JP2023021731A - Immunostimulatory composition - Google Patents

Abstract

To provide a component having an immunostimulatory action, particularly a component that is easy for oral administration.SOLUTION: An immunostimulatory composition contains a heat-sterilized cell of Bifidobacterium breve MCC1274 (FERM BP-11175). Herein, the immunostimulatory action may be derived from promoted production of IL-12 or MIP-1α or from activated cytotoxicity.SELECTED DRAWING: None

Description

The present invention relates to compositions for immunostimulation.

There is a great deal of interest in enhancing immune function and enhancing viral defense activity. Cytokines such as IL-12 (interleukin-12) and MIP-1α (macrophage inflammatory protein α) are considered to be involved in immunity and play an important role in preventing infectious diseases (Non-Patent Documents 1 and 2). . Therefore, various studies have been conducted on components that produce cytokines involved in immunity, and it has been reported that specific lactic acid bacteria promote IL-12 production and have an immunostimulatory effect (Non-Patent Documents 3 to 5).

In recent years, Bifidobacterium genus bacteria (so-called Bifidobacterium), which is a type of intestinal bacteria, have been found to have various effects useful for the health of humans and the like, and are utilized in foods, beverages, and pharmaceuticals.
Immunomodulatory action is also attracting attention as an action of bifidobacteria, and Non-Patent Document 1 discloses that live cells of Bifidobacterium longum BB536 are useful as probiotics.

S. Zundler et. al., Cytokine Growth Factor Rev., 26, 5, 559-568(2015) H. Takahashi et. al., J. Leukoc Biol., 72, 6, 1190-1197 (2002) S. Arai et al., PLoS One., 13, 6, :e0199018(2018) S. Murosaki et al., J. Allergy Clin Immunol., 102, 1, 57-64 (1998) S. Segawa et al. Int J Food Microbiol., 121, 1, 1-10 (2008) K. Iwabuchi, Journal of Gut Bacteria, 28: 141-146 (2014)

It is largely unknown how ingestion of Bifidobacterium spp. affects the immune system. In addition, in the case of viable cells, there may be restrictions on the form of ingestion and the processing of the composition.
An object of the present invention is to provide a component having an immunostimulatory action, particularly a component that can be easily orally ingested.

As a result of intensive research to solve the above problems, the present inventors have found that the heat-killed body of a specific Bifidobacterium bacterium has the effect of promoting the production of high IL-12 and MIP-1α. and completed the present invention.

That is, the present invention is a composition for immunostimulation containing heat-killed Bifidobacterium breve MCC1274 (FERM BP-11175).
The immunostimulation in the present invention may be by promotion of IL-12 production.
The immunostimulation in the present invention may be by promotion of MIP-1α production.
Promotion of IL-12 production or promotion of MIP-1α production in the present invention may involve the nucleic acid of Bifidobacterium breve.
The immunostimulation in the present invention may be by cytotoxic activation.
The composition of the present invention is preferably food or drink.
The compositions of the invention are preferably pharmaceuticals.

According to the present invention, the production of IL-12 and MIP-1α can be promoted. In addition, immunostimulation can thereby be achieved. Since the composition of the present invention can be orally ingested, it can be conveniently used in the form of foods, beverages, and pharmaceuticals.

The present invention will now be described in detail. However, the present invention is not limited to the following embodiments, and can be freely modified within the scope of the present invention.

The compositions of the present invention contain heat-killed bodies of Bifidobacterium breve MCC1274.
Bifidobacterium breve MCC1274 was established on August 25, 2009 by the Independent Administrative Institution National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (currently Independent Administrative Institution National Institute of Technology and Evaluation Patent Organism Depositary Center (NITE-IPOD) ( It has been internationally deposited under the Budapest Treaty under the accession number FERM BP-11175 at Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818.

