Composition for newborns or infants

Abstract

The present invention addresses the problem of providing a novel functionality of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623).　In a composition to be ingested by healthy newborns who are born in full term or healthy infants, one or more components selected from cells of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of the bacterium, and a treated product of the bacterium are contained as active ingredients. The problem is solved by subjecting the composition to an application for reducing the number of bacteria belonging to the family Enterobacteriaceae in the intestinal flora, an application for promoting the proliferation of bacteria belonging to the genus Bifidobacterium, or an anti-inflammatory application.

Description

Composition for newborns or infants

[0001] The present invention relates to a composition to be ingested by newborns or infants, which contains a specific Bifidobacterium bacterium as an active ingredient.

[0002] The human intestine is home to numerous bacteria, forming a complex microflora. It is known that Bifidobacterium bacteria (hereafter referred to as "bifidobacteria") are the most dominant species in the intestinal microflora of infants, and this is thought to be closely related to the maintenance of infant health. While the intestinal microflora generally forms a relatively stable ecosystem, the intestinal microflora of newborns and infants is still unstable. This is particularly true during the neonatal period, when the intestinal microflora is prone to disruption, raising concerns about infection. Therefore, establishing and maintaining a Bifidobacterium-dominated intestinal microflora in newborns and infants and maintaining a healthy intestinal environment are important for maintaining infant health.

[0003] In recent years, Bifidobacterium longum subsp. infantis, a type of intestinal bacterium, has attracted attention as a probiotic, and it has been reported that when administered to newborns and infants, it proliferates and colonizes in the intestine, resulting in a bifidobacteria-dominated intestinal flora and reducing inflammatory cytokines in the stool, thereby improving the intestinal environment (Patent Document 1, Non-Patent Documents 1 to 7, etc.). Furthermore, the M-63 strain of Bifidobacterium longum subsp. infantis is known to promote intestinal development in infants and improve mental health in those who consume it (Patent Documents 2 to 3).

[0004] International Publication No. 2012 / 092155 Japanese Patent Application Laid-Open No. 2021-183574 International Publication No. 2018 / 155565

[0005] Ishizeki et al., Anaerobe, 23, 38-44, (2013)Roze et al., British Journal of Nutrition, 107, 1616-1622 (2012)Smilowitz et al., BMC Pediatr., 17, 133, (2017)Frese et al., mSphere, 2 (6), e00501-17, (2017)Henrick et al., Pediatr. Res., 86, 749-757, (2019)J. De Andres et al., Benef Microbes, 9 (4), 573-384, (2018)Escribano et al., Pediatr. Res. 83 (6), (2018)

[0006] An object of the present invention is to provide new functionality when Bifidobacterium longum subsp. infantis M-63 strain is used as a probiotic for newborns or infants.

[0007] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that in healthy full-term newborns that ingested Bifidobacterium longum subsp. infantis M-63 strain, bacteria belonging to the family Enterobacteriaceae were reduced, and the bacterial diversity in the intestinal flora was reduced, resulting in the proliferation of Bifidobacterium bacteria, and the anti-inflammatory effects were exerted due to the increase in anti-inflammatory factors metabolized by Bifidobacterium bacteria, which led to the completion of the present invention.

[0008] That is, a first aspect of the present invention is a composition to be ingested by a healthy newborn or infant born at term, the composition containing one or more species selected from bacterial cells of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of said bacteria, and a processed product of said bacteria, and the composition is for reducing bacteria belonging to the family Enterobacteriaceae in the intestinal bacterial flora. The first aspect of the present invention can be rephrased as a composition for reducing bacteria belonging to the family Enterobacteriaceae in the intestinal bacterial flora of a healthy newborn or infant born at term, the composition containing one or more species selected from bacterial cells of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of said bacteria, and a processed product of said bacteria.

[0009] A second aspect of the present invention is a composition for promoting the growth of Bifidobacterium bacteria in the intestinal flora of a healthy newborn or infant born at term, the composition containing one or more bacteria selected from Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of the bacteria, and a processed product of the bacteria, to be ingested by a healthy newborn or infant born at term. The second aspect of the present invention can also be described as a composition for promoting the growth of Bifidobacterium bacteria in the intestinal flora of a healthy newborn or infant born at term, the composition containing one or more bacteria selected from Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of the bacteria, and a processed product of the bacteria.

[0010] A third aspect of the present invention is an anti-inflammatory composition to be ingested by healthy newborns or infants born at term, the composition comprising one or more selected from the group consisting of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of said bacterium, and a processed product of said bacterium. In the third aspect of the present invention, the anti-inflammatory effect is preferably achieved by an increase in one or more selected from phenylalanine, tryptophan, and tryptophan metabolites, and / or a decrease in one or two selected from IFN-γ and IL-1β in the intestine. That is, a preferred form of the third aspect of the present invention may be rephrased as a composition for increasing one or more types selected from phenylalanine, tryptophan, and tryptophan metabolites, and / or a composition for decreasing one or two types selected from IFN-γ and IL-1β in the intestine of a healthy newborn or infant born at term, which comprises one or more types selected from bacterial cells of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of said bacterium, and a processed product of said bacterium.

[0011] The composition of these embodiments preferably further comprises human milk oligosaccharides, which are preferably one or more selected from 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, and difucosyllactose. The composition of these embodiments preferably contains 1.0 x 10 β-lactosyl lactose in terms of the amount of the bacterial cells. ８ ~5.0 x 10 ９ The composition of these embodiments is preferably ingested at a dose of 0.01 cfu / kg body weight / day. The composition of these embodiments is preferably ingested in combination with breastfeeding. The composition of these embodiments is preferably a food or drink, more preferably an infant formula. The composition of these embodiments is preferably a pharmaceutical product.

[0012] According to the present invention, various new functionalities are provided when Bifidobacterium longum subsp. infantis M-63 strain is used as a probiotic for newborns or infants.

