WO2025005236A1 - Fusobacterium controlling composition and use thereof - Google Patents

Abstract

The present invention addresses the problem of controlling Fusobacterium with a method that is considered to have little influence on beneficial bacteria. Provided is a Fusobacterium controlling composition comprising water soluble dietary fiber. The water soluble dietary fiber is preferably isomaltodextrin.

Description

Composition for controlling Fusobacterium genus bacteria and use thereof

The present invention relates to a composition for controlling bacteria of the genus Fusobacterium and its use.

Fusobacterium bacteria are present in the human oral cavity and intestines, and are known to be the causative agent of periodontal disease. Fusobacterium nucleatum is also known to cause opportunistic infections, and its involvement in colon cancer has recently been suggested (Non-Patent Document 1, etc.). Furthermore, F. nucleatum has been isolated from gastrointestinal infections such as appendicitis (Non-Patent Document 2), and it has recently been suggested to be associated with inflammatory bowel disease (Non-Patent Documents 3, 4, 5).

On the other hand, isomaltodextrin is a type of water-soluble dietary fiber. Water-soluble dietary fiber or isomaltodextrin, sometimes in combination with other dietary fibers, is known to have several actions related to the digestive tract and defecation. For example, Patent Document 1 describes a maximum digestive tract transit time improver that contains one or more of indigestible sugars, low-viscosity water-soluble dietary fibers, and magnesium compounds. Patent Document 2 describes a composition for promoting the growth of Fusicatenibacter bacteria, which contains water-soluble dietary fiber as an active ingredient, and lists isomaltodextrin as an example of water-soluble dietary fiber. Furthermore, Patent Document 3 describes a composition for preventing, treating, or suppressing intestinal disorders, which is characterized by containing three or more types of indigestible carbohydrate ingredients selected from oligosaccharides (excluding chitin oligosaccharides) and/or dietary fibers, and preferred examples of indigestible dietary fibers include inulin, guar gum hydrolyzate, psyllium seed gum, indigestible dextrin, isomaltodextrin, cellulose, β-glucan, glucomannan, and agarose.

It has been reported that the anti-inflammatory effect of isomaltodextrin on dextran sulfate sodium (DSS)-induced colitis was evaluated in a mouse model (Non-Patent Document 6). This report stated that, although isomaltodextrin was unable to prevent weight loss or colon shortening induced by DSS, it did tend to reduce the inflammatory cytokines TNF-α and IL-6, inhibit the expression of IL-1β, MCP-1, and IL-17, and increase the anti-inflammatory cytokine IL-10, and further stated that the anti-inflammatory effect of isomaltodextrin is mediated by the inhibition of the expression of Toll-like receptor-4.

Furthermore, Patent Document 4 describes a preparation for dieting and/or improving intestinal flora, characterized in that Bifidobacterium breve, Bifidobacterium longum, and N-acetylglucosamine are encapsulated in an acid-resistant hard capsule, and describes that in the group administered the preparation of the examples, a significant increase in Lachnospiraceae sp. and Roseburia sp. and a significant decrease in Fusobacterium sp., Prevotella sp., and Enterobacteriaceae sp. were confirmed. Patent Document 5 describes a composition containing Bifidobacterium bacteria as an active ingredient for inhibiting the adsorption of Fusobacterium bacteria to mucosal cells, and a pharmaceutical composition containing Bifidobacterium bacteria as an active ingredient for preventing or treating a disease or symptom caused by inflammation of mucosal cells and selected from the group consisting of ulcerative colitis, colon cancer, esophageal cancer, premature birth, and periodontal disease. Non-Patent Document 7 describes that when subjects were given 15 g/day of oligofructose for 15 days, bacteroides, clostridia, and fusobacteria were reduced. Patent Document 6 describes a dietary fiber preparation that contains a first soluble fiber and a second soluble fiber and exhibits a prebiotic activity greater than the individual prebiotic activities of the first soluble fiber and the second soluble fiber, and fructooligosaccharides (FOS) are given as an example of the second soluble fiber. This document also describes that FOS can promote intestinal health by enhancing positive immune responses by promoting the growth of bifidobacteria and suppressing the growth of pathogenic bacteria that may cause diarrhea.

International Publication WO2006/054710 JP 2020-83830 A JP 2020-178685 A JP 2016-166169 A International Publication No. 2019/117212 (Patent Publication No. 2020-178685) JP 2012-111771 A

C. A. Brennan et ai. Fusobacterium nucleatum - symbiont, opportunist and oncobacterium. Nat Rev Microbiol. 2019;17(3):156-166. doi: 10.1038/s41579-018-0129-6. Metzger Z et al. Synergistic pathogenicity of Porphyromonas gingivalis and Fusobacterium nucleatum in the mouse subcutaneous chamber model. J. Endod 35, 86-94 (2009). [PubMed: 19084132] Strauss J et al. Invasive potential of gut mucosa-derived Fusobacterium nucleatum positively correlates with IBD status of the host. Inflamm. Bowel Dis 17, 1971-1978 (2011). [PubMed: 21830275] Gevers D et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 15, 382-392 (2014). [PubMed: 24629344] Pascal V et al. A microbial signature for Crohn’s disease. Gut 66, 813-822 (2017). [PubMed: 28179361] K. Majumder et. al. Intervention of Isomaltodextrin Mitigates Intestinal Inflammation in a Dextran Sodium Sulfate-Induced Mouse Model of Colitis via Inhibition of Toll-like Receptor-4. J Agric Food Chem. 2017;65(4):810-817. doi: 10.1021/acs.jafc.6b04903. Glenn R. Gibson et. al. Selective Stimulation of Bifidobacteria in the Human Colon by Oligofructose and Inulin. Gastroenterology 1995;108:975-982

There are no known effective materials that can control Fusobacterium bacteria.

In the intestines, Fusobacterium bacteria form a complex symbiotic system with other intestinal bacteria, making it difficult to selectively control Fusobacterium bacteria. For example, administration of antibiotics is thought to reduce not only Fusobacterium bacteria but also the number of beneficial bacteria. It is desirable to control Fusobacterium bacteria using a method that is thought to have minimal impact on beneficial bacteria.

