Composition for intestinal regulation containing lactic acid–producing bacteria and butyrate-producing bacteria

Abstract

The purpose of the present invention is to provide a novel means capable of increasing the butyrate concentration in the intestines. The present invention provides a composition containing lactic acid–producing bacteria and butyrate-producing bacteria of the Anaerostipes genus.

Description

Intestinal regulation composition containing lactic acid-producing bacteria and butyric acid-producing bacteria

[0001] The present invention relates to a composition comprising lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Anaerostipes.

[0002] Butyric acid is a type of short-chain fatty acid that plays an important role in regulating intestinal function in humans and animals, including strengthening intestinal barrier function and providing anti-inflammatory effects. For this reason, attempts have been made to administer or ingest butyric acid for the purpose of intestinal regulation; however, butyric acid has a very strong, unpleasant odor, making it difficult to administer or ingest itself. Meanwhile, butyric acid-producing bacteria exist in the intestines of humans and animals, and these bacteria can produce butyric acid in the intestines. For this reason, efforts have been made to increase the butyric acid concentration in the intestines using butyric acid-producing bacteria. For example, Patent Document 1 discloses an agent for increasing intestinal butyric acid-producing bacteria that uses spore-forming bacteria (Bacillus subtilis) to increase butyric acid bacteria (Feacalibacterium prausnitzii). Patent Document 2 also discloses a food composition containing D-mannitol or D-sorbitol as an active ingredient and having the effect of increasing intestinal butyric acid concentration, and describes its administration or ingestion to humans or animals. Furthermore, Non-Patent Document 1 discloses that lactic acid-producing bacteria (Bacillus coagulans SANK70258) increased bacteria related to the Lachnospiraceae family in a microbiota model of a healthy individual, thereby promoting butyric acid production.

[0003] However, efforts to increase the intestinal butyric acid concentration have not yet been sufficient, and there is a strong demand in this field for new methods that can increase the intestinal butyric acid concentration.

[0004] Anaerostipes caccae, a butyric acid-producing bacterium of the genus Anaerostipes, was isolated from human feces as a bacterium capable of producing butyric acid by utilizing lactic acid (Non-Patent Document 2).

[0005] JP 2013-147469 A JP 2004-49093 A

[0006] K. Sasaki et al., Appl Microbiol Biotechnol. 2020 May; 104(9):3859-3867. S. H. Duncan et al., Appl Environ Microbiol. 2004 Oct;70(10):5810-7.

[0007] An object of the present invention is to provide a new means by which the concentration of butyric acid in the intestine can be increased.

[0008] As a result of intensive research to solve the above problems, the inventors have discovered that the amount of butyric acid produced by Anaerostipes butyric acid-producing bacteria in the intestines can be increased by administering or ingesting a combination of lactic acid-producing bacteria and Anaerostipes butyric acid-producing bacteria.

[0009] The present invention is based on these novel findings and includes the following inventions: [1] A composition comprising lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Anaerostipes. [2] The composition of [1], wherein the butyric acid-producing bacteria is Anaerostipes caccae. [3] The composition of [1] or [2], wherein the lactic acid-producing bacteria is a spore-forming bacterium. [4] Any of the compositions of [1] to [3], wherein the lactic acid-producing bacteria is Weizmannia coagulans. [5] Any of the compositions of [1] to [4], which are oral compositions. [6] Any of the compositions of [1] to [5], which are foods, beverages, supplements, feed, feed additives, or pharmaceuticals. [7] Any of the compositions of [1] to [6], which are used to increase butyric acid concentration in the intestines. [8] Any of the compositions of [1] to [7], which are used for intestinal regulation. [9] Any of the compositions of [1] to [7], used for immunomodulation.

