Immunostimulator using brautia genus bactria

Abstract

To provide a microbial cell material having excellent property as an immunostimulation material.SOLUTION: The dead bacterial cells of the genus Brautia are the active ingredient in an immune activator. It is preferable that these dead cells are obtained by heat treatment under acidic conditions. Furthermore, it is preferable that they are obtained by heat treatment under conditions of pH 3.0 to 7.0 and temperature of 70 to 121°C.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the use of Blautia bacteria, and more particularly to the use of Blautia bacteria as an immunostimulant. [Background technology]

[0002] Lactic acid bacteria supplements are used to improve the body's immunity and maintain health. For example, Patent Document 1 describes a preparation of lactic acid bacteria that can be used as such lactic acid bacteria and that is microparticulated into nano-sized particles.

[0003] Meanwhile, recent data analysis of humans has revealed that Blautia bacteria, a type of intestinal bacteria, are inversely correlated with BMI and diabetes risk (see Non-Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4621218 [Non-patent literature]

[0005] [Non-Patent Document 1] Koji Hosomi1 et al., “Oral administration of Blautia wexlerae meliorates obesity and type 2 diabetes via etabolic remodeling of the gut microbiota” Nature Communications | (2022) 13:4477 Summary of the Invention [Problem to be solved by the invention]

[0006] Conventionally, little research has been done on the use of bacteria belonging to the genus Blautia.

[0007] An object of the present invention is to provide a microbial cell material that utilizes Blautia bacteria and has excellent properties as an immunopotentiating material. [Means for solving the problem]

[0008] Means of the Invention The present inventors have conducted various studies to achieve the above object and have found that killed cells of bacteria of the genus Blautia have excellent immunostimulating properties, which has led to the completion of the present invention.

[0009] That is, the present invention provides an immunostimulant containing killed cells of bacteria of the genus Blautia as an active ingredient.

[0010] In the above-mentioned immunostimulant, the killed cells of the bacterium belonging to the genus Blautia are preferably obtained by heat treatment under acidic conditions.

[0011] The killed cells of the Blautia bacteria are preferably obtained by heat treatment under conditions of pH 3.0 to 7.0 and temperature of 70 to 121°C.

[0012] Furthermore, it is preferable that the killed cells of the Blautia bacteria have an ability to induce IL-12 production measured by a method using mouse spleen cells that is 10 times or more higher than the ability to induce IL-12 production by live cells of the Blautia bacteria under the same conditions.

[0013] Furthermore, in addition to the above-mentioned ability to induce IL-12 production, it is preferable that the killed cells of the Blautia bacteria further have an ability to induce IL-10 production measured by a method using mouse spleen cells that is at least twice as high as the IL-10 production induction ability of live cells of the Blautia bacteria under the same conditions.

[0014] Moreover, the immunostimulant provided by the present invention is preferably one that promotes intestinal IgA production. Effect of the Invention

[0015] According to the present invention, a microbial cell material having excellent properties as an immunopotentiating material can be provided by utilizing bacteria of the genus Blautia. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1(a) is a graph showing the results of investigating the effect of adding Blautia bacteria (live bacteria or killed bacteria) to the culture medium in a culture system for mouse spleen cells in Test Example 1, where FIG. 1(a) is a graph showing the results of investigating the ability of spleen cells to induce IL-12 production, and FIG. 1(b) is a graph showing the results of investigating the ability of spleen cells to induce IL-10 production. [Diagram 2] FIG. 2(a) is a graph showing the results of investigating the effect of adding Blautia bacteria (killed bacteria in a pH 6 environment or a pH 4 environment) to the culture medium in a mouse spleen cell culture system in Test Example 1, where FIG. 2(a) is a graph showing the results of investigating the ability of spleen cells to induce IL-12 production, and FIG. 2(b) is a graph showing the results of investigating the ability of spleen cells to induce IL-10 production. [Diagram 3] FIG. 1 is a graph showing the results of investigating the effect of adding Blautia bacteria (live bacteria or killed bacteria in a pH 4.0 environment) to the culture medium in a mouse spleen cell culture system in Test Example 2, and also shows the results of investigating the expression levels of cytokines. [Figure 4] FIG. 1 shows the results of an investigation into the effects of adding Blautia bacteria (live bacteria or killed bacteria in an environment of pH 4.0) to feed and allowing BALB / c mice to ingest it ad libitum in Test Example 3, and also shows the results of an investigation into the concentration of short-chain fatty acids in the cecal contents. [Diagram 5] FIG. 1 shows the results of investigating the effects of adding Blautia bacteria (live bacteria or killed bacteria in an environment of pH 4.0) to feed and allowing BALB / c mice to ingest it ad libitum in Test Example 3, and also shows the results of investigating the IgA concentration in the feces. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] As used herein, the term "bacteria of the genus Blautia" refers to bacteria belonging to the genus Blautia, which is classified into the phylum Firmicutes. Specifically, Blautia caecimuris, Blautia glucerasea, Blautia coccoides, Blautia schinkii, Blautia stercoris, Blautia hydrogenotrophica, Blautia faecis, Blautia producta, Blautia hansenii, Blautia luti, Blautia wexlerae, etc. may be used alone or in combination of two or more. Among these, Blautia producta is particularly preferably selected from the viewpoint of immunostimulatory effect.

