Immunostimulator

Abstract

To provide a novel method for further enhancing the immunostimulating effect of lactic acid bacteria.SOLUTION: The present invention provides an immunostimulator for use in physical fatigue, the immunostimulator comprising lactic acid bacteria and glutamine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an immunostimulant.

Background Art

[0002] Immunity plays a very important role in maintaining and improving health, and its research has been conducted in various fields. There are innate immunity and acquired immunity in the immune system, which are responsible for the body's defense function. With the increasing health awareness, means to enhance these immune functions are highly desired. A method of ingesting lactic acid bacteria is known as a means to enhance such immune functions. However, the development of a method to maximize the immunostimulatory effect of lactic acid bacteria is highly desired.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a new method for further enhancing the immunostimulatory effect of lactic acid bacteria.

Means for Solving the Problems

[0006] Under such circumstances, as a result of extensive trial and error on various conditions, the present inventors have found that glutamine plays a very important role among various elements in order to bring about the immunostimulatory effect of lactic acid bacteria. As a result of intensive studies based on such new findings, the present inventors have arrived at the idea that when using a combination of lactic acid bacteria and glutamine, it functions effectively particularly during physical fatigue in which the glutamine in the body is reduced, and completed the present invention. Therefore, the present invention provides the following items: Item 1. An immunostimulant containing lactic acid bacteria and glutamine.

[0007] Item 2. An enhancer for enhancing the immunostimulatory effect of lactic acid bacteria, containing glutamine.

[0008] Item 3. An IgA production inducer containing lactic acid bacteria and glutamine.

[0009] Item 4. An oral composition containing lactic acid bacteria and glutamine, which acts on immune cells to maintain or improve the immune function of healthy individuals.

[0010] Item 5. The agent according to any one of Items 1 to 3 or the composition according to Item 4 for administration to a subject during physical fatigue.

[0011] Item 6. The agent according to any one of Items 1 to 3 or the composition according to Item 4, wherein the lactic acid bacteria belong to the genus Lactobacillus.

[0012] Item 7. The agent according to any one of Items 1 to 3 or the composition according to Item 4, wherein the lactic acid bacteria are lactic acid bacteria capable of producing IgA under normal environmental conditions.

[0013] Item 8. The agent according to any one of Items 1 to 3 or the composition according to Item 4, which is a food.

[0014] Item 9. A method using glutamine as an index for the immunostimulatory effect by lactic acid bacteria administration.

[0015] Item 10. Use of lactic acid bacteria and glutamine for producing an immunostimulant during physical fatigue.

[0016] Item 11. Use of glutamine for producing an enhancer for the immunostimulatory effect by lactic acid bacteria during physical fatigue.

[0017] Item 12. Use of lactic acid bacteria and glutamine for producing an IgA production inducer during physical fatigue.

[0018] Item 13. The use according to any one of Items 10 to 12, wherein the agent is for administration to a subject during physical fatigue.

[0019] Item 14. Use of lactic acid bacteria and glutamine for producing an oral composition for acting on immune cells and maintaining or improving the immune function of healthy people.

[0020] Item 15. The use according to Item 14, wherein the oral composition is for administration to a subject during physical fatigue.

Effects of the Invention

[0021] According to the present invention, it is possible to provide a method for exerting the immunostimulatory effect by lactic acid bacteria, particularly a method for exerting the immunostimulatory effect even under a glutamine-deficient condition such as during physical fatigue.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0023] Immunopotentiator The present invention provides an immunostimulant containing lactic acid bacteria and glutamine. The immunostimulant of the present invention is preferable because it can exhibit an effect when administered to a subject (preferably a mammal such as a human) in a state of physical fatigue. In such an embodiment, the present invention provides an immunostimulant at the time of physical fatigue, containing lactic acid bacteria and glutamine. In the present invention, the time of physical fatigue includes, in addition to the ordinary meaning in the fields of pharmaceuticals, quasi-drugs, foods, etc. to which the present invention belongs, situations similar to fatigue caused by excessive physical activities such as exercise, when undergoing surgical operations or suffering from extensive injuries such as burns over the whole body (Non-Patent Documents 1 to 3). In a preferred embodiment, the glutamine concentration in the blood of the subject ingesting the immunostimulant of the present invention is preferably 0.0095% by mass or less, more preferably 0.0080% by mass or less, and even more preferably 0.0066% by mass or less.

