Novel yeast with immunostimulatory properties

Abstract

This provides a novel material with immunostimulatory properties. [Solution] An immunostimulant comprising the cells of Pichia yeast or Torraspora yeast as an active ingredient; an immunostimulant food composition, an immunostimulant pharmaceutical composition, or an immunostimulant cosmetic composition comprising the immunostimulant; Pichia yeast with receipt number NITE AP-04248; Pichia yeast with receipt number NITE AP-04249; Torraspora yeast with receipt number NITE AP-04250; Torraspora yeast with receipt number NITE AP-04251.

Description

Technical Field

[0001] The present invention relates to a novel yeast having immunostimulatory activity.

Background Art

[0002] In recent years, the average lifespan of Japanese people has been increasing, and Japan has entered a rapidly aging society. On the other hand, as people age, their immunity declines, increasing the probability of contracting infectious diseases. To address the aging society, extending the healthy lifespan has become an issue for the future, and as one solution, the development of functional foods that contribute to extending the healthy lifespan has been urgently needed. For example, products using lactic acid bacteria materials with immunostimulatory activity have been developed (see, for example, Patent Document 1). In recent years, food materials have been applied not only to food compositions but also to pharmaceutical compositions and cosmetic compositions.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a new material having immunostimulatory activity.

Means for Solving the Problems

[0005] The present invention includes the following aspects. [[ID=4s3]] [1] An immunostimulant containing cells of yeast belonging to the genus Pichia or Torulaspora as an active ingredient. [2] The immunostimulant according to [1], wherein the cells are disrupted. [3] The immunostimulant according to [1] or [2], wherein the yeast belonging to the genus Pichia is yeast with an accession number of NITE AP-04248 or NITE AP-04249. [4] The immunostimulant according to any one of [1] to [3], wherein the toruspora yeast is a yeast with receipt number NITE AP-04250 or NITE AP-04251. An immunostimulant food composition, immunostimulant pharmaceutical composition, or immunostimulant cosmetic composition comprising an immunostimulant described in any of [5][1] to [4]. [6] Pichia yeast with receipt number NITE AP-04248. [7] Pichia yeast with receipt number NITE AP-04249. [8] Torraspora yeast with receipt number NITE AP-04250. [9] Torraspora yeast with receipt number NITE AP-04251. [Effects of the Invention]

[0006] According to the present invention, it is possible to provide a novel material having immunostimulatory properties. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 is a graph showing the measurement results of IFN-α in Experimental Example 2. [Figure 2] Figure 2 is a graph showing the measurement results of IFN-γ in Experiment Example 3. [Figure 3] Figure 3 is a graph showing the measurement results of IL-12 in Experiment Example 4. [Figure 4] Figure 4 shows a representative fluorescence microscope image from Experimental Example 5. [Figure 5] Figure 5 is a graph showing the results of Experiment Example 5. [Modes for carrying out the invention]

[0008] [Immunostimulant] In one embodiment, the present invention provides an immunostimulant containing the cells of a Pichia or Torraspora yeast as an active ingredient. As will be described later in the examples, the immunostimulant of this embodiment is of natural origin and has immunostimulatory activity equivalent to or better than NBRC 100933 (Lactococcus lactis subsp. lactis NBRC 100933), a lactic acid bacteria strain known to have immunostimulatory activity.

[0009] In this specification, immunostimulation refers to activating various immune functions, such as the innate immune system and the adaptive immune system. The immunostimulant of this embodiment only needs to activate any of these immune functions.

[0010] The immunostimulant of this embodiment is primarily intended to activate the innate immune system. However, as a result of the activation of the innate immune system, macrophages, natural killer cells, dendritic cells, etc., may produce cytokines, which could further activate the adaptive immune system.