[0013] Boxplots showing alpha diversity indices of fecal microbiota in the B. infantis M-63 group (left) and the placebo group (right) during the study period. Each graph shows the differences in fecal microbiota diversity indices between groups using the faith_PD index, Shannon index, Chao1 index, and observed_OTUs index. Each boxplot represents the median, interquartile range, minimum, and maximum. Between-group differences in alpha diversity were analyzed using the Wilcoxon rank-sum test. *p < 0.05 indicates statistical significance. Plots showing beta diversity indices of fecal microbiota in the B. infantis M-63 group (left) and the placebo group (right) during the study period. Each chart shows principal coordinate analysis (PCoA) plots of bacterial beta diversity based on Bray-Curtis dissimilarity, Jaccard distance, unweighted UniFrac distance, and weighted UniFrac distance, analyzed by time point. Beta diversity was analyzed using PERMANOVA. Principal coordinate analysis (PCoA) based on the Jensen-Shannon distance (JSD) shows the characteristics of the subjects' gut microbiota types. Partitioning Around Medoids (PAMs) clustering using JSD showed the optimal number of clusters for the subjects' gut microbiota types based on the Calinski-Harabasz index. Fecal microbiota composition for each gut microbiota type: Type 1: Bifidobacterium-dominant flora 1; Type 2: Bifidobacterium-dominant flora 2; Type 3: Bifidobacterium- and Bacteroides-mixed flora; Type 4: Enterobacteriaceae-dominant flora; Type 5: Enterococcus- and Clostridium-mixed flora; Type 6: Streptococcus-dominant flora. Graph showing the distribution of each intestinal flora type in the B. infantis M-63 group (left) and placebo group (right) (before ingestion of the test food, one week after ingestion of the test food, one month after birth, and three months after birth). Graph showing the amount of fecal metabolites for each intestinal flora type (types 1 to 6 from left): (a) Trp, (b) ILA, (c) IAld, (d) IAA, (e) IPA, (f) Tyr, (g) HPLA, (h) Phe. Data from before ingestion of the test food and one month after birth were used for analysis.Comparisons between types were performed using the Kruskal-Wallis test with Dunn post hoc test, and p values ​​were corrected with the Bonferroni test. *p<0.05 indicates statistical significance. Graph showing fecal metabolite levels for each intestinal microbiota type (Types 1 to 6, from left to right): (i) PLA, (j) Leu, (k) LeuA, (l) N-Lac-Phe, (m) MTCA, (n) Kyn, (o) KynA, (p) Ser. Analysis was performed using data from before ingestion of the test food and at one month of age. Comparisons between types were performed using the Kruskal-Wallis test with Dunn post hoc test, and p values ​​were corrected with the Bonferroni test. *p<0.05 indicates statistical significance. Graph showing the number of Bifidobacterium bacteria in the culture sediment after 24 hours of incubation of feces containing the test sample (n=4). Multiple comparisons were performed using the Tukey-Kramer HSD test, and a p-value of 0.05 or less was considered statistically significant. Graph (n=4) showing the concentration of a metabolite (acetic acid) in the culture supernatant after 24 hours of incubation of feces containing the test sample. Multiple comparisons were performed using the Tukey-Kramer HSD test, and a p-value of 0.05 or less was considered statistically significant. Graph (n=4) showing the concentration of a metabolite (4-hydroxyphenyllactic acid (HPLA)) in the culture supernatant after 24 hours of incubation of feces containing the test sample. Multiple comparisons were performed using the Tukey-Kramer HSD test, and a p-value of 0.05 or less was considered statistically significant. Graph (n=4) showing the concentration of a metabolite (indole-3-lactic acid (ILA)) in the culture supernatant after 24 hours of incubation of feces containing the test sample. Multiple comparisons were performed using the Tukey-Kramer HSD test, and a p-value of 0.05 or less was considered statistically significant.

[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 modified within the scope of the present invention. The compositions of the first to third aspects of the present invention will be collectively referred to as the "composition of the present invention."

[0015] The composition of the present invention contains, as an active ingredient, one or more selected from the group consisting of cells of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of said bacterium, and a processed product of said bacterium (these may be collectively referred to as "cells of B. infantis M-63 strain").

[0016] Bifidobacterium longum subsp. infantis M-63 strain was deposited on January 26, 2018, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under the accession number NITE BP-02623. It is a bacterium that was internationally deposited under the Budapest Treaty.

[0017] In this specification, the Bifidobacterium longum subsp. infantis M-63 strain is not limited to the strain deposited or registered under that name at a designated institution (hereinafter, for convenience of explanation, also referred to as the "deposited strain"), but also includes substantially equivalent strains thereto (also referred to as "derived strains" or "derived strains"). In other words, it is not limited to the strain deposited at the depository institution under the above accession number, but also includes substantially equivalent strains thereto. With regard to bacteria, "a strain substantially equivalent to the deposited strain" refers to a strain that belongs to the same species as the deposited strain, has a genome sequence similarity (average nucleotide identity value) with the deposited strain of preferably 99.0% or more, more preferably 99.5% or more, even more preferably 99.9% or more, and particularly preferably 100% identity, and preferably has the same bacteriological properties as the deposited strain. For bacteria, a strain substantially equivalent to the deposited strain may be, for example, a derivative strain derived from the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that have arisen naturally from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include irradiation with X-rays, irradiation with ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. Strains that have arisen naturally from the deposited strain include strains that have arisen naturally during use of the deposited strain. Such strains include mutant strains that have arisen naturally through cultivation (e.g., subculturing) of the deposited strain. Derivative strains may be constructed by one type of modification, or by two or more types of modifications.

[0018] The bacteria contained in the composition of the present invention may be commercially available, may be obtained by appropriate production, or may be obtained by culturing the aforementioned bacteria. The culture method is not particularly limited as long as it allows the bacteria to grow. As a culture method, for example, a method generally used for culturing lactic acid bacteria can be used as is, or with appropriate modifications. The culture temperature may be, for example, 25 to 50°C, and preferably 35 to 42°C. The culture can be preferably carried out under anaerobic conditions, for example, while aerating with anaerobic gas such as carbon dioxide. The culture can also be carried out under microaerobic conditions, such as liquid static culture. The culture can be carried out, for example, until the bacteria grow to a desired level.

[0019] The medium used for the culture is not particularly limited as long as it allows the bacteria to grow. For example, a medium commonly used for culturing lactic acid bacteria can be used as is or with appropriate modifications. Specifically, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysates, and blackstrap molasses can be used as carbon sources depending on the assimilation potential. Nitrogen sources include ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, as well as nitrates. Inorganic salts include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract can also be used. Specific examples of media commonly used for culturing Bifidobacterium longum subsp. infantis M-63 strain include reinforced clostridial medium, de Man, Rogosa, and Sharpe (MRS) medium, modified MRS medium (mMRS), TOS propionate (TOSP) medium, TOS propionate mupirocin (TOSP Mup) medium, Gifu Anaerobic Medium (GAM), and Yeast Extract-casein Hydrolysate Acid (YCFA) medium.

[0020] The Bifidobacterium longum subsp. infantis M-63 strain contained in the composition of the present invention may be in the form of any of bacterial cells, a bacterial culture, or a treated bacterial product. The bacterial cells may be live bacterial cells, killed bacterial cells, or a mixture of live and killed bacterial cells, but preferably contain live bacterial cells. A particularly preferred form of Bifidobacterium longum subsp. infantis M-63 strain contained in the composition of the present invention is a powder of live bacterial cells. A powder of live bacterial cells can be obtained by drying live bacterial cells under conditions that do not kill the bacteria.

[0021] As a bacterial culture, for example, the culture obtained by culturing may be used as is, or the culture may be diluted or concentrated before use, or bacterial cells recovered from the culture may be used. Furthermore, culture supernatants or culture fractions may also be used as the culture. When using culture supernatants, the supernatant of the culture solution obtained by culturing in GAM medium at 37°C for 16 hours is preferably used. As a processed bacterial product, bacterial cells or cultures may be crushed, heated, dried, or their diluted, dried, or fractionated products may be used. Heat-treated bacterial cells (heat-killed bacterial cells) can be obtained, for example, by treating the bacteria at 70 to 100°C for 10 to 40 minutes or at 90 to 150°C for 5 to 30 seconds. Pressure may also be applied during the heat treatment. The temperature does not necessarily need to be constant during the heat treatment, as long as it remains within the temperature range for the specified time. The heat-killed bacterial cells may be used as is after the heat treatment, or may be subjected to a process such as crushing, heat drying, freeze drying, or spray drying.

[0022] The content of the B. infantis M-63 strain cells in the composition of the present invention is not particularly limited and is set appropriately depending on the form of the composition. For example, the amount of the bacteria (equivalent amount of bacteria) is 1 x 10 ４ ~1 x 10 １３ cfu / g or 1 x 10 ４ ~1 x 10 １３ cfu / mL, preferably 1 x 10 ５ ~1 x 10 １２ cfu / g or 1 x 10 ５ ~1 x 10１２ cfu / mL, more preferably 1 x 10 ６ ~1 x 10 １１ cfu / g or 1 x 10 ６ ~1 x 10 １１ It is more preferable that the concentration is cfu / mL. In this specification, "cfu" stands for colony forming unit. When killed bacterial cells are used, cfu / g or cfu / mL may be interpreted as individual cells / g or individual cells / mL. When a culture supernatant is used as the bacterial culture, the content is preferably 0.1 to 100% by mass of the entire composition, more preferably 1 to 90% by mass, and even more preferably 10 to 80% by mass. In the composition of this embodiment, the content of the bacterial cells of the B. infantis M-63 strain relative to the entire composition is preferably 0.001% by mass or more but less than 100% by mass, more preferably 0.005 to 95% by mass, and even more preferably 0.01 to 85% by mass. These may be within the range of the content typically used when the composition is distributed as an oral composition.

