Novel butyric acid bacterium and use thereof

Abstract

To provide a novel butyric acid bacterium having excellent butyric acid productivity and immunostimulatory activity, as well as compositions, Nukadoko, Nukazuke, and immunostimulants containing the novel butyric acid bacterium.SOLUTION: A novel butyric acid bacterium according to the present invention is Clostridium beijerinckii TK211201 strain (Accession No.: NITE P-03982).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel butyric acid bacterium having excellent butyric acid production and immunostimulating properties, and a composition, rice bran bed, rice bran pickles, and an immunostimulant each containing the novel butyric acid bacterium. [Background technology]

[0002] Butyrate is not only the main energy source for the large intestine, but in recent years it has been reported that it is one of the factors that suppress colon cancer development and induce differentiation into regulatory T cells by inhibiting histone deacetylase, and is involved in health functions. However, even if butyrate is orally ingested, it is absorbed in the stomach or upper part of the small intestine, and may not reach the large intestine where the intestinal flora is formed. Therefore, attempts have been made to increase butyrate in the large intestine by using growth promoters for butyrate bacteria that grow in the intestine, or by directly ingesting butyrate bacteria in the form of a supplement.

[0003] For example, Patent Document 1 discloses butyric acid bacteria and / or butyric acid bacteria proliferation agents containing barley leaves and / or stems. Patent Document 2 discloses an intestinal environment improvement agent containing HMPA [3-(4-hydroxy-3-methoxyphenyl)propionic acid] and butyric acid bacteria as active ingredients. Patent Document 3 discloses a supplement that contains spore-forming butyric acid bacteria, butyric acid bacteria, and kestose to regulate the intestinal flora.

[0004] As a butyric acid bacterium, Clostridium butyricum is mainly known, but Clostridium beijerinckii is also known. For example, Patent Document 4 discloses a C. beijerinckii strain that produces a large amount of butyric acid and small amounts of lactic acid and acetic acid.

[0005] Butyric acid bacteria are a type of bacteria that have the ability to produce butyric acid using carbohydrates, including dietary fiber, as a substrate. These bacteria are generally obligate anaerobes, and therefore can only grow in limited environments, and are often found in the large intestine in particular.

[0006] The presence of butyric acid bacteria has been suggested in some foods. In particular, rice bran pickles, a traditional Japanese fermented food, are thought to be one of the few foods in which butyric acid bacteria can be ingested. However, there are no documents showing the detection of butyric acid bacteria in rice bran pickles or the rice bran bed used for pickling, and the growth of butyric acid bacteria is inferred based on the analysis of the growth of a wide variety of bacteria and aroma components in the rice bran bed. Therefore, it is not clear whether or not butyric acid bacteria are contained in rice bran pickles.

[0007] In recent years, interest in health functionality has been increasing with the progress of the aging society. In particular, there is a demand for the development of foods or medicines with immunostimulatory functions that contribute to the prevention or improvement of various diseases. Probiotics, including lactic acid bacteria and bifidobacteria, are one of the functional ingredients that have an immunostimulatory effect. Furthermore, butyric acid bacteria have begun to attract attention as a new probiotic in recent years.

[0008] For example, Patent Document 5 discloses a live feed in which killed bacteria having immunostimulatory activity for aquacultured animals are fed to the animals, and gives as an example of the killed bacteria Clostridium butyricum MIYAIRI, a butyric acid bacterium. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2022-140551 A [Patent Document 2] JP 2022-130294 A [Patent Document 3] Patent Publication No. 2022-126152 [Patent Document 4] International Publication No. 2021 / 166901 Brochure [Patent Document 5] Japanese Patent Application Publication No. 10-165108 Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, butyric acid bacteria have begun to attract attention in recent years with the increasing health consciousness, but there is still a demand for bacteria with excellent butyric acid production ability and immunostimulatory ability. Therefore, an object of the present invention is to provide a novel butyric acid bacterium having excellent butyric acid production ability and immunostimulating ability, and a composition, rice bran bed, rice bran pickles, and immunostimulants containing the novel butyric acid bacterium. [Means for solving the problem]

[0011] The present inventors have conducted extensive research to solve the above problems, and as a result, have isolated and identified a novel butyric acid bacterium having excellent butyric acid production and immunostimulatory activity from a rice bran bed prepared by the present inventors, thereby completing the present invention. The present invention will now be described.