[0011] As described later in the examples, the immunostimulant of this embodiment induces cytokine production in dendritic cells or immune cells. Examples of cytokines include type I interferons such as IFN-α, type II interferons such as IFN-γ, and interleukins such as IL-12. The immunostimulant of this embodiment has cytokine production induction ability equivalent to or greater than that of known immunostimulant lactic acid bacteria. Therefore, the immunostimulant of this embodiment can also be referred to as a cytokine production inducer, type I interferon production inducer, type II interferon production inducer, interleukin production inducer, etc.

[0012] As will be described later in the examples, the immunostimulant of this embodiment also increases the phagocytic activity of macrophages. The immunostimulant of this embodiment has a higher ability to increase the phagocytic activity of macrophages compared to known lactic acid bacteria that have immunostimulant effects. Therefore, the immunostimulant of this embodiment can also be described as a macrophage phagocytic activity enhancer, etc.

[0013] The immunostimulant of the present embodiment contains, as an active ingredient, the cells of yeast belonging to the genus Pichia or yeast belonging to the genus Torulaspora. The cells may be live cells or dead cells. When used as dead cells, they may be sterilized and inactivated by an autoclave or the like. The live cells or dead cells may be dried products. The drying of the cells can be carried out, for example, by freeze-drying, spray-drying or the like.

[0014] In the immunostimulant of the present embodiment, the cells of yeast belonging to the genus Pichia or yeast belonging to the genus Torulaspora may be disrupted. The cells may be those obtained by fractionating the disrupted cells by centrifugation or the like. The cells may be the precipitate when the disrupted cells are centrifuged. The precipitate is considered to be a fraction containing components mainly derived from the cell wall. The cells may be the supernatant when the disrupted cells are centrifuged. The supernatant is considered to be a fraction containing components mainly derived from the cytoplasm. The cells may be a mixture of the precipitate and the supernatant when the disrupted cells are centrifuged. Alternatively, the disrupted cells may be used without fractionation. The cells may be a processed product obtained by physical or chemical treatment. Examples of the processed product include those obtained by enzymatically treating the cells and those obtained by heat-treating the cells.

[0015] The yeast belonging to the genus Pichia or yeast belonging to the genus Torulaspora may be cultured in a normal medium. Examples of the medium include YPD medium, YM medium, PD medium and the like. After culturing, the cells may be collected by centrifugation or filtration and used.

[0016] In the immunostimulant of the present embodiment, the yeast belonging to the genus Pichia is preferably yeast with an accession number of NITE AP-04248 or NITE AP-04249. The yeast belonging to the genus Pichia with an accession number of NITE AP-04248 is the Pichia rarasimilans UT-2214 strain described later in the examples. The yeast belonging to the genus Pichia with an accession number of NITE AP-04249 is the Pichia rarasimilans UT-2216 strain described later in the examples.

[0017] In the immunostimulant of the present embodiment, the yeast of the genus Torulaspora is preferably yeast with an accession number of NITE AP-04250 or NITE AP-04251. The yeast of the genus Torulaspora with an accession number of NITE AP-04250 is the Torulaspora delbrueckii UT-222 strain described later in the examples. The yeast of the genus Torulaspora with an accession number of NITE AP-04251 is the Torulaspora delbrueckii UT-3058 strain described later in the examples.

[0018] [Food composition for immunostimulation] In one embodiment, the present invention provides a food composition for immunostimulation, which contains the above-described immunostimulant. By ingesting the food composition for immunostimulation of the present embodiment, it is expected to improve the immune function of the subject. The subject may be a human or a non-human animal.

[0019] When the subject is a non-human animal, the food composition for immunostimulation of the present embodiment can be referred to as a feed for immunostimulation. Examples of non-human animals include pets, livestock, and cultured fish. Examples of pets include cats, dogs, rabbits, etc. Examples of livestock include cows, horses, pigs, sheep, goats, chickens, etc. Examples of cultured fish include fish of the family Tetraodontidae (pufferfish, tiger pufferfish, etc.), the family Pleuronectidae (flatfish, halibut, etc.), the family Salmonidae (king salmon, red salmon, silver salmon, rainbow trout, iwana, etc.), the family Plecoglossidae (ayu, smelt, shishamo, etc.), the family Anguillidae (Japanese eel, European eel, etc.), the family Seriolae (amberjack, yellowtail, sea bass, etc.).

