Novel anaerostipes hadrus bacterium and use thereof

Abstract

To provide a butyric-acid-producing bacterium having high butyric acid production ability and suitable for medical application, and a composition containing the same. Provided is a butyric-acid-producing bacterium belonging to Anaerostipes hadrus that has a ratio of butyric acid production during mixed culture with a synbiotic to butyric acid production during monoculture of 2 or more and has no drug resistance gene. The synbiotic contains Lacticaseibacillus paracasei YIT9029 (FERM BP-1366), Bifidobacterium breve YIT12272 (FERM BP-11320), and a galactooligosaccharide.

Description

A novel bacterium of the genus Anaerostipes hadras and its utilization

[0001] The present invention relates to a new butyric acid-producing bacterium and a food or drink composition, pharmaceutical composition, or feed composition containing the same.

[0002] Short-chain fatty acids, such as acetic acid, propionic acid, and butyric acid, are produced in the lower gastrointestinal tract through the fermentation of indigestible carbohydrates derived from food by intestinal bacteria. Short-chain fatty acids are not only utilized as a major energy source for colonic mucosal epithelial cells but also exhibit numerous physiological functions. In particular, butyric acid is thought to exhibit diverse physiological activities, such as promoting epithelial cell proliferation and intestinal motility, as well as acting on the immune system and promoting the differentiation of regulatory T cells, which have anti-inflammatory properties (Non-Patent Documents 1-3). It has also been suggested that butyric acid produced by intestinal bacteria is important for the prevention of colon cancer and ulcerative colitis (Patent Document 1). In recent years, it has also been reported that butyric acid produced by intestinal bacteria may affect brain function and stress, reducing microglia-mediated inflammation in the brain due to aging (Non-Patent Document 4) and alleviating the symptoms of irritable bowel syndrome (IBS) (Non-Patent Document 5).

[0003] Certain bacteria belonging to the genus Lactobacillus and Bifidobacterium are known to promote the increase of intestinal butyric acid concentration (Patent Document 1). In addition, bacteria belonging to the genus Anaerostipes, particularly Anaerostipes hadrus (Eubacterium hadrum) DSM 3319, are known to produce butyric acid. T , YIT 12355, butylate-producing bacterium SSC / 2, SS2 / 1, etc. are known (Patent Document 2, Non-Patent Documents 6 to 8).

[0004] Bacteria often acquire resistance to antibiotics by transmitting drug-resistance genes from other bacteria. The emergence and increase of drug-resistant bacteria makes the prevention and treatment of infectious diseases more difficult and increases the likelihood of infection spreading or becoming more severe. Therefore, when applying bacteria to medical applications, it is desirable to use bacteria that are less likely to transmit drug-resistance genes.

[0005] JP 10-084909 A International Publication No. 2015 / 147277

[0006] Hamwe HM et al. Review article: the role of butyrate on colonic function. Aliment Pharmacol Ther. 27:104-119 (2008).Roy CC et al. Short-chain fatty acids: ready for prime time?Nutr Clin Pract. 21:351-366 (2006).Furusawa Y et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 504:446-450 (2013).Matt SM et al. Butyrate and Dietary Soluble Fiber Improve Neuroinflammation Associated With Aging in Mice. Front Immunol. 9:1832 (2018).Sun YY et al. The effect of Clostridium butyricum on symptoms and fecal microbiota in diarrhea-dominant irritable bowel syndrome: a randomized, double-blind, placebo-controlled trial. Sci Rep. 8:2964 (2018).Allen-Vercoe et al. Anaerostipes hadrus comb. nov., a dominant species within the human colonic microbiota; reclassification of Eubacterium hadrum Moore et al. 1976. Anaerobe 18:523-529 (2012).Duncan SH et al.Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product. Applied and Environmental Microbiology 70:5810-5817 (2004). Louis P et al. Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon. J. Bacteriology 186:2099-2106 (2004).

[0007] The present invention relates to providing a new butyric acid-producing bacterium having high butyric acid-producing ability and suitable for medical applications, and a food and drink composition, pharmaceutical composition, or feed composition containing the same.

[0008] The present inventors have screened a sample library held by the applicant, and as a result, have identified a bacterium belonging to Anaerostipes hadrus, the type strain Anaerostipes hadrus DSM 3319. T The present inventors have discovered a new butyric acid-producing bacterium that has superior butyric acid production ability compared to that of a bacterium containing a synbiotic, that produces more butyric acid when co-cultured with a synbiotic than when cultured alone, and that does not contain a drug resistance gene. The present inventors have also discovered that the amount of butyric acid produced by the butyric acid-producing bacterium increases when cultured in the presence of a galactooligosaccharide, particularly 3'-galactosyllactose, compared to when cultured in its absence, and that the amount of butyric acid produced increases when cultured in the presence of acetate compared to when cultured in its absence, and have completed the present invention.