[0018] Preferred examples of Blautia products include the Blautia RD014892 strain (accession number: NITE BP-03954) deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary (NPMD), an independent administrative institution, and bacteria substantially identical thereto. Here, the term "substantially identical" bacteria has the same meaning as understood by those skilled in the art, and refers to, for example, a bacterium whose 16SrRNA gene sequence for identifying the genus and species of the bacterium has a homology of 98% or more, preferably 99% or more, with the 16SrRNA gene sequence of the Blautia RD014892 strain, and which has the same bacteriological properties as the Blautia RD014892 strain.

[0019] The cultivation of Blautia bacteria and the maintenance of the bacteria can be carried out by known means. For example, the medium may be a liquid medium containing yeast extract, peptone, meat extract, amino acids, salts, minerals, etc. A commercially available medium such as "Modified GAM" (product name, Modified GAM Bouillon, Nissui Co., Ltd.) may be used. The cultivation can be carried out by inoculating the bacteria into the above-mentioned medium and then performing static cultivation or aeration and agitation cultivation under conditions of, for example, 25 to 40°C. For the preservation of live bacteria, they can be refrigerated while suspended in the medium for a short period of time, or frozen and preserved after being suspended in an antifreeze such as a glycerol solution for a long period of time.

[0020] When preparing bacteria of the genus Blautia, a bacterial cell concentrate can be prepared by concentrating the culture solution after culturing, or by collecting the bacteria by means of centrifugation, filtration, or the like, washing the bacteria with purified water, etc., and suspending the bacteria in purified water, etc. to a predetermined bacterial cell concentration. The content of bacteria of the genus Blautia in 100 parts by mass of the bacterial cell concentrate may be in the range of 0.1 to 50 parts by mass, 0.5 to 25 parts by mass, or 1 to 10 parts by mass, calculated as dry bacteria. This bacterial cell concentrate may contain an excipient. This makes it easier to maintain the properties of the bacteria after freezing or lyophilization, and after reconstitution with water.

[0021] The excipient is not particularly limited, and examples thereof include sugar alcohols such as dextrin, maltodextrin, cyclodextrin, xanthan gum, xylitol, sorbitol, maltitol, mannitol, and lactitol; sugars such as glucose, sucrose, fructose, lactose, dextrose, and lactose; and organic acids such as adipic acid, citric acid, glutaric acid, succinic acid, tartaric acid, fumaric acid, and malic acid.

[0022] In another embodiment, the bacteria of the genus Blautia may be prepared by pulverization and dispersion. The pulverization and dispersion may be performed, for example, by pulverizing and dispersing the above-mentioned concentrated bacterial cell solution using a means such as stirring, a mixer, a homogenizer, a ball mill, a bead mill, a jet mill, or a generator. In this case, depending on the case or as required, the above-mentioned excipient may be added and then pulverized and dispersed to prevent reagglomeration of the obtained bacterial powder. When an excipient is contained, the content thereof may be in the range of 1 to 99% by mass, 10 to 95% by mass, or 20 to 90% by mass calculated on a dry matter basis.

[0023] In yet another embodiment, when preparing the Blautia bacteria, a process of drying and powdering may be carried out. Examples of the drying and powdering method include freeze-drying, reduced pressure spray drying, spray drying using hot air, etc. By carrying out spray drying using hot air, the activity of live bacteria is usually destroyed, and killed bacteria can be obtained.

