JP6978621B1 - Composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for further promoting the growth of a plurality of major species of Bifidobacterium in the intestinal flora. SOLUTION: Oligosaccharides containing galactooligosaccharides, lactulose, and raffinose, including bifidobacteria longum, bifidobacteria breve, bifidobacteria infantis, and bifidobacteria bifidum. Prebiotics for Bifidobacterium. A composition comprising the oligosaccharide and the Bifidobacterium spp. Is provided. Further, a composition for promoting the growth of Bifidobacterium spp. Containing the oligosaccharide is provided. [Selection diagram] None

Description

The present invention relates to compositions containing galactooligosaccharides, lactulose, and raffinose.

Bifidobacterium (hereinafter also referred to as bifidobacteria) is known to predominate in the intestinal flora of infants, and this is thought to be closely related to the maintenance of infant health. ing. Therefore, it is important to develop foods that promote the growth of bifidobacteria in order to maintain the health of infants.

In recent years, research on prebiotics for growing bifidobacteria has been actively conducted, and it is considered that the sugar source is particularly important for the growth of bifidobacteria, and various oligosaccharides are actively used. It has also been proposed to combine various oligosaccharides and use them as prebiotics.
In Patent Document 1, bifidobacteria longum and bifidobacteria breve are described when bifidobacteria are independently cultured in combination with raffinose and oligosaccharides selected from lactulose, fructooligosaccharides and galactooligosaccharides. , Bifidobacterium addresscentis, respectively, it is described that a proliferative effect was observed.
Non-Patent Document 1 describes that the combination of lactulose, raffinose and galactooligosaccharide significantly increased the cell numbers of bifidobacteria breve and bifidobacteria.

Japanese Unexamined Patent Publication No. 10-175867

T. Ehara et al., British Journal of Nutrition (2016), 116, 270-278

In order to effectively utilize the intake of prebiotics, it is necessary that the prebiotics are assimilated into the intestinal bacteria and the intestinal bacteria proliferate. However, many species of Bifidobacterium are included, and their composition in the intestinal flora varies from individual to individual. In addition, there are various species of bifidobacteria that easily promote growth depending on prebiotics. Therefore, even if prebiotics are ingested, some individuals do not have bifidobacteria in the intestine that can assimilate the prebiotics, that is, in the "responder" of the prebiotics. It may not be (= non-responder), and the effect of prebiotics may not be fully obtained.
An object of the present invention is to provide a technique for further promoting the growth of a plurality of major species of Bifidobacterium in the intestinal flora.

As a result of diligent research to solve the above problems, the present inventors have been able to evenly propagate all four major species of bifidobacteria by combining galactooligosaccharide, lactulose, and raffinose. The present invention was completed with the idea that such a composition could be a prebiotic that is less likely to cause non-responders.

That is, the first aspect of the present invention is a composition containing an oligosaccharide and a bacterium belonging to the genus Bifidobacterium, wherein the oligosaccharide contains a galactooligosaccharide, lactulose, and raffinose, and the bifidobacteria. The genus bacterium is a composition comprising Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.
In this embodiment, the mass ratio of the contents of lactulose and raffinose is preferably 1: 9 to 9: 1.
In this embodiment, the mass ratio of the content of galactooligosaccharide and lactulose is preferably 1: 9 to 9: 1.
In this embodiment, the mass ratio of the content of galactooligosaccharide and raffinose is preferably 1: 9 to 9: 1.
In this embodiment, the total number of viable bacteria of the genus Bifidobacterium contained in the composition is preferably 1 × 10 ⁸ to 1 × 10 ¹¹ cfu per 1 g of the oligosaccharide contained in the composition.
The composition of this embodiment is preferably a nutritional composition, more preferably a prepared milk.

A second aspect of the present invention is a composition for promoting the growth of a bacterium of the genus Bifidobacterium containing an oligosaccharide, wherein the oligosaccharide contains a galactooligosaccharide, lactulose, and raffinose, and the genus Bifidobacterium. The bacterium is a composition comprising Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.
In this embodiment, the mass ratio of the contents of lactulose and raffinose is preferably 1: 9 to 9: 1.
In this embodiment, the mass ratio of the content of galactooligosaccharide and lactulose is preferably 1: 9 to 9: 1.
In this embodiment, the mass ratio of the content of galactooligosaccharide and raffinose is preferably 1: 9 to 9: 1.
The composition of this embodiment is preferably a nutritional composition, more preferably a prepared milk.
The composition of this embodiment is preferably prepared milk for infants.

A third aspect of the present invention is a composition containing an oligosaccharide and a bacterium belonging to the genus Bifidobacterium, wherein the oligosaccharide is a galactooligosaccharide, a lactulose, a raffinose, a fructooligosaccharide, a soybean oligosaccharide, or a milk fruit oligo. The bifidobacteria include three or more species selected from the group consisting of sugar, xylooligosaccharide, isomaltooligosaccharide, human milk oligosaccharide, coffee bean mannooligosaccharide, gluconic acid, polydextrose, and inulin, and the bifidobacteria are the bifidobacteria. A composition comprising Umm Longum, Bifidobacterium Breve, Bifidobacterium Infantis, and Bifidobacterium Bifidum.