[0012] [1] Clostridium beijerinckii strain TK211201 (accession number: NITE P-03982). [2] A composition comprising Clostridium beijerinckii strain TK211201 (accession number: NITE P-03982). [3] The composition described in [2] above, which is in the form of a food or beverage, a medicine, a supplement, or an animal feed. [4] A rice bran bed characterized by containing Clostridium beijerinckii strain TK211201 (accession number: NITE P-03982). [5] A bran pickle containing Clostridium beijerinckii strain TK211201 (accession number: NITE P-03982). [6] An immunostimulant characterized by containing Clostridium beijerinckii strain TK211201 (accession number: NITE P-03982). [7] The immunostimulant described in [6] above, which promotes the production of inflammatory cytokines. [8] The immunostimulant described in [7] above, wherein the inflammatory cytokine is TNFα and / or IL-6. Effect of the Invention

[0013] The novel butyric acid bacteria according to the present invention are useful as probiotics because of their excellent butyric acid production and immunostimulating properties, and are also effective as an active ingredient of immunosuppressants. Furthermore, they may produce butyric acid in the intestine, which may serve as food for useful intestinal bacteria, improving the intestinal environment. Furthermore, since the novel butyric acid bacteria according to the present invention have been isolated and identified from a rice bran bed, they may be able to grow even if they are re-added to rice bran or a rice bran bed containing discarded vegetables. As a result, rice bran pickles rich in butyric acid and also having immunostimulating properties can be produced from the rice bran bed to which the novel butyric acid bacteria according to the present invention have been re-added. Therefore, the novel butyric acid bacteria according to the present invention are industrially very excellent as a technology related to foods and preparations useful for improving the intestinal environment and stimulating the immune system. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a simplified molecular phylogenetic tree of the TK211201 strain according to the present invention. [Diagram 2] FIG. 2 is a graph showing the concentration of n-butyric acid in samples obtained by culturing the type strain of C. beijerinckii or the TK211201 strain of the present invention using (1) an enriched Clostridia medium, which is a microbiological test medium, or (2) an edible medium. [Diagram 3] FIG. 3 is a graph showing the concentration of n-butyric acid in samples prepared from bran beds containing the C. beijerinckii type strain or the TK211201 strain of the present invention, and from bran beds not containing these strains. [Figure 4] FIG. 4 is a graph showing (1) the TNFα concentration when the C. beijerinckii type strain or the TK211201 strain of the present invention is cultured in a medium containing 10 μg / mL, (2) the TNFα concentration when cultured in a medium containing 100 μg / mL, and (3) the IL-6 concentration when cultured in a medium containing 10 μg / mL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The Clostridium beijerinckii TK211201 strain (hereinafter abbreviated as "TK211201 strain"), which is a novel butyric acid bacterium according to the present invention, has been deposited at the depository institution as follows. (i) The name and address of the depositary institution; Name: National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Deposit Center (NPMD) Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (ii) Date of deposit: September 14, 2023 (iii) Accession number: NITE P-03982

[0016] The culture conditions of the TK211201 strain may be appropriately adjusted. For example, the components of the medium for culturing the TK211201 strain include carbon sources such as glucose, starch, and fructose; nitrogen sources or amino acid sources such as tryptone and peptone; general nutritional components such as meat extract, yeast extract, and protein hydrolysates; amino acids and salts thereof such as cysteine ​​and sodium glutamate; mineral components such as magnesium sulfate; pH adjusters such as trisodium citrate, dipotassium hydrogen phosphate, and anhydrous sodium acetate; salts such as sodium chloride; and vitamins such as ascorbic acid. For example, the pH of the medium can be adjusted to 6 or more and 8 or less, and the culture temperature can be adjusted to 10° C. or more and 40° C. or less, preferably 20° C. or more or 30° C. or more. The gas phase is preferably anaerobic conditions such as nitrogen, carbon dioxide, hydrogen, argon, and a mixture of two or more of these gases. The culture may be performed in two stages: preculture and mass culture.

