Lactic acid bacteria and immunostimulant

Abstract

The present invention provides an immunologic stimulant which contains Lactobacillus helveticus, and wherein the Lactobacillus helveticus has three functions: the activation of dendritic cell-like cells, the induction of the production of immunoglobulin A (IgA), and the induction of the production of interleukin-12 (IL-12).

Description

Technical Field

[0001] The present invention relates to lactobacillus ( Lactobacillus ) are lactic acid bacteria and immune stimulants. Background Art

[0002] The immune system plays a vital role in maintaining and improving health, and research on immunity has been conducted in many fields. There are two types of immunity: innate immunity and acquired immunity, both of which are responsible for the body's defense function. With the continuous improvement of health awareness, there is a strong demand for methods to enhance immune function. It is known that taking lactic acid bacteria can enhance such immune function. There are many types of lactic acid bacteria, and lactic acid bacteria of the genus Lactobacillus have industrial applicability due to their use in various food products, such as yogurt production. Therefore, there is a strong demand for the development of lactic acid bacteria of the genus Lactobacillus with high immunostimulatory activity.

[0003] Reference List Patent Literature (PTL) PTL1: Japanese Patent No. 6170190 PTL2: Japanese Patent No. 6652331 PTL3: Japanese Patent No. 6705628 PTL4: Japanese Patent No. 6796299 PTL5: Japanese Patent No. 5968655 Summary of the Invention

[0004] Technical issues One object of the present invention is to provide a method for Lactobacillus ) is a novel immunostimulant of the genus Lactobacillus, which has high immunostimulatory activity. In a preferred embodiment, an object of the present invention is to provide a novel Lactobacillus ( Lactobacillus ) species, which exhibits higher immunostimulatory activity than the known lactic acid bacteria strain JCM 5805, and an immunostimulatory agent using the lactic acid bacteria species.

[0005] Solution Under such circumstances, the present inventors have studied various lactic acid bacteria from the viewpoint of immunostimulation and have found a variety of lactic acid bacteria species with high immunostimulatory activity from numerous existing lactic acid bacteria. In addition, the present inventors have found that in Lactobacillus helveticus ( Lactobacillus helveticus ) species, some strains exhibited high activity. The present invention is based on this new discovery. Therefore, the present invention provides immunostimulants, lactic acid bacteria, and other subjects as shown below: Item 1. An immunostimulant comprising Lactobacillus helveticus ( Lactobacillus helveticus ), wherein the Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of immunoglobulin A (IgA) production, and induction of interleukin-12 (IL-12) production.

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

[0007] Item 3. The immunostimulant according to item 1 or 2, wherein the Lactobacillus helveticus satisfies at least one of the following (1) to (3): (1) When 250 μL of 8×10 5 cells / mL of human peripheral blood mononuclear cell solution was added to the wells at 1×10 7 When the Lactobacillus helveticus is added to the cells / well, 100 ng / mL or more of IgA is produced; (2) When 200 μL of a solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus is added to the cells / well, the expression intensity of CD86 becomes 1.5 times or higher than that of the control group without any lactic acid bacteria; and (3) When 200 μL of the solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus was added to the cells / well, 300 pg / mL or more of IL-12 was produced.

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

[0009] Item 5. The immunostimulant according to any one of items 1 to 4, wherein at least one of the following three immunostimulatory functions possessed by Lactobacillus helveticus is higher than that of Lactococcus lactis JCM 5805 ( Lactococcus lactis JCM 5805) strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0010] Item 6. The immunostimulant according to any one of items 1 to 5, wherein the following three immunostimulatory functions possessed by Lactobacillus helveticus are all higher than those of Lactococcus lactis JCM 5805 ( Lactococcus lactis JCM 5805) strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0011] Item 7. The immunostimulatory agent according to any one of items 1 to 6, wherein the immunostimulation is to maintain the immune function of a healthy individual.

[0012] Item 8. An oral composition for immunostimulation, comprising the immunostimulatory agent 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 or beverage.

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

[0015] Item 11. A species of Lactobacillus ( Lactobacillus ) genus, and its deposit number in the Patent Microorganisms Collection (NPMD) of the National Institute of Technology Evaluation (NITE) of Japan is NITE BP-03804.

[0016] Item 12. A species of Lactobacillus ( Lactobacillus ) genus, and its deposit number in the Patent Microorganisms Depository (NPMD) of the National Institute of Technology Evaluation (NITE) of Japan is NITE BP-03805.

[0017] Item 13. A lactic acid bacterium for immunostimulation, wherein the lactic acid bacterium is Lactobacillus helveticus ( Lactobacillus helveticus ): Activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

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

[0019] Item 15. The lactic acid bacteria for use according to Item 13 or 14, wherein the Lactobacillus helveticus satisfies at least one of the following (1) to (3): (1) When 250 μL of 8×10 5 cells / mL of human peripheral blood mononuclear cell solution was added to the wells at 1×10 7 When the Lactobacillus helveticus is added to the cells / well, 100 ng / mL or more of IgA is produced; (2) When 200 μL of a solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus is added to the cells / well, the expression intensity of CD86 becomes 1.5 times or higher than that of the control group without any lactic acid bacteria; and (3) When 200 μL of the solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus was added to the cells / well, 300 pg / mL or more of IL-12 was produced.

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

[0021] Item 17. The lactic acid bacteria for use according to any one of items 13 to 16, wherein at least one of the following three immunostimulatory functions possessed by Lactobacillus helveticus is higher than that of Lactococcus lactis JCM 5805 strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0022] Item 18. The lactic acid bacteria for use according to any one of items 13 to 17, wherein the following three immunostimulatory functions possessed by the Lactobacillus helveticus are higher than those of the Lactococcus lactis JCM 5805 strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

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

[0024] Item 20. A composition for immune stimulation, comprising 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.

