Immunostimulating composition

Abstract

To provide an immunostimulating composition.SOLUTION: In one form, an immunostimulating composition comprises a yeast body obtained by removing a soluble component from a yeast cell without involving autodigestion or cell wall degradation. In another form, an immunostimulating composition comprises (1) a yeast body obtained by removing a soluble component from a yeast cell without involving autodigestion or cell wall degradation, and (2) a biologically derived component with health-preserving capabilities, other than the yeast body with the removed soluble component.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for immunostimulation, a combination for immunostimulation, a kit for immunostimulation, or a method for immunostimulation. [Background technology]

[0002] About immune activation Immune function refers to the ability of the animal or human body to eliminate or kill disease-causing bacteria or viruses when it comes into contact with them (natural immunity). Immunostimulating effect refers to the effect of activating the immune function of the living body and enhancing weakened defenses, and an immunostimulating substance refers to a substance that has such an immunostimulating effect.

[0003] Immunostimulator using yeast cells Immunostimulants (immune enhancers) using yeast cells have been reported. Japanese Patent Publication No. 8-283175 (Patent No. 3744020) describes an immune enhancing agent containing feed for crustaceans, fish and livestock, which is characterized in that the yeast cells of Torula yeast or Saccharomyces cerevisiae are inactivated by heating, then cell wall-lytic enzymes and proteases are simultaneously allowed to act on the cells for 15 to 18 hours to elute proteins, and then the cells are centrifuged to remove the residue, and the protein components contained in the supernatant are used as active ingredients. The document describes that β-glucan, which is a cell wall component of conventional yeast cells, does not have sufficient activity and is expensive, making it difficult to widely use as a feed additive (

[0006] ), but that by heating the cells in advance to inactivate all the enzymes in the cells, and then allowing cell wall-lytic enzymes and proteases to act on the cells, an immune enhancing agent with extremely excellent immune enhancing effects was obtained (

[0010] ). In other words, the document uses yeast cells that have been treated with enzymes to decompose the cell walls and elute proteins in order to obtain an immune enhancing effect.

[0004] JP 2004-99580 A describes an immune-enhancing composition prepared by subjecting yeast cells to autolysis or by combining autolysis with protease and endo-β-1,3 glucanase, or enzymes containing at least endo-β-1,3 glucanase. By subjecting yeast cells to autolysis, alkali-insoluble polysaccharides are significantly reduced and the reactivity of β-1,3 glucanase is increased. By further treating the autolyzed yeast cells with enzymes such as protease and endo-β-1,3 glucanase, a composition containing monosaccharides, oligosaccharides, etc. is obtained. The composition exhibits antibacterial properties against fish. From this document, it is understood that glucan, etc. excluding alkali-insoluble polysaccharides, is the active ingredient for the immune-enhancing effect.

[0005] Furthermore, it has been reported that yeast-derived β-glucan activates macrophages, neutrophils, and NK cells (J. Immunol. Vol. 173, 2004, p. 797-806).

[0006] However, in order to make yeast cells usable as an immunostimulant, it is necessary to decompose the cells themselves to a state in which β-glucan can be utilized, and no method has been presented for providing yeast cells that are edible and do not contain unpleasant flavors that arise during decomposition.

[0007] Immunostimulatory activity of lactic acid bacteria Lactic acid bacteria have also been reported to have immunostimulatory effects. It was suggested that the immunostimulatory effect of 1073R-1 yogurt is due to the involvement of immunostimulatory polysaccharides produced by the 1073R-1 strain. (Milk Science Vol.58, No.2, 2009, p.35-39) When sterilized lactobacilli were added to spleen cells derived from BALB / c mice, including MCC1849, the concentration of IL-12 in the culture supernatant was significantly increased compared to other lactobacilli. (Milk Science Vol.68, No.3,2009, p.180-187) Exopolysaccharides (EPS) produced by Lactobacillus plantarum No. 14 strain (LP14) have immunostimulatory activity (Jpn. J. Lactic Acid Bact. Vol. 22, No. 2, 2011, p. 100-105) [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent Publication No. 8-283175 [Patent Document 2] Patent Publication No. 2004-99580 [Non-patent literature]

[0009] [Non-Patent Document 1] J. Immunol. Vol. 173, 2004, p. 797-806). [Non-Patent Document 2] Milk Science Vol.58, No.2, 2009, p.35-39 When sterilized lactobacillus bacteria were added to spleen cells derived from BALB / c mice, including MCC1849, [Non-Patent Document 3] Milk Science Vol.68,No.3,2009,p.180-187 [Non-Patent Document 4] Jpn.J.Lactic Acid Bact. Vol.22,No.2,2011,p.100-105 Summary of the Invention [Problem to be solved by the invention]

[0010] Yeast cells have excellent nutritional value and palatability, so yeast cells themselves have been used as feed additives for some time. However, there is a problem that it is difficult to obtain an immune-enhancing effect by ingesting yeast cells as they are, and it has been necessary to process them to a state in which the immune-stimulating active ingredients can be used by decomposing the cell walls through autolysis or enzyme treatment, by separating proteins or decomposing the glucan portion.

[0011] In addition, there are moves both in Japan and overseas to prohibit or restrict the use of antibacterial substances in the livestock industry as a measure against drug-resistant bacteria. Therefore, there is a demand for the development of feed ingredients that have an immunostimulating effect as an alternative to antibacterial substances. [Means for solving the problem]

[0012] The present inventors have made intensive research efforts to solve the above problems, and have found that yeast cells obtained after removing soluble components from yeast cells can be used as they are to contain a certain amount of immunostimulatory substances without special treatment such as autolysis or enzyme treatment, leading to the present invention. Furthermore, yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment maintain the shape of the yeast cells compared to conventional yeast decomposition products, and are in a powdered state when dried, so that they can be used as an excipient. Therefore, they can also be used in combination with other bio-derived components having health-maintaining functions, such as dried cells of lactic acid bacteria and butyric acid bacteria.

[0013] The present invention includes, but is not limited to, the following aspects. [1] A composition for immunostimulation comprising yeast cells from which soluble components have been removed without subjecting the yeast cells to autolysis or cell wall decomposition treatment. [2] The immunostimulating composition according to [1], wherein the removal of the soluble components is selected from the group consisting of hot water treatment, alkali treatment and protease treatment. [3] The composition for immunostimulation described in [1], wherein the yeast cells are cells of a yeast selected from the group consisting of Torula yeast or yeast species belonging to the genus Saccharomyces. [4] (1) Yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) A component derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed; A composition for immunostimulation comprising: [5] The immunostimulating composition according to [4], wherein the removal of the soluble components is selected from the group consisting of hot water treatment, alkali treatment and protease treatment. [6] The composition described in [4], wherein the health-maintaining function is a function attributable to components produced by physiologically active microorganisms, algae, or cells. [7] The composition described in [4], wherein the biological component having health-maintaining function is selected from the group consisting of lactic acid bacteria, bifidobacteria, butyric acid bacteria, and natto bacteria. [8] The composition described in [4], wherein the biological component having health-maintaining function is a lactic acid bacterium. [9] The composition described in [4], further comprising nutritional components derived from living organisms that have health-maintaining functions.

[10] The composition described in [9], wherein the nutritional component of the component derived from a living organism having a health-maintaining function is a culture medium for the component derived from a living organism having a health-maintaining function.

[11] The composition described in [1] or [4], which does not contain an antibacterial substance.

[12] A combination for activating the immune system includes (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism that has a health-maintaining function other than the yeast cells from which the soluble components have been removed.

[13] A kit for immunostimulation, comprising yeast cells from which soluble components have been removed without autolysis or cell wall decomposition.

[14] (1) Yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) A component derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed; A kit for immunostimulation comprising:

[15] A method for activating the immune system by administering to a subject yeast cells from which soluble components have been removed without subjecting the yeast cells to autolysis or cell wall decomposition treatment.