According to the present invention, there is provided a composition capable of promoting the growth of all four major species of bifidobacteria evenly in the intestine. The composition of the present invention can be an excellent prebiotic that is less likely to produce a "non-responder" in which the growth effect of bifidobacteria is less likely to be obtained by ingestion thereof.
Such a composition may be in the form of an oral composition such as a food or drink or a pharmaceutical product, or may 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
It is expected to improve the intestinal bacterial flora and help maintain the health of eaters, especially infants.

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.

The composition of the present invention contains an oligosaccharide, and the oligosaccharide essentially contains galactooligosaccharide, lactulose, and raffinose.
In the composition of the present invention, the total content of oligosaccharides is preferably 0.1 to 20% by mass, more preferably 0.5 to 10% by mass, still more preferably, with respect to the entire composition (per solid content). It is 1 to 5% by mass. Further, galactooligosaccharide, lactulose, and raffinose are preferably 20% by mass or more, more preferably 50% by mass or more, and further preferably 80% by mass or more of the total oligosaccharide contained in the composition of the present invention.

Lactulose is a disaccharide consisting of fructose and galactose (4-O-β-D-galactopyranosyl-D-fructose, Gal β1 → 4 Fru). Lactulose can be produced by a known method, for example, the methods described in JP-A-3-169888 and JP-A-6-228179. Further, as the lactulose, a commercially available product (for example, manufactured by Morinaga Milk Industry Co., Ltd.) can also be used.
In the composition of the present invention, the content of lactulose is preferably 0.02 to 10% by mass, more preferably 0.05 to 5% by mass, still more preferably 0. It is 1 to 2% by mass.

Raffinose is a trisaccharide in which fructose, galactose, and glucose are bound one by one (β-D-fructose furanosyl-α-D-galactopyranosyl- (1-6) -α-D-glucopyranoside, Gal α1. → 6 Glc α1 → 2β Fru). Raffinose can be produced by a known method, for example, the method described in "Food New Material Effective Utilization Technology Series No. 6," Raffinose ", Page 2, Japan Confectionery Research Center, 1996". .. Further, as raffinose, a commercially available product (for example, manufactured by Nippon Beet Sugar Mfg. Co., Ltd.) can also be used.
In the composition of the present invention, the content of raffinose is preferably 0.02 to 10% by mass, more preferably 0.1 to 5% by mass, still more preferably 0. It is 2 to 2% by mass.

Galactooligosaccharide (GOS) is an oligosaccharide having a structure represented by Gal- (Gal) n-Glc (n is 1-3, β1 → 4 bond or β1 → 6 bond) or a mixture thereof. Galactooligosaccharides are industrially produced from lactose as a raw material by a transfer reaction with β-galactosidase, and the main component is 4'-galactosyllactose (4'-GL), which is a trisaccharide in which one galactose is bound to the non-reducing end of lactose. ). As the galactooligosaccharide, a commercially available product (for example, manufactured by Yakult Pharmaceutical Co., Ltd.) can also be used. The galactooligosaccharide may be one kind or a mixture of two or more kinds.
In the composition of the present invention, the content of galactooligosaccharide is preferably 0.02 to 10% by mass, more preferably 0.1 to 5% by mass, still more preferably 0, based on the whole composition (per solid content). .2 to 2% by mass.

The composition of the present invention may contain one or more of other oligosaccharides as long as it contains galactooligosaccharides, lactulose, and raffinose. Other oligosaccharides include fructooligosaccharides, soybean oligosaccharides, milk fruit oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, human milk oligosaccharides, coffee bean mannooligosaccharides, gluconic acid, polydextrose, etc.
Inulin and the like can be mentioned.

In the composition of the present invention, the mass ratio of the contents of lactulose and raffinose is preferably 1: 9 to 9: 1, more preferably 2: 8 to 8: 2, and even more preferably 3: 7 to 7: 3. be.
In the composition of the present invention, the mass ratio of the content of galactooligosaccharide and lactulose is preferably 1: 9 to 9: 1, more preferably 2: 8 to 8: 2, still more preferably 3: 7 to 7: 3. Is.
In the composition of the present invention, the mass ratio of the content of galactooligosaccharide and raffinose is preferably 1: 9 to 9: 1, more preferably 2: 8 to 8: 2, still more preferably 3: 7 to 7: 3. Is.

The composition of the present invention can promote the growth of Bifidobacterium spp.
Bifidobacterium genus bacteria whose growth is promoted by the composition of the present invention are at least Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis. (Reclassified as Bifidobacterium infantis, Bifidobacterium longum subspecies infantis), and Bifidobacterium bifidum. These bifidobacteria are four species that are particularly important for the health of the intestinal flora in infancy (hereinafter, also referred to as "infant-type bifidobacteria"), and the composition of the present invention can promote their growth evenly. ..
Further, as long as the growth of the above four species is promoted, the growth of other bifidobacteria may also be promoted. Other bacteria of the genus Bifidobacterium include Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, and Bifidobacterium. Examples thereof include Bifidobacterium animalis, Bifidobacterium lactis, and Bifidobacterium pseudolongum.