[0009] That is, the present invention relates to the following [1] to [8]. [1] A butyric acid-producing bacterium belonging to Anaerostipes hadrus, which has a ratio of the amount of butyric acid produced when co-cultured with a synbiotic to the amount of butyric acid produced when cultured alone of 2 or more, and which does not have a drug resistance gene, and the synbiotic contains Lacticaseibacillus paracasei YIT 9029 (FERM BP-1366), Bifidobacterium breve YIT 12272 (FERM BP-11320), and galactooligosaccharides. [2] The butyric acid-producing bacterium according to [1], which has a ratio of the amount of butyric acid produced when cultured alone in the presence of 3'-galactosyllactose to the amount of butyric acid produced in the absence of 3'-galactosyllactose of 2 or more. [3] The butyric acid-producing bacterium according to [1], which is Anaerostipes hadrus YIT 13235 (NITE BP-04056). [4] A food or drink composition, pharmaceutical composition, or feed composition, comprising the butyric acid-producing bacterium according to any one of [1] to [3] and at least one selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts, wherein the synbiotics include Lacticaseibacillus paracasei YIT 9029 (FERM BP-1366), Bifidobacterium breve YIT 12272 (FERM BP-11320), and galactooligosaccharides. [5] A butyric acid production enhancer, comprising the butyric acid-producing bacterium according to any one of [1] to [3] and at least one selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts. [6] Use of the butyric acid-producing bacterium according to any one of [1] to [3] and at least one species selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts for the manufacture of a butyric acid production enhancer. [7] Use of the butyric acid-producing bacterium according to any one of [1] to [3] and at least one species selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts for the enhancement of butyric acid production. [8] A method for enhancing butyric acid production, comprising administering to a subject in need thereof the butyric acid-producing bacterium according to any one of [1] to [3] and at least one species selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts.

[0010] The butyric acid-producing bacteria of the present invention have excellent butyric acid production ability and do not contain drug resistance genes. Therefore, by using the butyric acid-producing bacteria of the present invention, butyric acid production in the intestine can be enhanced while the risk of transmitting drug resistance genes to other bacteria can be avoided. If the amount of butyric acid in the intestine is increased, various physiological actions based on butyric acid will be enhanced as described above.

[0011] Strain identification using the Random Amplified Polymorphic DNA (RAPD) method. Amount of butyric acid produced by A. hadrus strains without the addition of galactooligosaccharides, or with the addition of galactooligosaccharides (GOS), 4'-galactosyllactose (4'-GL), or 3'-galactosyllactose (3'-GL). Amount of butyric acid produced by A. hadrus strains without the addition of acetate and lactate, or with the addition of acetate or lactate. Amount of butyric acid produced by A. hadrus strains without the addition of synbiotics or with the addition of synbiotics.

[0012] The butyric acid-producing bacterium of the present invention is a bacterium belonging to Anaerostipes hadrus (hereinafter referred to as A. hadrus), which has a ratio of the amount of butyric acid produced when co-cultured with a synbiotic to the amount of butyric acid produced when cultured alone of 2 or more, and which does not have a drug resistance gene.

[0013] Here, "synbiotics" refers to a combination of probiotics and prebiotics. "Probiotics" refer to live microorganisms that, when ingested in appropriate amounts, have a beneficial effect on the health of the host. "Prebiotics" refer to substances that serve as "food" for bifidobacteria and lactic acid bacteria, and have a beneficial effect by increasing the number of these bacteria. "Monoculture" of the butyric acid-producing bacteria of the present invention refers to culturing essentially only the butyric acid-producing bacteria of the present invention, and "mixed culture" of the butyric acid-producing bacteria of the present invention with synbiotics refers to culturing the butyric acid-producing bacteria of the present invention and probiotics in the presence of prebiotics.

[0014] In the present invention, probiotics specifically refer to Lacticaseibacillus paracasei YIT 9029 (FERM BP-1366) and Bifidobacterium breve YIT 12272 (FERM BP-11320). The above-mentioned Lacticaseibacillus paracasei YIT 9029 has been internationally deposited as Lactobacillus casei YIT 9029 (FERM BP-1366, date of deposit: May 1, 1981) at the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, which is the international depository authority under the Budapest Treaty (currently the Patent Organism Depositary, National Institute of Technology and Evaluation, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan). Lacticaseibacillus paracasei was previously classified as Lactobacillus casei, but in recent years the genus Lactobacillus has been reclassified as Lacticaseibacillus paracasei (Zheng et al., A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 2020 Apr; 70(4):2782-2858 DOI 10.1099 / ijsem.0.004107). In this specification, lactic acid bacteria are classified according to the new classification based on the above literature.

[0015] In the present invention, prebiotics specifically refer to galactooligosaccharides. Galactooligosaccharides are a general term for oligosaccharides having at least one galactose residue in the molecule, and examples include sugars in which 2 to 9, preferably 3 to 4, monosaccharides are linked together. Examples of galactooligosaccharides include those in which galactose is linked via β1-2, β1-3, β1-4, or β1-6 bonds, with galactooligosaccharides having β1-3 bonds being particularly preferred. More preferred galactooligosaccharides include 3'-galactosyllactose (3'-GL; Galβ1-3Galβ1-4Glc), 4'-galactosyllactose (4'-GL; Galβ1-4Galβ1-4Glc), and 6'-galactosyllactose (6'-GL; Galβ1-6Galβ1-4Glc). From the viewpoint of the effect on butyric acid production, galactooligosaccharides containing 3'-galactosyllactose are more preferred, galactooligosaccharides containing 3'-galactosyllactose as the main component are more preferred, and 3'-galactosyllactose is even more preferred. 3'-Galactosyllactose can be produced by known methods, for example, according to the method described in Japanese Patent No. 3871371.

