Intestinal modulating composition comprising lactic acid-producing bacteria and butyric acid-producing bacteria

Abstract

An object of the present application is to provide a novel means capable of increasing the concentration of butyric acid in the intestinal tract, and to provide a composition comprising a lactic acid-producing bacterium and a butyric acid-producing bacterium of the genus Anaerostipes.

Description

Technical Field

[0001] This invention relates to compositions comprising lactic acid-producing bacteria and butyric acid-producing anaerobic corynebacteria. Background Technology

[0002] Butyric acid is a short-chain fatty acid that plays an important role in regulating the intestinal environment in humans and animals, primarily through its strengthening of the intestinal barrier and its anti-inflammatory effects. Therefore, attempts have been made to administer or ingest butyric acid for intestinal regulation. However, its strong, unpleasant odor makes administration or ingestion difficult. On the other hand, butyric acid-producing bacteria exist in the intestines of humans and animals, capable of generating butyric acid. Therefore, efforts have been made to increase the concentration of butyric acid in the intestines using butyric acid-producing bacteria. For example, Patent Document 1 discloses an intestinal butyric acid-producing bacteria increaser that uses Bacillus subtilis to increase the concentration of butyric acid-producing bacteria (Feacalibacterium prausnitzii). Patent Document 2 discloses a food composition that increases the concentration of butyric acid in the intestines, with D-mannitol or D-sorbitol as the active ingredient, and describes its administration to humans and animals or its ingestion. In addition, Non-Patent Literature 1 discloses that lactic acid-producing bacteria (Bacillus coagulans) SANK70258 increases bacteria associated with the Lachnospiraceae family in a microbial community model of healthy individuals, thereby promoting butyric acid production.

[0003] However, efforts to increase butyrate concentration in the gut are still insufficient, and there is an urgent need in this field for new methods to increase butyrate concentration in the gut.

[0004] Anaerostipescaccae, a butyric acid-producing bacterium belonging to the genus Anaerostipes, is a bacterium that can utilize lactic acid to produce butyric acid. It is a bacterium isolated from human feces (Non-Patent Document 2).

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Application Publication No. 2013-147469

[0008] Patent Document 2: Japanese Patent Application Publication No. 2004-49093

[0009] Non-patent literature

[0010] Non-patent literature 1: K. Sasaki et al., Appl Microbiol Biotechnol. 2020 May;104(9):3859-3867.

[0011] Non-patent literature 2: SHDuncan et al., Appl Environ Microbiol. 2004 Oct;70(10):5810-7. Summary of the Invention

[0012] The problem the invention aims to solve

[0013] The purpose of this invention is to provide a new method for increasing the concentration of butyric acid in the intestine.

[0014] Solution for solving the problem

[0015] In order to solve the above-mentioned problems, the inventors conducted in-depth research and found that by giving or ingesting a combination of lactic acid-producing bacteria and anaerobic corynebacteria butyrate-producing bacteria, the amount of butyrate produced by anaerobic corynebacteria butyrate-producing bacteria in the intestine can be increased.

[0016] Based on these new insights, this invention includes the following invention.

[0017] [1] A composition comprising lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Corynebacterium.

[0018] [2] According to the composition of [1], wherein the aforementioned butyric acid producing bacteria is Anaerostipescaccae.

[0019] [3] According to the composition of [1] or [2], wherein the aforementioned lactic acid producing bacteria are spore-forming bacteria.

[0020] [4] The composition according to any one of [1] to [3], wherein the aforementioned lactic acid producing bacteria is Weizmannia coagulans.

[0021] [5] The composition according to any one of [1] to [4] is an oral composition.

[0022] [6] The composition according to any one of [1] to [5] is a food, beverage, supplement, feed, feed additive or medicine.

[0023] [7] A composition according to any one of [1] to [6] is used to increase the concentration of butyric acid in the intestine.

[0024] [8] A composition according to any one of [1] to [7] is used for intestinal regulation.

[0025] [9] A composition according to any one of [1] to [7] is used for immunomodulation.

[0026]

[10] A composition according to any one of [1] to [7] is used to improve allergies.

[0027] [11a] A method for increasing butyrate concentration in the intestine of a subject, comprising: administering lactic acid-producing bacteria and anaerobic corynebacteria butyrate-producing bacteria to the subject.