The present invention provides the following:
[1] A composition for controlling bacteria belonging to the genus Fusobacterium, comprising water-soluble dietary fiber.
[2] The composition according to 1, wherein the water-soluble dietary fiber is a low-viscosity water-soluble dietary fiber.
[3] The composition according to 1 or 2, wherein the water-soluble dietary fiber is a hyperbranched α-glucan.
[4] The composition according to any one of claims 1 to 3, wherein the water-soluble dietary fiber is isomaltodextrin.
[5] The composition according to any one of claims 1 to 4, wherein the control of bacteria belonging to the genus Fusobacterium is a reduction in the number of bacteria belonging to the genus Fusobacterium.
[6] The composition according to any one of claims 1 to 5, for use as a prebiotic or synbiotic.
[7] The composition described in any one of 1 to 6 for treating at least one of inflammatory bowel disease and colorectal cancer via control of bacteria belonging to the genus Fusobacterium.
[8] A composition for treating colon cancer, comprising a water-soluble dietary fiber, preferably an α-glucan, more preferably a hyperbranched α-glucan.
[9] The composition according to 8, wherein the hyperbranched α-glucan is isomaltodextrin.
[10] The composition according to 8 or 9, for administration to a subject having a bacterium belonging to the genus Fusobacterium in its intestines.
[11] A pharmaceutical composition comprising isomaltodextrin.
[12] The pharmaceutical composition according to 13, for use in an intestinal disease.
[13] An enteric formulation comprising isomaltodextrin.

[14] A method for controlling bacteria belonging to the genus Fusobacterium in the intestine of a subject, or a method for reducing the risk of at least one of inflammatory bowel disease and colorectal cancer in a subject, comprising the steps of:
A step of having a subject ingest a composition containing water-soluble dietary fiber based on the presence or absence or amount of bacteria belonging to the genus Fusobacterium in the intestine of the subject.
[15] A method for suggesting ingestion of a composition to a subject, a method for supporting a subject's dietary habits, or a method for supporting a subject's health, comprising the steps of:
A step of suggesting intake of a composition containing water-soluble dietary fiber based on the presence or absence or amount of bacteria belonging to the genus Fusobacterium in the subject's intestine.

[21] A composition comprising water-soluble dietary fiber for use in a method for controlling bacteria belonging to the genus Fusobacterium. Use of water-soluble dietary fiber in the manufacture of a composition for controlling bacteria belonging to the genus Fusobacterium. A method or non-therapeutic method for controlling bacteria belonging to the genus Fusobacterium, comprising a step of administering a composition comprising water-soluble dietary fiber to a subject. Use or non-therapeutic use of a composition comprising water-soluble dietary fiber for controlling bacteria belonging to the genus Fusobacterium.
[22] The composition, use in manufacture, method or non-therapeutic method, or use or non-therapeutic use according to 21, wherein the water-soluble dietary fiber is a low-viscosity water-soluble dietary fiber.
[23] The composition, use in production, method or non-therapeutic method, or use or non-therapeutic use according to 21 or 22, wherein the water-soluble dietary fiber is a hyperbranched α-glucan.
[24] The composition, use in manufacture, method or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 23, wherein the water-soluble dietary fiber is isomaltodextrin.
[25] The composition, use in production, method or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 24, wherein the control of bacteria belonging to the genus Fusobacterium is a reduction in the number of bacteria belonging to the genus Fusobacterium.
[26] The composition, use in manufacture, method or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 25, wherein the composition is for use as a prebiotic or synbiotic.
[27] A composition, use in manufacture, method or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 26 for the treatment of at least one of inflammatory bowel disease and colorectal cancer via control of bacteria belonging to the genus Fusobacterium.
[28] A composition comprising water-soluble dietary fiber, preferably α-glucan, more preferably hyperbranched α-glucan, for use in a method for treating colorectal cancer. Use of water-soluble dietary fiber, preferably α-glucan, more preferably hyperbranched α-glucan, in the manufacture of a composition for treating colorectal cancer. A method or non-therapeutic method for treating colorectal cancer, comprising the step of administering to a subject a composition comprising water-soluble dietary fiber, preferably α-glucan, more preferably hyperbranched α-glucan. Use or non-therapeutic use of a composition comprising water-soluble dietary fiber, preferably α-glucan, more preferably hyperbranched α-glucan, for treating colorectal cancer.
[29] The composition, use in production, method or non-therapeutic method, or use or non-therapeutic use according to 28, wherein the hyperbranched α-glucan is isomaltodextrin.
[30] The composition, use in production, method or non-therapeutic method, or use or non-therapeutic use according to 28 or 29, wherein the composition is for administration to a subject having a bacterium belonging to the genus Fusobacterium in its intestine.
[31] A pharmaceutical composition comprising isomaltodextrin.
[32] The pharmaceutical composition according to 11, for use in an intestinal disease.
[33] An enteric formulation comprising isomaltodextrin.

Using isomaltodextrin as a prebiotic can provide a means to reduce Fusobacterium. Furthermore, reducing Fusobacterium can help prevent and improve colon cancer and inflammatory bowel disease.

Number of Fusobacterium bacteria after cultivation *P < 0.05 versus Control, Wilcoxon rank sum test

The present invention relates to a composition for controlling Fusobacterium bacteria, which contains water-soluble dietary fiber as an active ingredient.

[Active ingredient]
The composition of the present invention contains water-soluble dietary fiber as an active ingredient. In the present invention, dietary fiber is defined as the total of indigestible components in food that are not digested by digestive enzymes. Proteins, lipids, and carbohydrates are decomposed (digested) by digestive enzymes in the digestive tract and absorbed into the body from the small intestine, but dietary fiber is a component that passes through the small intestine without being affected by the action of digestive enzymes and reaches the large intestine. Dietary fiber includes starch (indigestible dextrin) and oligosaccharides that are difficult to digest. Dietary fiber can be broadly classified into water-soluble and insoluble. The composition of the present invention contains water-soluble dietary fiber. The composition may contain only one type of water-soluble dietary fiber as an active ingredient, or may contain two or more types. It has been reported that water-soluble dietary fiber is easily utilized by intestinal bacteria. Water-soluble dietary fiber has many advantages, such as being easily dispersed in the system during production and being prevented from remaining on the production line or adhering to the filter. Containing an active ingredient means that the ingredient is used in the composition in an effective amount to exert the intended function, and that it is specified in the labeling as an ingredient that contributes to the intended purpose, etc. In functional food products, active ingredients are sometimes called functional ingredients (ingredients that contribute to specific health purposes (excluding those related to reducing the risk of disease)).

