New strains of clostridium butyricum with butyric acid-producing ability

Abstract

An object of the present application is to provide a Clostridium butyricum which can be used more safely as an effective ingredient of a whole bowel agent or the like. The present application relates to a Clostridium butyricum which produces n-butanoic acid and has all of the following (1) to (3), (1) does not have D-xylose assimilation ability; (2) does not have D-trehalose assimilation ability; and (3) produces less formic acid in a culture solution after anaerobic culture at 37°C for 72 hours, as compared with NBRC 13949T strain which is a standard strain of Clostridium butyricum.

Description

Technical Field

[0001] The present invention relates to a novel Clostridium butyricum strain having butyric acid-producing ability, a intestinal regulator composition using the same, and the like. Background Art

[0002] In recent years, the usefulness of butyric acid bacteria has attracted attention. Butyric acid bacteria, which are intestinal bacteria, decompose dietary fiber in the large intestine to produce butyric acid. Since butyric acid is utilized as an energy source for the normal operation of the large intestine, butyric acid bacteria are used as an active ingredient of intestinal regulators (Non-Patent Documents 1 and 2).

[0003] As a representative example of butyric acid bacteria, Clostridium butyricum is known (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).

[0004] Description of Related Art Non-Patent Documents Non-Patent Document 1: Yuji Naito, "Increase Butyric Acid Bacteria to Achieve Health and Longevity - Understand All About the Trending Butyric Acid and Butyric Acid Bacteria!", Asa Publishing Co., Ltd., February 11, 2022 Non-Patent Document 2: Masaru Eda, "Amazing Butyric Acid Bacteria - The Dividing Line Between People Who Get Sick and People Who Do Not", Gentosha Inc., 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 for Intestinal Bacteria, "Glossary, Entry for Clostridium butyricum", [Online], [Retrieved on August 5, 5th year of Reiwa], 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], [Retrieved on November 1, 5th year of Reiwa], Internet <URL: https: / / www.ilo.org / dyn / icsc / showcard.listCards3> Non-Patent Document 6: Search results for "acetone" in the International Chemical Substances Safety Card (ICSC) database, [online], [searched on November 1, 2006], Internet <URL: https: / / www.ilo.org / dyn / icsc / showcard.listCards3> Non-patent literature 7: Journal of Intestinal Bacteriology, 31:15-22, 2017. Summary of the Invention

[0005] The problem that the invention aims to solve However, formic acid is known to be toxic when ingested orally, causing abdominal pain, stomach cramps and diarrhea (Non-Patent Literature 5).

[0006] In addition, acetone is known to be toxic when ingested orally, causing nausea and vomiting (Non-Patent Literature 6).

[0007] Therefore, the inventors set the goal of providing butyric acid bacteria that produce less formic acid and acetone and can be used more safely as an effective ingredient in intestinal regulators, etc.

[0008] Methods for solving problems The inventors conducted in-depth research on this topic and found that the *Clostridium butyricum* strain SIID29215-B6 (accession number: NITEBP-03916), isolated during the fermentation of organically cultivated Japanese soybeans to produce food raw materials, (1) is a new strain with different physiological and biochemical properties (differentiated D-xylose and D-trehalose) compared to previous *Clostridium butyricum* strains; and (2) compared to the standard strain of *Clostridium butyricum* (strain NBRC13949T), the aforementioned new strain produces less formic acid and acetone. This invention is based on these insights.

[0009] That is, the present invention relates to the following [1] to

[19] .

[0010] [1] Clostridium butyricum, which produces butyric acid and has all the characteristics of (1) to (3) below, (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

[0011] [2] Clostridium butyricum SIID29215-B6 strain (accession number: NITE BP-03916) or its mutant strain, wherein the aforementioned mutant strain produces butyric acid and possesses all of the following characteristics (1) to (3), (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

[0012] [3] Clostridium butyricum SIID29215-B6 strain (accession number: NITE BP-03916).

[0013] [4] Clostridium butyricum SIID49520-01-B1 strain (accession number: NITE BP-04190) or its mutant strain, wherein the aforementioned mutant strain produces butyric acid and has all of the following characteristics (1) to (3), (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

[0014] [5] Clostridium butyricum SIID49520-01-B1 strain (preservation number: NITE BP-04190).

[0015] [6] Clostridium butyricum SIID50030-B1 strain (accession number: NITE BP-04191) or its mutant strain, wherein the aforementioned mutant strain produces butyric acid and has all of the following characteristics (1) to (3), (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

[0016] [7] Clostridium butyricum SIID50030-B1 strain (accession number: NITE BP-04191).

[0017] [8] An intestinal regulating composition comprising, as an active ingredient, the cells, spores or culture medium of Clostridium butyricum as described in any one of [1] to [7].

[0018] [9] A composition for inhibiting the proliferation of Clostridium difficile, comprising, as an active ingredient, the cells, spores or culture medium of Clostridium butyricum as described in any one of [1] to [7].

[0019]

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

[0020]

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

[0021]

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

[0022]

[13] The composition described in [9] above is in the form of a pharmaceutical product.

[0023]

[14] The composition described in

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

[0024]

[15] The composition described in

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

[0025]

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

[0026]

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

[0027]

[18] The composition described in

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

[0028]

[19] The composition described in

[11] above is in the form of a food or beverage.

[0029] Invention Effects As shown in Examples 2 and 3 below, the *Clostridium butyricum* strain of the present invention produces less formic acid and acetone compared to conventional strains. Furthermore, as shown in Examples 8 and 10 below, the *Clostridium butyricum* strain of the present invention produces less formic acid compared to conventional strains. Therefore, the strain of the present invention can be used as an active ingredient in products utilizing butyric acid bacteria (intestinal regulators, etc.). Attached Figure Description

[0030] Figure 1 : Figure 1 A simplified molecular phylogenetic tree is shown based on a partial 16S rDNA sequence of Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916).

[0031] Figure 2 : Figure 2 A simplified molecular phylogenetic tree is shown based on a partial 16S rDNA sequence of Clostridium butyricum strain SIID49520-01-B1 (accession number: NITE BP-04190).

[0032] Figure 3 : Figure 3 A simplified molecular phylogenetic tree is shown based on a partial 16S rDNA sequence of Clostridium butyricum strain SIID50030-B1 (accession number: NITE BP-04191). Detailed Implementation

[0033] The strain of this invention is Clostridium butyricum.

[0034] As disclosed in Bergey's Manual of Systematic Bacteriology, 2nd Edition, Volume 3, The Firmicutes, pp. 739-742 (2009) (Non-Patent Document 3), and the homepage of the Institute of Enteric Bacteriology Foundation ("Term Collection: Clostridium butyricum", URL: https: / / bifidus-fund.jp / keyword / kw002.shtml) (Non-Patent Document 4), *Clostridium butyricum* is a representative species (type species) of the genus *Clostridium*.

[0035] The strain belonging to the preferred embodiment of the present invention has been deposited internationally at the Patent Microbial Collection Center of the Technical Base for Product Evaluation, an independent administrative agency, as stipulated by the Budapest Treaty (Room 122, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) (Identification mark: SIID29215-B6. Accession number: NITE BP-03916. Deposit date (acceptance date): June 15, 2023).

[0036] Another preferred strain of the present invention has been deposited internationally at the Patent and Microbial Collection Center of the Technical Base for Product Evaluation, an independent administrative agency, as stipulated by the Budapest Treaty (Room 122, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) (Identification mark: SIID49520-01-B1. Accession number: NITE BP-04190. Deposit date (acceptance date): October 29, 2024).