As used herein, "proliferation" of a bacterium includes an increase in the absolute number of bacteria, and "promotion of growth" means that the degree of the increase is larger when the composition of the present invention is applied than when it is not applied. .. That is, the effect of increasing the number of Bifidobacterium spp. When the composition of the present invention is applied in vivo or in vitro can be obtained as compared with the case where the composition 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, the number of the bacteria when the composition of the present invention is not applied. More preferably, it means that the number of bacteria is three times or more.
Such an increase in the number of bacteria is not only a direct measurement of the number of bacteria, but also a short period of turbidity (absorbency) or acetic acid of the gastrointestinal contents of animals such as humans who have ingested a medium or composition in which bacteria are cultured. It can be confirmed by measuring the amount of chain fatty acid and increasing the value, measuring the pH in the medium and decreasing the value, and the like.

Further, "proliferation" includes an increase in the abundance ratio of the bacterium in the intestinal flora, and "promotion of proliferation" has a larger degree of increase than when the composition of the present invention is applied, as compared with the case where it is not applied. Say that. That is, it includes increasing the abundance ratio of bifidobacteria present in the digestive tract of animals such as humans who have ingested the composition of the present invention. Here, the "presence rate" can also be rephrased as the "occupancy rate" for the entire bacterial group detected in the intestinal flora. "Increase in abundance" means that as long as the abundance of bifidobacteria in the intestinal flora increases, the abundance of other bacteria in the intestinal flora may increase or decrease at the same time. good.
The degree of increase in the abundance ratio is not particularly limited, but is preferably 2% or more, more preferably 5% or more, still more preferably 20 with respect to the abundance ratio of the bacterium when the composition of the present invention is not applied. It means that it is larger than%.

Further, "promotion of growth" may be an increase in the rate at which the absolute number of bacteria increases, or the rate at which the abundance ratio of the bacteria in the intestinal flora increases. The degree of increase in the rate is not particularly limited, but is preferably 10% or more, more preferably 20% or more, still more preferably 50% or more with respect to the rate of the bacterium when the composition of the present invention is not applied. It means big.
The increase in the rate can be confirmed, for example, by the fact that the time required to reach an arbitrary number or abundance ratio is shorter when the composition of the present invention is applied than when it is not applied.

The composition of the present invention may itself be in the form of foods and drinks, pharmaceuticals and the like, or may be contained in foods and drinks and pharmaceuticals as an additive.
The route of ingestion (administration) of the composition of the present invention may be oral or parenteral, but is usually oral. In addition, as parenteral ingestion (administration), rectal administration and the like can be mentioned.

As described above, since the composition of the present invention can promote the growth of Bifidobacterium spp., It is preferable to include Bifidobacterium spp. In the composition together with the oligosaccharide. That is, it is the composition of the first aspect of the present invention containing an oligosaccharide and a bacterium belonging to the genus Bifidobacterium.
Here, the Bifidobacterium bacterium contained in the composition of the first aspect of the present invention includes Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifido. Includes Bacterium Bifidum.

As Bifidobacterium longum, Bifidobacterium longum NITE
BP-02621 (also known as BB536 or Bifidobacterium longum subsp. Longum ATCC BAA-999) can be used. Bifidobacterium longum BB536 was deposited with NPMD on January 26, 2018 under the accession number of NITE BP-02621 under the Budapest Treaty. Bifidobacterium longum subsp. Longum ATCC BAA-999 (number: ATCC BAA-999), which is the same bacterium as this, is A.
merican Type Culture Collection (ATCC: USA, 2)
0110, Virginia, Manassas, University Boulevard 10801 (108)
01 University Boulevard, Manassas, VA 2011
It is available as ATCC BAA-999 from 0, United States of America) (see, for example, Japanese Patent Application Laid-Open No. 2012-223134).
Further, as the bifidobacteria longum, ATCC 15707, which is available from ATCC, can be used.

As Bifidobacterium breve, Bifidobacterium breve M-1
6V can be mentioned. Bifidobacterium Breve M-16V was released on January 2, 2018.
On the 6th, at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture 292-0818), with the accession number NITE BP-02622, Budapest International deposits have been made under the treaty. Available as a commercial product, for example, "Bifidobacterium Breve M-16" manufactured by Morinaga Milk Industry Co., Ltd.
"V" may be used.
In addition, as Bifidobacterium bleve, Bifidobacterium bleve
MCC1274 is also mentioned. Bifidobacterium Breve MCC1274 is 2
As of August 25, 2009, Incorporated Administrative Agency Industrial Technology Research Institute Patent Biology Deposit Center (currently Incorporated Administrative Agency Product Evaluation Technology Infrastructure Organization Patent Biology Deposit Center (IPOD) (〒292-0818 Kazusakamatari, Kisarazu City, Chiba Prefecture) 2-5-8 Room 120), accession number FERM BP-
At 11175, an international deposit was made under the Budapest Treaty.