[0016] The ratio of the amount of butyric acid produced when the butyric acid-producing bacteria of the present invention are co-cultured with a synbiotic to the amount of butyric acid produced when the butyric acid-producing bacteria of the present invention are cultured alone is preferably 2 to 5, more preferably 2 to 4, and even more preferably 2 to 3. The culture conditions may be any conditions suitable for culturing ordinary butyric acid-producing bacteria, as long as the conditions for the monoculture and the mixed culture are the same except for the presence or absence of a synbiotic. The inoculation amount of the butyric acid-producing bacteria of the present invention, or the butyric acid-producing bacteria of the present invention and a probiotic, in the medium is 1.0 x 10 ４ ~1.0 x 10 ８ cells / mL is preferred, and 1.0 x 10 ６ ~1.0 x 10 ８cells / mL is more preferable. The concentration of prebiotics added to the culture medium is preferably 0.1 to 2.0% (w / v), more preferably 0.5 to 1.0% (w / v), and even more preferably 1.0% (w / v). The culture is preferably anaerobic culture at 30 to 40°C for 12 to 48 hours, more preferably anaerobic culture at 37°C for 18 to 24 hours, and even more preferably anaerobic culture at 37°C for 24 hours. In one example, the culture conditions are as follows: A frozen storage solution of the strain (a solution in which the bacterial cells are suspended in 20% glycerol) (number of bacteria: 0.5 to 5.5 x 10 ９ The culture medium (1000 cells / mL) was thawed and inoculated at 1% into 4 mL of PY (peptone-yeast extract) liquid medium (PYGA medium) supplemented with 33 mM sodium acetate and 1% (w / v) glucose. After 24 hours of anaerobically culturing at 37°C, the culture medium was inoculated at 0.5% into PYGA medium and anaerobically cultured at 37°C for 18 hours. 1 mL of the culture medium was sampled and centrifuged to obtain bacterial cells, which were then suspended in 1 mL of PY liquid medium (m-PY medium) supplemented with PIPES (100 mM), a Good's buffer, to prepare a diluted culture medium with a turbidity (OD600) of 0.1. Separately, after culturing the probiotics appropriately, 1 mL of the culture medium was sampled and centrifuged to obtain bacterial cells, which were then suspended in 1 mL of m-PY medium to prepare a diluted culture medium with a turbidity (OD600) of 0.05. For monoculture, a diluted culture solution of the strain is inoculated at 1% into m-PY medium supplemented with 0.1% (w / v) glucose, and anaerobically cultured for 24 hours at 37° C. On the other hand, for mixed culture, a diluted culture solution of the strain and a diluted culture solution of the probiotic are each inoculated at 1% into m-PY medium supplemented with 0.1% (w / v) glucose and 1% (w / v) prebiotic, and anaerobically cultured for 24 hours at 37° C. Thereafter, the butyric acid concentration in the culture solution is measured for each of the monoculture and mixed culture.

[0017] The method for measuring the butyric acid concentration in the culture medium is not particularly limited as long as it allows measurement of the butyric acid concentration, and for example, it can be measured by ion exclusion high performance liquid chromatography (HPLC).

[0018] The butyric acid-producing bacteria of the present invention do not have a drug resistance gene. The absence of a drug resistance gene in a bacterium means that a gene that confers resistance to an antimicrobial agent is not detected in the genomic sequence of the bacterium. When the genomic sequence of the bacterium is compared with drug resistance genes listed in a publicly known database, if no genomic sequence with 95% or more sequence identity to the drug resistance gene is found in the genomic sequence of the bacterium, the bacterium can be determined to have no drug resistance gene. Specifically, when the genomic sequence of the bacterium is searched for the presence or absence of a drug resistance gene using ResFinder (ver. 4.1), if no genomic sequence with 95% or more sequence identity to the drug resistance gene listed in ResFinder (ver. 4.1) is found in the genomic sequence of the bacterium, the bacterium can be determined to have no drug resistance gene. Examples of such drug resistance genes include tetracycline resistance genes (tet(40), tet(O), tet(O / 32 / O), tet(M)), lincosamide resistance genes (lnu(c)), macrolide resistance genes (erm(B), vat(E), etc.), aminoglycoside resistance genes (ant(2'), aac(1)), etc.), β-lactam resistance genes (blaOXA, blaCTX, blaTEM, etc.), glycopeptide resistance genes (VanHDX, VanHAX, etc.), quinolone resistance genes (qnrB17, qnrB6, etc.), and the butyric acid-producing bacteria of the present invention do not have any of the drug resistance genes listed in ResFinder (ver. 4.1), including these genes. The butyric acid-producing bacteria of the present invention do not have a drug resistance gene, and therefore can avoid the risk of the drug resistance gene being transmitted to other bacteria, resulting in the emergence of antibiotic-resistant bacteria, and are therefore highly safe.

[0019] The butyric acid-producing bacteria of the present invention have a ratio of the amount of butyric acid produced when cultured together with synbiotics to the amount of butyric acid produced when cultured alone of 2 or more, and do not have a drug resistance gene. In addition, the ratio of the amount of butyric acid produced when cultured alone in the presence of 3'-galactosyllactose to the amount of butyric acid produced when cultured alone in the absence of 3'-galactosyllactose is 2 or more, or the ratio of the amount of butyric acid produced when cultured alone in the presence of acetate to the amount of butyric acid produced when cultured alone in the absence of 3'-galactosyllactose is 2 or more, or the ratio of the amount of butyric acid produced when cultured alone in the presence of 3'-galactosyllactose to the amount of butyric acid produced when cultured alone in the absence of 3'-galactosyllactose is 2 or more, and the ratio of the amount of butyric acid produced when cultured alone in the presence of acetate to the amount of butyric acid produced when cultured alone in the absence of acetate is 2 or more. It is preferable that the bacterium belongs to A. hadrus.

