Immunostimulant using bacteria of genus blautia

Abstract

Provided is a microbial cell material having exceptional properties as an immunostimulating material. This immunostimulant has dead cell of bacteria belonging to the genus Blautia as an active ingredient. The dead cells are preferably obtained by means of heat treatment under acidic conditions. Additionally, the dead cells are preferably obtained by means of heat treatment at a pH of 3.0-7.0 and a temperature of 70-121°C.

Description

Immunostimulant using Blautia bacteria

[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 an immunostimulant.

[0002] Lactic acid bacteria supplements are used to enhance 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, which are microparticulated to nanosize 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).

[0004] Patent No. 4621218

[0005] 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

[0006] Hitherto, there has been little research into the use of bacteria of 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.

[0008] As a result of various studies conducted by the present inventors to achieve the above object, they discovered that killed cells of bacteria of the genus Blautia have excellent immunopotentiating properties, which 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 immunopotentiator, the killed cells of the bacterium of the genus Blautia are preferably obtained by heat treatment under acidic conditions.

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

[0012] Furthermore, it is preferable that the killed cells of the bacterium of the genus Blautia 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 bacterium of the genus Blautia under the same conditions.

[0013] Furthermore, it is preferable that the killed cells of the Blautia bacteria have, in addition to the above-mentioned ability to induce IL-12 production, 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] Furthermore, the immunostimulant provided by the present invention is preferably one that promotes intestinal IgA production.

[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.

[0016] 1(a) and 1(b) are diagrams 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 diagram showing the results of investigating the ability of spleen cells to induce IL-12 production, and FIG. 1(b) is a diagram showing the results of investigating the ability of spleen cells to induce IL-10 production. FIG. 2(a) and 2(b) are diagrams 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 culture system for mouse spleen cells in Test Example 1, where FIG. 2(a) is a diagram showing the results of investigating the ability of spleen cells to induce IL-12 production, and FIG. 2(b) is a diagram showing the results of investigating the ability of spleen cells to induce IL-10 production.

[0033] Figure 1 is a graph showing the results of investigating the effect of adding Blautia bacteria (live bacteria or killed bacteria in an environment of pH 4.0) to a culture medium in a mouse spleen cell culture system, and showing the results of investigating the expression level of cytokines, in Test Example 3.

[0034] Figure 1 is a graph showing the results of investigating the effect of adding Blautia bacteria (live bacteria or killed bacteria in an environment of pH 4.0) to a feed and allowing BALB / c mice to freely ingest the feed, and showing the results of investigating the concentration of short-chain fatty acids in the cecal contents, in Test Example 2.

[0035] Figure 1 is a graph showing the results of investigating the effect of adding Blautia bacteria (live bacteria or killed bacteria in an environment of pH 4.0) to a feed and allowing BALB / c mice to freely ingest the feed, and showing the results of investigating the fecal IgA concentration, in Test Example 2.

[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 hensenii, Blautia Examples of suitable species include Blautia hansenii, Blautia luti, and Blautia wexlerae. These may be used alone or in combination of two or more. Among these, Blautia producta is particularly preferred from the viewpoint of immunostimulatory effect.

[0018] [Correction based on Rule 91 26.09.2024] Preferred examples of the Blautia product include the Blautia RD014892 strain (accession number: NITE BP-03954, date of deposit: August 1, 2023) deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD) (National Institute of Technology and Evaluation Patent Microorganisms Depositary, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Postal Code 292-0818), or bacteria substantially identical thereto. Here, "substantially the same" bacterium has the same meaning as understood by those skilled in the art, and refers to, for example, a bacterium whose 16S rRNA gene base sequence for identifying the genus and species of the bacterium has 98% or more, preferably 99% or more, homology with the 16S rRNA gene base sequence of the Blautia strain RD014892, and which also has the same mycological properties as the Blautia strain RD014892.

[0019] Cultivation and maintenance of Blautia bacteria can be carried out by well-known means. Examples of culture media include liquid media containing yeast extract, peptone, meat extract, amino acids, salts, minerals, etc. Commercially available media such as "Modified GAM" (trade name: Modified GAM Bouillon, Nissui Co., Ltd.) may also be used. Culture can be carried out by inoculating the cells into the medium and then culturing them statically or by culturing them with aeration and agitation at temperatures ranging from 25 to 40°C. Live cells can be stored in a suspension in the medium for short periods of time by refrigeration, or suspended in an antifreeze solution such as glycerol and frozen for long periods of time.

[0020] When preparing bacteria of the genus Blautia, a bacterial cell concentrate can be prepared by directly concentrating the culture solution after cultivation, or by collecting the bacteria by means of centrifugation, filtration, or the like, washing the bacteria with purified water, etc., and suspending them in purified water, etc., to a predetermined bacterial cell concentration. The content of bacteria of the genus Blautia per 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 allows the properties of the bacteria to be more easily maintained after reconstitution with water, even after freezing or lyophilization.