[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 of the following (1) to (3): (1) When 250 μL of 8×10 5 cells / mL of human peripheral blood mononuclear cell solution was added to the wells at 1×10 7 When the Lactobacillus helveticus is added to the cells / well, 100 ng / mL or more of IgA is produced; (2) When 200 μL of a solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus is added to the cells / well, the expression intensity of CD86 becomes 1.5 times or higher than that of the control group without any lactic acid bacteria; and (3) When 200 μL of the solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus was added to the cells / well, 300 pg / mL or more of IL-12 was produced.

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

[0028] Item 24. The composition according to any one of items 20 to 23, wherein at least one of the following three immunostimulatory functions possessed by Lactobacillus helveticus is higher than that of Lactococcus lactis JCM 5805 strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0029] Item 25. The composition according to any one of items 20 to 24, wherein the following three immunostimulatory functions possessed by the Lactobacillus helveticus are higher than those of the Lactococcus lactis JCM 5805 strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0030] Item 26. The composition according to any one of items 20 to 25, wherein the immune stimulation is to maintain the immune function of a healthy individual.

[0031] Item 27. A lactic acid bacterium belonging to Lactobacillus helveticus, wherein the lactic acid bacterium has three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0032] Item 28. The lactic acid bacteria according to item 27, wherein the lactic acid bacteria is Lactobacillus helveticus GCL1815 or Lactobacillus helveticus GCJ5-4B.

[0033] Item 29. The lactic acid bacteria according to item 27 or 28, wherein the Lactobacillus helveticus satisfies at least one of the following (1) to (3): (1) When 250 μL of 8×10 5 cells / mL of human peripheral blood mononuclear cell solution was added to the wells at 1×10 7 When the Lactobacillus helveticus is added to the cells / well, 100 ng / mL or more of IgA is produced; (2) When 200 μL of a solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus is added to the cells / well, the expression intensity of CD86 becomes 1.5 times or higher than that of the control group without any lactic acid bacteria; and (3) When 200 μL of the solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus was added to the cells / well, 300 pg / mL or more of IL-12 was produced.

[0034] Item 30. The lactic acid bacteria according to item 29, wherein the Lactobacillus helveticus satisfies the three requirements of (1) to (3).

[0035] Item 31. The lactic acid bacteria for use according to any one of items 13 to 16, wherein at least one of the following three immunostimulatory functions possessed by Lactobacillus helveticus is higher than that of Lactococcus lactis JCM 5805 strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0036] Item 32. The lactic acid bacteria according to any one of items 27 to 31, wherein the Lactobacillus helveticus has higher three immunostimulatory functions than Lactococcus lactis JCM 5805: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0037] Item 33. An oral composition for maintaining immune function in a healthy individual, comprising 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.

[0038] Item 34. Use of Lactobacillus helveticus in preparing an immunostimulant, wherein the Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0039] Item 35. A method for immune stimulation, comprising administering an effective amount of Lactobacillus helveticus to a subject in need thereof, wherein the Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0040] Item 36. A method for preventing or treating viral infection, comprising administering an effective amount of Lactobacillus helveticus to a subject in need thereof, wherein the Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0041] 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.

[0042] 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 of the following (1) to (3): (1) When 250 μL of 8×10 5 cells / mL of human peripheral blood mononuclear cell solution was added to the wells at 1×10 7 When the Lactobacillus helveticus is added to the cells / well, 100 ng / mL or more of IgA is produced; (2) When 200 μL of a solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus is added to the cells / well, the expression intensity of CD86 becomes 1.5 times or higher than that of the control group without any lactic acid bacteria; and (3) When 200 μL of the solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus was added to the cells / well, 300 pg / mL or more of IL-12 was produced.

[0043] Item 39. The use or method according to item 38, wherein the Lactobacillus helveticus satisfies the three requirements of (1) to (3).

[0044] 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 following three immunostimulatory functions possessed by Lactobacillus helveticus is greater than that of Lactococcus lactis JCM 5805 strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0045] 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 Lactobacillus helveticus has higher three immunostimulatory functions than the Lactococcus lactis JCM 5805 strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0046] 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 immune stimulation is to maintain immune function in a healthy individual.

[0047] Item 43. The method of any one of items 35 to 42, wherein the Lactobacillus helveticus is administered orally.

[0048] Item 44. The use according to any one of items 34 to 42, wherein the immunostimulant is an oral composition.

[0049] Item 45. The method according to any one of items 35 to 43, wherein said Lactobacillus helveticus is administered as a food or a beverage.

[0050] 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.

[0051] Item 47. The use according to any one of items 34 to 42, 44 and 46, wherein the immunostimulant is a preventive or therapeutic agent for viral infection.

