New strain of clostridium butyricum having butyric acid production ability

Abstract

The purpose of the present invention is to provide a butyric acid bacterium that can be utilized more safely as an active ingredient of an intestinal regulator and the like. The present invention relates to Clostridium butyricum that produces n-butyric acid and has all of the following features (1)-(3).　(1) No D-xylose assimilation ability. (2) No D-trehalose assimilation ability. (3) The amount of formic acid contained in the culture broth after 72 hours of anaerobic culture at 37°C is less than that of NBRC 13949T, which is the Clostridium butyricum reference strain.

Description

A new strain of Clostridium butyricum capable of producing butyrate

[0001] The present invention relates to a new strain of Clostridium butyricum capable of producing butyric acid, and a composition for intestinal regulation using the same.

[0002] In recent years, the usefulness of butyric acid bacteria has been attracting attention. Butyric acid bacteria, which are intestinal bacteria, produce butyric acid by breaking down dietary fiber in the large intestine. Butyric acid is used as an energy source for the normal functioning of the large intestine, and so butyric acid bacteria are used as an active ingredient in intestinal regulators (Non-Patent Documents 1 and 2). Clostridium butyricum is known as a representative example of butyric acid bacteria (Non-Patent Documents 1 to 4). Clostridium butyricum produces formic acid in addition to butyric acid and assimilates D-xylose and D-trehalose (Non-Patent Document 3).

[0003] Yuji Naito, "Increasing butyric acid bacteria will lead to health and longevity: Learn everything about the popular butyric acid and butyric acid bacteria!", Asa Publishing Co., Ltd., February 11, 2022. Akira Eda, "Amazing butyric acid bacteria: The dividing line between those who get sick and those who don't," Gentosha Co., Ltd., March 25, 2022. Bergey's Manual of Systematic Bacteriology, Second Edition, Volume Three, The Firmicutes, pp. 739-742 (2009). Japanese Society of Enterobacteriaceae, "Glossary: ​​Clostridium butyricum," [online], [searched August 5, 2023], Internet <URL: https: / / bifidus-fund.jp / keyword / kw002.shtml>. International Chemical Safety Cards (ICSC) Database, search results for "formic acid," [online], [searched November 1, 2023], Internet <URL: https: / / www.ilo.org / dyn / icsc / showcard.listCards3>International Chemical Safety Cards (ICSC) Database, search results for "acetone", [online], [searched November 1, 2023], Internet <URL: https: / / www.ilo.org / dyn / icsc / showcard.listCards3>Journal of Enterobacteriaceae, 31:15-22, 2017

[0004] However, formic acid is known to be toxic, causing abdominal pain, stomach cramps, and diarrhea when orally ingested (Non-Patent Document 5). Acetone is also known to be toxic, causing nausea and vomiting when orally ingested (Non-Patent Document 6). Therefore, the present inventors set out to provide a butyric acid bacterium that produces small amounts of formic acid and acetone and can be safely used as an active ingredient in intestinal regulators and the like.

[0005] As a result of intensive research into this problem, the present inventors have discovered that Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916), which was isolated during the process of fermenting organically grown domestic soybeans to produce a food ingredient, is (1) a novel strain with physiological and biochemical properties different from those of conventional Clostridium butyricum (it does not assimilate D-xylose or D-trehalose), and (2) when compared with the type strain of Clostridium butyricum (strain NBRC13949T), the new strain produces less formic acid and acetone. The present invention was made based on these findings.

[0006] That is, the present invention relates to the following [1] to

[19] . [1] Clostridium butyricum that produces n-butyric acid and has all of the following characteristics (1) to (3): (1) It does not have the ability to utilize D-xylose; (2) It does not have the ability to utilize D-trehalose; and (3) It produces less formic acid in the culture medium after anaerobic culture at 37°C for 72 hours, compared to the type strain of Clostridium butyricum, NBRC13949T. [2] Clostridium butyricum SIID29215-B6 strain (accession number: NITE BP-03916) or a mutant thereof, wherein the mutant produces n-butyric acid and has all of the following characteristics (1) to (3). (1) Inability to utilize D-xylose (2) Inability to utilize D-trehalose (3) Compared to the type strain of Clostridium butyricum, NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic culture at 37°C for 72 hours is lower [3] Clostridium butyricum SIID29215-B6 strain (Accession No.: NITE BP-03916). [4] Clostridium butyricum SIID49520-01-B1 strain (Accession No.: NITE BP-04190) or a mutant thereof, wherein the mutant produces n-butyric acid and has all of the following characteristics (1) to (3): (1) Inability to utilize D-xylose (2) Inability to utilize D-trehalose (3) Compared to the type strain of Clostridium butyricum, NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic culture at 37°C for 72 hours is lower. [5] Clostridium butyricum SIID49520-01-B1 strain (Accession No.: NITE BP-04190). [6] Clostridium butyricum SIID50030-B1 strain (Accession No.: NITE BP-04191) or a mutant thereof, wherein the mutant produces n-butyric acid and has all of the following characteristics (1) to (3):(1) Inability to utilize D-xylose (2) Inability to utilize D-trehalose (3) Compared to the type strain NBRC13949T of Clostridium butyricum, the amount of formic acid produced in the culture medium after anaerobic culture at 37°C for 72 hours is lower. [7] Clostridium butyricum SIID50030-B1 strain (Accession number: NITE BP-04191). [8] A composition for intestinal regulation, comprising as an active ingredient the bacterial cells, spores, or culture medium of Clostridium butyricum described in any one of [1] to [7] above. [9] A composition for inhibiting the growth of Clostridioides difficile, comprising as an active ingredient the bacterial cells, spores, or culture medium of Clostridium butyricum described in any one of [1] to [7] above.

[10] An anti-inflammatory composition comprising, as an active ingredient, the bacterial cells, spores, or culture medium of Clostridium butyricum according to any one of [1] to [7] above.

[11] A composition for inducing regulatory T cells comprising, as an active ingredient, the bacterial cells, spores, or culture medium of Clostridium butyricum according to any one of [1] to [7] above.

[12] The composition according to [8] above, in the form of a pharmaceutical product.

[13] The composition according to [9] above, in the form of a pharmaceutical product.

[14] The composition according to

[10] above, in the form of a pharmaceutical product.

[15] The composition according to

[11] above, in the form of a pharmaceutical product.

[16] The composition according to [8] above, in the form of a food or drink.

[17] The composition according to [9] above, in the form of a food or drink.

[18] The composition according to

[10] above, in the form of a food or drink.

[19] The composition according to

[11] above, in the form of a food or drink.

[0007] As shown in Examples 2 and 3 below, the Clostridium butyricum strain of the present invention produces less formic acid and acetone than conventional strains. Furthermore, as shown in Examples 8 and 10 below, the Clostridium butyricum strain of the present invention produces less formic acid than conventional strains. Therefore, the strain of the present invention is useful as an active ingredient in products (such as intestinal regulators) that utilize butyric acid bacteria.

[0008] Figure 1 shows a simplified molecular phylogenetic tree based on the 16S rDNA partial base sequence of the Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916). Figure 2 shows a simplified molecular phylogenetic tree based on the 16S rDNA partial base sequence of the Clostridium butyricum strain SIID49520-01-B1 (accession number: NITE BP-04190). Figure 3 shows a simplified molecular phylogenetic tree based on the 16S rDNA partial base sequence of the Clostridium butyricum strain SIID50030-B1 (accession number: NITE BP-04191).

[0009] The bacterial strain of the present invention is Clostridium butyricum. Clostridium butyricum is a representative species (type species) of the genus Clostridium, as described in Bergey's Manual of Systematic Bacteriology, Second Edition, Volume Three, The Firmicutes, pp. 739-742 (2009) (Non-Patent Document 3) and on the website of the Society of Intestinal Microbiology, Public Interest Foundation ("Glossary: ​​Clostridium butyricum," URL: https: / / bifidus-fund.jp / keyword / kw002.shtml) (Non-Patent Document 4).