[0020] The intake amount of the food composition for immunostimulation of the present embodiment can be appropriately determined according to the symptoms, body weight, age, gender, etc. of the subject, and it may be ingested once a day or divided into about 2 to 4 times a day. For example, in the case of humans, for adults, 1×10 9 ~1×10 12You should ingest an amount of bacterial cells equivalent to a single cell. Alternatively, an adult should ingest 1 to 1000 mg of bacterial cells per day on a dry weight basis.

[0021] The food composition of this embodiment may be in the form of a supplement, a beverage, a solid, semi-solid, or gel-like food, or any other form of cooked food. Examples of supplement forms include capsules.

[0022] Note that "1 x 10 per day" 9 ~1 × 10 12 The terms "an amount equivalent to one cell" and "1 to 1000 mg in dry weight" refer to the amount contained in a single serving, which varies depending on the form of the food composition, but are based on the recommended daily intake indicated on the label, or, in the case of a form that is usually consumed in one sitting, the amount contained in a single serving.

[0023] The food composition of this embodiment may be a functional food. A "functional food" refers to a food in which a business operator has submitted scientific evidence regarding the safety and functionality of the food to the Consumer Affairs Agency in accordance with rules established by the government. Examples of such claims include, but are not limited to, "Helps maintain the immune function of healthy people."

[0024] The food composition of this embodiment may be a food for special dietary uses. A food for special dietary uses is a food that has been approved by the government and is labeled with a special purpose, such as being suitable for the development of infants, or for maintaining and restoring the health of pregnant women, breastfeeding women, people with swallowing difficulties, or sick people.

[0025] Foods for special dietary uses include foods for sick people, powdered milk for pregnant and lactating women, infant formula, foods for people with swallowing difficulties, and foods for specified health uses. The food composition of this embodiment may be any of these.

[0026] Foods for Specified Health Uses (FOSHU) are foods that contain health-promoting ingredients (active ingredients) that affect the body's physiological functions, and that are labeled with a statement indicating that their consumption can be expected to achieve a specific health purpose. In order to sell a food as a FOSHU, each food product must undergo a review by the government regarding its effectiveness and safety, and receive approval.

[0027] Furthermore, even if a product is not classified as a functional food or a food for special dietary uses, it is possible to manufacture and sell it while promoting its functionality through websites, social networking services (SNS), flyers, or verbal announcements.

[0028] [Immunostimulant Pharmaceutical Composition] In one embodiment, the present invention provides an immunostimulatory pharmaceutical composition comprising the immunostimulant described above. By administering the immunostimulatory pharmaceutical composition of this embodiment to a patient, the patient's immune function can be improved.

[0029] The immunostimulant pharmaceutical composition of this embodiment may contain the above-mentioned immunostimulant and a pharmaceutically acceptable carrier. The pharmaceutical composition of this embodiment can be administered orally, for example, in the form of tablets, capsules, elixirs, microcapsules, etc., or parenterally, in the form of injections, suppositories, topical preparations, etc. More specifically, topical preparations include dosage forms such as ointments and patches.

[0030] Pharmaceutically acceptable carriers can be those commonly used in the formulation of pharmaceutical compositions without particular limitations. More specifically, examples include binders such as dimethylcellulose, polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, gelatin, hydroxypropylcellulose, polyvinylpyrrolidone, corn starch, tragacanth gum, and gum arabic; excipients such as glucose, lactose, corn starch, sorbitol, and crystalline cellulose; leavening agents such as alginic acid; disintegrants such as starch, sodium alginate, calcium carbonate, calcium citrate, and dextrin; lubricants such as talc, magnesium stearate, polyethylene glycol, and hydrogenated vegetable oil; solvents for injections such as water, ethanol, and glycerin; and adhesives such as rubber-based adhesives and silicone-based adhesives.