[0037] The strain belonging to another preferred embodiment of the present invention has been deposited internationally at the Patent and Microbial Collection Center of the Technical Base for Product Evaluation, an independent administrative agency, as stipulated by the Budapest Treaty (Room 122, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) (Identification mark: SIID50030-B1. Accession number: NITE BP-04191. Deposit date (acceptance date): October 29, 2024).

[0038] [Characteristics of the strains of the present invention] The characteristics of the strain of the present invention will be described below.

[0039] (1) Production of butyric acid The strain of this invention produces n-butyric acid.

[0040] The amount of butyric acid produced can be determined by analyzing the culture filtrate of the strain using organic acid analysis (e.g., high performance liquid chromatography).

[0041] (2) Sugar assimilation The strain of the present invention possesses one of the following characteristics (1) to (2) regarding sugar assimilation ability (the ability to utilize sugar as a nutrient source), preferably both of them. (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose.

[0042] Sugar assimilation capacity can be determined using commercially available microbial identification test kits (e.g., API20A (bioMerieux, FRA)).

[0043] The assimilation capacity of Clostridium butyricum is described in Bergey's Manual of Systematic Bacteriology, 2nd Edition, Volume 3, Firmicutes, pp. 739-742 (2009) (Non-Patent Literature 3).

[0044] Furthermore, as shown in Example 1 (Morphological, Physiological and Biochemical Tests) described later, the NBRC13949T strain, which is the standard strain of Clostridium butyricum, also has the ability to assimilate D-xylose and D-trehalose.

[0045] Therefore, the strain of the present invention, which differs from previous strains in its sugar assimilation ability, is a novel strain of Clostridium butyricum.

[0046] (3) Production of formic acid In a preferred embodiment, the formic acid produced by the strain of the present invention is less than that produced by the NBRC13949T strain, which serves as the standard strain of Clostridium butyricum.

[0047] Specifically, when comparing the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C, the amount produced by the strain of the present invention is less than that of the NBRC13949T strain, preferably less than 12% of the amount produced by the NBRC13949T strain.

[0048] The amount of formic acid produced can be determined by analyzing the culture filtrate of the strain using organic acid analysis (e.g., high performance liquid chromatography).

[0049] (4) Production of acetone In a preferred embodiment, the strain of the present invention produces less acetone than the NBRC13949T strain, which is the standard strain of Clostridium butyricum.

[0050] Specifically, when comparing the amount of acetone produced in the culture medium after 48 hours of anaerobic culture at 37°C, the amount produced by the strain of the present invention is less than that of the NBRC13949T strain, preferably less than 70% of the amount produced by the NBRC13949T strain.

[0051] The amount of acetone produced can be determined by GC / MS analysis of the culture supernatant of the strain.

[0052] (5) 16S rDNA and genomic DNA The strain of the present invention, as a partial base sequence of 16S rDNA (the gene encoding 16S rRNA), may have a base sequence that has at least 99.6%, preferably 99.8%, and more preferably 99.9% sequence identity with the base sequences shown in SEQ ID NO:1, SEQ ID NO:174, or SEQ ID NO:175 (described below).

[0053] Furthermore, the strain of the present invention, as a genome sequence, may have a base sequence having at least 99.6%, preferably 99.8%, more preferably 99.9% or more sequence identity with the base sequence shown in any of SEQ ID NO:2 to 173 described below.

[0054] (6) Spore forming ability In a preferred embodiment, the strain of the present invention is spore-forming. In this embodiment, the spores of the strain of the present invention can be used as an active ingredient in products utilizing butyric acid bacteria (intestinal regulators, etc.).

[0055] (7) Other characteristics In addition to the features described in (1) to (6) above, the strains of the present invention may also have one or more of the features of the Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916) shown in the following examples (e.g., heat resistance, Clostridium difficile proliferation inhibition, etc.).

[0056] In addition, the strains of the present invention may also have one or more of the characteristics of Clostridium butyricum SIID49520-01-B1 strain (accession number: NITE BP-04190) or Clostridium butyricum SIID50030-B1 strain (accession number: NITE BP-04191) as described in the following examples.

[0057] In a preferred embodiment, the strain of the present invention is Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916) or a mutant strain thereof. This mutant strain produces butyric acid and possesses all of the characteristics described in (1) to (3) below. (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

[0058] In a preferred embodiment, the strain of the present invention is a mutant strain of Clostridium butyricum SIID49520-01-B1 (accession number: NITEBP-04190). This mutant strain produces butyric acid and has all of the characteristics described in (1) to (3) below. (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

[0059] In another preferred embodiment, the strain of the present invention is a mutant strain of Clostridium butyricum SIID50030-B1 (accession number: NITEBP-04191). This mutant strain produces butyric acid and has all the characteristics described in (1) to (3) below. (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

[0060] The mutant strains of the aforementioned preserved strains were obtained by screening after inducing mutations through methods such as chemical treatment and / or ultraviolet irradiation, natural mutation (e.g., natural mutations occurring during repeated subculture), morphological mutations, transfection, and other genetic engineering methods to alter their physical and chemical properties. The mutant strains produce butyric acid and possess all of the following characteristics (1) to (3). (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

[0061] Furthermore, the mutant strain of the preserved strain, as a partial base sequence of the 16S rDNA of the preserved strain, may have a base sequence that has at least 99.6%, preferably 99.8%, and more preferably 99.9% sequence identity with the base sequence shown in SEQ ID NO:1, SEQ ID NO:174, or SEQ ID NO:175 (described below).

[0062] It should be noted that Clostridium butyricum strain SIID29215-B6 (accession number: NITE BP-03916) can be referred to as "butyric acid bacterium UniRAQU strain".

[0063] [Cultivation conditions of the strain of the present invention] No special conditions are required to cultivate the strains of this invention; they can be cultured under the conditions commonly used for Clostridium butyricum.

[0064] The culture medium is preferably a medium for anaerobic bacteria. The culture medium can be either a natural culture medium or a synthetic culture medium. When using the strains of the present invention for food applications, a culture medium consisting only of food raw materials and food additives can be used.

[0065] Cultivation can be carried out, for example, at a temperature of 30–45°C (preferably 36–38°C) under anaerobic conditions. Anaerobic conditions refer to a low-oxygen environment to which Clostridium butyricum can proliferate. Anaerobic conditions can be established using sealed containers or bags containing an oxygen absorber.

[0066] Examples of cultivation methods include static culture, stirred culture, and tank culture.

[0067] The optimal incubation time is 24 to 72 hours (inclusive of 48 hours).

[0068] It should be noted that spore formation does not require special conditions. Spores are formed by culturing the strain of the present invention under the aforementioned conditions, preferably by heating at 55-60°C for 30 minutes.

[0069] [Method for obtaining the strain of the present invention] As described above, three strains were preserved as strains of the present invention, all of which can be obtained from international depositary institutions.

[0070] Furthermore, the strain of this invention is the same as the three strains that have been internationally deposited, and can also be isolated from soybeans.

[0071] The strains of this invention belong to the Clostridium genus, which are soil bacteria. However, soybean is preferred as the source of the strains for this invention, compared to soil inhabited by various soil bacteria. This invention is not limited by any specific theory, and the reasons for preferring soybean as the source are as follows.