[0028] [11b] According to the method of [11a], wherein the aforementioned butyric acid producing bacteria are fecal anaerobic rod-shaped bacteria.

[0029] [11c] According to the method of [11a] or [11b], wherein the aforementioned lactic acid producing bacteria are spore-forming bacteria.

[0030] [11d] The method according to any one of [11a] to [11c], wherein the aforementioned lactic acid producing bacteria is Weizmann's coagulans.

[0031] [11e] The method according to any one of [11a] to [11d], wherein the aforementioned administration is oral administration.

[0032] [11f] The method according to any one of [11a] to [11e], wherein the aforementioned object is a non-human animal.

[0033] [11g] According to any one of [11a] to [11e], wherein the aforementioned object is a person.

[0034] [11h] is a method of intestinal regulation according to any one of [11a] to [11g].

[0035] [11i] The method according to any one of [11a] to [11g] is a method for immune regulation.

[0036] [11j] The method according to any one of [11a] to [11g] is a method for improving allergies.

[0037] [12a] A method for increasing butyrate concentration in the intestine of a subject, comprising ingesting lactic acid-producing bacteria and anaerobic corynebacteria butyrate-producing bacteria from the subject.

[0038] [12b] According to the method of [12a], wherein the aforementioned butyric acid producing bacteria are fecal anaerobic rod-shaped bacteria.

[0039] [12c] According to the method of [12a] or [12b], wherein the aforementioned lactic acid producing bacteria are spore-forming bacteria.

[0040] [12d] The method according to any one of [12a] to [12c], wherein the aforementioned lactic acid producing bacteria is Weizmann's coagulans.

[0041] [12e] The method according to any one of [12a] to [12d], wherein the aforementioned intake is administered orally.

[0042] [12f] The method according to any one of [12a] to [12e], wherein the aforementioned object is a non-human animal.

[0043] [12g] According to any one of [12a] to [12e], wherein the aforementioned object is a person.

[0044] [12h] is a method of intestinal regulation according to any one of [12a] to [12g].

[0045] [12i] The method according to any one of [12a] to [12g] is a method for immune regulation.

[0046] [12j] The method according to any one of [12a] to [12g] is a method for improving allergies.

[0047] [13a] Use of lactic acid-producing bacteria and butyric acid-producing bacteria of the genus Corynebacterium in the manufacture of compositions for increasing butyric acid concentration in the intestine.

[0048] [13b] According to the use of [13a], wherein the aforementioned butyric acid producing bacteria are fecal anaerobic rod-shaped bacteria.

[0049] [13c] According to the use of [13a] or [13b], wherein the aforementioned lactic acid producing bacteria are spore-forming bacteria.

[0050] [13d] According to the uses of [13a] to [13c], the aforementioned lactic acid producing bacteria is Weizmann's coagulans.

[0051] [13e] According to any one of [13a] to [13d], wherein the aforementioned composition is an oral preparation.

[0052] [13f] According to any one of [13a] to [13e], wherein the aforementioned composition is an intestinal regulator.

[0053] [13g] According to the use of any one of [13a] to [13e], wherein the aforementioned composition is an immunomodulator.

[0054] [13h] According to any one of [13a] to [13e], wherein the aforementioned composition is an allergy improver.

[0055] This specification includes the contents described in the specification of Japanese Patent Application No. 2024-028581, filed on February 28, 2024, which forms the basis of the priority of this application.

[0056] All publications, patents and patent applications referenced in this specification are incorporated herein by reference directly.

[0057] The effects of the invention

[0058] According to the present invention, a new means is provided that can increase the concentration of butyric acid in the intestine. Attached Figure Description

[0059] [ Figure 1 ] Figure 1 This graph represents the analysis results of butyrate concentration in the culture medium after in vitro culture of feces from patients with ulcerative colitis, where lactic acid-producing bacteria (Weizmannia coagulans) and anaerobic corynebacterium butyrate-producing bacteria (Anaerostipes caccae) or butyrate-producing bacteria (Clostridium butyricum) were added together. Controls were used, with either no bacteria added (Control) or any bacteria added alone.