[0024] In the present invention, among the Blautia bacteria that can be prepared as described above, a preparation obtained by heat treatment under acidic conditions is provided. According to the preparation obtained through such treatment, the activity of the live bacteria is usually lost and the bacteria are killed, so that the quality change associated with live bacteria can be suppressed. Furthermore, as shown in the examples described below, the killed bacteria of the Blautia genus have the ability to induce the production of immunostimulatory factors (IL-12, IL-10, TGF-β, IL-6, IFN-γ, etc.) in immune cells, and the functionality of improving the intestinal environment and inducing the production of IgA, an immune molecule. Therefore, for example, it can be suitably used as an active ingredient of a functional composition for improving the body's immune power and maintaining health. In particular, it can be suitably used in functional foods for maintaining the health of healthy people. From another perspective, it can be said that the present invention provides functional materials and participating ingredients that can be used in health foods and supplements for promoting health. Furthermore, it can be said that the present invention provides an immunostimulant containing the killed bacteria of the Blautia genus as an active ingredient.

[0025] Heat treatment of Blautia bacteria can be performed by treating the culture solution after culture as it is, or after preparing the above-mentioned bacterial cell concentrated solution as necessary, for example, in a jacketed tank, a thermostatic bath, or an autoclave. In this case, the pH is not limited, but may be, for example, pH 3.0 to 7.0, pH 3.0 to 6.0, or pH 3.0 to 5.0. The temperature condition of the heat treatment is not limited, but may be, for example, 70 to 121°C, 80 to 110°C, or 80 to 100°C. The heat treatment time is not limited, but may be, for example, 30 minutes to 120 hours, 30 minutes to 90 minutes, or 30 minutes to 60 minutes. If the treatment under such an environment is insufficient, the desired functionality such as immunostimulatory activity tends to be poor, which is not preferable.

[0026] In order to determine whether the Blautia bacteria provided by the present invention have been sufficiently treated under the above-mentioned environment, it is sufficient to check the preparation history. Alternatively, in some cases, it is also possible to make a determination from the viewpoint of functionality. For example, it is also possible to make a determination from the viewpoint of functionality by determining whether the IL-12 production induction ability measured by a method using mouse spleen cells is 10 times or more higher than the IL-12 production induction ability of live Blautia bacteria under the same conditions, or whether the IL-10 production induction ability measured by a method using mouse spleen cells is 2 times or more higher than the IL-10 production induction ability of live Blautia bacteria under the same conditions.

[0027] The killed cells of the Blautia bacteria provided by the present invention can be used, as desired, in various product forms such as foods and beverages, functional foods, pharmaceuticals, cosmetics, and animal feed.

[0028] As for the food and drink, the type of food to be blended is not particularly limited, and it can be blended with, for example, coffee, fruit juice, soft drinks, alcoholic beverages such as beer, milk, miso soup, soup, black tea, tea, powdered drinks, nutrients, liquid (fluid) foods such as syrup, margarine, paste, jam, and solid foods such as rice, bread, potato products, mochi, furikake, ham, sausage, candy, chocolate, gum, gummy, snacks, baked goods, and other staple foods, side dishes, confectioneries, and seasonings. Depending on the application, it may be molded into the form of powder, granules, tablets, etc. Also, it can be blended with excipients, bulking agents, binders, thickeners, emulsifiers, colorants, flavorings, food additives, seasonings, etc., as necessary.

[0029] In addition to foods for human consumption, when the killed cells of Blautia bacteria are mixed into feed and administered to animals such as livestock and pets, they can be mixed into the raw materials of the feed in advance to prepare a functional feed. That is, the killed cells of Blautia bacteria can be added as an active ingredient to feed for livestock such as pigs, chickens, cows, horses, sheep, pets (dogs, cats, birds), and farmed fish such as sea bream, yellowtail, tuna, eel, and pufferfish, and used as functional feed.

[0030] Examples of functional foods include supplements, health drinks, health foods, nutritional supplements, health functional foods, nutritional functional foods, foods for specified health uses, foods with functional claims, food additive materials, etc. The product form of these products is not particularly limited, but they can be commercialized as tablets, capsules, granules, powders, and beverages, for example.