[0017] The novel butyric acid bacteria according to the present invention may be used as is, but may also be separated from the culture medium. For example, after culturing the novel butyric acid bacteria, the bacterial cells may be collected by filtration, centrifugation, or the like. The collected novel butyric acid bacteria may be freeze-dried. Some bacteria die when freeze-dried, but some bacteria may enter a state of suspended animation and survive in the body or even in the intestines when ingested. An excipient such as lactose may be used during freeze-drying.

[0018] The novel butyric acid bacteria according to the present invention can be used as a composition for food, beverages, medicines, supplements, feed, and the like. In the composition, the novel butyric acid bacteria may be live bacteria, killed bacteria, or a mixture of live bacteria and killed bacteria. Even a composition containing killed bacteria of the novel butyric acid bacteria according to the present invention may contain useful butyric acid, and the products or killed bacteria of the novel butyric acid bacteria may exhibit immunostimulatory effects, and the killed bacteria themselves may act as prebiotics, be delivered to the intestine by ingestion, and become a substrate for bifidobacteria and the like, improving the intestinal flora. However, among the products of the novel butyric acid bacteria, butyric acid in particular may be absorbed in the stomach or upper part of the small intestine even if orally ingested, and may not reach the large intestine. In addition, the live bacteria of the novel butyric acid bacteria according to the present invention can directly improve the intestinal flora as a probiotic, so live bacteria or a mixture of live bacteria and killed bacteria are preferred.

[0019] Food and drink refers to liquid or solid substances that are primarily used for human consumption, and examples of beverages include milk drinks such as liquid yogurt, soft drinks, tea, coffee, cocoa, alcoholic beverages, soy milk, soup, etc. Examples of foods include rice bran beds, rice bran pickles, yogurt, etc.

[0020] Since the novel butyric acid bacteria according to the present invention are isolated from a rice bran bed, they can also be cultured in the rice bran bed. For example, a rice bran bed is generally made by adding salt water to rice bran and stirring until it becomes moderately hard. In this case, the novel butyric acid bacteria according to the present invention may be added as part of the salt water or in addition to the salt water. Next, discarded vegetables such as vegetable peels, stems, and cores are added. Also, kelp, chili pepper, Japanese pepper, etc. may be added. It is preferable to replace the discarded vegetables 3 to 4 times every 1 to 2 weeks when they become soft. It is also preferable to stir the rice bran bed from the bottom about 1 to 2 times a day.

[0021] Vegetables may be pickled in the rice bran bed to produce rice bran pickles. The vegetables used as the raw material for rice bran pickles are not particularly limited as long as they are commonly used as raw materials for rice bran pickles. For example, the pickles may include cucumber, bitter melon, zucchini, winter melon, and other Cucurbitaceae fruit vegetables; Solanaceae fruit vegetables such as chili pepper, tomato, eggplant, and bell pepper; Liliaceae stem vegetables such as garlic, green onion, and scallion; Convolvulaceae stem vegetables such as water spinach; Zingiberaceae stem vegetables such as ginger; Poaceae stem vegetables such as bamboo shoots; Cruciferae root vegetables such as turnip, szechuan mustard greens, and radish; Umbelliferae root vegetables such as carrots; Zingiberaceae flower vegetables such as myoga; Cruciferae leaf vegetables such as greens, cabbage, komatsuna, Shandong cabbage, tatsoi, takana, bok choy, nozawana, Chinese cabbage, spinach, mizuna, and mibuna; Liliaceae leaf vegetables such as chives; and Asteraceae leaf vegetables such as lettuce. Other meats such as beef, pork, and chicken, and fish may also be pickled.