[0023] The composition of the first aspect of the present invention has the effect of reducing bacteria belonging to the Enterobacteriaceae family in the intestinal microflora, and is therefore used to achieve this effect. This is because, in the intestines of a subject who ingests the composition of the present invention, bacteria of the genus Bifidobacterium become predominant, resulting in a relative decrease in bacteria belonging to the Enterobacteriaceae family. Therefore, in a second aspect, the present invention provides a composition used to achieve the effect of promoting the growth of bacteria of the genus Bifidobacterium. The inventors have found that the composition of the present invention reduces the diversity of bacterial species inhabiting the intestinal microflora, as shown in the examples described below. Specifically, the composition inhibits the presence of bacteria other than Bifidobacterium bacteria, thereby reducing the number of species. Meanwhile, the presence of Bifidobacterium bacteria is maintained or enhanced. As a result, Bifidobacterium bacteria become predominant in the intestinal microflora, while bacteria belonging to the Enterobacteriaceae family are reduced.

[0024] Bacteria belonging to the Enterobacteriaceae family that are reduced by the composition of the present invention include the genus Arsenophonus, the genus Biostraticola, and the genus Candidatus. Blochmannia, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Cosenzaea, Cronobacter, Dickeya, Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella Genus, Gibbsiella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Lonsdalea, Mangrovibacter, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Candidatus Examples of bacteria include those belonging to the genera Phlomobacter, Phaseolibacter, Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia, Rahnella, Raoultella, Saccharobacter, Salmonella, Samsonia, Serratia, Shigella, Shimwellia, Sodalis, Tatumella, Thorsellia, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia, and Yokenella. It is sufficient that the number or proportion of species or strains belonging to one or more of these genera in the intestinal flora is reduced. It is known that a predominance of bacteria belonging to the Enterobacteriaceae family in the intestinal flora is undesirable for the health of infants and young children. Furthermore, some bacteria belonging to the Enterobacteriaceae family are pathogenic. Therefore, the composition of the present invention, which has the effect of reducing bacteria belonging to the Enterobacteriaceae family, contributes to the health of those who consume it.

[0025] Here, "reduction" of bacteria refers to a decrease in the number of bacteria belonging to the Enterobacteriaceae family or their proportion relative to the entire bacterial flora when the composition of the present invention is applied in vivo or in vitro compared to when the composition of the present invention is not applied. The degree of such reduction is not particularly limited, but refers to a decrease of preferably 95% or less, more preferably 90% or less, and even more preferably 85% or less, compared to the number of bacteria or their proportion relative to when the composition of the present invention is not applied. In this specification, "presence ratio" can also be rephrased as "occupancy rate" relative to the entire bacterial group detected in the intestinal flora. Here, as long as the proportion of bacteria belonging to the Enterobacteriaceae family in the intestinal flora decreases, it may also include an increase in the proportion of other bacteria in the intestinal flora.

[0026] Bifidobacterium bacteria whose growth is promoted by the composition of the present invention are not limited to Bifidobacterium longum subsp. infantis M-63 strain, but also include other Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium pseudolongum, etc. may be included, and it is sufficient if some species or strains of these can grow. Among these, Bifidobacterium longum subsp. infantis is usually particularly likely to grow.

[0027] Here, "promotion of growth" refers to an increase in the number of Bifidobacterium bacteria or their proportion relative to the entire bacterial flora when the composition of the present invention is applied in vivo or in vitro, compared to when the composition is not applied. The degree of increase in bacterial count is not particularly limited, but refers to an increase that is preferably 1% or more, more preferably 5% or more, and even more preferably 10% or more relative to the number of Bifidobacterium bacteria when the composition of the present invention is not applied. Here, so long as the proportion of Bifidobacterium bacteria in the intestinal bacterial flora increases, this may also include a decrease in the proportion of other bacteria in the intestinal bacterial flora.

[0028] The composition of the present invention reduces bacteria belonging to the Enterobacteriaceae family in the intestinal flora and promotes the growth of bacteria of the genus Bifidobacterium, thereby quickly establishing a favorable intestinal flora environment. When the composition of the present invention establishes a favorable intestinal flora environment, a subject who ingests (administers) the composition may experience effects such as suppressing the growth of bacteria such as Escherichia coli in the intestine, regulating the intestines, relieving constipation, improving intestinal barrier function, suppressing intestinal inflammation, reducing, alleviating, or improving the symptoms of irritable bowel syndrome (IBS) and ulcerative colitis (UC), improving dietary fiber metabolism, promoting normal immune function development, and improving atopic eczema symptoms in infancy.

[0029] The composition of the third aspect of the present invention has an anti-inflammatory effect and is therefore used to achieve this effect. The term "anti-inflammatory" as used herein refers to an effect that suppresses inflammation (anti-inflammatory effect) so as to prevent an excessive immune response. This effect can be achieved by an increase in anti-inflammatory factors and / or a decrease in inflammatory factors in the intestine, particularly in feces. Specifically, this effect can be achieved by an increase in one or more substances selected from phenylalanine, tryptophan, and tryptophan metabolites, and / or a decrease in one or two substances selected from IFN-γ and IL-1β in the intestine. Tryptophan metabolites include substances metabolized by bacteria using tryptophan as a precursor, such as indole-3-lactic acid (ILA), indole-3-aldehyde (IAd), and indole-3-acetic acid (IAA). IFN-γ and IL-1β are known to be inflammatory factors, and phenylalanine, tryptophan, and tryptophan metabolites are known to contribute to the production of substances involved in suppressing inflammation. As shown in the examples below, a large amount of anti-inflammatory factors is detected in the intestines of infants whose intestinal flora is dominated by Bifidobacterium bacteria. Also, as mentioned above, Bifidobacterium bacteria become dominant in the intestinal flora of a subject who has ingested the composition of the present invention, and anti-inflammatory factors are more likely to be secreted and increase in the intestines. Therefore, an increase in anti-inflammatory factors is expected in the intestines of a subject who has ingested the composition of the present invention, and an anti-inflammatory effect can be expected.

[0030] The subject to which the composition of the present invention is administered (ingested) is not particularly limited as long as it is an animal, but is usually a mammal, preferably a human. Furthermore, the subject to which the composition of the present invention is administered (ingested) is a healthy newborn or infant born at full term, preferably a newborn or infant, more preferably a newborn. When targeting an animal other than a human, the term "baby" may be synonymous with "offspring." Here, a healthy full-term infant refers to an infant born after 37 weeks of gestation and weighing 2500 g or more at birth. That is, it refers to an infant other than a low-birth-weight infant or a preterm infant, whose intestinal development differs. Furthermore, a newborn refers to an infant less than 4 weeks old, an infant refers to an infant 28 days or older but less than 1 year old, and a toddler refers to an infant between 1 year old and preschool age or less than 7 years old.