[16] (1) Yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) A component derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed; A method of administering this to a subject to activate the immune system. Effect of the Invention

[0014] By using the yeast cells obtained after removing the soluble components as they are, it has become possible to easily prepare effective immunization compositions, kits, combinations, methods, etc. without carrying out special treatments such as autolysis or enzyme treatment. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the results of measuring the in vitro expression level of IL-12 p40 gene using J774.1 cells when yeast cells A to D having different types of origin and extraction processes and soluble components A to D extracted from yeast cells were added. [Diagram 2] FIG. 2 shows the results of measuring the in vitro expression level of IL-12 p40 gene using J774.1 cells when a composition containing lactic acid bacteria, a lactic acid bacteria medium, and / or dextrin was further added to yeast body A. [Diagram 3]FIG. 3 shows the results of measuring the amount of TNF-α gene expression in vitro using J774.1 cells when yeast cells A to D were added. [Figure 4] Figure 4 shows the results of measuring the amount of IL-12 protein produced ex vivo using mouse spleen cells when a composition containing yeast cells A, lactic acid bacteria, and a lactic acid bacteria medium was added. The graph shows the average value and standard deviation, and a significance test was performed using a multiple comparison test (Bonferroni method). **: p<0.01, *p<0.05 [Diagram 5] Figure 5 shows the results of measuring the amount of IFN-γ protein produced ex vivo using mouse spleen cells when a composition containing yeast cells A, lactic acid bacteria, and a lactic acid bacteria medium was added. The graph shows the average value and standard deviation, and a significance test was performed using a multiple comparison test (Bonferroni method). **: p<0.01, *p<0.05 [Figure 6] Figure 6 shows the results of measuring the amount of TNF-α protein produced ex vivo using mouse spleen cells when a composition containing yeast cells A, lactic acid bacteria, and a lactic acid bacteria medium was added. The graph shows the average value and standard deviation, and a significance test was performed using a multiple comparison test (Bonferroni method). **: p<0.01, *p<0.05 [Figure 7] FIG. 7 shows the results of measuring the amount of IL-12 protein produced in vivo in mice when a composition containing yeast cells A, lactic acid bacteria, and a lactic acid bacteria medium was added. [Figure 8] FIG. 8 shows the results of measuring the in vitro expression level of IL-12 p40 gene using J774.1 cells when yeast cells A to D, a commercially available bifidobacteria product, a commercially available lactic acid bacteria product, or a β-glucan product were added. [Figure 9] FIG. 9 shows the results of measuring the level of IL-12 p40 gene expression in vitro using J774.1 cells when yeast cells A to C and their soluble components, and each yeast in an unextracted state, were added. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention includes the following embodiments without limitation. Unless otherwise specified in the present specification, technical and scientific terms used in the present specification have the same meaning as those commonly understood by those skilled in the art. The substances, materials and examples disclosed in the present specification are merely illustrative and are not intended to be limiting. When referring to "in one embodiment" in the present specification, it means that the embodiment is not limited, i.e., it is not limited.

[0017] 1. Immunostimulating composition (A) In one aspect, the present invention relates to an immunostimulating composition (A). The immunostimulating composition includes, but is not limited to, yeast cells from which soluble components have been removed without autolysis or cell wall decomposition. The immunostimulating composition includes, but is not limited to, yeast cells from which soluble components have been removed without autolysis or cell wall decomposition, as an active ingredient.

[0018] The yeast cells do not undergo autolysis or cell wall decomposition. "Autolysis" generally refers to the decomposition of the organism's own tissues by enzymes that it possesses. Examples of enzymes that work in autolysis include protease, glucanase, lipase, and amylase. Enzymes that are mainly involved in autolysis are proteases that act on proteins and glucanases that decompose cell walls. "Cell wall decomposition treatment" refers to a treatment other than autolysis in which the yeast cell walls are artificially decomposed, and the treatment method is not particularly limited. In one embodiment, the cell wall decomposition treatment is a treatment in which the cell walls of the yeast cells are destroyed by enzymes or a treatment in which the cell walls of the yeast cells are physically destroyed. In one embodiment, the cell wall decomposition treatment is a treatment with an enzyme consisting of glucanase or a combination of glucanase and other enzymes, or a physical treatment such as ultrasonic treatment or grinding treatment. The yeast cells are obtained by removing soluble components from the yeast cells without performing autolysis or cell wall decomposition treatment. The method for removing the soluble components is not particularly limited as long as it is a method that is strong enough to destroy the cell walls of the yeast cells, but it should not be a method that is strong enough to destroy the cell walls of the yeast cells. Those skilled in the art can appropriately select a method for removing soluble components without destroying the cell walls of the yeast cells based on the disclosure of this specification and known techniques.

[0019] The removal of the soluble components is, without limitation, selected from the group consisting of hot water treatment, alkali treatment, and protease treatment. In the "hot water treatment", for example, the yeast cells are treated with water at 30°C to 80°C for 10 minutes to 24 hours to remove the soluble components and extract only the yeast cells. In the hot water treatment, amino acids and the like are mainly extracted. In the alkali treatment, the yeast cells are treated with, for example, a weak alkali (pH 8.0 to pH 10.0) solution for 10 minutes to 24 hours to remove the soluble components and extract only the yeast cells. The alkali treatment is particularly useful for extracting nucleic acids in the yeast cells without decomposing them. The protease treatment is not performed under strong conditions that destroy the cell walls of the yeast cells, but under weak conditions, for example, to remove residues in the yeast cells that have been treated with hot water or alkali. For example, the protease treatment refers to the above conditions in which the cell walls are not destroyed and the soluble components are extracted while the shape of the yeast cells is maintained.

[0020] The type of the yeast cell is not particularly limited. In one embodiment, the yeast cell may be a torula yeast, baker's yeast, sake yeast, etc. The yeast cell is a yeast cell selected from the group consisting of yeast species belonging to the genus Saccharomyces, such as baker's yeast and sake yeast.

[0021] The scientific name of "torula yeast" is Cyberlindnera jadini, which is derived from the genus name Torula utilis, of which it is a synonym. Torula yeast is used as an additive for processed foods and as a raw material for biochemical products.

[0022] "The genus Saccharomyces is a type of fungus that includes many species of yeast. Many species of this genus are considered to be very important in food production. Saccharomyces includes, but is not limited to, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces florentinus, Saccharomyces kluyveri, Saccharomyces martiniae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, Saccharomyces zonatus and other species.

[0023] For example, the budding yeast Saccharomyces cerevisiae is necessary for producing wine, bread, beer, etc., and is called baker's yeast, brewer's yeast, etc. Also, Saccharomyces bayanus is used to make wine, and Saccharomyces boulardii is used to make medicines. In one embodiment, the yeast cell is a yeast cell of Saccharomyces cerevisiae.

[0024] The immune function refers to the function (natural immunity) of the animal or human body to eliminate or kill disease-causing bacteria or viruses when it comes into contact with them. "Immunostimulating effect" refers to the effect of activating the immune function of the living body and enhancing the established defense action, and an immunostimulating composition refers to a composition having such an immunostimulating effect.

[0025] The immune system's attacks against pathogens mainly involve phagocytosis by phagocytes of the innate immune system, such as neutrophils and macrophages, the destruction of host cells by the release of cytotoxic substances by killer T cells, and the inactivation of pathogens by antibodies produced by B cells. Physiologically active proteins collectively known as cytokines play an important role in activating and suppressing the function of such immune cells. There are various types of cytokines, including interleukins, which are secreted by white blood cells and function to regulate the immune system, chemokines, which induce the migration of white blood cells, and interferons, which suppress the proliferation of viruses and cells.

