Intestinal regulation composition containing human milk oligosaccharide and heyndrickxia coagulans

Abstract

The purpose of the present invention is to provide a new means for increasing the butyric acid concentration in the intestine. Provided is a composition containing a human milk oligosaccharide and Heyndrickxia coagulans.

Description

Intestinal regulating composition containing human milk oligosaccharides and Heyndrickxia coagulans

[0001] The present invention relates to a composition comprising human milk oligosaccharides and Hendrickxia coagulans.

[0002] Butyric acid is a type of short-chain fatty acid that plays an important role in regulating the intestinal environment in humans and animals, including strengthening the intestinal barrier function and providing anti-inflammatory effects. Therefore, attempts have been made to administer or ingest butyric acid for the purpose of improving intestinal health. However, because butyric acid has a very strong, unpleasant odor, administering or ingesting butyric acid itself is not easy. On the other hand, butyric acid-producing bacteria exist in the intestines of humans and animals, and these bacteria can produce butyric acid in the intestines. Therefore, efforts are being made to increase the butyric acid concentration in the intestines using butyric acid-producing bacteria. For example, Patent Document 1 describes bacterial strains capable of producing butyric acid, including Anaerostipes caccae, Roseburia intestinalis, and Roseburia hominis, in the presence of human milk oligosaccharides, and Bifidobacterium longum subsp., which can consume human milk oligosaccharides. It has been disclosed that higher concentrations of butyrate were observed when co-culturing with *Infantis*.

[0003] Furthermore, Patent Document 2 describes that a nutritional composition comprising an oligosaccharide mixture containing at least one N-acetylated oligosaccharide, at least one galactooligosaccharide, and at least one sialylated oligosaccharide increases the production of short-chain fatty acids such as butyric acid in infants or young children, that the oligosaccharides in the oligosaccharide mixture are bovine milk oligosaccharides, and that the nutritional composition may further contain probiotics such as those belonging to the genera Lactobacillus, Streptococcus, Enterococcus, Bifidobacterium, and Saccharomyces.

[0004] Furthermore, Non-Patent Document 1 discloses that the lactic acid-producing bacterium Bacillus coagulans SANK70258 increased bacteria related to the Lachnospiraceae family in a microbiome model of healthy individuals, thereby promoting butyrate production.

[0005] However, efforts to increase butyrate levels in the intestines are still insufficient, and there is a strong need for new methods that can increase butyrate levels in the intestines.

[0006] Patent Document 3 discloses a method for preparing probiotics to improve the balance of the intestinal flora, which includes uniformly mixing a polysaccharide enzyme fermentation product using Lactobacillus acidophilus, Bifidobacterium lactis, and Bacteroides thetaiotaomicron, a short-chain fatty acid composition, a prebiotic composition, and Bacillus coagulans TQ33 to obtain an active composition, and it is also described that the prebiotic composition is prepared by mixing inulin, human milk oligosaccharides, and blueberry polyphenols.

[0007] Special table 2023-537608 publication WO2017 / 129644 publication CN115944665 publication

[0008] K. Sasaki et al., Appl Microbiol Biotechnol. 2020 May; 104(9):3859-3867.

[0009] The present invention aims to provide a novel means capable of increasing the concentration of butyrate in the intestines.

[0010] As a result of diligent research to solve the above problems, the present inventors have found that administering or ingesting a combination of human milk oligosaccharides and Heyndrickxia coagulans (hereinafter referred to as "H. coagulans") can significantly increase the butyrate concentration in the intestines.

[0011] The present invention is based on these novel findings and encompasses the following inventions: [1] A composition comprising human milk oligosaccharide and H. coagulans, used to increase butyrate concentration in the intestines. [2] The composition of [1], wherein the human milk oligosaccharide is one or more selected from the group consisting of acidic human milk oligosaccharide and salts thereof. [3] The composition of [1] or [2], wherein the human milk oligosaccharide is one or more selected from the group consisting of 3'-sialyl lactose, 6'-sialyl lactose and salts thereof. [4] Any of the compositions of [1] to [3] that are oral compositions. [5] Any of the compositions of [1] to [4] that are foods, beverages, supplements, feeds, feed additives, or pharmaceuticals. [6] Any of the compositions of [1] to [5] that are for regulating bowel function.