[0060] [ Figure 2 ] Figure 2 This graph represents the percentage of lactic acid-producing bacteria in an in vitro stool culture system using stool from patients with ulcerative colitis, after co-culturing with lactic acid-producing bacteria (Weizmann's coagulans) and anaerobic corynebacteria (fecal anaerobic corynebacteria) or butyrate-producing bacteria (Clostridium butyricum). Controls are provided, either using a culture system without any bacteria (control) or with any bacteria added alone.

[0061] [ Figure 3 ] Figure 3 This graph represents the percentage of Bifidobacterium bacteria in an in vitro stool culture system using stool from patients with ulcerative colitis, after co-culturing with lactic acid-producing bacteria (Weizmann's coagulans) and anaerobic corynebacteria (fecal anaerobic corynebacteria) or butyrate-producing bacteria (Clostridium butyricum). Controls were provided using culture systems with no added bacteria (control) or with any bacteria added alone. Detailed Implementation

[0062] This invention relates to compositions comprising lactic acid-producing bacteria and butyric acid-producing anaerobic corynebacteria.

[0063] In this invention, "lactic acid-producing bacteria" refers to intestinal bacteria capable of assimilating carbohydrates to produce lactic acid. As for lactic acid-producing bacteria usable in this invention, any bacteria capable of producing lactic acid in the intestine is acceptable; there are no particular limitations, but spore-forming bacteria (sometimes also called "spore-producing bacteria") are preferred. Lactic acid-producing bacteria that form spores (sometimes also called "spores") are highly resistant to drying, heat, and acid. When administered orally, they are not killed by gastric acid or bile acid, reach the intestine, germinate, become vegetative cells, and further proliferate to produce lactic acid; therefore, they are preferred.

[0064] As lactic acid-producing bacteria that can be used in this invention, examples more preferably include lactic acid-producing bacteria belonging to the genera *Weizmannia* (Bacillus), *Lactobacillus*, *Bifidobacterium*, and *Enterococcus*, but these are not limited to. Examples of bacteria belonging to the genus *Weizmannia* (Bacillus) include *Weizmannia coagulans* (sometimes also called "Bacillus coagulans"), *Bacillus subtilis*, *Bacillus licheniformis*, *Bacillus amyloliquefaciens*, and *Bacillus cereus*, but these are not limited to. Examples of bacteria belonging to the genus *Lactobacillus* include *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, *Lactobaillus gaceli*, and *Lactobacillus plantrum*, but the list is not limited to these. Examples of bacteria belonging to the genus *Bifidobacterium* include *Bifidobacterium adolescentis*, *Bifidobacterium bifidum*, *Bifidobacterium breve*, *Bifidobacterium longum*, and *Bifidobacterium pseudochain*, but the list is not limited to these. Examples of bacteria belonging to the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium, but are not limited to these.

[0065] As a lactic acid-producing bacterium usable in this invention, *Weizmannia coagulans* is particularly preferred. Examples of *Weizmannia coagulans* include strains SANK70258, P-22, lilac-01, SIM-7 DSM14043, C101, NBRC12583, GBI-1, GBI-20, GBI-30, and GBI-40, among which strain SANK70258 is preferred from the viewpoint of supply stability and ease of acquisition. Furthermore, any mutant strain derived from these strains can be used as long as it has the effect of promoting butyric acid production by anaerobic corynebacteria butyric acid-producing bacteria. Additionally, *Weizmannia coagulans* can be bacteria isolated from animal (preferably human) feces or commercially available bacteria. Commercially available products containing *Weizmannii coagulans* include, for example, Mitsubishi Chemical's "LACRIS-S", "LACRIS-S Granules", "LACRIS-15", and "LACRIS-10 for Feed", as well as products from Kerry Inc., SABINSA, Arterio-Bio, UNIQUEBIOTECH, and ASAHI BIOCYCLE.

[0066] In this invention, the "lactic acid producing bacteria" can be used in the form of spores, vegetative cells, mixtures thereof, as well as cultures containing them, culture media, and extracts thereof. Furthermore, in this invention, the "lactic acid producing bacteria" can be in any form, such as powder, granules, or liquid.

