Immunopotentiator using bacteria of the genus Blautia

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 bacteria of the genus Blautia, and more particularly to the use of bacteria of the genus Blautia as immunostimulants.

Background Art

[0002] The use of lactic acid bacteria supplements has been carried out to enhance the body's immunity and maintain health. For example, Patent Document 1 describes a preparation of lactic acid bacteria micronized to a nanosize, which can be used as such lactic acid bacteria.

[0003] On the other hand, in recent years, as a result of data analysis targeting humans, there is a finding that Blautia bacteria, which are one of the intestinal bacteria, are inversely correlated with BMI and diabetes risk (see Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Conventionally, the use of bacteria belonging to the genus Blautia has not been studied much.

[0007] An object of the present invention is to provide a microbial cell material having excellent properties as an immunostimulatory material by using bacteria belonging to the genus Blautia.

Means for Solving the Problems

[0008] As a result of various studies to achieve the above object, the present inventors have found that dead cells of bacteria belonging to the genus Blautia are excellent in immunostimulatory ability, and have completed the present invention.

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

[0010] In the above immunostimulant, it is preferable that the dead cells of the bacteria belonging to the genus Blautia are obtained by heat treatment under acidic conditions.

[0011] Also, it is preferable that the dead cells of the bacteria belonging to the genus Blautia are obtained by heat treatment under the conditions of pH 3.0 to 7.0 and temperature 70 to 121°C.

[0012] Also, it is preferable that the dead cells of the bacteria belonging to the genus Blautia 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 bacteria of the genus Blautia under the same conditions.

[0013] Also, in addition to the ability to induce IL-12 production, the dead cells of the bacteria belonging to the genus Blautia preferably have an ability to induce IL-10 production measured by a method using mouse spleen cells that is 2 times or more higher than the ability to induce IL-10 production by live bacteria of the genus Blautia under the same conditions.

[0014] Also, the immunostimulant provided by the present invention preferably promotes intestinal IgA production.

Effects of the Invention

[0015] According to the present invention, by using bacteria of the genus Bruitia, it is possible to provide a microbial cell material having excellent properties as an immune activating material.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

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Mode for Carrying Out the Invention

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

[0018] Examples of Blautia producta preferably include Blautia strain RD014892 (Accession No.: NITE BP-03954) deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NPMD), or bacteria substantially identical thereto. Here, "substantially identical" bacteria have the same meaning as understood by those skilled in the art. For example, the nucleotide sequence of the 16S rRNA gene for identifying the genus and species of bacteria has 98% or more, preferably 99% or more homology with the nucleotide sequence of the 16S rRNA gene of Blautia strain RD014892, and also has the same mycological properties as Blautia strain RD014892, etc.

[0019] The cultivation of bacteria belonging to the genus Blautia, the maintenance of bacterial cells, etc. can be carried out by well-known means. For example, as the culture medium, a liquid medium containing yeast extract, peptone, meat extract, amino acids, salts, minerals, etc. can be mentioned. A commercially available medium such as "Modified GAM" (trade name, Modified GAM Broth, Nissui Pharmaceutical Co., Ltd.) may also be used. Cultivation can be carried out by inoculating the bacterial cells into the above medium and then, for example, performing static cultivation or aeration agitation cultivation under conditions of 25 to 40 °C. For the preservation of live bacterial cells, in the case of a short period, it can be refrigerated while suspended in the medium, or in the case of a long period, it can be suspended in an antifreeze liquid such as a glycerol solution and then cryopreserved.

[0020] When preparing bacteria belonging to the genus Blautia, depending on the state of the culture broth after cultivation, the culture broth can be concentrated as it is, or the bacteria can be collected by means such as centrifugation or filtration, and the bacterial cells are further washed with purified water or the like and then suspended in purified water or the like so as to reach a predetermined bacterial cell concentration, whereby a bacterial cell concentrate can be prepared. The content of the bacterial cells of bacteria belonging to 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 in terms of dry bacterial cells, may be in the range of 0.5 to 25 parts by mass, or may be in the range of 1 to 10 parts by mass. An excipient may be contained in this bacterial cell concentrate. According to this, even after freezing or freeze-drying and after reconstitution with water, the properties of the bacterial cells are likely to be maintained.

