ease of cultivation, and were used in subsequent tests. The selected lactic acid bacteria are marked with an asterisk (*) in Table 1.

[0088] Evaluation of IgA production induction Isolation of peripheral blood mononuclear cells from human peripheral blood With the cooperation of 12 healthy adults, 20 mL of blood was collected and each was placed in a blood collection tube containing an anticoagulant (heparin sodium). First, the Lymphoprep® Tube (manufactured by AXS) was centrifuged at 400 × G for 1 minute to settle the Lymphoprep® inside before use. Next, blood and physiological saline were mixed in a 1:1 ratio in a 50 mL centrifuge tube, and 20-30 mL of this mixture was transferred to the Lymphoprep® Tube and centrifuged at 800 × G for 20 minutes at room temperature. From the layer formed by centrifugation, a solution of the peripheral blood mononuclear cell layer was collected using a Pasteur pipette into a new 50 mL centrifuge tube. 20 mL of physiological saline was added to the 50 mL centrifuge tube containing the peripheral blood mononuclear cells, and the peripheral blood mononuclear cells were pelletized by centrifuging 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 and suspended to obtain a peripheral blood mononuclear cell solution.

[0089] Seeding of peripheral blood mononuclear cells, addition of lactic acid bacteria, and cultivation. 90 μL of PBMC medium and 10 μL of peripheral blood mononuclear cell solution were measured into 1.5 mL microcentrifuge tubes and thoroughly mixed. Then, 20 μL of this mixture was transferred to a new 1.5 mL microcentrifuge tube, and 2 μL of Acridion Orange (Logos Biosystems) was added and thoroughly mixed. Subsequently, the cell concentration was calculated using a cell counter (Logos Biosystems, L20001). Peripheral blood mononuclear cell solution was then mixed in PBMC medium at a rate of 8 × 10⁶. 5The lactic acid bacteria suspensions were diluted to a concentration of cells / mL and seeded in 250 μL portions in 96-well plates (TPP). Furthermore, 1 × 10⁶ of the lactic acid bacteria suspensions from Preparation Example 1 and Preparation Example 2 were added. 7 Cells were added to each well of a 96-well plate, and lipopolysaccharide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as LPS) was added to each well to a final concentration of 10 ng / mL as a positive control. In addition, wells without added lactic acid bacteria were prepared as a control. The cells were incubated statically for 120 hours in a CO2 incubator at 37°C and 5% CO2.

[0090] IgA measurement A 96-well plate containing peripheral blood mononuclear cells cultured for 120 hours was removed, and 250 μL of the culture medium was transferred to an 8-strap microtube. Peripheral blood mononuclear cells were precipitated by centrifugation at 200×G for 10 minutes, and 220 μL of the culture supernatant was transferred to a new 8-strap microtube. The supernatant was further centrifuged at 1500×G for 10 minutes, and the culture supernatant was subjected to IgA quantification by ELISA. IgA quantification was performed using the Abcam Human IgA ELISA Kit, and the IgA concentration was measured according to the instructions in the kit's Protocol Booklet.

[0091] Evaluation of dendritic cell-like cell activation Preparation of differentiation medium Mylc-specific culture medium B (manufactured by Mycan Technologies Co., Ltd.) and Mylc-specific culture medium supplement B (manufactured by Mycan Technologies Co., Ltd.) were mixed in 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 Example 1 and Preparation Example 2 were diluted 10-fold with physiological saline to obtain a lactic acid bacteria suspension (1 mg / mL). The bacterial count concentration of the lactic acid bacteria suspension (1 mg / mL) was measured using the bleed method, resulting in 4 × 10⁻⁶ values. 7 The mixture was diluted with differentiation medium to achieve a cell / mL concentration.