[0020] The ratio of the amount of butyric acid produced by the butyric acid-producing bacteria of the present invention when cultured alone in the presence of 3'-galactosyllactose to the amount of butyric acid produced by the butyric acid-producing bacteria of the present invention when cultured alone in the absence of 3'-galactosyllactose is preferably 2 to 5, more preferably 2 to 4, and even more preferably 2 to 3. The culture conditions may be any suitable conditions for culturing ordinary butyric acid-producing bacteria, as long as the conditions other than the presence or absence of 3'-galactosyllactose are the same for the cultures alone in the absence of 3'-galactosyllactose and in the presence of 3'-galactosyllactose. The amount of butyric acid-producing bacteria of the present invention inoculated into the medium is 1.0 x 10 ４ ~1.0 x 10 ８ cells / mL is preferred, and 1.0 x 10 ６ ~1.0 x 10 ８ cells / mL is more preferable. The concentration of 3'-galactosyllactose added to the medium is preferably 0.1 to 2.0% (w / v), more preferably 0.5 to 1.2% (w / v), and even more preferably 1.0% (w / v). In addition, the culture is preferably anaerobic culture at 30 to 40°C for 12 to 48 hours, more preferably anaerobic culture at 37°C for 18 to 24 hours, and even more preferably anaerobic culture at 37°C for 24 hours. In one example, the culture conditions include the following: A frozen storage solution of the strain (a solution in which the bacterial cells are suspended in 20% glycerol) (number of bacteria: 0.5 to 5.5 x 10９ The culture medium (1 mL, 1000 cells / mL) was thawed and inoculated at 1% into 4 mL of PYGA medium supplemented with 33 mM sodium acetate and 1% (w / v) glucose. After 24 hours of anaerobically culturing at 37°C, the culture medium was inoculated at 0.5% into PYGA medium and cultured anaerobically at 37°C for 18 hours. 1 mL of the culture medium was sampled and centrifuged to obtain bacterial cells, which were suspended in 1 mL of m-PY medium to prepare a diluted culture medium so that the turbidity measured at OD600 was 0.1. The diluted culture medium was inoculated at 1% into m-PY medium supplemented with 0.1% (w / v) glucose (corresponding to the absence of 3'-galactosyllactose) and m-PY medium supplemented with 0.1% (w / v) glucose and 1% (w / v) 3'-galactosyllactose (corresponding to the presence of 3'-galactosyllactose), and cultured anaerobically at 37°C for 24 hours. Thereafter, the butyric acid concentration in the culture medium can be measured in both the absence and presence of 3'-galactosyllactose.

[0021] The ratio of the amount of butyric acid produced by the butyric acid-producing bacteria of the present invention when cultured alone in the presence of acetate to the amount of butyric acid produced by the butyric acid-producing bacteria of the present invention when cultured alone in the absence of acetate is preferably 2 to 5, more preferably 2 to 4, and even more preferably 2 to 3. Acetate salts are not particularly limited, but include sodium acetate, potassium acetate, calcium acetate, etc., with sodium acetate being preferred. The culture conditions may be any conditions suitable for culturing ordinary butyric acid-producing bacteria, as long as the conditions other than the presence or absence of acetate are the same for the culture in the absence of acetate and the culture in the presence of acetate. The inoculation amount of the butyric acid-producing bacteria of the present invention to the medium is 1.0 x 10 ４ ~1.0 x 10 ８ cells / mL is preferred, and 1.0 x 10 ６ ~1.0 x 10 ８The concentration of acetate added to the medium is preferably 5 to 50 mM, more preferably 10 to 30 mM, and even more preferably 20 mM. The culture is preferably anaerobic culture at 30 to 40°C for 12 to 48 hours, more preferably anaerobic culture at 37°C for 18 to 24 hours, and even more preferably anaerobic culture at 37°C for 24 hours. In one example, the culture conditions include the following: A frozen storage solution of the strain (a solution in which the bacterial cells are suspended in 20% glycerol) (number of bacteria: 0.5 to 5.5 x 10 ９ The culture (1000 cells / mL) was thawed and inoculated at 1% into 4 mL of PYGA medium supplemented with 33 mM sodium acetate and 1% (w / v) glucose. After 24 hours of anaerobically culturing at 37°C, the culture was inoculated at 0.5% into PYGA medium and cultured anaerobically at 37°C for 18 hours. 1 mL of the culture was sampled and centrifuged, and the resulting bacterial cells were suspended in 1 mL of m-PY medium to prepare a diluted culture solution so that the turbidity measured at OD600 was 0.1. The diluted culture solution was inoculated at 1% into m-PY medium supplemented with 0.1% (w / v) glucose (corresponding to the absence of acetate) and m-PY medium supplemented with 0.1% (w / v) glucose and 20 mM sodium acetate (corresponding to the presence of acetate), and cultured anaerobically at 37°C for 24 hours. The butyric acid concentration in the culture solution was then measured in both the absence and presence of acetate.

[0022] A specific example of the butyric acid-producing bacterium of the present invention is A. hadrus YIT 13235 (NITE BP-04056).