Examples of water-soluble dietary fiber include isomaltodextrin, barley beta-glucan, polydextrose, resistant dextrin, resistant glucan, alginic acid, fucoidan, guar gum hydrolysate, inulin, and acacia dietary fiber.

In one embodiment, the composition contains, as an active ingredient, low-viscosity water-soluble dietary fiber from among water-soluble dietary fibers. Low-viscosity water-soluble dietary fiber refers to water-soluble dietary fiber that has low viscosity in an aqueous solution. Specifically, it refers to water-soluble dietary fiber that has a viscosity of 100 mPa·s or less when measured in a 1% aqueous solution at a product temperature of 20°C using a B-type viscometer.

In one embodiment, the composition contains, as an active ingredient, a highly branched α-glucan, which is a low-viscosity water-soluble dietary fiber. An example of this is isomaltodextrin.

Isomaltodextrin is a water-soluble dietary fiber produced by the action of enzymes on starch. Compared to regular dextrin obtained by hydrolyzing starch, it is a dextrin that has a higher proportion of branched structures with α-1,6 glucoside bonds and does not contain glucose with β-glucosidic bonds. Isomaltodextrin is composed only of glucose with α-bonds, and since it has more branches than the starch that is the raw material, it is considered a type of hyperbranched α-glucan.

In a preferred embodiment, isomaltodextrin with a DE of 10 or less is used. 9 or less is more preferable, 8 or less is even more preferable, and 7.0 to 7.5 is even more preferable. Here, DE means dextrose equivalent, and is a value that represents a relative scale when the reducing power of dextrose (glucose) is set to 100. Isomaltodextrin has a lower viscosity than maltodextrin with the same DE.

The weight average molecular weight of the isomaltodextrin used is preferably 500 to 10,000, more preferably 3,000 to 7,000, and even more preferably 4,500 to 5,500. The number average molecular weight of the isomaltodextrin is preferably 1,000 to 5,000, and more preferably 2,000 to 3,000.

[Application]
(Function/Action)
The composition of the present invention can be used to control bacteria belonging to the genus Fusobacterium (sometimes simply referred to as Fusobacterium bacteria). In a preferred embodiment, the composition can be used to control Fusobacterium bacteria in the intestinal flora. In a more preferred embodiment, the composition can be used to control Fusobacterium bacteria in the human intestinal flora. Controlling Fusobacterium bacteria in the intestinal flora refers to controlling bacteria belonging to the genus Fusobacterium in a state in which various bacteria living in the intestine are present. Controlling bacteria refers to controlling the number of bacteria, regulating the intestinal flora including Fusobacterium bacteria, and helping to regulate the intestinal flora including Fusobacterium bacteria. In a preferred embodiment, the composition is for reducing the number of Fusobacterium bacteria and for helping to reduce the number of Fusobacterium bacteria.

The intestine refers to the digestive organ in humans and animals where bacteria reside and where ingested food is digested and absorbed. The intestine includes the small intestine and large intestine, and is preferably the large intestine.

Examples of Fusobacterium include:
Fusobacterium nucleatum subsp. nucleated,
Fusobacterium nucleatum subsp. animal,
Fusobacterium nucleatum subsp. vincentius (fusiform),
Fusobacterium nucleatum subsp. polymorphic,
Fusobacterium periodonticum,
Fusobacterium russii,
Fusobacterium canifelinum,
Fusobacterium simiae,
Fusobacterium necrophorum,
Fusobacterium equinus,
Fusobacterium gonidiaformans,
Fusobacterium deadly,
Fusobacterium necrogenes,
Fusobacterium ulcerans,
Fusobacterium varium,
Fusobacterium perfoetens

In the context of the present invention, the term "Fusobacterium" refers to bacteria that are identified as belonging to the genus Fusobacterium by molecular phylogenetic analysis based on the 16S rRNA gene. The criteria for determining genera by molecular phylogenetic analysis based on the 16S rRNA gene are well known to those skilled in the art (Stackebrandt E, Ebers J. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 2006;33:152-155.).

Whether or not a certain component controls Fusobacterium bacteria in the intestinal flora can be evaluated as follows. Feces provided by a healthy individual is added to an appropriate medium, and after culturing for a certain period of time if necessary, the component to be evaluated is added and cultured under appropriate conditions (for example, culturing at 37 ° C under anaerobic conditions similar to the conditions in the intestine for 16 hours). At this time, if the feces does not contain Fusobacterium bacteria, any available Fusobacterium bacteria is added at the start of the culture and cultured. The Fusobacterium bacteria to be added is, for example, F. nucleatum JCM8532T. The amount of addition can be appropriately determined depending on the conditions of the experiment, for example, 0.1 to 10 × 10 ⁴ cfu / mL. Then, the number of Fusobacterium bacteria in the flora in the culture is measured. The measurement results can be compared with the measurement results of a culture cultured under the same conditions except that a control (for example, sterilized water) was added instead of the component to be evaluated, and judgment can be made.