[0067] In this invention, "butyric acid-producing bacteria" refers to intestinal bacteria capable of assimilating lactic acid to produce butyric acid. As for butyric acid-producing bacteria usable in this invention, any bacteria capable of producing butyric acid in the intestine is acceptable; there are no particular limitations. Butyric acid-producing bacteria belonging to the genus *Corynebacterium* are preferred. Butyric acid-producing bacteria belonging to the genus *Corynebacterium* can increase butyric acid production in combination with the aforementioned lactic acid-producing bacteria and do not inhibit the growth of other intestinal bacteria (such as the aforementioned lactic acid-producing bacteria, Bifidobacteria, etc.). Furthermore, butyric acid-producing bacteria belonging to the genus *Corynebacterium* are spore-forming bacteria, highly resistant to drying, heat, and acid. When administered orally, they are not killed by gastric acid or bile acids, reach the intestine, germinate, become vegetative cells, and further proliferate to produce butyric acid; therefore, they are also preferred.

[0068] In this invention, examples of butyrate-producing bacteria belonging to the genus *Anaerostipes* include, but are not limited to, *Anaerostipes caccae*, *Anaerostipes hadrus*, *Anaerostipes butyraticus*, *Anaerostipes hominis*, *Anaerostipes rhamnosivorans*, and *Anaerostipes amylophilus*.

[0069] Butyrate-producing bacteria usable in this invention are particularly preferred to be fecal anaerobic corynebacteria. Examples of fecal anaerobic corynebacteria include strains DSM14662, L1-92, P127-A10a, 3_2_56FAA, and mgYG-HGUT-00080. Furthermore, mutant strains derived from these strains may be used as long as they enhance butyrate production in combination with the aforementioned lactic acid-producing bacteria. Commercially available bacteria or bacteria isolated from animal (preferably human) feces can also be used.

[0070] In this invention, the butyric acid-producing bacteria can be used in the form of spores, vegetative cells, mixtures thereof, as well as cultures containing them, culture media, and extracts thereof. Furthermore, in this invention, the butyric acid-producing bacteria can be in any form, such as powder, granules, or liquid.

[0071] Regarding the content of lactic acid-producing bacteria in the composition of the present invention, from the viewpoint of increasing butyric acid production by butyric acid-producing bacteria, a higher content of lactic acid-producing bacteria in the composition of the present invention is preferred, for example, 1 × 10⁻⁶. 3 cfu / g or higher, preferably 1×10 4 cfu / g or higher, preferably 1×10⁻⁶ 5 cfu / g or higher, more preferably 1×10 6 cfu / g or higher, preferably 1×10⁻⁶ 7 For values ​​above cfu / g, there is no specific upper limit; for example, it can be set to 1×10. 10 cfu / g or less, preferably 1×10 9 cfu / g or less, preferably 1×10⁻⁶ 8 Below CFU / g.

[0072] Regarding the content of butyric acid-producing bacteria in the composition of the present invention, from the viewpoint of increasing butyric acid production, a higher content of butyric acid-producing bacteria in the composition of the present invention is preferred, for example, 1 × 10⁻⁶. 3cfu / g or higher, preferably 1×10 4 cfu / g or higher, preferably 1×10⁻⁶ 5 cfu / g or higher, more preferably 1×10 6 cfu / g or higher, preferably 1×10⁻⁶ 7 For values ​​above cfu / g, there is no specific upper limit; it can be set to 1×10. 10 cfu / g or less, preferably 1×10 9 cfu / g or less, preferably 1×10⁻⁶ 8 Below CFU / g.

[0073] In the compositions of this invention, lactic acid-producing bacteria and anaerobic corynebacteria butyrate-producing bacteria can be contained in the same composition, or each bacterium can be contained in a separately prepared composition, as a single package suitable for administration or ingestion. When each bacterium is contained in a separate composition, "administered or ingested together" not only includes simultaneous administration or ingestion of each bacterium, but also includes administration or ingestion of each bacterium sequentially at predetermined intervals, provided that both bacteria are present in the intestine and butyrate production by the butyrate-producing bacteria is enhanced (the order of administration or ingestion is not limited).

[0074] The composition of the present invention can be configured in any form that can deliver lactic acid-producing bacteria and butyric acid-producing bacteria into the intestine, without particular limitation. From the viewpoint of ease of administration or ingestion, the oral composition is preferred.

[0075] In this invention, the oral composition may specifically include, but is not limited to, food, beverage, food additives, supplements, feed, feed additives, pharmaceuticals (including quasi-pharmaceuticals).