[0031] Pharmaceutical compositions may contain additives. Examples of additives include sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and red ginger oil; stabilizers such as benzyl alcohol and phenol; buffering agents such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives.

[0032] The dosage of the pharmaceutical composition can be appropriately determined based on the patient's symptoms, weight, age, sex, etc., and should be administered once a day or in 2 to 4 divided doses. For example, 1 x 10 per day for an adult. 9 ~1 × 10 12 Administering a quantity of bacterial cells equivalent to the number of individual cells is sufficient. Alternatively, administering 1 to 1000 mg of bacterial cells per day (based on dry weight) is appropriate for adults.

[0033] [Immunotherapy Cosmetic Composition] In one embodiment, the present invention provides an immunostimulatory cosmetic composition comprising the immunostimulant described above. By using the immunostimulatory cosmetic composition of this embodiment, it is expected that the immune function of the target will be improved.

[0034] The immunostimulatory cosmetic composition of this embodiment may contain the above-mentioned immunostimulators and additives commonly used in the field of cosmetics. Examples of additives include humectants, preservatives, glossing agents, antioxidants, pigments, thickeners, pH adjusters, fragrances, antibacterial agents, various oily components, UV absorbers, free radical scavengers, antioxidants, minerals, amino acids, and known whitening ingredients.

[0035] The dosage form of the immunostimulatory cosmetic composition of this embodiment is not limited as long as it has properties that can be applied to the skin, and examples include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, sprays, and the like.

[0036] [yeast] In one embodiment, the present invention provides a Pichia yeast whose certification number is NITE AP-04248.

[0037] In one embodiment, the present invention provides a Pichia yeast whose certification number is NITE AP-04249.

[0038] In one embodiment, the present invention provides a Torraspora yeast whose certification number is NITE AP-04250.

[0039] In one embodiment, the present invention provides a Torraspora yeast whose certification number is NITE AP-04251.

[0040] As described later in the examples, these yeast cells induce cytokine production in immune cells and dendritic cells at a level equivalent to or greater than that of known immunostimulant lactic acid bacteria. Furthermore, as described later in the examples, these yeast cells have a higher ability to enhance macrophage phagocytosis compared to known immunostimulant lactic acid bacteria. Therefore, these yeast cells can be used as immunostimulants. [Examples]

[0041] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0042] [Experimental Example 1] (Collection of natural yeast) Various plant samples were collected in spring and summer in Kamakura City, Kanagawa Prefecture, and on the Yayoi Campus of the University of Tokyo. A total of 65 different plant samples were collected.

[0043] The collected plants were placed in YPD medium in a 50 mL tube and cultured anaerobically at 30°C for 10 days (primary culture). Subsequently, the culture from the primary culture was placed in a fresh 15 mL tube of YPD medium and cultured anaerobically at 30°C for 10 days (secondary culture). After the secondary culture, sediment was observed at the bottom of the tube, indicating that yeast was growing in the culture medium.

[0044] Next, the cultures after secondary culture were seeded onto YPD agar and cultured anaerobically at 30°C for 1-3 days. Then, colonies were isolated and placed in fresh YPD medium in 15 mL tubes and cultured anaerobically at 30°C for 10 days. A total of 1044 colonies were isolated. Next, the tubes were centrifuged at 8,000 × g for 8 minutes, the excess supernatant was discarded, and the contents were transferred to storage tubes with 2D barcodes. Subsequently, they were pre-frozen in liquid nitrogen. Finally, they were freeze-dried at -100°C for 24 hours. The freeze-dried yeast was stored at room temperature.