The number of Fusobacterium bacteria contained in the bacterial flora can be determined by a known method. One of the preferred methods is to perform 16S metagenomic analysis (sequence analysis of 16S rRNA gene amplicon) on DNA extracted from the culture. When performing 16S metagenomic analysis, DNA can be extracted using a commercially available kit. The genomic region to be analyzed is not particularly limited as long as it can identify bacteria, but the V3-V4 region of the 16S rRNA gene can be used. Methods such as primers, amplification conditions, and amplicon purification for analyzing bacteria can also be methods well known to those skilled in the art. It is preferable to decipher the sequence using a next-generation sequencer with higher performance. A next-generation microbiome bioinformatics platform such as QIIME 2 ^TM can be used to analyze the obtained data. For 16S metagenomic analysis, those skilled in the art can refer to information such as Sanschagrin S, Yergeau E. Next-generation sequencing of 16S ribosomal RNA gene amplicons. J Vis Exp. 2014;(90):51709. Published 2014 Aug 29. doi:10.3791/51709.

In general, the intestinal flora contained in the flora may include, but is not limited to, the following bacteria. In one embodiment, the composition can maintain the numbers of Parabacteroides bacteria and Bifidobacterium bacteria while controlling the number of Fusobacterium bacteria. In another embodiment, the composition does not affect any of the following bacteria, or has only a small effect, while controlling the number of Fusobacterium bacteria.
Bacteroides uniformis, Alistipes putredinis, Parabacteroides merdae, Dorea longicatena, Ruminococcus bromii L2-63, Bacteroides caccae, Clostridium sp SS2-1, Bacteroides thetaiotaomicron VPI-5482, Eubacterium hallii, Ruminococcus torques L2-14, unknown sp SS3 4, Ruminococcus sp SR1 5, Faecalibacterium prausnitzii SL3 3, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Bacteroides vulgatus ATCC 8482, Roseburia intestinalis M50 1, Bacteroides sp. 2_1_7, Eubacterium siraeum 70 3, Parabacteroides distasonis ATCC 8503, Bacteroides sp. 9_1_42FAA, Bacteroides ovatus, Bacteroides sp. 4_3_47FAA, Bacteroides sp. 2_2_4, Eubacterium rectale M104 1, Bacteroides xylanisolvens XB1A, Coprococcus comes SL7 1, Bacteroides sp. D1, Bacteroides sp. D4, Eubacterium ventriosum, Bacteroides dorei, Ruminococcus obeum A2-162, Subdoligranulum variabile, Bacteroides capillosus, Streptococcus thermophilus LMD-9, Clostridium leptum, Holdemania filiformis, Bacteroides stercoris, Coprococcus eutactus, Clostridium sp M62 1, Bacteroides eggerthii, Butyrivibrio crossotus, Bacteroides finegoldii, Parabacteroides johnsonii, Clostridium sp L2-50, Clostridium nexile, Bacteroides pectinophilus, Anaerotruncus colihominis, Ruminococcus gnavus, Bacteroides intestinalis, Bacteroides fragilis 3_1_12, Clostridium asparagiforme, Enterococcus faecalis TX0104, Clostridium scindens, Blautia hansenii (Nature 464, 59-65 (2010) doi:10.1038/nature08821)

The Fusobacterium bacteria of the present invention are bacteria present in the human oral cavity and intestines, and are known to be causative bacteria of periodontal disease. Fusobacterium nucleatum is also known to cause opportunistic infections, and its involvement in colon cancer has recently been pointed out (Non-Patent Document 1, etc.). Furthermore, F. nucleatum has been isolated from gastrointestinal infections such as appendicitis (Non-Patent Document 2), and it has recently been suggested that it is also associated with inflammatory bowel disease (Non-Patent Documents 3, 4, 5). Therefore, in one embodiment, the composition can be used for the treatment of any of the following conditions selected from the group consisting of inflammatory bowel disease and colon cancer.

Inflammatory bowel diseases include ulcerative colitis and Crohn's disease.

When used for the treatment of inflammatory bowel disease, the composition can be configured to be free of any of the following:
Oligosaccharides (e.g., raffinose, stachyose, galactooligosaccharides, isomaltooligosaccharides, lactofructose oligosaccharides, lactulose, xylooligosaccharides, agarooligosaccharides, mannooligosaccharides, or fructooligosaccharides), inulin, pectin, processed pectin, guar gum, decomposed guar gum, psyllium seed gum, karaya gum, tragacanth gum, gum arabic, resistant starch, resistant dextrin, polydextrose, cellulose, hemicellulose, soybean polysaccharides, β-glucan, glucomannan, galactomannan, chondroitin sulfate, hyaluronic acid, levan, lignin, alginic acid and its salts, agarose, and chitosan. Fructooligosaccharides are also called short-chain fructose polymers or oligofructose (Patent Document 6, Non-Patent Document 7).

When used to treat inflammatory bowel disease, in a particularly preferred embodiment, the composition contains isomaltodextrin as an active ingredient and may contain no other dietary fiber.

When used to treat colon cancer, the composition may contain various water-soluble dietary fibers as active ingredients, but in a particularly preferred embodiment, the composition contains isomaltodextrin as an active ingredient and may contain no other dietary fibers.

The composition can also be used to treat diseases or conditions that can be improved by controlling the proliferation of Fusobacterium bacteria in the intestine. There can be a variety of such diseases or conditions.

In the present invention, when referring to a disease or condition, the term "treatment" includes reducing the risk of onset, delaying onset, prevention, treatment, and halting or delaying progression. It also includes helping to improve from a disease or condition, alleviating a disease or condition, and reducing the risk of developing a disease or condition. Treatment includes radical treatment (treatment to remove the cause) and symptomatic treatment (treatment to improve symptoms). Actions for improvement or treatment include medical actions performed by doctors and nurses and midwives under the instructions of doctors, as well as non-therapeutic actions performed by persons other than doctors, such as pharmacists, nutritionists (including registered dietitians and sports nutritionists), public health nurses, midwives, nurses, clinical laboratory technicians, sports instructors, pharmaceutical manufacturers, pharmaceutical distributors, food manufacturers, food distributors, etc. Prevention or reduction of the risk of onset also includes recommendations for the intake of specific foods and nutritional guidance (including nutritional guidance necessary for the medical treatment of injured or sick people, and nutritional guidance for maintaining and promoting health).

