A new strain of Clostridium butyricum capable of producing butyrate

Abstract

The present invention aims to provide a butyric acid bacterium that can be safely used as an active ingredient in intestinal regulators, etc. The present invention relates to Clostridium butyricum that 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 Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower.

Description

[Technical Field]

[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. [Background technology]

[0002] In recent years, the usefulness of butyric acid bacteria has been attracting attention. Butyric acid bacteria, which are intestinal bacteria, break down dietary fiber in the large intestine to produce butyric acid. Because butyric acid is used as an energy source for the normal functioning of the large intestine, 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). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] 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 [Non-patent document 2] Sho Eda, "Amazing Butyric Acid Bacteria: The Crossroads Between Those Who Get Sick and Those Who Don't," Gentosha Publishing, March 25, 2022 [Non-patent document 3] Bergey's Manual of Systematic Bacteriology, Second Edition, Volume Three, The Firmicutes, p.739-742 (2009) [Non-patent document 4] Japanese Society of Enterobacteriaceae, "Glossary: ​​Clostridium butyricum," [online], [Retrieved August 5, 2023], Internet<URL: https: / / bifidus-fund.jp / keyword / kw002.shtml> [Non-Patent Document 5] 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> [Non-patent document 6] 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> [Non-Patent Document 7] Journal of Enterobacteriaceae, 31:15-22, 2017 Summary of the Invention [Problem to be solved by the invention]

[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 an object to provide a butyric acid bacterium that produces less formic acid and acetone and can be used more safely as an active ingredient in intestinal regulators and the like. [Means for solving the problem]

[0005] As a result of intensive research into this problem, the present inventors have found 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) the production of formic acid and acetone by this new strain is lower than that of the type strain of Clostridium butyricum (NBRC13949T strain).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, which 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 Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower. [2] Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916) or a mutant strain thereof, wherein the mutant strain 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 Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower. [3] Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916). [4] Clostridium butyricum SIID49520-01-B1 strain (accession number: NITE BP-04190) or a mutant strain thereof, wherein the mutant strain 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 Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower. [5] Clostridium butyricum strain SIID49520-01-B1 (accession number: NITE BP-04190). [6] Clostridium butyricum SIID50030-B1 strain (accession number: NITE BP-04191) or a mutant strain thereof, wherein the mutant strain 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 Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower. [7] Clostridium butyricum SIID50030-B1 strain (accession number: NITE BP-04191). [8] A composition for intestinal regulation, comprising, as an active ingredient, the cells, spores, or culture medium of Clostridium butyricum according to any one of [1] to [7] above. [9] A composition for inhibiting the growth of Clostridioides difficile, comprising the cells, spores, or culture medium of Clostridium butyricum according to any one of [1] to [7] above as an active ingredient.

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

[12] The composition described in [8] above, in the form of a pharmaceutical.

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

[14] The composition according to

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

[15] The composition according to

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

[16] The composition described in [8] above, which is in the form of a food or beverage.

[17] The composition described in [9] above, which is in the form of a food or beverage.

[18] The composition described in

[10] above, which is in the form of a food or beverage.

[19] The composition described in

[11] above, which is in the form of a food or beverage. [Effects of the Invention]

[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. [Brief explanation of the drawings]

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

[0009] The 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 Japanese Society of Intestinal Microbiology (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 (accession date): 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 (for example, high performance liquid chromatography).

[0012] (2) Sugar utilization The strain of the present invention has one, preferably all two of the following characteristics (1) to (2) regarding sugar assimilation ability (ability to utilize sugar as a nutrient source). (1) Inability to utilize D-xylose (2) Inability to utilize D-trehalose The sugar assimilation ability can be determined using a commercially available microbial identification test kit (for example, 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 has the ability to utilize 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 utilize sugars, is a new strain of Clostridium butyricum.

[0014] (3) Formic acid production In a preferred embodiment, the amount of formic acid produced by the strain of the present invention is less than the amount produced by the NBRC13949T strain, which is 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 the amount 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 (for example, high performance liquid chromatography).