[10] Any of the compositions of [1] to [7], used for allergy relief. [11a] A method for increasing butyric acid concentration in the intestines of a subject, comprising administering lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Anaerostipes to the subject. [11b] The method of [11a], wherein the butyric acid-producing bacteria is Anaerostipes caccae. [11c] The method of [11a] or [11b], wherein the lactic acid-producing bacteria is spore-forming bacteria. [11d] Any of the methods of [11a] to [11c], wherein the lactic acid-producing bacteria is Weizmannia coagulans. [11e] Any of the methods of [11a] to [11d], wherein the administration is oral administration. [11f] Any of the methods [11a] to [11e], wherein the subject is a non-human animal. [11g] Any of the methods [11a] to [11e], wherein the subject is a human. [11h] Any of the methods [11a] to [11g], wherein the subject is a method for intestinal regulation. [11i] Any of the methods [11a] to [11g], wherein the subject is a method for immunomodulation. [11j] Any of the methods [11a] to [11g], wherein the subject is a method for alleviating allergies. [12a] A method for increasing butyric acid concentration in the intestines of a subject, comprising having a subject ingest lactic acid-producing bacteria and Anaerostipes butyric acid-producing bacteria.[12b] The method of [12a], wherein the butyric acid-producing bacterium is Anaerostipes caccae. [12c] The method of [12a] or [12b], wherein the lactic acid-producing bacterium is a spore-forming bacterium. [12d] Any of the methods of [12a] to [12c], wherein the lactic acid-producing bacterium is Weizmannia coagulans. [12e] Any of the methods of [12a] to [12d], wherein the ingestion is oral administration. [12f] Any of the methods of [12a] to [12e], wherein the subject is a non-human animal. [12g] Any of the methods of [12a] to [12e], wherein the subject is a human. [12h] Any of the methods of [12a] to [12g], wherein the method is a method for intestinal regulation. [12i] Any of the methods of [12a] to [12g], wherein the method is a method for immunomodulation. [12j] Any of the methods [12a] to [12g], which is a method for improving allergies. [13a] Use of lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Anaerostipes in the manufacture of a composition for increasing butyric acid concentration in the intestine. [13b] Use of [13a], wherein the butyric acid-producing bacteria is Anaerostipes caccae. [13c] Use of [13a] or [13b], wherein the lactic acid-producing bacteria is a spore-forming bacterium. [13d] Use of [13a] to [13c], wherein the lactic acid-producing bacteria is Weizmannia coagulans. [13e] Any of the use of [13a] to [13d], wherein the composition is an oral preparation. [13f] Any of the use of [13a] to [13e], wherein the composition is an intestinal regulator. [13g] Use of any of [13a] to [13e], wherein the composition is an immunomodulator. [13h] Use of any of [13a] to [13e], wherein the composition is an allergy-relieving agent. This specification includes the contents of the specification, etc. of Japanese Patent Application No. 2024-028581, filed February 28, 2024, which is the priority basis of this application. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

[0010] According to the present invention, a new means for increasing the butyric acid concentration in the intestine can be provided.

[0011] 1 is a graph showing the results of analyzing the butyric acid concentration in the medium after co-administration of lactic acid-producing bacteria (Weizmannia coagulans) and butyric acid-producing bacteria of the genus Anaerostipes (Anaerostipes caccae) or butyric acid-producing bacteria (Clostridium butyricum) to an in vitro fecal culture system using feces from a patient with ulcerative colitis and culturing. As controls, a medium containing no bacteria (Control) or one of the bacteria alone was used. 2 is a graph showing the results of analyzing the occupancy rate of lactic acid-producing bacteria in the bacterial flora after co-administration and cultivation of lactic acid-producing bacteria (Weizmannia coagulans) and butyric acid-producing bacteria of the genus Anaerostipes (Anaerostipes caccae) or butyric acid-producing bacteria (Clostridium butyricum) in an in vitro fecal culture system using feces from a patient with ulcerative colitis. As controls, a system in which neither bacterium was added (Control) or either bacterium was added alone was used. 3 is a graph showing the results of analyzing the occupancy rate of Bifidobacterium bacteria in the bacterial flora after co-administration and cultivation of lactic acid-producing bacteria (Weizmannia coagulans) and butyric acid-producing bacteria of the genus Anaerostipes (Anaerostipes caccae) or butyric acid-producing bacteria (Clostridium butyricum) in an in vitro fecal culture system using feces from a patient with ulcerative colitis. As controls, a system in which neither bacterium was added (Control) or either bacterium was added alone was used.