Bifidobacterium infantis also includes Bifidobacterium infantis M-63. Bifidobacterium Infantis M-63 has been deposited with NPMD on January 26, 2018 under the accession number NITE BP-02623 under the Budapest Treaty.
In addition, as Bifidobacterium Infantis, Bifidobacterium Longum Subspecies Infantis ATCC, which is available from ATCC.
15697 can be used.

Examples of the Bifidobacterium Bifidum include the Bifidobacterium Bifidum MCC1092. Bifidobacterium Bifidum MCC1092 has been deposited with NPMD on February 21, 2017 under the accession number NITE BP-02429 under the Budapest Treaty.
Further, examples of the bifidobacteria bifidum include bifidobacteria bifidum MCC1319. Bifidobacterium Bifidum MCC1319 has been deposited with NPMD on February 21, 2017 under the accession number NITE BP-02431 under the Budapest Treaty.
Further, examples of the bifidobacteria bifidum include bifidobacteria bifidum MCC1868. Bifidobacterium Bifidum MCC1868 has been deposited with NPMD on February 21, 2017 under the accession number NITE BP-02432 under the Budapest Treaty.
Moreover, as a bifidobacteria bifidum, a bifidobacteria bifidum MCC1870 can be mentioned. The Bifidobacterium Bifidum MCC1870 has been deposited with NPMD on February 21, 2017 under the accession number NITE BP-02433 under the Budapest Treaty.

Further, the composition of the first aspect of the present invention may also contain other bifidobacteria as long as it contains the above-mentioned four kinds of bifidobacteria. Other Bifidobacterium spp. Are Bifidobacterium addressntis, Bifidobacterium catenuratam, Bifidobacterium pseudocatenuratam, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium, pseudoron gum and the like can be mentioned.

The bacterium specified by the above-exemplified bacterial name is not limited to the strain itself (hereinafter, also referred to as “deposited strain” for convenience of explanation) that has been deposited or registered with a predetermined institution under the bacterial name. Substantially equivalent strains (also referred to as "derivative strains" or "derivative strains") are also included. That is, the stock itself is not limited to the stock itself deposited with the depositary institution with the deposit number, and the stock substantially equivalent thereto is also included. For each bacterium, the "strain substantially equivalent to the above-mentioned deposit strain" belongs to the same species as the above-mentioned deposit strain, has an effect of improving the intestinal flora, and further, the base sequence of the 16S rRNA gene thereof is the above-mentioned deposit. It has preferably 99.86% or more, more preferably 99.93% or more, still more preferably 100% identity with respect to the base sequence of the 16S rRNA gene of the strain, and is preferably the same as the above-mentioned deposited strain. A strain having mycological properties. For each bacterium, a strain substantially equivalent to the above-mentioned deposit strain may be, for example, a derivative strain having the deposit strain as a parent strain. Derivative strains include strains bred from deposited strains and strains naturally generated from deposited strains. Examples of the breeding method include modification by a genetic engineering method and modification by mutation treatment. Mutation treatment includes irradiation with X-rays, irradiation with ultraviolet rays, and treatment with mutant agents such as N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfonate, and methylmethanesulfonate. Will be. Examples of naturally occurring strains from the deposited strains include strains naturally occurring during the use of the deposited strains. Examples of such a strain include a mutant strain naturally generated by culturing a deposited strain (for example, subculture). The derivative strain may be constructed by one kind of modification, or may be constructed by two or more kinds of modifications.

As the bacterial cells of the genus Bifidobacterium contained in the composition of the first aspect of the present invention, a commercially available product may be used, or a commercially available product may be used.
In addition, the bacterial cells of the genus Bifidobacterium contained in the composition of this embodiment can be easily obtained by culturing the above-mentioned bacteria of the genus Bifidobacterium. The culture method is not particularly limited as long as Bifidobacterium spp. Can grow. As the culturing method, for example, a method usually used for culturing a bacterium belonging to the genus Bifidobacterium can be used as it is or modified as appropriate. The culture temperature may be, for example, 25 to 50 ° C, preferably 35 to 42 ° C. The culture can be preferably carried out under anaerobic conditions, and can be carried out, for example, while aerating an anaerobic gas such as carbon dioxide gas. In addition, the culture can also be carried out under slightly aerobic conditions such as liquid static culture. Culturing can be carried out, for example, until Bifidobacterium spp. Grow to the desired degree.

The medium used for culturing is not particularly limited as long as Bifidobacterium can grow. As the medium, for example, a medium usually used for culturing bifidobacteria can be used as it is or after being appropriately modified. That is, as the carbon source, for example, saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolyzate, and waste sugar honey can be used depending on the assimilation property. .. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate and nitrates can be used. Further, as the inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate and the like can be used. Further, organic components such as peptone, soybean flour, defatted soybean meal, meat extract and yeast extract may be used. In addition, as a medium usually used for culturing Bifidobacterium spp., Specifically, a fortified Clostridia medium (Reinforced Clostridial medium), an MRS medium (de Man, Rogosa, and Sharpe medium), and an mMRS medium (modified MRS medium). ), TOSP medium (TOS propionate medium), TOSP Mup
A medium (TOS propionate mupirocin medium) can be mentioned.