[0045] The immunostimulatory activity of the obtained yeast strains was evaluated, and the UT-2214, UT-2216, UT-2226, and UT-3058 strains, which demonstrated immunostimulatory activity, were deposited on December 26, 2024, with receipt numbers NITE AP-04248, NITE AP-04249, NITE AP-04250, and NITE AP-04251, respectively, at the Patent Organism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan) (Principal depositor name: Koji Nagata, Principal depositor address: 7-3-1 Hongo, Bunkyo-ku, Tokyo, within the University of Tokyo).

[0046] Strain UT-2214 was a yeast derived from dandelion, strain UT-2216 was a yeast derived from ajuga, strain UT-2226 was a yeast derived from muscari, and strain UT-3058 was a yeast derived from ajuga.

[0047] Based on the nucleotide sequences of the D1 / D2 domains and the internal transcribed spacer region (ITS region) of the ribosomal DNA (rDNA), it was estimated that strain UT-2214 is Pichia rarasimilans, strain UT-2216 is Pichia rarasimilans, strain UT-2226 is Tolulaspora delbrueckii, and strain UT-3058 is Tolulaspora delbrueckii.

[0048] [Experimental Example 2] (Measurement of IFN-α expression induction ability in dendritic cells) A bacterial cell extract was prepared from the yeast collected in Experimental Example 1, and its ability to induce IFN-α expression was measured when added to the culture medium of dendritic cells.

[0049] Preparation of bacterial cell extracts Cellular extracts were prepared from the yeast collected in Experimental Example 1. The yeast strains used were UT-2214, UT-2216, UT-2226, and UT-3058. For comparison, a lactic acid bacterium known to possess immunostimulatory properties was also used. The immunostimulatory lactic acid bacterium used was NBRC 100933 (Lactococcus lactis subsp. lactis NBRC 100933), obtained from the National Institute of Technology and Evaluation.

[0050] Each yeast cell and lactic acid bacteria was suspended in phosphate-buffered saline (PBS). A suspension containing at least 10 mg (dry weight) of cells was ground twice at 4,000 rpm for 15 seconds each using a bead mill (catalog number "BHA-6", AS ONE) and 3 mm diameter zirconia beads.

[0051] Next, a portion of the pulverized material was taken, centrifuged, and separated into supernatant and precipitate. PBS was added to the precipitate to adjust the dry mass of the bacterial cells to 100 mg / mL (hereinafter sometimes referred to as "bacterial cells (precipitate)"). PBS was also added to the supernatant to adjust the dry mass of the bacterial cells to 100 mg / mL (hereinafter sometimes referred to as "bacterial cells (supernatant)").

[0052] Furthermore, PBS was added to the remaining pulverized material to adjust the dry mass of the bacterial cells to 100 mg / mL (hereinafter sometimes referred to as "bacterial cells (unisolated material)").

[0053] Preparation of dendritic cells Human monocyte cell line THP-1 was differentiated into dendritic cells. THP-1 cell culture medium was supplemented with 20 nM Phorbol 12-myristate 13-acetate (PMA) and 10 nM vitamin D3, and incubated for 48 hours. As a result, the cells differentiated into macrophages. Subsequently, the medium was replaced with a medium containing 20 nM IL-4, and incubated for another 48 hours. As a result, the cells differentiated into dendritic cells.

[0054] Addition of bacterial cell extract to dendritic cells Cellular extracts (supernatant, precipitate, and unseparated material) were added to the dendritic cell culture medium to a final concentration of 1 mg / mL, and incubated at 37°C under 5% CO2 conditions for 24 hours. Subsequently, IFN-α in the culture medium was quantified by ELISA.

[0055] Figure 1 is a graph showing the measurement results for IFN-α. In Figure 1, "PBS" shows the results for dendritic cells without bacterial cell addition, "Supernatant" shows bacterial cells (supernatant), "Precipitate" shows bacterial cells (precipitate), "Unisolated" shows bacterial cells (unisolated), "NBRC 100933" shows the lactic acid bacterium strain NBRC 100933, and "UT-2214", "UT-2216", "UT-2226", and "UT-3058" show yeast strains UT-2214, UT-2216, UT-2226, and UT-3058, respectively. The values ​​in the graph represent the mean ± standard deviation (n=3).