[0031] Another aspect of the first aspect of the present invention is a composition for reducing bacteria belonging to the family Enterobacteriaceae in the intestinal flora of healthy newborns or infants born at term, comprising bacterial cells of the B. infantis M-63 strain. Another aspect of the first aspect of the present invention is use of bacterial cells of the B. infantis M-63 strain in the manufacture of a composition for reducing bacteria belonging to the family Enterobacteriaceae in the intestinal flora of healthy newborns or infants born at term. Another aspect of the first aspect of the present invention is use of bacterial cells of the B. infantis M-63 strain in promoting the reduction of bacteria belonging to the family Enterobacteriaceae in the intestinal flora of healthy newborns or infants born at term. Another aspect of the first aspect of the present invention is bacterial cells of the B. infantis M-63 strain used for reducing bacteria belonging to the family Enterobacteriaceae in the intestinal flora of healthy newborns or infants born at term. Another aspect of the first aspect of the present invention is use of bacterial cells of the B. infantis M-63 strain in promoting the reduction of bacteria belonging to the family Enterobacteriaceae in the intestinal flora of healthy newborns or infants born at term. The method for reducing bacteria belonging to the family Enterobacteriaceae in the intestinal flora comprises administering bacterial cells of the M-63 strain of B. infantis to healthy full-term newborns or infants.

[0032] Another aspect of the second aspect of the present invention is a composition for promoting the growth of Bifidobacterium bacteria in the intestinal flora of healthy newborns or infants born at term, comprising bacterial cells of the B. infantis M-63 strain. Another aspect of the second aspect of the present invention is use of bacterial cells of the B. infantis M-63 strain in the manufacture of a composition for promoting the growth of Bifidobacterium bacteria in the intestinal flora of healthy newborns or infants born at term. Another aspect of the second aspect of the present invention is use of bacterial cells of the B. infantis M-63 strain in promoting the growth of Bifidobacterium bacteria in the intestinal flora of healthy newborns or infants born at term. Another aspect of the second aspect of the present invention is bacterial cells of the B. infantis M-63 strain used for promoting the growth of Bifidobacterium bacteria in the intestinal flora of healthy newborns or infants born at term. Another aspect of the second embodiment of the present invention is a method for promoting the proliferation of Bifidobacterium bacteria in the intestinal bacterial flora, which comprises administering bacterial cells of the B. infantis M-63 strain to a healthy full-term newborn or infant.

[0033] Another aspect of the third aspect of the present invention is the use of bacterial cells of the B. infantis M-63 strain in the manufacture of an anti-inflammatory composition to be ingested by healthy newborns or infants born at full term. Another aspect of the third aspect of the present invention is the use of bacterial cells of the B. infantis M-63 strain for anti-inflammatory effects in healthy newborns or infants born at full term. Another aspect of the third aspect of the present invention is bacterial cells of the B. infantis M-63 strain used for anti-inflammatory effects in healthy newborns or infants born at full term. Another aspect of the third aspect of the present invention is an anti-inflammatory method comprising administering bacterial cells of the B. infantis M-63 strain to healthy newborns or infants born at full term.

[0034] Note that "administering bacterial cells of the B. infantis M-63 strain to a subject" may be synonymous with "allowing a subject to ingest bacterial cells of the B. infantis M-63 strain." The ingestion may be voluntary (ad libitum intake) or forced (forced intake). That is, the administration step may specifically be, for example, a step of incorporating bacterial cells of the B. infantis M-63 strain into food, drink, or feed and providing the bacterial cells to the subject, thereby allowing the subject to ad libitum ingest the bacterial cells of the B. infantis M-63 strain.

[0035] The timing and duration of ingestion (administration) of the composition of the present invention are not particularly limited, but it is preferably started from 7 days after birth or later, and is preferably taken (administered) continuously for at least one week, more preferably at least one year, and even more preferably at least three months.

[0036] The composition of the present invention may be in the form of a food or drink, a medicine, etc., or may be in the form of an additive contained in a food or drink, a medicine, etc. The route of ingestion (administration) of the composition of the present invention may be either oral or parenteral, but is usually oral. Examples of parenteral ingestion (administration) include transdermal, intravenous, rectal administration, and inhalation.

[0037] The intake (administration) amount of the composition of the present invention is appropriately selected depending on the age (months), sex, condition, other conditions, etc. of the subject to be ingested (administered). The intake (administration) amount of the composition of the present invention is, for example, 1.0 × 10 as the intake (administration) amount converted into the bacterial cells of Bifidobacterium longum subsp. infantis M-63 strain according to the present invention. ８ ~5.0 x 10 ９ cfu / kg body weight / day is preferred, and 5.0 x 10 ８ ~5.0 x 10 ９ cfu / kg body weight / day, more preferably 1.7 x 10 ９ ~3.3 x 10 ９cfu / kg body weight / day is even more preferable. Compared to conventional probiotics using Bifidobacterium bacteria, the composition of the present invention can achieve the above-mentioned effects even at such a low intake (administration) amount, thereby reducing the burden on the target child and making it useful. Regardless of the intake (administration) amount and period, the composition of the present invention can be taken (administered) once a day or in multiple divided doses.

[0038] The composition of the present invention preferably further contains an oligosaccharide. The oligosaccharide is assimilated by Bifidobacterium bacteria, thereby enhancing the activity of B. infantis M-63 strain and promoting the growth of Bifidobacterium bacteria. Here, oligosaccharide refers to a saccharide oligomer in which disaccharide or higher sugars are linked by glycosidic bonds. The number of sugar residues linked to each other by glycosidic bonds in the oligosaccharide of the present invention is 2 or more, preferably 2 to 10, and particularly preferably 3 to 5. The oligosaccharide is not particularly limited, but is typically one that can be assimilated by Bifidobacterium bacteria, and specific examples include raffinose, lactulose, maltotriose, stachyose, galactooligosaccharides, fructooligosaccharides, kestose, nystose, soybean oligosaccharides, lactose oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, coffee bean mannooligosaccharides, gluconic acid, polydextrose, inulin, human milk oligosaccharides, etc. Among these, preferred examples of oligosaccharides having 3 to 5 sugar residues include maltotriose, stachyose, galactooligosaccharides, fructooligosaccharides, kestose, nystose, and human milk oligosaccharides.

[0039] Human milk oligosaccharides are particularly preferred as oligosaccharides that can be contained in the composition of the present invention. It is believed that ingesting human milk oligosaccharides together with the B. infantis M-63 strain enhances the activity of the B. infantis M-63 strain, reduces bacteria belonging to the Enterobacteriaceae family, and enhances the proliferation of Bifidobacterium bacteria in the intestinal flora, thereby enhancing the anti-inflammatory effect. Human milk oligosaccharides are not particularly limited as long as they are oligosaccharides normally contained in human milk, and examples include fucosylated oligosaccharides, sialylated oligosaccharides, acetylated oligosaccharides, and galactosyllactose. Examples of fucosylated oligosaccharides include 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-fucopentaose-I, lacto-N-fucopentaose-II, lacto-N-fucopentaose-III, difucosyllactose, lactodifucotetraose, and difucosyllacto-N-tetraose. Examples of sialylated oligosaccharides include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose-N-tetraose a, sialyllactose-N-tetraose b, sialyllactose-N-tetraose c, and disialyllacto-N-tetraose. Examples of acetylated oligosaccharides include lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and lacto-N-hexaose (LNH). Examples of galactosyllactose include 3'-galactosyllactose, 4'-galactosyllactose, and 6'-galactosyllactose. Among these, from the viewpoint of the assimilation-promoting effect, one or more selected from 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, and difucosyllactose are particularly preferred.

