Composition

Abstract

The present invention addresses the problem of providing a technique for promoting various bacteria of the genus Bifidobacterium in intestinal flora. A prebiotic composition for bacteria of the genus Bifidobacterium is obtained by combining 1-kestose and galactooligosaccharide. The composition can proliferate one or two or more kinds of bacteria of the genus Bifidobacterium selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.

Description

Technical Field

[0001] The present invention relates to a composition containing 1-kestose and galactooligosaccharide.

Background Art

[0002] It is known that the intestinal flora of infants is most dominant with bacteria belonging to the genus Bifidobacterium (hereinafter also referred to as bifidobacteria), and this is considered to be closely related to the maintenance of the health of infants. Therefore, developing foods that promote the growth of bifidobacteria is important for maintaining the health of infants.

[0003] In recent years, research on prebiotics that grow bifidobacteria has been actively conducted. In particular, it is said that the sugar source is important for the growth of bifidobacteria, and various oligosaccharides are actively used. As one of them, 1-kestose has attracted attention as it grows beneficial bacteria such as bifidobacteria and suppresses the growth of bacteria that are not preferable for health (Non-Patent Document 1). Specifically, it has been reported that 1-kestose reduces Clostridium and grows bifidobacteria even in an intestinal environment dominated by harmful bacteria where the number of Clostridium bacteria is larger than the number of bifidobacteria (Patent Document 1). In addition, it has been reported that 1-kestose suppresses the growth of bacteria that are not preferable for health, such as bacteria belonging to the genus Tyzzerella and bacteria belonging to the genus Luminococcus (Patent Documents 2 and 3).

[0004] In recent years, it has also been proposed to use a combination of various oligosaccharides as prebiotics. For example, it is known that a combination of fructooligosaccharide and galactooligosaccharide promotes the growth of bifidobacteria such as Bifidobacterium breve (Patent Documents 4 to 6).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] [Non-Patent Document 1] A. Endo et al., Anaerobe, 61 (2020) 102076. [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] In order to effectively utilize the intake of prebiotics, it is necessary for the prebiotics to be assimilated by intestinal bacteria and for the intestinal bacteria to grow. However, many species are included in the genus Bifidobacterium, and its composition in the intestinal flora varies from individual to individual. In addition, various species of the genus Bifidobacterium that are likely to be promoted to grow are affected by prebiotics. Therefore, even if prebiotics are ingested, some individuals may not have the genus Bifidobacterium that can assimilate the prebiotics in their intestines, that is, they are not "responders" (= non-responders) to the prebiotics, and the effects of the prebiotics may not be fully obtained. In view of such a situation, an object of the present invention is to provide a technique for further promoting various species of the genus Bifidobacterium in the intestinal flora. [Means for Solving the Problems]

[0008] As a result of intensive studies to solve the above problems, the present inventors have found that by combining 1-kestose and galactooligosaccharide, it is possible to evenly grow all four major types of Bifidobacterium bacteria, and have conceived that such a composition can be a prebiotic that is less likely to produce non-responders, thus completing the present invention.

[0009] That is, a first aspect of the present invention is a composition containing 1-kestose and galactooligosaccharide. In this aspect, it is preferable that the mass ratio of the contents of 1-kestose and galactooligosaccharide is 1:9 to 9:1. The composition of this aspect preferably further contains Bifidobacterium bacteria including one or more selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum. The composition of this aspect is preferably a nutritional composition, and more preferably a prepared milk.

[0010] A second aspect of the present invention is a composition for promoting the growth of Bifidobacterium bacteria containing 1-kestose and galactooligosaccharide, wherein the Bifidobacterium bacteria include one or more selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.

[0011] A third aspect of the present invention is a composition for improving the intestinal flora containing 1-kestose and galactooligosaccharide, wherein the intestinal flora includes Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum. Another aspect of this embodiment is a composition containing 1-kestose and galactooligosaccharide, which is administered or ingested to a subject with a disease or condition that can be prevented or improved by improving the intestinal flora, or a subject with a disease or condition caused by the deterioration of the intestinal flora, and contains Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum. The composition of this embodiment is preferably used for intestinal regulation, immunomodulation, anti-allergy, prevention of bacterial and viral infections, reduction of oxidative stress, prevention or improvement of diarrhea, prevention or improvement of constipation, prevention or improvement of inflammatory bowel disease, or prevention of colorectal cancer.

[0012] In the second and third embodiments, the mass ratio of the contents of 1-kestose and galactooligosaccharide is preferably 1:9 to 9:1. The compositions of the second and third embodiments are preferably nutritional compositions, and more preferably prepared milk.

Advantages of the Invention

[0013] According to the present invention, there is provided a composition capable of promoting the uniform growth of various Bifidobacterium bacteria, particularly the four main types important for the health of infants, in the intestine. The composition of the present invention can be an excellent prebiotic that is less likely to produce "non-responders" in which the growth effect of Bifidobacterium bacteria is difficult to obtain by ingestion. Such a composition can be in the form of an oral composition such as a food or drink or a pharmaceutical product, or can be contained in a food or drink or a pharmaceutical product in the form of an additive or the like. By ingesting the composition of the present invention, an effect of improving the intestinal flora can be obtained, and it is expected to be useful for maintaining the health of the consumer, particularly infants.