[0076] "Food" includes health foods, functional foods, health-promoting functional foods (foods for specific health purposes, nutritional functional foods, functionally labeled foods, etc.), health supplements, and nutritional supplements. Additionally, the form of food can be appropriately chosen, such as solid, liquid, or paste.

[0077] "Beverages" include soft drinks, dairy drinks, alcoholic drinks, etc.

[0078] "Supplements" can take any form, including tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc., but are not limited to these. Additionally, to protect lactic acid-producing and butyric acid-producing bacteria from the effects of gastric acid and bile acid, allowing them to function effectively in the intestine, enteric solvents with coatings that exhibit different solubility at different pH levels can be formulated.

[0079] "Feed" includes feed for livestock or pets, and its form can be selected appropriately, such as solid, liquid or paste.

[0080] "Food additives" can be used as additives in food and beverages. Additionally, "feed additives" can be used as additives in livestock or pet feed. Furthermore, they can be in any form, including tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc.

[0081] "Pharmaceuticals (including quasi-pharmaceuticals)" can 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 liquids and suspensions are provided in a state that allows for freeze-drying and preservation. When used, they can also be prepared to an appropriate concentration by dissolving them in a buffer solution containing water, physiological saline, etc. In addition, preparations with solid dosage forms such as tablets can be coated as needed (e.g., sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, etc.), and can also be prepared using known techniques to achieve controlled release, such as sustained-release, delayed-release, or immediate-release formulations. Furthermore, in order to protect lactic acid-producing bacteria and butyric acid-producing bacteria from the effects of gastric acid and bile acid and enable them to function in the intestine, enteric solvents with coatings that have different solubilities at different pH levels can be prepared.

[0082] In the compositions of the present invention, in addition to the lactic acid-producing bacteria and butyric acid-producing bacteria described above, other ingredients commonly used in manufacturing the target utilization form may be appropriately combined in amounts corresponding to the desired utilization form, as needed and without impairing the effects of the present invention. Examples of such other ingredients include, but are not limited to, sugars, excipients, disintegrants, lubricants, binders, diluents, buffers, suspending agents, thickeners, preservatives, antibacterial agents, preservatives, antioxidants, ultraviolet absorbers, colorants, pigments, dyes, colorants, lubricants, plasticizers, solvents, co-solvents, isotonic agents, flavorings, taste and odor modifiers, vitamins, pH adjusters, chelating agents, etc.

[0083] Examples of subjects to which the compositions of the present invention are given or ingested include humans and non-human animals (mammals (e.g., primates (monkeys, chimpanzees, etc.), livestock (cattle, 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.

[0084] The dosage or intake of the composition of the present invention is not particularly limited and can be appropriately set according to the type, age, and number of days of the subject. For example, it can be administered or ingested at least once a day, preferably 1 to 5 times a day. The composition of the present invention can exert its effect in trace amounts and for a short time, but can be administered or ingested continuously or for a long period of time. For example, the composition of the present invention can be administered or ingested continuously for more than 2 days, preferably more than 10 days, more preferably more than 30 days. In addition, it can be administered or ingested continuously for more than 1 month, more than 2 months, more than 6 months, more than 1 year, or more than 1 year.

[0085] By administering or ingesting the composition of the present invention to a subject, compared to not administering or ingesting the composition of the present invention, or administering or ingesting only lactic acid-producing bacteria or anaerobic corynebacteria butyrate-producing bacteria, the butyrate production by butyrate-producing bacteria in the intestine can be increased, thus increasing the butyrate concentration in the intestine. The butyrate produced in the intestine regulates the intestinal environment by making the intestine weakly acidic, creating an environment conducive to the survival of so-called beneficial bacteria such as lactic acid bacteria and bifidobacteria, and inhibiting the growth of harmful bacteria. Furthermore, it can strengthen the intestinal barrier function, improve the absorption of minerals such as calcium and magnesium, and promote anti-inflammatory effects, thus regulating the intestinal condition of the subject. In other words, the composition of the present invention has an intestinal regulatory effect and can be used as an intestinal regulator.

[0086] Because of the above-mentioned effects, the composition of the present invention can be used in methods for increasing butyrate concentration in the intestine of a subject and / or in methods for regulating the intestine of a subject.