In addition, Bifidobacterium breve MCC1274 is not limited to the strain itself that has been deposited or registered with a predetermined institution under the name of the bacterium (hereinafter, for convenience of explanation, also referred to as the "deposited strain"). Also included are strains (also referred to as "derivative strains" or "derived strains") that are equivalent to With respect to bacteria, the "substantially equivalent strain" to the deposited strain belongs to the same species as the deposited strain, and the similarity of the genome sequence (Average Nucleotide Identity value) to the deposited strain is
It refers to a strain having preferably 99.0% or more, more preferably 99.5% or more, still more preferably 100% identity, and preferably having the same mycological properties as the above-deposited strain. For bacteria, a strain that is substantially equivalent to the above deposited strain may be, for example, a derivative of the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that arise naturally from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and treatment with mutating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. be done. Strains naturally occurring from the deposited strain include strains naturally occurring during use of the deposited strain. Such strains include mutants naturally occurring by culturing (eg, subculturing) the deposited strain. Derivative strains may be constructed with one modification, or may be constructed with two or more modifications.

Bifidobacterium breve MCC1274 to be contained in the composition of the present invention may be a commercially available product, may be obtained by appropriately producing it, or may be obtained by culturing the above-mentioned bacteria. may be used.
The culture method is not particularly limited as long as Bifidobacterium breve MCC1274 can grow. As a culture method, for example, Bifidobacterium breve MCC
Methods commonly used for culturing 1274 can be used as they are or with appropriate modifications. The culture temperature may be, for example, 25-50°C, preferably 35-42°C. Culturing is preferably carried out under anaerobic conditions, for example, while passing anaerobic gas such as carbon dioxide. Cultivation can also be performed under microaerobic conditions such as liquid stationary culture. Culturing can be carried out, for example, until the bacteria have grown to the desired extent.

The medium used for culture is not particularly limited as long as Bifidobacterium breve MCC1274 can grow. As the medium, for example, a medium commonly used for culturing the bacteria can be used as it is or after being appropriately modified. That is, as carbon sources, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, blackstrap molasses, etc. can be used depending on the assimilation. . As the nitrogen source, for example, ammonia, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic ingredients such as peptone, soybean flour, defatted soybean meal, meat extract and yeast extract may also be used. In addition, as a medium usually used for culturing the bacteria, specifically, a reinforced Clostridial medium
, MRS medium (de Man, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium), TOSP medium (TOS propionate medium), TOSP Mup medium (TOS propionate mupirocin medium), GAM (Gifu Anaerobic Medium) medium, YCFA ( Yeast Extract-casein Hydrolysate Acid) medium and the like.

In the present invention, Bifidobacterium breve MCC1274 is contained in the composition in the form of a heat-sterilized body.
The heat-sterilized body is obtained by heat-treating Bifidobacterium breve MCC1274, specifically, for example, at 60-100° C. for 10-40 minutes, or at 90-150° C. for 5-30 seconds. It is obtained by Note that pressure may be applied during the heat treatment. During the heat treatment, the temperature does not necessarily have to be constant, and it is sufficient that the temperature is within the above temperature range for a predetermined period of time.
The heat-sterilized body of Bifidobacterium breve MCC1274 may be used as it is after the heat treatment, or may be used after being crushed, heat-dried, freeze-dried, or spray-dried.
By including it in the composition as a heat-sterilized body, the options for processing the composition and ingestion form are expanded.

The content of the heat ^- sterilized Bifidobacterium breve MCC1274 in the composition of the present invention is not particularly limited. It may contain 0×10 ⁷ cells or more of heat-sterilized bodies. In addition, the cells (individual cells) may replace the colony forming unit (cfu) of bacteria cultured at 38° C. in a solid medium containing 10% by mass of reconstituted skim milk, for example.
These may be in the range of content when they are usually distributed as a composition for immunostimulation.

The composition of the present invention has an immunostimulatory effect. Here, "immunostimulatory" refers to maintenance, improvement, or enhancement of immune function. Maintenance of immune function includes maintaining a state in which normal or good immune function is exhibited and preventing deterioration of immune function, improvement of immune function includes restoring lowered immune function to normal state, Enhancing the immune function means enhancing the immune function (which may be rephrased as a biological defense function). It may also include prevention of infection by viruses, bacteria, etc., and prevention of infectious diseases by stimulating immunity. As used herein, "prevention" means preventing or delaying the onset of a disease or condition in a subject, or reducing the risk of a disease or condition in a subject.