Modes for Carrying Out the Invention

[0014] Next, the present invention will be described in detail. However, the present invention is not limited to the following embodiments and can be freely changed within the scope of the present invention.

[0015] The composition of the present invention essentially contains 1-kestose and galactooligosaccharide.

[0016] 1-Kestose is a trisaccharide formed by the binding of one glucose and two fructoses (1-O-(β-D-fructofuranosyl)-β-D-fructofuranosyl-α-D-glucopyranoside, Glc α1→2 Fru β1→2 Fru). 1-Kestose can be produced by allowing various enzymes to act on sucrose by a known method, for example, the method described in JP-A-58-201980. Further, it can be purified by the method using chromatography described in JP-A-2000-232878, and high-purity 1-kestose can be obtained by crystallization by the method described in JP-B-6-70075. In addition, commercially available products (for example, manufactured by Busan Food Science Co., Ltd., etc.) can also be used as 1-kestose. The purity of 1-kestose in the crystal or raw material is preferably 95% or more, but may be 80% or more, or may be 70% or more. In the composition of the present invention, the content of 1-kestose is preferably 0.1 to 20% by mass, more preferably 0.2 to 5% by mass, and still more preferably 0.3 to 3% by mass based on the whole composition (per solid content). In addition, from the viewpoint of obtaining a desired growth promoting effect of Bifidobacterium bacteria, the composition of the present invention may not contain other fructooligosaccharides, for example, fructooligosaccharides having a longer sugar chain such as nystose (tetrasaccharide) and 1-fructofuranosyl-D-nystose (pentasaccharide).

[0017] Galactooligosaccharide (GOS) is an oligosaccharide or a mixture thereof having a structure represented by Gal-(Gal)n-Glc (n is 1 to 3, β1→4 bond or β1→6 bond). Industrially, galactooligosaccharide is produced by a transfer reaction using lactose as a raw material and β-galactosidase, and the main component is 4'-galactosyllactose (4'-GL), a trisaccharide in which one galactose is bonded to the non-reducing end of lactose. Commercial products (for example, those manufactured by Yakult Pharmaceutical Industry Co., Ltd., etc.) can also be used. Galactooligosaccharide may be of one kind or a mixture of two or more kinds. In the composition of the present invention, the content of galactooligosaccharide is preferably 0.1 to 20% by mass, more preferably 0.2 to 5% by mass, and still more preferably 0.3 to 3% by mass, based on the whole composition (per solid content).

[0018] As long as the composition of the present invention contains 1-kestose and galactooligosaccharide, it may contain one or more other oligosaccharides. Examples of other oligosaccharides include lactulose, raffinose, fructooligosaccharide, soy oligosaccharide, lactofructose, xylooligosaccharide, isomaltooligosaccharide, human milk oligosaccharide, coffee bean manno-oligosaccharide, gluconic acid, polydextrose, inulin, and the like.

[0019] In the composition of the present invention, the mass ratio of the content of 1-kestose to galactooligosaccharide is preferably 1:9 to 9:1, more preferably 3:7 to 7:3, and still more preferably 4:6 to 6:4.

[0020] The composition of the present invention can promote the growth of bacteria belonging to the genus Bifidobacterium. The Bifidobacterium bacteria whose growth is promoted by the composition of the present invention include one or more selected from Bifidobacterium longum (reclassified as Bifidobacterium longum subsp. longum), Bifidobacterium breve, Bifidobacterium infantis (reclassified as Bifidobacterium longum subsp. infantis), and Bifidobacterium bifidum. These Bifidobacterium bacteria are four types that are particularly important for the health of the intestinal flora in infancy (hereinafter also referred to as "infant-type Bifidobacterium bacteria"), and the composition of the present invention can uniformly promote the growth of these bacteria. Also, as long as the growth of one or more selected from the above four types is promoted, the growth of other Bifidobacterium bacteria may also be promoted. Examples of other Bifidobacterium bacteria include Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium pseudolongum, Bifidobacterium longum subsp. suis, and the like.

[0021] In this specification, the "growth" of bacteria includes an increase in the absolute number of bacteria, and the "promotion of growth" means that the degree of such increase is greater when the composition of the present invention is applied than when it is not applied. That is, when the composition of the present invention is applied in vivo or in vitro, the number of Bifidobacterium bacteria increases compared to when it is not applied. The degree of increase in the number of such bacteria is not particularly limited, but is preferably 1.1 times or more, more preferably 1.5 times or more, and even more preferably 3 times or more the number of such bacteria compared to the number of the bacteria when the composition of the present invention is not applied. In addition to directly measuring the number of bacteria, such an increase in the number of bacteria can be confirmed, for example, by measuring the turbidity (absorbance) of the digestive tract contents of animals such as humans who have ingested the medium or composition in which the bacteria are cultured or the amount of short-chain fatty acids such as acetic acid, and the value increases, or by measuring the pH in the medium and the value decreases, etc.