[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 milk sugar; 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 preparation of Blautia bacteria may involve pulverization and dispersion. The pulverization and dispersion can be carried out, for example, by pulverizing 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, or a generator. In this case, depending on the case or as needed, the above-described excipient may be added before the pulverization and dispersion, thereby preventing reagglomeration of the resulting bacterial powder. When an excipient is contained, its content 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 Blautia bacteria, they may be subjected to a dry powdering process. Examples of the dry powdering method include freeze-drying, reduced pressure spray drying, spray drying using hot air, etc. Spray drying using hot air usually destroys the activity of live bacteria, and dead bacteria can be obtained.

[0024] The present invention provides a preparation of Blautia bacteria that can be prepared as described above, particularly a preparation obtained by heat treatment under acidic conditions. Preparations obtained through such treatment typically lose the activity of live bacteria, resulting in killed Blautia bacteria, thereby suppressing the quality changes associated with live bacteria. Furthermore, as shown in the examples below, killed Blautia bacteria 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 ability to improve the intestinal environment and induce the production of IgA, an immune molecule. Therefore, for example, they can be suitably used as an active ingredient in functional compositions for enhancing the body's immune system and maintaining health. In particular, they can be suitably used in functional foods for maintaining the health of healthy individuals. From another perspective, the present invention can be said to provide functional materials and components that can be used in health foods and supplements for promoting health. Furthermore, it can be said to provide an immunostimulant containing killed Blautia bacteria as an active ingredient.

[0025] Heat treatment of Blautia bacteria can be carried out by subjecting the culture solution after cultivation directly, or, if necessary, after preparing the culture solution into the above-mentioned bacterial cell concentrate, for example, in a jacketed tank, a thermostatic bath, or an autoclave. The pH is not limited, but may be, for example, 3.0 to 7.0, 3.0 to 6.0, or 3.0 to 5.0. The temperature conditions for the heat treatment are 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. Insufficient treatment under such conditions is undesirable, as it tends to result in a lack of desired functionality, such as immunostimulatory activity.

[0026] To determine whether the Blautia bacteria provided by the present invention have been sufficiently treated under the above-mentioned environment, the preparation history of the bacteria can be confirmed. Alternatively, in some cases, it is also possible to make a determination from the standpoint of functionality. For example, it is also possible to make a determination from the standpoint of functionality by determining whether the IL-12 production induction ability measured by a method using mouse spleen cells is 10-fold or more compared to 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-fold or more compared to the IL-10 production induction ability of live Blautia bacteria under the same conditions.

[0027] The 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, and animal feed, as desired.

[0028] There are no particular limitations on the type of food or beverage to be blended, and the composition can be blended into, for example, coffee, fruit juice, soft drinks, alcoholic beverages such as beer, milk, miso soup, soup, black tea, green tea, powdered drinks, nutritional supplements, liquid (fluid) foods such as syrup, margarine, paste, and jam, and solid foods such as cooked rice, bread, potato products, rice cakes, furikake (rice toppings), ham, sausage, candy, chocolate, chewing gum, gummy candy, snacks, and baked goods, as well as staple foods, side dishes, confectioneries, and seasonings. Depending on the application, the composition may be molded into powder, granules, tablets, or the like. Furthermore, the composition can be blended into excipients, bulking agents, binders, thickeners, emulsifiers, colorants, flavorings, food additives, seasonings, and the like, as needed.

[0029] In addition to foods for human consumption, when 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 functional feed. That is, killed cells of Blautia bacteria can be used as functional feed by adding them as an active ingredient to feed for livestock such as pigs, chickens, cattle, horses, and sheep, pets (dogs, cats, and birds), and farmed fish such as sea bream, yellowtail, tuna, eels, and pufferfish.

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

[0031] As a pharmaceutical product, it can be appropriately combined with a pharmaceutically acceptable base material or the like to form a pharmaceutical preparation, such as a tablet, chewable tablet, capsule, granule, powder, pill, syrup, tincture, decoction, or liquid.

[0032] Cosmetics can be made into cosmetic preparations by appropriately combining them with pharmaceutically acceptable base materials, etc. Examples include lotions, skin lotions, creams, emulsions, powders, foundations, packs, gels, jellies, aerosols, soaps, cleansing foams, bath additives, body soaps, sunscreens, ointments, patches, adhesive bandages, etc.

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

[0034] As described above, the killed cells of Blautia bacteria provided by the present invention can be made into various forms as probiotic materials or functional materials. Furthermore, according to the results of the Examples described below, they can be made into various forms when used as a material that exerts immunopotentiating activity (the active ingredient of an immunostimulant). In such cases, the content of the killed cells of Blautia bacteria can be appropriately determined in consideration of the relationship between the amount used in the various forms and the effective amount for exerting functionality. Typically, the content of the killed cells of Blautia bacteria, calculated on a dry matter basis, 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. Furthermore, the content calculated as the number of cells is 2.0 x 10 ７ ~2.0 x 10 １２ cells / g, and ７ ~1.0 x 10 １２ cells / g, and７ ~2.0 x 10 １１ The range may be in the range of 1000 to 10000 cells / g.