[0093] Seeding of dendritic cells, addition of lactic acid bacteria, and cultivation. Immortalized dendritic cells aMylc-2-A differentiated cells (hereinafter referred to as dendritic cells) purchased from Mycan Technologies, Inc. and differentiated for 3 days were collected. Next, the dendritic cells were centrifuged at 300×G for 5 minutes to precipitate, 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 divided into 2.5 × 10⁶ cells in the differentiation medium. 5 The lactic acid bacteria suspension was diluted to a concentration of cells / mL and seeded in 100 μL portions on Nunclon Sphera 96-well round-bottom plates (Thermo Fisher Scientific). 7 100 μL of lactic acid bacteria (cells / 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 without lactic acid bacteria was also prepared. The samples were incubated statically for 24 hours in a CO2 incubator at 37°C and 5% CO2.

[0094] Collection of dendritic cells and culture supernatant A 96-well plate in which dendritic cells had been cultured for 24 hours was removed, and 200 μL of the culture medium was transferred to an 8-channel microcentrifuge tube. The dendritic cells were precipitated by centrifugation at 500×G for 10 minutes, and 140 μL of the culture supernatant was transferred to a new 8-channel microcentrifuge tube. The culture supernatant remaining in the 8-channel microcentrifuge tube was removed with an aspirator to obtain the dendritic cell precipitate.

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

[0096] 2. Antibody solution An antibody solution was obtained by mixing staining buffer and PE anti-human CD86 Antibody in a 49:1 ratio.

[0097] Measurement of cell surface marker (CD86) The dendritic cell precipitate was suspended in 200 μL of staining buffer. The cells were centrifuged at 500×G for 5 minutes, the supernatant was removed using an aspirator, 20 μL of FcR blocking solution was added and the cells were suspended. The mixture was then allowed to stand on ice for 20 minutes. Next, 20 μL of antibody solution was added and the cells were suspended again. The mixture was then allowed to stand on ice for 30 minutes. After standing, 160 μL of staining buffer was added and the cells were suspended. The cells were centrifuged at 500×G for 5 minutes, and the supernatant was removed using an aspirator. Then, 200 μL of staining buffer was added and the cells were suspended again. The expression intensity of the cell surface marker (CD86) was measured using a flow cytometer (Sony Corporation, SA3800) (laser wavelength: 488 nm) on the solution in which the cells were resuspended with 200 μL of staining buffer.

[0098] Evaluation of NK cell activation As mentioned above, Collection of dendritic cells and culture supernatant Using the culture supernatant obtained by the method described in [reference], IL-12 levels were measured to evaluate NK cell activation. IL-12 levels were measured using the Abcam Human IL-12 p70 ELISA Kit, and the IL-12 concentration was measured according to the instructions (Protocol Booklet) included in the kit.

[0099] Evaluation of IgA production induction Figure 1 shows the results of evaluating the IgA production capacity of Preparation Example 1 and Preparation Example 2. As shown in Figure 1, compared to the control group without added lactic acid bacteria, adding and culturing the lactic acid bacteria from Preparation Example 1 significantly increased the IgA production induction capacity of peripheral blood mononuclear cells, except for cr2, la44, cr11, and la15. Similarly, compared to the case with added JCM5805 strain from Preparation Example 2, adding and culturing the lactic acid bacteria from Preparation Example 1 significantly increased the IgA production induction capacity of peripheral blood mononuclear cells, except for cr2, la44, cr11, and la15. It is expected that in animals, including humans, ingesting these lactic acid bacteria will enhance IgA production and improve infection defense by preventing the invasion of viruses and bacteria into the body.

[0100] Evaluation of dendritic cell-like cell activation Figure 2 shows the results of evaluating the activation of dendritic cell-like cells in Preparation Example 1 and Preparation Example 2. The activation of dendritic cell-like cells was evaluated by measuring the expression intensity of the cell surface marker (CD86). As shown in Figure 2, compared to the control group without added lactic acid bacteria, adding the lactic acid bacteria from Preparation Example 1 resulted in significant activation of dendritic cells, except for pe6. Similarly, compared to the case with added JCM5805 strain from Preparation Example 2, adding the lactic acid bacteria from Preparation Example 1 resulted in significant activation of dendritic cells, except for je1, fe20, pe5, la23, la24, la44, pe6, and la15. In animals, including humans, it is expected that ingesting these lactic acid bacteria will activate dendritic cells, thereby stimulating overall immunity.