[0087] In addition, the compositions of the present invention can be used for immunomodulation and / or improvement of allergies. By administering or ingesting the compositions of the present invention to a subject, compared with not administering or ingesting the compositions of the present invention, or administering or ingesting lactic acid-producing bacteria or anaerobic corynebacteria butyrate-producing bacteria alone, the butyrate production of butyrate by butyrate-producing bacteria in the intestine can be increased, thus increasing the butyrate concentration in the intestine. The butyrate generated in the intestine is ingested and acts on cells of the immune system, which can promote immunomodulation and / or improve allergies (Siddiqui MT et al., “The Immunomodulatory Functions of Butyrate.” J Inflamm Res. 2021 Nov 18;14:6025-6041.; Theiler A et al., “Butyrateameliorates allergic airway inflammation by limiting eosinophil trafficking and survival.” J Allergy Clin Immunol. 2019 Sep;144(3):764-776.). That is, the compositions of the present invention have the effect of immunomodulation and / or allergy improvement, and can be used as immunomodulators and / or allergy improvers.

[0088] "For immune regulation" refers to the function of activating stable or suppressed immune function (immune activation activity) or suppressing excessive immune function or overreaction to an appropriate level (immunosuppressive activity), such as functions aimed at stabilizing the overall balance of the immune response by controlling T cells, B cells, macrophages, etc., which are immune cells.

[0089] "Using to improve allergies" refers to suppressing harmful immune responses in the host through the physiological state of the defensive immune system. Common allergies include those caused by IgE-mediated immediate anaphylaxis and T-cell-mediated delayed anaphylaxis. Examples of allergic activity include allergic rhinitis, allergic asthma, food allergies, allergic dermatitis, allergic eye diseases, and anaphylaxis. Improving allergies means stimulating immune activity to reverse the aforementioned allergic symptoms. Examples include blocking Th-2-mediated immunity, reducing the production of Th-2-mediated cytokines (IL-4, IL-5, IL-13), increasing Th-1-mediated cytokines (IFN-γ), and inhibiting IgE. + Activation or differentiation of memory B cells and eosinophils, etc.

[0090] The present invention will be described below through examples, but the present invention is not limited to these examples.

[0091] Example

[0092] 1. Preparation of fecal suspension

[0093] On the day of culture, fecal samples were collected from seven patients with ulcerative colitis as inoculum sources. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by BD Corporation) and transported to the laboratory. To prepare the inoculum, a fecal suspension was prepared by adding 0.5 g of fecal sample to 0.1 M phosphate-buffered saline (PBS) buffer (pH 6.5, composed of a 68.5:31.5 molar ratio of 0.1 M NaH2PO4 and 0.1 M Na2HPO4) containing 1.0% L-ascorbic acid (manufactured by Fujifilm and Koden Pharmaceutical Co., Ltd.), bringing the total volume to 2 mL.

[0094] 2. Preparation of culture medium in tanks

[0095] The culture medium was prepared by mixing GAM medium [Code 05422] (manufactured by Nissui Pharmaceutical Co., Ltd.): 59 g / L and antifoaming agent: 50 μL / L. 100 mL of the culture medium was added to a 250 mL Jar fermenter (manufactured by ABLE, BJR-25NAIS-8M, hereinafter sometimes simply referred to as the jar), and the mixture was autoclaved at 115°C for 15 minutes.

[0096] 3. Tank culture conditions

[0097] After sterilization and before incubation, the culture vessel was exposed to a mixture of nitrogen and carbon dioxide (N2:CO2 = 80:20 (volume ratio)) at 37°C (15 mL / min) for 1 hour.

[0098] 4. Start of tank culture

[0099] 100 μL of the above-mentioned fecal suspension was inoculated into a container containing culture medium and anaerobic culture was initiated (0 hours). During the culture, the culture medium was continuously bubbled with a filtered, sterilized carbon dioxide mixture (N2:CO2 = 80:20 (volume ratio)) to maintain the anaerobic state in the culture tank. The culture temperature was maintained at 37°C, and the culture was carried out with continuous stirring at approximately 300 rpm.

[0100] 5. Single-cell culture and cell acquisition of *Weizmann's coagulation*

[0101] Sterilize the GAM medium supplemented with minerals in a flask. Inoculate with *Weizmannii coagulans* and begin culturing. Maintain the culturing temperature at 37°C and continuously stir at approximately 160 rpm for 18 hours aerobic incubation to obtain bacterial cells.