[0010] A bacterial strain corresponding to a preferred embodiment of the present invention has been internationally deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), an international depository institution under the provisions of the Budapest Treaty (identification: SIID29215-B6. Accession number: NITE BP-03916. Date of deposit (deposit): June 15, 2023). A bacterial strain according to another preferred embodiment of the present invention has been internationally deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), an international depository institution under the provisions of the Budapest Treaty (identification: SIID49520-01-B1; accession number: NITE BP-04190; date of deposit (accession): October 29, 2024). A bacterial strain according to another preferred embodiment of the present invention has been internationally deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), an international depository institution under the provisions of the Budapest Treaty (identification: SIID50030-B1; accession number: NITE BP-04191; date of deposit (accession): October 29, 2024).

[0011] [Characteristics of the strain of the present invention] The characteristics of the strain of the present invention are described below. (1) Production of n-butyric acid The strain of the present invention produces n-butyric acid. The amount of n-butyric acid produced can be measured by subjecting the culture filtrate of the strain to organic acid analysis (e.g., high performance liquid chromatography).

[0012] (2) Sugar assimilation The bacterial strain of the present invention has one, preferably two of the following characteristics (1) to (2) regarding sugar assimilation ability (the ability to utilize sugar as a nutrient source): (1) Inability to assimilate D-xylose (2) Inability to assimilate D-trehalose The sugar assimilation ability can be determined using a commercially available microbial identification test kit (e.g., API20A (bioMerieux, FRA)).

[0013] Bergey's Manual of Systematic Bacteriology, Second Edition, Volume Three, The Firmicutes, pp. 739-742 (2009) (Non-Patent Document 3) describes that Clostridium butyricum is capable of assimilating D-xylose and D-trehalose. Furthermore, as shown in Example 1 (morphological, physiological, and biochemical property tests) described below, the NBRC13949T strain, which is the type strain of Clostridium butyricum, also had the ability to assimilate D-xylose and D-trehalose. Therefore, the strain of the present invention, which differs from conventional strains in its ability to assimilate sugars, is a new strain of Clostridium butyricum.

[0014] (3) Production of Formic Acid In a preferred embodiment, the amount of formic acid produced by the strain of the present invention is less than that produced by the NBRC13949T strain, the type strain of Clostridium butyricum. Specifically, when compared based on the amount of formic acid produced in the culture medium after anaerobic culture at 37°C for 72 hours, the amount produced by the strain of the present invention is less than that produced by the NBRC13949T strain, and is preferably 12% or less of the amount produced by the NBRC13949T strain. The amount of formic acid produced can be measured by subjecting the culture filtrate of the strain to organic acid analysis (e.g., high-performance liquid chromatography).

[0015] (4) Production of Acetone In a preferred embodiment, the amount of acetone produced by the strain of the present invention is less than that produced by the NBRC13949T strain, which is the type strain of Clostridium butyricum. Specifically, when compared based on the amount of acetone produced in the culture medium after anaerobic culture at 37°C for 48 hours, the amount produced by the strain of the present invention is less than that produced by the NBRC13949T strain, and is preferably 70% or less of the amount produced by the NBRC13949T strain. The amount of acetone produced can be measured by subjecting the culture supernatant of the strain to GC / MS measurement.

[0016] (5) 16S rDNA and Genomic DNA The strain of the present invention may have, as a partial base sequence of 16S rDNA (a gene encoding 16S rRNA), a base sequence that has at least 99.6% or more, preferably 99.8% or more, and more preferably 99.9% or more sequence identity to the base sequence shown in SEQ ID NO: 1, SEQ ID NO: 174, or SEQ ID NO: 175 (described below). Furthermore, the strain of the present invention may have, as a genome sequence, a base sequence that has at least 99.6% or more, preferably 99.8% or more, and more preferably 99.9% or more sequence identity to the base sequence shown in any of SEQ ID NOs: 2 to 173 (described below). (6) Spore Formation In a preferred embodiment, the strain of the present invention has spore formation ability. In this embodiment, spores of the strain of the present invention can be used as an active ingredient in products (such as intestinal regulators) that utilize butyric acid bacteria.

[0017] (7) Other Features In addition to the features (1) to (6) above, the strain of the present invention may have one or more of the features (e.g., heat resistance, growth inhibition of Clostridioides difficile, etc.) possessed by the Clostridium butyricum strain SIID29215-B6 (Accession Number: NITE BP-03916) shown in the Examples described later. Furthermore, the strain of the present invention may have one or more of the features possessed by the Clostridium butyricum strain SIID49520-01-B1 (Accession Number: NITE BP-04190) or the Clostridium butyricum strain SIID50030-B1 (Accession Number: NITE BP-04191) shown in the Examples described later.

[0018] In a preferred embodiment, the strain of the present invention is Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916) or a mutant thereof. This mutant strain produces n-butyric acid and has all of the following characteristics (1) to (3): (1) it does not have the ability to utilize D-xylose; (2) it does not have the ability to utilize D-trehalose; and (3) it produces less formic acid in the culture medium after anaerobic culture at 37°C for 72 hours, compared to the type strain of Clostridium butyricum, NBRC13949T.

[0019] In a preferred embodiment, the strain of the present invention is a mutant of Clostridium butyricum strain SIID49520-01-B1 (accession number: NITE BP-04190). This mutant strain produces n-butyric acid and has all of the following characteristics (1) to (3): (1) it does not have the ability to utilize D-xylose; (2) it does not have the ability to utilize D-trehalose; and (3) it produces less formic acid in the culture medium after anaerobic culture at 37°C for 72 hours, compared to the type strain of Clostridium butyricum, NBRC13949T.

[0020] In another preferred embodiment, the strain of the present invention is a mutant of Clostridium butyricum strain SIID50030-B1 (accession number: NITE BP-04191). This mutant strain produces n-butyric acid and has all of the following characteristics (1) to (3): (1) it does not have the ability to utilize D-xylose; (2) it does not have the ability to utilize D-trehalose; and (3) it produces less formic acid in the culture medium after anaerobic culture at 37°C for 72 hours, compared to the type strain of Clostridium butyricum, NBRC13949T.

[0021] The mutant strains of the deposited strain described above have been modified in terms of their physical and chemical properties by screening the deposited strain after inducing mutations by drug treatment and / or ultraviolet irradiation, etc., natural mutations (for example, natural mutations that occur during repeated subculture), morphological mutations, or genetic engineering techniques such as transfection, and they produce n-butyric acid and have all of the following characteristics (1) to (3): (1) No D-xylose assimilation ability (2) No D-trehalose assimilation ability (3) Compared to the NBRC13949T strain, the type strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after anaerobic culture at 37°C for 72 hours is lower.

[0022] Furthermore, the mutant strain of the deposited strain may have a nucleotide sequence that has at least 99.6% or more, preferably 99.8% or more, and more preferably 99.9% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 174, or SEQ ID NO: 175 (described below) as a partial nucleotide sequence of the 16S rDNA of the deposited strain.

[0023] The Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916) can be referred to as the "clostridium butyricum unilac strain."

[0024] [Culture Conditions for the Strain of the Present Invention] No special conditions are required for culturing the strain of the present invention, and it can be cultured under conditions commonly used for Clostridium butyricum. The medium is preferably a medium for anaerobic bacteria. The medium may be either a natural medium or a synthetic medium. When using the strain of the present invention for food applications, a medium composed only of food ingredients and food additives may be used. Culture can be carried out under anaerobic conditions, for example, at a temperature of 30 to 45°C (preferably 36 to 38°C). Anaerobic conditions refer to a low-oxygen environment sufficient for the growth of Clostridium butyricum. Anaerobic conditions can be achieved using a sealed container or bag containing an oxygen absorber. Examples of culture methods include static culture, agitation culture, and tank culture. The culture time is preferably 24 to 72 hours (including 48 hours). No special conditions are required for spore formation; spores are formed by culturing the strain of the present invention under the aforementioned conditions, preferably by heating at 55 to 60°C for 30 minutes.