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

[0022] As another aspect, during the preparation of bacteria of the genus Bifidobacterium, a grinding and dispersion treatment may be performed. The grinding and dispersion treatment can be carried out, for example, by grinding and dispersing the above-described bacterial cell concentrate using means such as stirring, a mixer, a homogenizer, a ball mill, a bead mill, a jet mill, a generator, etc. In this case, depending on the circumstances or as necessary, by adding the above-described excipient and then performing the grinding and dispersion treatment, re-aggregation of the obtained bacterial powder can be prevented. When containing an excipient, the content thereof may be in the range of 1 to 99% by mass in terms of dry matter, may be in the range of 10 to 95% by mass, and may be in the range of 20 to 90% by mass.

[0023] Furthermore, as yet another aspect, during the preparation of bacteria of the genus Bifidobacterium, a drying and powdering treatment may be performed. Examples of the drying and powdering method include freeze-drying, vacuum spray drying, spray drying using hot air, etc. By performing spray drying (spray dry) using hot air, usually, the activity of live bacteria is lost and dead bacterial cells can be obtained.

[0024] In the present invention, among the bacteria of the genus Bifidobacterium that can be prepared as described above, in particular, a preparation obtained by heat treatment under acidic conditions is provided. According to the preparation obtained through such treatment, usually, the activity of live bacteria is lost and it has become dead bacterial cells of the genus Bifidobacterium, so that changes in quality associated with live bacteria can be suppressed. Also, as shown in the examples described later, dead bacterial cells of the genus Bifidobacterium have the ability to induce the production of immune activation factors (IL-12, IL-10, TGF-β, IL-6, IFN-γ, etc.) in immune cells and the functionality to improve the intestinal environment and induce the production of IgA, which is an immune molecule. Therefore, for example, it can be suitably used as an active ingredient of a functional composition for enhancing the body's immunity and maintaining health. In particular, it can be suitably used for functional foods, etc. for maintaining the health of healthy individuals. From another perspective, it can be said that the present invention provides a functional material and a related component that can be used for health foods and supplements for health promotion. Furthermore, it can be said that the present invention provides an immune activator containing dead bacterial cells of the genus Bifidobacterium as an active ingredient.

[0025] The heat treatment of the bacteria of the genus Blautia can be carried out by subjecting the culture broth after culturing as it is or, if necessary, preparing it into the above-mentioned cell concentrate, and then subjecting it to treatments such as placing it in a jacketed tank, a thermostatic bath, an autoclave, etc. At this time, the pH is not limited, but for example, it may be pH 3.0 to 7.0, it may be pH 3.0 to 6.0, or it may be pH 3.0 to 5.0. The temperature conditions for the heat treatment are not limited, but for example, it may be 70 to 121 °C, it may be 80 to 110 °C, or it may be 80 to 100 °C. The heat treatment time is not limited, but for example, it may be 30 minutes to 120 hours, it may be 30 minutes to 90 minutes, or it may be 30 minutes to 60 minutes. If the treatment under such an environment is not sufficient, the desired functionality such as immunostimulating ability tends to be poor, which is not preferable.

[0026] In order to determine whether the bacteria of the genus Blautia provided by the present invention have been sufficiently treated under the above-mentioned environment, the preparation history may be confirmed. Alternatively, in some cases, it is also possible to make a determination from the perspective of functionality. For example, whether the ability to induce IL-12 production measured by a method using mouse spleen cells is 10 times or more compared to the ability to induce IL-12 production by the live bacteria of the genus Blautia under the same conditions, or whether the ability to induce IL-10 production measured by a method using mouse spleen cells is 2 times or more compared to the ability to induce IL-10 production by the live bacteria of the genus Blautia under the same conditions. It is also possible to make a determination from such a perspective of functionality.

[0027] The heat-killed cells of the bacteria of the genus Blautia provided by the present invention can be used in various product forms such as foods and drinks, functional foods, pharmaceuticals, cosmetics, animal feeds, etc., if desired.

[0028] As for food and drink products, there are no particular restrictions on the types of foods to be blended. For example, coffee, fruit juice, soft drinks, alcoholic beverages such as beer, milk, miso soup, soup, black tea, tea, powdered drinks, nutritional supplements, syrups, margarine, paste, jams and other liquid (flowing) foods, staple foods such as cooked rice, bread, potato products, mochi, furikake, ham, sausage, candies, chocolate, gum, gummies, snack foods, baked confectioneries and other solid-shaped foods, as well as side dishes, confectioneries and seasonings can also be blended. Depending on the application, it may be formed into powders, granules, tablets and other forms. Also, if necessary, it can be blended with excipients, bulking agents, binders, thickeners, emulsifiers, colorants, fragrances, food additives, seasonings and the like.