[0012] [7] A method for increasing the butyrate concentration in the intestines of a subject, comprising administering human milk oligosaccharide and H. coagulans to the subject. [8] The method of [7], wherein the human milk oligosaccharide is one or more selected from the group consisting of acidic human milk oligosaccharide and salts thereof. [9] The method of [7] or [8], wherein the human milk oligosaccharide is one or more selected from the group consisting of 3'-sialyl lactose, 6'-sialyl lactose and salts thereof.

[10] Any method of [7] to [9], wherein the administration is oral.

[11] Any method of [7] to

[10] , wherein the subject is a non-human animal.

[12] Any method of [7] to

[10] , wherein the subject is a human.

[13] Any method of [7] to

[12] , wherein the method is a method for regulating bowel function.

[0013]

[14] A method for increasing the butyrate concentration in the intestines of a subject, comprising administering human milk oligosaccharide and H. coagulans to the subject.

[15] The method of

[14] , wherein the human milk oligosaccharide is one or more selected from the group consisting of acidic human milk oligosaccharide and salts thereof.

[16] The method of

[14] or

[15] , wherein the human milk oligosaccharide is one or more selected from the group consisting of 3'-sialyl lactose, 6'-sialyl lactose, and salts thereof.

[17] Any method of

[14] to

[16] , wherein the administration is oral administration.

[18] Any method of

[14] to

[17] , wherein the subject is a non-human animal.

[19] Any method of

[14] to

[17] , wherein the subject is a human.

[20] Any method of

[14] to

[19] , which is a method for regulating bowel function.

[21] Any method of

[14] to

[20] , excluding medical procedures.

[0014]

[22] Use of human milk oligosaccharides and H. coagulans in the manufacture of a composition for increasing butyrate concentration in the intestines.

[23] Use of

[22] , wherein the human milk oligosaccharide is one or more selected from the group consisting of acidic human milk oligosaccharides and salts thereof.

[24] Use of

[22] or

[23] , wherein the human milk oligosaccharide is one or more selected from the group consisting of 3'-sialyl lactose, 6'-sialyl lactose and salts thereof.

[25] Use of any of

[22] to

[24] , wherein the composition is an oral preparation.

[26] Use of any of

[22] to

[25] , wherein the composition is an intestinal regulator.

[0015]

[27] A pharmaceutical product comprising human milk oligosaccharide and H. coagulans for use in a method for increasing the butyrate concentration in the intestines of a target.

[28] The pharmaceutical product of

[27] , wherein the human milk oligosaccharide is one or more selected from the group consisting of acidic human milk oligosaccharide and salts thereof.

[29] The pharmaceutical product of

[27] or

[28] , wherein the human milk oligosaccharide is one or more selected from the group consisting of 3'-sialyl lactose, 6'-sialyl lactose and salts thereof.

[30] Any pharmaceutical product of

[27] to

[29] , wherein the administration is by oral administration.

[31] Any pharmaceutical product of

[27] to

[30] , wherein the target is a non-human animal.

[32] Any pharmaceutical product of

[27] to

[30] , wherein the target is a human.

[33] Any pharmaceutical product of

[27] to

[32] , wherein the intestinal regulator.

[0016]

[34] Use of a composition comprising human milk oligosaccharide and H. coagulans to increase the butyrate concentration in the intestines of a subject.

[35] Use of

[34] wherein the human milk oligosaccharide is one or more selected from the group consisting of acidic human milk oligosaccharide and salts thereof.

[36] Use of

[34] or

[35] wherein the human milk oligosaccharide is one or more selected from the group consisting of 3'-sialyl lactose, 6'-sialyl lactose and salts thereof.

[37] Use of any of

[34] to

[36] , which includes administering the composition to a subject.

[38] Use of any of

[34] to

[37] , wherein the subject is a non-human animal.

[39] Use of any of

[34] to

[37] , wherein the subject is a human.

[40] Use of any of

[34] to

[39] , wherein the composition is an intestinal regulator.