[0026] The major cytokines currently known include interleukins (IL-1 to IL-15), which act to promote functional differentiation of lymphoid cells, interferons (IFN-α, β, γ), which are produced in response to viral infections and are named after their interference with other viruses, and inflammatory cytokines such as IL-1, IL-6, and TNF, which are produced during LPS stimulation and in the early stages of inflammatory responses.

[0027] IL-12 is one of the cytokines of the interleukin group. IL-12p70 is a heterodimer consisting of two subunits, IL-12 p35 and IL-12 p40, and is mainly produced by phagocytes and dendritic cells. IL-12 is a stimulatory factor for NK cells and T cells, and its main function is to induce the production of interferon-gamma in NK cells and T cells and to enhance the activity of NK cells and cytotoxic T cells. It is involved in immune activation (defense against infection).

[0028] TNF-α activates macrophages and NK cells and damages tumor cells (anti-tumor effect). Interferon (IFN) is a member of the mammalian cytokine family that was identified as a factor that suppresses viral infection. Interferons are classified into types I, II, and III based on their protein structure and recognition of receptor complexes. Human type II interferon consists only of the IFN-γ gene and binds to the type II receptor consisting of the IFNGR1 (IFN-γR1) chain and the IFNGR2 (IFN-γR2) chain. IFN-γ is produced by immune system cells such as T cells and NK cells.

[0029] The immune activation effect can be examined using a known method for examining immune activity. For example, as described above, the expression levels of cytokines involved in immune activation (protection against infection), such as IL-12, TNF-α, IFN-γ, etc., can be examined, and it can be determined that immunity has been activated when the expression levels of cytokines increase. The expression levels may be examined as either the transcription amount (mRNA) or the translation amount (protein). The transcription amount and translation amount can be examined using a known method. For example, the transcription amount can be measured by extracting total RNA from cells to which (or not) the immune activation composition has been added, synthesizing cDNA, amplifying the cytokine gene by reverse transcriptase PCR (RT-PCR), electrophoresing the amplified gene, and analyzing the image. For example, the translation amount can be measured by measuring the production amount of a specific cytokine protein produced in the culture supernatant of cells to which (or not) the immune activation composition has been added, using an ELISA kit for the corresponding cytokine protein.

[0030] In one embodiment, when the transcription amount or translation amount is increased compared to when the composition for immunostimulation is not used, it is judged to have an immunostimulating effect. Non-limiting examples of the immunostimulating effect include when the transcription amount or translation amount is increased by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 35% or more, 50% or more, 80% or more, or 100% or more compared to when the composition for immunostimulation is not used.

[0031] In one embodiment, the composition for immunostimulation may be judged to have an immunostimulating effect when the transcription amount or translation amount is increased when the composition for immunostimulation is used compared to when the corresponding untreated yeast cells are used. Non-limiting examples of the composition for immunostimulation are when the transcription amount or translation amount is increased by 1% or more, 3% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more when the composition for immunostimulation is used compared to when the corresponding untreated yeast cells are used.

[0032] The composition for immunostimulation exhibits an immunostimulating effect similar to or greater than that achieved by using lactic acid bacteria known to have an immunostimulating effect. In one embodiment, the composition for immunostimulation does not contain an antibacterial substance. From the viewpoint of measures against drug-resistant bacteria, it is preferable for the composition for immunostimulation not to contain an antibacterial substance. "Antibacterial substance" is a general term for antibiotics and synthetic antibacterial agents. Antibiotics are natural substances produced by fungi such as penicillin, and synthetic antibacterial agents are chemically synthesized substances such as sulfa drugs and oxolinic acid. Most of the antibiotics available on the market are now completely chemically synthesized, and are collectively referred to as antibacterial substances unless there is a need to distinguish between the two. Antibacterial substances selectively act on a part of the metabolism or growth mechanism of microorganisms to inhibit the growth and growth of microorganisms or kill them. Substances that have high selective toxicity and are not very toxic to living cells are used as antibacterial substances. However, in the case of a composition to be ingested by a living body, it is preferable that they do not contain antibacterial substances.

[0033] Antibacterial substances are classified into two categories: those based on chemical structure and those based on action. When classified based on chemical structure, they are broadly divided into β-lactams, aminoglycosides, macrolides, tetracyclines, peptides, nucleic acids, polyenes, etc., but may also be further subdivided into penicillins, cephalosporins, and monobactams. When classified based on action, they are divided into antibacterial, antifungal, antitumor, etc. When emphasis is placed on use, they are classified into medical, veterinary, agricultural, etc.

[0034] In one embodiment, the immunostimulatory composition (A) may be a food or beverage composition or a pharmaceutical composition. Food or beverage compositions can be prepared by blending conventionally known ingredients such as colorants, preservatives, fragrances, flavorings, and coating agents, as necessary. Pharmaceutical compositions may contain known excipients, granulating agents, and other processing aids suitable for pharmaceutical use.

[0035] The subject that takes the immunostimulatory composition (A) is not particularly limited as long as it is an animal that requires immunostimulation. Examples include pet animals, industrial animals, etc. In one embodiment, the subject is a human. In another embodiment, the subject may be a non-human mammal. Examples of non-human mammals include non-human primates (monkeys, chimpanzees, gorillas, etc.), dolphins, sea lions, pigs, cows, horses, sheep, dogs, cats, rats, mice, guinea pigs, rabbits, etc.

[0036] The amount of yeast cells from which soluble components have been removed without autolysis or cell wall decomposition in the composition is not particularly limited. Preferably, the yeast cells from which soluble components have been removed without autolysis or cell wall decomposition are ingested (in this specification, "ingestion" also includes the meaning of "administration") in an amount effective for immunostimulation. The dosage can be appropriately adjusted depending on the type, body weight, body length, age, and body length of the target animal.

[0037] In one aspect, the present invention relates to yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, for use in an immunostimulatory composition. In one aspect, the present invention relates to the use of yeast cells from which soluble components have been removed without subjecting the yeast cells to autolysis or cell wall decomposition treatment in a composition for immunostimulation.

[0038] 2. Immunostimulating Composition (B) In one aspect, the present invention relates to an immunostimulating composition (B). The immunostimulating composition includes, but is not limited to, (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition, and (2) a component derived from a living organism having a health-maintaining function, other than the yeast cells from which the soluble components have been removed. The immunostimulating composition includes, but is not limited to, (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition, and (2) a component derived from a living organism having a health-maintaining function, other than the yeast cells from which the soluble components have been removed, as active ingredients.

[0039] Regarding "immunostimulation," "(1) Yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment," etc., they are as described in the section "1. Composition for immunostimulation (A)."

[0040] The matters described in "1. Immunostimulating composition (A)" can also be applied to the immunostimulating composition (B) unless there is any particular contradiction. In one embodiment, the removal of soluble components is selected from the group consisting of hot water treatment, alkali treatment and protease treatment.

[0041] The "biologically derived component having a health-maintaining function" is not particularly limited as long as it has a function of maintaining health and is derived from a biological body. The "health-maintaining function" may be any function that is favorable for maintaining the health of a biological body, and includes, for example, good sleep, intestinal regulation, prevention of cognitive decline, cancer prevention, prevention of lifestyle-related diseases, beauty effects, etc. The health-maintaining function may include an immune function.

[0042] In one embodiment, the health-maintaining function is a function attributable to a component produced by a physiologically active microorganism, algae, or cell. The physiologically active microorganism is selected from the group consisting of microorganisms other than yeast, such as lactic acid bacteria, bifidobacteria, butyric acid bacteria, natto bacteria, koji mold, and zygomycetes.

[0043] In one embodiment, the health-maintaining biological component is selected from the group consisting of lactic acid bacteria, bifidobacteria, butyric acid bacteria, and natto bacteria. In one embodiment, the health-maintaining biological component is lactic acid bacteria.