(subject)
The composition of the present invention is suitable for administration to subjects for whom it is desirable or necessary to control Fusobacterium bacteria in the intestine, particularly to reduce the number of bacteria; subjects with any one selected from the group consisting of inflammatory bowel disease and colorectal cancer; and subjects with a disease or condition that can be improved by controlling the proliferation of Fusobacterium bacteria in the intestine. In the present invention, the term "administer" is used not only to mean administering a pharmaceutical product to a subject, but also to mean having a subject ingest a food product other than a pharmaceutical product. "Administer" can be read as "intake" (or "ingestion"), and "administer" can be read as "administer".

Subjects for whom it is desirable or necessary to control Fusobacterium bacteria in the intestine include subjects in whom Fusobacterium bacteria have been detected in at least one of the intestine and feces, and subjects in whom a relatively high number of Fusobacterium bacteria have been detected in at least one of the intestine and feces. Subjects for whom it is desirable or necessary to control Fusobacterium bacteria in the intestine also include subjects who have been found to have a relatively high number of Fusobacterium bacteria by any test or analysis such as an intestinal flora test, a test of substances in the feces, or fecal metabolome analysis, subjects in whom Fusobacterium bacteria have been detected, subjects who have been found to be at high risk of inflammatory bowel disease, subjects who have been found to be at high risk of colorectal cancer, and subjects who have been suggested to take foods, beverages, or medicines capable of controlling Fusobacterium bacteria by a doctor, nurse, pharmacist, nutritionist, or the like, or by a computer program based on the test or analysis results. The subject also includes a subject who wishes to reduce the number of Fusobacterium bacteria in at least one of the intestines and feces, a subject who wishes to reduce the risk of colon cancer, and a subject who wishes to reduce the risk of inflammatory bowel disease. In one embodiment, the subject is a subject in which Fusobacterium bacteria have been detected in at least one of the intestines and feces, or a subject who has been recognized to have a relatively large number of Fusobacterium bacteria. The number of Fusobacterium bacteria detected or relatively large in at least one of the intestines and feces refers to, for example, the case where a culture solution containing 0.1% (w/v) concentration of stool collected from a subject is cultured under anaerobic conditions at 37°C for 16 hours, and the number of Fusobacterium bacteria in the culture solution is 1 x ¹⁰³ cells/mL or more, preferably 1 x ¹⁰⁴ cells/mL or more, more preferably 1 x ¹⁰⁴ cells/mL or more, and even more preferably 5 x ¹⁰⁵ cells/mL or more. Alternatively, it refers to the case where the number of Fusobacterium bacteria in feces collected from a subject is 1 x ¹⁰⁴ cells/g or more, preferably 5 x ¹⁰⁴ cells/g or more, more preferably 1 x ¹⁰⁵ cells/g or more, and even more preferably 5 x ¹⁰⁵ cells/g or more. The number of bacteria is measured, for example, by quantitative PCR.

In one embodiment, the subject is healthy (not diagnosed with a disease by a physician). The subject may be a human or a non-human animal.

The age of the subject is not particularly limited, and the subject may be, for example, a newborn (within 28 days of birth); an infant (less than 1 year of age); a toddler (1 to 6 years of age); a child (7 years or older, but less than 15 years of age); an adult (15 years or older); or a person 65 years or older. In a preferred embodiment, the subject is a relatively elderly person, for example, a person 60 years or older, a person 65 years or older, a person 70 years or older, or a person 75 years or older. It has been reported that the number of Fusobacterium bacteria in the intestine increases with age (e.g., Odamaki et al. BMC Microbiology (2016) 16:90
DOI 10.1186/s12866-016-0708-5).

[Composition]
(Food compositions, etc.)
The composition of the present invention can be a food composition or a pharmaceutical composition. Food and pharmaceutical products include not only those for humans but also those for animals other than humans, unless otherwise specified. Food includes general food, functional food, nutritional composition, and also therapeutic food (for the purpose of treatment. Prepared based on a menu prepared by a nutritionist or the like according to a doctor's meal prescription), dietary therapy food, ingredient-adjusted food, nursing care food, and food for medical support, unless otherwise specified. Food includes not only solid food but also liquid food, such as beverages, energy drinks, liquid food, and soup, unless otherwise specified. Functional foods refer to foods that can impart a certain functionality to the living body, and include, for example, foods for specified health uses (including conditionally designated health foods [specified health foods]), foods with functional claims, health functional foods including foods with nutritional functions, foods for special dietary uses, dietary supplements, dietary supplements, supplements (for example, tablets, coated tablets, sugar-coated tablets, enteric-coated agents, enteric preparations, enteric preparation capsules (enteric soft capsules, enteric hard capsules, colon delivery capsules, etc.), liquids, and other dosage forms), dietary supplements, food supplements, medical foods (following the definition of the U.S. Food and Drug Administration (FDA)), beauty foods (for example, diet foods), and other health foods in general. In addition, in the present invention, "functional foods" includes health foods to which a health claim based on the food standards of Codex (FAO/WHO Joint Food Standards Commission) is applied. A food supplement is a concentrated form of nutrients or other substances, either alone or in combination, that supplements the normal diet and has a nutritional or physiological effect, and is labeled as a food supplement. A dietary supplement is a product (other than tobacco) intended to supplement the diet, that contains one or more of the ingredients covered, and is labeled as a dietary supplement.

(Route of administration, etc.)
The composition of the present invention may be administered orally, parenterally, for example, via a tube (gastrostomy, enterostomy), or intranasally, but is preferably administered orally.

The composition can be administered to a subject repeatedly, and can be administered to a subject continuously for an extended period of time. The period is not particularly limited, but in order to fully demonstrate the effect, it is preferable to administer the composition continuously for a relatively long period of time, for example, 3 days or more, 1 week or more, 2 weeks or more, 1 month or more, 3 months or more, 6 months or more, or 1 year or more.

The composition may be administered routinely, proactively, such as when there is a high risk, or when the need arises. The composition may be administered with a meal, before, after, or between meals, or at the onset of the disease or condition that it is desired to ameliorate with the composition.

(Dosage, Content)
The dosage of the composition of the present invention may be any amount that exerts the desired effect. The dosage may be appropriately determined taking into consideration various factors such as the age, weight, and symptoms of the subject.