[0022] The novel butyric acid bacteria of the present invention and useful compounds such as butyric acid produced by the bacteria are thought to be present mainly in the bran bed, but even after the bran pickles are removed from the bran bed, they may be present in the bran bed adhering to the bran pickles, as well as on the surface of the bran pickles or inside the bran pickles.

[0023] The novel butyric acid bacteria according to the present invention may be used as an active ingredient in compositions such as medicines, supplements, and feed. The form of the medicine or supplement is not particularly limited, and examples thereof include solid, gel, and liquid forms. More specifically, examples thereof include powders, fine granules, granules, tablets, coated tablets, capsules, lozenges, and liquids. The novel butyric acid bacteria according to the present invention can be added to compositions such as medicines, supplements, and feed in the form of a culture solution or a dried form.

[0024] The composition of the present invention may contain additives other than the novel butyric acid bacteria of the present invention depending on the dosage form. Examples of additives include excipients, binders, lubricants, disintegrants, emulsifiers, stabilizers, absorption promoters, differentiation agents, pH adjusters, and preservatives. The amounts of the additives are not particularly limited and may be appropriately adjusted.

[0025] The dosage of the novel butyric acid bacteria according to the present invention may be adjusted as appropriate. For example, the dosage is about 0.2 to 5 times per day, with each dosage being 5×10 5 cfu or more, 5×10 9 It is sufficient to administer the dose at or below cfu.

[0026] The novel butyric acid bacteria according to the present invention are highly effective in producing butyric acid. In addition, butyric acid bacteria are generally more resistant to gastric acid and heat than lactic acid bacteria and are said to easily reach the intestine alive. Therefore, the novel butyric acid bacteria according to the present invention can reach the intestine and produce butyric acid. It is also said that butyric acid provides about 80% of the energy required for intestinal peristalsis. In addition, by keeping the intestine weakly acidic, the bad bacteria that prefer a relatively strong acidic environment are suppressed and the absorption of minerals is promoted, thereby improving the intestinal environment. Therefore, the novel butyric acid bacteria according to the present invention are considered to be useful for improving the intestinal environment.

[0027] In addition, the novel butyric acid bacteria of the present invention stimulate macrophage cells and promote the secretion of TNFα and IL-6, which are inflammatory cytokines that promote immune responses, and are therefore useful as active ingredients of immunostimulants. EXAMPLES

[0028] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the present invention by making appropriate modifications within the scope of the above and below-mentioned aims, and all of these modifications are included in the technical scope of the present invention.

[0029] Example 1: Isolation and identification of strain TK211201 (1) Isolation of strain TK211201 Rice bran from Shizuoka or Ishikawa was mixed with water and salt to a moisture content of 55% by mass and salt content of 5% by mass. After mixing, cucumber or cabbage was soaked for 24 hours. After soaking, 20 g of the bran bed was taken and suspended in an equal amount of RO water. The resulting suspension was filtered under anaerobic conditions, and the resulting filtrate was diluted and spread on a strengthened Clostridia medium ("CM0151B", Oxoid) and cultured at 37°C for 2 days. After culture, the obtained colonies were picked and spread on a new strengthened Clostridia medium and cultured under the same conditions. Among the isolated colonies, the one with the highest butyric acid production ability during cultivation was selected and subjected to simple identification. That is, the colony precipitate was added to 50 μL of achromopeptidase (Fujifilm Wako Pure Chemical Industries, Ltd.) and heated at 50 ° C for 10 minutes. Then, 100 μL of InstaGene DNA purification matrix (Bio-Rad) was added and heated at 55 ° C for 10 minutes. After heating, the mixture was stirred for 10 seconds using a vortex mixer, heated at 100 ° C for 10 minutes, and stirred again for 10 seconds using a vortex mixer. After stirring, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was used as a DNA sample. Bacterial DNA was amplified by repeating 30 cycles of reactions at 94 ° C for 10 seconds, 55 ° C for 5 seconds, and 72 ° C for 1 minute using 9F as a forward primer, 518R as a reverse primer, and a PCR premix reagent ("TaKaRa Ex Taq HS" Takara Bio Inc.). The obtained amplified product was purified using a PCR product purification kit (QIAquick PCR Purification Kit, Qiagen). The purified product was analyzed using a DNA sequencing service (FASMAC), and a homology search was performed using the Techno Suruga Lab Microbial Identification System. The colony obtained (TK211201 strain) was presumed to belong to Clostridium beijerinckii.