[0023] The butyric acid-producing bacteria of the present invention can be isolated from the library, for example, by inoculating a portion of the library, microorganisms collected from nature, etc., into a liquid medium using a sugar source as a selective factor, culturing the culture, and then inoculating and culturing the culture on plates containing and not containing the sugar source. Colonies grown on the sugar source-containing plates that have morphologies different from those grown on the sugar source-free plates are examined for their ability to produce butyric acid and for the presence or absence of a drug resistance gene. More specifically, as shown in the Examples below, a dilution of the library or microorganisms collected from nature, etc., is inoculated into a liquid medium supplemented with L-sorbose or D-xylitol and anaerobically cultured. The culture is then inoculated onto plates containing and not containing L-sorbose or D-xylitol, and anaerobically cultured. Among colonies grown on plates supplemented with L-sorbose or D-xylitol, colonies with morphologies different from those grown on plates not supplemented with L-sorbose or D-xylitol are confirmed to be A. hadrus by colony PCR targeting the 16S rRNA gene. For colonies confirmed to be A. hadrus, the amount of butyric acid produced in monoculture and in coculture with synbiotics is measured. Furthermore, the genomic DNA of colonies confirmed to be A. hadrus is sequenced, and the obtained genomic DNA sequence is searched for drug resistance genes using publicly available databases. From these results, colonies in which the ratio of butyric acid production in coculture with synbiotics to that in monoculture is 2 or greater and in which drug resistance genes are not detected are selected.

[0024] As shown in the Examples below, the butyric acid-producing bacteria of the present invention are classified as A. hadrus, and when co-cultured with synbiotics, butyric acid production is enhanced compared to when cultured alone. Furthermore, since the butyric acid-producing bacteria of the present invention do not have a drug resistance gene, the risk of transmitting the drug resistance gene to other bacteria is extremely low. In the above respects, A. hadrus YIT 13235 discovered in the Examples below is similar to A. hadrus DSM 3319, the type strain of A. hadrus. TThis is a novel strain that differs from A. hadrus YIT 12355 (NITE BP-01832) disclosed in Patent Document 2. A. hadrus YIT 13235 was deposited on January 10, 2024, at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) under the accession number NITE BP-04056.

[0025] The butyric acid-producing bacteria of the present invention have no safety issues and have excellent butyric acid production ability, particularly excellent butyric acid production ability in the presence of synbiotics. Therefore, compositions containing the butyric acid-producing bacteria of the present invention are useful as food and beverage compositions, pharmaceutical compositions, or feed compositions. From the viewpoint of enhancing butyric acid production, the composition preferably further contains at least one selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts. Specific examples of synbiotics, galactooligosaccharides, and acetate salts are as described above. The composition produces butyric acid in the intestines of animals, including humans, and is therefore useful as a butyric acid production enhancer. As described above, butyric acid is not only utilized as an energy source for colonic mucosal epithelial cells, but also has the effects of promoting epithelial cell proliferation, stimulating intestinal motility, promoting the differentiation and induction of regulatory T cells, and suppressing inflammation. It also has preventive and therapeutic effects against colon cancer and ulcerative colitis, improving brain function, alleviating stress, suppressing microglial inflammation, and improving irritable bowel syndrome. Therefore, the composition of the present invention is particularly useful as a pharmaceutical, food and beverage, or feed having these physiological activities.

[0026] The content of the butyric acid-producing bacteria of the present invention in the composition of the present invention is not particularly limited. However, the content of the butyric acid-producing bacteria of the present invention in terms of viable cells per 100 g of the composition of the present invention is preferably 10. ４ ~10 １４ The content of the synbiotics in the composition of the present invention is not particularly limited, but it is preferable that the composition of the present invention contains 10 cfu of live probiotics per 100 g of the composition. ４ ~10 １４cfu, preferably 0.01-90% (w / v) of prebiotics, more preferably 0.05-50% (w / v), preferably 0.01-90% (w / v), more preferably 0.05-50% (w / v), preferably 0.01-90% (w / v), more preferably 0.05-50% (w / v), preferably 5-50 mM, more preferably 10-30 mM of acetate.

[0027] The composition of the present invention can be formulated into a form suitable for each of foods, beverages, medicines, and feeds. When used as a medicine, it can be mixed with a solid or liquid non-toxic pharmaceutical carrier to form a conventional pharmaceutical formulation. Examples of such formulations include solid preparations such as tablets, granules, powders, and capsules; liquid preparations such as solutions, suspensions, and emulsions; and lyophilized preparations. These preparations can be prepared by conventional pharmaceutical methods. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and physiological saline. Conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonicity agents, and excipients can also be added as needed.

[0028] Furthermore, when used as a food or drink, it can be in any form, such as solid or liquid. When used as a food or drink, it may be contained as is or together with various nutritional components. Specifically, additives that can be used as a food or drink may be appropriately used, and the product may be formed into an edible form, i.e., granules, particles, tablets, capsules, paste, etc., using conventional means. Examples of types of food or drink include processed meat foods such as ham and sausage, processed seafood foods such as kamaboko and chikuwa, foods such as bread, confectionery, butter, and powdered milk, beverages such as water, fruit juice, milk, soft drinks, and tea drinks, and supplements. The same applies when used as feed.

[0029] When the composition of the present invention is administered to animals including humans, it is preferably administered orally in order to produce butyric acid in the intestines. The dosage may be adjusted appropriately taking into consideration the condition, weight, sex, age, etc. of the subject. For example, the dosage may be adjusted to 1.0 x 10 viable cell count of the butyric acid-producing bacteria of the present invention per day for an adult human. ４ cfu or more, and more preferably 1.0 x 10 ８ ~1.0 x 10 １２ cfu is more preferred.

[0030] The present invention will be described in more detail by the following examples, but the scope of the present invention is not limited thereto.