[0025] [Method of Obtaining the Strain of the Present Invention] As mentioned above, three strains of the present invention have been deposited internationally, and all are available from international depository institutions. Furthermore, like the three internationally deposited strains, the strain of the present invention can also be isolated from soybeans. Although the Clostridium bacteria to which the strain of the present invention belongs are soil bacteria, soybeans are a preferable source of obtaining the strain of the present invention, rather than soil, where many types of soil bacteria live. While the present invention is not limited by any particular theory, the reason why soybeans are a preferable source is as follows. At the time of harvest, the soybean pods split open, exposing the kernels inside. Clostridium bacteria (or their spores), blown up from the soil by wind or other factors, adhere to the exposed kernels. The thin skin of soybeans is primarily composed of dietary fiber. Furthermore, the kernels themselves are rich in protein. Both dietary fiber and protein are favorable substances for the growth of Clostridium bacteria. Therefore, there is a high possibility that Clostridium bacteria (or their spores) are attached to harvested soybeans (especially soybeans grown organically without the use of pesticides). Clostridium bacteria can be obtained from soybeans, for example, according to the following procedure: Soybeans are fermented together with water, honey, and indigestible dextrin in an aerobic environment at 37°C for 72 hours. After fermentation is complete, the resulting fermentation liquid and soybeans are made into a paste. This fermented soybean paste is subjected to the anaerobic conditions described above in the section "Cultivation conditions for the strain of the present invention." From the obtained Clostridium bacteria, Clostridium butyricum is identified using sequence information (e.g., the known base sequence of the 16S rDNA portion) of the type strain of Clostridium butyricum (NBRC13949T strain). The identified Clostridium butyricum can be further screened using the characteristics of the strain of the present invention (such as the ability to assimilate D-xylose and D-trehalose, or the ability to produce formic acid) as indicators to obtain the strain of the present invention.

[0026] [Uses of the strain of the present invention] The strain of the present invention can be used in the same applications as known butyric acid bacteria (particularly Clostridium butyricum), for example, as an active ingredient of an intestinal regulator, anti-inflammatory agent, or regulatory T cell inducer.

[0027] [Composition for Intestinal Regulation] Butyric acid bacteria can maintain normal intestinal flora through butyric acid production (Non-Patent Documents 1 and 2), and are therefore used as active ingredients in intestinal regulators. The strain of the present invention has the ability to produce butyric acid (see Examples 2, 8, and 10 below). Therefore, one aspect of the present invention is a composition for intestinal regulation containing the bacterial cells, spores, or culture solution of the bacterial strain of the present invention (hereinafter also referred to as "the bacterial cells of the present invention") as an active ingredient. The active ingredient may be either the bacterial cells or spores, or a combination of the bacterial cells and spores. The culture solution may contain either the bacterial cells or spores, or both the bacterial cells and spores. "Containing as an active ingredient" means that the composition contains the bacterial cells of the present invention in an amount (effective amount) sufficient to exert the desired intestinal regulation effect (effect of regulating the intestinal environment). The content of the bacterial cells of the present invention can be appropriately determined taking into account the form of the composition (e.g., food, beverage, pharmaceutical, etc.). For example, in the case of human foods, the content (number of bacteria) of "bacterial cells" as an active ingredient can be set to the same level as the number of butyric acid bacteria contained in general intestinal regulators. For example, the content of bacterial cells in human foods can be set so that the daily intake of bacterial cells is preferably 300 million to 2.5 billion cells, more preferably 1 billion to 2 billion cells. The intestinal regulation composition may contain one or more optional ingredients to the extent that the action of the active ingredient is not impaired. The optional ingredients can be selected appropriately depending on the form of the composition, etc. The optional ingredients may be additives for foods, beverages, or pharmaceuticals. The optional ingredients are known substances that are easily available on the market or can be prepared. A single type of optional ingredient may be used, or multiple types may be used in combination. The content of the optional ingredients can be set appropriately depending on the purpose of the intestinal regulation composition, etc. The intestinal regulation composition can be applied to a wide range of animal species without particular restrictions. The target subjects are preferably mammals (humans and non-human mammals (e.g., dogs and cats)), more preferably humans. Furthermore, the target subjects can be used regardless of gender or age. The intake amount of the composition for intestinal regulation can be appropriately set depending on the age and weight of the subject, the number of intakes, the route of administration, etc. The intake interval can be appropriately set depending on the intake amount, etc., and may be once a day or may be taken in several divided doses. The composition for intestinal regulation can be prepared by mixing the active ingredient (the bacterial cells of the present invention, etc.) and an optional ingredient (for example, an additive for food, drink, or medicine).

[0028] [Composition for Inhibiting the Growth of Clostridioides difficile] It is known that antibiotic-associated diarrhea (diarrhea, loose stools, etc.) occurs when the intestinal environment is disrupted by the administration of antibiotics, leading to the abnormal proliferation of certain bacteria. Clostridioides difficile is a typical causative bacterium of antibiotic-associated diarrhea. The bacterial strain of the present invention can inhibit the growth of Clostridioides difficile (see Example 6 below). Therefore, one aspect of the present invention is a composition for inhibiting the growth of Clostridioides difficile, comprising the bacterial cells of the present invention as an active ingredient. The active ingredient may be either bacterial cells or spores, or a combination of bacterial cells and spores. The culture medium may contain either bacterial cells or spores, or both bacterial cells and spores. The phrase "containing as an active ingredient" means that the composition contains the bacterial cells of the present invention in an amount sufficient (effective amount) to exert the desired growth inhibitory effect. The content of the bacterial cells of the present invention can be appropriately determined taking into account the form of the composition (e.g., food or beverage, pharmaceutical, etc.). For example, in the case of a food for human use, the content (number of bacteria) of "bacterial cells" as an active ingredient can be set to the same amount as the number of butyric acid bacteria contained in a general intestinal regulator. For example, the content of bacterial cells in a human food can be set so that the daily intake of bacterial cells is preferably 300 million to 2.5 billion cells, more preferably 1 billion to 2 billion cells. The proliferation-inhibitory composition may contain one or more optional ingredients to the extent that the action of the active ingredient is not impaired. The optional ingredients can be appropriately selected depending on the form of the composition, etc. The optional ingredients may be additives for food or beverage or pharmaceutical use. The optional ingredients are known substances that are easily available on the market or can be prepared. A single type of optional ingredient may be used, or multiple types may be used in combination. The content of the optional ingredients can be appropriately determined depending on the purpose of the formulation, etc. The proliferation-inhibitory composition can be applied to a wide range of animal species without particular limitation. The target of application is preferably mammals (humans and non-human mammals (e.g., dogs and cats)), more preferably humans. The amount of the proliferation-inhibitory composition to be ingested can be determined appropriately depending on the age and weight of the subject, the frequency of ingestion, the route of administration, etc.The intake interval can be appropriately set depending on the intake amount, etc., and may be once a day or may be divided into several doses. The growth-inhibiting composition can be prepared by mixing the active ingredient (the bacterial cells of the present invention, etc.) and an optional ingredient (for example, an additive for food, beverage, or pharmaceutical use).

