JP7706191B2 - Immunostimulant using Blautia bacteria - Google Patents

Description

NPMD NITE BP-03954

The present invention relates to the use of Blautia bacteria, and more specifically, to the use of Blautia bacteria as an immunostimulant.

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

On the other hand, recent data analysis of humans has revealed that the Blautia genus, a type of intestinal bacteria, is inversely correlated with BMI and diabetes risk (see Non-Patent Document 1).

Patent No. 4621218

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

To date, there has not been much research into the use of Blautia bacteria.

The object of the present invention is to provide a microbial cell material that has excellent properties as an immunostimulatory material by utilizing Blautia bacteria.

As a result of various studies conducted by the inventors to achieve the above object, they discovered that killed bacteria of the genus Blautia have excellent immunostimulatory properties, which led to the completion of the present invention.

That is, the present invention provides an immunostimulant that contains killed cells of Blautia bacteria as an active ingredient.

In the above immunostimulant, the killed cells of the Blautia bacteria are preferably obtained by heat treatment under acidic conditions.

The killed bacteria of the genus Blautia are preferably obtained by heat treatment at a pH of 3.0 to 7.0 and a temperature of 70 to 121°C.

In addition, it is preferable that the killed bacteria 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 bacteria of the genus Blautia under the same conditions.

In addition to the above-mentioned ability to induce IL-12 production, it is also preferable that the killed cells of the Blautia bacteria 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.

In addition, it is preferable that the immunostimulant provided by the present invention promotes intestinal IgA production.

According to the present invention, it is possible to provide a microbial cell material that has excellent properties as an immunostimulatory material by utilizing Blautia bacteria.

FIG. 1( a ) is a graph showing the results of investigating the effect of adding Blautia bacteria (live bacteria or killed bacteria) to a 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. FIG. 2( a ) is a graph showing the results of investigating the effect of adding Blautia bacteria (killed bacteria in an environment of pH 6 or pH 4) to a culture medium in a culture system of mouse spleen cells in Test Example 1. 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. FIG. 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 culture system of mouse spleen cells in Test Example 2, and a graph showing the results of investigating the expression level of cytokines. FIG. 13 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 feed and allowing BALB/c mice to ingest it ad libitum in Test Example 3, and also shows the results of investigating the concentration of short-chain fatty acids in the cecal contents. FIG. 13 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 feed and allowing BALB/c mice to freely ingest the feed in Test Example 3, and also shows the results of investigating the IgA concentration in the feces.

As used herein, "Blautia bacteria" refers to bacteria belonging to the genus Blautia, which is classified in the phylum Firmicutes. Specifically, Blautia caecimuris, Blautia gluceracea, Blautia coccoides, Blautia schinkii, Blautia stercoris, Blautia hydrogenotrophica, Blautia faecis, Blautia producta, Blautia hensenii ... hansenii), Blautia luti, Blautia wexlerae, etc. These may be used alone or in combination of two or more. Among these, Blautia producta is particularly preferably selected from the viewpoint of immunoactivation effect.

Preferred examples of Blautia products include Blautia RD014892 strain (accession number: NITE BP-03954) deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (NPMD), an independent administrative institution, and bacteria substantially identical thereto. Here, "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 base sequence for identifying the genus and species of bacteria has a homology of 98% or more, preferably 99% or more, to the 16SrRNA gene base sequence of Blautia RD014892 strain, and which also has the same bacteriological properties as Blautia RD014892 strain.

The cultivation of Blautia bacteria and the maintenance of the bacteria can be carried out by known means. For example, the medium can 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.) can also be used. The cultivation can be carried out by inoculating the bacteria into the medium and culturing it statically or culturing it with aeration and agitation at conditions of, for example, 25 to 40°C. For preservation of live bacteria, they can be refrigerated while suspended in the medium for a short period of time, or frozen and preserved for a long period of time after suspending them in an antifreeze such as a glycerol solution.

When preparing Blautia bacteria, a bacterial cell concentrate can be prepared by concentrating the culture liquid after cultivation as is, 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 Blautia bacteria cells 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 cells. This bacterial cell concentrate may contain an excipient. This makes it easier for the properties of the bacteria to be maintained after freezing or lyophilization, and after reconstitution with water.

The excipients are 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.

In another embodiment, the preparation of Blautia bacteria may involve pulverization and dispersion. The pulverization and dispersion may be carried out, 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 re-aggregation of the resulting bacterial powder. When an excipient is contained, the content may be in the range of 1 to 99% by mass, 10 to 95% by mass, or 20 to 90% by mass in terms of dry matter.

In yet another embodiment, the Blautia bacteria may be prepared by drying and powdering. Examples of the drying and powdering method include freeze-drying, reduced pressure spray drying, and spray drying using hot air. Note that spray drying using hot air usually destroys the activity of live bacteria, and dead bacteria can be obtained.

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. In addition, 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 that contains the killed bacteria of the Blautia genus as an active ingredient.

Heat treatment of Blautia bacteria can be performed by treating the culture solution after culture as is, or after preparing the above-mentioned bacterial cell concentrate as necessary, for example, in a jacketed tank, thermostatic bath, or 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 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. If the treatment is not sufficient under such an environment, it is not preferable because it tends to lack desired functionality such as immunostimulatory activity.