[0031] Example 1 (Isolation of Anaerostipes hadrus) (1) Isolation of bacteria using selective medium Three types of samples were selected from the sample library held by the applicant, and 300 mg of each sample was transferred to a small test tube. Anaerobic transport medium was added to the sample to prepare a 10-fold diluted solution. 100% CO was used to inject the solution. ２ Under gas injection, anaerobic dilution solution was used for 10 ３－６ The mixture was diluted 2x and inoculated onto a PY agar plate supplemented with 1% (w / v) glucose, 33 mM sodium acetate, 2 μg / mL levofloxacin, and 4 μg / mL azithromycin. The mixture was then stored in a glove box (gas phase: N ２ :CO ２ :O ２The culture medium was cultured anaerobically at 37°C for 48 hours in a PY agar medium (pH 7.5, pH 7.5, pH 7.5, pH 7.5) containing 1% (w / v) glucose and 33 mM sodium acetate. The culture medium composition is shown in Table 1. Characteristic colonies that grew were picked and subcultured on PY (PYGA) agar plates supplemented with 1% (w / v) glucose and 33 mM sodium acetate, followed by anaerobically culturing at 37°C for 48 hours. After repeating the same procedure once more, the grown colonies were identified by colony PCR to determine whether they were A. hadrus. A portion of the grown colonies was suspended in 50 μL of TE buffer and heated at 95°C for 10 minutes to extract DNA, which was used as a template DNA solution. A 7500 Real-Time PCR System (Thermo Fisher Scientific) was used for PCR. The total volume of the reaction solution was 20 μL, and TB Green® Premix EX Taq® II (Takara Bio Inc.) containing 0.4 μM of each primer was mixed with 2 μL of template DNA solution, followed by PCR. The reaction conditions were heating at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, 55°C for 30 seconds, and 72°C for 34 seconds. After 1 minute of reaction at 60°C, the temperature was increased to 95°C at a temperature gradient of 0.2°C / sec, and the fluorescence of SYBR Green I at this time was measured to determine the temperature (Tm) at which the double strand of the amplified product dissociated. The sequences of the A. hadrus -specific primers used in quantitative PCR are shown in Table 2. The positive control was A. hadrus YIT 10092, a type strain of A. hadrus. T (DSM3319 T DNA extracted from the cells of YIT 13225 was used as a negative control, and water was used as a negative control. Colonies that showed significant amplification compared to the negative control and had the same Tm value as the positive control were selected. As a result, three isolated strains (YIT 13225, YIT 13228, and YIT 13229) were selected.

[0032]

[0033]

[0034] (2) Isolation of bacteria using a sugar source as a selective factor Using the same sample as above, bacteria were isolated according to the same method as in Patent Document 2. That is, a portion of the sample dilution was inoculated at 0.5% into 0.5% (w / v) L-sorbose-supplemented PY (PYS) and 0.5% (w / v) D-xylitol-supplemented PY (PYX) liquid media, and anaerobically cultured at 37°C for 24 hours in a glove box. After the culture, the culture solution was diluted with the anaerobic dilution solution for 10 min. ６－７ The solution was diluted 2:1 and inoculated onto PY, PYS, and PYX agar plates and cultured anaerobically at 37°C for 72 hours. Among the colonies grown on the PYS and PYX plates, some colonies with different morphologies compared to those on the PY plates were picked, and strains determined to be A. hadrus were selected by colony PCR using A. hadrus -specific primers in the same manner as described above. As a result, two isolates (YIT 13233 and YIT 13235) were selected.

[0035] (3) Phylogenetic analysis of isolates based on 16S rRNA gene sequences. DNA was extracted from the culture medium of each isolate using the FastDNA™ SPIN Kit (MP Biomedicals). The full length of the 16S rDNA was amplified by PCR, and the entire sequence was sequenced. The obtained sequences were subjected to a FASTA search of the DNA Data Bank of Japan (DDBJ) and compared with the sequence database of known bacterial species.

[0036] (4) Strain identification by RAPD method Isolates and existing strain YIT 10092 T DNA was extracted from the culture medium of each of the strains, YIT 12355 (Patent Document 2), using a FastDNA SPIN Kit. Random Amplified Polymorphic DNA (RAPD) was performed using the extracted DNA as a template, and the strains were identified based on differences in band patterns. Primers A and B shown in Table 3 were used.

[0037]

[0038] (5) Results The results are shown in Table 4 and Figure 1. Table 4 shows that the isolated strains YIT 13225, YIT 13228, YIT 13229, YIT 13233, and YIT 13235 all showed significant amplification by colony PCR. Furthermore, the 16S rRNA gene sequence (approximately 1,450 bp) was identical to that of A. hadrus YIT 10092, the type strain of A. hadrus. T All five strains showed a homology of 99.5% or more with the sequence of YIT 10092. From these results, it was found that all five strains are classified as A. hadrus. T , YIT 12355 all showed different band patterns. These results indicated that the above five strains and the two existing strains were all different strains. All five strains were Gram-positive rods. From these results, YIT 13225, YIT 13228, YIT 13229, YIT 13233, and YIT 13235 were all determined to be novel strains.

[0039]

[0040] Example 2 (Assimilation of Galactooligosaccharides by A. hadrus) (1) Galactooligosaccharides Galactooligosaccharides (GOS) and its major components, 4'-galactosyllactose and 3'-galactosyllactose, were used as galactooligosaccharides. GOS was prepared by removing monosaccharide and lactose fractions from commercially available Oligomate 55N (Yakult Pharmaceutical Industry) using an activated carbon column. Specifically, a sugar solution diluted with purified water was added to a column packed with activated carbon (Fujifilm Wako Pure Chemical Industries) swollen with purified water. The column was washed with 2% ethanol, and the indigestible fraction was eluted with 50% ethanol. The eluate was then dried under reduced pressure to obtain the GOS sugar solution. This GOS sugar solution contained 3'-galactosyllactose in addition to 4'-galactosyllactose. 4'-Galactosyllactose prepared according to the method described in International Publication No. 2015 / 166903 was used. 3'-Galactosyllactose prepared according to the method described in Japanese Patent No. 3871371 was used.