[0029] [Anti-inflammatory Composition] Butyric acid bacteria are known to have anti-inflammatory effects (e.g., inhibitory effects on enteritis, ulcerative colitis, and Crohn's disease) through butyric acid production (Non-Patent Document 1). The bacterial strain of the present invention has butyric acid production ability (see Example 2 below). Therefore, one aspect of the present invention is an anti-inflammatory composition containing the bacterial cells of the present invention as an active ingredient. The active ingredient may be either bacterial cells or spores, or a combination of bacterial cells and spores. The culture medium may contain either bacterial cells or spores, or both bacterial cells and spores. "Containing as an active ingredient" means that the composition contains the bacterial cells of the present invention in an amount (effective amount) sufficient to exert the desired anti-inflammatory effect. The content of the bacterial cells of the present invention can be appropriately determined taking into account the form of the composition (e.g., food, beverage, pharmaceutical, etc.). For example, in the case of human food, the content (number of bacteria) of "bacterial cells" as an active ingredient can be set to the same amount as the number of butyric acid bacteria contained in a typical intestinal regulator. For example, the content of bacterial cells in human foods can be set so that the daily intake of bacterial cells is preferably 300 million to 2.5 billion cells, more preferably 1 billion to 2 billion cells. The anti-inflammatory composition may contain one or more optional ingredients to the extent that the action of the active ingredient is not impaired. The optional ingredients can be selected appropriately depending on the form of the composition, etc. The optional ingredients may be additives for foods, beverages, or pharmaceuticals. The optional ingredients are known substances that are easily available on the market or can be prepared. A single type of optional ingredient may be used, or multiple types may be used in combination. The content of the optional ingredient can be set appropriately depending on the purpose of incorporation, etc. The anti-inflammatory composition can be applied to a wide range of animal species without particular restrictions. The target of application is preferably mammals (humans and non-human mammals (e.g., dogs and cats)), more preferably humans. Furthermore, the gender and age of the target of application are not important. The intake amount of the anti-inflammatory composition can be set appropriately depending on the age and weight of the target of application, the frequency of intake, the route of administration, etc. The intake interval can be appropriately set depending on the intake amount, etc., and the composition may be taken once a day or in divided doses. The anti-inflammatory composition can be prepared by mixing the active ingredient (the bacterial cells of the present invention, etc.) and an optional ingredient (for example, an additive for food, beverage, or pharmaceutical use).

[0030] [Composition for Inducing Regulatory T Cells] Butyric acid bacteria are known to induce regulatory T cells through butyric acid production (Non-Patent Documents 1 and 7). Induction includes the induction of differentiation into regulatory T cells and the induction of proliferation of regulatory T cells (Non-Patent Documents 1 and 7). Regulatory T cells (Treg) play a role in suppressing immune responses against the self (immune tolerance). The bacterial strain of the present invention has the ability to produce butyric acid (see Example 2 below). Therefore, one aspect of the present invention is a composition for inducing regulatory T cells, comprising the bacterial cells of the present invention as an active ingredient. The active ingredient may be either bacterial cells or spores, or a combination of bacterial cells and spores. The culture medium may contain either bacterial cells or spores, or may contain both bacterial cells and spores. "Containing as an active ingredient" means that the composition contains the bacterial cells of the present invention in an amount (effective amount) sufficient to exert the desired regulatory T cell induction effect. The content of the bacterial cells of the present invention can be appropriately determined taking into account the form of the composition (e.g., food, beverage, pharmaceutical, etc.). For example, in the case of human foods, the content (number of bacteria) of "bacterial cells" as an active ingredient can be set to the same level as the number of butyric acid bacteria contained in general intestinal regulators. For example, the content of bacterial cells in human foods can be set so that the daily intake of bacterial cells is preferably 300 million to 2.5 billion cells, more preferably 1 billion to 2 billion cells. The composition for inducing regulatory T cells may contain one or more optional ingredients to the extent that the action of the active ingredient is not impaired. The optional ingredients can be selected appropriately depending on the form of the composition, etc. The optional ingredients may be additives for foods, beverages, or pharmaceuticals. The optional ingredients are known substances that are readily available on the market or can be prepared. A single type of optional ingredient may be used, or multiple types may be used in combination. The content of the optional ingredients can be set appropriately depending on the purpose of incorporation, etc. The composition for inducing regulatory T cells can be applied to a wide range of animal species without particular limitations. The target of application is preferably mammals (humans and non-human mammals (e.g., dogs and cats)), more preferably humans. Furthermore, the target of application is not limited to gender or age. The amount of intake of the composition for inducing regulatory T cells can be appropriately determined depending on the age and weight of the subject, the number of intakes, the administration route, etc. The interval between intakes can be appropriately determined depending on the intake amount, etc., and the composition may be taken once a day or in several divided doses.The composition for inducing regulatory T cells can be prepared by mixing an active ingredient (such as the bacterial cells of the present invention) with an optional ingredient (for example, an additive for food, drink, or medicine).

[0031] [Use of the composition containing the bacterial cells of the present invention] The above-mentioned composition for intestinal regulation, composition for inhibiting the growth of Clostridioides difficile, anti-inflammatory composition, and composition for inducing regulatory T cells can be used as a food or drink or a pharmaceutical product.

[0032] [Food and drink] The form of the food and drink is not particularly limited as long as it can be taken orally. Specific examples include liquid drinks and jelly drinks. Food and drink include health foods, functional foods, nutritional supplements, foods for specified health uses, foods for patients, and foods and drink with disease risk reduction labeling. Food and drink may be sports supplement products (including products certified by Informed Sports) or cereal products. Food and drink may contain food additives as optional ingredients. Examples of additives for liquid drinks include pH adjusters, emulsifiers, stabilizers, flavorings, sweeteners, etc. Examples of additives for jelly drinks include gelatin, food colorings, thickening polysaccharides, etc.

[0033] [Pharmaceuticals] The dosage form of the pharmaceutical is preferably one that can be administered orally or rectally (e.g., enema). Specific examples include liquids, capsules, and powders. The pharmaceutical may contain pharmaceutical additives as optional components. Examples of pharmaceutical additives include excipients, stabilizers, preservatives, humectants, emulsifiers, lubricants, sweeteners, colorants, flavorings, buffers, antioxidants, and pH adjusters.

[0034] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0035] [Test strain 1] Clostridium butyricum SIID29215-B6 strain (hereinafter also referred to as "B6 strain") was used. The B6 strain has been internationally deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, an international depository institution under the provisions of the Budapest Treaty (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) (Identification: SIID29215-B6. Accession number: NITE BP-03916. Deposit date: June 15, 2023).

[0036] As the type strain of Clostridium butyricum, Clostridium butyricum NBRC13949T strain (hereinafter also referred to as "type strain") was used.

[0037] Example 1: Morphological, physiological, and biochemical property tests Using an agar medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar) and an anaerobic culture kit, the B6 strain and the type strain were anaerobically cultured at 37°C for 72 hours using the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan). The morphology of the cultured B6 strain and the type strain was observed using an optical microscope. Furthermore, the physiological properties of the B6 strain and the type strain (catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F)) were tested according to the method of Barrow & Feltham et al. (Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edition, Cambridge: University Press; 1993). The results are shown in Table 1-1.

[0038] The B6 strain and the type strain were anaerobically cultured at 37°C for 24 hours using an agar medium (GAM Bouillon "Nissui" (Nissui Pharmaceutical, Japan) + agar) and an anaerobic culture kit, the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan). The physiological and biochemical properties of the cultured B6 strain and the type strain were identified using an anaerobic bacteria biochemical identification kit (API20A (bioMerieux, FRA)). The results are shown in Table 1-2.

[0039] Furthermore, additional physiological and biochemical properties of the B6 strain were tested using an API ZYM kit (bioMerieux, FRA), and the results are shown in Tables 1-3.

[0040] The B6 strain did not have the ability to assimilate D-xylose and D-trehalose (Table 1-2). On the other hand, the type strain had the ability to assimilate D-xylose and D-trehalose. Therefore, the B6 strain was determined to be a different strain from the type strain.