[0044] "Lactic acid bacteria" is a general term for bacteria that produce lactic acid through metabolism. Lactic acid bacteria are normally present in the digestive tract (intestinal bacteria) such as the intestines and the vagina, and are thought to help maintain homeostasis of the intestinal environment by living symbiotically or antagonistically with other microorganisms. In the narrow sense, lactic acid bacteria refers to those belonging to the order Lactobacillales. The order Lactobacillales includes the genera Lactobacillus, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, and Streptococcus (Streptococcus).

[0045] The genus Lactobacillus is a gram-positive rod-shaped bacillus belonging to the order Lactobacillales, class Bacillaceae, and family Firmicutes, and is also called Lactobacillus. In general, when the term "lactic acid bacillus" is used, this genus is often referred to in the narrow sense. Some species produce only lactic acid (homolactic fermentation) while others produce other substances besides lactic acid (heterolactic fermentation). The genus Lactobacillus is easily isolated from the field and has been used for yogurt production for a long time. Many species belonging to the genus Lactobacillus, such as Lactobacillus bulgaricus, Lactobacillus gasseri, and Lactobacillus acidophilus, are used for yogurt production. In one embodiment, the lactic acid bacterium is Lactobacillus plantarum, Lactobacillus pentosus, or Lactobacillus gasseri.

[0046] The Enterococcus genus is a gram-positive coccus belonging to the family Enterococcus of the order Lactobacillales, which performs homolactic fermentation. It inhabits the ileum, cecum, and large intestine. There are species such as E. faecalis and E. faecium. Of these, E. faecalis is often used in combination with Bifidobacterium (bifidobacteria) and Lactobacillus (acidophilus) as an intestinal regulator.

[0047] The genus Lactococcus is a gram-positive cocci belonging to the family Streptococcus in the order Lactobacillales. It is a "lactic acid cocci" in the narrow sense. It undergoes homolactic fermentation. It is commonly found in milk and dairy products, and is used in fermented dairy products made from these ingredients. Examples include L. lactis and L. cremoris. It is used in commercially available Caspian Sea yogurt. Nisin is a polycyclic antibacterial peptide that is used as a food preservative and is produced by the fermentation of Lactococcus lactis.

[0048] The genus Pediococcus is a gram-positive coccus belonging to the family Lactobacillaceae of the order Lactobacillales, which performs homolactic fermentation. It is often isolated from fermented plant products such as pickles. Examples include P. damnosus.

[0049] The genus Leuconostoc is a gram-positive coccus belonging to the family Leuconostaceae in the order Lactobacillales, and has a chain or diplococcal arrangement. It undergoes heterolactic fermentation. It is isolated from fermented plant products such as sauerkraut. Examples of the genus Leuconostoc include L. mesenteroides.

[0050] Streptococcus (Streptococcus), a gram-positive coccus belonging to the family Streptococcus of the order Lactobacillales, is classified as a lactic acid bacterium. Streptococcus thermophilus is found in fermented dairy products and is commonly used in the production of yoghurt (e.g. Bulgarian yoghurt).

[0051] "Bifidobacteria are gram-positive, obligately anaerobic rod-shaped bacteria belonging to the genus Bifidobacterium (Actinobacteria, Actinobacteriaidae, Bifidobacteriales, Bifidobacteriaceae, genus Bifidobacterium), a type of heterolactic acid bacteria, and are sometimes included in the broad definition of lactic acid bacteria because they produce lactic acid and acetic acid. Examples include B. bifidum and B. adolescentis.

[0052] "Butyric acid bacteria" is a general term for bacteria that exist in Escherichia coli and produce butyric acid. A representative example of butyric acid bacteria is Clostridium butyricum. Clostridium butyricum is an obligately anaerobic, spore-forming, gram-positive rod-shaped bacterium. The most well-known Clostridium butyricum strain is called Miyairi bacteria, and its spores are formulated and used as an intestinal regulator.

[0053] The scientific name for "natto bacteria" is Bacillus substilis var. natto, a type of bacillus subtilis. Natto bacteria are known to produce useful substances such as nattokinase, vitamin K2, and amino acids. Natto bacteria are also known to regulate the intestines.

[0054] "Algae is a general term for organisms that perform oxygenic photosynthesis, excluding mosses, ferns, and seed plants, which are primarily terrestrial. In other words, it includes evolutionarily distinct groups such as the true bacteria cyanobacteria (blue-green algae), the eukaryotic unicellular organisms (diatoms, xanthophytes, dinoflagellates, etc.), and the multicellular seaweeds (red algae, brown algae, green algae). For example, edible seaweeds include kombu, wakame, nori, green laver, tengusa, and fine soy sauce. Edible freshwater algae include Aphanothece sacrum.

[0055] When the "bio-derived component having a health-maintaining function" is a "component produced by cells," it includes, but is not limited to, polysaccharides such as α-glucan and β-glucan, peptides, amino acids, vitamins, minerals, other organic components, etc. The "component produced by cells" may be a component produced by yeast cells, particularly a soluble component produced by yeast cells, or other yeast cells, other than the yeast cells themselves that were originally added.

[0056] In one embodiment, the immunostimulating composition (B) further contains nutritional components of the component derived from a living organism having a health-maintaining function. The "nutritional components of the component derived from a living organism having a health-maintaining function" are not particularly limited as long as they are useful for increasing the amount and amplifying the function (effect) of (2) the component derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed. Those skilled in the art can select appropriate nutritional components depending on the type of the component derived from a living organism. For example, when the component is a microorganism having a physiological activity, the nutritional components include a growth medium for the growth of the microorganism, an additive, or a culture medium or additive suitable for the microorganism to exert a health-maintaining function. Alternatively, when the component is a component produced by a cell, the nutritional components include a medium or additive that allows the cell to produce the component in greater amounts. In one embodiment, for example, when the component derived from a living organism having a health-maintaining function is a lactic acid bacterium, the nutritional components of the lactic acid bacterium include dextrin, milk, peptone, salt, minerals, surfactants, etc.

[0057] In one embodiment, the nutritional components of the organism-derived component having a health-maintaining function are a medium for the organism-derived component having a health-maintaining function. The medium may be either a growth medium or a culture medium. In one embodiment, for example, when the organism-derived component having a health-maintaining function is a lactic acid bacterium, the medium for culturing the lactic acid bacterium includes a medium containing glucose, yeast extract, etc. In one embodiment, the medium composition of the culture medium for the lactic acid bacterium is 5.1% glucose, 3.1% yeast extract, and 91.8% water.

[0058] The immunostimulating composition (B) preferably has a function of maintaining health. The "function of maintaining health" is not particularly limited as long as it is a function favorable for maintaining the health of a living organism, as described above with respect to the "organism-derived component having a health-maintenance function." For example, it includes a good night's sleep, intestinal regulation, prevention of cognitive decline, cancer prevention, stress relief, prevention of lifestyle-related diseases, etc. The health-maintenance function may include an immune function.

[0059] In one embodiment, the immunostimulatory composition does not contain an antibacterial agent. The immunostimulatory composition (B) may be a food or beverage composition or a pharmaceutical composition. The food or beverage composition may be prepared by blending conventionally known ingredients such as colorants, preservatives, fragrances, flavorings, and coating agents, as necessary. The pharmaceutical composition may contain excipients, granulating agents, emulsifiers, and the like suitable for pharmaceutical use.

[0060] The subject that takes the immunostimulatory composition (B) is not particularly limited as long as it is an animal that requires immunostimulation. Examples include pet animals, industrial animals, etc. In one embodiment, the subject is a human. In another embodiment, the subject may be a non-human mammal. Examples of non-human mammals include non-human primates (monkeys, chimpanzees, gorillas, etc.), dolphins, sea lions, pigs, cows, horses, sheep, dogs, cats, rats, mice, guinea pigs, rabbits, etc.