[0012] The present invention relates to a composition comprising lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Anaerostipes.

[0013] In the present invention, "lactic acid-producing bacteria" refers to intestinal bacteria capable of assimilating sugars to produce lactic acid. Lactic acid-producing bacteria that can be used in the present invention are not particularly limited as long as they are capable of producing lactic acid in the intestine, but are preferably spore-forming bacteria (sometimes also referred to as "spore-forming bacteria"). Lactic acid-producing bacteria that are spore-forming bacteria form spores (sometimes also referred to as "spores"), and are highly resistant to dryness, heat, and acid. When orally administered, they reach the intestine without being killed by gastric acid or bile acid, germinate, become vegetative cells, and further grow to produce lactic acid, making them preferable.

[0014] More preferred examples of lactic acid-producing bacteria that can be used in the present invention include, but are not limited to, those belonging to the genus Weizmannia (Bacillus), Lactobacillus, Bifidobacterium, Enterococcus, etc. Examples of bacteria belonging to the genus Weizmannia (Bacillus) include, but are not limited to, Weizmannia coagulans (sometimes referred to as "Bacillus coagulans"), Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, etc. Examples of bacteria belonging to the genus Lactobacillus include, but are not limited to, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus gaseli, and Lactobacillus plantrum. Examples of bacteria belonging to the genus Bifidobacterium include, but are not limited to, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, etc. Examples of bacteria belonging to the genus Enterococcus include, but are not limited to, Enterococcus faecalis, Enterococcus faecium, etc.

[0015] A particularly preferred lactic acid-producing bacterium that can be used in the present invention is Weizmannia coagulans. Examples of Weizmannia coagulans include Weizmannia coagulans strains SANK70258, P-22, lilac-01, SIM-7 DSM14043, C101, NBRC12583, GBI-1, GBI-20, GBI-30, and GBI-40. Among these, Weizmannia coagulans strain SANK70258 is preferred in terms of stable supply and ease of availability. Furthermore, mutants derived from these strains may be used as long as they have the effect of promoting butyric acid production in butyric acid-producing bacteria of the genus Anaerostipes. Furthermore, Weizmannia coagulans isolated from animal (preferably human) feces or commercially available products can be used. Commercially available Weizmannia coagulans products include, for example, "Lacris (registered trademark)-S," "Lacris (registered trademark)-S Granules," "Lacris (registered trademark)-15," and "Feed Lacris (registered trademark)-10" from Mitsubishi Chemical Corporation, as well as products from Kerry Inc., SABINSA, Aterio Bio, UNIQUEBIOTECH, Asahi Biocycle, and the like.

[0016] In the present invention, the "lactic acid-producing bacteria" may be in the form of spores, vegetative cells, mixtures thereof, cultures containing them, culture solutions, or extracts thereof. Furthermore, the "lactic acid-producing bacteria" may be in any form, such as powder, granules, or liquid.

[0017] In the present invention, "butyric acid-producing bacteria" refers to intestinal bacteria capable of assimilating lactic acid to produce butyric acid. The butyric acid-producing bacteria that can be used in the present invention are not particularly limited as long as they are capable of producing butyric acid in the intestine, but preferably include those belonging to the genus Anaerostipes. Butyric acid-producing bacteria belonging to the genus Anaerostipes are preferred because they can improve butyric acid production in combination with the above-mentioned lactic acid-producing bacteria and do not inhibit the growth of other intestinal bacteria (e.g., the above-mentioned lactic acid-producing bacteria and bifidobacteria). Furthermore, butyric acid-producing bacteria belonging to the genus Anaerostipes are spore-forming bacteria that are highly resistant to dryness, heat, and acid. When orally administered, they reach the intestine, germinate, become vegetative cells, and further grow to produce butyric acid without being killed by gastric acid or bile acid, making them preferred.

[0018] In the present invention, examples of butyric acid-producing bacteria belonging to the genus Anaerostipes include Anaerostipes caccae, Anaerostipes hadrus, Anaerostipes butyraticus, Anaerostipes hominis, Anaerostipes rhamnosivorans, Anaerostipes amylophilus, and the like, but are not limited to these.