The daily dosage of the composition may be at least 0.1 g of active ingredient (total amount if multiple active ingredients are included), preferably at least 0.3 g, more preferably at least 0.6 g, and even more preferably at least 1 g. The upper limit of the active ingredient per day, whatever the lower limit, may be no more than 30 g, no more than 25 g, no more than 20 g, no more than 18 g, no more than 17 mg, no more than 16 g, or no more than 15 g. When multiple active ingredients are included in the composition, the amount of active ingredient refers to the total amount of active ingredients included.

Dosage may be once a day or multiple times a day, for example 2 to 10 times. The amount of active ingredient per dose may be, for example, 0.01 g or more, preferably 0.3 g or more, more preferably 0.6 g or more, and even more preferably 1 g or more. The upper limit of the amount of active ingredient per dose, whatever the lower limit, may be 20 g or less, 18 g or less, 16 g or less, 14 g or less, 12 g or less, 10 g or less, or 5 g or less.

The content of the active ingredient in the composition may be appropriately determined depending on the form of the composition. For example, the content of the active ingredient per solid content of the composition may be 0.5% or more, preferably 1% or more, more preferably 2% or more, and even more preferably 3% or more. The upper limit of the active ingredient per solid content, whatever the lower limit, may be 20% or less, 18% or less, 16% or less, 13% or less, or 10% or less. In the present invention, % means % by mass, unless otherwise specified.

When the composition is a liquid such as a beverage, the content of the active ingredient may be, for example, 0.1% or more, preferably 0.3% or more, more preferably 0.6% or more, even more preferably 1% or more, and even more preferably 2% or more. The upper limit of the content of the active ingredient when it is a liquid, whatever the lower limit, may be 15% or less, 10% or less, 8% or less, 6% or less, 4% or less, or 3% or less.

When the composition is in the form of a tablet or a granular powder, the content of the active ingredient may be, for example, 10% or more, preferably 30% or more, more preferably 50% or more, and even more preferably 60% or more. When the composition is in the form of a tablet or a granular powder, the upper limit of the content of the active ingredient may be 95% or less, 90% or less, or may be 80% or less, regardless of the lower limit.

When the composition is a fermented milk such as yogurt, the content may be, for example, 2% or more, preferably 4% or more, and more preferably 10% or more. When the composition is a fermented milk such as yogurt, the upper limit of the content of the active ingredient may be 30% or less, 25% or less, or may be 15% or less, regardless of the lower limit.

(Other ingredients, additives)
In the present invention, the composition may contain other active ingredients or nutritional ingredients that are acceptable as foods or pharmaceuticals. Examples of such ingredients include lipids (e.g., milk fat, vegetable oils and fats, and medium-chain fatty acid-containing oils and fats), proteins (e.g., milk protein, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), heat-denatured whey protein, and enzyme-treated whey protein), amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, and valine), and sugars other than water-soluble dietary fiber. These include carbohydrates (glucose, sucrose, fructose, maltose, trehalose, erythritol, maltitol, palatinose, xylitol, dextrin), electrolytes (e.g., sodium, potassium, calcium, magnesium), vitamins (e.g., vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folic acid, pantothenic acid, and nicotinic acids), minerals (e.g., copper, zinc, iron, cobalt, manganese), antibiotics, and dietary fiber.

　In the present invention, the composition may contain a prebiotic other than water-soluble dietary fiber. Prebiotics can be defined as indigestible food ingredients that have a beneficial effect on the host and improve the host's health by selectively altering the growth and activity of specific bacteria in the large intestine. The composition may contain one type or two or more types of prebiotics other than water-soluble dietary fiber.

　There are no particular limitations on prebiotics other than water-soluble dietary fiber, so long as they do not interfere with the effects of the active ingredients contained in the composition. Examples of prebiotics other than water-soluble dietary fiber include galactooligosaccharides, fructooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, raffinose, lactulose, lactosucrose, soybean oligosaccharides, coffee oligosaccharides, insoluble dietary fiber, and gluconic acid.

The composition may further contain additives that are acceptable for use as food or medicine. Examples of such additives include inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, preservatives, buffers, pH adjusters, emulsifiers, stabilizers, sweeteners, antioxidants, flavors, acidulants, and natural products. More specifically, these include water, other aqueous solvents, pharma- ceutical acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium alginate, dextrin, water-soluble dextran, water-soluble dextrin, corn starch, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, magnesium stearate, precipitated calcium carbonate, talc, human serum albumin, mannitol, sorbitol, lactose, sucralose, lactose hydrate, candy powder, white sugar, stevia, aspartame, acesulfame potassium, citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, acetic acid, fruit juice, vegetable juice, etc.

(Dosage form/shape)
The food composition of the present invention may be prepared in any form, such as solid, liquid, mixture, suspension, powder, granule, paste, jelly, gel, capsule, etc. The food composition of the present invention may be prepared in any form, such as dairy products, supplements, confectionery, beverages, drinks, seasonings, processed foods, side dishes, soups, etc. More specifically, the composition of the present invention may be in the form of liquid food, semi-liquid food, jelly, gel, powder, prepared milk powder, prepared liquid milk, powdered milk/liquid milk for pregnant women and lactating women, fermented milk, bar, mousse, chocolate, biscuit, ice cream, fermented milk, lactic acid bacteria beverage, dairy beverage, milk beverage, soft drink, tablet, cheese, bread, biscuit, cracker, pizza crust, food for sick people, nutritional food, frozen food, processed food, etc., and may be in the form of granule, powder, paste, thick liquid, etc. for mixing with beverages or foods for administration. The granules and powders may be in the form of cubes or sticks (packed in a single serving). In the present invention, modified powdered milk, as defined in the Ministerial Ordinance on Compositional Standards, etc. of Milk and Dairy Products (hereinafter referred to as the "Milk, etc. Ordinance"), refers to raw milk, cow's milk, special milk, raw buffalo milk, or foods produced using these as ingredients, processed or as the main ingredient, to which nutrients necessary for infants have been added, and made into a powder. In the present invention, modified liquid milk, as defined in the Milk, etc. Ordinance, refers to raw milk, cow's milk, special milk, raw buffalo milk, or foods produced using these as ingredients, processed or as the main ingredient, to which nutrients necessary for infants have been added, and made into a liquid.