[0061] In the composition, the amount of (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition is not particularly limited. Preferably, yeast cells from which soluble components have been removed without autolysis or cell wall decomposition are ingested (as used herein, "ingestion" also includes the meaning of "administration") in an amount effective for immunostimulation. The dose can be appropriately adjusted depending on the type, body weight, body length, age, and body length of the target animal.

[0062] The amount of (2) the components derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed in the composition is not particularly limited. Preferably, the components derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed are ingested (as used herein, "ingestion" also includes the meaning of "administration") in an amount effective for exerting the function of maintaining health. The dosage can be appropriately adjusted depending on the type, weight, body length, age, and body length of the target animal.

[0063] In one aspect, the present invention relates to (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism having a health-maintaining function, other than the yeast cells from which the soluble components have been removed, for use in an immunostimulating composition.

[0064] In one aspect, the present invention relates to (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) the use of a biological component having health-maintaining function other than the yeast cells from which the soluble components have been removed, in an immunostimulating composition.

[0065] 3. Combination In one aspect, the present invention includes a combination for stimulating immunity. The combination includes, but is not limited to, (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism having a health-maintaining function, other than the yeast cells from which the soluble components have been removed. The combination includes, but is not limited to, (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism having a health-maintaining function, other than the yeast cells from which the soluble components have been removed, as active ingredients.

[0066] Regarding "immunostimulation," "(1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition," and "(2) components derived from living organisms having health-maintaining functions other than yeast cells from which soluble components have been removed," etc., these are as described in the sections "1. Composition for immunostimulation (A)" and "2. Composition for immunostimulation (B)."

[0067] The matters described in "1. Immunostimulating composition (A)" and "2. Immunostimulating composition (B)" can also be applied to the above combination, unless there is any particular contradiction. The combination does not need to consist of (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living body that has a health-maintaining function other than the yeast cells from which the soluble components have been removed; it is sufficient that the combination be ingested by the living body at a time when the effects of both (1) and (2) can be obtained.

[0068] In one embodiment, "(2) a component derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed" is ingested by the living organism simultaneously with "(1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment", or before (1), or after (1). In one embodiment, (2) is ingested within 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or 10 minutes before ingestion of (1). In one embodiment, (2) is ingested within 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or 10 minutes after ingestion of (1). In one embodiment, (2) is taken within 3 hours before and within 3 hours after ingestion of (1), within 2 hours before and within 2 hours after ingestion, within 1 hour before and within 1 hour after ingestion, within 30 minutes before and within 30 minutes after ingestion, within 15 minutes before and within 15 minutes after ingestion, or within 10 minutes before and within 10 minutes after ingestion.

[0069] In one aspect, the present invention relates to (1) yeast cells from which soluble components have been removed without subjecting yeast cells to autolysis or cell wall decomposition treatment, which are used in combination with (2) a component derived from a living organism having a health-maintaining function other than the yeast cells from which soluble components have been removed, for immunostimulation.

[0070] 4. Kit In one aspect, the present invention relates to a kit for immunostimulation. As a non-limiting example, the kit includes yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment.As a non-limiting example, the kit includes yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment as an active ingredient.

[0071] As a non-limiting example, the kit includes (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism having a health-maintaining function, other than the yeast cells from which the soluble components have been removed.As a non-limiting example, the kit includes, as active ingredients, (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism having a health-maintaining function, other than the yeast cells from which the soluble components have been removed.

[0072] Regarding "immunostimulation," "(1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition," and "(2) components derived from living organisms having health-maintaining functions other than yeast cells from which soluble components have been removed," etc., these are as described in the sections "1. Composition for immunostimulation (A)" and "2. Composition for immunostimulation (B)."

[0073] The matters described in "1. Immunostimulating composition (A)", "2. Immunostimulating composition (B)", and "3. Combination" can also be applied to the above kit, unless there is any particular contradiction. The kit may further include nutritional components, physiologically active substances, etc., which are components derived from living organisms and have the health-maintaining function.

[0074] The kit may contain (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed, in one container. Alternatively, the kit may contain (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed, in two or more containers.

[0075] In one aspect, the present invention relates to the use of yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment in a kit for immunostimulation. In one aspect, the present invention relates to (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) the use of a biological component having a health-maintaining function other than the yeast cells from which the soluble components have been removed, in an immune activation kit.

[0076] 5. Methods for activating the immune system In one aspect, the present invention relates to a method for stimulating immunity, the method including, but not limited to, administering to a subject yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment. The method including, but not limited to, administering to a subject (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a biologically derived component having a health-maintaining function other than the yeast cells from which the soluble components have been removed.

[0077] Regarding "immunoactivation," "(1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition," and "(2) components derived from living organisms having health-maintaining functions other than yeast cells from which soluble components have been removed," these are as described in the sections "1. Immunoactivation composition (A)" and "2. Immunoactivation composition (B)."

[0078] The matters described in "1. Immunostimulating composition (A)", "2. Immunostimulating composition (B)", "3. Combination", and "4. Kit" may also be applied to the above-mentioned kit, unless there is any particular contradiction.

[0079] The dosage of "(1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment" and "(2) components derived from living organisms having health-maintaining functions other than the yeast cells from which the soluble components have been removed" is as described as the dosage in the sections "1. Immunostimulating composition (A)" and "2. Immunostimulating composition (B)."

[0080] The subject of the method is not particularly limited as long as it is an animal that requires immunostimulation. For example, it includes pet animals, industrial animals, etc. In one embodiment, the subject is a human. In one embodiment, the subject may be a non-human mammal. Examples of non-human mammals include non-human primates (monkeys, chimpanzees, gorillas, etc.), dolphins, sea lions, pigs, cows, horses, sheep, dogs, cats, rats, mice, guinea pigs, rabbits, etc.

[0081] The method of administration to the subject is not particularly limited. It may be either oral administration or parenteral administration. Oral administration is preferred. The method may be in vivo, in vitro or ex vivo. EXAMPLES

[0082] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0083] (Acquisition and preparation of reagents and samples) In the Examples herein, unless otherwise specified, reagents and samples were obtained or prepared as follows.

[0084] The PBS used was Dulbecco's PBS(-) powder "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.). The FBS used was S1810-500, ID number: S00Hl10001 (BioWest).

[0085] Preparation of RPMI-1640: 50 mL of heat-inactivated FBS was added to 500 mL of RPMI-1640 (containing L-glutamine, phenol red, and HEPES) to adjust the volume to 10% of the total medium volume. In addition, 50 mg of streptomycin sulfate and 30 mg of penicillin G potassium salt were dissolved in a small amount of medium, transferred to a 10 mL syringe equipped with a membrane filter unit, and added to the entire medium while filtering to prepare RPMI-1640 medium.

[0086] Lipopolysaccharide (LPS) from Escherichia coli O55:B5 (sigma) was used. ELISA kits The following ELISA kits were used for each measurement. (IL-12 p40 / 70 measurement) "Mouse IL-12 p40 / 70 ELISA" (RayBiotech Life) (TNF-α measurement) "Mouse TNF-alpha ELISA kit" (Proteintech Japan) (INF-γ measurement) "Mouse IFN-gamma ELISA kit" (Proteintech Japan)

[0087] Example 1: In vitro measurement of IL-12 p40 gene expression level using J774.1 cells (1) In this example, the correlation between the type of yeast, the extraction process, and the immunostimulatory activity was examined. Specifically, as described below, yeast cells A to D with different types of yeast origin and extraction processes and soluble components A to D extracted from the yeast cells were used in the test. The yeast cells obtained after removing the soluble components and the extracted soluble components were added to J774.1 cells, and the gene expression level of interleukin 12 (IL-12), which is one of the macrophage activation indicators, was confirmed.