[0030] (2) Identification of strain TK211201 DNA of the TK211201 strain was extracted using Cica Geneus DNA Extraction Reagent ST (Kanto Chemical). 16S rDNA was amplified using 9F and 1510R as primers for 16S rDNA amplification, Tks Gflex DNA Polymerase (Takara Bio), and BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). In addition, the base sequence of about 1500 bp of 16S rDNA was determined using 9F, 515F, 1099F, 536R, 926R, and 1510R as primers, ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems), and DNA sequencing software ("ChromaspRO 2.1" Technelysium). A BLAST homology search was performed using ENKI v3.2 (TechnoSuruga Laboratory) software and DB-BA17.0 (TechnoSuruga Laboratory) and the international nucleotide sequence database (DDB / ENA / GenBank) as databases. As a result, the highest homology rate was 99.5% with the DSM791 strain, the type strain of Clostridium beijerinckii. In addition, a simplified molecular phylogenetic analysis was performed using the neighbor-joining method and the Kimura-2-parameter model to correct the number of base substitutions. The reliability of the tree was evaluated using the bootstrap method (1000 iterations). The simplified molecular phylogenetic tree is shown in Figure 1. In Figure 1, "SIID45781-02" indicates the TK211201 strain.

[0031] Furthermore, when the 16S rDNA sequences of the TK211201 strain and the type strain of C. beijerinckii (DSM791 strain) were compared using analytical software ("ENKI v3.2" TechnoSuruga Laboratory), the number of divergent bases was found to be 7.

[0032] The TK211201 strain was cultured for 24 hours at 37°C under anaerobic conditions using enriched Clostridia agar medium (Oxoid), and its morphology was observed using an optical microscope (Olympus BX50F4) and a stereomicroscope (Nikon SMZ800N). In addition, the Gram stainability of the TK211201 strain was tested using a Gram staining solution (Faber G "Nissui", Nissui Pharmaceutical Co., Ltd.). The results are shown in Table 1. The spore formation results are for a culture time of up to 24 hours.

[0033] [Table 1]

[0034] From these results, it was concluded that strain TK211201 belongs to C. beijerinckii.

[0035] Example 2: Butyric acid production test (1) Microbiological test medium The TK211201 strain was added to a microbiological testing medium, enriched Clostridia medium ("CM0151B", Oxoid), and cultured for 24 hours under anaerobic conditions at 37°C (N2: 90%, CO2: 5%, H2: 5%). The resulting culture solution was centrifuged at 5,000 rpm for 15 minutes. The resulting supernatant was filtered using a Cellulose acetate filter (pore size: 0.45 μm), and the filtrate was used as the analytical sample. The concentration of butyric acid contained in the analytical sample was measured by the direct detection method under the following conditions. Column: Ion exclusion column ("Agilent Hi Plex H" Agilent Technologies), 8 μm, 7.7 × 300 mm Detector: Diode array detector ("HS", Agilent Technologies) Equipment: High-performance liquid chromatograph ("1260InfinityII" Agilent Technologies) For comparison, a similar experiment was performed except that the C. beijerinckii type strain (NBRC109359) was used instead of the TK211201 strain. The results are shown in Figure 2(1).

[0036] (2) Edible medium The experiment was conducted in the same manner, except that the microorganism test medium was changed to an edible medium with the composition shown in Table 2. The results are shown in Figure 2 (2). In Figure 2, "*" indicates a significant difference at p<0.05 in the Student's t-test.