As the bifidobacteria bacterium that can be contained in the composition of this embodiment, the bacterium or the fraction containing the bacterium can be used without particular limitation. That is, as the Bifidobacterium genus bacterium, for example, the culture obtained by culturing may be used as it is, the culture may be diluted or concentrated and used, and the cells recovered from the culture may be used. May be good. In addition, various additional operations such as heating and freeze-drying can be performed after culturing as long as the effect of improving the intestinal bacterial flora is not impaired. It is preferable that the additional operation is such that the survivability of the bacterial cells is high. That is, examples of the bifidobacteria that can be contained in the composition of this embodiment include cultures of bifidobacteria, cells recovered from the cultures, and processed products thereof. Examples of the treated product include diluted products, concentrated products, dried products and the like. The bacterial cells are usually preferably used in a form containing live bacterial cells. The bacterial cells may be, for example, those consisting of live bacterial cells or a mixture of live bacterial cells and dead bacterial cells.

In this embodiment, the bifidobacteria contained in the composition include at least viable bacteria, and the total amount, preferably 1 × 10 ⁴ to 1 × 10 ¹³ cfu /, is contained in the composition.
g or 1 × 10 ⁴ to 1 × 10 ¹³ cfu / mL, more preferably 1 × 10 ⁵ to 1 × 10 ¹² cfu / g or 1 × 10 ⁵ to 1 × 10 ¹² cfu / mL, still more preferably 1 × 10 ^It contains 6 to 1 × 10 ¹¹ cfu / g or 1 × 10 ⁶ to 1 × 10 ¹¹ cfu / mL of viable Bifidobacterium spp.
Further, in this embodiment, the total number of viable bacteria of the genus Bifidobacterium contained in the composition is preferably 1 × 10 ⁸ to 1 × 10 ¹¹ cfu per 1 g of oligosaccharides contained in the composition. It is preferably 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu, and more preferably 1 × 10 ^{8 to} 3 × 10 ⁹ cfu.
As long as it contains live bacteria, it may also contain dead bacteria.
Here, cfu refers to a colony forming unit. In the present specification, for example, it can be the value when cultured at 38 ° C. in a solid medium containing 10% by mass of reduced skim milk powder.
These ranges may usually be the content when distributed as a composition or the content when ingested orally.

As described above, since the composition of the present invention can promote the growth of bifidobacteria including infant-type bifidobacteria, it is preferably applicable to the use of promoting the growth of bifidobacteria. That is, it is a composition for growing Bifidobacterium spp., Which is the second aspect of the present invention and contains oligosaccharides.

Here, the bifidobacteria to be propagated are not limited to the bifidobacteria contained in the composition as in the first aspect of the present invention, but are contained in the digestive tract of an animal such as a human being orally ingested. Bifidobacterium spp. That are present are also included. In addition, the composition of the second aspect of the present invention is used for growing all four infant-type bifidobacteria.
Therefore, the composition of the second aspect of the present invention can be used to improve the intestinal flora. Here, "improvement of bacterial flora" includes increasing the number of bacteria present in the intestinal flora of Bifidobacterium spp., In particular, infant-type bifidobacteria, and the abundance ratio thereof. In addition, "improvement of bacterial flora" means increasing the abundance ratio of other good bacteria in the gut microbiota as long as the abundance ratio of Bifidobacterium spp. It may include reducing its abundance in the gut flora. Examples of other good bacteria include lactic acid bacteria. Examples of bad bacteria include Clostridium perfringens, Salmonella spp., Staphylococcus aureus, pathogenic Escherichia coli and the like.
The above-mentioned "presence ratio" can also be rephrased as "occupancy rate" with respect to the entire bacterial group detected in the intestinal flora.

The composition of the present invention is intended for a subject of a disease or pathological condition that can be prevented or ameliorated by improving the intestinal flora, or a subject of a disease or pathological condition caused by deterioration of the intestinal flora. Can be useful. For example, for intestinal regulation, immunomodulation, antiallergic, bacterial / viral infection protection, oxidative stress reduction, diarrhea prevention / improvement, constipation prevention / improvement, inflammatory bowel disease, colorectal cancer It can be used for prevention.

A second aspect of the present invention is the use of oligosaccharides in the production of a composition for promoting the growth of bifidobacteria, wherein the oligosaccharides include galactooligosaccharides, lactulose, and raffinose. Bifidobacterium spp. Can be paraphrased as use, including Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.
A second aspect of the present invention is the use of oligosaccharides in promoting the growth of bifidobacteria, wherein the oligosaccharides include galactooligosaccharides, lactulose, and raffinose, and the bifidobacteria. Can be rephrased as use, including Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.
A second aspect of the present invention is an oligosaccharide used to promote the growth of a bacterium belonging to the genus Bifidobacterium, wherein the oligosaccharide contains a galactooligosaccharide, lactulose, and raffinose, and the bifidobacteria. The genus bacterium can also be rephrased as an oligosaccharide containing Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum.