[0102] 6. Single-cell culture and cell acquisition of fecal anaerobic corynebacteria

[0103] Add GAM culture medium to a glass vial with a butyl rubber stopper, seal, and sterilize. Bubble the medium with filtered, sterilized anaerobic gas to establish an anaerobic environment. Inoculate the vial with fecal anaerobic rod-shaped bacteria (NBRC114412). Maintain the culture temperature at 37°C and incubate under anaerobic static conditions for 18 hours to obtain bacterial cells.

[0104] 7. Single-cell culture and cell acquisition of Clostridium butyricum

[0105] RCM culture medium was added to a glass vial with a butyl rubber stopper and sealed for sterilization. The medium was bubbled with filtered, sterilized anaerobic gas to establish an anaerobic environment. Clostridium butyricum (ATCC19398) was inoculated into the vial. The culture temperature was maintained at 37°C, and the vial was incubated under anaerobic static conditions for 18 hours to obtain bacterial cells.

[0106] 8. Add probiotics (Prebio bacteria) to the container after 0 hours of incubation.

[0107] At 0 hours of incubation, *Weizmannii coagulans*, *Corynebacterium fecal microbiota*, and *Clostridium butyricum* were added to each container to begin culturing. Additionally, *Corynebacterium fecal microbiota* was added to *Weizmannii coagulans* in another container to begin culturing, and then *Clostridium butyricum* was added to *Weizmannii coagulans* in yet another container to begin culturing. It should be noted that the added probiotics were all at a concentration of 4 × 10⁻⁶ for each strain. 7 Add cells per mL.

[0108] 9. Obtaining the culture medium in the tank

[0109] The culture medium was recovered 48 hours after the start of incubation, and a portion (1 mL) of this culture medium was taken as the test sample. Organic acid analysis and bacterial community analysis were performed using the test sample.

[0110] 10. Organic acid analysis

[0111] The culture medium recovered 48 hours after the start of incubation was used to determine the concentration of organic acids (short-chain fatty acids: acetic acid, propionic acid, and butyric acid). The filtrate obtained after filtering the culture medium was subjected to high-performance liquid chromatography (HPLC) (manufactured by Shimadzu Corporation) using an Aminex HPX-87H column (manufactured by Bio-Rad Laboratories, Inc.) and a RID-10A differential refractive index detector (manufactured by Shimadzu Corporation) to determine the amounts of butyric acid, propionic acid, and acetic acid. HPLC was performed using 5 mM H₂SO₄ as the mobile phase at a flow rate of 0.6 mL / min at 65 °C.

[0112] 11. Bacterial community analysis

[0113] Genomic DNA of the bacterial community was extracted from the culture medium recovered at various time points before and after the start of culture. The V3-V4 region of the bacterial 16S rRNA gene was amplified from the extracted genomic DNA, and the sequence was analyzed using a next-generation sequencer for bacterial diversity and composition analysis. The steps are shown below.

[0114] Using primer pairs SD-Bact-0341-bS-17 (Sequence No. 1) and SD-Bact-0785-aA-21 (Sequence No. 2), the bacterial 16S rRNA gene was amplified using extracted genomic DNA as a template. Illumina adapter overhang nucleotide sequences (manufactured by Illumina, Inc.) were appended to the gene-specific sequence. PCR cycles were performed according to the manufacturer's instructions. The amplicon was purified using validated AMPure XPDNA purification beads (manufactured by Beckman Coulter) and eluted to 25 μl of 10 mM Tris (pH 8.5). The amplicon was quantified in an Agilent Bioanalyzer 2100 DNA 1000 array (manufactured by Agilent Technologies) at equimolar concentrations. Using a MiSeq sequencer (manufactured by Illumina, Inc.), the 16S rRNA gene product (together with an internal control (PhiX control V3; manufactured by Illumina, Inc.)) and a 600-cycle MiSeq kit (manufactured by Illumina, Inc.) were subjected to paired-end sequencing.

[0115] [Table 1]

[0116]

[0117] For paired-end reads with a Q score of 20 or higher obtained by excising PhiX sequences using Basespace Sequence Hub (https: / / basespace.illumina.com / ), merging was performed using QIIME version 2022.11. After quality control and correction using the DADA2 workflow, OTUs were inferred. Alpha diversity was estimated using the obtained OTUs, and the Shannon index was calculated. Furthermore, the obtained OTUs were classified using a naive Bayes classifier learned from the Silva 138_99% OTU full-length sequence database to determine bacterial species. The relative occupancy of each bacterial community was calculated using Excel (Microsoft Japan) from the classification data of species attribution.