[0019] A particularly preferred butyric acid-producing bacterium that can be used in the present invention is Anaerostipes caccae. Examples of Anaerostipes caccae include DSM14662 strain, L1-92 strain, P127-A10a strain, 3_2_56FAA strain, and mgYG-HGUT-00080 strain. Mutant strains derived from these strains may also be used, as long as they have the effect of improving butyric acid production in combination with the above-mentioned lactic acid-producing bacteria. Anaerostipes caccae may be commercially available or may be isolated from animal (preferably human) feces.

[0020] In the present invention, the butyric acid-producing bacteria can be used in the form of spores, vegetative cells, mixtures thereof, cultures containing them, culture solutions, and extracts thereof. In addition, the butyric acid-producing bacteria can be used in any form, such as powder, granules, or liquid.

[0021] The content of lactic acid-producing bacteria in the composition of the present invention is preferably as high as possible from the viewpoint of improving butyric acid production by the butyric acid-producing bacteria. For example, ３ cfu / g or more, preferably 1 x 10 ４ cfu / g or more, more preferably 1 x 10 ５ cfu / g or more, more preferably 1 x 10 ６ cfu / g or more, particularly preferably 1 x 10 ７ cfu / g or more, and the upper limit is not particularly limited, but for example, 1 × 10 １０ cfu / g or less, preferably 1 x 10 ９ cfu / g or less, more preferably 1 x 10 ８ cfu / g or less.

[0022] The content of butyric acid-producing bacteria in the composition of the present invention is preferably as high as possible from the viewpoint of enhancing butyric acid production. For example, ３ cfu / g or more, preferably 1 x 10 ４ cfu / g or more, more preferably 1 x 10 ５ cfu / g or more, more preferably 1 x 10 ６ cfu / g or more, particularly preferably 1 x 10 ７ cfu / g or more, and the upper limit is not particularly limited, but is preferably 1 x 10 １０ cfu / g or less, preferably 1 x 10 ９ cfu / g or less, more preferably 1 x 10 ８ cfu / g or less.

[0023] In the composition of the present invention, the lactic acid-producing bacteria and the Anaerostipes butyric acid-producing bacteria may be contained in the same composition, or each bacterium may be contained in a separate composition prepared separately and manufactured, packaged, and distributed as a single package suitable for combined administration or combined ingestion. When each bacterium is contained in a separate composition, "combined administration" or "combined ingestion" does not only mean that the bacteria are administered or ingested simultaneously, but also means that the bacteria are administered or ingested sequentially at a predetermined interval (the order of administration or ingestion is not limited), as long as both bacteria are present in the intestine and can improve butyric acid production by the butyric acid-producing bacteria.

[0024] The form of the composition of the present invention may be any form that allows the lactic acid-producing bacteria and butyric acid-producing bacteria to be delivered to the intestine, and is not particularly limited. However, from the viewpoint of ease of administration or ingestion, an oral composition form is preferred.

[0025] In the present invention, specific examples of oral compositions include foods, beverages, additives for foods and beverages, supplements, feed, additives for feed, pharmaceuticals (including quasi-drugs), etc., but are not limited to these.

[0026] "Food" includes health foods, functional foods, health claim foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.), health supplements, nutritional supplements, etc. The form of the food can be selected appropriately, such as solid, liquid, or paste.

[0027] "Beverages" includes soft drinks, dairy drinks, alcoholic drinks, etc.

[0028] The "supplement" may be in any form, including, but not limited to, tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. In addition, in order to protect the lactic acid-producing bacteria and butyric acid-producing bacteria from gastric acid and bile acid and allow them to act in the intestine, they may be in the form of an enteric-coated formulation that is coated with a coating that differs in solubility at different pH levels.

[0029] "Feed" includes feed for livestock or pets, and its form can be selected appropriately, such as solid, liquid, or paste.