[0022] Furthermore, "growth" includes an increase in the proportion of the bacteria in the intestinal flora, and "promotion of growth" means that the degree of such increase is greater when the composition of the present invention is applied than when it is not applied. That is, it includes increasing the proportion of Bifidobacterium bacteria present in the digestive tract of animals such as humans who have ingested the composition of the present invention. Here, the "proportion" can also be referred to as the "occupancy rate" with respect to the entire flora detected in the intestinal flora. The "increase in proportion" may occur simultaneously with an increase or decrease in the proportion of other bacteria in the intestinal flora as long as the proportion of Bifidobacterium bacteria in the intestinal flora increases. The degree of increase in such proportion is not particularly limited, but is preferably 2% or more, more preferably 5% or more, and even more preferably 20% or more greater than the proportion of the bacteria when the composition of the present invention is not applied.

[0023] In addition, "promotion of growth" may mean that the rate at which the absolute number of bacteria increases or the rate at which the proportion of the bacteria in the intestinal flora increases is increased. The degree of increase in such a rate is not particularly limited, but it preferably means that it is 10% or more, more preferably 20% or more, and even more preferably 50% or more greater than the rate of the bacteria when the composition of the present invention is not applied, relative to the rate of the bacteria when the composition of the present invention is not applied. The increase in the rate can be confirmed, for example, by the fact that the time required to reach any number of bacteria or proportion of presence is shorter when the composition of the present invention is applied than when it is not applied.

[0024] The composition of the present invention may itself be in the form of foods and drinks, pharmaceuticals, etc., or may be in the form of being contained in foods and drinks, pharmaceuticals, etc. as an additive. The intake (administration) route of the composition of the present invention may be either oral or parenteral, but is usually oral. Examples of parenteral intake (administration) include rectal administration.

[0025] As described above, since the composition of the present invention can promote the growth of Bifidobacterium bacteria, it is preferable to contain Bifidobacterium bacteria in the composition together with 1-kestose and galactooligosaccharides. Here, the Bifidobacterium bacteria to be contained in the composition of the present invention are not particularly limited, and examples include Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium pseudolongum, etc. Among these, it is more preferable to contain one or more selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.

[0026] As Bifidobacterium longum, Bifidobacterium longum NITE BP-02621 (alias: BB536 or Bifidobacterium longum subsp. longum ATCC BAA-999) can be used. Bifidobacterium longum BB536 was deposited internationally under the Budapest Treaty with the accession number NITE BP-02621 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) on January 26, 2018. Bifidobacterium longum subsp. longum ATCC BAA-999 (number: ATCC BAA-999), which is the same bacterium, is available from the American Type Culture Collection (ATCC: 10801 University Boulevard, Manassas, VA 20110, United States of America, Manassas, Virginia 20110, USA) as ATCC BAA-999 (see, for example, Japanese Unexamined Patent Application Publication No. 2012-223134, etc.). Also, as Bifidobacterium longum, Bifidobacterium longum subsp. longum ATCC 15707, which is available from ATCC, can be used.

[0027] As Bifidobacterium breve, Bifidobacterium breve M-16V can be mentioned. Bifidobacterium breve M-16V was deposited internationally under the Budapest Treaty with the accession number NITE BP-02622 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture 292-0818) on January 26, 2018. A commercially available product, for example, "Bifidobacterium breve M-16V" manufactured by Morinaga Milk Industry Co., Ltd. may be used. In addition, as Bifidobacterium breve, Bifidobacterium breve MCC1274 can also be mentioned. Bifidobacterium breve MCC1274 was deposited internationally under the Budapest Treaty with the deposit number FERM BP-11175 at the Patent Microorganisms Depositary, National Institute of Advanced Industrial Science and Technology (currently, the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (IPOD), Room 120, 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818) on August 25, 2009.

[0028] As Bifidobacterium infantis, Bifidobacterium infantis M-63 can also be mentioned. Bifidobacterium infantis M-63 was deposited internationally under the Budapest Treaty with the deposit number NITE BP-02623 at NPMD on January 26, 2018. In addition, as Bifidobacterium infantis, Bifidobacterium longum subsp. infantis ATCC 15697, which is available from ATCC, can be used.

[0029] As Bifidobacterium bifidum, Bifidobacterium bifidum MCC1092 can be mentioned. Bifidobacterium bifidum MCC1092 was deposited internationally under the Budapest Treaty with the deposit number NITE BP-02429 at NPMD on February 21, 2017. In addition, as Bifidobacterium bifidum, Bifidobacterium bifidum MCC1319 can be mentioned. Bifidobacterium bifidum MCC1319 was deposited internationally under the Budapest Treaty with the deposit number NITE BP-02431 at NPMD on February 21, 2017. In addition, as Bifidobacterium bifidum, Bifidobacterium bifidum MCC1868 can be mentioned. Bifidobacterium bifidum MCC1868 was deposited internationally under the Budapest Treaty with the deposit number NITE BP-02432 at NPMD on February 21, 2017. In addition, examples of Bifidobacterium bifidum include Bifidobacterium bifidum MCC1870. Bifidobacterium bifidum MCC1870 was deposited with the NPMD on February 21, 2017, under the accession number NITE BP-02433, with an international deposit based on the Budapest Treaty.