[0118] 12. Results of organic acid analysis

[0119] No difference in butyric acid production was found between the use of Clostridium butyricum alone and the combination of Clostridium butyricum and Weizmannii coagulans. However, an increase in butyric acid production was observed in the combination of Clostridium butyricum and Weizmannii coagulans compared to the use of fecal anaerobic bacteria alone (median, reference). Figure 1 ).

[0120] 13. Results of cultured *Weizmannii* in bacterial community analysis.

[0121] Compared to the use of *Clostridium butyricum* alone, no difference was found in the relative abundance of *Weizmann's coagulans* after culture when *Clostridium butyricum* and *Weizmann's coagulans* were combined. However, compared to the use of fecal anaerobic corynebacteria alone, the relative abundance of *Weizmann's coagulans* was higher (median, reference) when *Ferrous anaerobic corynebacteria* and *Weizmann's coagulans* were combined. Figure 2 ).

[0122] 14. Results of cultured Bifidobacterium bacteria in bacterial community analysis

[0123] The relative abundance of Bifidobacterium spp. was low when Clostridium butyricum was used alone or in combination with Weizmann's coagulans. On the other hand, the relative abundance of Bifidobacterium spp. was high when Corynebacterium feces alone or in combination with Weizmann's coagulans (median, reference). Figure 3 ).

[0124] Based on the above results, it was confirmed that combining fecal anaerobic Corynebacterium and Weizmannii coagulans can increase the butyrate production of fecal anaerobic Corynebacterium. In addition, the proliferation of Weizmannii coagulans was not inhibited in this combination, nor was the proliferation of other intestinal bacteria, which helps to efficiently regulate the intestinal environment.

Claims

1. A composition comprising lactic acid-producing bacteria and butyric acid-producing anaerobic corynebacteria.

2. The composition according to claim 1, wherein, The butyric acid-producing bacteria are fecal anaerobic rod-shaped bacteria.

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

4. The composition according to claim 1, wherein, The lactic acid-producing bacteria is *Weizmannii* coagulans.

5. The composition according to claim 1, wherein it is an oral composition.

6. The composition according to claim 1, wherein it is a food, beverage, supplement, feed, feed additive, or drug.

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

8. The composition according to any one of claims 1 to 7, used for intestinal regulation.

9. The composition according to any one of claims 1 to 7, used for immunomodulation.

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

11. A method for increasing butyrate concentration in the intestine of a non-human animal, comprising: The non-human animals were given lactic acid-producing bacteria and butyric acid-producing anaerobic corynebacteria.

12. A method for increasing butyrate concentration in the intestine of a subject, the method excluding medical procedures, the method comprising: The subject ingests lactic acid-producing bacteria and butyric acid-producing anaerobic corynebacteria.

13. A method for increasing butyrate concentration in the intestine of a subject, comprising: The subject was given lactic acid-producing bacteria and butyric acid-producing anaerobic corynebacteria.

14. The method according to claim 13, wherein, The butyric acid-producing bacteria are fecal anaerobic rod-shaped bacteria.

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

16. The method according to claim 13, wherein, The lactic acid-producing bacteria is *Weizmannii* coagulans.

17. The method according to claim 13, wherein, The administration is oral.

18. The method according to claim 13, wherein, The subject can be a non-human animal or a human.

19. The method according to claim 13, wherein it is an intestinal regulation method.

20. The method according to claim 13, wherein it is a method for immune regulation.

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

22. Use of lactic acid-producing bacteria and anaerobic corynebacteria of the butyric acid-producing genus in the manufacture of compositions for increasing butyric acid concentration in the intestine.

23. The use according to claim 22, wherein, The butyric acid-producing bacteria are fecal anaerobic rod-shaped bacteria.

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 bacteria is *Weizmannii* coagulans.

26. The use according to claim 22, wherein, The composition is an immunomodulator.

27. The use according to claim 22, wherein, The composition is an intestinal regulator.

28. The use according to claim 22, wherein, The composition is an immunomodulator.

29. The use according to claim 22, wherein, The composition is an allergy improver.

Images