[0015] (4) Acetone production In a preferred embodiment, the amount of acetone produced by the strain of the present invention is less than the amount 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 the amount 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 its 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 any of the base sequences shown in SEQ ID NOs: 2 to 173 described below. (6) Spore-forming In a preferred embodiment, the strain of the present invention is capable of forming spores, and 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 characteristics (1) to (6) above, the strain of the present invention may have one or more of the characteristics (e.g., heat resistance, growth inhibition of Clostridioides difficile, etc.) of the Clostridium butyricum SIID29215-B6 strain (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 characteristics of Clostridium butyricum strain SIID49520-01-B1 (accession number: NITE BP-04190) or Clostridium butyricum strain SIID50030-B1 (accession number: NITE BP-04191), which are shown in the Examples described below.

[0018] In a preferred embodiment, the strain of the present invention is Clostridium butyricum SIID29215-B6 (Accession No.: NITE BP-03916) or a mutant thereof. This 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 Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower.

[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), which 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 Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower.

[0020] In another preferred embodiment, the strain of the present invention is a mutant of Clostridium butyricum SIID50030-B1 (Accession No.: NITE BP-04191), which 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 Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower.

[0021] The mutant strains of the above-mentioned deposited strains have their physical and chemical properties altered by methods such as screening for mutations induced by drug treatment and / or ultraviolet irradiation, natural mutations (e.g., natural mutations that occur during repeated subculture), morphological mutations, or genetic engineering methods such as transfection, and are capable of producing n-butyric acid and have all of the following characteristics (1) to (3). (1) Inability to utilize D-xylose (2) Inability to utilize D-trehalose (3) Compared to the Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture medium after anaerobic incubation at 37°C for 72 hours was lower.

[0022] Furthermore, the mutant strain of the deposited strain may have a partial base sequence of the 16S rDNA of the deposited strain 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).

[0023] The Clostridium butyricum SIID29215-B6 strain (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 the strain of the present invention is used for food applications, a medium composed only of food materials and food additives may be used. The culture can be carried out under anaerobic conditions at a temperature of, for example, 30 to 45°C (preferably 36 to 38°C). Anaerobic conditions refer to a low-oxygen environment in which Clostridium butyricum can grow. Anaerobic conditions can be achieved using a sealed container or bag containing an oxygen absorber. Examples of the culture format 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 conditions described above, but spores are preferably formed by heating at 55 to 60°C for 30 minutes.

[0025] [Method for obtaining the strain of the present invention] As mentioned above, three strains of the present invention have been deposited internationally and are all available from international depository institutions. Furthermore, the strain of the present invention can also be isolated from soybean, as with the three strains deposited internationally. The genus Clostridium, to which the strain of the present invention belongs, is a soil bacterium, but soybeans are a more preferable source of the strain of the present invention than soil, which is home to many different types of soil bacteria. While the present invention is not limited by any particular theory, the reason why soybeans are a preferred source is as follows. When it's time to harvest soybeans, the pods split open, exposing the kernels inside. Clostridium bacteria (or their spores) that have been blown up from the soil by wind or other factors become attached to the exposed kernels. The thin skin of soybeans is mainly composed of dietary fiber. The kernels are also 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 with water, honey, and indigestible dextrin in an aerobic environment at 37°C for 72 hours. After fermentation is complete, the resulting fermented liquid and soybeans are made into a paste. This fermented soybean paste is then subjected to the anaerobic conditions described in the section entitled "Cultivation Conditions for the Strain of the Present Invention" above. From the obtained Clostridium bacteria, Clostridium butyricum is identified using sequence information (e.g., the known nucleotide 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, and 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 for the same purposes 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] [Intestinal regulation composition] 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 strains of the present invention have the ability to produce butyric acid (Examples 2, 8, and 10 described 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 strains of the present invention (hereinafter also referred to as "the bacterial cells, etc. 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. The phrase "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 regulating effect (effect of regulating the intestinal environment). The content of the bacterial cells of the present invention can be appropriately determined taking into consideration the form of the composition (food, drink, 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 anti-intestinal agent. For example, the content of bacterial cells in a food for human use 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 intestinal regulation 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 ingredient may be an additive for food, beverage, or pharmaceutical use. The optional ingredient is a known substance and is 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 ingredient can be appropriately set depending on the purpose of incorporation, etc. The composition for intestinal regulation can be applied to a wide range of animal species without particular limitations. The subject of application is preferably mammals (humans and non-human mammals (e.g., dogs and cats)), more preferably humans. Furthermore, the subject of application can be any gender or age. The amount of intake of the composition for intestinal regulation can be appropriately determined depending on the age and weight of the subject, the number of intakes, the route of administration, etc. The intake interval can be set appropriately depending on the intake amount, etc., and it may be taken once a day or in several divided doses. The composition for intestinal regulation 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).