A second aspect of the present invention is a method for promoting the growth of bifidobacteria, which comprises administering an oligosaccharide to a subject, wherein the oligosaccharide comprises a galactooligosaccharide, lactulose, and raffinose. , The method, wherein the Bifidobacterium bacterium comprises 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. In addition, "administering oligosaccharide to a subject" may be synonymous with "to ingest oligosaccharide to a subject". Ingestion may be voluntary (free ingestion) or compulsory (compulsory ingestion). That is, specifically, the administration step may be, for example, a step of blending oligosaccharides with food or drink or feed and supplying them to the subject, thereby allowing the subject to freely ingest the oligosaccharides.

The ingestion (administration) timing of the composition of the present invention is not particularly limited, and can be appropriately selected according to the condition of the administration target.

The ingestion (administration) amount of the composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the ingestion (administration) target.
The drug can be administered once a day or in multiple divided doses, regardless of the amount and duration of ingestion (administration).

The present invention has, as a third aspect, a composition containing an oligosaccharide and a bacterium belonging to the genus Bifidobacterium, wherein the oligosaccharide is a galactooligosaccharide, a lactulose, a raffinose, a fructooligosaccharide, a soybean oligosaccharide, or a milk fruit. Also provided are those comprising three or more selected from the group consisting of oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, human milk oligosaccharides, coffee bean mannooligosaccharides, gluconic acid, polydextrose and inulin. The three or more oligosaccharides may be in any combination, but preferably include one or more selected from galactooligosaccharides, lactulose, and raffinose.
Here, the bacterium belonging to the genus Bifidobacterium includes Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidam, and is the first of the present invention. According to the description in the above embodiment.

The composition of the present invention is preferably in the form of food and drink.
The form and properties of the food and drink are not particularly limited as long as the effects of the present invention are not impaired, and the food and drink can be produced by a usual method using raw materials usually used for food and drink.
The composition of the present invention also includes aspects of additives added to foods and drinks and pharmaceutical products. Examples of such an embodiment include additives added to milked breast milk and prepared milk, and it is assumed that the added milk is ingested by newborns and infants.
The food and drink are usually taken orally, but are not limited to this, and may be, for example, nasal ingested, gastrostomy or intestinal fistula. For example, it is assumed that a newborn baby or an infant is ingested the prepared milk which is the composition of the present invention described later or the breast milk to which the composition of the present invention is added by a nasal gastric feeding tube or the like.

The food and drink may be in the form of liquid, paste, gel-like solid, powder, etc., for example, beverages; bread, macaroni, spaghetti, noodles, cake mix, fried flour, bread flour, and other wheat flour products; instant noodles. , Cup noodles, retort / cooked foods, canned foods, microwave foods, instant soups / stews, instant miso soups / beverages, canned soups, freeze-dried foods, and other instant foods such as instant foods; canned agricultural products, canned fruits, jams・ Agricultural processed products such as marmalades, pickles, boiled beans, dried agricultural products, cereals (processed grain products);・ Processed livestock products such as pastes, livestock hams and sausages; milk and dairy products such as processed milk, dairy beverages, yogurt, lactic acid bacteria beverages, cheese, ice creams, creams and other dairy products; , Vegetable oils and other fats and oils; Soy sauce, miso, sauces, tomato processing seasonings, mirins, vinegars and other basic seasonings; Other complex seasonings and foods such as complex seasonings; frozen foods such as frozen foods, semi-cooked frozen foods, and cooked frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, Confectionery such as crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, and other confectionery; carbonated beverages, natural fruit juice, fruit juice beverages, refreshing beverages with fruit juice, fruit beverages, fruit beverages with fruit grains, vegetable beverages, Beverages such as soy milk, soy milk beverages, coffee beverages, tea beverages, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages, other favorite beverages, baby foods, sprinkles, other commercial foods such as tea pickled paste, etc. ; Nutritional compositions such as prepared milk (including powdered milk, liquid milk, etc.), liquid foods, supplements; functional foods (foods for specified health use, nutritionally functional foods) and the like.

Of these, nutritional compositions are preferred.
In the present invention, the "nutrition composition" is not particularly limited as one aspect of food and drink, but is preferably prepared milk, liquid food, or the like, and more preferably prepared milk. The ingestion target may be infants, toddlers, children, and adults, but is preferably infants and toddlers.
The prepared milk includes prepared milk powder and prepared liquid milk.
Prepared milk powder is required for infants by processing raw milk, milk, special milk, or foods manufactured from these as the main raw material, according to the ministerial ordinance (Ministry Ordinance on Milk, etc.) concerning the component standards of milk and dairy products. It is defined as "powdered with nutrients".
Prepared liquid milk is defined in the Ministerial Ordinance as "raw milk, milk, special milk, or processed foods made from these as raw materials, or made into liquid by adding nutrients necessary for infants." ..
Further, the prepared milk is a mixture of various proteins, fats and oils, carbohydrates, minerals, vitamins and other nutritional components, and includes those processed into powder or liquid.
In addition, the prepared milk further includes "prepared milk for infants", "prepared liquid milk for infants" and "milk powder for pregnant women and lactating women" in special-purpose foods specified by the Health Promotion Law. Aspects such as nutritional powder for adults and nutritional powder for elderly people are also included.

