Brouettia bacteria

Abstract

To provide a microbial cell material having excellent property.SOLUTION: This is a dead cell body of bacteria from the Blautia genus, characterized by being obtained by heat treatment of Blautia genus bacteria under acidic conditions. Additionally, it is a dead cell body of Blautia genus bacteria, where the IL-12 production induction capability measured using mouse spleen cells is more than ten times greater compared to the IL-12 production induction capability by live Blautia genus bacteria under the same conditions. It also refers to a method for producing the dead cell body of Blautia genus bacteria, characterized by subjecting live Blautia genus bacteria to heat treatment under acidic conditions.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 heat-treated cells of bacteria of the genus Blautia.

Background Art

[0002] By using lactic acid bacteria supplements, attempts are made to enhance the body's immunity and maintain health. For example, Patent Document 1 describes a preparation of lactic acid bacteria micronized to a nano size that 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 gut 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 the cells of bacteria belonging to the genus Bifidobacterium has not been studied much.

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

Means for Solving the Problems

[0008] As a result of various studies to achieve the above object, the present inventors have found that a preparation of bacteria belonging to the genus Bifidobacterium prepared under specific conditions is excellent in the ability to induce the production of immunostimulatory factors and the function of improving the intestinal environment, and have completed the present invention.

[0009] That is, in a first aspect of the present invention, there is provided a dead cell of a bacterium belonging to the genus Bifidobacterium, which is obtained by heat-treating the bacterium belonging to the genus Bifidobacterium under acidic conditions.

[0010] In the above-mentioned dead cell of the bacterium belonging to the genus Bifidobacterium, it is preferably obtained by heat treatment under the conditions of pH 3.0 to 7.0 and temperature 70 to 121°C.

[0011] Further, in a second aspect of the present invention, there is provided a dead cell of a bacterium belonging to the genus Bifidobacterium, wherein 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 live bacteria of the bacterium belonging to the genus Bifidobacterium under the same conditions.

[0012] In the above-mentioned dead cell of the bacterium belonging to the genus Bifidobacterium, it is preferable that 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 live bacteria of the bacterium belonging to the genus Bifidobacterium under the same conditions.

[0013] Furthermore, in its third aspect, the present invention provides a method for producing dead cells of B. bacterium, characterized by heat-treating viable cells of B. bacterium under acidic conditions.

[0014] In the method for producing dead cells of B. bacterium, it is preferable to perform heat treatment under the conditions of pH 3.0 to 7.0 and a temperature of 70 to 121°C.

Advantages of the Invention

[0015] According to the present invention, it is possible to provide a microbial cell material having excellent properties by using B. bacterium.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

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Embodiments for Carrying Out the Invention

[0017] As used herein, the term "bacteria of the genus Blautia" refers to bacteria belonging to the genus Blautia, which is 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 individually 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 the B. producta may preferably include the B. RD014892 strain (accession number: NITE BP-03954) deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NPMD), or bacteria substantially identical thereto. Here, the term "substantially identical" bacteria is synonymous with the meaning 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 the B. RD014892 strain, and still has the same mycological properties as the B. RD014892 strain, etc.

[0019] The culture of B. bacteria, the maintenance of the bacterial cells, etc. can be carried out by well-known means. For example, as the 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. The culture can be carried out by inoculating the bacterial cells into the above medium and then, for example, statically culturing or aerobically stirring and culturing under the conditions of 25 to 40 °C. For the preservation of live bacterial cells, for short periods, they can be refrigerated while suspended in the medium, or for long periods, they can be suspended in an antifreeze solution such as a glycerol solution and then cryopreserved.

[0020] When preparing B. bacteria, depending on the state of the culture solution after culturing, the culture solution 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 to a predetermined cell concentration to prepare a cell concentrate. The content of the bacterial cells of B. bacteria in 100 parts by mass of the 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 in terms of dry bacterial cells. An excipient may be contained in this cell concentrate. According to this, the properties of the bacterial cells are likely to be maintained after freezing, freeze-drying, or reconstitution with water.

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

[0022] Also, as another aspect, when preparing the bacteria of the genus Bruitia, a pulverization / dispersion treatment may be performed. The pulverization / dispersion treatment can be carried out, for example, by pulverizing and dispersing the above-mentioned bacterial cell concentrate using means such as stirring, mixer, homogenizer, ball mill, bead mill, jet mill, generator, etc. In this case, depending on the situation or as necessary, by adding the above-mentioned excipient and then performing the pulverization / dispersion treatment, re-aggregation of the obtained bacterial powder can be prevented. When containing the 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, or may be in the range of 20 to 90% by mass.