[0041] Example 2: Organic acid production ability test Using a liquid medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan)) and an anaerobic culture kit, AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan), the B6 strain and the type strain were anaerobically cultured at 37°C for 72 hours (culture volume: 5 mL). The culture solution was filtered through a membrane filter with a pore size of 0.20 µm to obtain a sample solution. The concentration of organic acids contained in the sample solution was measured by high-performance liquid chromatography. The measurement conditions were as follows: System: Shimadzu Organic Acid Analysis System (Shimadzu, Japan) Column: Shim-pack Fast-OA, 100 mm x 7.8 mm ID, 3 columns used in series Guard column: Shim-pack Fast-OA, 10 mm x 4.0 mm ID Eluent: 5 mmol / L p-toluenesulfonic acid Reaction solution: 5 mmol / L p-toluenesulfonic acid, 100 μmol / L EDTA, 20 mmol / L Bis-Tris Flow rate: 0.8 mL / min Oven temperature: 50°C Detector: Electrical conductivity detector CDD-10Avp The nine organic acids measured were succinic acid, lactic acid, formic acid, acetic acid, propionic acid, iso-butyric acid, n-butyric acid, iso-valeric acid, and n-valeric acid. The results are shown in Table 2.

[0042] In the table, blank cells indicate values ​​below the lower limit of quantitation. The lower limit of quantitation was 5 μg / mL for succinic acid, lactic acid, acetic acid, and propionic acid, and 10 μg / mL for formic acid, iso-butyric acid, n-butyric acid, iso-valeric acid, and n-valeric acid. The value for each organic acid is the average of three samples.

[0043] The B6 strain produced n-butyric acid. Furthermore, the amount of formic acid produced in the culture medium after anaerobic culture of the B6 strain at 37°C for 72 hours was less than that of the reference strain (NBRC13949T).

[0044] Example 3: Acetone and Alcohol Production Ability Test Using a liquid medium (GAM Bouillon "Nissui" (Nissui Pharmaceutical, Japan)) and an anaerobic culture kit (Anelopouch Kenki System, Mitsubishi Gas Chemical, Japan), the B6 strain and the type strain were anaerobically cultured at 37°C for 48 hours. After 24 and 48 hours of culture, the culture solution was aliquoted into sterile tubes. The culture solution was centrifuged, and the resulting supernatant (10 mL or more) was filtered through a 0.2 μm cellulose acetate filter (sterilized). The filtrate was stored refrigerated at 4°C and used as a sample. Approximately 1 g of the sample was placed in a volumetric flask, and the volume was adjusted to 10 mL with methanol. The mixture was stirred on a shaker for 1 hour and then allowed to stand for 3 hours. 1 μL of the supernatant was then subjected to GC / MS measurement. The measurement conditions are as follows. CG / MS: Agilent Technologies, 6890N / 5973inert Column: HP-1 (0.25 mmφ×30 m, df=1.00 μm) Column temperature: 40°C (3 min) → 10°C / min → 100°C → 20°C / min → 300°C (10 min) Column pressure: constant flow mode (51 kPa, Vac) Column flow rate: 1 mL / min (He) Injection port temperature: 250°C Injection volume: 1 μL Injection method: split (10:1) Detector: MS Ion source temperature: 230°C Ionization method: EI (70 eV) Scan range: SIM (m / z: 31, 41, 43, 45, 58, 59, 2.0 to 3.5 min) : SIM (m / z: 41, 43, 45, 56, 59, 74, 3.5 to 29.0 min) Gain: 976 V

[0045] The pure chemicals of each component shown in Table 3 were diluted with methanol to prepare standard samples of a fixed concentration. 1 μL of the standard sample was measured in the same manner as the above-mentioned samples, and a calibration curve was created from the peak area in the mass chromatogram of the base ion or molecular ion of each quantified component and the preparation concentration, and the content of each component per 1 g of sample was calculated. The results are shown in Table 3.

[0046]

[0047] The amount of acetone produced in the culture medium after anaerobic culture of the B6 strain at 37°C for 48 hours was less than that of the type strain (NBRC13949T).

[0048] Example 4: Spore formation confirmation test Using a liquid medium (GAM Bouillon "Nissui" (Nissui Pharmaceutical, Japan) or an agar medium (GAM Bouillon "Nissui" (Nissui Pharmaceutical, Japan) + agar), and an anaerobic culture kit, AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan), the B6 strain was anaerobically cultured at 37°C for 24 hours, 72 hours, 7 days, and 14 days. After the culture, the bacterial cells were Gram stained (Faber G "Nissui" (Nissui Pharmaceutical, Japan)). The spore-forming ability was confirmed by microscopic observation (optical microscope: BX50F4 (Olympus, Japan)). The results are shown in Table 4.

[0049]

[0050] Strain B6 was a spore-forming, Gram-positive bacterium.

[0051] Example 5: Heat resistance test Using an agar medium (GAM Bouillon "Nissui" (Nissui Pharmaceutical, Japan) + agar) and an Anaerobic Pouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, the B6 strain was pre-cultured under anaerobic conditions at 37°C for 24 or 72 hours. The culture was suspended in 10 mL of physiological saline to prepare a bacterial solution. Five mL of the bacterial solution was dispensed into a sterile medium-sized test tube (18 x 170 mm) and subjected to heat treatment at 60°C for 30 minutes in a water bath. Five mL of the bacterial solution before heat treatment served as a control. The viable cell counts before and after heat treatment were measured according to the following conditions. The results are shown in Table 5. Culture medium: GAM bouillon "Nissui" (Nissui Pharmaceutical, Japan) and agar. Culture temperature: 37°C. Culture time: 24 to 48 hours. Diluent: physiological saline. Dilution ratio: undiluted to 10. 5Double dilution Measurement method: Dilution plate method (0.1 mL surface smear CFU method) Automatic dilution / smearing device: easySpiralDilute (registered trademark) (Interscience, France) Number of smears: 3 plates of the same dilution Other: Anaerobic culture (Anelopouch Kenki System (Mitsubishi Gas Chemical, Japan)) Colony observation: Stereomicroscope (SMZ800N (Nikon, Japan))

[0052]

[0053] Since live bacteria grew in the bacterial solution after the heat treatment, the B6 strain was determined to be heat-resistant.

[0054] Example 6: Growth Inhibition Test of Clostridioides difficile The effect of the B6 strain on the growth of Clostridioides difficile, a typical causative bacterium of antibiotic-associated diarrhea (diarrhea and loose stools that occur as a side effect of antibiotics), was evaluated. (1) Preparation of Sterile Filtrate of Clostridium butyricum B6 Strain Using a liquid medium (GAM Bouillon "Nissui" (Nissui Pharmaceutical, Japan)) and an anaerobic culture kit, the B6 strain was cultured at 37°C for 72 hours under anaerobic conditions using the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan). The total number of bacteria in the culture solution was counted according to the following conditions. The results are shown in Table 6-1. Dilution solution: Physiological saline Dilution ratio: Undiluted to 10 4 Dilution times: Hemocytometer: Disposable hemocytometer. C-Chip (DHC-N01: Neubawell improved type) (NanoEntek, Korea). Dilution rates where the number of cells in the large compartment was 100 or more and 1000 or less were counted. Microscope: Optical microscope (BX50 (Olympus, Japan))

[0055]

[0056] This culture solution was sterilized by filtration through a 0.2 μm cellulose acetate filter to obtain a sterile filtrate.

[0057] (2) Preparation of test medium The sterile filtrate prepared in (1) was mixed with equal amounts of 2x concentrated GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) to prepare the test medium. GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) was used as a control.

[0058] (3) Preparation of Clostridioides difficile Bacterial Solution Using an agar medium (GAM Bouillon "Nissui" (Nissui Pharmaceutical, Japan) + agar) and an anaerobic culture kit, the type strain of Clostridioides difficile, JCM1296T, was cultured under anaerobic conditions at 37°C for 24 hours using the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan). The culture solution was suspended in sterile saline to a concentration equivalent to McFarland turbidity standard solution No. 0.5. The suspension was counted using a hemocytometer, and the bacterial solution volume was determined to be approximately 1 x 10 8 The concentration was adjusted to 1000 cells / mL.