[0072] When soybeans are harvested, the pods split open, exposing the seeds. These exposed seeds can harbor Clostridium bacteria (or their spores) blown from the soil by wind or other means. The thin skin of the soybean seed pod is primarily composed of dietary fiber. Furthermore, the seed pod is rich in protein. Both dietary fiber and protein are preferred substances for the proliferation of Clostridium bacteria. Therefore, harvested soybean seeds (especially those from organically grown soybeans without pesticides) are highly likely to harbor Clostridium bacteria (or their spores).

[0073] For example, Clostridium bacteria can be obtained from soybeans by following the steps below.

[0074] Soybeans were fermented with water, honey, and indigestible dextrin at 37°C for 72 hours under aerobic conditions. After fermentation, the resulting fermentation broth was mixed with soybeans to form a paste. This fermented soybean paste was then subjected to anaerobic conditions as described in the section on "Cultivation Conditions of the Strains of the Invention".

[0075] Clostridium butyricum was identified from the obtained Clostridium species using the sequence information (e.g., the known 16S rDNA base sequence) of a standard strain of Clostridium butyricum (NBRC13949T). The strains of the present invention can be obtained by further screening identified Clostridium butyricum strains based on characteristics possessed by the strains of the present invention (assimilation capacity of D-xylose and D-trehalose, formic acid production capacity, etc.).

[0076] [Uses of the strain of the present invention] The strains of the present invention can be used for the same purposes as known butyric acid bacteria (especially Clostridium butyricum), for example, as active ingredients in intestinal regulators, anti-inflammatory agents, and regulatory T-cell inducers.

[0077] [Composition for Intestinal Regulation] Butyric acid bacteria can maintain normal intestinal flora by producing butyric acid (Non-Patent Literature 1 and 2), and therefore can be used as an active ingredient in intestinal regulators. The strains of the present invention have the ability to produce butyric acid (Examples 2, 8 and 10 described later). Therefore, one aspect of the present invention is an intestinal regulator composition comprising the bacterial cells, spores or culture medium of the strains of the present invention (hereinafter also referred to as "the bacterial cells, etc. of the present invention") as an active ingredient.

[0078] The active ingredient can be either bacterial cells or spores, or a combination of both. The culture medium can contain either bacterial cells or spores, or both.

[0079] "Contains as an active ingredient" means that the composition contains the bacteria of the present invention, etc., in an amount (effective amount) sufficient to exert the desired intestinal regulating effect (the effect of adjusting the intestinal environment).

[0080] The content of the bacteria in this invention can be appropriately set considering the form of the composition (food, beverage, or pharmaceutical, etc.). For example, in the case of food for human consumption, the content (number of bacteria) of the "bacteria" as the active ingredient can be set in the same way as the number of butyric acid bacteria contained in a typical intestinal regulator. For example, the content of bacteria in the food for human consumption can be set such that the daily intake of bacteria is preferably 300 million to 2.5 billion, more preferably 1 billion to 2 billion.

[0081] Intestinal composition may contain one or more ingredients without impairing the action of the active ingredient. The ingredients may be selected appropriately based on the composition's form, etc.

[0082] Any ingredient can be an additive for food, beverage, or pharmaceutical use. Any ingredient is a known substance, readily available on the market, or can be prepared. Any ingredient can be a single type or in combination. The content of any ingredient can be appropriately set according to its intended purpose.

[0083] The intestinal composition can be applied to a wide range of animal species without particular limitation. The preferred target population is mammals (humans and non-human mammals such as dogs and cats), more preferably humans. Furthermore, the sex and age of the target population are not limited.

[0084] The dosage of the intestinal regulating composition can be appropriately set according to the age and weight of the target population, the frequency of administration, and the route of administration.

[0085] The intake interval can be set appropriately according to the intake amount, such as once a day or in several portions.

[0086] Compositions for intestinal regulation can be prepared by mixing an active ingredient (such as the bacterial cells of the present invention) with any ingredient (e.g., additives for food or medicine).

[0087] [A composition for inhibiting the proliferation of Clostridium difficile] It is known that antibiotic-associated diarrhea (antibiotic-associated diarrhea) can be caused by an imbalance in the intestinal environment due to the use of antibiotics, leading to the abnormal proliferation of certain bacteria. *Clostridioides difficile* is a representative pathogen causing antibiotic-associated diarrhea.

[0088] The strains of the present invention can inhibit the proliferation of Clostridium difficile (Example 6 described later). Therefore, one aspect of the present invention is a composition for inhibiting the proliferation of Clostridium difficile, which contains the bacterial cells of the present invention as an active ingredient.

[0089] The active ingredient can be either bacterial cells or spores, or a combination of both. The culture medium can contain either bacterial cells or spores, or both.

[0090] "Contains as an active ingredient" means that the composition contains the bacteria of the present invention, etc., in an amount sufficient to exert the desired growth inhibition effect (effective amount).

[0091] The content of the bacteria in this invention can be appropriately set considering the form of the composition (food, beverage, or pharmaceutical, etc.). For example, in the case of food for human consumption, the content (number of bacteria) of the "bacteria" as the active ingredient can be set in the same way as the number of butyric acid bacteria contained in a typical intestinal regulator. For example, the content of bacteria in the food for human consumption can be set such that the daily intake of bacteria is preferably 300 million to 2.5 billion, more preferably 1 billion to 2 billion.

[0092] The proliferation-inhibiting composition may contain one or more arbitrary components without impairing the effect of the active ingredient. The arbitrary components may be selected appropriately based on the composition's form, etc. The arbitrary components may be food additives or pharmaceutical additives. The arbitrary components may be known substances, readily available on the market, or prepared. A single type of arbitrary component may be used, or multiple types may be used in combination. The content of any arbitrary component may be appropriately set according to its intended purpose, etc.

[0093] The proliferation-inhibiting composition can be applied to a wide range of animal species without particular limitation. The preferred subjects are mammals (humans and non-human mammals such as dogs and cats), more preferably humans. Furthermore, the sex and age of the subjects are not limited.

[0094] The intake of the proliferation-inhibiting composition can be appropriately set according to the age and weight of the target population, the frequency of intake, the route of administration, etc.

[0095] The intake interval can be set appropriately according to the intake amount, such as once a day or in several portions.

[0096] The proliferation inhibition composition can be prepared by mixing the active ingredient (the bacterial cells of the present invention, etc.) with any ingredient (e.g., an additive for food or medicine).

[0097] [Anti-inflammatory composition] Butyric acid bacteria are known to have anti-inflammatory effects (e.g., inhibition of enteritis, ulcerative colitis, and Crohn's disease) by producing butyric acid (Non-Patent Literature 1). The strains of the present invention have the ability to produce butyric acid (Example 2 described later). Therefore, one aspect of the present invention is an anti-inflammatory composition containing the bacterial cells of the present invention as an active ingredient.

[0098] The active ingredient can be either bacterial cells or spores, or a combination of both. The culture medium can contain either bacterial cells or spores, or both.

[0099] "Contains as an active ingredient" means that the composition contains the bacteria or the like of the present invention in an amount (effective amount) sufficient to exert the desired anti-inflammatory effect.

[0100] The content of the bacteria in this invention can be appropriately set considering the form of the composition (food or medicine, etc.). For example, in the case of food for human consumption, the content (number of bacteria) of the "bacteria" as the active ingredient can be set in the same way as the number of butyric acid bacteria contained in a typical intestinal regulator. For example, the content of bacteria in the food for human consumption can be set such that the daily intake of bacteria is preferably 300 million to 2.5 billion, more preferably 1 billion to 2 billion.