[41] Use of any of

[34] to

[40] , excluding medical procedures. This specification incorporates the contents of the specification of Japanese Patent Application No. 2024-224886, filed on December 20, 2024, which forms the basis of the priority claim of this application. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0017] According to the present invention, a novel method is available that can increase the butyrate concentration in the intestines.

[0018] Figure 1 is a box plot showing the results of analyzing the butyrate concentration in the culture medium of in vitro fecal culture systems using the feces of healthy individuals, when 3'-SL, 6'-SL, or 2'-FL were added individually (labeled "3'-SL", "6'-SL", and "2'-FL"), and when 3'-SL, 6'-SL, or 2'-FL were co-added with H. coagulans SANK70258 (labeled "3'-SL + H. coagulans", "6'-SL + H. coagulans", and "2'-FL + H. coagulans"). The control group consists of a fecal culture system without any additives (labeled "Control") and H. coagulans. The culture medium was used after culturing a fecal culture system (labeled "H. coagulans") to which coagulans SANK70258 had been added alone. The horizontal line in the box indicates the median, and the black diamond indicates the mean. n=8.

[0019] This invention relates to a composition comprising human milk oligosaccharide and H. coagulans. Conventionally, H. coagulans has been known to promote butyrate production (Non-Patent Literature 1), but its effect has not always been sufficient. In order to further enhance the effect of improving the intestinal environment, a further increase in the butyrate concentration in the intestines has been desired. In this invention, by using human milk oligosaccharide in combination with H. coagulans, the amount of butyrate produced in the intestines can be increased compared to when H. coagulans or human milk oligosaccharide is used alone, and a significant increase in the butyrate concentration in the intestines can be achieved.

[0020] This increase in butyrate concentration may be due to human milk oligosaccharides activating one of the metabolic pathways of H. coagulans, and / or the formation of a metabolic network between H. coagulans and the intestinal flora coexisting with it. For example, in the presence of human milk oligosaccharides, it is possible that a metabolic pathway is activated in one of the bacteria of the intestinal flora in a manner different from that of normal sugar sources, and the production ratio of intermediate metabolites such as acetic acid changes. Furthermore, it is possible that lactic acid production by H. coagulans increases, and that the coexisting butyrate-producing bacteria utilize the produced lactic acid as a substrate. It is presumed that such actions, as a result, activate the butyrate production system of the butyrate-producing bacteria, thereby enhancing butyrate production and producing the aforementioned effect. In other words, the increase in butyrate concentration of the present invention is due to the administration of human milk oligosaccharides alone or H. coagulans. This is thought to be due to a synbiotic synergistic effect based on the interaction between the two, which cannot be achieved with coagulans alone.

[0021] In this invention, "human milk oligosaccharide" refers to any oligosaccharide normally found in human milk and is not particularly limited. Typical examples include acidic human milk oligosaccharides such as 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), sialyl-lacto-N-tetraose a (LSTa), sialyl-lacto-N-tetraose b (LSTb), sialyl-lacto-N-tetraose c (LSTc), and disialyl lacto-N-tetraose (DS-LNT), as well as 2'-fucosyl lactose (2'-FL), 3-fucosyl lactose (3-FL), difucosyl lactose (DF-L (LDFT)), and lacto-n Examples include neutral human milk oligosaccharides such as -tetraose (LNT), lacto-n-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II), lacto-N-neohexaose (LNnH), lacto-N-hexaose (LNH) (F-LNH-I), F-LNH-II, DF-LNH-I, and DF-LNH-II. In the present invention, acidic human milk oligosaccharides are preferred, and among them, 3'-SL, 6'-SL, etc., can be used particularly suitably. Furthermore, in the present invention, human milk oligosaccharides also include salts thereof. In this specification, "salt" refers to alkali metal salts such as sodium, potassium, and lithium, alkaline earth metal salts such as magnesium and calcium, ammonium salts, substituted ammonium salts, etc., but is not limited to these.