[0028] [Composition for inhibiting the growth of Clostridioides difficile] It is known that antibiotic-associated diarrhea (diarrhea and loose stools) occurs when the intestinal environment is disrupted by antibiotic use, leading to the overgrowth 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, which comprises 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. The phrase "containing as an active ingredient" means that the composition contains the bacterial cells of the present invention in an amount sufficient to exert the desired growth-inhibiting effect (effective amount). The content of the bacterial cells of the present invention can be appropriately determined taking into consideration the form of the composition (food, drink, 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 anti-intestinal agent. For example, the content of bacterial cells in a food for human use 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 growth-inhibiting 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 appropriately set depending on the purpose of its incorporation, etc. The proliferation-inhibitory composition 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 the proliferation-inhibiting composition to be ingested can be appropriately determined depending on the age and weight of the subject, the number of times of ingestion, the route of administration, etc. The intake interval can be set appropriately depending on the intake amount, etc., and it may be taken once a day or in several divided doses. The growth-inhibiting composition 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).

[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 strain of the present invention has the ability to produce butyric acid (Example 2 described 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 may contain 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 to exert the desired anti-inflammatory effect (an effective amount). The content of the bacterial cells of the present invention can be appropriately determined taking into consideration the form of the composition (food, drink, 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 anti-intestinal agent. For example, the content of bacterial cells in a food for human use 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 as long as the optional ingredients do not impair the action of the active ingredient. The optional ingredients can be appropriately selected depending on the form of the composition, etc. The optional ingredient may be an additive for food, beverage, or pharmaceutical use. The optional ingredient is a known substance and is 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 ingredient can be appropriately set depending on the purpose of incorporation, etc. The anti-inflammatory composition 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 sex or age. The amount of the anti-inflammatory composition to be ingested can be appropriately determined depending on the age and weight of the subject, the frequency of ingestion, the route of administration, etc. The intake interval can be set appropriately depending on the intake amount, etc., and it may be taken once a day or in several divided doses. The anti-inflammatory composition 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).

[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). The 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 (Tregs) play a role in suppressing immune responses against oneself (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, which contains 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. 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 regulatory T cell inducing effect. The content of the bacterial cells of the present invention can be appropriately determined taking into consideration the form of the composition (food, drink, 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 anti-intestinal agent. For example, the content of bacterial cells in a food for human use 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 as long as the optional ingredients do not impair the action of the active ingredient. The optional ingredients can be appropriately selected depending on the form of the composition, etc. The optional ingredient may be an additive for food, beverage, or pharmaceutical use. The optional ingredient is a known substance and is 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 ingredient can be appropriately set 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 sex 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 route of administration, etc. The intake interval can be set appropriately depending on the intake amount, etc., and it 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 proliferation 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 beverages] The form of the food or drink is not particularly limited as long as it can be taken orally, and examples include liquid drinks and jelly drinks. Food and beverage products include health foods, functional foods, nutritional supplements, foods for specified health uses, foods for the sick, and foods and beverages labeled as reducing disease risk. The food or beverage may be a sports supplement product (including an Informed Sport certified product) or a cereal product. The food and drink may contain food additives as optional ingredients. Additives for liquid drinks include pH adjusters, emulsifiers, stabilizers, flavorings, sweeteners, etc. Additives for jelly drinks include gelatin, food colorings, thickening polysaccharides, etc.