[0024] In the present invention, examples of the lactic acid bacteria include Lactobacillus, Lactococcus, Lactiplantibacillus, Limosilactobacillus, etc., and Lactobacillus is preferred. Examples of Lactobacillus include Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus cryptos, Lactobacillus gasseri, etc., and Lactobacillus helveticus, Lactobacillus rhamnosus, etc. are preferred. As Lactobacillus helveticus, preferably, Lactobacillus helveticus GCJ5-4B, etc. are mentioned. In a preferred embodiment, examples of Lactobacillus helveticus include those having an accession number of NITE BP-03805, etc. in the NPMD of NITE.

[0025] In the present invention, as the lactic acid bacteria which are the active ingredient of the immunostimulant, those having at least one (preferably at least two, more preferably all three) functions selected from the group consisting of induction of immunoglobulin A (IgA) production, activation of dendritic cell-like cells, and induction of interleukin-12 (IL-12) production under normal environmental conditions are preferred. In particular, it is preferable that at least one function is the IgA production-inducing function. In the present invention, the normal environmental conditions mean a state in which the content of glutamine is not decreased, such as blood during physical fatigue, and more typically, a state in which the concentration of glutamine is 0.0095 - 0.0117% by mass.

[0026] Specifically, in one embodiment of the present invention, the lactic acid bacteria which are the active ingredient of the immunostimulant preferably have the ability to induce IgA production under normal environmental conditions. IgA secreted on mucosal surfaces such as the intestinal tract, oral cavity, and nasal cavity plays a major role in inhibiting the invasion of pathogenic bacteria and viruses. More specifically, for example, IgA is considered to contribute to the prevention of a wide range of infectious diseases because, unlike other immunoglobulins, it has low specificity and acts on many types of bacteria and viruses. In addition, in one embodiment of the present invention, the lactic acid bacteria, which are the active ingredient of the immunostimulant, preferably have the ability to activate dendritic cell-like cells under normal environmental conditions. Dendritic cells play a major role in the immune function. More specifically, for example, dendritic cells play an important role in innate immunity together with macrophages and NK cells, etc. When foreign substances such as bacteria and viruses invade the body, they first show the function of eliminating them. In addition, dendritic cells transmit information about the invading foreign substances to T cells and B cells by antigen presentation, etc., and also play an important role in acquired immunity such as promoting antibody production that specifically acts on foreign substances and activating cytotoxic T cells.

[0027] Thus, dendritic cells are cells involved in both innate and acquired immunity and have a great influence on the overall immunity. There are various types of dendritic cells, which are roughly classified into plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). pDC shows antiviral growth inhibitory activity and is the main producer of type I interferon that also contributes to the activation of NK cells, but has a weak antigen-presenting ability. On the other hand, in addition to producing IL-12 that activates NK cells, cDC has a strong antigen-presenting ability and thus plays an important role in the induction of acquired immunity. cDC, which activates both innate and acquired immunity, is one of the central cells responsible for preventing infectious diseases. In the present invention, "dendritic cell-like cells" is a general term for cells having the functions of dendritic cells collected from a living body and cDCs induced from stem cells such as monocytes, iPS cells, and ES cells.

[0028] In the present invention, the induction of IgA production by lactic acid bacteria is, for example, in a well seeded with 250 μL of human-derived peripheral blood mononuclear cell solution at a concentration of 8×10 5 cells / mL, 1×10 7It can be evaluated by adding the lactic acid bacteria to the cells / wells and measuring the produced IgA. More specifically, the above measurement can be carried out according to the method described in the examples of the present application. In the present invention, among the lactic acid bacteria, those in which IgA of 100 ng / mL or more, more preferably 150 ng / mL or more is detected when measured by the above method are preferred. Also, in the present invention, among the lactic acid bacteria, the IgA production amount when measured by the above method is, compared with a control without adding lactic acid bacteria (in the ratio of [IgA production amount (ng / mL) in the lactic acid bacteria-added group of the active ingredient of the present invention] / [IgA production amount (ng / mL) in the control]), for example, 105% or more, preferably 110% or more, preferably 120% or more, preferably 140% or more, preferably 170% or more. Those having such values are preferred.