As shown in the Examples below, the heat-killed form of Bifidobacterium breve MCC1274 promotes the production of cytokines such as IL-12 and MIP-1α. Since these cytokines are immune factors known to be involved in immunostimulation, the composition of the present invention exerts an immunostimulatory effect by promoting the production of IL-12 and/or the production of MIP-1α. It is considered that Here, "promotion of production" means that the production amount or production rate of IL-12 or MIP-1α is higher after application than before application of the composition of the present invention, or when the composition of the present invention is not applied. As applied in comparison, to increase. Promotion of cytokine production is not particularly limited, but can be confirmed by increasing the blood concentration of these cytokines.
The immunostimulatory action of the composition of the present invention may include mechanisms other than promotion of IL-12 production and mechanisms other than promotion of MIP-1α production.

Also, as shown in the examples below, Bifidobacterium breve MCC12
The promotion of IL-12 production and MIP-1α production by administration of the heat-killed body of 74 occurs via nucleic acid recognition TLRs (Toll-like receptors). Therefore, in the composition of the present invention, the nucleic acid of Bifidobacterium breve MCC1274 is considered to be a component involved in promotion of IL-12 and MIP-1α production and immunostimulation.
Therefore, in the present invention, the heat-sterilized form of Bifidobacterium breve MCC1274 is usually contained in the composition in a form containing the nucleic acid of Bifidobacterium breve MCC1274.

As shown in the examples below, the heat-killed form of Bifidobacterium breve MCC1274 enhances the cytotoxic activity of immune cells. Since high cytotoxic activity can prevent invasion and infection by external enemies such as viruses and cancer cells, the composition of the present invention is thought to exert an immunostimulatory effect by cytotoxic activation. Here, "activation" of cytotoxicity means that the cytotoxic activity increases after application of the composition of the present invention compared to before application, or when the composition of the present invention is applied compared to when it is not applied. It means to increase. Cytotoxic activity is not particularly limited, but can be confirmed by evaluating the mortality rate of other cells coexisting with NK cells.

Another aspect of the invention is the use of a heat-killed form of Bifidobacterium breve MCC1274 in the manufacture of an immunostimulatory composition.
Another aspect of the invention is Bifidobacterium breve MCC1 in immunostimulatory
274 is the use of heat sterilizers.
Another aspect of the invention is a heat-killed form of Bifidobacterium breve MCC1274 used for immunostimulation.
Another aspect of the invention is a method of stimulating immunity comprising administering a heat-killed form of Bifidobacterium breve MCC1274 to a subject.

Subjects to whom the composition of the present invention is administered (administered person) and subjects to be ingested (consumer) are not particularly limited as long as they are animals, but are usually humans. Also, it may be an adult, a child, an infant, a newborn, or the like. Moreover, sex is not specifically limited.

In the present specification, "administering the heat-killed Bifidobacterium breve MCC1274 to the subject" is synonymous with "giving the subject the heat-killed Bifidobacterium breve MCC1274." you can The intake is usually voluntary (free intake), but may be forced (forced intake). That is, specifically, the administration step is, for example, a step of mixing the heat-sterilized Bifidobacterium breve MCC1274 into food, drink or feed and supplying it to the subject, thereby allowing the subject to freely ingest it. may

The timing of ingestion (administration) of the composition of the present invention is not particularly limited, and can be appropriately selected according to the condition of the subject of ingestion (administration).

The intake (administration) amount of the composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the intake (administration) target.
The ingestion (administration) amount of the composition of the present invention is
The ingestion (administration) amount of MCC1274 cells is, for example, preferably in the range of 1×10 ⁷ to 1×10 ¹² cells/day, and more preferably in the range of 1×10 ⁸ to 1×10 ¹¹ cells/day. , 1×10 ⁹ to 1×10 ¹¹ cells/day are more preferred.
The composition of the present invention can be ingested (administered) once a day or divided into multiple times a day, regardless of the amount and duration of ingestion (administration).

The intake (administration) period of the composition of the present invention is not particularly limited. In addition, there is no particular upper limit for the intake (administration) period, and continuous long-term intake (administration) is possible.

The intake (administration) route of the composition of the present invention may be oral or parenteral, but oral is preferred. In addition, parenteral intake (administration) includes percutaneous, intravenous, rectal administration, inhalation, and the like.

