IL-10 production-inducing activator, anti-inflammatory agent, regulatory T cell production-inducing activator, and immunosuppressant for hyperimmune disease

Abstract

This invention provides novel applications for Lentinctobacillus cosonis, including an IL-10 production inducer activator, an anti-inflammatory agent, a regulatory T cell production inducer activator, and an immunosuppressant for diseases with hyperimmune responses. [Solution] An IL-10 production induction activator containing at least one of the bacterial cells or bacterial cell treatment products of lactic acid bacteria belonging to Lentilactobacillus kosonis as an active ingredient, which activates the induction of interleukin-10 (IL-10) production in dendritic cells. Also an anti-inflammatory agent that activates the anti-inflammatory properties of the body. Also a regulatory T cell production induction activator containing at least one of the bacterial cells or bacterial cell treatment products of lactic acid bacteria belonging to Lentilactobacillus kosonis as an active ingredient, which activates the induction of regulatory T cell (Treg) production. Also an immune hyperreaction disease suppressant that suppresses diseases caused by excessive immune responses in the body by activating the induction of regulatory T cell production.

Description

[Technical Field]

[0001] This invention relates to an IL-10 production-inducing activator, an anti-inflammatory agent, a regulatory T cell production-inducing activator, and an immunosuppressant for diseases using Lentilactobacillus kosonis. [Background technology]

[0002] Health drinks made primarily from enzyme liquid produced by fermenting and aging raw materials such as vegetables, fruits, and seaweed are known to have the effect of boosting immunity and improving physical condition. Vegetable brown sugar fermented liquid (for example, the product name "Georina® Enzyme") is an enzyme drink obtained by natural fermentation by a complex community of microorganisms using raw materials such as unrefined sugar, leafy vegetables, root vegetables, brown sugar, galactooligosaccharides, stem vegetables, potatoes, mushrooms, flowering vegetables, kelp, and wakame. It has been reported that the microbial flora that appears and disappears during the fermentation process of this vegetable brown sugar fermented liquid is dominated by lactic acid bacteria in a short period of time due to natural fermentation, that the microbial flora pattern is stable from batch to batch, and that there is a change in bacterial species even among lactic acid bacteria (see, for example, Non-Patent Literature 1).

[0003] The inventors of this invention, while conducting research on fermented vegetable brown sugar liquid, have discovered novel strains of useful lactic acid bacteria, such as Apilactobacillus kosoi 10H strain (see, for example, Patent Document 1) and Lentilactobacillus kosonis C06_No.73 strain (see, for example, Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Tai-Ying Chiou et al., “Changes in the bacterial community in the fermentation process of koso, a Japanese sugar-vegetable fermented beverage”, Biosci Biotechnol Biochem., The Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2017, 81(2), 403-410. [Patent Documents]

[0005] [Patent Document 1] Patent No. 7362081 [Patent Document 2] Patent No. 7179343 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Incidentally, the inventors of this invention have found that Lenticularbacillus cousonis strain C06_No.73 can be used as an IgA production promoter and an immunostimulant (see Patent Document 2). However, the Lenticularbacillus cousonis species itself was registered as a new species upon the discovery of Lenticularbacillus cousonis strain C06_No.73, and many aspects of the useful effects of Lenticularbacillus cousonis remain unclear.

[0007] This invention has been made in view of the above circumstances, and aims to provide a novel application for Lentilactobacillus cosonis: an IL-10 production inducing activator and an anti-inflammatory agent. [Means for solving the problem]

[0008] The inventors of the present invention have discovered that lactic acid bacteria belonging to Lentractobacillus cousonis activate the induction of dendritic cell interleukin-10 (IL-10) production and the induction of regulatory T cell (Treg) production, and have completed the present invention. The present invention includes the following embodiments.

[0009] (1) An IL-10 production induction activator containing at least one of the bacterial cells or bacterial cell treatment products of a lactic acid bacterium belonging to Lentilactobacillus kosonis as an active ingredient, which activates the induction of interleukin-10 (IL-10) production in dendritic cells. (2) The lactic acid bacterium is the IL-10 production inducing activator described in (1) above, which is Lentinctobacillus cosonis C06 No. 73 strain. (3) The IL-10 production-inducing activator described in (1) or (2) above, in the form of food or beverages, pharmaceuticals, animal feed, or active ingredient compositions to be incorporated therein. (4) An anti-inflammatory agent containing at least one of the bacterial cells or bacterial cell treatment products of a lactic acid bacterium belonging to Lentilactobacillus kosonis as an active ingredient, which activates the anti-inflammatory properties of the body by activating the induction of interleukin-10 (IL-10) production in dendritic cells. (5) The lactic acid bacterium is Lentinctobacillus cosonis C06 No. 73 strain, which is the anti-inflammatory agent described in (4) above. (6) The anti-inflammatory agent described in (4) or (5) above, in the form of food or beverage, pharmaceuticals, animal feed, or active ingredient compositions to be incorporated therein. (7) A regulatory T cell production induction activator containing at least one of the bacterial cells or bacterial cell treatment products of a lactic acid bacterium belonging to Lentilactobacillus kosonis as an active ingredient, which activates the induction of regulatory T cell (Treg) production. (8) The lactic acid bacterium is Lentilactobacillus cosonis C06 No. 73 strain, which is the regulatory T cell production inducing activator described in (7) above. (9) A regulatory T cell production inducing activator according to (7) or (8) above, in the form of food or beverage, pharmaceutical, animal feed, or an active ingredient composition to be incorporated therein. (10) An immunosuppressant containing at least one of the bacterial cells or bacterial cell treatment products of a lactic acid bacterium belonging to Lentilactobacillus kosonis as an active ingredient, which suppresses diseases caused by an excessive immune response in the body by activating the induction of regulatory T cell (Treg) production. (11) The lactic acid bacterium is Lentinctobacillus cosonis C06 No. 73 strain, which is the immunosuppressant for hyperimmune disease described in (10) above. (12) An immunosuppressant for hyperimmune disease as described in (10) or (11) above, in the form of food or beverage, pharmaceuticals, animal feed, or an active ingredient composition to be incorporated therein. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide new applications for Lentinctobacillus cousonis, such as an IL-10 production-inducing activator, an anti-inflammatory agent, a regulatory T cell production-inducing activator, and an immunosuppressant for diseases with hyperimmune reactions. [Brief explanation of the drawing]