[0023] Also, as yet another aspect, when preparing the bacteria of the genus Bruitia, a drying and powdering treatment may be performed. Examples of the drying and powdering method include techniques such as freeze-drying, vacuum spray drying, spray drying using hot air, etc. Note that by performing spray drying (spray dry) using hot air, usually, the activity of live bacteria is lost and the dead bacterial cells can be obtained.

[0024] In the present invention, among the Bifidobacterium bacteria that can be prepared as described above, in particular, a preparation obtained by heat-treating under acidic conditions is provided. According to the preparation obtained through such treatment, usually, the activity of live bacteria is lost and it becomes dead cells of Bifidobacterium bacteria, so that changes in quality associated with live bacteria can be suppressed. Also, as shown in the examples described later, the dead cells of Bifidobacterium bacteria have the ability to induce the production of immune activation factors (such as 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 and the like for maintaining the health of healthy individuals. From another perspective, it can be said that the present invention provides functional materials and related components that can be used in health foods and supplements for promoting health.

[0025] The heat treatment of Bifidobacterium bacteria 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, for example, treating it in a jacketed tank, a constant temperature bath, an autoclave, etc. At that 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. As the temperature conditions for the heat treatment, although not limited, for example, it may be 70 to 121°C, it may be 80 to 110°C, or it may be 80 to 100°C. As the heat treatment time, although not limited, 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, there is a tendency for the desired functionality such as immune activation ability to be poor, which is not preferable.

[0026] To determine whether the Bacteroides bacteria provided by the present invention have been sufficiently processed in the above-described environment, it is sufficient to check their preparation history. 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 viable Bacteroides bacteria 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 viable Bacteroides bacteria under the same conditions. It is also possible to make a determination from such a perspective of functionality.

[0027] The dead cells of the Bacteroides bacteria provided by the present invention can be used, if desired, in various product forms such as foods and drinks, functional foods, pharmaceuticals, cosmetics, animal feeds, and the like.

[0028] There are no particular restrictions on the types of foods and drinks to be formulated. For example, liquid (flowing) foods such as coffee, fruit juice, soft drinks, alcoholic beverages such as beer, milk, miso soup, soup, black tea, tea, powdered drinks, nutritional supplements, syrups, margarine, paste, jam, etc., staple foods such as cooked rice, bread, potato products, mochi, furikake, ham, sausage, candy, chocolate, gum, gummy, snack foods, baked confectioneries, etc., side dishes, confectioneries, and seasonings can also be formulated. Depending on the use, it may be formed into powders, granules, tablets, etc. Further, if necessary, it can also be formulated with excipients, bulking agents, binders, thickeners, emulsifiers, coloring agents, fragrances, food additives, seasonings, etc.

[0029] In addition to foods for human consumption, when dead cells of bacteria belonging to the genus Blautia 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 a functional feed. That is, using dead cells of bacteria belonging to the genus Blautia 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, yellowtail, tuna, eel, pufferfish, etc., and can be used as a 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 labeling, 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, granules, powders, and beverages.

[0031] As pharmaceuticals, they can be formulated into pharmaceutical preparations in appropriate combination with pharmaceutically acceptable substrates, etc. For example, they can be in the form of tablets, chewables, capsules, granules, powders, pills, syrups, tinctures, decoctions, liquids, etc.

[0032] As cosmetics, they can be formulated into cosmetic preparations in appropriate combination with pharmaceutically acceptable substrates, etc. For example, lotions, toners, creams, milks, powders, foundations, packs, gels, jellies, aerosols, soaps, cleansing foams, bath agents, body soaps, sun care products, ointments, patches, band-aids, etc.

[0033] As described above, the dead cells of the Blautia bacteria 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 Blautia bacteria. Examples of other components include microbial cell materials such as those of the genera Lactobacillus, Lactiplantibacillus, Lactococcus, Bifidobacterium, Streptococcus, Enterococcus, Akkermansia, Christensenella, Clostridium, Bacteroides, Bacillus, Paraprevotella, Faecalibacterium, Lentilactobacillus, Eubacterium, and Veillonella, preferably dead cell materials thereof, and the like.

[0034] As described above, the dead cells of the Blautia bacteria provided by the present invention can be made into various forms as probiotic materials or functional materials. In that case, the content of the dead cells of such Blautia bacteria 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, the content in terms of the dry matter of the dead cells of the Blautia bacteria may be in the range of 0.001 to 100% by mass, may be in the range of 0.001 to 50% by mass, or may be in the range of 0.001 to 10% by mass. 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, may be in the range of 2.0×10 7 ~1.0×10 12 cells / g, or may be in the range of 2.0×10 7 ~2.0×10 11 cells / g.