When the composition of the present invention is to be orally ingested (administered), it is preferably in the form of a food or drink.
As food and drink, the form and properties are not particularly limited as long as they can be orally ingested (administered) without impairing the effects of the present invention. It can be produced by a normal method using raw materials that are usually used for food and drink.

Food and drink, regardless of the form such as liquid, paste, gel-like solid, powder, etc. Wheat flour products such as fried flour and bread crumbs; instant noodles, cup noodles, retort/cooked foods, cooked canned foods, microwave oven foods, instant soups/stews, instant miso soups/soups, canned soups, freeze-dried foods, other instant foods, etc. Instant foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (processed grains) and other processed agricultural products; canned marine products, fish hams and sausages, fish paste products, marine delicacies Processed marine products such as tsukudani; processed livestock products such as canned livestock/pastes, livestock meat hams/sausages; Milk and dairy products such as other dairy products; fats and oils such as butter, margarine, and vegetable oils; basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, and vinegar; cooking mixes and curry Complex seasonings and foods such as ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings; Frozen foods such as frozen foods, half-cooked frozen foods, and cooked frozen foods; caramels, candies , chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, and other confectionery; Beverages, fruit drinks, fruit drinks with fruit, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic beverages, and other favorite drinks , baby food, furikake, ochazuke seaweed and other commercially available foods, etc.; infant formula; enteral nutrition food; A supplementary food etc. are mentioned.

In the case of supplements as food and drink, solid preparations such as powders, granules, tablets, capsules, etc., which may be enteric treated by enteric coating, etc.; solutions, syrups, suspensions, Liquid formulations such as emulsions; and the like can be formulated. Such formulations can be made in accordance with the description of the components, carriers, and methods relating to the formulation of pharmaceuticals, which will be described later.

Moreover, it can also be set as feed as one aspect|mode of food-drinks. For feed, pet food,
Examples include livestock feed and fish feed.
The form of feed is not particularly limited, and Bifidobacterium breve MCC127
In addition to the heat sterilized body of 4, for example, cereals such as corn, wheat, barley, rye, and milo; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, coconut oil cake, and linseed oil cake; bran, wheat bran, rice bran, defatted rice bran Rice brans such as corn gluten meal, corn jam meal, etc.; Animal feeds such as fish meal, skimmed milk powder, whey, yellow grease, tallow; Yeasts such as torula yeast, brewer's yeast; Mineral feed such as calcium carbonate; oils and fats; simple amino acids; sugars and the like may be contained.

When the composition of the present invention is in the form of a food or drink (including feed), it can be provided and sold as a food or drink labeled as an immunostimulatory application. In addition, the heat-sterilized body of Bifidobacterium breve MCC1274 according to the present specification can be used for producing these foods and drinks.

Such "display" acts include all acts for informing consumers of the above-mentioned use. Regardless of the object, medium, etc. to be displayed, all of them fall under the act of "display" in the present invention.
In addition, it is preferable that the "display" be performed in an expression that allows the consumer to directly recognize the use. Specifically, the act of transferring, handing over, displaying for the purpose of transfer or delivery, importing products related to food and beverages or product packaging that describes the above-mentioned use, advertisements related to products, price lists or transaction documents Examples include the act of displaying or distributing information with the above-mentioned use described, or providing information containing such information with the above-mentioned use by electromagnetic means (Internet, etc.).

On the other hand, the content of the display is preferably a display approved by the government (for example, a display that is approved based on various systems established by the government and performed in a manner based on such approval). In addition, it is preferable to attach such display contents to packaging, containers, catalogs, pamphlets, POP and other advertising materials at sales sites, other documents, and the like.

In addition, "labeling" includes health foods, functional foods, enteral nutrition foods, foods for special dietary uses, foods with health claims (foods for specified health uses, foods with nutrient function claims, foods with function claims), nutritional supplements, and pharmaceuticals. Display as an off-the-shelf item is also possible. Among these, in particular, the labeling approved by the Consumer Affairs Agency, for example, the labeling approved by the system related to food for specified health use, food with nutrient function claims, or food with function claims, or similar system. Specifically, labeling as a food for specified health use, labeling as a food for specified health use with certain conditions, labeling to the effect that it affects the structure and function of the body, labeling to reduce the risk of disease, labeling for functionality based on scientific evidence. Labeling, etc. can be mentioned, more specifically, the Cabinet Office Ordinance Concerning Permission for Special Use Labeling as stipulated in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009) A typical example is labeling as a food for specified health use (especially labeling for health use) and similar labeling.
Such claims include, for example, "working on immune function", "helping to maintain immune function in healthy people", "enhancing immune function", "supporting immune function", "maintaining immune function in healthy people". "Supports", "Increases resistance to infectious diseases", "Increases biological defense function", etc.