[0011] [Figure 1] This bar graph shows the results of gene expression analysis for dendritic cells cultured with lactic acid bacteria in Example 1. [Figure 2] This bar graph shows the measurement results of secreted cytokine levels in dendritic cells cultured with lactic acid bacteria in Example 1. [Figure 3] This box plot shows the results of the analysis of the induced production of regulatory T cells (CD4-positive Foxp3-positive cells) in mouse spleen cells cultured with lactic acid bacteria in Example 2. [Modes for carrying out the invention]

[0012] Hereinafter, embodiments of the present invention will be described. Note that the embodiments described below do not limit the invention according to the claims, and not all of the elements and combinations thereof described in the embodiments are essential for the solution means of the present invention.

[0013] (I) Lactic acid bacteria belonging to Lentilactobacillus kisonensis, cells, and cell-treated products The active ingredient in one embodiment of the present invention is at least one of cells and cell-treated products of lactic acid bacteria belonging to Lentilactobacillus kisonensis. Lentilactobacillus kisonensis is a lactic acid bacterium belonging to the genus Lentilactobacillus of the family Lactobacillaceae (Lactic acid bacteria family) in the order Lactobacillales. More preferably, the above lactic acid bacterium is Lentilactobacillus kisonensis C06_No.73 strain or a mutant strain thereof.

[0014] The "cells" in this specification include both live cells and dead cells. The cells may be frozen or dried. In addition, the "cell-treated product" in this specification refers to a product containing components derived from cells obtained by performing a treatment for destroying cells or a treatment for extracting components from cells. The cell-treated product includes not only those containing part or all of the cells, such as cytoplasm and cell wall components obtained by treating the cells by physical means, but also those not containing the cells themselves, such as extracts from the cells and culture supernatants.

[0015] The "mutant strain" in this specification means those obtained by mutating a specific strain by a method well known to those skilled in the art within a range not affecting its main properties, or those that those skilled in the art can confirm to be equivalent thereto.

[0016] (II) Lentilactobacillus kisonensis C06_No.73 strain Lentractobacillus cousonis strain C06_No.73 was deposited with the National Institute of Technology and Evaluation (NITE), Patent Microorganism Depository Center (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) on November 16, 2021 (original deposit date). The accession number is NITE P-03559. Hereafter, this strain may be simply referred to as "C06_No.73 strain". The NBRC number for C06_No.73 strain is "NBRC 111893", and this number is also shown in Figure 3 below.

[0017] Strain C06_No.73 is the first strain obtained from a vegetable brown sugar fermentation liquid produced at the Arsoa Saku Factory in Saku City, Nagano Prefecture, Japan. Strain C06_No.73 is Gram-positive and catalase-negative, and observations using a scanning electron microscope (JSM-6010LA, JEOL Ltd.) show that it has a rod-like shape with a size of approximately 0.7-0.8 × 1.3-3.0 μm. The colonies of strain C06_No.73 are approximately 1-3 mm in diameter, white, opaque, circular, and generally raised and moist.

[0018] The mycological characteristics of strain C06_No.73 are as follows: Growing pH: 4.0~8.0 (Optimal pH is 6.5) Growth temperature: 18-39°C (optimal temperature is 33°C for 24-hour incubation and 27°C for 48-hour incubation) NaCl resistance: Growth inhibited at 6% (w / v) or higher.

[0019] Compared to typical Lentractobacillus strains, strain C06_No.73 has unique characteristics regarding sugar metabolism, such as being able to utilize glycerol but being unable to utilize L-arabinose and D-xylose.

[0020] The taxonomic characteristics of strain C06_No.73 are described in a paper published by the inventors (Tai-Ying Chiou et al., “Lentilactobacillus kosonis sp. Nov., isolated from koso, a Japanese sugar-vegetable fermented beverage”, Int. J. Syst. Evol. Microbiol., (English), 2021, 71:005128).

[0021] (III) IL-10 production inducing activators, anti-inflammatory agents, regulatory T cell production inducing activators, and immunosuppressants for hyperimmune disease In this specification, "IL-10 production induction activator" refers to a substance that activates the induction of interleukin-10 (IL-10) production in dendritic cells (DCs).

[0022] Dendritic cells are antigen-presenting cells present in the body that secrete various substances (cytokines, etc.) in response to the antigens they take in. IL-10 is a representative anti-inflammatory cytokine and is mainly involved in maintaining homeostasis through its immunomodulatory function. Diseases that are directly or indirectly associated with a decrease in IL-10 include irritable bowel disease, Crohn's disease, depression, rheumatoid arthritis, sclerosis, asthma, allergies, sleep apnea syndrome, peripheral neuropathy, psoriasis, and eczema.

[0023] In this specification, "anti-inflammatory agent" refers to a substance that activates the anti-inflammatory properties of a living organism by activating the induction of IL-10 production in dendritic cells. Furthermore, in this specification, an "anti-inflammatory agent" can also be defined as a substance that, when added to a culture medium containing dendritic cells and cultured for a predetermined period, increases the amount of IL-10 and IL-10 gene induced by the dendritic cells compared to when the anti-inflammatory agent is not added.