[0052] Beneficial effects of the present invention The present invention provides a novel immunostimulant comprising lactic acid bacteria of the genus Lactobacillus having high immunostimulatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The results of the evaluation of the IgA production ability in Preparation Example 1 and Preparation Example 2 are shown. The abbreviations in the figure are as follows: je1: Lactobacillus jensenii 1 ( Lactobacillus jensenii 1), fe6: fermented Lactobacillus mucilaginosus 6 ( Limosilactobacillus fermentum 6), fe20: fermented Lactobacillus mucilaginosus 20 ( Limosilactobacillus fermentum 20), cr2: Lactobacillus crispatus 2 ( Lactobacillus crispatus 2), pe5: Pentosaceus lactobacillus 5 ( Lactiplantibacillus pentosus 5), la23: Lactococcus lactis 23 ( Lactococcus lactis 23), la24: Lactococcus lactis 24 ( Lactococcus lactis 24), he11: Lactobacillus helveticus GCJ5-4B ( Lactobacillus helveticus GCJ5-4B) (Accession No.: NITE BP-03805), he12: Lactobacillus helveticus GCL1815 ( Lactobacillus helveticus GCL1815) (Accession No.: NITE BP-03804), ga8: Lactobacillus gasseri 8 ( Lactobacillus gasseri 8), la44: Lactococcus lactis 44 ( Lactococcus lactis 44), pe6: Pentosaceus lactobacillus 6 ( Lactiplantibacillus pentosus 6), cr11: Lactobacillus crispatus 11 ( Lactobacillus crispatus 11), la15: Lactococcus lactis 15 ( Lactococcus lactis 15).

[0054] Figure 2 The evaluation results of activation of dendritic cell-like cells in Preparation Examples 1 and 2 are shown.

[0055] Figure 3 The results of evaluation of natural killer cell (NK cell) activation in Preparation Examples 1 and 2 are shown. DETAILED DESCRIPTION

[0056] Immunostimulants The present invention provides an immunostimulant comprising Lactobacillus helveticus ( Lactobacillus helveticus ), wherein the Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of immunoglobulin A (IgA) production, and induction of interleukin-12 (IL-12) production.

[0057] Examples of Lactobacillus helveticus include Lactobacillus helveticus GCL1815 ( Lactobacillus helveticus GCL1815) (including the genus described as "Lactobacillus helveticus GCD8-9D ( Lactobacillus helveticus GCD8-9D)" type, the same below), Lactobacillus helveticus GCJ5-4B ( Lactobacillus helveticus GCJ5-4B), among which Lactobacillus helveticus GCL1815 and Lactobacillus helveticus GCJ5-4B are preferred. In a preferred embodiment, examples of Lactobacillus helveticus include strains deposited on January 20, 2023, at the Patent Microorganisms Depository of the National Institute for Technology Evaluation (NITE) of Japan (Address: Room 122, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, 292-0818, Japan), with deposit numbers NITE BP-03804 and NITE BP-03805.

[0058] In the present invention, Lactobacillus helveticus, which is an active ingredient of the immunostimulant, has all three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0059] Specifically, first, Lactobacillus helveticus, the active ingredient of the immunostimulant, activates dendritic cell-like cells. Dendritic cells play an important role in immune function. More specifically, for example, dendritic cells, together with macrophages and NK cells, play an important role in innate immunity. When foreign substances such as bacteria or viruses enter the body, dendritic cells are the first to act to eliminate them. In addition, dendritic cells play an important role in acquired immunity by presenting antigens to T cells and B cells to convey information about invading foreign substances, promoting the production of antibodies that specifically act on these foreign substances, and activating cytotoxic T cells.

[0060] Dendritic cells are therefore involved in both innate and adaptive immunity and have a significant impact on the entire immune system. There are multiple types of dendritic cells, which can be broadly divided into plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs). pDCs are the primary producers of type I interferons, which have activity that inhibits viral growth and also contribute to the activation of natural killer (NK) cells, but their antigen-presenting ability is relatively weak. On the other hand, cDCs not only produce IL-12, which activates NK cells, but also play a key role in inducing adaptive immunity due to their strong antigen-presenting ability. IgA, a type of adaptive immunity, is believed to contribute to the protection against a variety of infectious diseases because, unlike other immunoglobulins, it has a low specificity and can act against a wide range of bacteria and viruses. cDCs can activate both innate and adaptive immunity, making them a key cell type in the prevention of infectious diseases. In the present invention, the term "dendritic cell-like cells" is a general term for cells, including dendritic cells collected from living organisms, as well as cells with cDC function derived from mononuclear cells or stem cells, such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). The effect of lactic acid bacteria on dendritic cells in vivo can be assessed by measuring the degree of activation of dendritic cell-like cells.

[0061] Specifically, the activation of dendritic cell-like cells by lactic acid bacteria can be evaluated as follows: 200 μL of a 1.25×10 5 4×10 cells / mL of human dendritic cell-like cells were added to the wells. 6Lactobacillus cells / well are added, and the expression intensity of CD86 is then measured. More specifically, the above-mentioned measurement can be performed according to the method described in the Examples of this specification. In the present invention, among Lactobacillus helveticus strains, preferred strains are those that have a CD86 expression intensity of, for example, 105% or higher, preferably 130% or higher, more preferably 145% or higher, even more preferably 150% or higher, still more preferably 180% or higher, and particularly preferably 200% or higher, as measured according to the above-mentioned method, compared to a control group not containing any lactic acid bacteria (based on the ratio represented by [CD86 expression intensity in the group containing lactic acid bacteria (active ingredient of the present invention)] / [CD86 expression intensity in the control group]). Although the dendritic cell-like cells used in the above-mentioned measurement are not limited, immortalized dendritic cells differentiated from immature dendritic cells (Mylc cells) used in the Examples of this specification can generally be used.

[0062] The induction of IgA production by lactic acid bacteria can be evaluated as follows: 250 μL of 8×10 5 1×10 cells / mL of human peripheral blood mononuclear cell solution was added to the wells. 7 cells / well of lactic acid bacteria, and then the produced IgA is measured. More specifically, the measurement can be performed according to the method described in the Examples of the present application. In the present invention, among Lactobacillus helveticus strains, strains that produce 100 ng / mL or more, more preferably 150 ng / mL or more of IgA as measured according to the above method are preferred. In the present invention, among Lactobacillus helveticus strains, strains that produce, as compared to a control not containing any lactic acid bacteria, an amount of IgA measured according to the above method of, for example, 105% or more, preferably 110% or more, preferably 120% or more, preferably 140% or more, and preferably 170% or more (based on the ratio shown as [IgA production (ng / mL) in the group to which lactic acid bacteria (active ingredient of the present invention) was added) / [IgA production (ng / mL) in the control group]).