The compositions of the invention can also be in the form of pharmaceuticals.
The route of administration of pharmaceuticals may be either oral or parenteral, but oral is preferred. In addition, parenteral intake (administration) includes percutaneous, intravenous, rectal administration, inhalation, and the like.
As for the pharmaceutical form, it can be appropriately formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, solid preparations such as powders, granules, tablets and capsules; and liquid preparations such as solutions, syrups, suspensions and emulsions can be formulated. In the case of parenteral administration, it can be formulated into suppositories, ointments, injections, and the like.
For formulation, in addition to heat-sterilized Bifidobacterium breve MCC1274, ingredients such as excipients, pH adjusters, colorants, and corrigents that are commonly used for formulation can be used. It is also possible to use other medicinal ingredients, known or future immunostimulatory ingredients, immune system cytokine production promoters, and the like.
In addition, formulation can be appropriately carried out by a known method depending on the dosage form. For formulation, a carrier that is usually used for formulation may be blended as appropriate to form the formulation. Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like.

Excipients include, for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose derivatives such as carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light silicic anhydride, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate;

Examples of binding agents include gelatin, polyvinylpyrrolidone, macrogol, etc., in addition to the excipients described above.

Examples of disintegrants include, in addition to the excipients described above, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.

Lubricants include, for example, talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gairou; ; carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acid anhydride and silicic acid hydrate; be done.

Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride;

Flavoring agents include, for example, sweeteners, acidulants, flavoring agents, and the like.
In addition, solvents such as water and the like can be used as carriers for liquid formulations for oral administration.

The timing of taking the drug of the present invention is not particularly limited, such as before meals, after meals, between meals, and before bedtime.

EXAMPLES The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

<Example 1> Animal administration test (1) Preparation of administration product Bifidobacterium breve MCC1274 freeze-dried powder
After diluting to a concentration of ×10 ⁸ cfu/mL, the solution was heated at 90° C. for 30 minutes to prepare a Bifidobacterium heat-killed body-administered product (hereinafter also referred to as a heat-killed body-administered product).

(2) Animal test
Using Crl:CD(SD) rats (Charles River Japan Co., Ltd.: healthy rats) as experimental animals, the effect of continuous administration of heat-killed Bifidobacterium breve MCC1274 administered for 4 weeks was evaluated.
Rats received at the age of 7 weeks were allowed free access to lab MR stock diet (manufactured by Nosan Kogyo Co., Ltd.) and tap water. After one week of acclimatization, the rats were divided into two groups (6 rats in each group), and the rats in the control group were orally administered 2 mL/rat of physiological saline using an oral probe once a day for 4 weeks, followed by heat sterilization. To rats in the body administration group, 2 mL/rat of the solution prepared in (1) was orally administered using an oral probe once a day for 4 weeks. One hour after the end of the final administration, the experimental animals were treated under sevoflurane anesthesia, blood was collected from the abdominal posterior vena cava, and the serum was recovered by centrifugation. Various immune mediators in the blood were quantified for the recovered serum using Cytokines array (RayBiotech).

(3) Results Table 1 shows factors for which a significant increase was confirmed by cytokine array analysis. Administration of Bifidobacterium heat-killed body increased blood MIP-1α, TIM-1, Notch-2, RANTES, TWEAK R, Nope, TCK-1, MCP-
A significant increase in the amount of 1 was confirmed. In particular, MIP-1α was significantly increased by 16.9 times in the heat-sterilized body administration group compared to the control group.
The above results suggested that the administration of heat-killed Bifidobacterium breve MCC1274 promoted the secretion of many immune factors such as MIP-1α. In particular, MIP-1α, RANTES, and MCP-1 are known to play important roles in preventing infectious diseases and avoiding aggravation due to complications. From this result, the administration of the heat-killed Bifidobacterium breve MCC1274 promotes the production of immune factors,
It was suggested that an immunostimulatory action is thereby obtained.