[0029] In addition, 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. Specifically, the activation of dendritic cell-like cells by lactic acid bacteria is, for example, in a well seeded with 200 μL of human-derived dendritic cell-like cells at a concentration of 1.25×10 5 cells / mL, adding 4×10 6 cells / well of lactic acid bacteria, and it can be evaluated by measuring the expression intensity of CD86. The above measurement can be carried out according to the method usually used in the study of immunostimulatory effects. In the present invention, among the lactic acid bacteria, the expression intensity of CD86 when measured by the above method is, compared with a control without adding lactic acid bacteria (in the ratio of [expression intensity of CD86 in the lactic acid bacteria-added group of the active ingredient of the present invention] / [expression intensity of CD86 in the control]), for example, 105% or more, preferably 130% or more, more preferably 145% or more, still more preferably 150% or more, yet still more preferably 180% or more, particularly preferably 200% or more. The dendritic cell-like cells used for the above measurement are not limited, and those used in the study of immunostimulatory effects can be widely used.

[0030] Then, the induction of IL-12 production by lactic acid bacteria can be evaluated, for example, by adding 4×10 5 cells / well of the lactic acid bacteria to a well seeded with 200 μL of human-derived dendritic cell-like cells at a concentration of 1.25×10 6 cells / mL and measuring the produced IL-12. The above measurement can be carried out according to a method commonly used in the study of immunostimulatory effects. In the present invention, among lactic acid bacteria, those in which IL-12 of preferably 300 pg / mL or more, more preferably 600 pg / mL or more, still more preferably 900 pg / mL or more is detected when measured by the above method are preferred.

[0031] Also, in the present invention, among lactic acid bacteria, the amount of IL-12 production when measured by the above method, compared with a control without addition of lactic acid bacteria (at a ratio of [IL-12 production (pg / mL) in the lactic acid bacteria-added group of the active ingredient of the present invention] / [IL-12 production (pg / mL) in the control]), for example, is preferably 110% or more, more preferably 180% or more, still more preferably 290% or more, yet even more preferably 500% or more, and particularly preferably 1000% or more.

[0032] In a more preferred embodiment, as the lactic acid bacteria, it is preferable to use those having at least one (preferably at least two, more preferably all three) immunostimulatory functions selected from the group consisting of IgA production induction, dendritic cell-like cell activation, and IL-12 production induction, particularly those having an IgA production induction function higher than that of Lactococcus lactis JCM5805 strain. The measurement methods of IgA production induction, dendritic cell-like cell activation, and IL-12 production induction in this embodiment are the same as above. The above functions of the JCM5805 strain lactic acid bacteria can be measured by performing the same operations as above except that the JCM5805 strain lactic acid bacteria are used instead of the lactic acid bacteria that is the active ingredient of the immunostimulant of the present invention.

[0033] In the present invention, as the above-mentioned lactic acid bacteria, dead bacteria bodies (heat-killed bacteria bodies, etc.) or live bacteria can be used. In the present invention, the above-mentioned lactic acid bacteria can be used alone or in combination of two or more kinds.

[0034] In the present invention, the above-mentioned lactic acid bacteria are used as an active ingredient of an immunopotentiator. Examples of the "immunopotentiating" use include uses such as inducing the production of the above-mentioned IgA, activating dendritic cell-like cells, and inducing the production of IL-12. In addition, in the present invention, the term "immunopotentiation" includes not only improving the immune function of a person with a reduced immune function but also maintaining the immune function of a healthy person.

[0035] The immunopotentiator of the present invention is characterized by using a combination of lactic acid bacteria and glutamine. The mixing ratio of lactic acid bacteria and glutamine can be set, for example, in the range of 1 to 10,000 parts by mass, preferably 5 to 6,000 parts by mass, more preferably 10 to 3,000 parts by mass of the latter with respect to 1 part by mass of the former powder raw material.