[0031] As a pharmaceutical product, it can be appropriately combined with a pharma- ceutical acceptable base material, etc., to form a pharmaceutical preparation, for example, in the form of a tablet, chewable tablet, capsule, granule, powder, pill, syrup, tincture, decoction, liquid, etc.

[0032] Cosmetics can be made into cosmetic preparations by appropriately combining with pharma- ceutically acceptable base materials, etc. Examples include lotions, skin lotions, creams, milky lotions, powders, foundations, packs, gels, jellies, aerosols, soaps, cleansing foams, bath agents, body soaps, sun care products, ointments, patches, bandages, etc.

[0033] As described above, the killed bacteria of the genus Blautia provided by the present invention can be made into various forms as probiotic materials or functional materials. In this case, other components can be contained in addition to the bacteria of the genus Blautia. Examples of other components include microbial cell materials of the genus Lactobacillus, Lacticaseibacillus, Lactiprantibacillus, Lactococcus, Bifidobacterium, Streptococcus, Enterococcus, Akkermansia, Christensenella, Clostridium, Bacteroides, Bacillus, Paraprevotella, Faecalibacterium, Lentilactobacillus, Eubacterium, Veillonella, etc., preferably killed bacterial materials thereof.

[0034] As described above, the killed cells of the Blautia bacteria provided by the present invention can be made into various forms as a probiotic material or functional material. According to the results of the Examples described later, the killed cells of the Blautia bacteria can be made into various forms when used as a material that exerts immunoactivating ability (active ingredient of an immunoactivator). In this case, the content of the killed cells of the Blautia bacteria in various forms may be appropriately determined in consideration of the relationship between the amount used in that form and the effective amount for exerting functionality. Typically, the content of the killed cells of the Blautia bacteria in terms of dry matter may be in the range of 0.001 to 100% by mass, 0.001 to 50% by mass, or 0.001 to 10% by mass. In addition, the content converted into the number of cells may be 2.0×10 7 ~2.0×10 12 cells / g, and may range from 2.0×10 7 ~1.0×10 12cells / g, and may range from 2.0×10 7 ~2.0×10 11 The cell density may range from 100 to 1000 cells / g.

[0035] When a human is to ingest the killed cells of the Blautia bacteria provided by the present invention, the dosage may be appropriately determined depending on the health condition and age of the subject, or the level of functionality required. Typically, the intake amount of lactic acid bacteria in terms of dry matter may be in the range of 0.0005 mg to 500 mg / day / kg body weight, 0.005 mg to 50 mg / day / kg body weight, or 0.05 mg to 5 mg / day / kg body weight. In addition, the content converted into the number of bacteria is 1.0×10 6 ~1.0×10 12 cells / day / kg body weight, and may range from 1.0×10 7 ~1.0×10 11 cells / day / kg body weight, and may range from 1.0×10 8 ~1.0×10 10 The range may be in the range of cells / day / kg body weight. EXAMPLES

[0036] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to the scope of these examples.

[0037] [Sample preparation] Blautia RD014892 strain was used as the Blautia bacteria and cultured in MRS medium at 37℃ under anaerobic conditions for 24 hours. The culture solution was centrifuged at 8000×g for 10 minutes, the supernatant was removed, and distilled water was added to suspend the bacteria, which was used as a sample (viable bacteria). On the other hand, the same culture solution was adjusted to pH 6.0 or pH 4.0 by adding acetic acid, and then heat-treated at 80℃ for 30 minutes. The bacteria were then similarly collected and suspended in distilled water to use as a heat-treated sample (pH 6.0 or pH 4.0).

[0038] [Test Example 1] (method) Spleens were harvested from BALB / cA mice (female, 10 weeks old) according to standard methods. Spleens were harvested in as sterile conditions as possible in a clean bench. The harvested spleens were then used to recover cells using a cell strainer (pore size: 100 μm), and the cells were then collected at a cell concentration of 2.5 × 10 6 The cells were prepared in liquid medium at a concentration of 1.0 μg / mL. The liquid medium used was RPMI-1640 (containing L-glutamine and phenol red, Fujifilm Wako Pure Chemical Industries) with a final concentration of 10% FBS (Thermo Fisher Scientific) and Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture (Thermo Fisher Scientific). Blautia bacteria (live bacteria), (heat-treated at pH 6.0), and (heat-treated at pH 4.0) were added to the cell solution at a final concentration of 1.0 μg / mL in terms of dry cells, and cultured at 37°C under a 5% CO2 environment. The concentrations of each cytokine in the supernatant after culture were measured by ELISA at 24 hours for IL-12 and 96 hours for IL-10. The mean and standard deviation of the measurements were calculated from 6 wells for IL-12 and 5 wells for IL-10. In addition, wells containing only spleen cells without adding Blautia bacteria served as controls.