[0088] (Experimental Materials) J774.1 cells: A mouse-derived monocyte-macrophage-like cell line (animal lineage: BALB / c mouse). The cells are registered with the JCRB Cell Bank under the cell number JCRB0018. (https: / / cellbank.nibiohn.go.jp / ~cellbank / cgi-bin / search_res_det.cgi?ID=216) J774.1 cells were cultured in RPMI-1640 medium supplemented with 10% inactivated FBS and 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Industries).

[0089] (yeast cells and soluble components) Yeast cells A to D having different types of yeast origins and extraction processes and soluble components A to D extracted from the yeast cells were prepared as follows.

[0090] Yeast A: Yeast cells obtained by removing soluble components from Torula yeast through hot water extraction and drying. The cell wall is not decomposed. Yeast B: Baker's yeast with a high succinic acid content is extracted with hot water to remove soluble components and then dried. The cell wall is not decomposed.

[0091] Yeast C: The soluble components were removed from baker's yeast using a weak alkaline solution. The yeast cells were then treated with protease to remove any residual material from within the cells, and then dried. The cell walls were not decomposed.

[0092] Yeast D: yeast cells (product name: DYP-SY-02, Fuji Food Industries) obtained by removing soluble components from baker's yeast using a weak alkaline solution and drying. The cell wall is not decomposed.

[0093] Soluble component A: A soluble component obtained by hot water extraction from Torula yeast. Soluble component B: A soluble component obtained by hot water extraction from baker's yeast containing a high amount of succinic acid. Soluble component C: A soluble component obtained from baker's yeast containing a high content of free amino acids using a weak alkaline solution. Soluble component D: A soluble component obtained from baker's yeast, which is rich in nucleic acids, in a weak alkaline solution.

[0094] (method) First, J774.1 cells were plated in 12-well cell culture plates at a final concentration of 1.0 × 10 per well. 6 The medium was dispensed so that the cells would become 1 mL per well.

[0095] Next, yeast cells A to D and soluble components A to D were each adjusted to a concentration of 10 mg / mL with PBS. Heat sterilization was performed at 100°C for 5 minutes. After sterilization, cells were dispensed from the suspension of yeast cells or soluble components to a final concentration of 1 mg / mL, and the resulting solution was added to each well.

[0096] In addition, RPMI-1640 medium supplemented with 20% PBS was used as a negative control, and lipopolysaccharide (LPS) (Sigma-Aldrich Co. Ltd., derived from E. coli. 055:B5 strain) was added to the cells at a final concentration of 100 ng / mL as a positive control.

[0097] After incubating in a CO2 incubator for 3 hours, the cells were detached by pipetting and collected. Total RNA was extracted from the collected cells using an Rneasy Mini Kit (QIAGEN). cDNA was synthesized using the extracted RNA as a template with the SuperScript IV First-Strand Synthesis System (Invitrogen).

[0098] Next, the IL-12 p40 gene was amplified by reverse transcriptase PCR (RT-PCR). The primers, reaction conditions, and reactive composition used in RT-PCR are as follows:

[0099] [Table 1]

[0100] [Table 2]

[0101] [Table 3]

[0102] Then, the cells were electrophoresed on a 2% agarose gel, and the expression level of IL-12 p40 gene was measured by image analysis using Image-J. The results are shown in Figure 1. The IL-12 p40 gene expression level in each sample was expressed as a relative value when the gene expression level in PBS was set to 1. Each measurement was performed three times, and the average value and standard deviation are shown.

[0103] Yeast cells A to D showed the same level of IL-12 p40 gene expression as the positive control LPS, and it was shown that when yeast cells were added, the gene expression level of IL-12 p40 was high, similar to that of LPS, which is known as an immunostimulant. On the other hand, the soluble components A to D extracted from yeast cells all showed the same gene expression level as the negative control PBS, suggesting that these samples do not have immunostimulant activity.

[0104] These results indicate that the expression level of IL-12 p40 gene was generally high in yeast cells from which soluble components were removed without autolysis or cell wall degradation, whereas the expression level of IL-12 p40 gene was low in soluble components.

[0105] Example 2: In vitro measurement of IL-12 p40 gene expression level using J774.1 cells (2) In this example, a composition containing lactic acid bacteria (Lactobacillus plantarum genus), lactic acid bacteria medium, and dextrin in addition to the yeast cells used in Example 1 was used to examine the immunostimulatory activity of each sample by examining the gene expression level of IL-12 in mouse-derived blood cells and lymphoid cells.

[0106] (Experimental Materials) Lactic acid bacteria medium composition: glucose 5.1%, yeast extract 3.1%, water 91.8%. Lactic acid bacteria: Lactobacillus plantarum genus Yeast cell A: same as in Example 1.

[0107] (method) Samples 1 to 4 were prepared as follows using yeast cells, lactic acid bacteria cells (Lactobacillus plantarum), a lactic acid bacteria medium, and dextrin.

[0108] Sample 1: Yeast A + lactic acid bacteria + lactic acid bacteria medium (composition: yeast A 66%, lactic acid bacteria 1%, lactic acid bacteria medium 33%) Sample 2: Yeast A + lactic acid bacteria + lactic acid bacteria medium + dextrin (composition: yeast A 16.5%, lactic acid bacteria 1%, lactic acid bacteria medium 33%, dextrin 49.5%) Sample 3: Yeast cell A Sample 4: Lactic acid bacteria + lactic acid bacteria medium + dextrin (composition: 1.2% lactic acid bacteria, 39.5% lactic acid bacteria medium, 59.3% dextrin) In addition, RPMI-1640 medium supplemented with 20% PBS was used as a negative control, and lipopolysaccharide (LPS) (Sigma-Aldrich Co. Ltd., derived from E. coli. 055:B5 strain) was added to the cells at a final concentration of 100 ng / mL as a positive control.

[0109] First, J774.1 cells were plated in a 12-well cell culture plate at 1.0 x 10 cells per well. 6 The medium was dispensed so that the cells were separated. The amount of medium per well was adjusted to 1 mL. The prepared samples 1 to 4 and LPS were suspended in phosphate buffered saline (PBS) and heat sterilized at 100° C. for 5 minutes. After heat sterilization, the suspension was diluted with RPMI-1640 medium to the following final concentrations and added to the cells.

[0110] Sample 1 and Sample 2: 1 mg / mL Sample 3: 165 μg / mL Sample 4: 835 μg / mL LPS: 100ng / mL Each sample was added to J774.1 cells, incubated in a CO2 incubator for 3 hours, and then the cells were detached by pipetting and collected. Total RNA was extracted from the collected cells in the same manner as in Example 1, cDNA was synthesized, and the IL-12 p40 gene was amplified by reverse transcriptase PCR (RT-PCR). Then, the resultant was electrophoresed on a 2% agarose gel, and the expression level of the IL-12 p40 gene was measured by image analysis using Image-J.

[0111] The results are shown in Figure 2. The IL-12 p40 gene expression level in each sample was expressed as a relative value when the gene expression level in PBS was set to 1. Each measurement was performed three times, and the average value and standard deviation are shown.

[0112] Samples 1, 2, and 3, which contained yeast cells A, all showed the same level of IL-12 p40 gene expression as LPS used as the positive control, demonstrating that they have immunostimulatory activity. On the other hand, sample 4, which did not contain yeast cells A and consisted only of other composition raw materials, showed almost the same level of IL-12 p40 gene expression as the negative control, confirming that the parts of the composition used in this test other than yeast cells A did not have an immunostimulatory effect.

[0113] The above results indicate that among the yeast cells, lactic acid bacteria cells, lactic acid bacteria medium, and dextrin used in this sample, the yeast cells in particular contributed to enhancing the immunostimulatory activity.

[0114] Example 3: Measurement of TNF-α gene expression level in vitro using J774.1 cells In this example, the yeast cells A to D used in Example 1 were used to carry out a test for measuring the gene expression level of TNF-α, an inflammatory cytokine.