[0059] (4) C. difficile Cultivation 20 μL of the C. difficile solution prepared in (3) was inoculated into a medium bottle containing 20 mL of test medium, and static culture was performed at 37°C under anaerobic conditions using an AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan) anaerobic culture kit. After 24 and 48 hours of culture, 2 mL of the culture medium was sampled, and the number of C. difficile bacteria was measured according to the following conditions. The results are shown in Table 6-2. Culture medium: GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar Culture temperature: 37°C Culture time: 24 to 48 hours Diluent: Physiological saline Dilution ratio: Undiluted to 10 7Double dilution Measurement method: Dilution plate method (0.1 mL surface smear, colony forming units (CFU) count) Automatic dilution / smearing device: easySpiralDilute (registered trademark) (Interscience, France) Number of smears: 3 plates for each dilution Other: Anaerobic culture (Anelopouch Kenki System (Mitsubishi Gas Chemical, Japan)) Colony observation: Stereomicroscope (SMZ800N (Nikon, Japan)) Evaluation: Count the colonies grown on plates at the appropriate dilution level

[0060]

[0061] Addition of the sterile filtrate of strain B6 inhibited the growth of C. difficile, suggesting that strain B6 produced a substance that inhibited the growth of C. difficile.

[0062] Reference Example 1: Analysis of partial 16S rDNA base sequence The partial base sequence (SEQ ID NO: 1) of the 16S rDNA (16S rRNA gene) of the B6 strain was analyzed to estimate its affiliation.

[0063]

[0064] The analysis was carried out under the following conditions: The results are shown in Tables 7-1 and 7-2.

[0065] DNA extraction: Cica Genesis DNA Extraction Reagent ST (Kanto Chemical, Japan) PCR amplification: TKs Gflex DNA Polymerase (Takara Bio, Japan) Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) Primers used: PCR amplification: 9F, 1510R Sequence (approximately 1500 bp): 9F, 515F, 1099F, 536R, 926R, 1510R Sequence: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems, USA) Biosystems) - Base sequence determination: ChromasPro 2.1 (Technelysium, AUS) - BLAST homology search: Analysis software: ENKI v3.2 (TechnoSuruga Laboratory, Japan) Database: DB-BA17.0 (TechnoSuruga Laboratory) International base sequence database (DDBJ / ENA / GenBank) - Simple molecular phylogenetic tree analysis: Phylogenetic tree estimation: Neighbor-joining method Base substitution model: Kimura-2-parameter Tree structure reliability evaluation: Bootstrap method (1,000 iterations)

[0066] Note 1) BSL (Biosafety Level) is Level 1 * (opportunistic pathogen) or higher, and a blank space means level 1. Note 2) An "_ (underscore)" in the strain name means a space. Note 3) The top 11 strains in the table show sequence data used for simple molecular phylogenetic analysis.

[0067]

[0068] A simplified molecular phylogenetic tree based on the 16S rDNA partial base sequence of the B6 strain is shown in Figure 1. In the figure, SIID29215-B6 indicates the B6 strain. SIID29215-04 indicates the type strain (Clostridium butyricum NBRC13949T strain). The line in the upper left indicates a scale bar. The numbers at the branches of the phylogenetic branches indicate bootstrap values, which are values ​​that indicate the reliability of the tree. The T at the end of the strain name indicates the type strain of that species. BSL indicates the biosafety level (BSL1). * (opportunistic pathogens) and above).

[0069] Reference Example 2: In silico DDH (DNA-DNA hybridization) analysis In silico DDH analysis is a method for evaluating the species similarity between two strains by comparing the whole genome sequences or draft genome sequences of a control strain and a comparison strain on a computer. In this reference example, ANI analysis and GGDC analysis, which use different calculation methods, were performed.

[0070] (1) ANI (Average Nucleotide Identity) Analysis In ANI analysis, the genome sequence of a control strain is fragmented into 1,020 bp fragments on a computer, and a homology search is performed for each fragment against the genome sequence of a comparison strain. The ANI value between the genome sequences is calculated from the average of these homology values. The publicly available program ANI Calculator (http: / / enve-omics.ce.gatech.edu / ani / index) is used to calculate the ANI value. In ANI analysis, an ANI value of 95% or higher is determined to be the same species. In this reference example, ANI analysis was performed using the B6 strain as the comparison strain and the type strain (Clostridium butyricum NBRC13949T strain) as the control strain. The ANI value between the B6 strain and the type strain was 100%.

[0071] (2) GGDC (Genome-to-Genome Distance Calculator) Analysis. GGDC analysis involves identifying highly homologous regions between the genome sequences of a control strain and a comparison strain on a computer, and then calculating the pairwise distance between them to determine the in silico DDH value (BMC Bioinformatics 2013;14:60). The publicly available program Genome-to-Genome Distance Calculator (http: / / ggdc.dsmz.de / ggdc.php#) is used to calculate the in silico DDH value. In GGDC analysis, if the in silico DDH value is 70% or higher, the strains are considered to be of the same species. In this Reference Example, GGDC analysis was performed using the B6 strain as a comparative strain and the type strain (Clostridium butyricum NBRC13949T strain) as a control strain, and the in silico DDH value between the B6 strain and the type strain was 99.9%.

[0072] Based on the results of Reference Examples 1 and 2, the B6 strain was determined to be Clostridium butyricum. The B6 strain does not have the ability to assimilate D-xylose or D-trehalose (Example 1). On the other hand, the NBRC13949T strain (the type strain of Clostridium butyricum) had the ability to assimilate D-xylose and D-trehalose (Example 1). Furthermore, known Clostridium butyricum are known to have the ability to assimilate D-xylose and D-trehalose (Bergey's Manual of Systematic Bacteriology, Second Edition, Volume Three, The Firmicutes, pp. 739-742 (2009) (Non-Patent Document 3)). Therefore, the B6 strain was determined to be a different strain from known Clostridium butyricum, including type strains.

[0073] Reference Example 3: Genome analysis of strain B6 Genome analysis of strain B6 was carried out according to the following method. 1. DNA extraction - DNA extraction and purification: NucleoSpin Plant II (MACHEREY-NAGEL, GER) 2. Library preparation - Kits used: Nextera DNA Flex Library Prep Kit (Illumina, USA) Nextera DNA CD Indexes (Illumina) 3. Genome sequencing Sequencing was performed according to the protocol provided with the sequencer. - Sequencer: iSeq 100 System (Illumina) - Sequencing kit: iSeq 100 i1 Reagent kit (Illumina) - Sequencing method: 2 x 151 bp paired-end sequencing 4. Data analysis: Quality filtering: trimmomatic ver 0.39; de novo assembly: Spades ver 3.15.4; misassembly correction (polishing): pilon ver 1.24; coverage (depth) calculation: bowtie2 ver 2.3.5.1, samtools ver 1.10. Depth coverage was calculated by mapping reads to the longest contig and calculating the average value per site. G+C content calculation: SeqKit ver 0.12.0

[0074] The results are shown below. a) Shows the total length of the contig / scaffold. b) Shows the value calculated from the assembled base sequence.

[0075] The 172 contig sequences of the obtained B6 strain are designated as SEQ ID NOs: 2 to 173. The sequences designated as SEQ ID NOs: 2 to 173 are described in the sequence listing included in the present international application at the time of filing.

[0076] [Reference to deposited biological material] Name of depository institution: Patent Microorganism Depositary, National Institute of Technology and Evaluation Contact: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan Accession number: NITE BP-03916 Identification marking: SIID29215-B6 Date of deposit: June 15, 2023 Place of origin: Japan Source of isolation: Isolated from a fermentation liquid containing a medium obtained at the food ingredient manufacturing plant of Hiremount Co., Ltd. in Kaizu City, Gifu Prefecture. The medium uses soybeans produced in Japan. The soybeans used are organically grown in Japan without the use of pesticides.

[0077] [Test strain 2] Clostridium butyricum SIID49520-01-B1 strain (hereinafter also referred to as "01-B1 strain") was used. The 01-B1 strain has been internationally deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), an international depository institution under the provisions of the Budapest Treaty (Room 122) (Identification: SIID49520-01-B1. Accession number: NITE BP-04190. Date of deposit (deposit): October 29, 2024).