　Formulated milk powder or liquid milk (these are sometimes collectively called formulated milk powders. In addition, when explaining formulated milk powders, formulated milk powder is sometimes used as an example, but the explanation also applies to formulated liquid milk.) can be for infants, follow-up use (to supplement weaning food), low birth weight infants, children, adults, pregnant women, breastfeeding women, the elderly, those with allergies, those with lactose intolerance, and those with congenital metabolic disorders. Preferred examples of formulated milk powders are for infants, follow-up use, low birth weight infants, and children.

The enteric formulation and pharmaceutical composition of the present invention can be in any dosage form suitable for oral administration, including solid preparations such as tablets, granules, powders, pills, and capsules, liquid preparations such as solutions, suspensions, and syrups, gels, and aerosols.

(others)
In the production of the composition of the present invention, the stage of blending the active ingredient can be appropriately selected. The stage of blending is not particularly limited as long as it does not significantly impair the properties of the active ingredient. For example, the active ingredient can be blended by mixing it with the raw material. Alternatively, the active ingredient can be added at the final stage of production to produce a composition containing the active ingredient.

The composition of the present invention may display the intended use (use), and in one embodiment, it may display that administration is recommended for a specific subject. Examples of subjects to be displayed are as described above. In one embodiment, the composition of the present invention may display the function of the composition or active ingredient, or the usage method based on that function. For example, it may display that the composition can control the proliferation of Fusobacterium bacteria in the intestine, that the composition can inhibit the proliferation of Fusobacterium bacteria in the intestine, that the composition can treat a disease or condition that is improved by controlling (preferably inhibiting) the proliferation of Fusobacterium bacteria in the intestine, that the composition can be used as a prebiotic, that the composition can be used as a synbiotic (a combination of probiotics and prebiotics), or that the composition can treat inflammatory bowel disease or colon cancer. Note that a period such as "temporary" or "long-term" may be displayed at the beginning of each wording as appropriate.

　Display can be explicit or implicit. An example of explicit display is a direct statement on a tangible object such as the product itself, packaging, container, label, tag, etc., while an example of implicit display (which can also be called implied) includes advertising and promotional activities by place or means such as websites, storefronts, pamphlets, exhibitions, seminars such as media seminars, books, newspapers, magazines, television, radio, mail, email, and voice.

In one embodiment, the recommendation to consume the composition is displayed personally. Such a display can be made using a document (whether written or electronic) addressed to the subject, the subject's tablet, smartphone, personal computer, the subject's SNS, etc. Furthermore, such a display can be made together with the display of the results of any test or analysis, such as an intestinal flora test, a test of substances in the feces, and a fecal metabolome analysis, for the subject. It can also be used for precision nutrition (individualized nutrition, which refers to proposing appropriate meals (nutrition) according to an individual's constitution).

In one aspect, a method for controlling bacteria belonging to the genus Fusobacterium in the intestine of a subject, or a method for reducing the risk of at least one of inflammatory bowel disease and colorectal cancer in a subject, is provided, comprising a step of having the subject ingest a composition comprising water-soluble dietary fiber based on the presence or absence or amount of bacteria belonging to the genus Fusobacterium in the intestine of the subject. In another aspect, a method for suggesting to a subject to ingest a composition comprising water-soluble dietary fiber, a method for supporting a subject's diet, or a method for supporting a subject's health is provided, comprising a step of suggesting to a subject to ingest a composition comprising water-soluble dietary fiber based on the presence or absence or amount of bacteria belonging to the genus Fusobacterium in the intestine of the subject. These methods may include other steps, for example, any of the following:
collecting a fecal sample from the subject, or having the subject collect a fecal sample;
A step of analyzing the presence or amount of Fusobacterium bacteria in a fecal sample; a step of displaying the analysis results; a step of determining whether it is desirable or necessary for the subject to reduce the number of Fusobacterium bacteria based on the analysis results; a step of displaying the determination results; and a step of displaying a product which is a composition containing water-soluble dietary fiber.

The present invention will be explained in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[Control of Fusobacterium by soluble dietary fiber]

(1) Preparation of test solution 90 mg of isomaltodextrin (Fibrixa (registered trademark), loss on drying 8.0% or less, pH 3.5-6.5 of 10% aqueous solution, dietary fiber (enzyme-HPLC method) 80.0% or more (per solids), nutritional values (per 100 g): 216 kcal, carbohydrates 12.7 g, dietary fiber 82.7 g) was weighed out and dissolved in 1 mL of pure water of pH 4.5 in an anaerobic chamber. The test material was then transferred through a 0.22 μm filter into a tube in which the gas phase had been replaced in advance in an anaerobic chamber.

(2) Preparation of culture medium In an anaerobic chamber, 12 mg of feces provided by six healthy adults was weighed into an Eppendorf tube in which the gas phase had been replaced beforehand. Then, 500 μL of YCFA medium was added, homogenized, and YCFA medium was added to prepare fecal juice. The fecal concentration in the fecal juice was 0.1% (w/v). Then, 290 μL of fecal juice was added to each well of a 96-well plate, and 10 μL of the isomaltodextrin solution (final concentration 0.3% (w/v)) prepared in (1) or pure water was added to each well to form the test group (isomaltodextrin group) and the control group (water group), respectively.

(3) Culture The test group and the control group prepared in (2) above were cultured for 16 hours under anaerobic conditions at 37° C. Then, DNA was extracted from each culture solution using a Maxwell® RSC Instrument (Promega Corporation).

(4) Measurement of bacterial count Quantitative PCR was performed on DNA extracted from the culture solution in which the number of Fusobacterium bacteria had been measured in advance, and a calibration curve was created. Quantitative PCR was performed on the DNA obtained in (3) above, and the number of Fusobacterium bacteria was calculated based on the calibration curve. Then, the number of bacteria per mL of culture solution was corrected. The primer sequences used for quantitative PCR were as shown in Table 1. DNA extracted from Fusobacterium nucleatum subsp. nucleatum JCM 8532T (type species of Fusobacterium bacteria) was used as the standard.