[0041] (2) Strain used: A. hadrus YIT 10092, the existing type strain of A. hadrus T (DSM 3319 T ), the existing YIT 12355, and five isolated strains were used.

[0042] (3) Evaluation of butyric acid production ability The above strains were frozen and stored (a solution in which the cells were suspended in 20% glycerol) (number of cells: 0.5 to 5 × 10 ９ The culture medium (100 mM PIPES) was thawed, and a portion of the culture was inoculated into 4 mL of PYGA liquid medium at 1%. The culture was then anaerobically cultured at 37°C for 24 hours. After incubation, a portion of the culture was inoculated into fresh PYGA liquid medium at 0.5% and cultured at 37°C for 18 hours. One mL of each cultured bacterial solution was sampled and centrifuged (4°C, 10,000 × g, 10 minutes). The supernatant was removed, and 1 mL of PY (m-PY) medium supplemented with 100 mM PIPES was added to suspend the pellet. The suspension was then diluted with m-PY medium to a turbidity (OD600) of 0.1. A portion of this bacterial solution was inoculated into m-PY medium supplemented with 0.1% (w / v) glucose or m-PY liquid medium supplemented with 0.1% (w / v) glucose and 1% (w / v) galactooligosaccharides at 1%. The culture was then anaerobically cultured at 37°C for 24 hours. All of the above operations were carried out in a glove box. After the cultivation was completed, the organic acid concentration in the culture medium was analyzed by ion exclusion HPLC.

[0043] (HPLC analysis conditions) Eluent: 15 mM perchloric acid - 7% acetonitrile pH adjuster: 15 mM perchloric acid - 60 mM trishydroxymethylaminomethane - 7% acetonitrile Separation column: Organic acid analysis column RSpak KC-811 x 2 (Showa Denko KK) Column temperature: 42°C Injected sample amount: 10 μL Flow rate: 1.0 mL / min Analysis time: 35 minutes Detector: Waters 432 electrical conductivity detector Cell temperature: 45°C

[0044] (4) Results The results are shown in Figure 2. The ratio of butyric acid production in the presence of galactooligosaccharides to that in the absence of galactooligosaccharides is shown in Table 5. When GOS was added, an increase in butyric acid production was observed in all strains, but the increase did not differ significantly between strains. When 4'-galactosyllactose, the main component of GOS, was added, no increase in butyric acid production was observed. However, when 3'-galactosyllactose was added, it was found that in all strains, the increase was equal to or greater than that observed when GOS was added. These results suggest that A. hadrus strains produce butyric acid using galactooligosaccharides and that they primarily utilize 3'-galactosyllactose among galactooligosaccharides. Furthermore, the amount of butyric acid produced by YIT 13235 when 3'-galactosyllactose was added was significantly higher than that of YIT 10092. T It was the second highest.

[0045]

[0046] Example 3 (Butyric acid production in the presence of acetate or lactate) (1) Evaluation of butyric acid production ability A portion of the bacterial solution prepared under the same conditions as those described in Example 2 was inoculated at 1% into m-PY medium supplemented with 0.1% (w / v) glucose, or into a medium containing 0.1% (w / v) glucose and 20 mM sodium acetate, sodium L-lactate, or sodium D-lactate, and anaerobically cultured at 37°C for 24 hours in a glove box. After the culture was completed, the organic acid concentration in the culture solution was analyzed by ion exclusion HPLC. The analysis was performed under the same conditions as in Example 2.

[0047] (2) Results The results are shown in Figure 3. Table 6 shows the ratio of the amount of butyric acid produced in the presence of acetic acid, L-lactic acid, or D-lactic acid to the amount of butyric acid produced in the absence of acetic acid, L-lactic acid, or D-lactic acid. YIT 10092 T However, all five strains isolated in this study, like YIT 12355, showed an increase in butyrate production when acetic acid was added, and no strains showed a significant increase in butyrate production when D-lactic acid was added. Furthermore, no significant difference in butyrate production was observed between YIT 12355 and the five strains when acetic acid was added.

[0048]

[0049] Example 4 (Butyric acid production by combining A. hadrus and synbiotics) (1) Synbiotics Lacticaseibacillus paracasei strain Shirota YIT 9029 (LcS) and Bifidobacterium breve strain Yakult YIT 12272 (BbrY) were used as probiotics contained in the synbiotics. GOS was used as prebiotics.

[0050] (2) Preparation of inoculation solution of LcS and BbrY. LcS and BbrY bead stocks stored in Microbank (trademark) (Pro-Lab Diagnostics) were added to 10 mL of MRS liquid medium or GAM liquid medium supplemented with 1% (w / v) glucose using a gas injector under 100% CO ２ After inoculation under gas injection and sealing with a butyl rubber stopper, the cells were cultured at 37°C for 18 hours. 1 mL of each cultured bacterial solution was sampled and centrifuged (4°C, 10,000 x g, 10 minutes). The supernatant was removed, and the pellet was suspended in 1 mL of m-PY medium and diluted to a turbidity (OD600) of 0.05.