[0040] The human milk oligosaccharides contained in the composition of the present invention may be purified or a mixture, as long as the effects of the present invention are not impaired. The recommended intake amount of human milk oligosaccharides is generally about 1 g to about 15 g per day, more preferably about 2 g to about 10 g per day, and even more preferably about 3 g to about 7 g per day. The composition of the present invention may contain such an amount. In certain embodiments, the range is:

[0041] The composition of the present invention is preferably taken in combination with breastfeeding when the infant is in the lactating stage. Breastfeeding provides breast milk components such as human milk oligosaccharides, which enhances the activity of the B. infantis M-63 strain, reduces bacteria belonging to the Enterobacteriaceae family, and further increases the proliferation of Bifidobacterium bacteria in the intestinal flora, presumably enhancing the anti-inflammatory effect. If breastfeeding is used in combination with breastfeeding, formula may be used in combination, but the higher the proportion of breastfeeding, the more likely the above-mentioned effects are to be achieved. Note that "breastfeeding" refers to breastfeeding a newborn or infant for nutritional purposes.

[0042] When the composition of the present invention is intended to be orally ingested, it is preferably in the form of a food or beverage. The form or properties of the food or beverage are not particularly limited as long as it does not impair the effects of the present invention and can be orally ingested, and can be produced by a conventional method using raw materials typically used for foods and beverages, except that the food or beverage contains cells of the B. infantis M-63 strain.

[0043] Food and drink should be selected appropriately according to the age of the child, regardless of the form, such as liquid, paste, gel-like solid, or powder, and includes, for example, tablets; liquid food (nutritional food for tube feeding); wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken powder, and breadcrumbs; instant noodles, cup noodles, retort / prepared foods, canned foods, microwaveable foods, instant soup / stew, instant miso soup / cleaning liquid, canned soup, freeze-dried foods, and other instant foods. Instant foods; processed agricultural products such as canned agricultural products, canned fruit, jams and marmalades, pickles, boiled beans, dried agricultural products, and cereals (processed grain products); processed seafood products such as canned seafood, fish ham and sausages, fish paste products, seafood delicacies, and tsukudani (simmered fish dishes); processed livestock products such as canned livestock products and pastes, and livestock ham and sausages; dairy products such as processed milk, milk drinks, yogurt (fermented milk), lactic acid bacteria drinks, cheese, ice cream, cream, and other dairy products; butter, margarine, vegetable oil, and other oils Fats; basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, vinegars; complex seasonings such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as frozen ingredients, semi-cooked frozen foods, and cooked frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, desserts, jellies, and other Examples of such beverages include sweets and confectionery; carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks containing fruit juice, fruit pulp drinks, fruit drinks containing fruit particles, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, and other beverages; other commercially available foods such as baby food, furikake rice seasoning, and ochazuke nori seaweed; nutritional compositions such as supplements and infant formula (including powdered milk, liquid milk, etc.); enteral nutritional foods; and functional foods (foods for specified health uses, foods with nutrient functions).

[0044] Among these, nutritional compositions are preferred. In the present invention, the "nutritional composition" is not particularly limited as a form of food or beverage, but is preferably a formula, liquid food, etc., and more preferably a formula. The intended recipient may be an infant, a toddler, a child, or an adult, but is preferably an infant or a toddler. Formula includes formula powder and liquid formula. Formula powder is defined in the Ministerial Ordinance on the Compositional Standards of Milk and Dairy Products (Milk Ministerial Ordinance) as "a powder obtained by processing raw milk, cow's milk, special cow's milk, or a food produced using these as an ingredient, or using these as the main ingredient, and adding nutrients necessary for infants." Formula liquid is defined in the Ministerial Ordinance as "a liquid obtained by processing raw milk, cow's milk, special cow's milk, or a food produced using these as an ingredient, or using these as the main ingredient, and adding nutrients necessary for infants." Formula is also a blend of nutritional components such as various proteins, carbohydrates, lipids (oils and fats), vitamins, minerals, etc., and includes formulas processed into powder or liquid form. Furthermore, prepared milk also includes "powdered infant formula," "liquid infant formula," and "powdered milk for pregnant and nursing women," which are special-use foods defined in the Health Promotion Act, as well as forms such as powdered infant formula, nutritional powder for adults, and nutritional powder for the elderly.

[0045] The compositions of the present invention, particularly nutritional compositions such as infant formula, may further contain proteins, carbohydrates, lipids (oils and fats), vitamins, and minerals. Protein sources preferably include milk protein, soy protein, rice protein, pea protein, and oat protein, or mixtures thereof. Milk protein may be in the form of milk protein concentrate, milk protein isolate, whey protein, or casein, or a mixture of both. Protein may be intact protein or hydrolyzed protein, either partially or extensively hydrolyzed, with hydrolyzed protein being preferred from the viewpoint of intestinal digestion. Protein may also be provided in the form of free amino acids. Protein may comprise about 5% to about 20% of the energy of the compositions of the present invention.

[0046] The protein source may be a source of glutamine, threonine, cysteine, serine, proline, or a combination of these amino acids. Glutamine can be used by enterocytes as an energy source. Threonine, serine, and proline are important amino acids for the production of mucins, which contribute to intestinal barrier function. Cysteine ​​is the primary precursor of glutathione, which is important for the body's antioxidant defense.

[0047] The carbohydrate is preferably easily digestible, such as lactose, maltodextrin, hydrolyzed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, high fructose corn syrup, rice-derived carbohydrates, pea-derived carbohydrates, potato-derived carbohydrates, tapioca, sucrose, glucose, fructose, sucrose, honey, sugar alcohols (e.g., maltitol, erythritol, sorbitol), or mixtures thereof, with low dextrose equivalent (DE) maltodextrin being particularly preferred. The carbohydrate included in the compositions of the present invention may be in an amount that provides about 35% to about 55% of the energy of the composition.

[0048] Lipids include medium-chain triglycerides (MCT) and long-chain triglycerides (LCT), preferably long-chain triglycerides (LCT). Generally, the lipids included in the compositions of the present invention may be in an amount that provides about 35% to about 50% of the energy of the composition. The lipids may contain certain essential fatty acids (omega-3 and omega-6 fatty acids). Preferably, these polyunsaturated fatty acids may be in an amount that provides less than about 30% of the total energy of the lipid source.

[0049] Suitable sources of LCTs may be rapeseed oil, sunflower seed oil, palm oil, soybean oil, milk fat, corn oil, high oleic oil, and soy lecithin. A suitable source of MCTs may be fractionated coconut oil. The lipid profile of the compositions of the present invention is preferably designed so that the ratio of polyunsaturated fatty acids omega-6 (n-6) to omega-3 (n-3) is about (n-6) / (n-3) = about 5 to about 15.

[0050] Examples of vitamins that the compositions of the present invention may contain include vitamins A, B-complex (such as B1, B2, B6 and B12), C, D, E and K, niacin and acid vitamins such as pantothenic acid, folic acid and biotin.

[0051] Examples of minerals that the compositions of the present invention may contain include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium, and boron.

[0052] The compositions of the present invention may also contain carotenoids, such as lutein, lycopene, zeaxanthin, and beta-carotene. The total amount of carotenoids included may vary from about 0.001 μg / mL to about 10 μg / mL. Lutein may be included in an amount of about 0.001 μg / mL to about 10 μg / mL, preferably about 0.044 μg / mL to about 5 μg / mL. Lycopene may be included in an amount of about 0.001 μg / mL to about 10 μg / mL, preferably about 0.0185 μg / mL to about 5 μg / mL. Beta-carotene may be included in an amount of about 0.001 μg / mL to about 10 μg / mL, for example, about 0.034 μg / mL to about 5 μg / mL beta-carotene.