[0035] When humans ingest the killed cells of Blautia bacteria provided by the present invention, the dosage may be appropriately determined depending on the subject's health condition, age, or the level of functionality required. Typically, the intake amount of lactic acid bacteria in dry matter equivalent 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. Furthermore, the content converted to the number of bacteria is 1.0 x 10 ６ ~1.0 x 10 １２ cells / day / kg body weight, and may range from 1.0 x 10 ７ ~1.0 x 10 １１ cells / day / kg body weight, and may range from 1.0 x 10 ８ ~1.0 x 10 １０ The dose may range from 1000 to 10000 cells / day / kg body weight.

[0036] The present invention will be explained in more detail below by way of 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 a Blautia bacterium and cultured in MRS medium at 37°C under an anaerobic environment for 24 hours. The culture was centrifuged at 8000 x g for 10 minutes, the supernatant was removed, and distilled water was added to suspend the cells, which was used as a sample (viable cells). Separately, the same culture was adjusted to pH 6.0 or pH 4.0 by adding acetic acid and then heat-treated at 80°C for 30 minutes. The cells were then collected 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) Spleens were collected from BALB / cA mice (female, 10 weeks old) according to a conventional method. The spleens were collected in as sterile a condition as possible in a clean bench. The collected spleens were then passed through a cell strainer (pore size: 100 μm) to recover cells at a cell concentration of 2.5 × 10 ６The liquid medium used was prepared by appropriately blending RPMI-1640 (containing L-glutamine and phenol red, Fujifilm Wako Pure Chemical Industries) with FBS (Thermo Fisher Scientific) and Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture (Thermo Fisher Scientific) at a final concentration of 10%. To the cell suspension, Blautia bacteria (live cells), (heat-treated at pH 6.0), and (heat-treated at pH 4.0) were added to a final concentration of 1.0 μg / mL in terms of dry cells, and the culture was incubated at 37°C and 5% CO ２ The cells were cultured under a 5% CO2-free environment. The concentrations of cytokines in the culture supernatant were measured by ELISA 24 hours after the start of culture for IL-12 and 96 hours after the start of culture 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. A well containing only spleen cells without Blautia bacteria was used as a control.

[0039] (Evaluation) As a result, as shown in Figure 1, the live bacteria of the genus Blautia produced IL-12 and IL-10 in amounts similar to those of the control without bacterial cells, whereas the heat-killed bacteria of the genus Blautia produced significantly higher amounts 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 heat treatment was performed in an environment of pH 4.0.

[0041] Test Example 2 (Method) Spleen cell suspensions from BALB / cA mice (female, 10 weeks old) were prepared in the same manner as in Test Example 1. Each Blautia bacterium (live bacteria) and each bacterium (heat-treated at pH 4.0 and 80°C) were added to the cell suspensions to a final concentration of 1.0 μg / mL in terms of dry cells, and the resulting mixture was incubated at 37°C and 5% CO ２The cells were cultured under a 5% CO2 environment. Six hours after the start of culture, the cells were harvested, 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, and IFN-γ) were measured by real-time PCR, and the obtained values ​​were normalized by the mRNA expression level of β-actin. Separately, real-time PCR was performed using spleen cells without the addition of Blautia bacteria, and the relative mRNA expression levels of various cytokines were calculated using this as a reference.

[0042] (Evaluation) As a result, as shown in Figure 3, when Blautia bacteria (pH 4.0, heat-treated at 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 when Blautia bacteria (live bacteria) were added.

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

[0044]

[0045] (Evaluation) - Concentration of short-chain fatty acids in cecal contents As shown in Figure 4, in the group administered with 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 to the control group and the group administered with Blautia bacteria (live bacteria), while a decrease in succinic acid, which is known to cause diarrhea, etc., was observed.

[0046] - Fecal IgA concentration As shown in Figure 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 revealed that the functionality of Blautia bacteria in improving the intestinal environment can be enhanced by heat-treating them under acidic conditions.

Claims

1. An immunostimulant whose active ingredient is killed cells of Blautia bacteria.

2. The immunostimulant according to claim 1, wherein the killed cells of the bacterium of the genus Blautia are obtained by heat treatment under acidic conditions.

3. The immunostimulant according to claim 2, wherein the killed cells of the Blautia bacterium are obtained by heat treatment under conditions of pH 3.0 to 7.0 and temperature of 70 to 121°C.

4. The immunostimulant described in claim 1, wherein the killed cells of the Blautia bacteria 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 bacteria under the same conditions.

5. The immunostimulant described in claim 4, wherein the killed cells of the Blautia bacteria 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 bacteria under the same conditions.

6. The immunostimulant according to any one of claims 1 to 5, which promotes intestinal IgA production.

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