[0030] "Food and beverage additives" can be used as additives for foods and beverages. "Feed additives" can be used as additives for livestock or pet feed. These may be in any form, such as tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc.

[0031] "Drugs (including quasi-drugs)" may be in any form, including, but not limited to, tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. Liquid preparations such as liquid preparations and suspensions may be provided in a freeze-dried and storable state, and may be dissolved in a buffer solution containing water or saline, etc., to adjust to an appropriate concentration before use. Solid dosage forms such as tablets may be coated as needed (e.g., sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, etc.), or may be formulated into controlled-release formulations such as sustained-release, delayed-release, or immediate-release formulations using known techniques. Furthermore, enteric-coated formulations may be coated with a coating that differs in solubility at different pH levels to protect lactic acid-producing bacteria and butyric acid-producing bacteria from gastric acid and bile acid and allow them to act in the intestine.

[0032] In addition to the lactic acid-producing bacteria and butyric acid-producing bacteria, the composition of the present invention may optionally contain other components commonly used in the production of the intended use in amounts appropriate for the desired use, provided that the effects of the present invention are not impaired. Examples of such other components include, but are not limited to, sugars, excipients, disintegrants, lubricants, binders, diluents, buffers, suspending agents, thickeners, preservatives, antibacterial agents, antiseptics, antioxidants, UV absorbers, colorants, pigments, dyes, pigments, lubricants, plasticizers, solvents, solubilizers, isotonicity agents, seasonings, flavorings, vitamins, pH adjusters, and chelating agents.

[0033] Subjects to which the composition of the present invention is administered or ingested include humans and non-human animals (mammals (e.g., primates (monkeys, chimpanzees, etc.), livestock (cows, horses, pigs, sheep, etc.), pets (dogs, cats, etc.), laboratory animals (mice, rats, etc.)), birds, reptiles, etc.), preferably humans and non-human mammals, and particularly preferably humans.

[0034] The dosage or intake of the composition of the present invention is not particularly limited and can be set appropriately depending on the type, age, and age of the subject. For example, the composition can be administered or ingested at least once per day, preferably 1 to 5 times per day. The composition of the present invention can be effective in a small amount and for a short period of time, but can also be administered or ingested continuously or over a long period of time. For example, the composition of the present invention can be administered or ingested for two or more consecutive days, preferably 10 or more days, and more preferably 30 or more days, and can be administered or ingested continuously for one month or more, two or more months, six or more months, one year or more, or longer.

[0035] When administered or ingested by a subject, the composition of the present invention can increase the amount of butyric acid produced by butyric acid bacteria in the intestine and increase the concentration of butyric acid in the intestine, compared to when the composition of the present invention is not administered or ingested, or when lactic acid-producing bacteria or Anaerostipes butyric acid-producing bacteria are administered or ingested alone. The butyric acid produced in the intestine improves the intestinal environment by making the intestine weakly acidic, creating an environment that is favorable for so-called beneficial bacteria such as lactic acid bacteria and bifidobacteria, while suppressing the growth of harmful bacteria. It can also promote physiological effects such as strengthening the intestinal barrier function, improving mineral absorption such as calcium and magnesium, and promoting anti-inflammatory effects, thereby regulating the intestinal condition of the subject. That is, the composition of the present invention has an intestinal regulating effect and can be used as an intestinal regulator.

[0036] Since the composition of the present invention has the above-mentioned effects, it can be used in a method for increasing the butyric acid concentration in the intestines of a subject and / or a method for regulating the intestines in a subject.

[0037] The compositions of the present invention can also be used for immunomodulation and / or allergy improvement. By administering or ingesting the compositions of the present invention to a subject, the amount of butyrate produced by butyric acid bacteria in the intestine can be increased, thereby increasing the concentration of butyric acid in the intestine, compared to when the compositions of the present invention are not administered or ingested, or when lactic acid-producing bacteria or Anaerostipes butyric acid-producing bacteria are administered or ingested alone. The butyric acid produced in the intestine is taken up by the body and acts on cells of the immune system, promoting effects such as immunomodulation and / or allergy improvement (Siddiqui MT et al., "The Immunomodulatory Functions of Butyrate." J. Inflamm Res. 2021 Nov 18;14:6025-6041; Theiler A et al., "Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival." J. Allergy Clin Immunol. 2019 Sep;144(3):764-776). That is, the composition of the present invention has the effect of regulating immunity and / or ameliorating allergies, and can be used as an immunomodulator and / or an allergy ameliorator.