[0063] The induction of IL-12 production by lactic acid bacteria can be evaluated as follows: for example, 200 μL of a 1.25×10 5 4×10 cells / mL of human dendritic cell-like cells were added to the wells. 6 Lactobacillus cells / well are added, and the IL-12 production is then measured. More specifically, the measurement can be performed according to the method described in the Examples of this specification. In the present invention, among Lactobacillus helveticus strains, preferred strains are those that preferably produce 300 pg / mL or more, more preferably 600 pg / mL or more, and even more preferably 900 pg / mL or more of IL-12 as measured according to the above method.

[0064] In the present invention, among Lactobacillus helveticus strains, preferred strains are those that produce IL-12 in an amount of, for example, 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 as compared to a control not containing any lactic acid bacteria as measured according to the above-mentioned method (based on the ratio shown as [IL-12 production (pg / mL) in the group to which lactic acid bacteria (active ingredient of the present invention) was added) / [IL-12 production (pg / mL) in the control group]).

[0065] In a more preferred embodiment, Lactobacillus helveticus is preferably used, exhibiting at least one (preferably at least two, and more preferably all three) immunostimulatory activity selected from the group consisting of dendritic cell-like cell activation, induction of IgA production, and induction of IL-12 production, which is superior to that of Lactococcus lactis JCM 5805. The method for measuring dendritic cell-like cell activation, induction of IgA production, and induction of IL-12 production in this embodiment is the same as described above. The function of the lactic acid bacteria JCM 5805 strain can be measured by performing the same procedures as described above, except that the lactic acid bacteria JCM 5805 strain is used in place of the Lactobacillus helveticus that is the active ingredient in the immunostimulant of the present invention.

[0066] In the present invention, the Lactobacillus helveticus used can be dead bacteria (such as heat-killed bacteria) or live bacteria. In the present invention, the Lactobacillus helveticus can be used alone or in combination of two or more strains.

[0067] In the present invention, the lactic acid bacteria are used as active ingredients in immunostimulants. The "immunostimulation" application can be used for activation of dendritic cell-like cells, induction of IgA production, induction of IL-12 production, etc. In the present invention, the term "immunostimulation" includes not only improving the immune function of people with weakened immune function, but also maintaining the immune function of healthy individuals.

[0068] In the present invention, the active ingredient Lactobacillus helveticus itself can be used as an immunostimulant, or Lactobacillus helveticus can be used in the form of a composition comprising a lactic acid bacteria powder in combination with various pharmaceutically acceptable or food-addable carriers (e.g., isotonic agents, chelating agents, stabilizers, pH regulators, preservatives, antioxidants, solubilizers, thickeners, excipients, and adhesives). In this embodiment, the content of Lactobacillus helveticus in the composition of the immunostimulant 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 in the Lactobacillus helveticus powder used for the immunostimulant is not limited and can be appropriately set within the range of, for example, 100 million to 100 trillion / g, preferably 10 billion to 10 trillion / g, more preferably 50 billion to 1 trillion / g, even more preferably 100 billion or more, and particularly preferably 300 billion or more.

[0069] Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyols such as glycerol, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonic agents can be used alone or in combination of two or more.

[0070] Examples of chelating agents include edetates such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate; as well as edetate, nitrilotriacetic acid or its salt, sodium hexametaphosphate, and citric acid. These chelating agents can be used alone or in combination of two or more.

[0071] Examples of stabilizers include sodium bisulfite.

[0072] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid; 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 trishydroxymethylaminomethane. These pH adjusters can be used alone or in combination of two or more.

[0073] Examples of preservatives include sorbic acid; potassium sorbate; parabens such as methyl paraben, ethyl paraben, propyl paraben, and butyl paraben; quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride; alkylpolyaminoethylglycine; chlorobutanol; polyquaternium salts; polyhexamethylene biguanide; and chlorhexidine. These preservatives may be used alone or in combination of two or more.

[0074] Examples of antioxidants include sodium bisulfite, dried sodium sulfite, sodium metabisulfite, and mixed tocopherol concentrates. These antioxidants may be used alone or in combination of two or more.

[0075] Examples of the solubilizing agent include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, polyethylene glycol and D-mannitol. These solubilizing agents may be used alone or in combination of two or more.

[0076] Examples of thickeners include polyethylene glycol, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol. These thickeners can be used alone or in combination of two or more.

[0077] Examples of excipients include lactose, corn starch, L-cysteine, trehalose, maltitol, and sorbitol. These excipients may be used alone or in combination of two or more.

[0078] Examples of binders include microcrystalline cellulose, starch, sucrose, hydroxypropyl cellulose, gelatin, powdered gum arabic, polyvinyl pyrrolidone, pullulan, dextrin, cyclodextrin, methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyethylene glycol. These binders can be used alone or in combination of two or more.

[0079] In addition to the above-mentioned Lactobacillus helveticus, the above-mentioned composition may also contain substances known to have immunostimulatory functions. Substances known to have immunostimulatory functions include vitamin C, vitamin A, and zinc. These substances may be used alone or in combination of two or more.