[0039] (evaluation) As a result, as shown in Figure 1, live Blautia bacteria produced the same amounts of IL-12 and IL-10 as the control without added bacteria, whereas killed Blautia bacteria obtained by heat treatment significantly increased the production of these immunostimulatory factors.

[0040] Furthermore, as shown in FIG. 2, the ability of killed cells of Blautia bacteria to induce the production of immunostimulatory factors was particularly remarkable when the cells were heat-treated in an environment of pH 4.0.

[0041] [Test Example 2] (method) A spleen cell solution from a BALB / cA mouse (female, 10 weeks old) was prepared in the same manner as in Test Example 1. Each Blautia genus bacterium (live bacteria) and (pH 4.0, heat treatment at 80°C) were added to the cell solution so that the final concentration in terms of dry cells was 1.0 μg / mL, and the cells were cultured at 37°C and in a 5% CO2 environment. Six hours after the start of culture, the cells were collected, and RNA was extracted using an RNeasy Mini Kit (QIAGEN). Next, cDNA was synthesized from the obtained RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO). Using the synthesized cDNA and iTaq Universal SYBR Green Supermix (BIO RAD), the mRNA expression levels of various cytokines (TGF-β, IL-6, IFN-γ) were measured by real-time PCR, and the measured values ​​obtained were normalized by the mRNA expression level of β-actin. Separately, real-time PCR was performed in the same manner using spleen cells to which no Blautia bacteria had been added, and the relative values ​​of the mRNA expression levels of various cytokines were calculated using this as a standard.

[0042] (evaluation) As a result, as shown in Figure 3, when Blautia bacteria (pH 4.0, heat-treated at 80°C) were added, increased gene expression levels of various cytokines (TGF-β, IL-6, IFN-γ) known as immunostimulatory factors were observed compared to when Blautia bacteria (live bacteria) were added.

[0043] [Test Example 3] (method) After acclimating BALB / c mice for one week, they were allowed to freely consume purified feed (AIN-93G) containing each sample at the dose shown in the table below for one week. After that, feces were collected from the colon of the euthanized mice and IgA was measured by ELISA, and the concentration of short-chain fatty acids (succinic acid, acetic acid, propionic acid, and butyric acid) in the cecal contents was measured.

[0044] [Table 1]

[0045] (evaluation) ·Cecal contents medium short chain fatty acid concentration As shown in Figure 4, in the group administered Blautia bacteria (pH 4.0, heat-treated at 80°C), increases were observed in acetic acid, propionic acid, and butyric acid, which are known to have functional properties for improving the intestinal environment, while a decrease was observed in succinic acid, which is known to cause diarrhea, when compared to the control group and the group administered Blautia bacteria (live bacteria).

[0046] ·Fecal IgA concentration As shown in FIG. 5, an increase in IgA, an immune molecule, was observed in the group administered Blautia bacteria (pH 4.0, heat-treated at 80°C) compared to the control group and the group administered Blautia bacteria (live bacteria).

[0047] From the above, it was clarified that the functionality of Blautia in improving the intestinal environment can be enhanced by heat treatment under acidic conditions.

Claims

1. An immunostimulant comprising, as an active ingredient, killed bacteria obtained by heat-treating Blautia RD014892 strain (accession number: NITEBP-03954) of the genus Blautia under acidic conditions of pH 3.0 to 5.0 and temperature of 70 to 121°C.

2. The immunostimulant according to claim 1, wherein the killed cells of the Blautia bacterium have an IL-12 production induction ability measured by a method using mouse spleen cells that is 10 times or more higher than the IL-12 production induction ability of live cells of the Blautia bacterium under the same conditions.

3. The immunostimulant according to claim 2, wherein the killed cells of the Blautia bacterium further have an IL-10 production induction ability measured by a method using mouse spleen cells that is at least twice as high as the IL-10 production induction ability of live cells of the Blautia bacterium under the same conditions.

4. The immunostimulant according to any one of claims 1 to 3, which promotes intestinal IgA production.

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