[0115] (method) J774.1 cells were plated in 12-well cell culture plates at 1.0 × 10 cells per well. 6 The medium was dispensed so that the cells would become 1 mL per well.

[0116] Next, the yeast cells A to D and the soluble components A to D extracted from the yeast cells used in Example 1 were each suspended and dissolved to a concentration of 10 mg / mL. They were sterilized by heating at 100°C for 5 minutes, diluted with RPMI-1640 medium to a concentration of 1 mg / mL, and added to the cells.

[0117] In addition, RPMI-1640 medium supplemented with 20% PBS was used as a negative control, and lipopolysaccharide (LPS) (Sigma-Aldrich Co. Ltd., derived from E. coli. 055:B5 strain) was added to the cells at a final concentration of 100 ng / mL as a positive control.

[0118] After incubating each sample for 3 hours in a CO2 incubator, the cells were detached by pipetting and collected. As in Example 1, total RNA was extracted from the collected cells, cDNA was synthesized, and the TNF-α gene was amplified by reverse transcriptase PCR (RT-PCR). Then, electrophoresis was performed on a 2% agarose gel, and the expression level of the TNF-α gene was measured by image analysis using Image-J.

[0119] The results are shown in Figure 3. The TNF-α gene expression level in each sample was expressed as a relative value when the gene expression level in PBS was set to 1. When yeasts A to D, which were made by removing soluble components from yeast and drying it, were added, the level of TNF-α gene expression increased, with yeasts A and B in particular showing high values, even higher than the positive control, LPS.

[0120] These results demonstrated that the use of yeast cells from which soluble components have been removed has an enhancing effect not only on IL-12 but also on TNF-α, which are factors related to enhanced immunostimulatory activity.

[0121] Example 4 Ex vivo measurement of IL-12, TNF-α, and IFN-γ protein production using mouse spleen cells In this example, Sample 1: Yeast A + lactic acid bacteria + lactic acid bacteria medium (composition: yeast A 66%, lactic acid bacteria 1%, lactic acid bacteria medium 33%) prepared in Example 2 was added to spleen cells extracted from mice, and the amounts of IL-12, TNF-α, and IFN-γ proteins produced were measured.

[0122] (method) BALB / c mice (6 weeks old, female: purchased from Japan SLC) were pre-bred for one week to acclimate them to the environment. They were bred in individual cages in a breeding room with a 12-hour light / dark cycle. They were allowed to freely consume sterilized water prepared by autoclaving pure water (105°C, 30 minutes) as drinking water, and solid feed (CE-2: manufactured by CLEA Japan) as feed.

[0123] After preliminary breeding, the rats were anesthetized by isoflurane inhalation (induction: 3%, maintenance: 1.5%) and the spleens were harvested. The harvested spleens were placed in a petri dish containing 5-10 mL of Hank's balanced salt solution (HBSS), dispersed using a cell strainer, and stored on ice. The HBSS containing the spleen cells was centrifuged (300 g, 8 min) and the supernatant was removed. The precipitated spleen cells were dispersed in 1 mL of red blood cell lysis buffer and stirred for 1 min, after which 9 mL of HBSS was added and centrifuged under the same conditions as above. The procedure of dispersing in red blood cell lysis buffer and centrifuging was repeated until the red blood cells were completely lysed. After red blood cell lysis, the obtained spleen cells were adjusted in number using RPMI-1640 medium and placed in a 12-well cell culture plate at 2 × 10 per well. 7 The cells were seeded.

[0124] Sample 1 of Example 2 was added to the spleen cells to a final concentration of 1 mg / mL, and PBS was added to the medium at 20% for a negative control. The cells were incubated in a CO2 incubator for 48 hours, and the culture supernatant was collected after incubation. The protein production amounts of IL-12, TNF-α, and IFN-γ produced in the culture supernatant were measured using ELISA kits for each protein.

[0125] The results are shown in Figures 4-6. The significance test was performed using a Bonferroni-corrected t-test. ** = significance level 1%. It was revealed that sample 1 resulted in significantly higher protein production levels for all of IL-12, TNF-α, and IFN-γ, compared with the negative control PBS.

[0126] These results demonstrated that the use of yeast cells from which soluble components had been removed increased the levels of proteins related to immunostimulatory activity (IL-12, TNF-α, and IFN-γ) not only in J774.1 cells, a mouse-derived monocyte-macrophage-like cell line, but also in cells derived from spleen cells extracted from the living body.

[0127] Example 5: In vivo IL-12 protein production measurement test in mice In this example, the amount of IL-12 protein produced in vivo in mice administered the composition of the present invention was measured. Specifically, Sample 1: yeast A + lactic acid bacteria + lactic acid bacteria medium (composition: yeast A 66%, lactic acid bacteria 1%, lactic acid bacteria medium 33%) prepared in Example 2 was orally administered to mice, and the spleen of the mouse was removed after administration, and the amount of IL-12 protein produced in the spleen cells was measured. As a negative control, the amount of IL-12 protein produced in the spleen cells of mice orally administered PBS was also measured.

[0128] (method) BALB / c mice (6 weeks old, female: purchased from Japan SLC) were pre-bred for one week to acclimate them to the environment. They were bred in individual cages in a breeding room with a 12-hour light-dark cycle. They were allowed to freely consume sterilized water prepared by autoclaving pure water (105°C, 30 minutes) as drinking water, and solid feed (CE-2: manufactured by Japan CLEA) as feed. After pre-breding, the negative control group was given 0.5 mL PBS per mouse, and the sample administration group was given sample 1 suspended in 0.5 mL PBS at 500 mg / kg body weight, and these were orally administered once a day for four days. After four days of oral administration, the abdomen was opened under isoflurane inhalation anesthesia (induction: 3%, maintenance 1.5%), and the spleen was collected. The collected spleen was placed in a petri dish containing 5 to 10 mL of Hanks' balanced salt solution (HBSS), dispersed with a cell strainer, and incubated on ice for five minutes. The HBSS containing the spleen cells was centrifuged (300g, 8 minutes) and the supernatant was removed. The precipitated spleen cells were dispersed in 1 mL of red blood cell lysis buffer and stirred for 1 minute, after which 9 mL of HBSS was added and centrifuged under the same conditions as above. The procedure of dispersing in red blood cell lysis buffer and centrifuging was repeated until the red blood cells were completely lysed.

[0129] After erythrocyte lysis, the number of spleen cells obtained was adjusted using RPMI-1640 medium, and the cells were cultured in a 12-well cell culture plate at 2 × 10 cells per well. 7 The cells were seeded in a medium containing 2 mL of medium per well, and 4 μg of concanavalin A was added. The cells were incubated in a CO2 incubator for 48 hours. After incubation, the culture supernatant was collected, and the amount of IL-12 protein produced in the culture supernatant was measured using an ELISA kit.

[0130] The results are shown in Figure 7. Sample 1 resulted in a significant increase in the amount of IL-12 protein produced compared to the negative control (PBS). This result demonstrated that in addition to the effects on cultured cells as previously described, oral administration also increased the amount of IL-12 protein produced.

[0131] Example 6 In vitro measurement of IL-12 gene expression level using J774.1 cells (3) The expression levels of IL-12 gene in J774.1 cells were compared among the yeast cells A to D used in Example 1, a commercially available bifidobacteria product, a commercially available lactic acid bacteria product, and a β-glucan product. (Commercially available bifidobacteria, lactic acid bacteria and β-glucan products) The commercially available bifidobacteria products, lactic acid bacteria products, and β-glucan products used are as follows. All of these have been reported to have immune-stimulating effects and IL-12 production-inducing effects.