<Example 2> Cell addition test 1
(1) Preparation of spiked sample Bifidobacterium breve MCC1274 lyophilized powder was diluted with physiological saline for 1 hour.
After suspending at 0 or 100 mg / mL, heat at 90 ° C. or 4 ° C. for 30 minutes, heat sterilized Bifidobacterium cell sample (HKB) and heat untreated Bifidobacterium cell sample (non-HKB ). The resulting Bifidobacterium cell sample was added to RPMI medium containing 10% Fetal Bovine Serum, 100 unit/mL Penicillin, and 100 μg/mL Streptomycin (hereinafter referred to as RPMI medium) to a final concentration of 10 μg/
0.1% (v/v) was added to give 1 mL or 100 µg/mL, and the following cell experiments were performed.

(2) Cell experiment Rat peripheral blood mononuclear cells (PBMC, iQ Biosciences) containing a wide variety of immune cells such as monocytes and lymphocytes isolated from peripheral blood were treated with Bifidobacterium prepared in (1). Cell sample-containing medium (HKB or non-HKB) was seeded in a 96-well plate at 1.0×10 ⁵ cells/well, and cultured at 37° C., 5% CO ₂ for 6 hours. PBMC were cultured in the same manner in RPMI medium containing 1% (v/v) PBS without Bifidobacterium, and this was used as a control.
(3) mRNA gene expression level analysis Total RNA was recovered from cultured nuclear cells using NucleoSpin RNA Plus XS, and cDNA was prepared using TakaRa PrimeScript RT reagent Kit. Using this cDNA as a sample, the expression levels of IL-12, MIP-1α and GAPDH were quantitatively analyzed by real-time PCR. Each expression level of IL-12 and MIP-1α was corrected with the expression level of GAPDH.

(4) Results Table 2 shows the expression level of IL-12 and Table 3 shows the expression level of MIP-1α in each group. A dose-dependent IL-12 and MIP-1α expression-enhancing activity was confirmed in Bifidobacterium cell samples with or without heat treatment. Furthermore, it was found that this activity was enhanced by heat-treating the cells. Specifically, when 100 μg / mL of Bifidobacterium was added, non-HKB promoted IL-12 by 391 times and MIP-1α by 18.8 million times. It was confirmed that -12 promoted gene expression by 629 times and MIP-1α by 450 million times.

IL-12 acts on T cells and NK cells, and is considered to play an important role related to innate immunity and adaptive immunity. In addition, it has been suggested that MIP-1α, the immune factor that was most altered by ingestion of heat-killed Bifidobacterium in Example 1, is essential for the host's resistance to bacterial sepsis (non-patent literature 2), it may contribute to the prevention of infectious diseases and the avoidance of aggravation due to complications.
From the above results, the cells of Bifidobacterium breve MCC1274 are IL-1
2 and MIP-1α. Furthermore, it was also shown that such an immunostimulatory effect is enhanced by heat-treating the cells.

<Example 3> Cell addition test 2
Chloroquine (CQ), an endosomal acidification inhibitor, has been reported to inhibit nucleic acid recognition Toll-like receptors (TLR3/7/8/9) (A. Kuznik et. al. J Immunol. 2011, 186, 8 , 4794-4804.). CQ was used to verify whether the promotion of immune factor production by heat-killed Bifidobacterium breve (HKB) shown in Example 2 was mediated by nucleic acid recognition TLRs.

(1) Preparation of added sample A heat-sterilized cell sample (HKB) of Bifidobacterium breve MCC1274 was prepared in the same manner as in Example 2(1).
(2) Cell experiment under TLR inhibition conditions Rat peripheral blood mononuclear cells (PBMC, iQ Biosciences) containing a wide variety of immune cells such as monocytes and lymphocytes isolated from peripheral blood were treated at 1.0 × 10 The cells were seeded at ⁵ cells/well in a 96-well plate containing RPMI medium supplemented with CQ (final concentration 100 μM), and cultured at 37° C., 5% CO ₂ for 2 hours. Thereafter, the Bifidobacterium cell sample (HKB) prepared in (1) was added to a final concentration of 100 μg/mL or the same volume of PBS, and cultured at 37° C. and 5% CO ₂ for 6 hours.
(3) mRNA Gene Expression Level Analysis Regarding the cultured cells, the nuclear expression levels of IL-12 and MIP-1α were measured in the same manner as in Example 2(3).