[0030] Note that the bacteria identified by the above-exemplified bacterial names are not limited to the strains themselves that have been deposited or registered with a predetermined institution under the relevant bacterial names (hereinafter, also referred to as "deposited strains" for convenience of explanation), but also include strains that are substantially equivalent thereto (also referred to as "derived strains" or "induced strains"). That is, it is not limited to the strains themselves deposited with the above depository institution under the above accession number, but also includes strains that are substantially equivalent thereto. For each bacterium, a "strain that is substantially equivalent to the above deposited strain" belongs to the same species as the above deposited strain, has an effect of improving the intestinal flora, and further, the nucleotide sequence of its 16S rRNA gene has an identity of preferably 99.86% or more, more preferably 99.93% or more, and even more preferably 100% with respect to the nucleotide sequence of the 16S rRNA gene of the above deposited strain, and preferably has the same mycological properties as the above deposited strain. For each bacterium, a strain that is substantially equivalent to the above deposited strain may be, for example, a derived strain having the above deposited strain as a parent strain. Examples of derived strains include strains bred from the deposited strain and strains that occur naturally from the deposited strain. Examples of breeding methods include modification by genetic engineering techniques and modification by mutagenesis treatment. Examples of mutagenesis treatment include irradiation with X-rays, irradiation with ultraviolet rays, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. Examples of strains that occur naturally from the deposited strain include strains that occur naturally during the use of the deposited strain. Such strains include mutant strains that occur naturally by culturing the deposited strain (e.g., subculture). Derived strains may be constructed by one kind of modification or by two or more kinds of modifications.

[0031] As the cells of the Bifidobacterium bacterium to be contained in the composition of the present invention, commercially available products may be used, or those appropriately produced and obtained may also be used. In addition, the cells of the Bifidobacterium bacterium to be contained in the composition of the present invention can be easily obtained by culturing the aforementioned Bifidobacterium bacterium. The culturing method is not particularly limited as long as the Bifidobacterium bacterium can grow. As the culturing method, for example, the method usually used for culturing the Bifidobacterium bacterium can be used as it is or appropriately modified. The culturing temperature may be, for example, 25 to 50°C, preferably 35 to 42°C. The culturing can preferably be carried out under anaerobic conditions, for example, while aerating an anaerobic gas such as carbon dioxide gas. Also, the culturing can be carried out under microaerobic conditions such as liquid static culture. The culturing can be carried out, for example, until the Bifidobacterium bacterium grows to a desired degree.

[0032] The medium used for cultivation is not particularly limited as long as Bifidobacterium bacteria can grow. As the medium, for example, a medium commonly used for culturing Bifidobacterium bacteria can be used as it is or modified as appropriate. That is, as the carbon source, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and molasses can be used according to their assimilability. As the nitrogen source, for example, ammonium salts and nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate can be used. Also, as inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, etc. can be used. Organic components such as peptone, soybean powder, defatted soybean meal, meat extract, and yeast extract may also be used. Specific examples of media commonly used for culturing Bifidobacterium bacteria include Reinforced Clostridial medium, MRS medium (de Man, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium), TOSP medium (TOS propionate medium), and TOSP Mup medium (TOS propionate mupirocin medium).

[0033] As the Bifidobacterium bacterium that can be contained in the composition of the present invention, the cells or fractions containing the same can be used without particular limitation. That is, as the Bifidobacterium bacterium, for example, a culture obtained by culturing may be used as it is, the culture may be diluted or concentrated and used, or cells recovered from the culture may be used. Further, as long as the effect of improving the intestinal flora is not impaired, various additional operations such as heating and freeze-drying can be performed after culturing. The additional operation is preferably one with high viability of the cells. That is, as the Bifidobacterium bacterium that can be contained in the composition of the present invention, specifically, a culture of the Bifidobacterium bacterium, cells recovered from the culture, or processed products thereof can be mentioned, and examples of the processed products include dilutions, concentrates, or dried products. The cells are preferably used in a form containing viable cells. The cells may be composed of viable cells, for example, or may be a mixture of viable cells and dead cells.

[0034] In the present invention, the Bifidobacterium bacterium contained in the composition contains at least viable bacteria, and in the composition, in total, preferably 1×10 4 ~1×10 13 cfu / g or 1×10 4 ~1×10 13 cfu / mL, more preferably 1×10 5 ~1×10 12 cfu / g or 1×10 5 ~1×10 12 cfu / mL, still more preferably 1×10 6 ~1×10 11 cfu / g or 1×10 6 ~1×10 11 cfu / mL of viable Bifidobacterium bacteria is contained. As long as viable bacteria are contained, dead bacteria may also be included. Note that cfu refers to Colony forming unit. In this specification, for example, it can be the value when cultured at 38°C on a solid medium containing 10% by mass of reduced skim milk powder. These ranges may be the contents when distributed as a composition or when orally ingested.