[0053] The composition of the present invention may contain additional probiotics in addition to the B. infantis strain M-63 cells. Further usable probiotics include, for example, B. animalis subsp. lactis BB-12, B. lactis HN019, B. lactis Bi07, B. infantis ATCC 15697, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus HNO01, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus LA-6, Lactobacillus acidophilus LA-8, Lactobacillus acidophilus LA-9, Lactobacillus acidophilus LA-10, Lactobacillus acidophilus LA-11, Lactobacillus acidophilus LA-12, Lactobacillus acidophilus LA-13, Lactobacillus acidophilus LA-14, Lactobacillus acidophilus LA-15, Lactobacillus acidophilus LA-16, Lactobacillus acidophilus LA-17, Lactobacillus acidophilus LA-18, Lactobacillus acidophilus LA-19 ... NCFM, Lactobacillus fermentum (L. fermentum) CECT5716, Bifidobacterium longum (B. longum) BB536, Bifidobacterium longum (B. longum) AH1205, Bifidobacterium longum (B. longum) AH1206, Bifidobacterium breve (B. breve) M-16V, MCC1274, L. reuteri (L. reuteri) ATCC 55730, Lactobacillus reuteri (L. reuteri) ATCC PTA-6485, Lactobacillus reuteri (L. reuteri) DSM 17938), and the like.

[0054] 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. When preparing such preparations, the components, carriers, and methods for formulating pharmaceuticals described below can be used in accordance with the description.

[0055] Furthermore, as an embodiment of the food and beverage, the feed can also be used as a feed. Examples of the feed include pet food, livestock feed, and fish feed. The form of the feed is not particularly limited, and may contain, in addition to the fungal cells of the B. infantis M-63 strain, for example, grains such as corn, wheat, barley, rye, and milo; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and linseed oil cake; bran such as wheat bran, rice bran, and defatted rice bran; manufacturing residues such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skim 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; simple amino acids; sugars, etc.

[0056] When the composition of the present invention is in the form of a food or beverage (including feed), it can be provided and sold as a food or beverage labeled with the purpose of improving the intestinal bacterial flora, etc. Furthermore, the bacterial cells of the B. infantis M-63 strain according to the present specification can be used for producing such a food or beverage.

[0057] Such "indication" includes all acts intended to inform consumers of the intended use, and any expression that can recall or infer the intended use falls under the category of "indication" in this invention, regardless of the purpose of the indication, the content of the indication, the object or medium on which it is displayed, etc. Furthermore, it is preferable that the "indication" be carried out using an expression that allows consumers to directly recognize the intended use. Specific examples include acts of transferring, delivering, displaying for transfer or delivery, or importing food and beverage products or product packaging with the intended use stated thereon; displaying or distributing product advertisements, price lists, or transaction documents with the intended use stated thereon; or providing information containing the intended use via electromagnetic means (such as the Internet).

[0058] On the other hand, the content of the labeling is preferably a labeling approved by the government, etc. (for example, a labeling approved based on various systems established by the government and made in a manner based on such approval.) It is also preferable that such a labeling content be attached to promotional materials at the point of sale such as packaging, containers, catalogs, pamphlets, and POP, as well as other documents.

[0059] "Labeling" also includes labeling as health food, functional food, enteral nutritional food, special dietary food, health functional food, food for specified health uses, food with nutrient functions, food with functional claims, and quasi-drugs. Among these, labeling approved by the Consumer Affairs Agency, such as labeling approved under systems related to foods for specified health uses, foods with nutrient functions, or foods with functional claims, or similar systems, can be cited. Specific examples include labeling as a food for specified health uses, labeling as a conditional food for specified health uses, labeling claiming to affect the structure or function of the body, labeling claiming to reduce disease risk, and labeling claiming functionality based on scientific evidence. More specifically, typical examples include labeling as a food for specified health uses (especially labeling of health uses) and similar labeling as defined in the Cabinet Office Ordinance on Permission for Special Use Labeling under the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009). Examples of such claims include "reduces bad bacteria in infants' stomachs," "increases good bacteria in infants' stomachs," "helps maintain healthy infant stomachs," and "suppresses inflammation in infants' stomachs."

[0060] The composition of the present invention can also be in the form of a pharmaceutical. The route of administration of the pharmaceutical may be oral or parenteral, with oral administration being preferred. Examples of parenteral ingestion (administration) include transdermal, intravenous, rectal, and inhalation. Pharmaceuticals can be formulated into a desired dosage form, as appropriate, depending on the administration method. For example, for oral administration, the pharmaceutical can be formulated into solid preparations such as powders, granules, tablets, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. Enteric-coated preparations can also be used with enteric coating. For parenteral administration, the pharmaceutical can be formulated into suppositories, ointments, injections, and the like. In addition to the B. infantis M-63 strain cells, ingredients commonly used in pharmaceutical formulations, such as excipients, pH adjusters, colorants, and flavoring agents, can be used in the formulation. It is also possible to use other medicinal ingredients, or known or future ingredients with intestinal flora-improving properties. Additionally, formulation can be carried out by known methods, as appropriate, depending on the dosage form. When preparing the formulation, carriers typically used in formulations may be appropriately blended to prepare the formulation, such as excipients, binders, disintegrants, lubricants, stabilizers, and flavoring agents.

[0061] Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and carboxymethyl cellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

[0062] Examples of binders include gelatin, polyvinylpyrrolidone, macrogol, and the like, in addition to the above-mentioned excipients.

[0063] Examples of disintegrants include the above-mentioned excipients as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone.

[0064] Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as Veegum and Gaifu; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates 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 silicic anhydride and silicic acid hydrate; and starch derivatives.

[0065] Examples of stabilizers 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.

[0066] Examples of flavoring agents include sweeteners, acidulants, fragrances, etc. In the case of liquid preparations for oral administration, examples of carriers used include solvents such as water.

[0067] The timing of taking the pharmaceutical of the present invention is not particularly limited, and may be before meals, after meals, between meals, or before going to bed.

[0068] The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

[0069] <Test Example 1> (1) Clinical trial of B. infantis M-63 strain administered to newborns This study complied with the Declaration of Helsinki and was approved by the Matsumoto City Hospital Research Ethics Committee on September 27, 2019. The study was registered in the UMIN Clinical Trials Registry (UMIN000038351). 111 full-term infants born at Matsumoto City Hospital participated as study subjects. Prior to the study, the principal investigator explained the study in detail, and consent and written consent were obtained from all study participants. Healthy newborns born at term were divided into two groups: the M-63 group (n = 56 subjects) and the placebo group (n = 53 subjects). The M-63 group received 1 billion live Bifidobacterium longum subsp. infantis M-63 bacterial powder (Morinaga Milk Industry Co., Ltd.) per day, and the placebo group received 1 g of sterilized dextrin per day until 3 months of age. These test foods were suspended in sterile water and placed in sterile bottles at room temperature for ingestion. Fecal samples were collected from the infants' diapers into collection tubes (Techno Suruga Research Institute Co., Ltd.) before ingestion (up to 7 days after birth), 1 week after initiation of ingestion, 1 month after birth, and 3 months after birth. The collected fecal samples were stored at -80°C until analysis.