[0035] When a human ingests the heat-killed cells of the bacteria of the genus Blautia provided by the present invention, the dosage may be appropriately set according to the health condition, age of the subject, or the degree of functionality required. Typically, it may be in the range of 0.0005 mg to 500 mg / day / kg body weight, in the range of 0.005 mg to 50 mg / day / kg body weight, or in the range of 0.05 mg to 5 mg / day / kg body weight, in terms of the intake amount of the dried product of lactic acid bacteria. Further, in terms of the content converted into 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, or 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] [Sample Preparation] Blautia sp. RD014892 strain was used as the bacteria of the genus Blautia 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, distilled water was added to suspend the cells, which was used as a sample (live cells). On the other hand, for the same culture solution, after adjusting the pH to 6.0 or pH 4.0 by adding acetic acid, it was heat-treated at 80°C for 30 minutes, and then the cells were recovered and suspended in distilled water in the same manner, which was used as a heat-treated sample (pH 6.0 or pH 4.0).

[0038] [Test Example 1] (Method) According to a conventional method, the spleen was collected from BALB / cA mice (female, 10 weeks old). The spleen was collected as aseptically as possible in a clean bench. The collected spleen was used to recover cells with a cell strainer (pore size: 100 μm), and then the cell concentration was adjusted to 2.5×106 It was prepared in a liquid medium to a concentration of cells / mL. The liquid medium used was prepared by appropriately mixing RPMI-1640 (containing L-glutamine and phenol red, manufactured by 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). To this cell suspension, live bacteria of the genus Blautia, (heat-treated at pH 6.0), and (heat-treated at pH 4.0) were added 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 an environment of 5% CO2. Then, for IL-12, the concentration of each cytokine contained in the supernatant after culture was measured by ELISA at 24 hours from the start of culture, and for IL-10, it was measured at 96 hours from the start of culture. For IL-12, the average value and standard deviation were calculated from 6 wells, and for IL-10, they were calculated from 5 wells. Also, wells containing only spleen cells without the addition of Blautia bacteria were used as controls.

[0039] (Evaluation) As a result, as shown in Figure 1, for live bacteria of the genus Blautia, the production amounts of both IL-12 and IL-10 were at the same level as those of the control without the addition of bacteria. In contrast, for heat-killed bacteria of the genus Blautia obtained by heat treatment, the production amounts of these immune-activating factors were significantly increased.

[0040] Also, as shown in Figure 2, the ability to induce the production of immune-activating factors by heat-killed bacteria of the genus Blautia was particularly remarkable when heat treatment was carried out in an environment of pH 4.0.

[0041] [Test Example 2] (Method) In the same manner as in Test Example 1, a splenocyte solution of BALB / cA mice (female, 10 weeks old) was prepared. Each bacterium of the genus Blautia (live bacteria) and (heat-treated at 80 °C, pH 4.0) was added to the cell solution 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 measurement 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 bacteria of the genus Blautia were not added, and based on this, the 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 80 °C, pH 4.0) 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 dosage shown in the following table for one week. Thereafter, 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 group administered with Blautia bacteria (heated at pH 4.0 and 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 group administered with 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 group administered with Blautia bacteria (heated at pH 4.0 and 80°C), an increase in IgA, an immune molecule, was observed when compared with the control group and the group administered with Blautia bacteria (live bacteria).

[0047] From the above, it was clarified that heating Blautia bacteria under acidic conditions enhances the functionality of improving the intestinal environment.

Claims

1. A dead cell of Bacteroides bacteria, characterized in that it is obtained by heat-treating Bacteroides sp. RD014892 strain (Accession No.: NITE BP-03954) as Bacteroides bacteria under acidic conditions of pH 3.0 to 5.0 and a temperature of 70 to 121°C.

2. The dead cell of Bacteroides bacteria according to Claim 1, wherein the ability to induce IL-12 production measured by a method using mouse spleen cells is 10 times or more as compared with the ability to induce IL-12 production by viable cells of Bacteroides bacteria under the same conditions.

3. The dead cell of Bacteroides bacteria according to Claim 2, wherein the dead cell of Bacteroides bacteria further has an ability to induce IL-10 production measured by a method using mouse spleen cells, which is 2 times or more as compared with the ability to induce IL-10 production by viable cells of Bacteroides bacteria under the same conditions.

4. A method for producing a dead cell of Bacteroides bacteria, characterized in that it comprises heat-treating viable cells of Bacteroides sp. RD014892 strain (Accession No.: NITE BP-03954) as Bacteroides bacteria under acidic conditions of pH 3.0 to 5.0 and a temperature of 70 to 121°C.

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