[0035] As described above, since the composition of the present invention can promote the growth of Bifidobacterium bacteria including infant-type Bifidobacterium bifidum, it can be preferably applied to the use of promoting the growth of Bifidobacterium bacteria.

[0036] Here, the Bifidobacterium bacteria to be grown are not limited to the Bifidobacterium bacteria contained in the composition as described above, but also include Bifidobacterium bacteria present in the digestive tract of animals such as humans who have ingested orally. In addition, the composition of the present invention is used to grow preferably one or more, more preferably all four types of infant-type Bifidobacterium bifidum. Therefore, the composition of the present invention can be used to improve the intestinal flora. Here, "improvement of the flora" includes increasing the number of bacteria and their proportion of existence in the intestinal flora of Bifidobacterium bacteria, particularly infant-type Bifidobacterium bifidum. In addition, "improvement of the flora" may include increasing the proportion of other beneficial bacteria in the intestinal flora and decreasing the proportion of harmful bacteria in the intestinal flora as long as the proportion of Bifidobacterium bacteria in the intestinal flora increases. Examples of other beneficial bacteria include lactic acid bacteria. Examples of harmful bacteria include Clostridium welchii, Salmonella bacteria, Staphylococcus aureus, pathogenic Escherichia coli, etc. The "proportion of existence" can also be referred to as the "occupation rate" with respect to the entire flora detected in the intestinal flora.

[0037] The composition of the present invention can be useful for subjects with diseases or conditions that can be prevented or improved by improving the intestinal flora, or subjects with diseases or conditions caused by the deterioration of the intestinal flora. For example, it can be used for intestinal regulation, immune regulation, anti-allergy, prevention of bacterial / viral infection, reduction of oxidative stress, prevention / improvement of diarrhea, prevention / improvement of constipation, inflammatory bowel disease, prevention of colorectal cancer, etc.

[0038] The present invention can be rephrased as the use of 1-kestose and galactooligosaccharides in the production of a composition for promoting the growth of Bifidobacterium bacteria, wherein the Bifidobacterium bacteria include one or more selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum. The present invention can be rephrased as the use of 1-kestose and galactooligosaccharides in promoting the growth of Bifidobacterium bacteria, wherein the Bifidobacterium bacteria include one or more selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum. The present invention can also be rephrased as a composition containing 1-kestose and galactooligosaccharides used for promoting the growth of Bifidobacterium bacteria, wherein the Bifidobacterium bacteria include one or more selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.

[0039] The present invention can also be rephrased as a method for promoting the growth of Bifidobacterium bacteria, which includes administering 1-kestose and galactooligosaccharides, wherein the Bifidobacterium bacteria include one or more selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum. Here, the subject is not particularly limited as long as it is an animal, but is usually a human. Note that "administering to the subject" may be synonymous with "causing the subject to ingest". Ingestion may be voluntary (free ingestion) or forced (forced ingestion). That is, specifically, the administration step may be, for example, a step of formulating 1-kestose and galactooligosaccharide in food and drink or feed and supplying it to the subject, thereby allowing the subject to freely ingest 1-kestose and galactooligosaccharide.

[0040] The timing of ingestion (administration) of the composition of the present invention is not particularly limited and can be appropriately selected according to the state of the administration subject.

[0041] The ingestion (administration) amount of the composition of the present invention is appropriately selected according to the age, sex, state, and other conditions of the ingestion (administration) subject. Note that regardless of the amount and period of ingestion (administration), the drug can be administered once a day or divided into multiple times.

[0042] The composition of the present invention is preferably in the form of food and drink. As the food and drink, the form and properties are not particularly limited as long as the effects of the present invention are not impaired, and it can be produced by a usual method using raw materials usually used in food and drink. Note that the form of additives added to food and drink or pharmaceuticals is also included in the composition of the present invention. Examples of such forms include additives added to expressed breast milk or prepared milk, and it is assumed that the milk after addition is ingested by newborns and infants. Food and drink are usually orally ingested, but are not limited thereto. For example, those ingested nasally or those ingested through a gastrostomy or enterostomy may also be acceptable. For example, it is assumed that for newborns and infants, prepared milk which is the composition of the present invention described later or breast milk added with the composition of the present invention is ingested through a nasogastric feeding tube or the like.