[0101] Anti-inflammatory compositions may contain one or more ingredients without impairing the action of the active ingredient. The ingredients may be selected appropriately based on the composition's form, etc.

[0102] Any ingredient can be an additive for food, beverage, or pharmaceutical use. Any ingredient is a known substance, readily available on the market, or can be prepared. Any ingredient can be a single type or in combination. The content of any ingredient can be appropriately set according to its intended purpose.

[0103] The anti-inflammatory composition can be applied to a wide range of animal species without particular limitation. The preferred target population is mammals (humans and non-human mammals such as dogs and cats), more preferably humans. Furthermore, the sex and age of the target population are not limited.

[0104] The intake of anti-inflammatory compositions can be appropriately set according to the age and weight of the target population, the frequency of administration, the route of administration, etc.

[0105] The intake interval can be set appropriately according to the intake amount, such as once a day or in several portions.

[0106] Anti-inflammatory compositions can be prepared by mixing an active ingredient (such as the bacterial cells of the present invention) with any ingredient (e.g., food additives or pharmaceutical additives).

[0107] [Composition for Inducing Regulatory T Cells] It is known that butyric acid bacteria induce regulatory T cells by producing butyric acid (Non-Patent Literature 1 and 7). Induction includes induction of differentiation into regulatory T cells and induction of proliferation of regulatory T cells (Non-Patent Literature 1 and 7).

[0108] Regulatory T cells (Tregs) have the function of suppressing the immune response against themselves (immune tolerance). The strains of the present invention have the ability to produce butyrate (Example 2 described later). 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.

[0109] The active ingredient can be either bacterial cells or spores, or a combination of both. The culture medium can contain either bacterial cells or spores, or both.

[0110] "Contains 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.

[0111] The content of the bacteria in this invention can be appropriately set considering the form of the composition (food or medicine, etc.). For example, in the case of food for human consumption, the content (number of bacteria) of the "bacteria" as the active ingredient can be set in the same way as the number of butyric acid bacteria contained in a typical intestinal regulator. For example, the content of bacteria in the food for human consumption can be set such that the daily intake of bacteria is preferably 300 million to 2.5 billion, more preferably 1 billion to 2 billion.

[0112] The composition for inducing regulatory T cells may contain one or more ingredients without impairing the effect of the active ingredient. The ingredients may be selected appropriately based on the composition's morphology, etc.

[0113] Any ingredient can be an additive for food, beverage, or pharmaceutical use. Any ingredient is a known substance, readily available on the market, or can be prepared. Any ingredient can be a single type or in combination. The content of any ingredient can be appropriately set according to its intended purpose.

[0114] The composition for inducing regulatory T cells can be applied to a wide range of animal species without particular limitation. The preferred subjects are mammals (humans and non-human mammals such as dogs and cats), more preferably humans. Furthermore, the sex and age of the subjects are not limited.

[0115] The dosage of the composition for inducing regulatory T cells can be appropriately set according to the age and weight of the target population, the frequency of administration, and the route of administration.

[0116] The intake interval can be set appropriately according to the intake amount, such as once a day or in several portions.

[0117] 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 any ingredient (e.g., an additive for food or medicine).

[0118] [How to use the composition containing the bacterial cells of the present invention] The aforementioned intestinal regulating composition, Clostridium difficile proliferation inhibitory composition, anti-inflammatory composition, and regulatory T cell induction composition can be used as food or medicine.

[0119] [Food and Drinks] There are no particular restrictions on the form of food and beverages as long as they can be ingested orally. Specific examples include liquid beverages and jelly drinks.

[0120] Food and beverage products include health foods, functional foods, nutritional supplements, foods for specific health purposes, foods for patients, and food and beverage products with disease risk reduction labels.

[0121] Food and beverage products can include sports nutrition supplements (including products certified by Informed Sport) and cereal products.

[0122] Food and beverage products may also contain food additives as any ingredient.

[0123] Examples of additives used in liquid beverages include pH adjusters, emulsifiers, stabilizers, flavorings, and sweeteners. Examples of additives used in jelly beverages include gelatin, food colorings, and thickening polysaccharides.

[0124] 〔drug〕 The preferred dosage form of the medicine is one that can be administered orally or rectally (e.g., via enema). Specific examples include liquids, capsules, and powders.

[0125] Pharmaceuticals may contain pharmaceutical additives as any ingredient. Examples of pharmaceutical additives include excipients, stabilizers, preservatives, humectants, emulsifiers, lubricants, sweeteners, colorants, flavorings, buffers, antioxidants, and pH adjusters. Example

[0126] The present invention will be described in more detail below through embodiments, but the present invention is not limited thereto.

[0127] [Test strain 1] The Clostridium butyricum SIID29215-B6 strain (hereinafter also referred to as "B6 strain") was used.

[0128] Strain B6 has been deposited internationally at the Patent and Microbial Collection Center of the Technical Base for Product Evaluation, an independent administrative agency, as stipulated by the Budapest Treaty (Room 2-5-8122, Kazusa-Kamazutsu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) (Identification mark: SIID29215-B6. Accession number: NITE BP-03916. Date of deposit (acceptance date): June 15, 2023).

[0129] As a standard strain of Clostridium butyricum, the Clostridium butyricum NBRC13949T strain (hereinafter also referred to as the "standard strain") was used.

[0130] [Example 1: Morphological, Physiological and Biochemical Tests] Using agar medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar), and the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, strain B6 and standard strains were anaerobically cultured at 37°C for 72 hours.

[0131] The morphology of the cultured B6 strain and the standard strain was observed using an optical microscope.

[0132] Furthermore, based on 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 physiological traits (catalase reaction, oxidase reaction, glucose acid production / gas production, glucose oxidation / fermentation (O / F)) of strain B6 and standard strain were tested. The results are shown in Table 1-1.

[0133] [Table 1-1]

[0134] Using agar medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar), and the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, strain B6 and standard strains were anaerobically cultured at 37°C for 24 hours.

[0135] The physiological and biochemical characteristics of the cultured B6 strain and standard strain were identified using an anaerobic bacterial biochemical identification kit (API20A (bioMerieux, FRA)). The results are shown in Tables 1-2.

[0136] [Table 1-2]

[0137] Further, additional physiological and biochemical traits of strain B6 were tested using the API ZYM kit (bioMerieux, FRA). The results are shown in Tables 1-3.

[0138] [Table 1-3]

[0139] Strain B6 does not have the ability to assimilate D-xylose and D-trehalose (Table 1-2).

[0140] On the other hand, the standard strain has the ability to assimilate D-xylose and D-trehalose.

[0141] Therefore, strain B6 was determined to be a strain different from the standard strain.

[0142] [Example 2: Organic Acid Production Capacity Test] Using liquid culture medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan)) and the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, strain B6 and standard strains were anaerobically cultured at 37°C for 72 hours (culture medium volume: 5 mL).

[0143] The culture medium was filtered through a membrane filter with a pore size of 0.20 μm to obtain the sample solution.

[0144] The concentration of organic acids in a sample solution was determined by high performance liquid chromatography.

[0145] The measurement conditions are as follows. • System: Shimadzu Organic Acid Analysis System (Shimadzu, Japan) • Column: Shim-pack Fast-OA, 100mm×7.8mmID, 3 in series • Protective pillars: Shim-pack Fast-OA, 10mm × 4.0mm 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 • Column oven temperature: 50℃ • Detector: Conductivity detector CDD-10Avp.