[0024] In this specification, "regulatory T cell production induction activator" refers to a substance that activates the induction of regulatory T cell (Treg) production. In this specification, a "regulatory T cell production induction activator" can also be described as a substance that, when added to a culture medium together with biological tissue containing naive T cells (e.g., spleen cells) and cultured for a predetermined period, increases the proportion of regulatory T cells among the T cells after culture compared to when the regulatory T cell production induction activator was not added.

[0025] Regulatory T cells are a type of T cell, but in the body, they play a role in suppressing excessive immune responses, such as inhibiting the activity of other T cells that have the ability to induce autoimmune diseases. Current research suggests that regulatory T cells possess the characteristic proteins CD4 and forkhead box P3 (Foxp3), and these can be detected using molecular markers. Therefore, regulatory T cells as used herein can also be said to be CD4-positive Foxp3-positive cells.

[0026] Immunosuppression by regulatory T cells is thought to be exerted through the following mechanisms: (a) inhibition of effector T cell proliferation by regulatory T cells consuming IL-2, (b) anergy of naive T cells by suppressing the expression of the antigen-presenting cell co-stimulatory molecule CD80 / CD86 due to high CTLA-4 expression by regulatory T cells, and (c) production of immunosuppressive cytokines (IL-10 and TGF-β) by regulatory T cells.

[0027] Furthermore, the induction and activation of regulatory T cell production by lactic acid bacteria is thought to be exerted through the following mechanisms: (a) the action of at least one of the lactic acid bacteria' cells, cell components (lipoteichoic acid, peptidoglycan, etc.), extracellular components (polysaccharides, exosomes, etc.), and produced components (short-chain fatty acids, etc.) on dendritic cells (dendritic cells produce immunosuppressive cytokines (IL-10 and TGF-β) and retinoic acid, which act on naive T cells to induce the production of regulatory T cells); (b) the action of lactic acid bacteria on intestinal epithelial cells (intestinal epithelial cells produce immunosuppressive cytokines and retinoic acid); and (c) the promotion of Foxp3 gene expression by inhibiting histone deacetylases in T cells through short-chain fatty acids produced by lactic acid bacteria.

[0028] In this specification, "immune hyperreaction disease suppressant" refers to a substance that suppresses diseases caused by an excessive immune response in the body by activating the induction of regulatory T cell (Treg) production.

[0029] Diseases resulting from an overreaction of the body's immune system typically include, but are not limited to, autoimmune diseases, inflammatory diseases, allergic diseases, and organ transplant rejection. The relationship between these diseases and regulatory T cells is gradually becoming clearer, for example, through findings such as the fact that a deficiency in the Foxp3 gene causes autoimmune diseases specific to Scurfy mice and causes human IPEX syndrome.

[0030] (IV) Food and beverages, pharmaceuticals, animal feed, or compositions containing active ingredients thereof (Active ingredient composition) The IL-10 production-inducing activator, anti-inflammatory agent, regulatory T cell production-inducing activator, and immunosuppressant for hyperimmune disease according to this embodiment can be used in the form of active ingredient compositions for incorporation into food and beverages, pharmaceuticals, or animal feed. When used as an active ingredient composition, at least one of the bacterial cells or bacterial cell treatment products of lactic acid bacteria belonging to Lentinctobacillus cosonis, which is the active ingredient, can be used as is, or the culture / fermentation liquid (including culture supernatant) of the lactic acid bacteria, or their crude or refined products can also be used. Furthermore, if the active ingredient is a bacterial cell treatment product, the substance used in the treatment of the bacterial cells (e.g., extraction solvent) can also be used as a material for the active ingredient composition.

[0031] Furthermore, the bacterial cells may be not only live cells, but also sterilized cells (dead cells) obtained by heat sterilization or other procedures. When lactic acid bacteria belonging to Lentilactobacillus cousonis are killed by heat treatment, the conditions are preferably 75°C for 1 minute or more, and more preferably 85°C for 1 minute or more. Dead cells have the advantage of reducing the possibility of morphological changes during delivery or display after product manufacturing.

[0032] The above culture medium can be obtained by culturing lactic acid bacteria belonging to Lentilactobacillus cousonis in a suitable medium, such as MRS medium, at 18-39°C for approximately 16-48 hours. After culturing, the cultured cells can be obtained, for example, by centrifugation of the culture medium at 3,000 rpm at 4°C for 10 minutes. These can be purified according to conventional methods. Furthermore, the cells can be freeze-dried or spray-dried. The cells obtained in this way can be used as an active ingredient.

[0033] Furthermore, the active ingredient composition of this embodiment may optionally contain an appropriate amount of nutrients suitable for maintaining and growing lactic acid bacteria belonging to Lentilactobacillus cousonis. Specific examples of these nutrients include carbon sources used in culture media for microbial cultivation, such as fructose, glucose, sorbose, ribose, lyxose, xylose, arabinose, lactulose, and sucrose; nitrogen sources, such as yeast extract and peptone; vitamins; minerals; trace metal elements; and other nutrients. Examples of vitamins include vitamin B, vitamin D, vitamin C, vitamin E, and vitamin K. Examples of trace metal elements include zinc and selenium. Examples of other nutrients include various oligosaccharides such as lactulose oligosaccharide, soybean oligosaccharide, lactitol, fructooligosaccharide, and galactooligosaccharide. The amount of these oligosaccharides included is not particularly limited, but it is preferable to select an amount that is typically in the range of 1 to 30% by weight in the composition of this embodiment.