When the composition of the present invention is in the form of a supplement, it may be formulated into a solid preparation such as a powder, a granule, a tablet or a capsule; a liquid preparation such as a solution, a syrup, a suspension or an emulsion; can. In such a formulation, the description of the components, carriers, and methods related to the formulation of the pharmaceutical product described later can be applied.

It can also be used as feed as an aspect of food and drink. Examples of the feed include pet food, livestock feed, fish feed and the like.
The form of the feed is not particularly limited, and for example, grains such as corn, wheat, barley, rye, and mylo; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and flaxseed oil cake; Bran such as defatted rice bran; Production cake such as corn gluten meal and corn jam meal; Animal feed such as fish flour, defatted milk powder, whey, yellow grease and tallow; Yeasts such as tollula yeast and beer yeast; It may contain mineral feeds such as calcium phosphate and calcium carbonate; oils and fats; simple amino acids; sugars and the like.

When the composition of the present invention is in the form of a food or drink (including feed), it can be provided and sold as a food or drink labeled for the purpose of growing bifidobacteria in the intestine.

Such "display" actions include all actions for informing the consumer of the use, and if the expression is such that the use can be recalled or inferred, the purpose of the display, the content of the display, and the like. Regardless of the object or medium to be displayed, all of them fall under the "display" act of the present invention.
Further, it is preferable that the "display" is performed by an expression that allows the consumer to directly recognize the above-mentioned use. Specifically, the act of transferring a product related to food and drink or the packaging of the product that describes the above-mentioned use, displaying it for delivery, transfer or delivery, and importing it, advertising on the product, price list or transaction documents Examples include the act of describing the above-mentioned use and displaying or distributing it, or describing the above-mentioned use in the information containing these and providing it by an electromagnetic (Internet, etc.) method.

On the other hand, as the display content, it is preferable that the display is approved by the government or the like (for example, a display obtained based on various systems established by the government and performed in a mode based on such approval). In addition, it is preferable to attach such display contents to packaging, containers, catalogs, pamphlets, promotional materials such as POPs at sales sites, and other documents.

In addition, "labeling" includes health foods, functional foods, enteral nutrition foods, special purpose foods, health functional foods, specified health foods, nutritional functional foods, functionally labeled foods, non-medicinal products, etc. The display is also mentioned. Among these, in particular, labeling approved by the Consumer Affairs Agency, for example, a system related to foods for specified health use, nutritionally functional foods, or foods with functional claims, or labeling approved by a system similar to these can be mentioned. Specifically, labeling as a food for specified health use, labeling as a food for specified health use conditionally, labeling to the effect that it affects the structure and function of the body, labeling for reducing the risk of illness, and functionality based on scientific evidence. Indications, etc. can be mentioned, and more specifically, the Cabinet Office Ordinance on the permission of special use indications stipulated in the Health Promotion Act (Cabinet Office Ordinance No. 57, August 31, 2001). Labeling as a food for specified health use (particularly labeling for health use) and similar labeling specified in the above are typical examples.
Such indications include, for example, "People who want to increase bifidobacteria", "To increase bifidobacteria that are useful for baby's health", "Improve intestinal flora", "For infant's tummy health", etc. Display is mentioned.

When the composition of the present invention is in the form of a pharmaceutical product, the route of administration thereof may be oral or parenteral, but oral is preferable. In addition, as parenteral ingestion (administration), rectal administration and the like can be mentioned.
As the form of the drug, it can be appropriately formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, it can be formulated into a solid preparation such as a powder, a granule, a tablet or a capsule; a liquid preparation such as a solution, a syrup, a suspension or an emulsion. In the case of parenteral administration, it can be formulated into a suppository, an ointment, an injection or the like.
At the time of formulation, components such as excipients, pH adjusters, colorants, and flavoring agents that are usually used for formulation can be used. It is also possible to use other medicinal ingredients, prebiotics against known or future bacteria of the genus Bifidobacterium, prebiotics against other bacteria, and the like in combination.
In addition, the formulation can be carried out by a known method as appropriate depending on the dosage form. At the time of formulation, a formulation carrier may be blended and formulated as appropriate.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannit, and sorbit; starch derivatives such as corn starch, horse bell starch, α-starch, dextrin, and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, and the like. Cellulose derivatives such as hydroxypropylmethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium; Arabic rubber; Dextran; Plulan; Silate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate; Phosphate derivatives such as calcium phosphate; Carbonated Carbonated carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate can be mentioned.

Examples of the binder include gelatin; polyvinylpyrrolidone; macrogol and the like in addition to the above-mentioned excipients.

Examples of the disintegrant include, in addition to the above-mentioned excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.

Examples of the lubricant include talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gay wax; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid. Carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic acid hydrate; starch derivatives and the like. Be done.

Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like.

Examples of the flavoring agent include sweeteners, acidulants, and flavors.
Examples of the carrier used in the case of a liquid preparation for oral administration include a solvent such as water.