[0043] Food and drink products include, regardless of form such as liquid, paste, gel-like solid, powder, etc., for example, confectionery; wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, tempura flour, breadcrumbs, etc.; instant noodles, cup noodles, retort foods, canned cooked foods, microwave foods, instant soups and stews, instant miso soup and broths, canned soups, freeze-dried foods, and other instant foods; agricultural processed products such as canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (processed grain products), etc.; fishery processed products such as canned fishery products, fish ham and sausage, fish paste products, seafood delicacies, tsukudani, etc.; livestock processed products such as canned livestock products and pastes, livestock meat ham and sausage, etc.; milk and dairy products such as processed milk, milk beverages, yogurts, lactic acid bacteria beverages, cheese, ice creams, cream, and other dairy products; fats and oils such as butter, margarines, vegetable oils, etc.; basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, vinegars, etc.; compound seasonings and food products such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other compound seasonings; frozen foods such as raw material frozen foods, semi-cooked frozen foods, cooked frozen foods, etc.; confectionery such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, and other confectionery; preferred beverages such as carbonated beverages, natural fruit juices, fruit juice beverages, fruit-flavored soft drinks, pulp beverages, fruit drinks with fruit pieces, vegetable-based beverages, soy milk, soy milk beverages, coffee beverages, tea beverages, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages, and other preferred beverages; other commercially available foods such as baby food, furikake, nori for ochazuke, etc.; nutritional compositions such as prepared milk (including powdered milk, liquid milk, etc.), liquid foods, supplements, etc.; health functional foods (foods for specified health use, nutritional functional foods, foods with functional claims), etc.

[0044] Among these, nutritional compositions are preferably mentioned. In the present invention, the "nutritional composition" is not particularly limited as one aspect of food and drink products, but is preferably prepared milk, liquid food, supplement, etc., and more preferably prepared milk. The intake targets include infants, young children, children, and adults, but preferably infants and young children. Prepared milk includes prepared powdered milk and prepared liquid milk. In the ordinance of the Ministry of Health, Labour and Welfare regarding the ingredient standards of milk and dairy products (Ordinance on Milk etc.), prepared powdered milk is defined as "raw milk, cow milk, special cow milk, or foods manufactured using these as raw materials, processed, or made into powder form with essential nutrients for infants added as the main ingredient". In the said ordinance, prepared liquid milk is defined as "raw milk, cow milk, special cow milk, or foods manufactured using these as raw materials, processed, or made into liquid form with essential nutrients for infants added as the main ingredient". In addition, prepared milk is a product in which various nutritional components such as proteins, fats and oils, carbohydrates, minerals, vitamins, etc. are blended, and also includes those processed into powder or liquid form. Furthermore, prepared milk further includes "prepared powdered milk for infants", "prepared liquid milk for infants", and "powdered milk for pregnant and lactating women" defined as special-purpose foods in the Health Promotion Act, and also includes forms such as prepared powdered milk for infants, nutritional powder for adults, and nutritional powder for the elderly.

[0045] When the composition of the present invention is in the form of a supplement, it can be formulated into solid preparations such as powders, granules, tablets, capsules, etc.; liquid preparations such as solutions, syrups, suspensions, emulsions, etc. In such formulation, it can follow the explanations of the components, carriers, and methods related to the formulation of pharmaceuticals described later.

[0046] Moreover, it can also be used as a feed as an aspect of food and drink. Examples of feeds include pet food, livestock feed, fish feed, etc. The form of the feed is not particularly limited. For example, it may contain grains such as corn, wheat, barley, rye, and millet; vegetable oil meals such as soybean oil meal, rapeseed oil meal, coconut oil meal, and linseed oil meal; bran such as bran, wheat bran, rice bran, and defatted rice bran; manufacturing by-products such as corn gluten meal and corn germ meal; animal feeds such as fish meal, skimmed milk powder, whey, yellow grease, and tallow; yeasts such as torula yeast and brewer's yeast; mineral feeds such as tricalcium phosphate and calcium carbonate; oils and fats; single amino acids; sugars, etc.

[0047] When the composition of the present invention is in the form of food or drink (including feed), it can be provided and sold as a food or drink with a label indicating the use of promoting the growth of Bifidobacterium in the intestine.

[0048] Such "labeling" acts include all acts for informing consumers of the above-mentioned use. As long as it is an expression that can make consumers recall or infer the above-mentioned use, regardless of the purpose of the label, the content of the label, the object or medium to be labeled, etc., all fall within the scope of the "labeling" acts of the present invention. In addition, the "labeling" is preferably carried out by an expression that allows consumers to directly recognize the above-mentioned use. Specifically, it includes acts such as transferring, delivering, displaying for transfer or delivery, importing a product related to food or drink or its packaging with the above-mentioned use described thereon, advertising related to the product, displaying, distributing, or publishing a price list or transaction document with the above-mentioned use described thereon, or providing information containing these by electromagnetic (such as the Internet) means with the above-mentioned use described therein.

[0049] On the other hand, the content of the label is preferably a label approved by the administration or the like (for example, a label approved based on various systems determined by the administration and carried out in a manner based on such approval). In addition, it is preferable to attach such label content to promotional materials at the point of sale such as packaging, containers, catalogs, pamphlets, POPs, and other documents.