[0034] The amount of lactic acid bacteria belonging to Lentinctobacillus cosonis in the active ingredient composition of this embodiment is generally such that there are 10 bacteria per 100g of the composition. 8 ~10 11 An amount of approximately 100 cells (not necessarily viable cells) can be appropriately selected. The active ingredient composition of this embodiment is preferably prepared in the form of food, beverages, pharmaceuticals, etc., as described later, by appropriately blending a suitable edible carrier (food material), a pharmaceutically acceptable carrier, etc.

[0035] (Pharmaceuticals) When the IL-10 production-inducing activator, anti-inflammatory agent, regulatory T cell production-inducing activator, and immunosuppressant of hyperimmune disease of this embodiment are put into pharmaceutical form, they are prepared and put into practical use using a suitable pharmaceutical carrier along with at least one of the bacterial cells or bacterial cell treatment products of lactic acid bacteria belonging to Lentinctobacillus cosonis, which are the active ingredients. Examples of such pharmaceutical carriers include fillers, bulking agents, binders, humectants, disintegrants, surfactants, lubricants, diluents, excipients, etc., that are commonly used in this field.

[0036] While various forms can be selected for the dosage unit of a pharmaceutical product, oral formulations are preferred. Typical oral formulations include tablets, pills, powders, liquids, suspensions, emulsions, granules, and capsules.

[0037] When forming the tablets, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, and potassium phosphate are used as formulation carriers; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone are used; sodium carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, dried starch, and argy Disintegrants such as sodium phosphate, agar powder, laminaran powder, sodium bicarbonate, and calcium carbonate; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, and monoglyceride stearate; disintegration inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oil; absorption enhancers such as quaternary ammonium salts and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid; and lubricants such as refined talc, stearate, boric acid powder, and polyethylene glycol can be used. The tablets may be coated with a conventional coating as needed, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, or film-coated tablets, or they may be double-coated or multi-layered tablets.

[0038] When forming the product into pill form, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc can be used as formulation carriers; binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol can be used; and disintegrants such as laminaran and agar can be used.

[0039] Furthermore, pharmaceuticals may contain colorants, preservatives, fragrances, flavorings, sweeteners, and other pharmaceuticals as needed.

[0040] There are no particular restrictions on the method of administering the pharmaceutical product of this embodiment, and it is determined according to the formulation, the patient's age, sex, other conditions, the severity of the disease, etc. The dosage is also appropriately selected according to the method of use, the patient's age, sex, other conditions, the severity of the disease, etc., but it is generally considered that the above active ingredient composition should be approximately 0.5 to 20 mg per kg of body weight per day. The pharmaceutical product can be administered to humans in 1 to 4 divided doses per day.

[0041] Furthermore, the pharmaceutical product may be a so-called topical preparation, specifically a cosmetic, topical drug, or quasi-drug. In this case, the active ingredient composition of the present invention is prepared in the form of a general topical preparation using a suitable pharmaceutical carrier that is pharmaceutically acceptable, and then put into practical use.

[0042] Examples of such formulation carriers include humectants such as glycerin, petrolatum, urea, hyaluronic acid, and heparin; PABA derivatives (para-aminobenzoic acid, Escalol 507, etc.), cinnamic acid derivatives (Neo-Heliopan, Parsol MCX, Sunguard B, etc.), salicylic acid derivatives (octyl salicylate, etc.), benzophenone derivatives (ASL-24, ASL-24S, etc.), dibenzoylmethane derivatives (Parsol A, Parsol DAM, etc.), heterocyclic derivatives (Tinuvin derivatives, etc.), and ultraviolet absorbers and scatterers such as titanium dioxide; disodium edetate, trisodium edetate, and citrate. Metal sequestering agents such as acids, sodium citrate, tartaric acid, sodium tartrate, lactic acid, malic acid, sodium polyphosphate, sodium metaphosphate, and gluconic acid; sebum inhibitors such as salicylic acid, sulfur, caffeine, and tannins; disinfectants such as benzalkonium chloride, benzethonium chloride, and chlorhexidine gluconate; anti-inflammatory agents such as diphenhydramine hydrochloride, tranexamic acid, guaiazulene, azulene, allantoin, hinokitiol, glycyrrhizic acid and its salts, glycyrrhizic acid derivatives, and glycyrrhetinic acid; vitamin A, vitamin B group (B1, B2, B6, Vitamins such as B12, B15), folic acid, nicotinic acid derivatives, pantothenic acid derivatives, biotin, vitamin C, vitamin D group (D2, D3), vitamin E, ubiquinone derivatives, vitamin K (K1, K2, K3, K4); amino acids and their derivatives such as aspartic acid, glutamic acid, alanine, lysine, glycine, glutamine, serine, cysteine, cystine, tyrosine, proline, arginine, pyrrolidone carboxylic acid; retinol, tocopherol acetate, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, ellagic acid, placental extract, etc. Whitening agents; antioxidants such as butylhydroxytoluene, butylhydroxyanisole, and propyl gallate; astringents such as zinc chloride, zinc sulfate, zinc carbohydrate, zinc oxide, and aluminum potassium sulfate; sugars such as glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, and lactitol; various plant extracts such as licorice, chamomile, horse chestnut, saxifrage, peony, quince, scutellaria baicalensis, Phellodendron amurense, Coptis japonica, Houttuynia cordata, and ginkgo biloba; as well as oily components, surfactants, thickeners, alcohols, powder components, and pigments.

[0043] Specific examples of topical formulations include cosmetic creams, lotions, toners, face masks, skin milks (emulsifiers), gels, powders, lip balms, lipsticks, under-makeup products, foundations, sun care products, bath additives, body shampoos, body rinses, soaps, cleansing foams, ointments, patches, jellies, aerosols, and the like.