[0056] As a result, when bacterial cell extracts (supernatant) were added to the culture medium of dendritic cells, strains UT-2214, UT-2216, UT-2226, and UT-3058 showed higher IFN-α induction ability than Lactobacillus NBRC 100933 strain. When bacterial cell extracts (precipitation) were added to the culture medium of dendritic cells, strains UT-2214 and UT-2226 showed higher IFN-α induction ability than Lactobacillus NBRC 100933 strain. When bacterial cell extracts (unisolated) were added to the culture medium of dendritic cells, strains UT-2214, UT-2226, and UT-3058 showed higher IFN-α induction ability than Lactobacillus NBRC 100933 strain, which is known to have immunostimulatory activity.

[0057] [Experimental Example 3] (Measurement of IFN-γ expression induction ability in dendritic cells) Cellular extracts were added to dendritic cell culture media prepared in the same manner as in Experimental Example 2 to a final concentration of 1 mg / mL, and incubated at 37°C in a 5% CO2 environment for 24 hours. Subsequently, IFN-γ in the culture media was quantified by ELISA.

[0058] For bacterial cell extracts, we used extracts from yeast strains UT-2214, UT-2216, UT-2226, and UT-3058. For comparison, we also used a bacterial cell extract from lactic acid bacterium strain NBRC 100933. For the bacterial cell extracts, we used the cell supernatant. We also prepared samples (PBS) without the addition of bacterial cell extracts.

[0059] Figure 2 is a graph showing the measurement results of IFN-γ. In Figure 2, "PBS" indicates the results for dendritic cells to which the same volume of PBS was added instead of bacterial cells, "NBRC 100933" refers to the lactic acid bacterium strain NBRC 100933, and "UT-2214", "UT-2216", "UT-2226", and "UT-3058" refer to yeast strains UT-2214, UT-2216, UT-2226, and UT-3058, respectively. The values ​​in the graph represent the mean ± standard deviation (n=3).

[0060] As a result, it was revealed that strains UT-2214, UT-2216, UT-2226, and UT-3058 exhibited higher IFN-γ induction ability than Lactobacillus NBRC 100933 strain, which is known to possess immunostimulatory properties.

[0061] [Experimental Example 4] (Measurement of IL-12 expression induction ability in dendritic cells) Cellular extracts were added to dendritic cell culture media prepared in the same manner as in Experimental Example 2 to a final concentration of 1 mg / mL, and incubated at 37°C in a 5% CO2 environment for 24 hours. Subsequently, IL-12 in the culture media was quantified by ELISA.

[0062] For bacterial cell extracts, we used extracts from yeast strains UT-2214, UT-2216, UT-2226, and UT-3058. For comparison, we also used a bacterial cell extract from lactic acid bacterium strain NBRC 100933. For the bacterial cell extracts, we used the cell supernatant. We also prepared samples (PBS) without the addition of bacterial cell extracts.

[0063] Figure 3 is a graph showing the measurement results for IL-12. In Figure 3, "PBS" indicates the results for dendritic cells to which the same volume of PBS was added instead of bacterial cells, "NBRC 100933" refers to the lactic acid bacterium strain NBRC 100933, and "UT-2214," "UT-2216," "UT-2226," and "UT-3058" refer to yeast strains UT-2214, UT-2216, UT-2226, and UT-3058, respectively. The values ​​in the graph represent the mean ± standard deviation (n=3).

[0064] As a result, it was revealed that strains UT-2214, UT-2216, UT-2226, and UT-3058 exhibited higher IL-12 induction ability than Lactobacillus NBRC 100933 strain, which is known to possess immunostimulatory properties.

[0065] [Experimental Example 5] (Measurement of macrophage phagocytic activity) We investigated the effect of adding bacterial cell extracts to macrophage culture media to enhance the phagocytic activity of macrophages.