[0080] By taking the immunostimulant of the present invention, a subject (preferably a mammal, such as a human) will experience an immunostimulatory effect. The intake amount of the immunostimulant of the present invention is not limited, and the daily intake of the active ingredient, Lactobacillus helveticus, can be appropriately set within 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. Although the intake amount of the immunostimulant of the present invention based on the weight of Lactobacillus helveticus is also not limited, the daily intake of Lactobacillus helveticus as the active ingredient can be appropriately set within the range of, for example, 1 mg to 10 g, preferably 10 mg to 1000 mg, more preferably 40 mg to 300 mg, and further preferably 200 mg or more.

[0081] Oral composition In another embodiment, the present invention provides an oral composition containing an immunostimulant. Accordingly, the present invention provides an oral composition containing Lactobacillus helveticus. Such oral compositions include food compositions or beverage compositions and pharmaceutical compositions. In the present invention, such food compositions or beverage compositions include health functional foods (foods with nutritional function claims, foods for specific health uses, and foods with functional claims).

[0082] Examples of the food or beverage compositions include beverages such as vegetable juice drinks, fruit juice drinks, mixed fruit and vegetable juice drinks, fermented milk drinks, and almond-containing beverages; as well as foods such as ice cream, sorbets, almond-containing foods, cookies (e.g., cream-filled biscuits), chocolate (including quasi-chocolate), and fermented dairy products (yogurt and cheese). The ice cream is preferably dairy ice cream (e.g., dairy ice cream containing fermented milk). The cream-filled biscuits are preferably biscuits with Lactobacillus helveticus added to the cream. Quasi-chocolate includes quasi-chocolate containing corn. In the present invention, dairy products and foods or beverages containing dairy products are preferred among these foods and beverages. Dairy products include fermented milk, ice cream, and milk beverages. Examples of foods or beverages containing dairy products include cream-filled biscuits and milk chocolate. Preferred sorbets include dairy-containing sorbets (including fermented milk-containing sorbets and similar desserts). The food or beverage compositions of the present invention may also include a supplement. The supplement is preferably a sports supplement (e.g., a sports supplement containing amino acids). Examples of sports supplements containing amino acids include sports supplements containing glutamine.

[0083] The specific details, intake amount, and other details of the active ingredient Lactobacillus helveticus in the embodiment of the oral composition are as described above in the description of the immunostimulant. In this embodiment, the content of Lactobacillus helveticus in the oral composition is not particularly limited and 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% or more by mass. The oral composition of the present invention can be used for immunostimulation purposes.

[0084] Preventive or therapeutic agent for viral infection Administration of the specific Lactobacillus helveticus as the active ingredient of the present invention can produce an immunostimulatory effect. Therefore, the immunostimulatory effect provided by the present invention is expected to provide a preventive or therapeutic effect on viral infections. Therefore, the present invention provides a viral infection preventive or therapeutic agent containing the above-mentioned immunostimulatory agent. The present invention also provides a viral infection preventive or therapeutic agent containing the above-mentioned Lactobacillus helveticus. Examples of target viruses include, but are not limited to, influenza virus, coronavirus, adenovirus, respiratory syncytial virus (RS virus), human metapneumovirus, and rhinovirus. The specific details, intake amount, and other details of the Lactobacillus helveticus as the active ingredient in the embodiments of the viral infection preventive or therapeutic agent are as described above in the description of the immunostimulatory agent and oral composition.

[0085] Lactobacillus helveticus The present invention utilizes the aforementioned Lactobacillus helveticus to provide immunostimulatory effects and preventive or therapeutic effects against viral infections. This Lactobacillus helveticus is novel. Therefore, the present invention also provides Lactobacillus helveticus itself. The methods and other details of using Lactobacillus helveticus in this embodiment are as described above in the description of the immunostimulant, oral composition, and agent for preventing or treating viral infections.

[0086] Example Preparation of inactivated lactic acid bacteria powder Preparation Example 1 In-house lactic acid bacteria strains were statically cultured in MRS medium (Merck Millipore) at 30°C or 37°C for 48 hours. After cultivation, the bacteria were harvested by centrifugation at 8000×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 the concentration was adjusted to 10 mg / mL with physiological saline to obtain a lactic acid bacteria suspension.

[0087] Preparation Example 2 Heat-inactivated cells of the Lactococcus lactis JCM5805 strain (hereinafter referred to as "JCM5805 strain") were prepared in the same manner as in Preparation Example 1. The heat-inactivated cells were then freeze-dried and the concentration was adjusted to 10 mg / mL with physiological saline to obtain a lactic acid bacteria suspension. Strains with "JCM" in their names indicate bacterial strains distributed by the RIKEN Institute of Physical and Chemical Research (RIKEN).

[0088] Screening based on the inducibility of IgA production Preparation of Peripheral Blood Mononuclear Cell Culture Medium (hereinafter referred to as "PBMC Culture Medium") PBMC culture medium was prepared by adding the following components to RPMI 1640 medium (Thermo Fisher Scientific) to achieve the specified percentages: 10% by mass heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific), 1% by mass MEM vitamin solution (Thermo Fisher Scientific), 1% by mass MEM non-essential amino acid solution (Thermo Fisher Scientific), 1% by mass penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd.), 1% by mass sodium pyruvate solution (Thermo Fisher Scientific), and 0.1% by mass 2-mercaptoethanol (Thermo Fisher Scientific).

[0089] Preparation of human peripheral blood mononuclear cells (PBMCs) Frozen human peripheral blood mononuclear cells (PBMCs) from healthy donors (Astarte Biologics) were rapidly thawed in a 37°C water bath, added to a 50-mL centrifuge tube containing 10 mL of RPMI1640 medium supplemented with 10% FBS, and gently mixed by inversion. PBMCs were pelleted by centrifugation at 200 × g for 5 minutes, the supernatant removed by aspiration, and 10 mL of PBMC medium was added to the pelleted PBMCs to resuspend them, creating a PBMC suspension.