[0038] "For immunomodulation" refers to a function of activating stable or suppressed immune function (immunostimulatory activity), or a function of suppressing excessive or over-reactive immune function to an appropriate level (immunosuppressive activity), and includes, for example, those that aim to stabilize the overall balance of the immune response by controlling immune cells such as T cells, B cells, and macrophages.

[0039] "For allergy improvement" refers to suppressing immune responses harmful to the host due to the physiological state of protective immunity. Common allergies are caused by, for example, IgE-mediated immediate-type hypersensitivity and T-cell-mediated delayed-type hypersensitivity. Allergic activities include allergic rhinitis, allergic asthma, food allergies, allergic dermatitis, allergic eye disease, and anaphylaxis. Allergy improvement refers to stimulating immune activity that reverses the above-mentioned allergic symptoms. For example, blocking Th-2-mediated immunity, reducing Th-2-mediated cytokine (IL-4, IL-5, IL-13) production, increasing Th-1-mediated cytokine (IFN-γ), and stimulating IgE ＋ These include blocking the activation or differentiation of memory B cells and eosinophils.

[0040] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0041] 1. Preparation of fecal suspension. Feces serving as an inoculum for intestinal bacterial flora were collected from seven patients with ulcerative colitis on the day of culture. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by Becton, Tickinson and Company) and transported to the laboratory. To prepare the inoculum, 0.5 g of feces was diluted with 0.1 M phosphate-buffered saline (PBS) buffer (pH 6.5, 0.1 M NaHCO3) containing 1.0% L-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ２ P.O. ４ and 0.1 M Na ２ HPO ４ A fecal suspension was prepared by adding a 68.5:31.5 (molar ratio) mixture of the above (a 68.5:31.5 mixture of the above) to a total volume of 2 mL.

[0042] 2. Preparation of jar medium GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.): 59 g / L and antifoaming agent: 50 μL / L were mixed to prepare a medium. 100 mL of the medium was added to a jar fermentor (manufactured by Able Co., Ltd., BJR-25NAIS-8M, hereinafter sometimes abbreviated as jar) with a capacity of approximately 250 mL, and sterilized in an autoclave at 115°C for 15 minutes.

[0043] 3. Jar Culture Conditions After sterilization, anaerobic conditions were established in the culture vessel by aerating (15 mL / min) a mixed gas of nitrogen and carbon dioxide (N:CO = 80:20 (volume ratio)) that had been sterilized by filtration through a 0.2 μm PTFE membrane (manufactured by Pall Corporation) for 1 hour at 37°C before cultivation.

[0044] 4. Start of jar culture 100 μL of the fecal suspension was inoculated into a medium-containing container, and anaerobic culture was initiated (culture time 0). During the culture, filtered and sterilized carbon dioxide mixed gas (N:CO ２ The medium was constantly bubbled at a volume ratio of 80:20 to maintain an anaerobic state in the culture tank. The culture temperature was maintained at 37°C, and the incubation was performed with continuous stirring at approximately 300 rpm.

[0045] 5. Single-cell culture of Weizmannia coagulans and bacterial cell collection. Mineral-supplemented GAM medium was placed in a flask and sterilized. Weizmannia coagulans was inoculated and cultured. The culture temperature was maintained at 37°C, and the medium was continuously stirred at approximately 160 rpm for 18 hours under aerobic conditions to collect bacterial cells.

[0046] 6. Single-cell culture of Anaerostipes caccae and bacterial cell collection GAM medium was placed in a glass vial with a butyl rubber stopper, which was then sealed and sterilized. Filter-sterilized anaerobic gas was bubbled through the medium to create an anaerobic condition. Anaerostipes caccae (NBRC114412) was inoculated into the vial. The culture temperature was maintained at 37°C, and the culture was allowed to stand anaerobically for 18 hours to collect bacterial cells.