[0029] In addition to foods for human consumption, when the heat-killed cells of B. bacterium are mixed into feed and administered to animals such as livestock and pets, they can be premixed into the raw materials of the feed and prepared as functional feed. That is, using the heat-killed cells of B. bacterium as an active ingredient, it can be added to feeds for livestock such as pigs, chickens, cows, horses, sheep, etc., pets (dogs, cats, birds), etc., and fish for aquaculture such as sea bream, tuna, eel, pufferfish, etc., and used as functional feed.

[0030] Examples of functional foods include supplements, health drinks, health foods, dietary supplements, foods with health claims, foods with nutritional claims, foods for specified health uses, foods with functional claims, raw materials for food additives, etc. These product forms are not particularly limited, but for example, they can be commercialized in the form of tablets, capsules, granule agents, powders, and drinks.

[0031] As for pharmaceuticals, they can be made into pharmaceutical preparations in combination with pharmaceutically acceptable substrates and the like as appropriate. For example, they can be in the form of tablets, chewables, capsules, granule agents, powders, pills, syrups, tinctures, decoctions, liquid preparations and the like.

[0032] As cosmetics, they can be formulated into cosmetic preparations as appropriate in combination with pharmaceutically acceptable base materials and the like. For example, lotions, toners, creams, milks, powders, foundations, packs, gels, jellies, aerosols, soaps, cleansing foams, bath agents, body soaps, sun care products, ointments, patches, bandages, etc. can be mentioned.

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

[0034] As described above, the dead cells of the bacteria of the genus Blautia provided by the present invention can be made into various forms as probiotic materials or functional materials. And according to the results of the examples described later, it can be made into various forms when used as a material that exhibits immune activation ability (the active ingredient of an immunostimulant). In that case, the content of the dead cells of the bacteria of the genus Blautia may be appropriately determined in consideration of the relationship between the amount used in that form and the effective amount for exerting the function when made into various forms. Typically, it 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 terms of the content of the dried product of the dead cells of the bacteria of the genus Blautia. Also, in terms of the content converted to the number of cells, it may be in the range of 2.0×10 7 ~2.0×10 12 cells / g, or 2.0×10 7 ~1.0×10 12It may be in the range of cells / g, 2.0×10 7 ~2.0×10 11 cells / g.

[0035] When a human ingests the dead cells of the bacteria of the genus Bruitia provided by the present invention, the dosage may be appropriately set according to the health status, age of the subject, or the degree of functionality required. Typically, in terms of the intake amount in terms of the dried product of lactic acid bacteria, it may be in the range of 0.0005 mg to 500 mg / day / kg body weight, may be in the range of 0.005 mg to 50 mg / day / kg body weight, and may be in the range of 0.05 mg to 5 mg / day / kg body weight. Also, in terms of the content converted to the number of cells, it may be in the range of 1.0×10 6 ~1.0×10 12 cells / day / kg body weight, may be in the range of 1.0×10 7 ~1.0×10 11 cells / day / kg body weight, and may be in the range of 1.0×10 8 ~1.0×10 10 cells / day / kg body weight. [Examples]

[0036] Hereinafter, the present invention will be described more specifically with reference to examples, but the scope of the present invention is not limited to the scope of these examples.

[0037] [Preparation of Samples] Bruitia sp. RD014892 strain was used as the bacteria of the genus Bruitia and cultured in MRS medium at 37°C under anaerobic conditions for 24 hours. The culture solution was centrifuged at 8000×g for 10 minutes, and after removing the supernatant, the cells were suspended in distilled water to obtain a sample (live bacteria). On the other hand, for the same culture solution, acetic acid was added to adjust the pH to 6.0 or pH 4.0, and then heat-treated at 80°C for 30 minutes. After that, the cells were recovered in the same manner and suspended in distilled water to obtain a heat-treated sample (pH 6.0 or pH 4.0).

[0038] [Test Example 1] [Method] Spleens were collected from BALB / cA mice (female, 10 weeks old) according to a conventional method. The spleen collection was performed under aseptic conditions as much as possible in a clean bench. After the collected spleen was passed through a cell strainer (pore size: 100 μm) to recover cells, it was prepared with a liquid medium so that the cell concentration became 2.5×10 6 cells / mL. The liquid medium used was prepared by appropriately mixing RPMI-1640 (containing L-glutamine and phenol red, FUJIFILM Wako Pure Chemical Corporation) with FBS (Thermo Fisher Scientific) at a final concentration of 10% and Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture (Thermo Fisher Scientific). Each of the bacteria of the genus Bifidobacterium (live bacteria), (heat-treated at pH 6.0), and (heat-treated at pH 4.0) was added to the cell suspension so that the final concentration in terms of dry cell weight became 1.0 μg / mL, and the cells were cultured at 37°C in an environment of 5% CO2. Then, for IL-12, the cytokine concentration contained in the supernatant after culture was measured by ELISA at the 24-hour time point from the start of culture, and for IL-10, the cytokine concentration was measured by ELISA at the 96-hour time point from the start of culture. For IL-12, the average value and standard deviation were calculated from 6 wells, and for IL-10, the average value and standard deviation were calculated from 5 wells. Also, wells with only spleen cells to which no bacteria of the genus Bifidobacterium were added were used as controls.