[0078] Example 7: Morphological, physiological, and biochemical property tests. Using an agar medium (Accudia GAM broth (Shimadzu Diagnostics Corporation, Japan) + agar) and an anaerobic culture kit (Anelopouch Kenki System (Mitsubishi Gas Chemical, Japan)), the 01-B1 strain was anaerobically cultured at 37°C for 24 hours. The morphology of the cultured 01-B1 strain was observed using an optical microscope. Furthermore, the physiological properties of the 01-B1 strain (catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F)) were tested according to the method of Barrow & Feltham et al. (Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edition, Cambridge: University Press; 1993). The results are shown in Table 8-1. The results for the type strain in Table 8-1 were obtained in Example 1.

[0079] The 01-B1 strain was anaerobically cultured at 37°C for 24 hours using an agar medium (Acudia GAM broth (Shimadzu Diagnostics Corporation, Japan) + agar) and an Anaerobic Pouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. The physiological and biochemical properties of the cultured 01-B1 strain were identified using an anaerobic bacteria biochemical identification kit (API20A (bioMerieux, FRA)). The results are shown in Table 8-2. The results for the type strain in Table 8-2 were obtained in Example 1.

[0080] Furthermore, additional physiological and biochemical properties of the 01-B1 strain were tested using an API ZYM kit (bioMerieux, FRA). The results are shown in Table 8-3.

[0081] The 01-B1 strain did not have the ability to assimilate D-xylose and D-trehalose (Table 8-2). On the other hand, the type strain had the ability to assimilate D-xylose and D-trehalose. Therefore, the 01-B1 strain was determined to be a different strain from the type strain.

[0082] Example 8: Organic acid production ability test Using a liquid medium (Acudia GAM Bouillon (Shimadzu Diagnostics Corporation, Japan)) and an Anaerobic Pouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, the 01-B1 strain, the B6 strain (test strain 1), and the reference strain were anaerobically cultured at 37°C for 72 hours (culture volume: 200 mL). The culture solution was filtered through a membrane filter with a pore size of 0.20 μm to obtain a sample solution. The concentration of organic acids contained in the sample solution was measured by high-performance liquid chromatography. The measurement conditions were as follows: System: Nexera Organic Acid Analysis System (Shimadzu Corporation) Model: LC-40D (Shimadzu Corporation) Detector: Electrical Conductivity Meter CDD-10Avp (Shimadzu Corporation) Column: Shim-pack SCR-102H x 2, φ8.0 mm x 300 mm (Shimadzu Corporation) Card column: SCR-102H, φ6.0 mm x 50 mm (Shimadzu Corporation) Column temperature: 45°C Mobile phase: 5 mmol / L p-toluenesulfonic acid Reaction solution: 5 mmol / L p-toluenesulfonic acid containing 0.1 mmol / L EDTA and 20 mmol / L Bis-Tris Flow rate: Mobile phase 0.8 mL / min, reaction solution 0.8 mL / min Injection volume: 10 μL The organic acids measured were four types: lactic acid, formic acid, acetic acid, and n-butyric acid. The results are shown in Table 9.

[0083] In the table, blank cells indicate values ​​below the lower limit of quantitation, which was 0.01 g / 100 g.

[0084] The 01-B1 strain produced n-butyric acid. Furthermore, the amount of formic acid produced in the culture medium after anaerobic culture of the 01-B1 strain at 37°C for 72 hours was less than that of the type strain (NBRC13949T).

[0085] Reference Example 4: Analysis of 16S rDNA partial base sequence The partial base sequence (SEQ ID NO: 174) of the 16S rDNA (16S rRNA gene) of the 01-B1 strain was analyzed to estimate its affiliation.

[0086]

[0087] The analysis was carried out under the following conditions: The results are shown in Tables 10-1 and 10-2.

[0088] DNA extraction: Achromopeptidase (FUJIFILM Wako Pure Chemical, Japan) PCR amplification: TKs Gflex DNA Polymerase (Takara Bio, Japan) Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) Primers used: PCR amplification: 9F, 1510R Sequence (approximately 1500 bp): 9F, 515F, 1099F, 536R, 926R, 1510R Sequence: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) - Nucleotide sequence determination: ChromasPro 2.1 (Technelysium, AUS) - BLAST homology search: Analysis software: ENKI v3.2 (TechnoSuruga Laboratory, Japan) Database: DB-BA17.0 (TechnoSuruga Laboratory) International Nucleotide Sequence Database (DDBJ / ENA / GenBank) - Simple molecular phylogenetic tree analysis: Phylogenetic tree estimation: Neighbor-joining method Base substitution model: Kimura-2-parameter Tree structure reliability evaluation: Bootstrap method (1,000 iterations)

[0089] Note 1) BSL (Biosafety Level) is Level 1 * (opportunistic pathogen) or higher, and a blank space means level 1. Note 2) An "_ (underscore)" in the strain name means a space. Note 3) The 11 strains numbered 1 to 3 and 5 to 12 from the top in the table show sequence data used for simple molecular phylogenetic analysis.

[0090]

[0091] A simplified molecular phylogenetic tree based on the partial 16S rDNA sequence of the 01-B1 strain is shown in Figure 2. In the figure, SIID49520-01-B1 indicates the 01-B1 strain. The line in the upper left indicates the scale bar. The numbers at the branches of the phylogenetic branches indicate the bootstrap value, which is a value that indicates the reliability of the tree structure. The T at the end of the strain name indicates the type strain of that species. BSL indicates the biosafety level (BSL1* (opportunistic pathogen) or higher).

[0092] [Reference to deposited biological material] Name of depository institution: Patent Microorganism Depositary, National Institute of Technology and Evaluation Contact: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, 292-0818, Japan Accession number: NITE BP-04190 Identification marking: SIID49520-01-B1 Date of deposit: October 29, 2024 Place of origin: Japan Source of isolation: Isolated from a fermentation liquid containing a medium fermented at Hiremount Co., Ltd.'s food ingredients manufacturing plant in Kaizu, Gifu Prefecture. The medium uses soybeans produced in Japan. The soybeans used were organically grown in Japan without the use of pesticides.

[0093] [Test strain 3] Clostridium butyricum SIID50030-B1 strain (hereinafter also referred to as "B1 strain") was used. The B1 strain has been internationally deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), an international depository institution under the provisions of the Budapest Treaty (identification: SIID50030-B1; accession number: NITE BP-04191; date of deposit (accession): October 29, 2024).

[0094] Example 9: Morphological, Physiological, and Biochemical Properties Tests Using an agar medium (Accudia GAM broth (Shimadzu Diagnostics Corporation, Japan) + agar) and an anaerobic culture kit, the B1 strain was anaerobically cultured at 37°C for 24 hours using the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan). The morphology of the cultured B1 strain was observed using an optical microscope. Furthermore, the physiological properties of the B1 strain (catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F)) were tested according to the method of Barrow & Feltham et al. (Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edition, Cambridge: University Press; 1993). The results are shown in Table 11-1. Note that the results for the type strain in Table 11-1 were obtained in Example 1.

[0095] The B1 strain was anaerobically cultured at 37°C for 24 hours using an agar medium (Acudia GAM broth (Shimadzu Diagnostics Corporation, Japan) + agar) and an anaerobic culture kit, AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan). The physiological and biochemical properties of the cultured B1 strain were identified using an anaerobic bacteria biochemical identification kit (API20A (bioMerieux, FRA)). The results are shown in Table 11-2. The results for the type strain in Table 11-2 were obtained in Example 1.

[0096] Furthermore, additional physiological and biochemical properties of the B1 strain were tested using an API ZYM kit (bioMerieux, FRA). The results are shown in Table 11-3.

[0097] The B1 strain did not have the ability to assimilate D-xylose and D-trehalose (Table 11-2). On the other hand, the type strain had the ability to assimilate D-xylose and D-trehalose. Therefore, the B1 strain was determined to be a different strain from the type strain.