[0047] 7. Single-cell culture of Clostridium butyricum and bacterial cell collection RCM medium was placed in a glass vial with a butyl rubber stopper, which was then sealed and sterilized. Filter-sterilized anaerobic gas was bubbled through the medium to create an anaerobic condition. Clostridium butyricum (ATCC19398) was inoculated into the vial. The culture temperature was maintained at 37°C, and the culture was allowed to stand anaerobically for 18 hours to collect bacterial cells.

[0048] 8. Addition of prebio bacteria to jars at 0 hours of culture At 0 hours of culture, Weizmannia coagulans, Anaerostipes caccae, and Clostridium butyricum were added to each jar to start the culture. In another jar, Anaerostipes caccae was added to Weizmannia coagulans to start the culture, and in yet another jar, Clostridium butyricum was added to Weizmannia coagulans to start the culture. All of the prebio bacteria added were 4 x 10 ７ The solution was added so as to give a concentration of 100 cells / mL.

[0049] 9. Obtaining jar culture fluid The culture fluid was collected 48 hours after the start of cultivation, and a portion (1 mL) of this culture fluid was taken out as a test sample. The test sample was subjected to organic acid analysis and bacterial flora analysis.

[0050] 10. Organic Acid Analysis The culture medium collected 48 hours after the start of cultivation was used to measure the concentration of organic acids (short-chain fatty acids: acetic acid, propionic acid, and butyric acid). The filtrate obtained by filtering the culture medium was subjected to high-performance liquid chromatography (HPLC) (Shimadzu Corporation) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories) and a RID-10A refractive index detector (Shimadzu Corporation) to measure the amounts of butyric acid, propionic acid, and acetic acid. HPLC was performed at a flow rate of 0.6 mL / min using 5 mM H as the mobile phase. ２ SO ４ The reactor was operated at 65°C using a 1000kJ / min reactor.

[0051] 11. Bacterial Flora Analysis Genomic DNA of the bacterial flora was extracted from the culture medium collected at various times before and after the start of cultivation. The V3-V4 region of the bacterial 16S rRNA gene was amplified from the extracted genomic DNA and sequenced using a next-generation sequencer to perform bacterial diversity and bacterial composition analyses. The procedure is as follows:

[0052] The bacterial 16S rRNA gene was amplified using the primer pair S-D-Bact-0341-b-S-17 (SEQ ID NO: 1) and S-D-Bact-0785-a-A-21 (SEQ ID NO: 2) with extracted genomic DNA as a template. An Illumina adapter overhang nucleotide sequence (Illumina, Inc.) was added to the gene-specific sequence. PCR cycling reactions were performed according to the manufacturer's instructions. Confirmed amplicons were purified using AMPure XP DNA purification beads (Beckman Coulter, Inc.) and eluted in 25 μl of 10 mM Tris (pH 8.5). Amplicons were quantified on an Agilent Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies, Inc.) and pooled at equimolar concentrations. The 16S rRNA gene product (together with an internal control (PhiX control V3; Illumina)) was subjected to paired-end sequencing using a MiSeq sequencer (Illumina) with a 600-cycle MiSeq reagent kit (Illumina).

[0053]

[0054] Paired-end reads with a Q score of 20 or more were obtained by extracting the PhiX sequence using Basespace Sequence Hub (https: / / basespace.illumina.com / ). The reads were then combined using QIIME 2 version 2022.11 and quality control and correction were performed using the DADA2 pipeline. The OTUs were then inferred. The resulting OTUs were used to estimate alpha diversity and calculate the Shannon index. The resulting OTUs were then classified using a naive Bayes classifier trained on the Silva 138_99% OTU full-length sequence database, and bacterial species assignment was performed. Using Excel (Microsoft Japan Co., Ltd.), the relative occupancy rate of each bacterial group was calculated from the classification data of bacterial species attribution.