[0036] In the present invention, even if the combination itself of lactic acid bacteria, which is the active ingredient of the present invention, and glutamine is used as an immunostimulant, the combination may be used as a composition combined with various carriers (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizing aids, thickening agents, excipients, binders, etc.) that are pharmaceutically acceptable or can be added to foods. In this embodiment, the total content of the combination of lactic acid bacteria and glutamine in the immunostimulant composition is not limited and can be appropriately set within the range of 0.0001% by mass to 100% by mass. The total content of the combination of lactic acid bacteria and glutamine in the immunostimulant composition can be appropriately set, for example, in the range of 0.0001% by mass or more, 0.0005% by mass or more, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 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. Although the upper limit of the total content of the combination of lactic acid bacteria and glutamine in the immunostimulant composition is not limited, it may be, for example, 100% by mass, and can be appropriately set in the range of 99% by mass or less, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, 0.1% by mass or less, etc. The number of lactic acid bacteria in the lactic acid bacteria powder used in the immunostimulant is not limited, but can be appropriately set, for example, in the range of 100 million to 100 trillion cells / g, preferably 10 billion to 10 trillion cells / g, more preferably 50 billion to 1 trillion cells / g, still more preferably 100 billion cells / g or more, and particularly preferably 300 billion cells / g or more.The content of the lactic acid bacteria powder in the immunostimulant can be appropriately set within a range such as, for example, 0.0001% by mass or more, 0.0005% by mass or more, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 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. Although the upper limit of the content of the lactic acid bacteria powder in the immunostimulant is not limited, it can be appropriately set within a range such as, for example, 99% by mass or less, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, 0.1% by mass or less, etc. The content of glutamine in the immunostimulant can be appropriately set within a range such as, for example, 0.0001% by mass or more, 0.0005% by mass or more, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 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. Although the upper limit of the content of glutamine in the immunostimulant is not limited, it can be appropriately set within a range such as, for example, 99% by mass or less, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, 0.1% by mass or less, etc.

[0037] 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; 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.

[0038] Examples of chelating agents include edetates such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate; ethylenediaminetetraacetate salts, nitrilotriacetic acid or its salts, sodium hexametaphosphate, and citric acid. These chelating agents can be used alone or in combination of two or more.

[0039] Examples of stabilizers include sodium bisulfite.

[0040] 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 trometamol. These pH adjusters can be used alone or in combination of two or more.

[0041] Examples of preservatives include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate; quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride; alkyl polyaminoethyl glycine, chlorobutanol, polyquat, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used alone or in combination of two or more.

[0042] Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, etc. These antioxidants can be used alone or in combination of two or more.

[0043] Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc. These solubilizing agents can be used alone or in combination of two or more.

[0044] Examples of thickening agents include polyethylene glycol, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. These thickening agents can be used alone or in combination of two or more.

[0045] Examples of excipients include lactose, corn starch, L-cysteine, trehalose, maltitol, sorbitol, etc. These excipients can be used alone or in combination of two or more.

[0046] Examples of binders include crystalline cellulose, starch, sucrose, hydroxypropylcellulose, gelatin, gum arabic powder, polyvinylpyrrolidone, pullulan, dextrin, cyclodextrin, methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyethylene glycol, etc. These binders can be used alone or in combination of two or more.

[0047] In addition to the above lactic acid bacteria, the composition may further contain a substance known to have an immunostimulatory function. Substances known to have an immunostimulatory function include, for example, vitamin C, vitamin A, zinc, and the like. These substances can be used alone or in combination of two or more.

[0048] When the immunostimulant of the present invention is ingested by a subject (preferably a mammal such as a human), an immunostimulatory effect is brought about. The intake amount of the immunostimulant of the present invention is not limited, but as the daily intake amount of the lactic acid bacteria as an active ingredient, for example, it can be appropriately set in the range of 10 million to 10 trillion, preferably 100 million to 1 trillion, more preferably 1 billion to 100 billion, still more preferably 5 billion to 30 billion, and particularly preferably 10 billion to 20 billion. The intake amount of the immunostimulant of the present invention based on the weight of the lactic acid bacteria is also not limited, but as the daily intake amount of the lactic acid bacteria as an active ingredient, for example, it can be appropriately set in the range of 1 mg to 10 g, preferably 10 mg to 1000 mg, more preferably 40 mg to 300 mg, still more preferably 200 mg or more. Also, as the daily intake amount of glutamine, for example, it can be appropriately set in the range of 500 mg to 100 g, preferably 300 mg to 70 g, more preferably 700 mg to 40 g, still more preferably 1 g to 15 g, and particularly preferably 3 g to 12 g. The specific timing of ingestion of the immunostimulant of the present invention is not particularly limited, but it is preferably ingested before, during, or after an act and / or event that causes physical fatigue. If it is before the act and / or event, it is preferably within 3 hours before the act and / or event, and if it is after the act and / or event, it is preferably within 3 hours after the act and / or event. For example, when the immunostimulant of the present invention is ingested for immunostimulation during physical fatigue due to training, it may be ingested at any timing before, during, or after training. Before training is preferred. If it is before training, it is preferably within 3 hours before training, and if it is after training, it is preferably within 3 hours after training. The number of intakes may be at least once at any timing, and it may be ingested multiple times at any timing during the above period.