[0050] In addition, "labeling" includes labeling as health foods, functional foods, enteral nutritional foods, special-purpose foods, health functional foods, foods for specified health use, nutritional functional foods, foods with functional claims, quasi-drugs, etc. Among these, in particular, labeling approved by the Consumer Affairs Agency, such as labeling related to the systems for foods for specified health use, nutritional functional foods, or foods with functional claims, or labeling approved under systems similar thereto, can be mentioned. Specifically, examples include labeling as a food for specified health use, labeling as a conditional food for specified health use, labeling indicating an effect on the structure or function of the body, disease risk reduction labeling, and labeling of functionality based on scientific evidence. More specifically, typical examples include labeling as a food for specified health use (particularly labeling of the use for health) and similar labeling as defined in the Cabinet Office Ordinance (Cabinet Office Ordinance No. 57 of August 31, 2009) regarding the permission of special-purpose labeling prescribed in the Health Promotion Act. Examples of such labeling include labeling such as "For those who want to increase Bifidobacterium", "To increase Bifidobacterium useful for the health of babies", "To improve the intestinal flora", "For the health of the stomach of infants", etc.

[0051] When the composition of the present invention is in the form of a pharmaceutical, the administration route may be either oral or parenteral, but oral administration is preferred. In addition, examples of parenteral intake (administration) include rectal administration. As the form of the pharmaceutical, depending on the administration method, it can be formulated into a desired dosage form as appropriate. For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, capsules, etc.; liquid preparations such as solutions, syrups, suspensions, emulsions, etc. In addition, in the case of parenteral administration, it can be formulated into suppositories, ointments, injections, etc. When formulating, components such as excipients, pH adjusters, coloring agents, flavoring agents, etc., which are usually used in formulation, can be used. In addition, it is also possible to use in combination other medicinal components, prebiotics for Bifidobacterium genus bacteria that are known or will be discovered in the future, and prebiotics for other bacteria. In addition, the formulation can be carried out by a known method as appropriate according to the dosage form. When formulating, a formulation carrier may be blended as appropriate for formulation.

[0052] Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol, etc.; starch derivatives such as corn starch, potato starch, α-starch, dextrin, carboxymethyl starch, etc.; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, etc.; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium metasilicate aluminate, etc.; phosphate derivatives such as calcium phosphate, etc.; carbonate derivatives such as calcium carbonate, etc.; sulfate derivatives such as calcium sulfate, etc.

[0053] Examples of binders include, for example, gelatin; polyvinylpyrrolidone; macrogol, etc. in addition to the above excipients.

[0054] Examples of disintegrants include, for example, chemically modified starches or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, etc. in addition to the above excipients.

[0055] Examples of lubricants include, for example, talc; stearic acid; metal stearates such as calcium stearate, magnesium stearate, etc.; colloidal silica; waxes such as beeswax, carnauba wax, etc.; boric acid; glycol; carboxylic acids such as fumaric acid, adipic acid, etc.; sodium carboxylate salts such as sodium benzoate, etc.; sulfates such as sodium sulfate, etc.; leucine; lauryl sulfates such as sodium lauryl sulfate, magnesium lauryl sulfate, etc.; silicic acids such as anhydrous silicic acid, hydrated silicic acid, etc.; starch derivatives, etc.

[0056] Examples of stabilizers include, for example, paraoxybenzoic acid esters such as methyl paraben, propyl paraben, etc.; alcohols such as chlorobutanol, benzyl alcohol, phenylethyl alcohol, etc.; benzalkonium chloride; acetic anhydride; sorbic acid, etc.

[0057] Examples of the flavoring and deodorizing agent include sweeteners, acidulants, fragrances, and the like. In addition, examples of the carrier used in the case of a liquid preparation for oral administration include solvents such as water.

[0058] The timing of ingesting the pharmaceutical of the present invention is not particularly limited, for example, before meals, after meals, between meals, before bedtime, and the like.

Examples

[0059] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0060] <Test Example 1> Evaluation of the growth promoting effect of various oligosaccharides on infant-type Bifidobacterium by fecal culture (1) Pretreatment of specimens Fecal samples were obtained from 7 healthy infants aged 1 to 12 months (designated as a, b, c, d, e, f, and g, respectively). The samples were collected immediately after defecation and immediately transferred to 10°C or lower under anaerobic conditions. Within 8 hours after defecation, the collected feces were diluted with 10 times the volume of physiological saline and stored at -80°C until use.

[0061] (2) Culture test To simulate the environment in the large intestine, a culture experiment was conducted according to the following procedure. A Bio Jr.8 100 mL × 8 multi-culture device (BJR-25NA1S-8M manufactured by Biott Co., Ltd.) was used, and the operation was carried out according to the device's instruction manual. 100 mL of YCFA medium with the composition shown in Table 1 was used as the medium. The total concentration of oligosaccharides in the medium was set to 1% by mass. The medium components other than oligosaccharides were dissolved in purified water and sterilized by autoclaving, and the oligosaccharides were dissolved in purified water and then filter-sterilized and added to the autoclaved medium. The culture temperature was set at 37 °C, and filtered CO2 was blown into each culture vessel to maintain an anaerobic state during the culture period. After adjusting the pH to 7.0, 100 μL (10 mg as feces) of a fecal sample diluted with physiological saline was added to start anaerobic culture. To simulate the pH in the large intestine of infants, when the pH dropped below 5.5 during the culture period, it was neutralized with 1 M Na2CO3 to control the pH. The medium was collected 24 hours after the start of the culture, centrifuged at 8000 rmp, 3 min, and 4 °C, and the supernatant and precipitate were collected respectively.