[0022] In the present invention, human milk oligosaccharides may be used individually or in combination of two or more types, but it is preferable to contain a preferred human milk oligosaccharide in a relatively high concentration. For example, in one embodiment, the human milk oligosaccharide contains 60% or more by mass, preferably 70% or more by mass, more preferably 80% or more by mass, and even more preferably 90% or more by mass, of acidic human milk oligosaccharides, with acidic human milk oligosaccharides being particularly preferable. In another embodiment, the human milk oligosaccharide contains 60% or more by mass, preferably 70% or more by mass, more preferably 80% or more by mass, and even more preferably 90% or more by mass, of 3'-SL and 6'-SL, with 3'-SL and 6'-SL being particularly preferable. In another embodiment, the human milk oligosaccharide contains 60% by mass or more, preferably 70% by mass or more, more preferably 80% by mass or more, and even more preferably 90% by mass or more, of 3'-SL as a percentage of 100% by mass of the whole, and is particularly preferably composed of 3'-SL only. In yet another embodiment, the human milk oligosaccharide contains 60% by mass or more, preferably 70% by mass or more, more preferably 80% by mass or more, and even more preferably 90% by mass or more, of 6'-SL as a percentage of 100% by mass of the whole, and is particularly preferably composed of 6'-SL only.

[0023] In the present invention, human milk oligosaccharides may be purified, crudely purified, or concentrated from human milk, or they may be produced from microorganisms, animal cells, transgenic animals, etc. by genetic engineering, or they may be chemically synthesized.

[0024] The amount of human milk oligosaccharide in the composition of the present invention is preferably as high as possible from the viewpoint of increasing the butyric acid concentration in the intestines by using it in combination with H. coagulans. For example, the amount can be preferably 0.001% by mass or more and less than 100% by mass (for example, 0.001% by mass or more and 99.5% by mass or less, 0.001% by mass or more and 99% by mass or less, 0.001% by mass or more and 98% by mass or less, 0.001% by mass or more and 97% by mass or less, 0.001% by mass or more and 95% by mass or less, or 0.001% by mass or more and 90% by mass or less) relative to 100% by mass of the entire composition of the present invention, more preferably 0.005 to 75% by mass, and even more preferably 0.01 to 50% by mass.

[0025] In this invention, "H. coagulans" refers to a lactic acid-producing bacterium capable of utilizing sugars to produce lactic acid, and is a type of intestinal bacterium. Furthermore, H. coagulans is a spore-forming bacterium (sometimes called a "spore-forming bacterium"), and by forming spores (sometimes called "spores"), it can increase its resistance to drying, heat, and acid. When administered orally, the spores reach the intestines without being destroyed by stomach acid or bile acid, germinate, become vegetative cells, and further proliferate to produce lactic acid. Note that H. coagulans may also be referred to as "Weizmannia coagulans" or "Bacillus coagulans." These are synonyms arising from a change in scientific name, and these names can be used interchangeably.

[0026] In the present invention, examples of H. coagulans include strains SANK70258, P-22, lilac-01, SIM-7 DSM14043, C101, NBRC12583, GBI-1, GBI-20, GBI-30, GBI-40, SC208, BC99, BC01, etc. Among these, strain SANK70258 of H. coagulans is preferred in terms of supply stability and ease of acquisition. Furthermore, any mutant strain derived from these bacterial strains may be used, as long as it has the effect of increasing the butyrate concentration in the intestines when used in combination with human milk oligosaccharides. In addition, H. coagulans can be isolated from animal (preferably human) feces or commercially available H. Examples of commercially available coagulans include Mitsubishi Chemical Corporation's "Lacris®-S", "Lacris®-S Granules", "Lacris®-15", and "Lacris®-10 for Feed", as well as products from Kerry Inc., SABINSA, Atelio Bio, UNIQUEBIOTECH, Asahi Biocycle, and others.

[0027] In the present invention, H. coagulans can be used in the form of spores, vegetative cells, mixtures thereof, cultures containing them, culture media, and extracts thereof. Furthermore, in the present invention, H. coagulans may be in any form, such as powder, granules, or liquid.

[0028] The H. coagulans content in the composition of the present invention is preferable to be higher, for example, 1 × 10⁻⁶, from the viewpoint of increasing the butyric acid concentration in the intestines when used in combination with human milk oligosaccharides. ３ cfu / g or more, preferably 1 × 10 ４ cfu / g or more, more preferably 1 × 10⁻⁶ ５ cfu / g or more, more preferably 1 × 10 ６ cfu / g or more, particularly preferably 1 × 10 ７ The cfu / g level is greater than or equal to 1 × 10⁻¹⁰, and there is no particular upper limit, but for example, 1 × 10⁻¹⁰ １０ cfu / g or less, preferably 1 × 10⁻⁶９ cfu / g or less, more preferably 1 × 10 ８ It can be set to cfu / g or less.