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

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

<Test Example 1> Evaluation of the growth-promoting effect of various oligosaccharides on infant-type bifidobacteria by stool culture (1) Sample pretreatment 7 healthy infants 1 to 12 months old (a, b, c, d, respectively) A stool sample was obtained from (e, f, g). The sample was collected immediately after defecation, and immediately transferred to an anaerobic condition at 10 ° C. or lower. Within 8 hours after defecation, the collected stool was diluted with 10-fold saline and stored at −80 ° C. until use.

(2) Culture test In order to imitate the environment in the large intestine, a culture experiment was conducted according to the following procedure. Bio Jr. 8 A 100 mL × 8 continuous culture device (BJR-25NA1S-8M manufactured by Biot Co., Ltd.) was used, and the operation was performed according to the instruction manual of the device. As the medium, 100 mL of YCFA medium having the composition shown in Table 1 was used. The total concentration of oligosaccharides in the medium was 1% by mass. Medium components other than oligosaccharides were dissolved in purified water and sterilized in an autoclave, and oligosaccharides were dissolved in purified water and then sterilized by filtration, and added to the product after the autoclave. The culture temperature was 37 ° C., and filtration-sterilized CO ₂ 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 stool) of a stool sample diluted with physiological saline was added, and anaerobic culture was started. In order to imitate the pH in the large intestine of an infant, when the pH fell below 5.5 during the culture period, it was _{neutralized with 1M Na 2} CO ₃ to control the pH. The medium was collected 24 hours after the start of the culture, centrifuged at 8000 rpm, 3 min, and 4 ° C., and the supernatant and the precipitate were collected, respectively.

(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, using the Applied Bio systems 7500 Fast & 7500 real-time PCR system (manufactured by Thermo Fisher), Bifidobacterium and various Bifidobacterium species (Bifidobacterium breve, Bifidobacterium) The bacterial counts of um longum, bifidobacteria infantis, and bifidobacteria bifidum) were measured by quantitative PCR. The primers and PCR conditions are shown in Table 2.

(4) Results Tables 3 to 7 show the growth degrees of various species of Bifidobacterium and Bifidobacterium after culturing for 24 hours, which were measured by quantitative PCR. The degree of proliferation is a logarithmic value of the rate of increase when the number of bacteria before culturing is 1. The species and numbers of bifidobacteria possessed by the test infants ranged from 1 to 4 species, consistent with previous reports, and the combinations varied from individual to individual. The growth promoting effect on the genus Bifidobacterium and each species of Bifidobacterium was analyzed by analysis of variance / analysis of covariance ("Fit to model" menu of statistical analysis software JMP), and each group (Example / Comparative example). ) Are ranked in descending order of proliferation effect, and the top group is placed in the upper part of the table. In addition, each sample was ranked in descending order of growth effect, and the top samples were placed in the left column of the table. In other words, each group and each sample were ranked by comprehensive evaluation by statistical processing, and arranged in order from the upper left to the lower right so that the growth effect was weakened.

In Example 1 comprising a combination of galactooligosaccharides, lactulose, and raffinose, four species, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum. The growth promoting effect was ranked first or second among various combinations of various oligosaccharides for all infant-type bifidobacteria. From this, it was clarified that the combination of galactooligosaccharide, lactulose, and raffinose has the effect of evenly promoting the growth of all infant-type bifidobacteria in all test infants. That is, the combination of galactooligosaccharides, lactulose, and raffinose can be excellent prebiotics that are less likely to produce non-responders.

Claims (13)

A composition containing an oligosaccharide and a bacterium belonging to the genus Bifidobacterium.
The oligosaccharides include galactooligosaccharides, lactulose, and raffinose.
A composition in which the Bifidobacterium bacterium comprises Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum. The composition according to claim 1, wherein the mass ratio of the contents of lactulose and raffinose is 1: 9 to 9: 1. The composition according to claim 1, wherein the mass ratio of the content of galactooligosaccharide and lactulose is 1: 9 to 9: 1. The composition according to claim 1, wherein the mass ratio of the content of galactooligosaccharide and raffinose is 1: 9 to 9: 1. The first aspect of the present invention, wherein the total viable cell count of the bifidobacteria contained in the composition is 1 × 10 ⁸ to 1 × 10 ¹¹ cfu per 1 g of the oligosaccharide contained in the composition. Composition. The composition according to any one of claims 1 to 5, which is a nutritional composition. The composition according to claim 6, which is a prepared milk. A composition for promoting the growth of bifidobacteria containing oligosaccharides.
The oligosaccharides include galactooligosaccharides, lactulose, and raffinose.
A composition in which the Bifidobacterium bacterium comprises Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium bifidum. The composition according to claim 8, wherein the mass ratio of the content of galactooligosaccharide and lactulose is 1: 9 to 9: 1. The composition according to claim 8, wherein the mass ratio of the content of galactooligosaccharide and raffinose is 1: 9 to 9: 1. The composition according to claim 8, wherein the mass ratio of the contents of lactulose and raffinose is 1: 9 to 9: 1. The composition according to any one of claims 8 to 11, which is a nutritional composition. The composition according to claim 12, which is a prepared milk .