[0090] PBMC inoculation, lactic acid bacteria addition, and culture 20 μL of PBMC suspension and 2 μL of acridine orange (Logos Biosystems) were added to a 1.5 mL microcentrifuge tube and mixed thoroughly. Subsequently, the cell concentration was calculated using a cell counter (Logos Biosystems, L20001). The PBMC suspension was diluted with PBMC culture medium to a cell concentration of 8 × 10 5 cells / mL, and then 250 μL of the PBMC suspension was inoculated into each well of a 96-well plate (TPP). Furthermore, the lactic acid bacteria suspension from Preparation Example 1 was added to each well of the 96-well plate to a concentration of 50 μg / mL. As a control, wells without any lactic acid bacteria were also prepared. The PBMCs were then placed in a CO2 incubator and statically cultured for 120 hours at 37°C and 5% CO2.

[0091] IgA assay After 120 hours of culture, the 96-well plate containing PBMCs was removed and 250 μL of the culture medium was transferred to an 8-tube microcentrifuge strip. The PBMCs were pelleted by centrifugation at 200 × g for 10 minutes, and 220 μL of the culture supernatant was transferred to another set of 8-tube microcentrifuge strips. The supernatant was then centrifuged again at 1500 × g for 10 minutes. The resulting supernatant was used for quantification of IgA using an enzyme-linked immunosorbent assay (ELISA). For IgA quantification, a human IgA ELISA kit (Abcam) was used, and IgA concentrations were determined according to the kit's protocol. To ensure experimental reproducibility, the entire process from PBMC plating to IgA measurement was repeated twice.

[0092] Test results Lactic acid bacteria in the culture medium (in which PBMCs were cultured using the lactic acid bacteria of Preparation Example 1) were screened for those that produced IgA concentrations at least twice that of the culture medium containing PBMCs cultured without lactic acid bacteria (control group). As a result of the screening under these conditions, 70 lactic acid bacteria strains were identified as having the ability to induce IgA production. Table 1 shows a list of these lactic acid bacteria. The IgA production rate was calculated using the following formula: IgA production rate = IgA concentration in PBMC culture medium containing lactic acid bacteria / IgA concentration in PBMC culture medium without lactic acid bacteria Table 1

[0093]

[0094]

[0095] Of these lactic acid bacteria, 14 strains were selected for subsequent testing based on their species, culturing difficulty, and other factors. The selected lactic acid bacteria are marked with an asterisk (*) in Table 1.

[0096] Evaluation of induction of IgA production Isolation of peripheral blood mononuclear cells from human peripheral blood 20 mL of blood samples were collected from 12 healthy adult volunteers and placed in blood collection tubes containing anticoagulant (sodium heparin). TM The tubes (AXS) were centrifuged at 400 × G for 1 minute to allow Lymphoprep TM Settling to Lymphoprep TMNext, mix the blood with saline in a 1:1 ratio in a 50 mL centrifuge tube and transfer 20 mL to 30 mL of the mixture to a Lymphoprep TM Tubes were centrifuged at 800×g for 20 minutes at room temperature. From each layer formed by centrifugation, the solution layer containing peripheral blood mononuclear cells was collected using a Pasteur pipette and transferred to another 50 mL centrifuge tube. 20 mL of physiological saline was added to the 50 mL centrifuge tube containing the collected peripheral blood mononuclear cells, followed by centrifugation at 250×g for 10 minutes to pellet the cells. The supernatant was removed using an aspirator, and 1 mL of PBMC medium was added to the pelleted peripheral blood mononuclear cells to resuspend the cells, thereby obtaining a peripheral blood mononuclear cell suspension.

[0097] Inoculation of peripheral blood mononuclear cells, addition of lactic acid bacteria, and culture Measure 90 μL of PBMC culture medium and 10 μL of peripheral blood mononuclear cell suspension, place them in a 1.5 mL microcentrifuge tube, and mix thoroughly. Then, transfer 20 μL of the mixture to another 1.5 mL microcentrifuge tube, add 2 μL of acridine orange (Logos Biosystems), and mix thoroughly. Subsequently, calculate the cell concentration using a cell counter (Logos Biosystems, L20001). Dilute the peripheral blood mononuclear cell suspension with PBMC culture medium to a concentration of 8 × 10 5 cells / mL, and then 250 μL of the dilution was inoculated into each well of a 96-well plate (TPP). In addition, the lactic acid bacteria suspension of Preparation Example 1 and the lactic acid bacteria suspension of Preparation Example 2 were inoculated at 1×10 7 Cells were added to each well of a 96-well plate. Lipopolysaccharide (Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "LPS") was also added to a final concentration of 10 ng / mL as a positive control. A well without lactic acid bacteria was also prepared as a control. The cells were statically cultured in a CO2 incubator at 37°C and 5% CO2 for 120 hours.

[0098] IgA assay After 120 hours of culture, the 96-well plate containing peripheral blood mononuclear cells was removed and 250 μL of the culture medium was transferred to an 8-well microtube strip. The peripheral blood mononuclear cells were pelleted by centrifugation at 200 × g for 10 minutes. Then, 220 μL of the culture supernatant was transferred to another set of 8-well microtube strips and centrifuged again at 1500 × g for 10 minutes. The resulting supernatant was used for quantification of IgA by ELISA. For IgA quantification, a human IgA ELISA kit (Abcam) was used, and IgA concentrations were determined according to the kit's protocol.