[0029] In the composition of the present invention, human milk oligosaccharides and H. coagulans may be contained in the same composition, or they may be contained in separate compositions prepared separately and manufactured, packaged, and distributed as a single package suitable for combined administration or combined intake. When they are contained in separate compositions, "combined administration" or "combined intake" includes not only cases where human milk oligosaccharides and H. coagulans are administered or taken at the same time, but also cases where human milk oligosaccharides and H. coagulans are administered or taken sequentially at predetermined intervals (the order of administration or intake is not limited), as long as both are present in the intestines and can increase the butyrate concentration in the intestines.

[0030] The composition of the present invention can be in any form that allows human milk oligosaccharides and H. coagulans to be delivered to the intestines, and is not particularly limited, but an oral composition is preferred from the viewpoint of ease of administration or ingestion.

[0031] In the present invention, oral compositions specifically include, but are not limited to, foods, beverages, food and beverage additives, supplements, animal feed, animal feed additives, pharmaceuticals (including quasi-drugs), etc.

[0032] "Food" includes health foods, functional foods, health functional foods (foods for specified health uses, nutrient function foods, foods with functional claims, etc.), health supplements, nutritional supplements, etc. Furthermore, the form of the food can be selected as appropriate, such as solid, liquid, or paste.

[0033] "Beverages" include soft drinks, dairy drinks, alcoholic beverages, etc.

[0034] The "supplement" may have any shape, including, but not limited to, tablets, granules, powders, dragees, capsules, syrups, suspensions, solutions, emulsions, etc. Also, in order to protect human milk oligosaccharide and H. coagulans from gastric acid and bile acids and allow them to act in the intestine, it may be an enteric solvent with a coating that has different solubility differences for different pH values.

[0035] The "feed" includes feed for livestock or pets, and its shape can be appropriately selected, such as solid, liquid or paste-like.

[0036] The "food and beverage additive" can be used as an additive for food and beverages. Also, the "feed additive" can be used as an additive for livestock or pet feed. Also, these may have any shape, such as tablets, granules, powders, dragees, capsules, syrups, suspensions, solutions, emulsions, etc.

[0037] The "pharmaceutical product (including quasi-drugs)" may have any shape, including, but not limited to, tablets, granules, powders, dragees, capsules, syrups, suspensions, solutions, emulsions, etc. Liquid preparations such as solutions and suspensions may be provided in a state that can be freeze-dried and stored, and may be used after being dissolved in a buffer solution containing water, physiological saline, etc. at the time of use and adjusted to an appropriate concentration. Also, those having a solid dosage form such as tablets may be coated as necessary (for example, dragees, gelatin-coated tablets, enteric tablets, etc.), and may also be a preparation with controlled release, such as a sustained-release preparation, a delayed-release preparation or an immediate-release preparation, using known techniques. Also, in order to protect human milk oligosaccharide and H. coagulans from gastric acid and bile acids and allow them to act in the intestine, it may be an enteric solvent with a coating that has different solubility differences for different pH values.

[0038] In addition to human milk oligosaccharide and H. coagulans, the composition of the present invention may further contain, as needed, components commonly used in the production of the intended form of use, in an amount appropriate for the desired form of use within the range that does not impair the effects of the present invention. Such other components include, for example, excipients, disintegrants, lubricants, binders, diluents, buffers, suspending agents, thickeners, preservatives, antibacterial agents, antiseptics, antioxidants, ultraviolet absorbers, colorants, pigments, dyes, coloring matters, lubricants, plasticizers, solvents, solubilizing agents, isotonic agents, saccharides, seasonings, flavoring agents, vitamins, pH adjusters, chelating agents, etc., but are not limited thereto.