[0146] The organic acids tested were succinic acid, lactic acid, formic acid, acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, and n-valeric acid. The results are shown in Table 2.

[0147] [Table 2]

[0148] In the table, empty columns indicate values ​​below the lower limit of quantification (LOQ). The LQ is 5 μg / mL for succinic acid, lactic acid, acetic acid, and propionic acid; and 10 μg / mL for formic acid, isobutyric acid, n-butyric acid, isovaleric acid, and n-valeric acid. The values ​​for each organic acid are the average of three samples.

[0149] Strain B6 produced butyric acid. In addition, the amount of formic acid produced in the culture medium of strain B6 after anaerobic culture at 37°C for 72 hours was less than that of the standard strain (NBRC13949T).

[0150] [Example 3: Acetone and Alcohol Production Capacity Test] Using liquid culture medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan)) and the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, strain B6 and standard strains were anaerobically cultured at 37°C for 48 hours.

[0151] After 24 hours and 48 hours of incubation, the culture medium was aliquoted into sterile tubes. The supernatant (more than 10 mL) obtained from centrifugation was filtered through a sterile 0.2 μm cellulose acetate filter. The filtrate was stored at 4°C as a sample.

[0152] Approximately 1 g of the sample was placed in a volumetric flask and diluted to 10 mL with methanol. The mixture was stirred with a shaker for 1 hour and then allowed to stand for 3 hours. 1 μL of the supernatant was then used for GC / MS analysis.

[0153] The measurement conditions are as follows. ・CG / MS: Agilent Technologies, 6890N / 5973inert ・Column: HP-1 (0.25mmφ×30m, df=1.00μm) • Column temperature: 40℃ (3 min) → 10℃ / min → 100℃ → 20℃ / min → 300℃ (10 min) • Column pressure: Constant current mode (51kPa, Vac) • Column flow rate: 1 mL / min (He) • Inlet temperature: 250℃ • Injection volume: 1 μL • Injection method: Split (10:1) • Detector: MS • Ion source temperature: 230℃ • Ionization method: EI (70eV) • Scan range: SIM (m / z: 31, 41, 43, 45, 58, 59, 2.0~3.5min) :SIM(m / z:41,43,45,56,59,74, 3.5~29.0min) • Gain: 976V.

[0154] The pure pharmaceutical samples of each component shown in Table 3 were diluted with methanol to prepare standards of a certain concentration. 1 μL of each standard was measured in the same manner as the aforementioned sample. A standard curve was constructed based on the peak area of ​​the fundamental or molecular ion of each quantitative component in the mass spectrometer and the prepared concentration. The content of each component relative to 1 g of the sample was then determined. The results are shown in Table 3.

[0155] [Table 3]

[0156] The amount of acetone produced in the culture medium of strain B6 after anaerobic culture at 37°C for 48 hours was less than that of the standard strain (NBRC13949T).

[0157] [Example 4: Spore formation confirmation test] Using liquid culture medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) or agar medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar), and with the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, strain B6 was anaerobically cultured at 37°C for 24 hours, 72 hours, 7 days, and 14 days.

[0158] After culturing, the bacterial cells were Gram-stained (Favor G "Nissui" (Nissui Pharmaceutical, Japan)) and their spore-forming ability was confirmed by microscopic observation (optical microscope: BX50F4 (Olympus, Japan)). The results are shown in Table 4.

[0159] [Table 4]

[0160] Strain B6 is a spore-forming Gram-positive bacterium.

[0161] [Example 5: Heat Resistance Test] Using agar medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar), and the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, strain B6 was pre-cultured at 37°C under anaerobic conditions for 24 or 72 hours.

[0162] The culture was suspended in 10 mL of physiological saline to prepare a bacterial suspension. 5 mL of the bacterial suspension was dispensed into a sterile medium-sized test tube (18×170 mm) and subjected to heat treatment at 60°C for 30 minutes in a water bath.

[0163] Use 5 mL of the bacterial culture before heat treatment as a control.

[0164] The viable bacterial count was determined before and after heat treatment under the following conditions. The results are shown in Table 5. • Culture medium: GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) and agar • Culture temperature: 37℃ • Culture time: 24 hours to 48 hours • Diluent: Physiological saline • Dilution ratio: stock solution ~ 10 5 Dilution • Assay method: Dilution plate method (0.1 mL surface-coated CFU method) • Automatic dilution and application device: easySpiralDilute (registered trademark) (Interscience, France) • Number of application patches: 3 patches of the same dilution • Other: Anaerobic culture (AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan)) • Colony observation: Stereomicroscope (SMZ800N (Nikon, Japan)).

[0165] [Table 5]

[0166] Since the live bacteria proliferated from the heat-treated bacterial solution, strain B6 was determined to be heat-resistant.

[0167] [Example 6: Assay for the inhibition of Clostridium difficile proliferation] To evaluate the effect of strain B6 on the proliferation of Clostridium difficile, a representative pathogen of antibiotic-associated diarrhea (diarrhea and soft stools caused as a side effect of antibiotics).

[0168] (1) Preparation of sterile filtrate of Clostridium butyricum strain B6 Using liquid culture medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan)) and the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, strain B6 was cultured at 37°C under anaerobic conditions for 72 hours. The total number of bacteria in the culture medium was counted under the following conditions. The results are shown in Table 6-1. • Diluent: Physiological saline • Dilution ratio: stock solution ~ 10 4 Dilution • Blood cell counter: A disposable blood cell counter. C-Chip (DHC-N01: Neubawell modified version) (NanoEntek, Korea). Counts cells at dilutions with a cell count of 100 to 1000 in large squares. • Microscope: Optical microscope (BX50 (Olympus, Japan)).

[0169] [Table 6-1]

[0170] The culture medium was sterilized by filtration through a 0.2 μm cellulose acetate filter to prepare a sterile filtrate.

[0171] (2) Preparation of test culture medium The sterile filtrate prepared in (1) was mixed with an equal amount of GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) at twice the concentration to prepare the test culture medium.

[0172] GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) was used as a control.

[0173] (3) Preparation of Clostridium difficile bacterial culture Using agar medium (GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar), and the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit, the standard strain JCM1296T of Clostridium difficile was cultured at 37°C under anaerobic conditions for 24 hours.

[0174] The culture medium was suspended in sterile physiological saline to prepare a concentration equivalent to McFarland No. 0.5 turbidity standard. The suspension was then counted using a hemocytometer to obtain a bacterial volume of approximately 1 × 10⁻⁶. 8 Cells / mL.

[0175] (4) Cultivation of Clostridium difficile In a medium bottle containing 20 mL of test culture medium, 20 μL of Clostridium difficile culture prepared in (3) was inoculated and statically cultured at 37 °C under anaerobic conditions using the AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan) as an anaerobic culture kit.

[0176] After 24 hours and 48 hours of incubation, 2 mL of culture medium was collected, and the number of Clostridium difficile bacteria was determined under the following conditions. The results are shown in Table 6-2. • Culture medium: GAM Broth "Nissui" (Nissui Pharmaceutical, Japan) + agar • Culture temperature: 37℃ • Culture time: 24 hours to 48 hours • Diluent: Physiological saline • Dilution ratio: stock solution ~ 10 7 Dilution • Assay method: Dilution plate method (0.1 mL surface smear for colony forming units (CFU) counting) • Automatic dilution and application device: easySpiralDilute (registered trademark) (Interscience, France) • Number of application patches: 3 patches of the same dilution • Other: Anaerobic culture (AneroPouch Kenki System (Mitsubishi Gas Chemical, Japan)) • Colony observation: Stereomicroscope (SMZ800N (Nikon, Japan)) • Judgment: Count the colonies grown on plates at the appropriate dilution stage.