[0070] (2) Fecal DNA Extraction and Microbial Analysis. Fecal DNA extraction and microbial analysis were performed using standard methods. Specifically, DNA was extracted from fecal samples using a GENE PREP STAR PI-480 instrument (Kurashiki Boseki Co., Ltd., Osaka, Japan) with glass bead lysis and sample homogenization. The V3-V4 region of the bacterial 16S rRNA gene was amplified and sequenced using an Illumina MiSeq instrument (Illumina, San Diego, CA, USA). After removing sequences aligned with GRCh38 and phiX reads, the remaining sequences were analyzed using the QIIME2 software package version 2017.10 (https: / / qiime2.org / ). DADA2 was employed to remove potential chimeric sequences and trim read bases. Taxonomic classification was performed using Greengenes 13.8 data, and alpha and beta diversity were calculated using QIIME2 software. Analysis was performed using PCoA and partitioning around medoid (PAM) clustering using R software version 3.6.0. Microbiota type clustering was performed at the genus level. Jensen-Shannon distance and PAM clustering were applied to cluster samples, and the optimal microbiota type clustering was determined using the Calinski-Harabasz (CH) index. Hierarchical clustering analysis was performed using MeV suite version 4.9 (https: / / sourceforge.net / projects / mev-tm4 / ), and distances were calculated based on Pearson correlation of the microbiota type transition data.

[0071] (3) Fecal Cytokine Analysis by Electrochemiluminescence. Interleukin-1β (IL-1β) and interleukin-8 (IL-8) levels were assessed using U-PLEX Biomarker Group 1 (hu) Assays (Meso Scale Discovery, Rockville, MD). Interferon-γ (IFN-γ), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were measured using the S-PLEX Proinflammatory Panel 1 (human) Kit (Meso Scale Discovery, Rockville, MD). Additionally, interleukin-5 (IL-5) was quantified using the S-PLEX Human IL-5 Kit (Meso Scale Discovery, Rockville, MD), and interleukin-22 (IL-22) was measured using the S-PLEX Human IL-22 Kit (Meso Scale Discovery, Rockville, MD). The procedures were performed according to the manufacturer's instructions. Briefly, 50–100 mg of fecal sample per subject, stored at −80°C, was diluted 10-fold with a 100:1 mixture of PBS and proteinase inhibitor (Nacalai Tesque, Kyoto, Japan). After dilution, the sample was crushed for three 2-minute cycles in a bead crusher (TAITEC, Saitama, Japan) using 5 mm stainless steel beads (QIAGEN, Valencia, CA). After centrifugation, the supernatant was collected and each cytokine was measured using each kit. Standards and samples were measured in duplicate using a Meso Scale Discovery (MSD) Quick Plex instrument and analyzed using MSD software. Cytokine values ​​were normalized by fecal weight, and values ​​below the detection limit of the kit were defined as zero.

[0072] (4) Extraction and Quantification of Fecal Metabolites: 10–40 mg of each fecal sample was collected and 200 μL of methanol solution was added. After crushing with 300 mg of zirconia beads at 2000 rpm for 30 seconds, the samples were centrifuged at 10,000 × g for 10 minutes at 4°C, and the supernatant was collected. The entire supernatant was filtered through a Nanosep 3 Komega microconcentrator (Pall) and then centrifuged at 9100 × g for 120 minutes at 4°C. The resulting filtrate was collected and dried in a centrifugal evaporator for 30–60 minutes. All pretreated samples were stored at −20°C before measurement. Fecal metabolite concentrations were measured using liquid chromatography-tandem mass spectrometry (LC-MS / MS) on a TSQ-FORTIS (Thermo Fisher Scientific) coupled to a Vanquish HPLC column. The metabolites measured were indole-3-lactic acid (ILA), indole-3-aldehyde (IAld), indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), 4-hydroxyphenyllactic acid (HPLA), 3-phenyllactic acid (PLA), tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). LC-MS / MS quantification was performed using an XBridge® C8 column (4.6 mm × 150 mm, 3.5 μm) (Waters Corporation, Milford, MA, USA). The mobile phase consisted of water (A) and methanol (B) containing 0.05% (v:v) formic acid, with a flow rate of 0.2 ml / min. The gradient elution profile was as follows: (i) 2% B for 2 min, (ii) increasing from 2% to 65% until 40 min, (iii) increasing from 65% to 99% until 45 min, (iv) holding at 99% until 55 min, (vii) decreasing from 99% to 2% until 60 min, (viii) holding at 2% until 75 min. All chemical reagents used were of analytical grade, and one sample from each experiment was analyzed.

[0073] (5) Statistical Analysis: ALDEx2 was used to assess differences in the microbiota between groups at the phylum and genus levels. Significance was determined by a q-value of <0.05. The Wilcoxon rank-sum test was used to compare two groups, and Fisher's exact test was used to analyze the distribution of infants within each microbiota type or cluster. The Kruskal-Wallis test confirmed the presence of groups with similar distributions, and Dunn's multiple comparison test with Bonferroni correction confirmed specific differences within groups. Statistical analysis was performed using R software (version 3.6.0) and IBM SPSS Statistics (version 28.0), with a p-value of <0.05.

[0074] (6) Results The results are shown in Tables 1 to 3 and Figures 1 to 8. As shown in Table 1, the B. infantis M-63 group showed a significant decrease in Enterobacteriaceae bacteria at 1 month of age compared to the placebo group. The alpha diversity of the intestinal microbiota in both groups was calculated using several indices. As shown in Figure 1, the B. infantis M-63 group showed significantly lower diversity in all indices at 1 month of age. Furthermore, when analyzing the beta diversity using a principal coordinate analysis (PCoA) plot, the intestinal microbiota composition between the B. infantis M-63 group and the placebo group was significantly different, as shown in Figure 2.

[0075] Principal coordinate analysis (PCoA) was performed to evaluate the evolution of the infants' gut microbiota during the intervention period, revealing six distinct microbiota types (Figs. 3 and 4). Summarizing the gut microbiota composition of each type (Fig. 5), types 1 and 2 were characterized by a Bifidobacterium-dominant gut microbiota, while type 3 was characterized by a mixed Bifidobacterium-Bacteroides gut microbiota, representing the gut microbiota of healthy infants. Conversely, types 4, 5, and 6 were dominated by facultative anaerobes, showing significant changes in gut microbiota composition. Specifically, type 4 showed a Enterobacteriaceae-dominant gut microbiota, type 5 showed a mixed Enterococcus-Clostridium gut microbiota, and type 6 showed a Streptococcus-dominant gut microbiota. Examination of the distribution of microbiota types between the two groups revealed significant disparities (Fig. 6). Before the test food intake, there was no significant bias in the distribution of bacterial flora types within each group. However, after one week of B. infantis M-63 administration, there was a clear concentration of bacterial flora types toward types 1 and 2, dominated by bifidobacteria. The B. infantis M-63 group consistently maintained type 1 or type 2, while almost all infants settled into type 1 or type 2, dominated by bifidobacteria, or type 3, a mixture of bifidobacteria and bacteroides.

[0076] Intestinal inflammation was assessed by measuring fecal cytokine levels (Table 2). Although there were no significant differences in the detection rates of the target cytokines between the two groups before and after one month of age, cytokine concentrations tended to differ between the two groups. Specifically, at one month of age, IFN-γ and IL-1β levels were significantly reduced in the B. infantis M-63 group compared with the placebo group. These results suggest that B. infantis M-63 exerts its anti-inflammatory effect by reducing IFN-γ and IL-1β levels. Analysis of metabolites in fecal samples revealed a significant increase in phenylalanine levels in the B. infantis M-63 group (Table 3). Although no statistically significant differences were observed, phenyllactic acid and tryptophan levels tended to increase in the B. infantis M-63 group at one month of age. However, no significant differences were observed between the two groups at one month of age for tryptophan metabolites such as indole-3-lactic acid and indole-3-aldehyde.