[0039] The subjects to which the composition of the present invention is administered or ingested include humans and non-human animals (mammals (e.g., primates (such as monkeys, chimpanzees, etc.), livestock (such as cows, horses, pigs, sheep, etc.), pets (such as dogs, cats, etc.), laboratory animals (such as mice, rats, etc.)), birds, reptiles, etc.), preferably humans and non-human mammalian animals, and particularly preferably humans.

[0040] The dosage or intake amount of the composition of the present invention is not particularly limited and can be appropriately set according to the type, age, day age, etc. 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 effects in a small amount and in a short period, but can be administered or ingested continuously or over a long period. For example, the composition of the present invention can be administered or ingested continuously for 2 days or more, preferably 10 days or more, more preferably 30 days or more, and can also be continuously administered or ingested over a period of 1 month or more, 2 months or more, 6 months or more, 1 year or more, or longer.

[0041] The composition of the present invention, when administered or ingested by a subject, can increase the amount of butyrate produced by butyrate-producing bacteria in the intestines and increase the butyrate concentration in the intestines, compared to cases where the composition of the present invention is not administered or ingested, or when human milk oligosaccharides or H. coagulans are administered or ingested alone. The butyrate produced in the intestines regulates the intestinal environment by making the intestines slightly acidic, creating an environment where so-called beneficial bacteria such as lactic acid bacteria and bifidobacteria can thrive, while suppressing the growth of harmful bacteria. Furthermore, it can promote physiological effects such as strengthening the intestinal barrier function, improving the absorption of minerals such as calcium and magnesium, and anti-inflammatory effects, thereby regulating the intestinal health of the subject. In other words, the composition of the present invention has an intestinal regulating effect and can be used as an intestinal regulator.

[0042] Because the composition of the present invention has the effects described above, it can be used in a method for increasing the butyrate concentration in the intestines of a target subject, and / or in a method for regulating bowel function in a target subject. The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0043] 1. Preparation of the culture medium: The culture medium for anaerobic bacteria was prepared based on the composition of Microorganisms Medium No. 104 (PYG MEDIUM modified; CATALOGUE) of the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, and according to its preparation method. After sterilization of the culture medium, a reducing agent (L-Cysteine ​​Hydrochloride, L-Ascorbic Acid Sodium Salt), which had been pre-filtered and sterilized using a 0.22 μm filter (Merck Millipore), was added.

[0044] 2. Preparation of Human Milk Oligosaccharides As human milk oligosaccharides, 3'-SL (manufactured by Biosynth, 3'-Sialyllactose sodium salt code OS04397), 6'-SL (manufactured by Biosynth, 6'-Sialyllactose sodium salt code OS04398), and 2'-FL (manufactured by Biosynth, 2'-Fucosyllactose-Synthetic code OF06739) were used. 3'-SL, 6'-SL, and 2'-FL were each dissolved in distilled water and then filter-sterilized through a 0.22 μm filter (manufactured by Merck Millipore). After filtration, they were each added to the medium so that the final concentration in the medium was 0.2%, and medium, 3'-SL + medium, 6'-SL + medium, and 2'-FL + medium were prepared. After preparation, they were anaerobically replaced overnight in an anaerobic chamber (Te-Her type ANAEROBOX ANX-4 model manufactured by Hirasawa Co., Ltd., using a mixed gas (N ２ base, CO ２ 10%, H ２ less than 4%)).

[0045] 3. Obtaining Cells of H. coagulans The spore cells of H. coagulans SANK70258 were suspended in sterile water and then spread on a previously prepared agar medium plate (Tryptose Soya agar medium (code 05516 manufactured by Shimadzu Diagnostics), 0.3% sucrose), and statically cultured at 40 °C for 20 hours under aerobic conditions using an incubator (Bioshaker BR-53FP manufactured by TAITEC). After culturing, the grown cells were suspended in sterilized physiological saline to obtain a cell suspension of H. coagulans.

[0046] 4. Preparation of Fecal Suspension Fecal samples were provided by 8 healthy Japanese people aged 20 to 59 years old who had not taken antibiotics or probiotic preparations for 2 weeks before stool collection, and those that had been cryopreserved were used (Ethical Review Committee Approval Number of Mitsubishi Chemical Group Human Tissue Research, etc.: Research Ethics C-23-001-03).