[0037] [Table 2]

[0038] As shown in the results in FIG. 2, the TK211201 strain of the present invention exhibited significantly higher butyric acid production ability than the same type of standard strain in both the microbiological test medium and the edible medium, and was found to produce approximately 2.5 times as much butyric acid.

[0039] Example 3: Butyric acid production test (1) HPLC analysis The TK211201 strain or the C. beijerinckii type strain (NBRC 109359) was added to the edible medium used in Example 2(2) and cultured for 24 hours under anaerobic conditions (N2: 90%, CO2: 5%, H2: 5%) at 37°C. The resulting culture solution was then added to a new edible medium at 1% and cultured for 24 hours under aerobic conditions at 37°C. The resulting culture solution or edible medium was used to prepare a rice bran bed with the composition shown in Table 3. In addition, a rice bran bed to which only the edible medium was added was also prepared for comparison.

[0040] [Table 3]

[0041] The rice bran bed prepared was vacuum-packed and sterilized at 94°C for 30 minutes. Then, 20g of sterilized RO water was added to 20g of the prepared rice bran bed, thoroughly suspended, and filtered using a filter ("Nonwoven Spunbond" AS ONE Corporation). The obtained filtrate was used as an analytical sample and analyzed by HPLC in the same manner as in Example 2(1). The results are shown in Figure 3. As shown in the results in FIG. 3, the type strain of C. beijerinckii also produces butyric acid in the rice bran bed, but the TK211201 strain of the present invention was found to be capable of producing more than twice as much butyric acid as the type strain after 24 hours of culture.

[0042] (2) Measurement of bacterial count by PCR Using a DNA extraction kit ("Nucleospin (registered trademark) Soil" from Takara Bio Inc.), DNA was extracted from the analysis sample obtained in Example 3 (1). The obtained DNA solution and standard reagent for real-time PCR ("TB Green (registered trademark) Premix Ex Taq TM A reaction solution was prepared by mixing the C. beijerinckii specific primer solution (Takara Bio Inc.) and sterilized ultrapure water in the composition shown in Table 4.

[0043] [Table 4]

[0044] Real-time PCR was performed using a Thermal Cycler Dice (registered trademark) Real Time System II (Takara Bio Inc.) as a real-time PCR device according to the program shown in Table 5.

[0045] [Table 5]

[0046] The number of C. beijerinckii bacteria was quantified using DNA extracted from the C. beijerinckii type strain as a standard sample and calculations were performed using the analytical software TaKaRa Food and Environmental Test (Takara Bio Inc.). The results are shown in Table 6.

[0047] [Table 6]

[0048] As shown in the results in Table 6, it was demonstrated that C. beijerinckii can grow effectively in rice bran bed, and that the TK211201 strain of the present invention can grow more vigorously than the reference strain.

[0049] Example 4: Immunostimulatory Activity Test (1) Preparation of freeze-dried powder of C. beijerinckii TK211201 strain or C. beijerinckii type strain (NBRC109359) was added to the edible medium used in Example 2(2) and cultured for 24 hours under anaerobic conditions at 37°C (N2: 90%, CO2: 5%, H2: 5%). The obtained culture solution was added to a new edible medium at 1% and cultured for 24 hours under aerobic conditions at 37°C. The obtained culture solution was centrifuged at 15,000 rpm and 4°C for 10 minutes, and the supernatant was removed. Ultrapure water was added to the residue to suspend it so that the total liquid volume was 20 mL, and then the mixture was centrifuged again at 15,000 rpm and 4°C for 10 minutes, and the supernatant was removed. 20 mL of ultrapure water was added to the residue to suspend it, and this was dispensed into sterilized vials in 2 mL portions. The mixture was then freeze-dried using a freeze dryer ("CHRiST ALPHA2-4LSCplus" Kubota Shoji Co., Ltd.). Separately, cell culture medium was prepared by adding 10% FBS (Biosera), 100 units / mL penicillin G, and 100 μg / mL streptomycin sulfate to D-MEM (Fujifilm Wako Pure Chemical Industries, Ltd.). The obtained lyophilized powder was suspended in cell culture medium to a concentration of 2 mg / mL and stored at -30°C until measurement.