[0055] 12. Results of organic acid analysis No difference in butyric acid production was observed between Clostridium butyricum alone and the combination of Clostridium butyricum and Weizmannia coagulans. However, increased butyric acid production was observed when Anaerostipes caccae and Weizmannia coagulans were combined compared to Anaerostipes caccae alone (median, see Figure 1).

[0056] 13. Results of Weizmannia coagulans after cultivation in bacterial flora analysis. When Clostridium butyricum and Weizmannia coagulans were combined, there was no difference in the relative abundance of Weizmannia coagulans after cultivation compared to when Clostridium butyricum was used alone. However, when Anaerostipes caccae and Weizmannia coagulans were combined, the relative abundance of Weizmannia coagulans was higher than when Anaerostipes caccae was used alone (median, see Figure 2).

[0057] 14. Results of Bifidobacterium genus bacteria after cultivation in bacterial flora analysis When Clostridium butyricum was used alone and when Clostridium butyricum was combined with Weizmannia coagulans, the relative abundance of Bifidobacterium genus bacteria was low, whereas when Anaerostipes caccae was used alone and when Anaerostipes caccae was combined with Weizmannia coagulans, the relative abundance of Bifidobacterium genus bacteria was high (median, see Figure 3).

[0058] From the above results, it was confirmed that by combining Anaerostipes caccae and Weizmannia coagulans, it is possible to increase the amount of butyric acid produced by Anaerostipes caccae, and that this combination does not inhibit the growth of Weizmannia coagulans or other intestinal bacteria, thereby contributing to efficiently improving the intestinal environment.

Claims

1. A composition comprising lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Anaerostipes.

2. The composition of claim 1, wherein the butyric acid-producing bacterium is Anaerostipes caccae.

3. The composition of claim 1, wherein the lactic acid-producing bacteria are spore-forming bacteria.

4. The composition of claim 1, wherein the lactic acid-producing bacterium is Weizmannia coagulans.

5. The composition of claim 1, which is an oral composition.

6. The composition of claim 1, which is a food, beverage, supplement, feed, feed additive, or medicine.

7. The composition according to claim 1, which is used to increase the concentration of butyric acid in the intestine.

8. A composition according to any one of claims 1 to 7, for use in regulating the intestines.

9. A composition according to any one of claims 1 to 7, for use in immunomodulation.

10. The composition according to any one of claims 1 to 7, which is used to improve allergies.

11. A method for increasing butyric acid concentration in the intestine of a non-human animal, comprising administering to the non-human animal lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Anaerostipes.

12. A method (excluding medical procedures) for increasing butyric acid concentration in the intestines of a subject, comprising administering to the subject lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Anaerostipes.

13. A method for increasing butyrate concentration in the intestine of a subject, comprising administering to the subject lactic acid-producing bacteria and butyrate-producing bacteria of the genus Anaerostipes.

14. The method of claim 13, wherein the butyric acid-producing bacterium is Anaerostipes caccae.

15. The method of claim 13, wherein the lactic acid-producing bacteria are spore-forming bacteria.

16. The method of claim 13, wherein the lactic acid-producing bacterium is Weizmannia coagulans.

17. The method of claim 13, wherein said administration is oral administration.

18. The method of claim 13, wherein the subject is a non-human animal or a human.

19. The method according to claim 13, which is a method for regulating the intestines.

20. The method of claim 13, which is a method for immunomodulation.

21. The method according to claim 13, which is a method for improving allergies.

22. Use of lactic acid-producing bacteria and Anaerostipes butyric acid-producing bacteria in the manufacture of a composition for increasing butyric acid concentration in the intestine.

23. The use according to claim 22, wherein the butyric acid-producing bacterium is Anaerostipes caccae.

24. The use according to claim 22, wherein the lactic acid-producing bacteria are spore-forming bacteria.

25. The use according to claim 22, wherein the lactic acid-producing bacterium is Weizmannia coagulans.

26. The use of claim 22, wherein the composition is an immunomodulator.

27. The use according to claim 22, wherein the composition is an anti-intestinal agent.

28. The use of claim 22, wherein the composition is an immunomodulator.

29. The use according to claim 22, wherein the composition is an allergy-relieving agent.

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