[0033] [Pharmaceuticals] The pharmaceutical dosage form is preferably one that can be administered orally or rectally (for example, by enema), and specific examples include liquids, capsules, and powders. Pharmaceuticals may contain pharmaceutical additives as optional ingredients, such as excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, flavorings, buffers, antioxidants, and pH adjusters. [Example]

[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] The 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 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 (accession): June 15, 2023).

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

[0037] Example 1: Morphological, physiological and biochemical property tests The B6 strain and the type strain were anaerobically cultured at 37°C for 72 hours using agar medium (GAM Broth “Nissui” (Nissui Pharmaceutical, Japan) + agar) and the Anaerobic Pouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. 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 reference 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. [Table 1-1]

[0038] The B6 strain and the type strain were anaerobically cultured at 37°C for 24 hours using agar medium (GAM bouillon "Nissui" (Nissui Pharmaceutical, Japan) + agar) and the Anaerobic Pouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. 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. [Table 1-2]

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

[0040] The B6 strain was unable to utilize D-xylose and D-trehalose (Table 1-2). On the other hand, the type strain was able to utilize D-xylose and D-trehalose. Therefore, the B6 strain was determined to be a strain different from the type strain.

[0041] Example 2: Organic acid production ability test The B6 strain and the type strain were anaerobically cultured for 72 hours at 37°C (culture volume: 5 mL) using liquid medium (GAM Broth “Nissui” (Nissui Pharmaceutical, Japan)) and the Anaerobic Pouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. The culture solution was filtered through a membrane filter with a pore size of 0.20 μm to prepare a sample solution. The concentrations of organic acids contained in the sample solutions were measured by high performance liquid chromatography. The measurement conditions are as follows. System: Shimadzu organic acid analysis system (Shimadzu, Japan) Column: Shim-pack Fast-OA, 100mm x 7.8mm ID, 3 columns in series Guard column: Shim-pack Fast-OA, 10mm x 4.0mm ID Eluent: 5mmol / L p-toluenesulfonic acid ·Reaction solution: 5mmol / L p-toluenesulfonic acid, 100μmol / L EDTA, 20mmol / L Bis-Tris ·Flow rate: 0.8mL / min Oven temperature: 50℃ 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] [Table 2] 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] Strain B6 produced n-butyric acid. Furthermore, the amount of formic acid produced in the culture medium after anaerobic culture of strain B6 at 37°C for 72 hours was less than that of the reference strain (NBRC13949T).

[0044] Example 3: Acetone and alcohol production ability test The B6 strain and the type strain were anaerobically cultured at 37°C for 48 hours using a liquid medium (GAM bouillon "Nissui" (Nissui Pharmaceutical, Japan)) and an anaerobic culture kit, the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan). The culture medium was separated into sterile tubes after 24 and 48 hours of cultivation. The supernatant (10 mL or more) obtained by centrifuging the culture medium 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 sample was placed in a measuring 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 mm diameter x 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 (51kPa, Vac) Column flow rate: 1 mL / min (He) ·Inlet temperature: 250℃ ·Injection volume: 1μL Injection method: Split (10:1) Detector: MS Ion source temperature: 230℃ Ionization method: EI (70 eV) Scan range: SIM (m / z: 31, 41, 43, 45, 58, 59, 2.0-3.5 min) :SIM(m / z:41,43,45,56,59,74, 3.5~29.0min) Gain: 976V

[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 sample described above, 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, to determine the content of each component per 1 g of sample. The results are shown in Table 3.

[0046] [Table 3]

[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 reference strain (NBRC13949T).