[0049] Other embodiments The above describes the present invention using the embodiment of an immunostimulant among various embodiments, but the present invention is not limited to such an embodiment. As described above, based on the new finding that glutamine plays a very important role in bringing about the immunostimulatory effect by lactic acid bacteria, and further based on the fact that the combination of the lactic acid bacteria and glutamine functions particularly effectively during physical fatigue when the glutamine in the body is reduced. Therefore, in another embodiment, the present invention is, for example, An agent for enhancing the immunostimulatory effect by lactic acid bacteria containing glutamine; An IgA production inducer containing lactic acid bacteria and glutamine; An oral composition containing lactic acid bacteria and glutamine, acting on immune cells, for maintaining or improving the immune function of healthy individuals; Use of lactic acid bacteria and glutamine for producing an immunostimulant; Use of glutamine for producing an agent for enhancing the immunostimulatory effect by lactic acid bacteria; Use of lactic acid bacteria and glutamine for producing an IgA production inducer, etc. are provided. In these embodiments, the type of lactic acid bacteria, the usage amounts of lactic acid bacteria and glutamine, the usage method, the types and amounts of other components, etc. may adopt the same conditions as those described above for the immunostimulant. Also, in these embodiments as well, similar to the immunostimulant, it is preferably applied to the subject during physical fatigue. Therefore, in such an embodiment, in terms of form, the present invention is, for example, An agent for enhancing the immunostimulatory effect by lactic acid bacteria containing glutamine during physical fatigue; An IgA production inducer containing lactic acid bacteria and glutamine during physical fatigue; An oral composition containing lactic acid bacteria and glutamine, acting on immune cells during physical fatigue, for maintaining or improving the immune function of healthy individuals; Use of lactic acid bacteria and glutamine for producing an immunostimulant during physical fatigue; Use of glutamine for producing an agent for enhancing the immunostimulatory effect by lactic acid bacteria during physical fatigue; Provided are the use of lactic acid bacteria and glutamine for producing an IgA production inducer during physical fatigue, etc.

[0050] In an embodiment of the oral composition of the present invention, examples of the oral composition include food and drink compositions, pharmaceutical compositions, etc. In the present invention, food and drink compositions include health functional foods (nutritional functional foods, foods for specified health use, foods with functional claims), etc.

[0051] Examples of food and drink compositions include beverages such as vegetable juice beverages, fruit juice beverages, vegetable juice and fruit juice mixed beverages, fermented milk beverages, almond-containing beverages, etc.; foods such as ice creams, frozen confections, almond-containing foods, biscuits (such as cream sandwich biscuits), chocolates (including semi-chocolates, etc.), fermented milk foods (yogurt, cheese), etc. Among ice creams, lacto ice (lacto ice containing fermented milk, etc.) is preferred. Among cream sandwich biscuits, those containing the above lactic acid bacteria in the cream are preferred. Examples of semi-chocolates include corn-containing semi-chocolates, etc. In the present invention, among these food and drink products, dairy products, food and drink products containing dairy products, etc. are preferred. Examples of dairy products include fermented milk, ice creams, milk beverages, etc. Examples of food and drink products containing dairy products include cream sandwich biscuits, milk chocolate, etc. Among frozen confections, frozen confections containing milk (including frozen confections containing fermented milk, etc.) are preferred. The food and drink compositions in the present invention also include supplements, etc. Among supplements, sports supplements (amino acid-containing sports supplements, etc.) are preferred. Examples of amino acid-containing sports supplements include, for example, glutamine-containing sports supplements, etc.