[0047] After thawing, the frozen fecal samples were centrifuged at 8,000 x g for 5 minutes using a centrifuge (MDX-310F, manufactured by Tommy Seiko Co., Ltd.). The precipitate was then centrifuged in an anaerobic chamber and treated with 0.1 M phosphate-buffered saline (pH 6.8, 0.1 M NaH). ２ PO ４ and 0.1 M Na ２ HPO ４ The suspension was suspended in a mixture of liquids in a volume ratio of 51.0:49.0. The turbidity of the suspension was measured at a wavelength of 600 nm using a spectrophotometer (Thermo Scientific GENESYS30).

[0048] 5. Initiating the culture, 0.6 mL each of anaerobic culture medium, 3'-SL, 6'-SL, or 2'-FL supplemented medium is dispensed into a 96-well plate (Treff deep-well plate code 0310200) in an anaerobic chamber. Then, either the prepared fecal sample alone is inoculated (hereinafter referred to as "Control", "3'-SL", "6'-SL", or "2'-FL"), or the prepared fecal sample and H. coagulans SANK70258 are inoculated (hereinafter referred to as "H. coagulans", "3'-SL + H. coagulans", "6'-SL + H. coagulans", or "2'-FL + H. coagulans"). The fecal suspension should be at a final concentration of OD. ６００ The vaccine was administered so that the ratio was 0.2, and H. coagulans SANK70258 had a final concentration of 1 × 10⁻¹⁰. ７ Inoculation was performed to a cell / mL ratio. After inoculation, a gas-permeable seal (Thermo Scientific AB-0718) was attached to the top surface of the 96-well plate, and it was placed in an aneurobox (square jar A-110). Anaerobic conditions were maintained by setting an aneuropack Kenki (A-06), and shaking culture was started at 37°C and 180 rpm in a constant temperature shaking incubator (TAITEC Bioshaker BR-53FP).

[0049] 6. Acquisition of Culture Medium and Organic Acid Analysis The culture medium was collected 48 hours after the start of culture, filtered, and the resulting filtrate was used for organic acid analysis. The filtrate of the obtained culture medium was subjected to high-performance liquid chromatography (HPLC, Hitachi High-Tech Corporation) equipped with an MCI GEL column (CK08EH, Mitsubishi Chemical Corporation) and an RI detector (L-2490, Hitachi High-Tech Corporation) to measure the concentration of organic acids (acetic acid, propionic acid, and butyric acid). The HPLC analysis was performed at a flow rate of 0.6 mL / min, using a 0.1 (wt)% phosphoric acid aqueous solution as the mobile phase, and at 50°C.

[0050] 7. Results of Organic Acid Analysis Figure 1 shows the results of the measurement of butyric acid concentration in the test samples. Compared to the addition of H. coagulans alone ("H. coagulans") or human milk oligosaccharide alone ("3'-SL", "6'-SL", or "2'-FL"), the addition of human milk oligosaccharide and H. coagulans together ("3'-SL + H. coagulans", "6'-SL + H. coagulans", "2'-FL + H. coagulans") showed an increase in butyric acid production (higher values ​​were observed for both the median (horizontal line in the box) and the mean (black diamond)).

Claims

1. A composition containing human milk oligosaccharides and Heyndrickxia coagulans, used to increase butyrate concentration in the intestines.

2. The composition according to claim 1, wherein the human milk oligosaccharide is one or more selected from the group consisting of acidic human milk oligosaccharides and salts thereof.

3. The composition according to claim 1, wherein the human milk oligosaccharide is one or more selected from the group consisting of 3'-sialyl lactose, 6'-sialyl lactose, and salts thereof.

4. The composition according to claim 1, which is an oral composition.

5. The composition according to claim 1, which is a food, beverage, supplement, animal feed, feed additive, or pharmaceutical.

6. A composition according to any one of claims 1 to 5, which is for regulating bowel function.

7. A method for increasing the butyrate concentration in the intestines of a non-human animal, comprising administering human milk oligosaccharides and Heyndrickxia coagulans to the non-human animal.

8. A method (excluding medical procedures) for increasing the butyrate concentration in the intestines of a subject, comprising administering human milk oligosaccharides and Heyndrickxia coagulans to the subject.

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