[0051] (3) Mixed Culture m-PY medium was prepared by adding 0.1% (w / v) glucose or 0.1% (w / v) glucose and 0.1% (w / v) GOS. The 0.1% (w / v) glucose-supplemented medium contained a bacterial suspension of each A. hadrus strain prepared in the same manner as in Example 2. The 0.1% (w / v) glucose and 1% (w / v) GOS-supplemented medium contained a bacterial suspension of each A. hadrus strain plus 1% (v / v) inoculation of each LcS or BbY inoculum solution. The medium was then anaerobically cultured at 37°C for 24 hours in a glove box. The organic acid concentration in the culture medium after the culture was completed was analyzed by ion-exclusion HPLC. The analysis conditions were the same as those in Example 2.

[0052] (4) Results The results are shown in Figure 4. The ratio of butyric acid production during mixed culture with synbiotics to the amount of butyric acid produced during monoculture is shown in Table 7. Strain differences were observed in the amount of butyric acid produced during mixed culture with synbiotics, and YIT 12355, YIT 13225, YIT 13228, and YIT 13235 showed a significant increase in butyric acid production during mixed culture with synbiotics. Because A. hadrus utilizes GOS and acetic acid that can be used by A. hadrus is produced by synbiotics, it is thought that butyric acid production is greatly enhanced in some A. hadrus strains by utilizing these.

[0053]

[0054] Example 5 (Search for Drug Resistance Genes) (1) Genome Sequencing Two milliliters of the culture medium from the above strain was sampled and centrifuged (10,000 × g, 10 minutes) to remove the supernatant. DNA was extracted from the resulting pellet using a FastDNA SPIN Kit (MP Biomedicals), and the concentration was quantified using a Quant-iT PicoGreen dsDNA Kit (Invitrogen). The DNA concentration was adjusted to 60 ng / μL, and then fragmented to 800 bp using a Covaris M220 (M&S Instruments). A library was then prepared using a TruSeq DNA PCR-Free Library Prep Kit (Illumina). The resulting library was subjected to paired-end sequencing at 2 × 250 bp using Miseq (Illumina) with Miseq reagent kit v2 (500 cycles).

[0055] (2) Construction of the draft genome After quality trimming of the obtained reads using fastp (ver. 0.23.4), assembly was performed using Unicycler (ver. 0.5.0). Of the obtained contigs, those of 500 bp or less were removed, and alignment was performed by homology search against the complete genome of A. hadrus BPB5 strain using progressiveMauve (ver. 2015-02-13). Furthermore, structural and functional annotation was performed using Prokka (ver. 1.14.5).

[0056] (3) Searching for drug resistance genes using a database Using the obtained genome sequence, antibiotic resistance genes of each A. hadrus strain were detected using ResFinder (ver. 4.1). If a base sequence with 95% or more sequence identity to a drug resistance gene listed in ResFinder was found in the genome sequence, it was determined that the strain contained a drug resistance gene. ResFinder lists 2,774 types of drug resistance genes.

[0057] (4) Results The results are shown in Table 8. Tetracycline resistance genes tet(40) (GenBank Accession No. AM419571), tet(O) (GenBank Accession No. M18896), and tet(M) (GenBank Accession No. FR671418) were detected in YIT 12355. tet(40) (GenBank Accession No. AM419571), tet(O / 32 / O) (GenBank Accession No. AIOQ01000025), and the lincosamide resistance gene lnu(C) (GenBank Accession No. AY928180) were detected in YIT 13225. tet(O) (GenBank Accession No. Y07780) was detected in YIT 13228 and YIT 13233. The strains in which none of the drug resistance genes searched for in ResFinder were detected were YIT 13229 and YIT 13235, and the risk of transmission of drug resistance genes was determined to be extremely low. Furthermore, no correlation was found between the presence or absence of drug resistance genes and increased butyrate production in mixed culture with synbiotics. YIT 13235 was the only strain in which drug resistance genes were not detected and butyrate production was enhanced by synbiotics.

[0058]

Claims

1. A butyric acid-producing bacterium belonging to Anaerostipes hadrus, in which the ratio of butyric acid production when co-cultured with a synbiotic to the amount of butyric acid produced when cultured alone is 2 or more, the bacterium does not have a drug resistance gene, and the synbiotic contains Lacticaseibacillus paracasei YIT 9029 (FERM BP-1366), Bifidobacterium breve YIT 12272 (FERM BP-11320), and galactooligosaccharides.

2. The butyric acid-producing bacterium according to claim 1, wherein the ratio of the amount of butyric acid produced when cultured alone in the presence of 3'-galactosyllactose to the amount of butyric acid produced when cultured alone in the absence of 3'-galactosyllactose is 2 or more.

3. The butyric acid-producing bacterium according to claim 1, which is Anaerostipes hadrus YIT 13235 (NITE BP-04056).

4. A food or drink composition, pharmaceutical composition, or feed composition comprising the butyric acid-producing bacterium according to any one of claims 1 to 3 and at least one selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts, wherein the synbiotics include Lacticaseibacillus paracasei YIT 9029 (FERM BP-1366), Bifidobacterium breve YIT 12272 (FERM BP-11320), and galactooligosaccharides.

5. A butyric acid production enhancer comprising the butyric acid-producing bacterium according to any one of claims 1 to 3 and at least one selected from the group consisting of synbiotics, galactooligosaccharides and acetate salts.

6. Use of the butyric acid-producing bacterium according to any one of claims 1 to 3 and at least one species selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts for the production of a butyric acid production enhancer.

7. A butyric acid-producing bacterium according to any one of claims 1 to 3, and at least one species selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts, for use in enhancing butyric acid production.

8. A method for enhancing butyric acid production, comprising administering to a subject in need thereof the butyric acid-producing bacterium according to any one of claims 1 to 3 and at least one species selected from the group consisting of synbiotics, galactooligosaccharides, and acetate salts.