[0052] Details of the lactic acid bacteria as the active ingredient in the embodiment of the oral composition, its intake amount, etc. are the same as those described above in the description of the immunopotentiator. In this embodiment, the lactic acid bacteria content in the oral composition is not particularly limited, but can be appropriately set, for example, in the range of 0.00001 to 99% by mass, preferably 0.0001 to 50% by mass, more preferably 0.001 to 10% by mass, still more preferably 0.01 to 1% by mass, and particularly preferably 0.1% by mass or more.

[0053] In another embodiment, the present invention provides a method using glutamine as an indicator of the immune activation effect by administering lactic acid bacteria. In a preferred embodiment of the method, when the concentration of glutamine in a sample (such as blood) collected from a subject is equal to or higher than a preset value, it can be determined that the immune activation effect by administering lactic acid bacteria works effectively. In this embodiment, when the preset concentration value of glutamine is T (mass %), the value T can be set, for example, in the range of T≤0.0120, preferably T≤0.0096, more preferably T≤0.0081, and even more preferably T≤0.0067. The lower limit of the value T is not limited, but it can be appropriately selected, for example, from 0.0050, 0.0067, 0.0081, 0.0096, etc.

Examples

[0054] Preparation Example 1 Preparation of heat-killed lactic acid bacteria powder The lactic acid bacteria strains owned by the self-insurance were statically cultured at 30 °C or 37 °C for 48 hours using MRS medium (manufactured by Merck Millipore). After culturing, the bacteria 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. Thereafter, 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. The types of lactic acid bacteria used were as follows: la23: Lactococcus lactis 23 pe5: Lactiplantibacillus pentosus 5 fe6: Limosilactobacillus fermentum GCE6-5I he11: Lactobacillus helveticus GCJ5-4B cr2: Lactobacillus crispatus 2 ga8: Lactobacillus gasseri 8

[0055] Example 1 Screening by IgA production-inducing ability Preparation of medium for peripheral blood mononuclear cells (hereinafter referred to as PBMC medium) Inactivated fetal bovine serum (manufactured by Thermo Fisher Scientific) (hereinafter referred to as FBS) at 10% by mass, MEM vitamin solution (manufactured by Thermo Fisher Scientific) at 1% by mass, MEM non-essential amino acid solution (manufactured by Thermo Fisher Scientific) at 1% by mass, penicillin-streptomycin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at 1% by mass, sodium pyruvate solution (manufactured by Thermo Fisher Scientific) at 1% by mass, and 2-mercaptoethanol (manufactured by Thermo Fisher Scientific) at 0.1% by mass were added to RPMI 1640 (manufactured by Thermo Fisher Scientific) to prepare a PBMC medium. The medium contains 300 mg / L of glutamine.

[0056] Preparation of glutamine-free medium for peripheral blood mononuclear cells (hereinafter referred to as PBMC(-)Gln medium) Inactivated fetal bovine serum (manufactured by Thermo Fisher Scientific) (hereinafter referred to as FBS) at 10% by mass, MEM vitamin solution (manufactured by Thermo Fisher Scientific) at 1% by mass, MEM non-essential amino acid solution (manufactured by Thermo Fisher Scientific) at 1% by mass, penicillin-streptomycin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at 1% by mass, sodium pyruvate solution (manufactured by Thermo Fisher Scientific) at 1% by mass, and 2-mercaptoethanol (manufactured by Thermo Fisher Scientific) at 0.1% by mass were added to RPMI 1640, no glutamine (manufactured by Thermo Fisher Scientific) to prepare a PBMC(-)Gln medium.

[0057] Preparation of human peripheral blood mononuclear cells (PBMC) Human peripheral blood mononuclear cells (PBMC) derived from frozen normal donors (manufactured by Astarte Biologics) were rapidly thawed in a 37°C water bath and added to a 50 mL centrifuge tube containing 10 mL of RPMI1640 medium supplemented with 10% FBS. Then, they were gently inverted and mixed. The PBMC were pelleted 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 pelleted PBMC and resuspended to obtain a PBMC suspension.