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.

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

As for food and beverage products, there is no particular limit to the type of food to be blended, 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, nutritional supplements, liquid (fluid) foods such as syrup, margarine, paste, jam, and solid foods such as cooked rice, bread, potato products, mochi, furikake, ham, sausage, candy, chocolate, gum, gummy candy, snacks, and baked goods, as well as staple foods, side dishes, confectioneries, and seasonings. Depending on the application, it may be molded into powder, granules, tablets, etc. Furthermore, it can be blended with excipients, bulking agents, binders, thickeners, emulsifiers, colorants, flavorings, food additives, seasonings, etc., as necessary.

In addition to food for human consumption, when the killed bacteria of the genus Blautia 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 bacteria of the genus Blautia can be used as a functional feed by adding the active ingredient to the feed of livestock such as pigs, chickens, cows, horses, sheep, pets (dogs, cats, birds), and farmed fish such as sea bream, yellowtail, tuna, eel, and pufferfish.

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 is not particularly limited, but can be manufactured as tablets, capsules, granules, powders, and beverages, for example.

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

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

As described above, the killed cells of the 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 of the genera Lactobacillus, Lacticaseibacillus, Lactiprantibacillus, Lactococcus, Bifidobacterium, Streptococcus, Enterococcus, Akkermansia, Christensenella, Clostridium, Bacteroides, Bacillus, Paraprevotella, Faecalibacterium, Lentilactobacillus, Eubacterium, Veillonella, etc., preferably killed cell materials thereof.

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 taking into consideration the relationship between the amount used in the 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. The content converted into the number of cells may be in the range of 2.0×10 ⁷ to 2.0×10 ¹² cells/g, 2.0×10 ⁷ to 1.0×10 ¹² cells/g, or 2.0×10 ⁷ to 2.0×10 ¹¹ cells/g.

When a human is to ingest the killed cells of the Blautia bacteria provided by the present invention, the dosage may be appropriately set 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 cells may be in the range of 1.0 x 10 ⁶ to 1.0 x 10 ¹² cells/day/kg body weight, 1.0 x 10 ⁷ to 1.0 x 10 ¹¹ cells/day/kg body weight, or 1.0 x 10 ⁸ to 1.0 x 10 ¹⁰ cells/day/kg body weight.

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

[Sample preparation]
Blautia RD014892 strain was used as the Blautia bacteria, and was cultured in MRS medium at 37°C under anaerobic conditions for 24 hours. The culture solution was centrifuged at 8000 x g for 10 minutes, the supernatant was removed, and distilled water was added to suspend the bacteria, which was used as a sample (live 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°C 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).

[Test Example 1]
(method)
Spleens were collected from BALB/cA mice (female, 10 weeks old) according to a conventional method. Spleens were collected in as sterile conditions as possible in a clean bench. The collected spleens were then collected using a cell strainer (pore size: 100 μm) and then prepared in a liquid medium so that the cell concentration was 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 Industries) with 10% final concentration of FBS (Thermo Fisher Scientific) and Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture (Thermo Fisher Scientific). To the cell solution, Blautia bacteria (live bacteria), (heat-treated at pH 6.0), and (heat-treated at pH 4.0) were added so that the final concentration calculated as dry cells was 1.0 μg/mL, and the cells were cultured in an environment of 37°C and 5% _CO2 . The concentrations of each cytokine contained in the supernatant after culture 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 average values and standard deviations of the measurements were calculated from 6 wells for IL-12 and 5 wells for IL-10. A well containing only spleen cells without the addition of Blautia bacteria was used as a control.

(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.

In addition, as shown in Figure 2, the ability of killed Blautia bacteria to induce the production of immunostimulatory factors was particularly significant when heat-treated in an environment of pH 4.0.

[Test Example 2]
(method)
In the same manner as in Test Example 1, spleen cell liquid from BALB/cA mice (female, 10 weeks old) was prepared. Each Blautia bacteria (live bacteria) and (pH 4.0, heat treatment at 80°C) were added to the cell liquid so that the final concentration in terms of dry cells was 1.0 μg/mL, and the cells were cultured at 37°C and 5% _CO2 . Six hours after the start of culture, the cells were collected, and RNA was extracted using 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 carried out 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.

(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.

[Test Example 3]
(method)
BALB/c mice were acclimated for one week, and then fed ad libitum for one week purified feed (AIN-93G) containing each sample in the doses shown in the table below. After that, feces were collected from the colon of the euthanized mice, 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.

(evaluation)
Concentration of short-chain fatty acids in cecal contents As shown in Figure 4, in the group administered Blautia (pH 4.0, heat-treated at 80°C), increases were observed in the levels of 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 (live bacteria).

- 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 groups administered Blautia bacteria (live bacteria).

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 (4)

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 . The immunostimulant according to claim 1, wherein the killed bacteria 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 bacteria of the genus Blautia under the same conditions. 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. The immunostimulant according to any one of claims 1 to 3 , which promotes intestinal IgA production.

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