For stool samples in which Fusobacterium genus bacteria were not detected prior to culture, Fusobacterium nucleatum JCM8532T was added at 1 x 10 ⁴ cfu/ml at the start of culture.

As a result, the number of Fusobacterium bacteria was reduced in all cultures to which isomaltodextrin was added compared to the control (Figure 1).

These results suggest that isomaltodextrin may reduce the number of Fusobacterium bacteria.

In this way, it can be said that water-soluble dietary fibers such as isomaltodextrin can be used to control Fusobacterium bacteria, particularly to reduce the number of bacteria in the intestinal flora. Fusobacterium bacteria have recently been suggested to be involved in colon cancer (Non-Patent Document 1, etc.), and F. nucleatum has been isolated from digestive infections such as appendicitis (Non-Patent Document 2), and it has recently been suggested to be associated with inflammatory bowel disease (Non-Patent Documents 3, 4, 5). Therefore, it can be said that water-soluble dietary fibers such as isomaltodextrin can be used to treat these diseases.

[Production Example 1: Tablet Candy]

Using Fibryxa, a tablet-type food composition for controlling bacteria belonging to the genus Fusobacterium was prepared according to the following formula in a conventional manner. Similar food compositions can be produced by adding indigestible dextrin or inulin instead of Fibryxa.

Granulated sugar 718 parts Concentrated fruit juice 50 parts Citric acid 60 parts Flavoring 20 parts Gelatin 10 parts Fiberixa 100 parts Calcium carbonate 15 parts Magnesium oxide 5 parts Potassium dihydrogen phosphate 22 parts

[Production Example 2: Powdered beverage]

Using Fiberixa, a powdered beverage-type food composition for controlling bacteria belonging to the genus Fusobacterium was prepared according to the following formula in a conventional manner. The composition can be prepared.

Anhydrous crystalline glucose 650 parts Citric acid granules 60 parts Tartaric acid 60 parts Sodium bicarbonate granules 55 parts Flavor 25 parts Fiberixa 100 parts Dolomite 50 parts

[Production Example 3: Beverage]

Using Fiberixa, the ingredients were mixed and dissolved in the following ratio, sealed in a container, and heated at 90°C for 5 minutes to obtain a prebiotic drink containing isomaltodextrin. Similar drinks can be produced by adding indigestible dextrin or inulin instead of Fiberixa.

Fiberixa 3 parts by weight 5 times concentrated peach juice 2 parts by weight Sugar 5 parts by weight Citric acid 0.3 parts by weight Sodium citrate 0.05 parts by weight Water 89.65 parts by weight

[Production Example 4: Fermented milk]

A yogurt base mix is prepared by mixing milk, a dairy product (derived from milk), and water so that the final product has a non-fat milk solid content of 9.5% and a milk fat content of 3.0%. The prepared yogurt base mix is homogenized, heat-sterilized at 95° C. for 5 minutes, and then cooled to about 40° C. This sterilized yogurt base mix is inoculated with Lactobacillus delbrueckii subsp. bulgaricus lactic acid bacteria and Streptococcus thermophilus lactic acid bacteria, and fermented, to which is added isomaltodextrin to produce a yogurt for controlling Fusobacterium bacteria in the intestinal flora, containing 200 mg of isomaltodextrin per product.

[Production Example 6: Capsules]

Fiberixa is filled into enteric soft capsules at 200 mg per capsule to manufacture medicines for intestinal disorders, intestinal regulation (regulating bowel movements), and the alleviation of loose stools, constipation, and abdominal bloating.

[Production Example 8: Enteric-coated supplement (tablets)]

The powder mixture containing isomaltodextrin is compressed and coated to obtain tablets containing 100-400mg of isomaltodextrin per tablet. The additives other than isomaltodextrin are corn starch, dextrin, lactose hydrate, precipitated calcium carbonate, candy powder, white sugar, talc, and magnesium stearate.

[Production Example 7: Enteric release supplement (capsule)]

Isomaltodextrin is loaded at 100-400 mg per capsule into pulse capsules (Pulsincap®, Scherer DDS, Ltd.) designed to release their contents approximately 8 hours after contact with the contents of the gastrointestinal tract.

The present invention supports the maintenance and improvement of people's health by providing a composition for controlling Fusobacterium bacteria. The present invention also provides a food composition and a method for producing food that supports the maintenance and improvement of people's health. Furthermore, the present invention can improve the nutrition of a wide variety of people, ensure healthy lifestyles, and promote welfare.

[Sequences listed in the sequence listing]
SEQ ID NO:1 PCR Forward Primer
SEQ ID NO:2 PCR Reverse Primer

Claims (13)

A composition for controlling bacteria belonging to the genus Fusobacterium, comprising water-soluble dietary fiber. The composition according to claim 1, wherein the water-soluble dietary fiber is a low-viscosity water-soluble dietary fiber. The composition according to claim 1, wherein the water-soluble dietary fiber is a hyperbranched α-glucan. The composition according to claim 1, wherein the water-soluble dietary fiber is isomaltodextrin. The composition according to claim 1, wherein the control of bacteria belonging to the genus Fusobacterium is a reduction in the number of bacteria belonging to the genus Fusobacterium. The composition according to any one of claims 1 to 5 for use as a prebiotic or synbiotic. The composition according to any one of claims 1 to 5 for treating at least one of inflammatory bowel disease and colon cancer through the control of bacteria belonging to the genus Fusobacterium. A composition for treating colon cancer, comprising α-glucan. The composition according to claim 8, wherein the α-glucan is isomaltodextrin. The composition according to claim 8 or 9, for administration to a subject having bacteria belonging to the genus Fusobacterium in the intestine. A pharmaceutical composition comprising isomaltodextrin. The pharmaceutical composition according to claim 11 for use in intestinal diseases. Enteric-coated formulation containing isomaltodextrin.

Images