[0039] (Evaluation) As a result, as shown in Fig. 1, for both live bacteria of the genus Bifidobacterium and IL-10, the production amounts were about the same as those of the control without adding the bacterial cells, whereas for the heat-treated dead bacterial cells of the genus Bifidobacterium, the production amounts of these immune activation factors were significantly increased.

[0040] Also, as shown in Fig. 2, the ability to induce the production of immune activation factors by the heat-treated dead bacterial cells of the genus Bifidobacterium was particularly remarkable when heat-treated in an environment of pH 4.0.

[0041] [Test Example 2] (Method) In the same manner as in Test Example 1, splenocyte lysates of BALB / cA mice (female, 10 weeks old) were prepared. Each bacterium of the genus Blautia (live bacteria) and (heat-treated at pH 4.0 and 80 °C) was added to the cell lysate so that the final concentration in terms of dry cell mass was 1.0 μg / mL, and the mixture was cultured at 37 °C in a 5% CO₂ environment. Six hours after the start of the culture, the cells were collected, and RNA was extracted using the 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 obtained measured values were normalized by the mRNA expression level of β-actin. On the other hand, separately, real-time PCR was performed in the same manner using splenocytes to which no bacterium of the genus Blautia was added, and based on this, relative values of the mRNA expression levels of various cytokines were calculated.

[0042] (Evaluation) As a result, as shown in Figure 3, when Blautia bacteria (heat-treated at pH 4.0 and 80 °C) were added, an increase in the gene expression levels of various cytokines (TGF-β, IL-6, IFN-γ) known as immunostimulatory factors was observed compared to the case where 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 a purified diet (AIN-93G) formulated to the dosages shown in the table below for each sample for one week. Then, feces in the colon were collected from the euthanized mice, and IgA was measured by the ELISA method, and the cecal contents were measured for the concentrations of short-chain fatty acids (succinic acid, acetic acid, propionic acid, butyric acid).

[0044]

Table 1

[0045] (Evaluation) · Concentration of short-chain fatty acids in cecal contents As shown in Fig. 4, in the administration group of Blautia bacteria (pH 4.0, heat-treated at 80°C), increases in acetic acid, propionic acid, and butyric acid, which are known to have the function of improving the intestinal environment, were observed when compared with the control group and the administration group of Blautia bacteria (live bacteria). On the other hand, a decrease in succinic acid, which is known to cause diarrhea and the like, was observed.

[0046] · Concentration of IgA in feces As shown in Fig. 5, in the administration group of Blautia bacteria (pH 4.0, heat-treated at 80°C), an increase in IgA, an immune molecule, was observed when compared with the control group and the administration group of Blautia bacteria (live bacteria).

[0047] From the above, it became clear that the functionality of improving the intestinal environment is enhanced by heat-treating Blautia bacteria under acidic conditions.

Claims

1. An immunopotentiator comprising a heat-killed cell body obtained by heat-treating Bacteroides RD014892 strain (accession number: NITE BP-03954), which is a bacterium belonging to the genus Bacteroides, under acidic conditions of pH 3.0 to 5.0 and a temperature of 70 to 121°C, as an active ingredient.

2. The immunopotentiator according to Claim 1, wherein the heat-killed cell body of the bacterium belonging to the genus Bacteroides has an ability to induce IL-12 production, measured by a method using mouse spleen cells, that is 10 times or more as compared with the ability to induce IL-12 production by the live bacterium of the bacterium belonging to the genus Bacteroides under the same conditions.

3. The immunopotentiator according to Claim 2, wherein the heat-killed cell body of the bacterium belonging to the genus Bacteroides further has an ability to induce IL-10 production, measured by a method using mouse spleen cells, that is 2 times or more as compared with the ability to induce IL-10 production by the live bacterium of the bacterium belonging to the genus Bacteroides under the same conditions.

4. The immunopotentiator according to any one of Claims 1 to 3, wherein the immunopotentiator promotes intestinal IgA production.

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