[0177] [Table 6-2]

[0178] The proliferation of Clostridium difficile was inhibited by adding sterile filtrate of strain B6. This suggests that strain B6 produces a substance that inhibits the proliferation of Clostridium difficile.

[0179] [Reference Example 1: Partial Base Sequence Analysis of 16S rDNA] A partial base sequence (SEQ ID NO:1) of the 16S rDNA (16S rRNA gene) of strain B6 was analyzed and its attribution was deduced. [SEQ ID NO:1]

[0180] The analysis was performed under the following conditions. The results are shown in Tables 7-1 and 7-2. • DNA extraction: Cica geneus DNA extraction reagent ST (Kanto Chemical, Japan) ・PCR amplification: TKs Gflex DNA Polymerase (Takara Bio, Japan) • Cyclic Sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, USA) • Use primers: PCR amplification: 9F, 1510R Sequencing (approximately 1500bp): 9F, 515F, 1099F, 536R, 926R, 1510R ・Sequencing: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) • Base sequence determined: 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 databases (DDBJ / ENA / GenBank) • Simplified molecular phylogenetic tree analysis: Prediction of a hierarchical tree: Adjacency method Base substitution model: Kimura-2-parameter Tree-based reliability assessment: Bootstrap method (1,000 replicates).

[0181] [Table 7-1] Note 1) BSL (Biosafety Level) marking level 1 * (Opportunistic pathogens) and above, an empty column means level 1. Note 2) The “_ (underscore)” in the plant name means a space. Note 3) The 11 strains from top to bottom in the table represent sequence data for simplified molecular systematic analysis.

[0182] [Table 7-2]

[0183] A simplified molecular phylogenetic tree based on a partial 16S rDNA sequence of strain B6 is shown below. Figure 1 In the diagram, SIID29215-B6 represents strain B6. Additionally, SIID29215-04 represents the standard strain (Clostridium butyricum NBRC13949T strain). The line in the upper left represents a scale bar. The numbers at the bifurcation points of the system branches represent the spread value, which indicates the reliability of the tree structure. The "T" at the end of the strain name indicates the standard strain (Type strain) for that species. BSL indicates the biosafety level (labeled BSL1). * (Opportunistic pathogens) above.

[0184] [Reference Example 2: In silico DDH (DNA-DNA hybridization) analysis] In silico DDH analysis is a method that compares the whole genome sequence and draft genome sequence of control and comparison strains on a computer and evaluates the similarities and differences between the two strains.

[0185] In this reference example, ANI analysis and GGDC analysis with different calculation methods were performed.

[0186] (1) ANI (Average Nucleotide Identity) Analysis In the ANI analysis, the genome sequence of the control strain was fragmented into 1,020 bp segments on a computer. Homology searches were performed on each segment of the genome sequence of the comparison strain, and the ANI value between the genome sequences was calculated based on the average of these homology values. The ANI value was calculated using the publicly available program ANI Calculator (http: / / enve-omics.ce.gatech.edu / ani / index). In the ANI analysis, sequences with an ANI value of 95% or higher were considered to be of the same species.

[0187] In this reference example, strain B6 was used as the comparison strain and the standard strain (Clostridium butyricum NBRC13949T strain) was used as the control strain for ANI analysis. The results showed that the ANI value between strain B6 and the standard strain was 100%.

[0188] (2) GGDC (Genome-to-Genome Distance Calculator) analysis In GGDC analysis, regions of high homology between the genome sequences of control and comparison strains are identified on a computer, and their pairwise distances are calculated to determine the in silico DDH value (BMC Bioinformatics 2013;14:60). The in silico DDH value is calculated using the publicly available program Genome-to-Genome Distance Calculator (http: / / ggdc.dsmz.de / ggdc.php#). In GGDC analysis, an in silico DDH value of 70% or higher is considered equivalent to that of the same species.

[0189] In this reference example, strain B6 was used as the comparison strain and the standard strain (Clostridium butyricum NBRC13949T strain) was used as the control strain for GGDC analysis. The results showed that the in silico DDH value between strain B6 and the standard strain was 99.9%.

[0190] Based on the results of Reference Examples 1 and 2, strain B6 was identified as Clostridium butyricum.

[0191] Of these, strain B6 does not have the ability to assimilate D-xylose and D-trehalose (Example 1).

[0192] On the other hand, strain NBRC13949T (the standard strain of Clostridium butyricum) has the ability to assimilate D-xylose and D-trehalose (Example 1).

[0193] Furthermore, Clostridium butyricum is known to possess the assimilation capacity for D-xylose and D-trehalose (Bergey's Manual of Systematic Bacteriology, Second Edition, Volume Three, The Firmicutes, p.739-742 (2009) (Non-Patent Literature 3)).

[0194] Therefore, strain B6 was determined to be a strain different from the well-known Clostridium butyricum strain, including the standard strain.

[0195] [Reference Example 3: Genome analysis of strain B6] The genome analysis of strain B6 was performed according to the following method.

[0196] 1. DNA extraction • DNA extraction and purification: NucleoSpin Plant II (MACHEREY-NAGEL, GER).

[0197] 2. Library preparation • Kit used: Nextera DNA Flex Library Prep Kit (Illumina, USA) Nextera DNA CD Indexes (Illumina).

[0198] 3. Genome sequencing Sequence determination was performed according to the protocol provided with the sequencer. • Sequencing instrument: iSeq 100 System (Illumina) • Sequencing kit: iSeq 100 i1 Reagent kit (Illumina) • Sequencing method: 2×151 bp paired-end sequencing.

[0199] 4. Data Analysis • Quality filtration: trimmomatic ver 0.39 • Reassemble from scratch: Spades ver 3.15.4 • Error correction (polishing): pilon ver 1.24 • Coverage (depth) calculation: bowtie2 ver 2.3.5.1, samtools ver 1.10 Deep coverage is calculated by comparing the longest contigs and taking the average value for each site. • Calculation of G+C content: SeqKit ver 0.12.0.

[0200] The results are shown below. a) Represents the sum of the lengths of the overlapping group / scaffold. b) represents the value calculated based on the assembled base sequence.

[0201] The 172 contig sequences of strain B6 obtained are designated as SEQ ID NO:2–173. It should be noted that the sequences shown in SEQ ID NO:2–173 are included in the sequence listing contained in the international application of this case at the time of filing.

[0202] [Explanation on the Preservation of Biological Materials] Name of depository: Patent and Microbial Collection Center, Technical Base for Product Evaluation, Independent Administrative Institution Address: Room 122, 2-5-8 Kazusa-Kamazutari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan Accession number: NITE BP-03916 Identification mark: SIID29215-B6 Date of preservation: June 15, 2023 Country of origin: Japan Source of isolation: Isolated from fermentation broth containing culture medium obtained from the food raw material manufacturing plant of Higher Mount Co., Ltd. in Kaizu City, Gifu Prefecture. The culture medium used was domestically grown soybeans from Japan. The soybeans used were organically grown in Japan without the use of pesticides.

[0203] [Test strain 2] The Clostridium butyricum strain SIID49520-01-B1 (hereinafter also referred to as "01-B1 strain") was used.