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

[0049] [Table 4]

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

[0051] Example 5: Heat resistance test The B6 strain was precultured under anaerobic conditions at 37°C for 24 or 72 hours using agar medium (GAM bouillon "Nissui" (Nissui Pharmaceutical, Japan) + agar) and the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. The culture was suspended in 10 mL of physiological saline to prepare a bacterial suspension. Five mL of the bacterial suspension was dispensed into a sterilized medium-sized test tube (18 × 170 mm) and heated in a water bath at 60°C for 30 minutes. 5 mL of the bacterial solution before heat treatment was used as a control. The viable cell count was measured before and after heat treatment under the following conditions, and the results are shown in Table 5. Culture medium: GAM bouillon "Nissui" (Nissui Pharmaceutical, Japan) and agar ·Culture temperature: 37℃ ·Culture time: 24 to 48 hours Diluent: Physiological saline Dilution ratio: undiluted to 10 5 2x dilution ·Measurement method: Dilution plate method (0.1mL surface smear CFU method) Automatic dilution and smearing device: easySpiralDilute (registered trademark) (Interscience, France) Number of smears: 3 of each dilution Other: Anaerobic culture (Anelopouch Kenki System (Mitsubishi Gas Chemical, Japan)) Colony observation: Stereo microscope (SMZ800N (Nikon, Japan))

[0052] [Table 5]

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

[0054] Example 6: Clostridioides difficile growth inhibition test 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 strain B6 The B6 strain was cultured under anaerobic conditions at 37°C for 72 hours using a liquid medium (GAM Bouillon "Nissui" (Nissui Pharmaceutical, Japan)) and an anaerobic culture kit, the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan). The total number of bacteria in the culture medium was counted according to the following conditions. The results are shown in Table 6-1. Diluent: Physiological saline Dilution ratio: undiluted to 10 4 2x dilution Hemocytometer: Disposable hemocytometer. C-Chip (DHC-N01: Neubawell improved type) (NanoEntek, Korea). Dilution ratios 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] [Table 6-1]

[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 test medium was prepared by mixing equal amounts of the sterile filtrate prepared in (1) and double-concentrated GAM Broth "Nissui" (Nissui Pharmaceutical, Japan). GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) was used as a control.

[0058] (3) Preparation of Clostridioides difficile bacterial suspension The type strain of Clostridioides difficile, JCM1296T, was cultured under anaerobic conditions at 37°C for 24 hours using agar medium (GAM broth "Nissui" (Nissui Pharmaceutical, Japan) + agar) and the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. The culture solution was suspended in sterile physiological saline to a concentration equivalent to McFarland No. 0.5 turbidity standard solution. The suspension was counted using a hemocytometer to determine the bacterial volume of approximately 1 x 10 8 The concentration was adjusted to 100 cells / mL.

[0059] (4) C. difficile culture 20 μL of the Clostridioides 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 the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. After 24 and 48 hours of incubation, 2 mL of the culture medium was sampled and the number of Clostridioides difficile bacteria was measured according to the conditions below. The results are shown in Table 6-2. Culture medium: GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar ·Culture temperature: 37℃ ·Culture time: 24 to 48 hours Diluent: Physiological saline Dilution ratio: undiluted to 10 7 2x dilution Measurement method: Dilution plate method (0.1 mL surface smear, colony forming unit (CFU) count) Automatic dilution and smearing device: easySpiralDilute (registered trademark) (Interscience, France) Number of smears: 3 of each dilution Other: Anaerobic culture (Anelopouch Kenki System (Mitsubishi Gas Chemical, Japan)) Colony observation: Stereo microscope (SMZ800N (Nikon, Japan)) Evaluation: Count the colonies grown on the plates at the appropriate dilution level.

[0060] [Table 6-2]

[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: 16S rDNA partial base sequence analysis] 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] [SEQ ID NO: 1]

[0064] The analysis was carried out under the following conditions, and 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) Primer used: PCR amplification: 9F, 1510R Sequence (approximately 1500 bp): 9F, 515F, 1099F, 536R, 926R, 1510R Sequencing: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) Sequencing: 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 Databases (DDBJ / ENA / GenBank) ·Simple molecular phylogenetic tree analysis: Phylogenetic tree estimation: neighbor-joining method Base substitution model: Kimura-2-parameter Reliability assessment of tree structure: Bootstrap method (1,000 iterations)

[0066] [Table 7-1] Note 1) BSL (Biosafety Level) is Level 1* (Opportunistic pathogen) or higher, and a blank space means level 1. Note 2) An "_" (underscore) in a strain name means a space. Note 3) The top 11 strains in the table show sequence data used for simple molecular phylogenetic analysis.