[0044] (food and drink) In this specification, "food and beverages" includes all forms of food and beverages that are used orally exclusively for consumption. In this specification, even if a product is in the form of a tablet or similar, it is included in the definition of food and beverages as long as it is used exclusively for consumption. For example, health foods, health supplements, foods for the sick, nutritional supplements, or foods with health claims as defined by the Ministry of Health, Labour and Welfare (foods for specified health uses, foods with nutritional function claims) are also included in the definition of food and beverages in this specification. Health foods are foods intended for health, health maintenance, and promotion, in a more positive sense than ordinary foods.

[0045] When the IL-10 production-inducing activator, anti-inflammatory agent, regulatory T cell production-inducing activator, and immunosuppressant for hyperimmune disease of this embodiment are put into the form of food or beverages, they are prepared and put into practical use using appropriate materials acceptable in the food and beverage field, together with at least one of the bacterial cells or bacterial cell treatment products of lactic acid bacteria belonging to Lentinctobacillus cosonis, which are the active ingredients.

[0046] Examples of food and beverage forms include fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, and fermented soy milk beverages. "Fermented milk" refers to a paste-like or liquid product made by fermenting milk or dairy products with lactic acid bacteria or yeast. Therefore, fermented milk includes both beverage and yogurt forms. "Lactic acid bacteria beverage" refers to a beverage made by diluting a paste-like or liquid product made by fermenting milk or dairy products with lactic acid bacteria or yeast as the main ingredient.

[0047] Other forms of food and beverages include, for example, fermented foods such as pickles, miso, fermented tea, and bread; infant foods such as baby food, powdered milk, and baby food; effervescent preparations; confectionery such as gum, gummies, and pudding; noodles; nutritional supplements such as capsules, granules, powders, and tablets; and dairy products other than fermented milk and lactic acid bacteria beverages.

[0048] The content of the active ingredient composition in the food and beverage of this embodiment is not particularly limited and can be determined as appropriate. From the viewpoint of achieving effects such as IL-10 production induction and activation, anti-inflammatory, regulatory T cell production induction and activation, and suppression of immune hyperreaction diseases, the content of the active ingredient composition is preferably, for example, 0.001% by mass or more, more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more, based on the total mass of each food and beverage. On the other hand, there is no particular upper limit to the content of the active ingredient composition in the food and beverage, and it can usually be adjusted as appropriate according to the form of the food and beverage.

[0049] (feed) When the IL-10 production-inducing activator, anti-inflammatory agent, regulatory T cell production-inducing activator, and immunosuppressant for hyperimmune disease of this embodiment are provided in the form of animal feed, they can be, for example, in the form of an orally administered formulation (aqueous solution, emulsion, granules, powder, capsule, tablet, etc.).

[0050] [Examples] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. In the following description, the "%" in numerical values ​​indicating the amount of various components added means mass percentage concentration (mass%) unless otherwise specified.

[0051] [Example 1] IL-10 production-inducing activity (Preparation of lactic acid bacteria) Lactobacillus was prepared for the gene expression analysis and measurement of secreted cytokine levels described later. For the Lentractobacillus cousonis C06_No.73 strain, a strain stored at the Matsuzaki Laboratory, Ishikawa Prefectural University was used. This stored strain originated from a strain obtained from vegetable brown sugar fermentation liquid manufactured at the Arsoa Saku Factory in Saku City, Nagano Prefecture, Japan.

[0052] For comparison, we also prepared Lacticaseibacillus rhamnosus GG strain (ATCC53103) (hereinafter sometimes simply referred to as "GG strain"). This GG strain was obtained from the American Type Culture Collection (ATCC) in the United States.

[0053] (Preparation of dendritic cells) For the test, bone marrow-derived dendritic cells were used, generated from bone marrow cells of the femur and tibia of 4-week-old female BALBc / A mice (Claire Japan Co., Ltd.). After washing the bone marrow cells collected from the mice, the cell count was reduced to 1 × 10⁶. 6 The cells were suspended in dendritic cell medium (described later) at a concentration of cells / mL and cultured at 37°C under 5% CO2 conditions.

[0054] For dendritic cell culture, we used Gibco RPMI-1640 medium (Thermo Fisher Scientific, USA) (RPMI-1640 COMP medium), which contains 100 U / mL penicillin, 100 μg / mL streptomycin, 55 μmol / L 2-mercaptoethanol, and 10% (v / v) fetal bovine serum, to which PeproTech granulocyte-macrophage colony-stimulating factor (Thermo Fisher Scientific, USA) was added to a concentration of 20 ng / mL.

[0055] On days 3 and 5 of culture, half of the dendritic cell culture medium was replaced with fresh medium. On day 6 of culture, cells containing dendritic cells were collected, magnetically labeled with anti-CD11c microbeads (Milteny Biotech, Germany), and isolated using AutoMACS (Milteny Biotech, Germany) by a standard method. Therefore, the dendritic cells used in the study can be considered bone marrow-derived dendritic cells.

[0056] (Gene expression analysis) For the dendritic cells obtained as described above, 1.0 × 10 6Cells were cultured for 12 hours in Gibco RPMI-1640 medium (Thermo Fisher Scientific, USA) (RPMI-1640 COMP medium) containing 100 U / mL penicillin, 100 μg / mL streptomycin, 55 μmol / L 2-mercaptoethanol, and 10% (v / v) fetal bovine serum in 3 ml of wells.

[0057] The above cultures were performed with no added lactic acid bacteria (control), with added C06_No.73 strain, and with added GG strain. The concentration of lactic acid bacteria was OD for all cases. 600 The value was set to 0.01. Subsequently, total RNA was isolated from dendritic cells using the RNA extraction kit Illustra® RNAspin (Global Life Science Technologies Japan Co., Ltd.), and cDNA was synthesized from the total RNA using the Invitrogen SuperScript® III reverse transcription kit (Thermo Fisher Scientific, USA).