[0204] Strain 01-B1 has been deposited internationally at the Patent and Microbial Collection Center of the Technical Base for Product Evaluation, an independent administrative agency, as stipulated by the Budapest Treaty (Room 2-5-8122, Kazusa-Kamazutsu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) (Identification mark: SIID49520-01-B1. Accession number: NITE BP-04190. Date of deposit (acceptance date): October 29, 2024).

[0205] [Example 7: Morphological, Physiological and Biochemical Tests] Using agar medium (AccuDia GAM Broth (Shimadzu Diagnostics Corporation, Japan) + agar), and with the AneroPouch Kenki System (Mitsubishi GasChemical, Japan) as an anaerobic culture kit, strain 01-B1 was anaerobically cultured at 37°C for 24 hours.

[0206] The morphology of the cultured 01-B1 strain was observed using an optical microscope.

[0207] Furthermore, based on 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 physiological traits (catalase reaction, oxidase reaction, glucose acid production / gas production, glucose oxidation / fermentation (O / F)) of strain 01-B1 were tested. The results are shown in Table 8-1.

[0208] It should be noted that the results for the standard strains in Table 8-1 were obtained in Example 1.

[0209] [Table 8-1]

[0210] Using agar medium (AccuDia GAM Broth (Shimadzu Diagnostics Corporation, Japan) + agar), and with the AneroPouch Kenki System (Mitsubishi GasChemical, Japan) as an anaerobic culture kit, strain 01-B1 was anaerobically cultured at 37°C for 24 hours.

[0211] The physiological and biochemical characteristics of the cultured 01-B1 strain were identified using an anaerobic biochemical identification kit (API20A (bioMerieux, FRA)). The results are shown in Table 8-2.

[0212] It should be noted that the results for the standard strains in Table 8-2 were obtained in Example 1.

[0213] [Table 8-2]

[0214] Further, additional physiological and biochemical traits of strain 01-B1 were tested using the API ZYM kit (bioMerieux, FRA). The results are shown in Table 8-3.

[0215] [Table 8-3]

[0216] The 01-B1 strain does not have the ability to assimilate D-xylose and D-trehalose (Table 8-2).

[0217] On the other hand, the standard strain has the ability to assimilate D-xylose and D-trehalose.

[0218] Therefore, strain 01-B1 was determined to be a strain different from the standard strain.

[0219] [Example 8: Organic Acid Production Capacity Test] Using liquid culture medium (AccuDia GAM Broth (Shimadzu Diagnostics Corporation, Japan)) and the AneroPouch Kenki System (Mitsubishi GasChemical, Japan) as an anaerobic culture kit, strains 01-B1, B6 (test strain 1), and standard strains were anaerobically cultured at 37°C for 72 hours (culture medium volume: 200 mL).

[0220] The culture medium was filtered through a membrane filter with a pore size of 0.20 μm to obtain the sample solution.

[0221] The concentration of organic acids in a sample solution was determined by high performance liquid chromatography.

[0222] The measurement conditions are as follows. • System: Nexera Organic Acid Analysis System (Shimadzu Corporation) • Model: LC-40D (Shimadzu Corporation) • Detector: Conductivity detector CDD-10Avp (Shimadzu Corporation) ・Column: Shim-pack SCR-102H×2, φ8.0mm×300mm (Shimadzu Corporation) • Protective pillar: SCR-102H, φ6.0mm×50mm (Shimadzu Corporation) • Column temperature: 45℃ • 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.

[0223] The organic acids tested were lactic acid, formic acid, acetic acid, and butyric acid. The results are shown in Table 9.

[0224] [Table 9] In the table, empty columns indicate values ​​below the lower limit of quantification (LVC). The LVC is 0.01 g / 100 g.

[0225] The 01-B1 strain produced butyric acid. In addition, the amount of formic acid produced in the culture medium of the 01-B1 strain after anaerobic culture at 37°C for 72 hours was less than that of the standard strain (NBRC13949T).

[0226] [Reference Example 4: Partial Base Sequence Analysis of 16S rDNA] A partial base sequence (SEQ ID No: 174) of the 16S rDNA (16S rRNA gene) of strain 01-B1 was analyzed and its attribution was presumed. [SEQ ID NO:174]

[0227] The analysis was performed under the following conditions. The results are shown in Tables 10-1 and 10-2. • DNA extraction: Achromopeptidase (FUJIFILM Wako Pure Chemical, Japan) ・PCR amplification: TKs Gflex DNA Polymerase (Takara Bio, Japan) • Cyclic Sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, USA) • Use primers: PCR amplification: 9F, 1510R Sequencing (approximately 1500bp): 9F, 515F, 1099F, 536R, 926R, 1510R ・Sequencing: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) • Base sequence determined: 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 databases (DDBJ / ENA / GenBank) • Simplified molecular phylogenetic tree analysis: Prediction of a hierarchical tree: Adjacency method Base substitution model: Kimura-2-parameter Tree-based reliability assessment: Bootstrap method (1,000 replicates).

[0228] [Table 10-1] Note 1) BSL (Biosafety Level) marking level 1 * (Opportunistic pathogens) and above, an empty column means level 1. Note 2) The “_ (underscore)” in the plant name means a space. Note 3) The 11 strains from top to bottom, namely strains 1 to 3 and strains 5 to 12, represent sequence data provided for simplified molecular systematic analysis.

[0229] [Table 10-2]

[0230] A simplified molecular phylogenetic tree based on a partial 16S rDNA base sequence of strain 01-B1 is shown below. Figure 2 In the diagram, SIID49520-01-B1 represents strain 01-B1. The line in the upper left corner represents a scale. The number at the branching point of the system represents the spread value, which indicates the reliability of the tree structure. The "T" at the end of the strain name indicates the standard strain (Type strain). BSL indicates the biosafety level (marked BSL1* (opportunistic pathogen) and above).

[0231] [Explanation on the Preservation of Biological Materials] Name of depository: Patent and Microbial Collection Center, Technical Base for Product Evaluation, Independent Administrative Institution Address: Room 122, 2-5-8 Kazusa-Kamazutari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan Accession number: NITE BP-04190 Identification mark: SIID49520-01-B1 Date of preservation: October 29, 2024 Country of origin: Japan Source of isolation: Isolated from fermentation broth containing culture medium at the food raw material manufacturing plant of Higher Mount Co., Ltd. in Kaizu City, Gifu Prefecture. The culture medium used was domestically grown soybeans from Japan. The soybeans used were organically grown in Japan without the use of pesticides.

[0232] [Test strain 3] The Clostridium butyricum SIID50030-B1 strain (hereinafter also referred to as "B1 strain") was used.

[0233] Strain B1 has been deposited internationally at the Patent and Microbial Collection Center of the Technical Base for Product Evaluation, an independent administrative agency, as stipulated by the Budapest Treaty (Room 2-5-8122, Kazusa-Kamazutsu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) (Identification mark: SIID50030-B1. Accession number: NITE BP-04191. Date of deposit (acceptance date): October 29, 2024).

[0234] [Example 9: Morphological, Physiological and Biochemical Tests] Using agar medium (AccuDia GAM Broth (Shimadzu Diagnostics Corporation, Japan) + agar), and with the AneroPouch Kenki System (Mitsubishi GasChemical, Japan) as an anaerobic culture kit, strain B1 was anaerobically cultured at 37°C for 24 hours.

[0235] The morphology of the cultured B1 strain was observed using an optical microscope.