[0067] [Table 7-2]

[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 the scale bar. The numbers at the forks 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 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 species differences 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 the 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 the 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, if the ANI value is 95% or higher, the strains are considered to be of the same species. In this reference example, ANI analysis was performed using the B6 strain as a comparison strain and the type strain (Clostridium butyricum NBRC13949T strain) as a control strain, and the ANI value between the B6 strain and the type strain was 100%.

[0071] (2) Genome-to-Genome Distance Calculator (GGDC) analysis GGDC analysis involves identifying regions of high homology between the genome sequences of a control strain and a comparison strain on a computer, and 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, an in silico DDH value of 70% or higher is considered to be homologous. In this reference example, GGDC analysis was performed using the B6 strain as a comparison 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. Here, the B6 strain does not have the ability to assimilate D-xylose and D-trehalose (Example 1). On the other hand, the NBRC13949T strain (the type strain of Clostridium butyricum) was able to assimilate D-xylose and D-trehalose (Example 1). Furthermore, known Clostridium butyricum strains are known to be able to utilize 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 strain distinct from known Clostridium butyricum strains, including the type strain.

[0073] [Reference Example 3: Genome analysis of B6 strain] Genome analysis of the B6 strain was carried out according to the following method. 1. DNA Extraction DNA extraction and purification: NucleoSpin Plant II (MACHEREY-NAGEL, GER) 2. Library Preparation Kit used: Nextera DNA Flex Library Prep Kit (Illumina, USA) Nextera DNA CD Indexes (Illumina) 3. Genome Sequencing Sequencing was performed according to the protocol attached to 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 assemble: Spades ver 3.15.4 -Fixed misassembly (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. Calculation of G+C content: SeqKit ver 0.12.0

[0074] The results are shown below. TIFF0007741601000012.tif15136 a) Shows the sum of contig / scaffold lengths. b) The value calculated from the assembled base sequence is shown.

[0075] The 172 contig sequences of the obtained B6 strain are respectively represented by SEQ ID NOs: 2 to 173. The sequences represented by 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: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Contact: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan Accession number: NITE BP-03916 Identification mark: SIID29215-B6 Deposit date: June 15, 2023 Origin: Japan Source: Isolated from fermentation liquid containing a culture medium obtained at Hiremount Co., Ltd.'s food ingredient manufacturing plant in Kaizu City, Gifu Prefecture. The culture medium uses soybeans grown in Japan organically without the use of pesticides.

[0077] [Test strain 2] The 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 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: SIID49520-01-B1; accession number: NITE BP-04190; date of deposit (accession): October 29, 2024).

[0078] Example 7: Morphological, physiological and biochemical property tests The 01-B1 strain was anaerobically cultured at 37°C for 24 hours using agar medium (Accudia GAM broth (Shimadzu Diagnostics Corporation, Japan) + agar) and the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. The morphology of the cultured 01-B1 strain was observed using an optical microscope. Furthermore, the physiological properties of strain 01-B1 (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. [Table 8-1]

[0079] The 01-B1 strain was anaerobically cultured at 37°C for 24 hours using agar medium (Accudia GAM broth (Shimadzu Diagnostics Corporation, Japan) + agar) and the AnaeroPouch 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. [Table 8-2]

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

[0081] The 01-B1 strain was unable to assimilate D-xylose and D-trehalose (Table 8-2). On the other hand, the type strain was able to utilize D-xylose and D-trehalose. Therefore, the 01-B1 strain was determined to be a strain different from the type strain.