[0058] Real-time PCR was performed using the Applied Biosystems StepOne Real-Time PCR System (Thermo Fisher Scientific, USA) and the Applied Biosystems Power SYBR® Green Master Mix (Thermo Fisher Scientific, USA).

[0059] The following primers were used to amplify the DNA. (IL-10 amplification primer: dendritic cell IL-10 PCR primer) Forward:5'-ATGCAGGACTTTAAGGGTTACTTG-3'(Sequence ID 1) Reverse:5'-GAATTCAAATGCTCCTTGATTTCT-3'(Sequence ID 2) (IL-12 amplification primer: dendritic cell IL-12 PCR primer) Forward:5'-ACTCACATCTGCTGCTCCACAAG-3'(Sequence ID 3) Reverse:5'-CACGTGAACCGTCCGGAGTA-3'(Sequence ID 4)

[0060] Furthermore, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control to evaluate the degree of gene expression related to IL-10 and IL-12. The following primers were used to amplify this gene. (GAPDH amplification primer: dendritic cell GAPDH PCR primer) Forward:5'-CTACACTGAGGACCAGGTTGTCT-3'(Sequence ID 5) Reverse:5'-ATTGTCATACCAGGAAATGAGCTT-3'(Sequence ID 6)

[0061] The results of the gene expression analysis are shown in Figure 1. Figure 1 is a bar graph showing the results of the gene expression analysis for dendritic cells cultured with lactic acid bacteria in Example 1. Figure 1(a) is a bar graph showing the results for IL-10, and Figure 1(b) is a bar graph showing the results for IL-12. The height of each bar graph in Figure 1 represents the ratio when the degree of gene expression in the control group without lactic acid bacteria is set to 1. The ** mark in Figure 1(a) indicates that there is a statistically significant difference P<0.01 between the groups.

[0062] In Figure 1, "L. kosonis" shows the results for strain C06_No.73, and "L. rhamnosus GG" shows the results for strain GG. The same applies to Figure 2, which will be discussed later.

[0063] As shown in Figure 1, the Lenticularbacillus cousonis strain C06_No.73 showed significantly higher gene expression of IL-10 (an interleukin mainly related to anti-inflammatory activity) in dendritic cells compared to the common lactic acid bacterium strain GG. Furthermore, the Lenticularbacillus cousonis strain C06_No.73 also showed significantly higher gene expression of IL-10 compared to IL-12 (an interleukin mainly related to immunostimulation) in dendritic cells.

[0064] (Measurement of secreted cytokine levels) For the dendritic cells obtained as described above, 1.0 × 10 6 Cells were cultured for 24 hours in Gibco RPMI-1640 medium (Thermo Fisher Scientific, USA) (RPMI-1640 COMP medium) containing 100 U / mL penicillin, 100 μg / mL streptomycin, 55 μmol / L 2-mercaptoethanol, and 10% (v / v) fetal bovine serum in 3 ml of wells.

[0065] The above cultures were performed with the addition of strain C06_No.73 and strain GG. The concentration of lactic acid bacteria was OD for all samples. 600 The value was set to 0.01. Subsequently, the supernatant obtained by centrifugation (400 × g, 10 min) was used to measure the amount of secreted cytokines from dendritic cells using the Mouse IL-10 ELISA MAX STANDARD Set (BioLegend, USA) and the Mouse IL-12 / IL-23 (p40) ELISA MAX STANDARD Set (BioLegend, USA).

[0066] Figure 2 shows the measurement results of secreted cytokine levels. Figure 2 is a bar graph showing the measurement results of secreted cytokine levels for dendritic cells cultured with lactic acid bacteria in Example 1. Figure 2(a) is a bar graph showing the results for IL-10, and Figure 2(b) is a bar graph showing the results for IL-12. The asterisk (*) in Figure 2(a) indicates a statistically significant difference (P<0.05) between the groups.

[0067] As shown in Figure 2, the Lenticularbacillus cousonis strain C06_No.73 was found to produce higher levels of IL-10 and IL-12 in dendritic cells compared to the common lactic acid bacterium strain GG. Furthermore, the Lenticularbacillus cousonis strain C06_No.73 was found to produce significantly higher levels of IL-10 compared to IL-12 in dendritic cells.

[0068] Specifically, the ratio of IL-10 production to IL-12 production (IL-10 / IL-12) was 3.05 for the GG strain, while it was 3.54 for the C06_No.73 strain. This indicates the high anti-inflammatory properties of the C06_No.73 strain, and by extension, Lentilactobacillus cousonis.

[0069] From the above results, it was found that the bacterial cells or processed products of lactic acid bacteria belonging to Lenticularbacillus cousonis can be used as IL-10 production activators or materials for activating IL-10 production induction in dendritic cells. Therefore, it is considered that the bacterial cells or processed products of lactic acid bacteria belonging to Lenticularbacillus cousonis can be used as anti-inflammatory agents or materials for activating the anti-inflammatory properties of living organisms by activating IL-10 production induction in dendritic cells.

[0070] [Example 2] Regulatory T cell production induction activity (Preparation of lactic acid bacteria) Lactobacillus was prepared to measure the amount of regulatory T cells (CD4-positive Foxp3-positive cells) induced, as described later. The same strain of Lentractobacillus cousonis C06_No.73 used in Example 1 was employed.