[0077] We examined the relationship between fecal metabolites and gut microbiota characteristics (Figs. 7 and 8). Although ILA did not show significant differences between the B. infantis M-63 group and the placebo group, it was significantly increased in infants with bifidobacterium-dominated gut microbiota types 1 and 2 and a mixed bifidobacterium- and bacteroides-dominated gut microbiota type 3 compared with non-bifidobacterium-dominated types. This suggests a relationship between bifidobacterial abundance and ILA. Furthermore, IAld and IAA also showed significant increases in bifidobacterium-dominated types 2 and 3. Other aromatic lactic acids (ALA), such as HPLA and PLA, were also significantly higher in the bifidobacterium-dominated gut microbiota. On the other hand, production of the amino acids Trp, Phe, and Tyr did not show significant differences according to gut microbiota characteristics. These results suggest that the type of intestinal flora has a significant impact on the production of metabolites, and that an intestinal flora dominated by bifidobacteria promotes the production of beneficial substances such as 5-ALA.

[0078]

[0079]

[0080]

[0081] Test Example 2: Evaluation of the Combination of B. infantis M-63 Strain and Human Milk Oligosaccharides by Fecal Culture (1) Sample Pretreatment Fecal samples were obtained from four healthy infants aged 3 to 5 years. The samples were collected immediately after defecation and immediately placed under anaerobic conditions at 10°C or below. Within 8 hours after defecation, the collected feces were diluted with 10 volumes of saline and stored at -80°C until use.

[0082] (2) Fecal Culture 2'-Fucosyllactose (2'-FL), a typical human milk oligosaccharide found most abundantly in breast milk, was used. To simulate the environment within the large intestine, a culture experiment was performed using the following procedure. A Bio Jr. 8 100 mL x 8-series culture device (BJR-25NA1S-8M, manufactured by Biot Co., Ltd.) was used, and the operation was performed according to the device's instructions. 100 mL of YCFA medium with the composition shown in Table 4 was used as the culture medium. All medium components except for the B. infantis M-63 strain and 2'-FL were dissolved in purified water and sterilized by autoclave. For the B. infantis M-63 strain, live bacterial powder (manufactured by Morinaga Milk Industry Co., Ltd.) was added to the autoclaved medium. 2'-FL (manufactured by Kyowa Hakko Bio Co., Ltd.) was dissolved in purified water, sterilized by filtration, and then added to the autoclaved medium. The culture temperature was 37°C, and filter-sterilized CO was added to each culture vessel. ２ The culture medium was then bubbled with 1M NaCl to maintain anaerobic conditions during the incubation period. After adjusting the pH to 7.0, 100 μL (10 mg of feces) of a stool sample diluted with saline was added, and anaerobic incubation was initiated. In order to simulate the pH in the large intestine of infants, 1M NaCl was added if the pH fell below 5.5 during the incubation period. ２ CO ３ The medium was collected 24 hours after the start of the culture and centrifuged at 8000 rpm for 3 minutes at 4°C, and the supernatant and precipitate were collected.

[0083]

[0084] (3) DNA extraction and bacterial count measurement 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, quantitative PCR was performed using the FW primer (SEQ ID NO: 1) and RV primer (SEQ ID NO: 2) shown in Table 5 on an Applied Biosystems 7500 Fast & 7500 real-time PCR system (Thermo Fisher Scientific) to measure the number of Bifidobacterium bacteria. The PCR conditions were denaturation at 95°C for 5 seconds, followed by 40 cycles of annealing at 55°C for 20 seconds and extension at 72°C for 30 seconds.

[0085]

[0086] (4) Quantification of Metabolites in Culture Supernatants. Metabolites (acetic acid, 4-hydroxyphenyllactic acid, and indole-3-lactic acid) in the culture supernatants collected in (2) were quantified as follows. Acetic acid concentration was quantified using HPLC (Shimadzu Organic Acid Analysis System; Shimadzu Corporation, Kyoto, Japan). Analysis was performed using a Shim-pack SCR-102H column (300 mm × 8 mm ID, three columns in series) with a Shim-pack SCR-102H (50 mm × 6 mm ID) as a guard column at a flow rate of 0.8 mL / min at 45°C for 30 min. 5 mmol / L p-toluenesulfonic acid was used as the solvent, and 5 mmol / L p-toluenesulfonic acid, 100 μmol / L EDTA, and 20 mmol / L Bis-Tris solution was used as the reaction mixture. Quantitative determination of 4-hydroxyphenyllactic acid (HPLA) and indole-3-lactic acid (ILA) concentrations was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) on a TSQ-FORTIS (Thermo Fisher Scientific) coupled to a Vanquish HPLC column. An XBridge® C8 column (4.6 mm × 150 mm, 3.5 μm) (Waters Corporation, Milford, MA, USA) was used for LC-MS / MS quantitation. The mobile phase consisted of water (A) and methanol (B) containing 0.1% (v:v) formic acid, with a flow rate of 0.2 ml / min. The gradient elution profile was as follows: (i) 30% B for 5 min, (ii) increasing from 30% to 95% B by 42 min, (iii) holding at 95% B until 47 min, (iv) decreasing from 95% to 30% B until 50.5 min, and (vii) holding at 30% B until 60 min. All chemical reagents used were of analytical grade.

[0087] (5) Results Figure 9 shows the number of Bifidobacterium bacteria after 24 hours of culture, as measured by quantitative PCR. 2'-FL alone did not exhibit any growth-promoting effect on Bifidobacterium bacteria, but when combined with B. infantis M-63 strain, a significant growth-promoting effect on Bifidobacterium bacteria was observed.

[0088] Figures 10 to 12 show the concentrations of metabolites in the supernatant after 24 hours of culture. Compared to 2'-FL alone, the combination of 2'-FL and B. infantis M-63 significantly increased the production of acetic acid, HPLA, and ILA. These results demonstrate that B. infantis M-63 enhances the effects of 2'-FL, a representative human milk oligosaccharide, and is useful for improving the intestinal environment, as indicated by the proliferation of bifidobacteria and the production of beneficial metabolites.

Claims

1. A composition to be ingested by healthy newborns or infants born at term, comprising one or more bacteria selected from the group consisting of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of said bacteria, and a processed product of said bacteria, said composition being used to reduce bacteria belonging to the family Enterobacteriaceae in the intestinal flora.

2. A composition for promoting the growth of Bifidobacterium bacteria, which is to be ingested by healthy newborns or infants born at full term, and which contains one or more bacteria selected from the group consisting of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623), a culture of said bacteria, and a processed product of said bacteria.

3. An anti-inflammatory composition to be ingested by healthy newborns or infants born at term, comprising one or more selected from the group consisting of Bifidobacterium longum subsp. infantis M-63 strain (NITE BP-02623) cells, a culture of said bacteria, and a processed product of said bacteria.

4. The composition according to claim 3, which has an anti-inflammatory effect by increasing one or more species selected from phenylalanine, tryptophan and tryptophan metabolites in the intestine, and / or by decreasing one or two species selected from IFN-γ and IL-1β.

5. A composition according to any one of claims 1 to 3, further comprising human milk oligosaccharides.

6. The composition of claim 5, wherein the human milk oligosaccharide is one or more selected from 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, and difucosyllactose.

7. The amount of the bacterial cells is 1.0 x 10 ８ ~5.0 x 10 ９ The composition of any one of claims 1 to 3, ingested at a dose of cfu / kg body weight / day.

8. A composition according to any one of claims 1 to 3, taken in combination with breastfeeding.

9. The composition according to any one of claims 1 to 3, which is a food or drink.

10. The composition of claim 8 which is a formula.

11. The composition according to any one of claims 1 to 3, which is a pharmaceutical product.

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