[0099] Evaluation of dendritic cell-like cell activation Preparation of differentiation medium Differentiation medium was obtained by mixing Mylc Specialty Medium B (MiCAN Technologies) and Mylc Specialty Medium Supplement B (MiCAN Technologies) at a ratio of 200:1.

[0100] Preparation of lactic acid bacteria (1 mg / mL) suspension The lactic acid bacteria suspensions obtained in Preparation Example 1 and Preparation Example 2 were diluted 10-fold with physiological saline to obtain a 1 mg / mL lactic acid bacteria suspension. The bacterial concentration of the 1 mg / mL lactic acid bacteria suspension was determined according to the Breed method, and then diluted with differentiation medium to a concentration of 4×10 7 cells / mL.

[0101] Inoculation of dendritic cells, addition of lactic acid bacteria, and culture aMylc-2-A differentiated cells (immortalized dendritic cells purchased from MiCAN Technologies and induced to differentiate for 3 days) were collected. The dendritic cells were then pelleted by centrifugation at 300×G for 5 minutes. 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 cells / mL with differentiation medium. 5 cells / mL, and then 100 μL of dendritic cells were seeded into each well of a Nunclon Sphera 96-well round-bottom plate (Thermo Fisher Scientific). In addition, 100 μL of lactic acid bacteria suspension (4 × 10 7 cells / mL). 100 μL of lipopolysaccharide (LPS) was added to each well as a positive control to a final concentration of 1 ng / mL. A well without lactic acid bacteria was also prepared as a control. The cells were incubated in a CO2 incubator at 37°C and 5% CO2 for 120 hours.

[0102] Collection of dendritic cells and culture supernatant Remove the 96-well plate containing dendritic cells after 24 hours of culture and transfer 200 μL of culture medium to an 8-tube microcentrifuge strip. Pellet the dendritic cells by centrifugation at 500 × g for 10 minutes, then transfer 140 μL of the culture supernatant to another 8-tube microcentrifuge strip. Remove the remaining culture supernatant from the 8-tube microcentrifuge strip using an aspirator to obtain the dendritic cell pellet.

[0103] Preparation of reagents for measuring cell surface markers (CD86) 1. FcR blocking solution eBioscience TM Flow cytometry staining buffer (Invitrogen) (hereafter referred to as “staining buffer”) and Fc receptor binding inhibitor (eBioscience) were mixed at a ratio of 19:1 to obtain FcR blocking solution.

[0104] 2. Antibody Solution The staining buffer and PE-labeled anti-human CD86 antibody were mixed at a ratio of 49:1 to obtain an antibody solution.

[0105] Determination of cell surface marker (CD86) 200 μL of staining buffer was added to the dendritic cell pellet to resuspend it. The suspension was centrifuged at 500 × G for 5 minutes, and the supernatant was removed with an aspirator. 20 μL of FcR blocking solution was added to the cell pellet to resuspend it, followed by incubation on ice for 20 minutes. Furthermore, 20 μL of antibody solution was added to the cells to resuspend them, followed by incubation on ice for 30 minutes. After incubation, 160 μL of staining buffer was added to the cells to resuspend them, followed by centrifugation at 500 × G for 5 minutes, and the supernatant was removed with an aspirator. Subsequently, 200 μL of staining buffer was added to the cells to resuspend them, followed by centrifugation at 500 × G for 5 minutes, and the supernatant was removed with an aspirator. The expression intensity of the cell surface marker (CD86) was measured using a flow cytometer (SA3800, Sony Corporation, laser wavelength: 488 nm) in the solution prepared by resuspending the cells with 200 μL of staining buffer.

[0106] Evaluation of NK cell activation For review price For NK cell activation, IL-12 levels were measured using the culture supernatant obtained as described above in the "Collection of Dendritic Cells and Culture Supernatant" section. IL-12 levels were measured using a human IL-12 p70 ELISA kit (Abcam) according to the kit's included protocol.

[0107] Evaluation of induction of IgA production Figure 1 The results of the evaluation of the IgA production ability in Preparation Example 1 and Preparation Example 2 are shown. Figure 1As shown, compared to the control group without lactic acid bacteria (control group), culture with the lactic acid bacteria of Preparation Example 1, with the exception of CR2, LA44, CR11, and LA15, significantly increased the ability to induce IgA production in peripheral blood mononuclear cells. Furthermore, compared to the case where the JCM5805 strain of Preparation Example 2 was added, culture with the lactic acid bacteria of Preparation Example 1, with the exception of CR2, LA44, CR11, and LA15, significantly increased the ability to induce IgA production in peripheral blood mononuclear cells. In animals, including humans, ingestion of these lactic acid bacteria is expected to enhance IgA production, thereby preventing viral and bacterial invasion and improving defenses against infection.

[0108] Evaluation of dendritic cell-like cell activation Figure 2 The results of evaluation of activation of dendritic cell-like cells in Preparation Examples 1 and 2 are shown. The activation of dendritic cells was evaluated by measuring the expression intensity of the cell surface marker (CD86). Figure 2 As shown, compared to the control group without lactic acid bacteria (control), cultures using the lactic acid bacteria of Preparation Example 1 significantly activated dendritic cells, with the exception of PE6. Furthermore, compared to the group with the JCM5805 strain of Preparation Example 2, cultures using the lactic acid bacteria of Preparation Example 1 significantly activated dendritic cells, with the exception of JE1, FE20, PE5, LA23, LA24, LA44, PE6, and LA15. In animals, including humans, ingestion of these lactic acid bacteria is expected to activate dendritic cells and thereby stimulate the entire immune system.