[0058] Seeding of PBMC, addition of lactic acid bacteria, and culture 20 μL of the PBMC suspension and 2 μL of acridine orange (manufactured by Logos Biosystems) were added to a 1.5 mL microtube and mixed well. Then, the cell concentration was calculated using a cell counter (manufactured by Logos Biosystems, L20001). The PBMC suspension was diluted with PBMC medium to a cell concentration of 8×10 5 cells / mL and seeded at 250 μL per well in a 96-well plate (manufactured by TPP). Furthermore, the lactic acid bacteria suspension of Preparation Example 1 was added to the wells of the 96-well plate so that the lactic acid bacteria concentration in the reaction solution was 10 μg / mL each. Also, wells without added lactic acid bacteria were prepared as controls. Static culture was performed in a CO2 incubator at 37°C and 5% CO2 for 120 hours.

[0059] IgA measurement The 96-well plate in which PBMC had been cultured for 120 hours was taken out, and 250 μL of the culture solution was aliquoted into an 8-well microtube. The PBMC were pelleted by centrifugation at 200×G for 10 minutes, and 220 μL of the culture supernatant was aliquoted into a new 8-well microtube. Further centrifugation was performed at 1,500×G for 10 minutes, and the culture supernatant was subjected to IgA quantification by ELISA. IgA quantification was performed using a Human IgA ELISA Kit manufactured by Abcam, and the IgA concentration was measured according to the protocol booklet in the kit. To confirm the reproducibility of the experiment, the operations from seeding the PBMC to measuring IgA were performed twice. The results are shown in Figure 1. As shown in Fig. 1, in any of the lactic acid bacteria, IgA production was much more induced in the presence of glutamine than in the absence of glutamine. Example 2 Using pe5 (Lactiplantibacillus pentosus 5) as the lactic acid bacterium, the PBMC medium was prepared, the PBMC(-) Gln medium was prepared (control), PBMC was prepared and seeded, the lactic acid bacterium was added, cultured, and IgA was measured in the same manner as in Example 1, except that glutamine was added to the PBMC medium to a concentration of 200 μM or 500 μM. For reference, a test with the same glutamine concentration (2.1 mM) as in Example 1 was also conducted. The results are shown in Fig. 2. As shown in Fig. 2, IgA production was induced by the combination with the lactic acid bacterium more at 500 μM than at 200 μM of the glutamine concentration. Also, a higher IgA concentration was shown by the combination with the lactic acid bacterium at 2.1 mM than at 500 μM of the glutamine concentration. It is expected that the difference in the IgA concentration in the test group with a glutamine concentration of 2.1 mM between Example 1 and Example 2 is due to lot differences of PBMC cells, etc. As is clear from the above tests, by combining glutamine with lactic acid bacteria, IgA production is much more induced than in the absence of glutamine.

Claims

1. An immunopotentiator comprising lactic acid bacteria and glutamine.

2. An agent for enhancing the immunopotentiating effect of lactic acid bacteria, which contains glutamine.

3. An IgA production inducer comprising lactic acid bacteria and glutamine.

4. An oral composition comprising lactic acid bacteria and glutamine, which acts on immune cells to maintain or improve the immune function of healthy individuals.

5. The agent according to any one of Claims 1 to 3 or the composition according to Claim 4, which is for administration to a subject during physical fatigue.

6. The agent according to any one of Claims 1 to 3 or the composition according to Claim 4, wherein the lactic acid bacteria belong to the genus Lactobacillus.

7. The agent according to any one of Claims 1 to 3 or the composition according to Claim 4, wherein the lactic acid bacteria are lactic acid bacteria capable of producing IgA under normal environmental conditions.

8. The agent according to any one of Claims 1 to 3 or the composition according to Claim 4, which is a food.

9. A method using glutamine as an index for the immunopotentiating effect by administration of lactic acid bacteria.

10. Use of lactic acid bacteria and glutamine for producing an immunopotentiator during physical fatigue.

11. Use of glutamine for producing an agent for enhancing the immunopotentiating effect of lactic acid bacteria during physical fatigue.

12. Use of lactic acid bacteria and glutamine for producing an IgA production inducer during physical fatigue.

13. The use according to any one of Claims 10 to 12, wherein the agent is for administration to a subject during physical fatigue.

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