[0062]

Table 1

[0063] (3) DNA extraction DNA was extracted from the precipitate collected in (2) using a GENE PREP STAR PI-480 automatic extractor (Kurabo Industries Ltd Japan). Using the extracted DNA as a template, the number of bacteria of the genus Bifidobacterium and various species of the genus Bifidobacterium (Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum) was measured by quantitative PCR using an Applied Bio systems 7500 Fast & 7500 real-time PCR system (manufactured by Thermo Fisher). The primers and PCR conditions are shown in Table 2. Either Table 2-1 or 2-2 may be adopted.

[0064]

Table 2-1

[0065] [Table 2-2]

[0066] (4) Results Tables 3 to 7 show the growth degrees of Bifidobacterium genus and various Bifidobacterium species after 24-hour culture measured by quantitative PCR. The growth degree is the value obtained by logarithmically expressing the growth rate when the number of bacteria before culture is set to 1. The types and numbers of Bifidobacterium genus bacteria possessed by the test infants were 1 to 4 species, consistent with previous reports, and the combinations were also diverse among individuals. The growth promotion effects on the Bifidobacterium genus and each Bifidobacterium species were analyzed by analysis of variance and covariance analysis (the "Fit Model" menu of statistical analysis software JMP), ranked in descending order of the growth effect for each group (Example / Comparative Example), and the upper-ranked groups were arranged in the upper row of the table. Also, the specimens were ranked in descending order of the growth effect, and the upper-ranked specimens were arranged in the left column of the table. That is, statistical processing was used to rank each group and each specimen in a comprehensive evaluation, and they were arranged in the order from the upper left to the lower right so that the growth effect became weaker.

[0067] Galactooligosaccharides alone showed excellent growth promoting effects on Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum, but did not show sufficient growth promoting effects on Bifidobacterium longum (Comparative Example 1). On the other hand, when 1-kestose and galactooligosaccharides were combined, the excellent growth promoting effects on Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum were not impaired, and furthermore, an excellent growth promoting effect was shown on Bifidobacterium longum. Among various combinations of various oligosaccharides, the growth promoting effect was ranked first or second for all four types of infantile bifidobacteria (Example 1). From this, it became clear that by combining 1-kestose and galactooligosaccharides, an effect of uniformly promoting the growth of all infantile bifidobacteria in all test infants can be obtained. That is, the combination of 1-kestose and galactooligosaccharides can be an excellent prebiotic that is less likely to produce non-responders.

[0068]

Table 3

[0069]

Table 4

[0070]

Table 5

[0071]

Table 6

[0072]

Table 7

Claims

1. A composition containing 1-kestose, galactooligosaccharide, and bacteria belonging to the genus Bifidobacterium, wherein the bacteria belonging to the genus Bifidobacterium include one or two selected from the group consisting of Bifidobacterium infantis and Bifidobacterium bifidum.

2. The composition according to claim 1, wherein the mass ratio of the content of 1-kestose to galactooligosaccharide is 1:9 to 9:

1.

3. The composition according to claim 1 or 2, which is a nutritional composition.

4. The composition according to claim 3, which is a prepared milk.

5. A composition for promoting the growth of bacteria belonging to the genus Bifidobacterium, containing 1-kestose and galactooligosaccharide, wherein the bacteria belonging to the genus Bifidobacterium include one or two selected from the group consisting of Bifidobacterium infantis and Bifidobacterium bifidum.

6. A composition for improving the intestinal flora, containing 1-kestose and galactooligosaccharide, wherein the intestinal flora includes one or two selected from the group consisting of Bifidobacterium infantis and Bifidobacterium bifidum.

7. A composition containing 1-kestose and galactooligosaccharide, which is administered or ingested to a subject with a disease or condition that can be prevented or improved by improving the intestinal flora, or a subject with a disease or condition caused by the deterioration of the intestinal flora, wherein the intestinal flora includes one or two selected from the group consisting of Bifidobacterium infantis and Bifidobacterium bifidum.

8. The composition according to claim 6 or 7, which is used for intestinal regulation, immune regulation, anti-allergy, defense against bacterial and viral infections, reduction of oxidative stress, prevention or improvement of diarrhea, prevention or improvement of constipation, prevention or improvement of inflammatory bowel disease, or prevention of colorectal cancer.

9. The composition according to any one of claims 5 to 7, wherein the mass ratio of the contents of 1-kestose and galactooligosaccharide is 1:9 to 9:

1.

10. The composition according to any one of claims 5 to 7, which is a nutritional composition.

11. The composition according to claim 10, which is a prepared milk.

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