[0066] Preparation of macrophages Human monocyte cell line THP-1 was differentiated into macrophages. THP-1 cell culture medium was mixed with 20 nM Phorbol 12-myristate 13-acetate (PMA) and 10 nM vitamin D3, and incubated for 48 hours. As a result, the cells differentiated into macrophages.

[0067] Addition of bacterial cell extract to macrophages A final concentration of 1 mg / mL of bacterial cell extract and a final concentration of 2 μg / mL of pHrodo red zymosan (Thermo Fisher Scientific) were added to macrophage culture medium, and incubated at 37°C in a 5% CO2 environment for 30 minutes. pHrodo red zymosan is nonfluorescent outside the cell, but emits red fluorescence when phagocytosed by macrophages and moved into phagosomes.

[0068] For bacterial cell extracts, we used extracts from yeast strains UT-2214, UT-2216, UT-2226, and UT-3058. For comparison, we also used a bacterial cell extract from lactic acid bacterium strain NBRC 100933. For the bacterial cell extracts, we used the cell supernatant. We also prepared a sample (PBS) without the addition of bacterial cell extracts.

[0069] Next, Hoechst33342 (Dojin Chemical Research Institute) at a final concentration of 33.3 μg / mL was added to the macrophage culture medium, and the fluorescence of Hoechst33342 and pHrodo red zymosan was observed using a fluorescence microscope. Hoechst33342 stains the nucleus.

[0070] Figure 4 shows a typical fluorescence microscope image. The scale bar represents 10 μm. In Figure 4, "PBS" indicates the results of macrophages to which the same volume of PBS was added instead of bacterial cells; "NBRC 100933" indicates the lactic acid bacterium strain NBRC 100933; and "UT-2214", "UT-2216", "UT-2226", and "UT-3058" indicate yeast strains UT-2214, UT-2216, UT-2226, and UT-3058, respectively. "xy" indicates an image in the xy plane, "yz" indicates an image in the yz plane, and "xz" indicates an image in the xz plane.

[0071] Figure 5 shows the average of three fields of view obtained by measuring the integrated value of red fluorescence in cells at 20x magnification. In Figure 5, "PBS" indicates the results for macrophages to which the same volume of PBS was added instead of bacterial cells, "NBRC 100933" refers to the lactic acid bacterium strain NBRC 100933, and "UT-2214", "UT-2216", "UT-2226", and "UT-3058" refer to yeast strains UT-2214, UT-2216, UT-2226, and UT-3058, respectively.

[0072] As a result, when bacterial cell extracts (supernatant) were added to macrophage culture medium, strains UT-2214, UT-2216, UT-2226, and UT-3058 showed a higher effect in promoting macrophage phagocytosis than Lactobacillus NBRC 100933, which is known to have immunostimulatory activity. [Industrial applicability]

[0073] According to the present invention, it is possible to provide a novel material having immunostimulatory properties. (Receipt No.)

[0074] NITE AP-04248 NITE AP-04249 NITE AP-04250 NITE AP-04251

Claims

1. An immunostimulant containing the cells of Pichia or Torraspora yeasts as an active ingredient.

2. The immunostimulant according to claim 1, wherein the bacterial cells are pulverized.

3. The immunostimulant according to claim 1, wherein the Pichia yeast is a yeast with receipt number NITE AP-04248 or NITE AP-04249.

4. The immunostimulant according to claim 1, wherein the Torraspora yeast is a yeast with receipt number NITE AP-04250 or NITE AP-04251.

5. An immunostimulant food composition, an immunostimulant pharmaceutical composition, or an immunostimulant cosmetic composition comprising an immunostimulant according to any one of claims 1 to 4.

6. Pichia yeast with receipt number NITE AP-04248.

7. Pichia yeast with receipt number NITE AP-04249.

8. Torraspora yeast with receipt number NITE AP-04250.

9. Torraspora yeast with receipt number NITE AP-04251.

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