[0109] Evaluation of NK cell activation Figure 3 The results of the evaluation of NK cell activation in Preparation Example 1 and Preparation Example 2 are shown. The activation of NK cells was evaluated by measuring the concentration of IL-12. Figure 3 As shown, compared to the control group (no lactic acid bacteria added), culture with the lactic acid bacteria of Preparation Example 1 significantly activated NK cells, with the exception of 1A44. In particular, he11 and he12 significantly activated NK cells. Furthermore, culture with the lactic acid bacteria of Preparation Example 1 significantly activated NK cells compared to the group with the JCM5805 strain of Preparation Example 2. In animals, including humans, ingestion of these lactic acid bacteria is expected to activate NK cells, thereby enhancing their ability to attack cells infected with bacteria or viruses, preventing the spread of infection and improving defenses against infection.

[0110] As shown in the above experiments, he11 and he12 exhibited all three functions: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production. Specifically, he11 and he12 exhibited all of the following functions (1) to (3): (1) When 250 μL of 8×10 5 cells / mL of human peripheral blood mononuclear cell solution was added to the wells at 1×10 7 When the Lactobacillus helveticus is added to the cells / well, 100 ng / mL or more of IgA is produced; (2) When 200 μL of a solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus is added to the cells / well, the expression intensity of CD86 becomes 1.5 times or higher than that of the control group without any lactic acid bacteria; and (3) When 200 μL of the solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus was added to the cells / well, 300 pg / mL or more of IL-12 was produced.

[0111] In addition, he11 and he12 showed that their following three immunostimulatory functions were higher than those of the JCM 5805 strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

[0112] Formulation Example 1 Ice cream containing inactivated lactic acid bacteria powder was obtained from the raw materials shown in Table 2. Specifically, first, egg yolks and sugar were placed in a bowl and thoroughly mixed. In another pot, cream, milk, and inactivated lactic acid bacteria powder were placed and heated. When bubbles began to appear at the edge of the pot, the pot was removed from the heat. While stirring, this heated mixture was gradually added to a bowl containing whipped egg yolks and sugar. The resulting mixture was placed in a metal container and cooled, covered with a lid, and refrigerated at -20°C. After 3 hours, the entire mixture was stirred and then stirred again 4 times every 30 minutes to obtain ice cream containing inactivated lactic acid bacteria powder.

[0113] Table 2

[0114] Formulation Example 2 Yogurt containing inactivated lactic acid bacteria powder was obtained from the raw materials shown in Table 3.

[0115] Table 3

[0116] Formulation Example 3 Chocolate containing inactivated lactic acid bacteria powder was obtained from the raw materials shown in Table 4. Specifically, the chocolate was melted in a water bath at approximately 50°C, and then the inactivated lactic acid bacteria powder was added and thoroughly mixed. The mixture was poured into a mold, shaped, and then left to solidify at room temperature, thereby obtaining chocolate containing inactivated lactic acid bacteria powder.

[0117] Table 4

Claims

1. An immunostimulant comprising Lactobacillus helveticus ( Lactobacillus helveticus ),in, The Lactobacillus helveticus has three functions: activation of dendritic cell-like cells, induction of immunoglobulin A (IgA) production, and induction of interleukin-12 (IL-12) production.

2. The immunostimulant according to claim 1, wherein The Lactobacillus helveticus is Lactobacillus helveticus GCL1815 ( Lactobacillus helveticus GCL1815) or Lactobacillus helveticus GCJ5-4B ( Lactobacillus helveticus GCJ5-4B).

3. The immunostimulant according to claim 1 or 2, wherein The Lactobacillus helveticus satisfies at least one of the following (1) to (3): (1) When 250 μL of 8×10 5 cells / mL of human peripheral blood mononuclear cell solution was added to the wells at 1×10 7 When the Lactobacillus helveticus is added to the cells / well, 100 ng / mL or more of IgA is produced; (2) When 200 μL of a solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When Lactobacillus helveticus was added to the cells / well, the expression intensity of CD86 became 1.5 times or higher than that of the control group without any lactic acid bacteria; as well as (3) When 200 μL of the solution with a concentration of 1.25×10 5 cells / mL of human dendritic cell-like cells were plated at 4×10 6 When the Lactobacillus helveticus was added to the cells / well, 300 pg / mL or more of IL-12 was produced.

4. The immunostimulant according to claim 3, wherein The Lactobacillus helveticus meets the three requirements (1) to (3).

5. The immunostimulant according to claim 1 or 2, wherein At least one of the following three immunostimulatory functions of the Lactobacillus helveticus is higher than that of Lactococcus lactis JCM 5805 ( Lactococcus lactis JCM 5805) strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

6. The immunostimulant according to claim 1 or 2, wherein The following three immune stimulating functions of Lactobacillus helveticus are all higher than those of Lactococcus lactis JCM 5805 ( Lactococcus lactis JCM 5805) strain: activation of dendritic cell-like cells, induction of IgA production, and induction of IL-12 production.

7. The immunostimulant according to claim 1 or 2, wherein The immune stimulation is to maintain the immune function of healthy individuals.

8. An oral composition for immunostimulation, comprising the immunostimulant according to claim 1 or 2. 9 . The oral composition according to claim 8 , which is a composition for food or beverage.

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

11. A species of Lactobacillus ( Lactobacillus ) genus, and its deposit number in the Patent Microorganisms Collection (NPMD) of the National Institute of Technology Evaluation (NITE) of Japan is NITE BP-03804.

12. A species of Lactobacillus ( Lactobacillus ) genus, and its deposit number in the Patent Microorganisms Depository (NPMD) of the National Institute of Technology Evaluation (NITE) of Japan is NITE BP-03805.

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