[0071] For comparison, we also prepared Lactobacillus acidophilus strain JCM1132 (reference strain; hereafter sometimes simply referred to as "JCM1132 strain"). This JCM1132 strain was obtained from the Microbial Materials Development Laboratory, BioResource Research Center, RIKEN (The Institute of Physical and Chemical Research). Furthermore, lactic acid bacteria belonging to the Lactobacillus acidophilus are known to have high regulatory T cell production induction activity among lactic acid bacteria (see, for example, "Fink LN, "Induction of regulatory T cells by probiotics: potential for treatment of allergy?", Clinical & Experimental Allergy, Blackwell Publishing (US), 2009, 40, 5-8" and "Park JS et al., "Lactobacillus acidophilus improves intestinal inflammation in an acute colitis mouse model by regulation of Th17 and Treg cell balance and fibrosis development", Journal of Medicinal Food, Mary Ann Liebert (US), 2018, 21, 215-224").

[0072] For the aforementioned C06_No.73 and JCM1132 strains, 4 mL of BD Difco® de Man-Rogosa-Sharpe (MRS) medium (Becton Dickinson, USA) was used as a container in a 5 mL glass vial (No. 1 from Maruemu Co., Ltd.) and incubated at 30°C for 48 hours with the vial tightly sealed.

[0073] After the cultivation, the culture broth was transferred to a 15 mL polypropylene centrifuge tube (Biolamo of AS ONE Corporation), and processed with a micro high-speed cooling centrifuge MX-301 (Tommy Seiko Co., Ltd.) (12,000 rpm, 1 minute) to remove the MRS medium and obtain a cell pellet. For the cell pellet, the steps of suspension in BD BBL (registered trademark) physiological saline (Becton Dickinson, USA) and centrifugation (12,000 rpm, 1 minute) were repeated three times to wash the cells. Then, the cells were prepared with RPMI-1640 COMP medium so that the OD value at 600 nm (OD 600 ) became 1.0.

[0074] (Preparation of spleen cells) The spleen of 7-week-old male BALB / cA mice (CLEA Japan Inc.) was excised and minced with an injection needle (22G) in RPMI-1640 medium COMP medium. Then, the minced spleen was passed through a 70 μm mesh strainer, and the cells were collected in a 50 mL conical tube (Thermo Fisher Scientific, USA).

[0075] Thereafter, a cell pellet was obtained by centrifugation (800 rpm, 8 minutes, 4 °C) using a tabletop cooling centrifuge Model 2800 (Kubota Shokai Co., Ltd.). The cell pellet was treated with ACK buffer (pH: 7.2) containing 8.3 g / L ammonium chloride (NH4Cl), 1.0 g / L potassium hydrogen carbonate (KHCO3), and 18.6 mg / L disodium ethylenediaminetetraacetate (EDTA·2Na) for 5 minutes. Then, the cells treated with RPMI-1640 COMP medium were washed, and the cells were suspended in RPMI-1640 COMP medium to obtain a spleen cell suspension containing immune cells.

[0076] (Analysis of regulatory T cell production induction activity) The cultivation for the analysis of regulatory T cell production induction activity was carried out using a 96-well U-bottom plastic culture plate 163320 (Thermo Fisher Scientific, USA). The cultivation (co-cultivation) was carried out at a final concentration of 5.0×10 5The study was conducted at 37°C and 5% CO2 using RPMI-1640 COMP medium (200 μL per well) containing cells / well of spleen cells, Lactobacillus cells (C06_No.73 strain or JCM1132 strain) at a final concentration of 0.01 (OD value at 600 nm), Dynabeads® mouse T-Activator CD3 / CD28 (Thermo Fisher Scientific, USA) at a final concentration of 0.25 μl / well, PeproTech transforming growth factor (TGF)-β1 (Thermo Fisher Scientific, USA) at a final concentration of 5 ng / well, and SIGMA-ALDRICH® L2630 lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Merck, Germany) at a final concentration of 5 μg / mL.

[0077] Splenocytes cultured for 5 days were harvested on a magnetic plate (ALPAQUA, USA) and transferred to a 96-well V-bottom plastic plate 277143 (Thermo Fisher Scientific, USA). Subsequently, a cell pellet was obtained by centrifugation (400 × g, 3 min) using a benchtop multi-tube centrifuge LC-220 (Tommy Seikou Co., Ltd.).

[0078] To analyze the expression of markers related to regulatory T cell production induction, the cell pellet was washed twice with BD Pharmingen Stain Buffer (FBS) (Becton Dickinson, USA) (hereinafter referred to as "Stain Buffer"). Then, 25 μL of BD Pharmingen Mouse BD Fc Block Reagent (Becton Dickinson, USA), diluted 100-fold with Stain Buffer (FBS), was added and allowed to stand for 10 minutes.

[0079] Furthermore, 25 μL of Stain Buffer containing 500-fold diluted Invitrogen® eBioscience® Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific, USA), 100-fold diluted BD Pharmingen FITC-labeled anti-mouse CD4 antibody (Becton Dickinson, USA), and 100-fold diluted PerCP-labeled anti-mouse CD45 antibody (BioLegend, USA) was added and allowed to stand for 20 minutes.

[0080] Subsequently, the cells were suspended and centrifuged (400 × g, 3 min) using Stain Buffer, and this process was repeated three times to obtain a washed cell pellet. 100 μL of BD Cytofix / Cytoperm Fixation and Permeabilization Solution (Becton Dickinson, USA) was added to the washed cell pellet, and it was allowed to stand for 45 minutes.

[0081] Next, the cells were suspended and centrifuged (700 × g, 5 min) three times using BD Perm / Wash Perm / Wash Buffer (Becton Dickinson, USA) (hereinafter referred to as "Perm / Wash Buffer"). To the resulting washed cell pellet, 25 μL of Perm / Wash Buffer containing 50-fold diluted Invitrogen® eBioscience® PE-labeled anti-mouse Foxp3 antibody (Clone Number FJK-16s) (Thermo Fisher Scientific, USA) was added and allowed to stand for 45 minutes.