[0082] Example 8: Organic acid production ability test Using a liquid medium (Accudia GAM broth (Shimadzu Diagnostics Corporation, Japan)) and an anaerobic culture kit (Anelopouch Kenki System (Mitsubishi Gas Chemical, Japan)), the 01-B1 strain, the B6 strain (test strain 1), and the reference strain were cultured anaerobically 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 prepare a sample solution. The concentrations of organic acids contained in the sample solutions were measured by high performance liquid chromatography. The measurement conditions are 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, φ6.0mm x 50mm (Shimadzu Corporation) Column temperature: 45℃ Mobile phase: 5mmol / L p-toluenesulfonic acid ·Reaction solution: Contains 0.1mmol / L EDTA and 20mmol / L Bis-Tris 5mmol / L p-toluenesulfonic acid Flow rate: Mobile phase 0.8 mL / min, reaction solution 0.8 mL / min ·Injection volume: 10μL The organic acids measured were lactic acid, formic acid, acetic acid, and n-butyric acid. The results are shown in Table 9.

[0083] [Table 9] 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 reference strain (NBRC13949T).

[0085] [Reference Example 4: 16S rDNA partial base sequence analysis] 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] [SEQ ID NO: 174]

[0087] The analysis was carried out under the following conditions, and 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) Primer used: PCR amplification: 9F, 1510R Sequence (approximately 1500 bp): 9F, 515F, 1099F, 536R, 926R, 1510R Sequencing: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) Sequencing: 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 Databases (DDBJ / ENA / GenBank) ·Simple molecular phylogenetic tree analysis: Phylogenetic tree estimation: neighbor-joining method Base substitution model: Kimura-2-parameter Reliability assessment of tree structure: Bootstrap method (1,000 iterations)

[0089] [Table 10-1] Note 1) BSL (Biosafety Level) is Level 1 * (Opportunistic pathogen) or higher, and a blank space means level 1. Note 2) An "_" (underscore) in a strain name means a space. Note 3) The 11 strains numbered 1 to 3 and 5 to 12 from the top of the table show sequence data used for simple molecular phylogenetic analysis.

[0090] [Table 10-2]

[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 forks 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: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Contact: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan Accession number: NITE BP-04190 Identification mark: SIID49520-01-B1 Deposit date: October 29, 2024 Origin: Japan Source: Isolated from fermentation liquid containing a culture medium fermented at Hiremount Co., Ltd.'s food ingredient manufacturing plant in Kaizu City, Gifu Prefecture. The culture medium uses soybeans grown in Japan organically without the use of pesticides.

[0093] [Test strain 3] The 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 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 property tests The B1 strain was anaerobically cultured for 24 hours at 37°C using agar medium (Acudia GAM broth (Shimadzu Diagnostics Corporation, Japan) + agar) and the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. The morphology of the cultured B1 strain was observed using an optical microscope. Furthermore, the physiological properties of strain B1 (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. The results for the type strain in Table 11-1 were obtained in Example 1. [Table 11-1]

[0095] The B1 strain was anaerobically cultured for 24 hours at 37°C using agar medium (Acudia GAM broth (Shimadzu Diagnostics Corporation, Japan) + agar) and the AnaeroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit. 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. [Table 11-2]

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

[0097] Strain B1 was unable to utilize D-xylose and D-trehalose (Table 11-2). On the other hand, the type strain was able to utilize D-xylose and D-trehalose. Therefore, the B1 strain was determined to be a strain different from the type strain.

[0098] Example 10: Organic acid production ability test Using a liquid medium (Acudia GAM broth (Shimadzu Diagnostics Corporation, Japan)) and an anaerobic culture kit (Anelopouch Kenki System (Mitsubishi Gas Chemical, Japan)), strains B1, B6 (test strain 1), and the reference strain were cultured anaerobically 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 prepare a sample solution. The concentrations of organic acids contained in the sample solutions were measured by high performance liquid chromatography. The measurement conditions are 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, φ6.0mm x 50mm (Shimadzu Corporation) Column temperature: 45℃ Mobile phase: 5mmol / L p-toluenesulfonic acid ·Reaction solution: Contains 0.1mmol / L EDTA and 20mmol / L Bis-Tris 5mmol / L p-toluenesulfonic acid Flow rate: Mobile phase 0.8 mL / min, reaction solution 0.8 mL / min ·Injection volume: 10μL The organic acids measured were lactic acid, formic acid, acetic acid, and n-butyric acid. The results are shown in Table 12.