[0236] Furthermore, based on 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 physiological traits (catalase reaction, oxidase reaction, glucose acid production / gas production, glucose oxidation / fermentation (O / F)) of strain B1 were tested. The results are shown in Table 11-1.

[0237] It should be noted that the results for the standard strains in Table 11-1 were obtained in Example 1.

[0238] [Table 11-1]

[0239] Using agar medium (AccuDia GAM Broth (Shimadzu Diagnostics Corporation, Japan) + agar), and with the AneroPouch Kenki System (Mitsubishi GasChemical, Japan) as an anaerobic culture kit, strain B1 was anaerobically cultured at 37°C for 24 hours.

[0240] The physiological and biochemical characteristics of the cultured B1 strain were identified using an anaerobic biochemical identification kit (API20A (bioMerieux, FRA)). The results are shown in Table 11-2.

[0241] It should be noted that the results for the standard strains in Table 11-2 were obtained in Example 1.

[0242] [Table 11-2]

[0243] Further, additional physiological and biochemical traits of strain B1 were tested using the API ZYM kit (bioMerieux, FRA). The results are shown in Tables 11-3.

[0244] [Table 11-3]

[0245] Strain B1 does not have the ability to assimilate D-xylose and D-trehalose (Table 11-2).

[0246] On the other hand, the standard strain has the ability to assimilate D-xylose and D-trehalose.

[0247] Therefore, strain B1 was determined to be a strain different from the standard strain.

[0248] [Example 10: Organic Acid Production Capacity Test] Using liquid culture medium (AccuDia GAM Broth (Shimadzu Diagnostics Corporation, Japan)) and the AneroPouch Kenki System (Mitsubishi GasChemical, Japan) as an anaerobic culture kit, strains B1, B6 (test strain 1), and standard strains were anaerobically cultured at 37°C for 72 hours (culture medium volume: 200 mL).

[0249] The culture medium was filtered through a membrane filter with a pore size of 0.20 μm to obtain the sample solution.

[0250] The concentration of organic acids in a sample solution was determined by high performance liquid chromatography.

[0251] The measurement conditions are as follows. • System: Nexera Organic Acid Analysis System (Shimadzu Corporation) • Model: LC-40D (Shimadzu Corporation) • Detector: Conductivity detector CDD-10Avp (Shimadzu Corporation) ・Column: Shim-pack SCR-102H×2, φ8.0mm×300mm (Shimadzu Corporation) • Protective pillar: SCR-102H, φ6.0mm×50mm (Shimadzu Corporation) • Column temperature: 45℃ • 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.

[0252] The organic acids tested were lactic acid, formic acid, acetic acid, and butyric acid. The results are shown in Table 12.

[0253] [Table 12] In the table, empty columns indicate values ​​below the lower limit of quantification (LVC). The LVC is 0.01 g / 100 g.

[0254] Strain B1 produced butyric acid. In addition, the amount of formic acid produced in the culture medium of strain B1 after anaerobic culture at 37°C for 72 hours was less than that of the standard strain (NBRC13949T).

[0255] [Reference Example 5: Partial Base Sequence Analysis of 16S rDNA] A partial base sequence (SEQ ID NO:175) of the 16S rDNA (16S rRNA gene) of strain B1 was analyzed and its attribution was deduced. [SEQ ID NO:175]

[0256] The analysis was performed under the following conditions. The results are shown in Tables 13-1 and 13-2. • DNA extraction: Achromopeptidase (FUJIFILM Wako Pure Chemical, Japan) ・PCR amplification: TKs Gflex DNA Polymerase (Takara Bio, Japan) • Cyclic Sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, USA) • Use primers: PCR amplification: 9F, 1510R Sequencing (approximately 1500bp): 9F, 515F, 1099F, 536R, 802R, 1510R ・Sequencing: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) • Base sequence determined: 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 databases (DDBJ / ENA / GenBank) • Simplified molecular phylogenetic tree analysis: Prediction of a hierarchical tree: Adjacency method Base substitution model: Kimura-2-parameter Tree-based reliability assessment: Bootstrap method (1,000 replicates).

[0257] [Table 13-1] Note 1) BSL (Biosafety Level) marking level 1 * (Opportunistic pathogens) and above, an empty column means level 1. Note 2) The “_ (underscore)” in the plant name means a space. Note 3) The 10 strains from top to bottom, namely strains 1 to 3 and strains 5 to 11, represent sequence data provided for simplified molecular systematic analysis.

[0258] [Table 13-2]

[0259] A simplified molecular phylogenetic tree based on a partial 16S rDNA sequence of strain B1 is shown below. Figure 3 In the diagram, SIID50030-B1 represents strain B1. The line in the upper left corner represents the scale. The number at the branching point of the system represents the spread value, which indicates the reliability of the tree structure. The "T" at the end of the strain name indicates the standard strain (Type strain). BSL indicates the biosafety level (marked BSL1* (opportunistic pathogen) and above).

[0260] [Explanation on the Preservation of Biological Materials] Name of depository: Patent and Microbial Collection Center, Technical Base for Product Evaluation, Independent Administrative Institution Address: Room 122, 2-5-8 Kazusa-Kamazutari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan Accession number: NITE BP-04191 Identification mark: SIID50030-B1 Date of preservation: October 29, 2024 Country of origin: Japan Source of isolation: Isolated from fermentation broth containing culture medium at the food raw material manufacturing plant of Higher Mount Co., Ltd. in Kaizu City, Gifu Prefecture. The culture medium used was domestically grown soybeans from Japan. The soybeans used were organically grown in Japan without the use of pesticides.

[0261] It should be noted that strain 01-B1 (tested strain 2) differs from strain B1 (tested strain 3) in the following aspects. Therefore, strain 01-B1 is determined to be a different strain from strain B1.

[0262] Industrial practicality This invention can be used in food, medicine, and other fields.

Claims

1. *Clostridium butyricum*, which produces butyric acid and possesses all of the characteristics described in (1) to (3) below, (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

2. Clostridium butyricum SIID29215-B6 strain (preservation number: NITE BP-03916) or its mutant strain, wherein, The aforementioned mutant strain produces butyric acid and possesses all of the characteristics described in (1) to (3) below. (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

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

4. Clostridium butyricum SIID49520-01-B1 strain (preservation number: NITE BP-04190) or its mutant strain, wherein, The aforementioned mutant strain produces butyric acid and possesses all of the characteristics described in (1) to (3) below. (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

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

6. Clostridium butyricum SIID50030-B1 strain (preservation number: NITE BP-04191) or its mutant strain, wherein, The aforementioned mutant strain produces butyric acid and possesses all of the characteristics described in (1) to (3) below. (1) It does not have the ability to assimilate D-xylose. (2) It does not have the ability to assimilate D-trehalose. (3) Compared with the NBRC13949T strain, which is the standard strain of Clostridium butyricum, the amount of formic acid produced in the culture medium after 72 hours of anaerobic culture at 37°C was less.

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

8. An intestinal regulating composition comprising, as an active ingredient, the cells, spores or culture medium of Clostridium butyricum as described in any one of claims 1 to 7.

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

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

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

12. The composition according to claim 8, wherein it is in the form of a pharmaceutical product.

13. The composition according to claim 9, wherein it is in the form of a pharmaceutical product.

14. The composition according to claim 10, wherein it is in the form of a pharmaceutical product.

15. The composition according to claim 11, wherein it is in the form of a pharmaceutical product.

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

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

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

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

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