[0082] The process of suspension and centrifugation (700×g, 5min) using Perm / Wash Buffer was repeated three times. The resulting washed cell pellet was suspended in 100 μL of Stain Buffer and analyzed using a NovoCyte® 2060 flow cytometer (ACEA Biosciences, USA). In this analysis, live cells were gated using forward scattering (FSC) and side scattering (SCC) characteristics, and eFluor780-negative and CD45-positive cells were gated. The proportion of CD4-positive and Foxp3-positive cells within the gated cells was then quantified.

[0083] Figure 3 shows the analysis results for regulatory T cells (CD4-positive Foxp3-positive cells). Figure 3 is a box plot showing the analysis results of the induced production of regulatory T cells (CD4-positive Foxp3-positive cells) in mouse spleen cells cultured with lactic acid bacteria in Example 2. The asterisk (*) in Figure 3 indicates a statistically significant difference (P < 0.01) between the groups. Note that "Foxp3+CD4+ T cells" in Figure 3 means the same as CD4-positive Foxp3-positive cells.

[0084] In Figure 3, within the boxes for each item, the thick line inside the box indicates the median, the top edge of the box indicates the 75th percentile (third quartile), the bottom edge of the box indicates the 25th percentile (first quartile), and the whiskers extending beyond the box indicate the maximum and minimum values.

[0085] As shown in Figure 3, the proportion of regulatory T cells induced by the Lentractobacillus cosonis strain C06_No.73 was significantly higher compared to that produced by the JCM1132 strain.

[0086] Specifically, regarding the proportion of regulatory T cells, the JCM1132 strain showed a minimum of 3.85%, a 25th percentile of 4.5525%, a median of 6.160%, a 75th percentile of 6.7100%, and a maximum of 7.12%. On the other hand, the C06_No.73 strain showed a minimum of 8.02%, a 25th percentile of 8.0225%, a median of 8.075%, a 75th percentile of 8.6075%, and a maximum of 9.08%. These results demonstrate the high regulatory T cell production induction activity of the C06_No.73 strain, and by extension, Lentinctopacillus cousonis, suggesting potential applications in suppressing hyperimmune diseases.

[0087] From the above results, it was found that the bacterial cells or processed products of lactic acid bacteria belonging to Lenticularbacillus cosonis can be used as regulatory T cell production activators or materials for activating the production of regulatory T cells (Tregs). Therefore, it is considered that the bacterial cells or processed products of lactic acid bacteria belonging to Lenticularbacillus cosonis can be used as immunosuppressants or materials for suppressing diseases caused by excessive immune responses in the body by activating the production of regulatory T cells (Tregs).

[0088] (Prescription example 1: Tablets) Tablets can be manufactured by mixing the following ingredients according to a conventional method and compressing them into tablets. The active ingredient composition used is the bacterial cell of strain C06_No.73 prepared in the example. (composition) Active ingredient composition 150 mg Cellulose 80mg Starch 20mg Sucrose fatty acid ester 2 mg

[0089] (Prescription example 2: Capsules) Soft capsules can be obtained by mixing the following components according to a conventional method and filling them into a capsule base containing gelatin and glycerin. The active ingredient composition used is the bacterial cells of strain C06_No.73 prepared in the example. (composition) Active ingredient composition 100 mg Beeswax 10mg Grape seed oil 110mg

Claims

1. An IL-10 production induction activator containing at least one of the bacterial cells or bacterial cell treatment products of a lactic acid bacterium belonging to Lentilactobacillus kosonis as an active ingredient, which activates the induction of interleukin-10 (IL-10) production in dendritic cells.

2. The IL-10 production inducing activator according to claim 1, wherein the lactic acid bacterium is Lentinctobacillus cousonis C06 No. 73 strain.

3. The IL-10 production-inducing activator according to claim 1 or 2, in the form of a food or beverage, pharmaceutical, animal feed, or an active ingredient composition to be incorporated therein.

4. An anti-inflammatory agent containing at least one of the bacterial cells or bacterial cell treatment products of a lactic acid bacterium belonging to Lentilactobacillus kosonis as an active ingredient, which activates the anti-inflammatory properties of the body by activating the induction of interleukin-10 (IL-10) production in dendritic cells.

5. The anti-inflammatory agent according to claim 4, wherein the lactic acid bacterium is Lentinactobacillus cosonis C06 No. 73 strain.

6. The anti-inflammatory agent according to claim 4 or 5, in the form of a food or beverage, a pharmaceutical, animal feed, or an active ingredient composition to be incorporated therein.

7. A regulatory T cell production induction activator containing at least one of the bacterial cells or bacterial cell treatment products of a lactic acid bacterium belonging to Lentilactobacillus kosonis as an active ingredient, which activates the induction of regulatory T cell (Treg) production.

8. The regulatory T cell production inducing activator according to claim 7, wherein the lactic acid bacterium is Lentinctobacillus cousonis C06 No. 73 strain.

9. The regulatory T cell production inducing activator according to claim 7 or 8, in the form of a food or beverage, a pharmaceutical, animal feed, or an active ingredient composition to be incorporated therein.

10. An immunosuppressant for diseases caused by excessive immune responses in the body, containing at least one of the bacterial cells or bacterial cell treatment products of a lactic acid bacterium belonging to Lentilactobacillus kosonis as an active ingredient, and activating the induction of regulatory T cell (Treg) production.

11. The immunosuppressant for hyperimmune disease according to claim 10, wherein the lactic acid bacterium is Lentinctobacillus cosonis C06 No. 73 strain.

12. An immunosuppressant for hyperimmune disease according to claim 10 or 11, in the form of a food or beverage, a pharmaceutical, animal feed, or an active ingredient composition to be incorporated therein.

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