[0101] Evaluation of NK cell activation Figure 3 shows the results of evaluating NK cell activation in Preparation Example 1 and Preparation Example 2. NK cell activation was evaluated by measuring IL-12 concentration. As shown in Figure 3, compared to the control group without added lactic acid bacteria, adding the lactic acid bacteria from Preparation Example 1 and culturing resulted in significant NK cell activation, except for la44. In particular, he11 and he12 significantly activated NK cells. Furthermore, compared to the case with added JCM5805 strain from Preparation Example 2, adding the lactic acid bacteria from Preparation Example 1 and culturing resulted in significant NK cell activation. In animals, including humans, it is expected that ingesting these lactic acid bacteria will activate NK cells, thereby enhancing their ability to attack cells infected with bacteria and viruses and prevent the spread of infection, thus improving infection defense capabilities.

[0102] As shown in the above tests, he11 and he12 demonstrated all three functions: dendritic cell-like cell activation, IgA production induction, and IL-12 production induction. Specifically, he11 and he12 demonstrated all of the following functions (1) to (3): (1) 8 × 10 5 1 × 10⁶ wells were seeded with 250 μL of human peripheral blood mononuclear cell solution at a concentration of cells / mL. 7 When the lactic acid bacteria are added in a cell / well quantity, they produce IgA at a concentration of 100 ng / mL or higher. (2) 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6 The CD86 expression level in cells / wells when the lactic acid bacteria are added is 1.5 times or more compared to the control without the lactic acid bacteria. (3) 1.25 × 10 5 200 μL of human-derived dendritic cell-like cells at a concentration of cells / mL were seeded in wells, and then 4 × 10 6 When the lactic acid bacteria in cells / well are added, they produce IL-12 at a concentration of 300 pg / mL or higher. Furthermore, he11 and he12 exhibited higher immunostimulatory functions in all three areas—dendritic cell-like cell activation, IgA production induction, and IL-12 production induction—than the JCM5805 strain.

[0103] Prescription Example 1 Ice cream containing dead lactic acid bacteria powder was obtained using the ingredients shown in Table 2. Specifically, first, egg yolks and sugar were mixed well in a bowl. Separately, heavy cream, milk, and dead lactic acid bacteria powder were placed in a saucepan and heated. When the mixture began to simmer around the edges of the saucepan, it was removed from the heat and gradually added to the bowl of egg yolks and sugar mixture while stirring. The resulting mixture was placed in a metal container, allowed to cool slightly, covered, and cooled at -20°C. After 3 hours, the mixture was stirred, and then stirred four more times at 30-minute intervals to obtain ice cream containing dead lactic acid bacteria powder.

[0104] [Table 2]

[0105] Prescription Example 2 Yogurt containing dead lactic acid bacteria powder was obtained using the materials shown in Table 3.

[0106] [Table 3]

[0107] Prescription Example 3 Chocolate containing dead lactic acid bacteria powder was obtained using the materials shown in Table 4. Specifically, chocolate was melted in a water bath at approximately 50°C, then the dead lactic acid bacteria powder was added and mixed well, the mixture was placed in a mold and shaped, and then allowed to solidify at room temperature to obtain chocolate containing dead lactic acid bacteria powder.

[0108] [Table 4]

Claims

1. An immunostimulant containing, as an active ingredient, Lactobacillus helveticus with accession number NITE BP-03804 at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), or Lactobacillus helveticus with accession number NITE BP-03805 at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE).

2. The immunostimulant according to claim 1, wherein the immunostimulation is the maintenance of the immune function of a healthy person.

3. An oral composition for immunostimulation containing the immunostimulant described in claim 1 or 2.

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

5. An agent for the prevention or treatment of viral infections, comprising the immunostimulant described in claim 1 or 2.

6. Lactobacillus helveticus, whose accession number at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE) is NITE BP-03804.

7. Lactobacillus helveticus, whose accession number at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE) is NITE BP-03805.

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