(4) Results Table 4 shows the expression level of IL-12 and Table 5 shows the expression level of MIP-1α in each group. Compared to the addition of PBS, the addition of HKB increased the expression levels of both IL-12 and MIP-1α, and the addition of CQ, a nucleic acid recognition TLR inhibitor, significantly promoted the expression of IL-12 and MIP-1α by HKB. Suppressed. Specifically, the activity of HKB to promote the expression of IL-12 and MIP-1α was suppressed by 99.2% and 59.0%, respectively, by the addition of CQ.
From the above results, the promotion of IL-12 and MIP-1α expression by the heat-killed Bifidobacterium breve MCC1274 is an effect mediated by nucleic acid recognition TLRs, and the nucleic acid of the bacterium is the component involved. It was suggested.

<Example 4> Cell addition test 3
Immunostimulation (activation of immune cells to protect themselves from invading foreign enemies and foreign substances) is induced by promoting the production of immune factors by the heat-killed Bifidobacterium breve (HKB) shown in Example 2. The cytotoxic activity (NK activity) was used as an index to evaluate whether or not.

(1) Preparation of added sample A heat-sterilized cell sample (HKB) of Bifidobacterium breve MCC1274 was prepared in the same manner as in Example 2(1).
(2) Addition experiment of PBMC Human peripheral blood mononuclear cells (PBMC, iQ Biosciences) containing a wide variety of immune cells such as monocytes and lymphocytes isolated from peripheral blood were added at 1.0 × 10 ⁶ cells/cells. The Bifidobacterium cell sample (HKB) prepared in (1) was seeded to a final concentration of 1.0 × 10 ⁸ cells/well, or An equal volume of PBS was added and incubated at 37°C, 5% _CO2 for 24 hours.
(3) K562 co-culture experiment The leukemia cell line K562, which is frequently used as a target cell for NK cells due to its high sensitivity to natural killer cells (NK cells), was cultured in RPMI medium at 37°C and 5% _CO2 . . After that, CFSE (Cayman Chemical Co.) was allowed to react for 15 minutes for labeling. The labeled K562 was co-cultured at 37° C., 5% CO ₂ for 4 hours together with the PBMC cultured for 24 hours in (2) and the culture medium so as to have 2.0×10 ⁴ cells/well.
(4) Dead Cell Staining and Measuring Method Co-cultured K562 and PBMC were subjected to dead cell staining using Fixable Viability Dye eFluor 780 (eBiosciinc). K562 dead cell percentage was measured using a flow cytometry BD FACSCanto. A group in which K562 was cultured alone without being co-cultured with PBMC during the co-culture was used as a negative control.

(5) Results Table 6 shows the live cell ratio and dead cell ratio of K562. In the K562 monoculture, the dead cell ratio was 0.15%, but the dead cell ratio was increased by co-culturing with PBMC. This is suggested to be due to the cytotoxic activity of PBMC. Comparing the cytotoxic activity of PBMC treated with PBS or HKB, HKB treatment increased the proportion of dead cells. Specifically, HKB increased the cytotoxic activity of PBMC containing various immune cell populations by 3.88-fold.
From the above results, it was suggested that the heat-killed form of Bifidobacterium breve MCC1274 enhances the cytotoxic activity, thereby enhancing the ability to defend the body against various infections.

Claims (7)

Bifidobacterium breve M
A composition for immunostimulation containing a heat-killed form of CC1274 (FERM BP-11175). 2. The composition of claim 1, wherein said immunostimulatory is by enhanced production of IL-12. 2. The composition of claim 1, wherein said immunostimulatory is by enhanced production of MIP-1α. 4. The composition according to claim 2 or 3, wherein said enhancement of production is effected by the involvement of said Bifidobacterium breve nucleic acid. 2. The composition of claim 1, wherein said immunostimulatory is by cytotoxic activation. The composition according to any one of claims 1 to 5, which is a food or drink. A composition according to any one of claims 1 to 5, which is a medicament.