[0050] (2) Immunostimulating activity test Mouse-derived macrophage-like cells (RAW264.7, RIKEN BRC) were cultured in the cell culture medium prepared in Example 4(1) at 37°C in a 5% CO environment for 24 hours. After the culture, the cells were harvested and then diluted to 2.5 x 10 4 A cell suspension of 100 μL / mL was prepared. After seeding 100 μL of the prepared cell suspension on a 96-well plate, the plate was cultured at 37° C. in a 5% CO2 environment for 24 hours. The culture medium was removed, and the bacterial suspension prepared in Example 4(1) was added to each well at 100 μL each so that the final concentration of each bacterium was 10 μg / mL or 100 μg / mL, and the plate was cultured under the same conditions for 24 hours. Thereafter, TNF-α and IL-6 in the culture supernatant were measured using a TNF alpha Mouse ELISA Kit (Invitrogen) and an IL-6 Mouse ELISA Kit (Invitrogen). The results are shown in FIG. 4. In FIG. 4, "*" indicates that there is a significant difference at p<0.05 in the Student's t-test.

[0051] TNFα plays an essential role in the early response to viral infection by inducing the expression of vascular endothelial cells as well as enhancing leukocyte adhesion molecules that stimulate immune cell infiltration, for example by promoting the infiltration of lymphocytes into the site of infection. IL-6 is a pleiotropic cytokine that plays an important role in the immune system, particularly in inducing the differentiation of B cells into antibody-producing cells. As shown in FIG. 4, the TK211201 strain of the present invention significantly enhanced the production of TNFα and IL-6 by macrophage-like cells compared to the reference strain.

[0052] To summarize the above experimental results, the C. beijerinckii TK211201 strain isolated from the rice bran bed showed significant butyric acid production and the ability to promote the secretion of TNF-α and IL-6 from macrophages compared to the same type strain. In addition to being the main energy source for the large intestine, butyrate contributes to maintaining a normal intestinal flora. In recent years, it has also been reported that it is one of the factors that suppress the development of colon cancer by inhibiting histone deacetylase and induces differentiation into regulatory T cells, and that it is involved in the health functions of the host. Furthermore, macrophages, a type of immune cell, play a central role as cells of the innate immune system. Macrophages are activated by recognizing foreign substances, including bacteria, with receptors on the cell surface. The activated macrophages then secrete inflammatory cytokines, including TNF-α and IL-6, activating the host's immune function. TNF-α enhances leukocyte adhesion molecules that stimulate the infiltration of immune cells into foreign substances, and IL-6 is known to be involved in inducing the differentiation of B cells into antibody-producing cells. Both are essential cytokines in the immune response and are strongly involved in the host's defense against infection and elimination of foreign substances. In other words, it was suggested that C. beijerinckii TK211201 strain is a strain that has excellent butyric acid production ability and also functions as an immunostimulant. It was also revealed that by adding the culture liquid of this strain to a rice bran bed, it is possible to produce a rice bran bed containing high concentrations of butyric acid and fungal bodies.

Claims

1. Clostridium beijerinckii TK211201 strain (accession number: NITE P-03982).

2. A composition comprising Clostridium beijerinckii TK211201 strain (accession number: NITE P-03982).

3. The composition according to claim 2, which is in the form of a food or drink, a medicine, a supplement, or a feed.

4. A rice bran bed characterized by containing Clostridium beijerinckii TK211201 strain (accession number: NITE P-03982).

5. A rice bran pickle containing Clostridium beijerinckii TK211201 strain (accession number: NITE P-03982).

6. An immunostimulant comprising Clostridium beijerinckii TK211201 strain (accession number: NITE P-03982).

7. The immunostimulant according to claim 6, which promotes the production of inflammatory cytokines.

8. The immunostimulant according to claim 7, wherein the inflammatory cytokine is TNFα and / or IL-6.

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