[0099] [Table 12] In the table, blank cells indicate values ​​below the lower limit of quantitation, which was 0.01 g / 100 g.

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

[0101] [Reference Example 5: 16S rDNA partial base sequence analysis] 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] [SEQ ID NO: 175]

[0103] The analysis was carried out under the following conditions, and 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) Primer used: PCR amplification: 9F, 1510R Sequence (approximately 1500 bp): 9F, 515F, 1099F, 536R, 802R, 1510R Sequencing: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) Sequencing: 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 Databases (DDBJ / ENA / GenBank) ·Simple molecular phylogenetic tree analysis: Phylogenetic tree estimation: neighbor-joining method Base substitution model: Kimura-2-parameter Reliability assessment of tree structure: Bootstrap method (1,000 iterations)

[0105] [Table 13-1] Note 1) BSL (Biosafety Level) is Level 1 * (Opportunistic pathogen) or higher, and a blank space means level 1. Note 2) An "_" (underscore) in a strain name means a space. Note 3) The 10 strains numbered 1 to 3 and 5 to 11 from the top of the table show sequence data used for simple molecular phylogenetic analysis.

[0106] [Table 13-2]

[0107] A simplified molecular phylogenetic tree based on the partial 16S rDNA sequence of strain B1 is shown in Figure 3. In the figure, SIID50030-B1 indicates strain B1. The line in the upper left indicates the scale bar. The numbers at the junctions of the phylogenetic branches indicate bootstrap values, which represent 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: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Contact: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan Accession number: NITE BP-04191 Identification mark: SIID50030-B1 Deposit date: October 29, 2024 Origin: Japan Source: Isolated from fermentation liquid containing a culture medium fermented at Hiremount Co., Ltd.'s food ingredient manufacturing plant in Kaizu City, Gifu Prefecture. The culture medium uses soybeans grown in Japan organically 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. TIFF0007741601000025.tif31123 [Industrial Applicability]

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

Claims

1. A Clostridium butyricum that 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 with the Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture solution after anaerobic culture at 37°C for 72 hours is low.

2. A Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916) or a mutant strain thereof, wherein the mutant strain produces n-butyric acid and has all of the following characteristics (1) to (4): (1) Inability to utilize D-xylose (2) Inability to utilize D-trehalose (3) Compared with the Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture solution after anaerobic culture at 37°C for 72 hours is low. (4) As a partial base sequence of 16S rDNA, it has a base sequence having at least 99.6% sequence identity to the base sequence shown in SEQ ID NO:

1.

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

4. A Clostridium butyricum strain SIID49520-01-B1 (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 (4): (1) Inability to utilize D-xylose (2) Inability to utilize D-trehalose (3) Compared with the Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture solution after anaerobic culture at 37°C for 72 hours is low. (4) As a partial base sequence of 16S rDNA, it has a base sequence having at least 99.6% sequence identity to the base sequence shown in SEQ ID NO:

174.

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

6. A Clostridium butyricum strain SIID50030-B1 (accession number: NITE BP-04191) or a mutant strain thereof, wherein the mutant strain produces n-butyric acid and has all of the following characteristics (1) to (4): (1) Inability to utilize D-xylose (2) Inability to utilize D-trehalose (3) Compared with the Clostridium butyricum type strain NBRC13949T, the amount of formic acid produced in the culture solution after anaerobic culture at 37°C for 72 hours is low. (4) As a partial base sequence of 16S rDNA, it has a base sequence having at least 99.6% sequence identity to the base sequence shown in SEQ ID NO:

175.

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

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

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 the cells, spores or culture medium of Clostridium butyricum according to any one of claims 1 to 7 as an active ingredient.

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

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

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

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

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

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

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

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

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