[0098] Example 10: Organic acid production ability test Using a liquid medium (Acudia GAM Bouillon (Shimadzu Diagnostics Corporation, Japan)) and an anaerobic culture kit (Anelopouch Kenki System (Mitsubishi Gas Chemical, Japan)), strain B1, strain B6 (test strain 1), and the reference strain were anaerobically cultured at 37°C for 72 hours (culture volume: 200 mL). The culture solution was filtered through a membrane filter with a pore size of 0.20 μm to obtain a sample solution. The concentration of organic acids contained in the sample solution was measured by high-performance liquid chromatography. The measurement conditions were as follows: System: Nexera Organic Acid Analysis System (Shimadzu Corporation) Model: LC-40D (Shimadzu Corporation) Detector: Electrical Conductivity Meter CDD-10Avp (Shimadzu Corporation) Column: Shim-pack SCR-102H x 2, φ8.0 mm x 300 mm (Shimadzu Corporation) Card column: SCR-102H, φ6.0 mm x 50 mm (Shimadzu Corporation) Column temperature: 45°C Mobile phase: 5 mmol / L p-toluenesulfonic acid Reaction solution: 5 mmol / L p-toluenesulfonic acid containing 0.1 mmol / L EDTA and 20 mmol / L Bis-Tris Flow rate: Mobile phase 0.8 mL / min, reaction solution 0.8 mL / min Injection volume: 10 μL The organic acids measured were four types: lactic acid, formic acid, acetic acid, and n-butyric acid. The results are shown in Table 12.

[0099] In the table, blank cells indicate values ​​below the lower limit of quantitation, which was 0.01 g / 100 g.

[0100] The B1 strain produced n-butyric acid. Furthermore, the amount of formic acid produced in the culture medium after anaerobic culture of the B1 strain at 37°C for 72 hours was less than that of the type strain (NBRC13949T).

[0101] Reference Example 5: Analysis of partial 16S rDNA base sequence The partial base sequence (SEQ ID NO: 175) of the 16S rDNA (16S rRNA gene) of the B1 strain was analyzed to estimate its affiliation.

[0102]

[0103] The analysis was carried out under the following conditions: The results are shown in Tables 13-1 and 13-2.

[0104] DNA extraction: Achromopeptidase (FUJIFILM Wako Pure Chemical, Japan) PCR amplification: TKs Gflex DNA Polymerase (Takara Bio, Japan) Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) Primers used: PCR amplification: 9F, 1510R Sequence (approximately 1500 bp): 9F, 515F, 1099F, 536R, 802R, 1510R Sequence: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) - Nucleotide sequence determination: ChromasPro 2.1 (Technelysium, AUS) - BLAST homology search: Analysis software: ENKI v3.2 (TechnoSuruga Laboratory, Japan) Database: DB-BA17.0 (TechnoSuruga Laboratory) International Nucleotide Sequence Database (DDBJ / ENA / GenBank) - Simple molecular phylogenetic tree analysis: Phylogenetic tree estimation: Neighbor-joining method Base substitution model: Kimura-2-parameter Tree structure reliability evaluation: Bootstrap method (1,000 iterations)

[0105] Note 1) BSL (Biosafety Level) is Level 1 * (opportunistic pathogen) or higher, and a blank space indicates level 1. Note 2) An "_ (underscore)" in the strain name means a space. Note 3) The 10 strains numbered 1 to 3 and 5 to 11 from the top in the table show sequence data used for simple molecular phylogenetic analysis.

[0106]

[0107] A simplified molecular phylogenetic tree based on the partial 16S rDNA sequence of the B1 strain is shown in Figure 3. In the figure, SIID50030-B1 indicates the B1 strain. The line in the upper left corner indicates the scale bar. The numbers at the branches of the phylogenetic branches indicate the bootstrap value, which is a value that indicates the reliability of the tree structure. The T at the end of the strain name indicates the type strain of that species. BSL indicates the biosafety level (BSL1* (opportunistic pathogen) or higher).

[0108] [Reference to deposited biological material] Name of depository institution: Patent Microorganism Depositary, National Institute of Technology and Evaluation Contact: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan Accession number: NITE BP-04191 Identification marking: SIID50030-B1 Date of deposit: October 29, 2024 Place of origin: Japan Source of isolation: Isolated from a fermentation liquid containing a medium fermented at Hiremount Co., Ltd.'s food ingredients manufacturing plant in Kaizu City, Gifu Prefecture. The medium uses soybeans produced in Japan. The soybeans used were organically grown in Japan without the use of pesticides.

[0109] The 01-B1 strain (test strain 2) and the B1 strain (test strain 3) differed in the following respects: Therefore, the 01-B1 strain was determined to be a different strain from the B1 strain.

[0110] The present invention can be used for foods, beverages, medicines, etc.

Claims

1. Clostridium butyricum that produces n-butyric acid and has all of the following characteristics (1) to (3): (1) It does not have the ability to utilize D-xylose; (2) It does not have the ability to utilize D-trehalose; (3) Compared with the NBRC13949T strain, which is the type strain of Clostridium butyricum, the amount of formic acid produced in the culture solution after anaerobic culture at 37°C for 72 hours is smaller.

2. Clostridium butyricum SIID29215-B6 strain (accession number: NITE BP-03916) or a mutant thereof, wherein the mutant produces n-butyric acid and has all of the following characteristics (1) to (3): (1) No D-xylose assimilation ability (2) No D-trehalose assimilation ability (3) Compared with the NBRC13949T strain, which is the type strain of Clostridium butyricum, the amount of formic acid produced in the culture solution after anaerobic culture at 37°C for 72 hours is smaller.

3. Clostridium butyricum SIID29215-B6 strain (accession number: NITE BP-03916).

4. Clostridium butyricum SIID49520-01-B1 strain (accession number: NITE BP-04190) or a mutant thereof, wherein the mutant produces n-butyric acid and has all of the following characteristics (1) to (3): (1) No D-xylose assimilation ability (2) No D-trehalose assimilation ability (3) Compared with the NBRC13949T strain, which is the type strain of Clostridium butyricum, the amount of formic acid produced in the culture solution after anaerobic culture at 37°C for 72 hours is smaller.

5. Clostridium butyricum SIID49520-01-B1 strain (accession number: NITE BP-04190).

6. Clostridium butyricum SIID50030-B1 strain (accession number: NITE BP-04191) or a mutant thereof, wherein the mutant produces n-butyric acid and has all of the following characteristics (1) to (3): (1) No D-xylose assimilation ability (2) No D-trehalose assimilation ability (3) Compared with the NBRC13949T strain, which is the type strain of Clostridium butyricum, the amount of formic acid produced in the culture solution after anaerobic culture at 37°C for 72 hours is smaller.

7. Clostridium butyricum SIID50030-B1 strain (accession number: NITE BP-04191).

8. A composition for intestinal regulation comprising the bacterial cells, spores or culture medium of Clostridium butyricum according to any one of claims 1 to 7 as an active ingredient.

9. A composition for inhibiting the proliferation of Clostridioides difficile, comprising as an active ingredient the bacterial cells, spores or culture medium of Clostridium butyricum according to any one of claims 1 to 7.

10. An anti-inflammatory composition comprising the cells, spores or culture medium of Clostridium butyricum according to any one of claims 1 to 7 as an active ingredient.

11. A composition for inducing regulatory T cells, comprising as an active ingredient the bacterial cells, spores or culture medium of Clostridium butyricum according to any one of claims 1 to 7.

12. The composition of claim 8 in the form of a pharmaceutical preparation.

13. The composition of claim 9 in the form of a pharmaceutical preparation.

14. The composition of claim 10 in the form of a pharmaceutical preparation.

15. The composition of claim 11 in the form of a pharmaceutical preparation.

16. The composition according to claim 8, which is in the form of a food or beverage.

17. The composition according to claim 9, which is in the form of a food or beverage.

18. The composition according to claim 10, which is in the form of a food or beverage.

19. The composition according to claim 11, which is in the form of a food or beverage.

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