[0132] Commercial product A: Bifidobacterium longum Commercial product B: Lactic acid bacteria (Lactobacillus plantarum) Commercial product C: Lactic acid bacteria (Lactobacillus pentosus) Commercial product D: Lactic acid bacteria (Lactobacillus gasseri) Commercial product E: Yeast-derived water-soluble β-glucan (Processing of commercially available bifidobacteria and lactic acid bacteria products) Commercially available bifidobacteria and lactic acid bacteria products contain not only pure bacteria but also food ingredients such as dextrin, so these were removed through processing.

[0133] First, about 2.7g to 6g of each commercial product was placed in a 50ml tube, and 25ml of distilled water was added to disperse it. If necessary, vortexed until the commercial product was sufficiently dispersed and dissolved. The mixture was centrifuged at 9000rpm for 5 minutes, and the supernatant was removed. Each commercial product was freeze-dried and used for the test.

[0134] (method) J774.1 cells were plated in 12-well cell culture plates at 1.0 × 10 cells per well. 6 The medium was dispensed so that the cells would become 1 mL per well.

[0135] Next, the yeast cells A to D of Example 1 and the commercially available bifidobacteria products, lactic acid bacteria products, and β-glucan products A to E prepared above were each suspended in PBS to a concentration of 10 mg / mL. They were sterilized by heating at 100°C for 5 minutes, diluted with RPMI-1640 medium to a concentration of 1 mg / mL, and added to the cells.

[0136] In addition, RPMI-1640 medium supplemented with 20% PBS was used as a negative control, and lipopolysaccharide (LPS) (Sigma-Aldrich Co. Ltd., derived from E. coli. 055:B5 strain) was added to the cells at a final concentration of 100 ng / mL as a positive control.

[0137] After 3 hours of incubation in a CO2 incubator, the cells were detached by pipetting and collected. As in Example 1, total RNA was extracted from the collected cells, cDNA was synthesized, and the IL-12 p40 gene was amplified by reverse transcriptase PCR (RT-PCR). Then, the resultant was electrophoresed on a 2% agarose gel, and the expression level of the IL-12 p40 gene was measured by image analysis using Image-J.

[0138] The results are shown in Figure 8. Results showed that yeasts A, B, and D expressed higher amounts of IL-12 p40 gene than any of the commercially available products A to E. It was also confirmed that yeast C expressed a higher amount of IL-12 p40 gene than commercial product E, and had the same level of IL-12 p40 gene expression as commercial products A and C.

[0139] These results demonstrate that the use of yeast cells from which soluble components have been removed has an IL-12 production inducing effect equal to or greater than that of commercially available bifidobacteria products, lactic acid bacteria products, and β-glucan products, which have been reported to have immune-stimulating effects and IL-12 production inducing effects.

[0140] Furthermore, yeast cells from which soluble components have been removed from yeast have a mechanism for expressing an immunostimulatory effect that is different from that of lactic acid bacteria preparations, so they can be expected to be effective even when lactic acid bacteria are ineffective. When β-glucan is used, a process for increasing the purity, such as by extracting β-glucan, is required. In contrast, yeast cells from which soluble components have been removed from yeast have the advantage that they can be easily prepared by simply removing the soluble components from the yeast cells.

[0141] Example 7 Measurement of IL-12 p40 gene expression level in vitro using J774.1 cells (4) The expression level of the IL-12 p40 gene was measured when the yeast cells A to C of Example 1 and their soluble components were used, and when each yeast was used in an unextracted state. (method) First, J774.1 cells were plated in a 12-well cell culture plate at 1.0 × 10 cells per well. 6 The medium was dispensed so that the cells would become 1 mL per well.

[0142] Next, the yeast cells A to C and the soluble components A to C extracted from the yeast cells of Example 1, as well as the yeast cells A to C from which soluble components were not extracted, were each adjusted to a concentration of 10 mg / mL with PBS. They were sterilized by heating at 100°C for 5 minutes, diluted with RPMI-1640 medium to a final concentration of 1 mg / mL, and added to the cells.

[0143] In addition, RPMI-1640 medium supplemented with 20% PBS was used as a negative control, and lipopolysaccharide (LPS) (Sigma-Aldrich Co. Ltd., derived from E. coli. 055:B5 strain) was added to the cells at a final concentration of 100 ng / mL as a positive control.

[0144] After incubation in a CO2 incubator for 3 hours, the cells were detached by pipetting and collected. As in Example 1, total RNA was extracted from the collected cells, cDNA was synthesized, and the IL-12 p40 gene was amplified by reverse transcriptase PCR (RT-PCR). Then, the cells were electrophoresed on a 2% agarose gel, and the expression level of the IL-12 p40 gene was measured by image analysis using Image-J.

[0145] The results are shown in Figure 9. It was confirmed that the IL-12 p40 gene expression levels were generally higher in the cases where soluble components were not extracted (unextracted yeasts A to C) compared to the soluble components A to C. It was also revealed that for the unextracted yeasts A and B, the IL-12 p40 gene expression levels were higher when the soluble components were extracted, i.e., the yeast cells A and B.

[0146] These results indicate that yeast cells can induce IL-12 p40 gene expression to a certain extent even without removing soluble components, but that extraction treatment to remove soluble components induces a higher level of IL-12 p40 gene expression. [Industrial Applicability]

[0147] In the present invention, by using yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment as is, it is possible to obtain immunostimulating activity without special treatment such as autolysis or enzyme treatment. The present invention provides compositions, kits, combinations, methods, etc. for immunostimulation that are simpler and more effective than conventional ones. The present invention makes it possible to provide food and feed ingredients, etc., that have immunostimulating effects by using the immunostimulating compositions, etc.

Claims

1. A composition for immunostimulation comprising yeast cells from which soluble components have been removed without subjecting the yeast cells to autolysis or cell wall decomposition treatment.

2. The immunostimulatory composition according to claim 1, wherein the removal of the soluble components is performed by a method selected from the group consisting of hot water treatment, alkali treatment and protease treatment.

3. The immunostimulatory composition according to claim 1 , wherein the yeast cells are cells of a yeast selected from the group consisting of Torula yeast or a yeast species belonging to the genus Saccharomyces.

4. (1) Yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) A component derived from a living body having a health-maintaining function other than the yeast cells from which the soluble components have been removed; A composition for immunostimulation comprising:

5. The immunostimulatory composition according to claim 4, wherein the removal of the soluble components is performed by a method selected from the group consisting of hot water treatment, alkali treatment and protease treatment.

6. The composition according to claim 4 , wherein the health-maintaining function is a function attributable to a component produced by a physiologically active microorganism, algae or cell.

7. The composition according to claim 4, wherein the biologically derived component having a health-maintaining function is selected from the group consisting of lactic acid bacteria, bifidobacteria, butyric acid bacteria, and natto bacteria.

8. The composition according to claim 4 , wherein the component derived from a living organism and having a health-maintaining function is a lactic acid bacterium.

9. The composition according to claim 4, further comprising nutritional components of biological origin having said health-maintaining function.

10. The composition according to claim 9 , wherein the nutritional component of the component derived from a living organism having a health-maintaining function is a culture medium for the component derived from a living organism having a health-maintaining function.

11. The composition of claim 1 or 4, which does not contain an antibacterial agent.

12. A combination for stimulating the immune system includes (1) yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) a component derived from a living organism having a health-maintaining function other than the yeast cells from which the soluble components have been removed.

13. A kit for immunostimulation, comprising yeast cells from which soluble components have been removed without autolysis or cell wall decomposition.

14. (1) Yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) A component derived from a living body having a health-maintaining function other than the yeast cells from which the soluble components have been removed; A kit for immunostimulation comprising:

15. A method for activating the immune system by administering to a subject yeast cells from which soluble components have been removed without subjecting the yeast cells to autolysis or cell wall decomposition treatment.

16. (1) Yeast cells from which soluble components have been removed without autolysis or cell wall decomposition treatment, and (2) A component derived from a living body having a health-maintaining function other than the yeast cells from which the soluble components have been removed; A method of administering this to a subject to activate the immune system.

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