Probiotic and composition comprising same

Abstract

The present invention addresses the problem of providing a technique for assisting the metabolism of dietary fiber in an infant in a weaning period.　The problem is solved by Bifidobacterium breve having the following protein (A), (B) or (C).　(A) is a protein comprising the amino acid sequence of SEQ ID NO: 2. (B) is a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 2, and having a glycoside hydrolase 59 (GH59) activity. (C) is a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and having a GH59 activity.

Description

Probiotics and compositions containing the same

[0001] This invention relates to probiotics possessing enzymes with newly discovered functions, and to compositions containing such enzymes or probiotics that can be used as food, beverages, or pharmaceuticals. Furthermore, this invention relates to a method for determining the degree of maturity of the intestinal microbiota of infants.

[0002] Infants transition to weaning around 5-6 months of age, beginning to consume foods other than breast milk or infant formula to supplement the nutrients necessary for growth. During weaning, infants' intestines digest and absorb substances they have not previously experienced digesting or absorbing, such as dietary fiber found in vegetables, which can put a strain on their immature intestines. Therefore, it is common practice to introduce solid foods gradually in small amounts, in accordance with the maturity of the infant's intestines.

[0003] Incidentally, bacteria belonging to the genus Bifidobacterium have traditionally been known to have a beneficial effect on the digestive system. Furthermore, various functionalities have been found in Bifidobacterium breve (for example, in Patent Documents 1 and 2), and it is being used as a prebiotic.

[0004] International Publication No. 2011 / 034166 Brochure International Publication No. 2017 / 209156 Brochure

[0005] The present invention aims to provide a technology that assists in the metabolism of dietary fiber in infants during the weaning period.

[0006] The human gut is home to a large number of bacteria, forming a complex microbiota (flora). The inventors focused on the fact that the gut microbiota in infants changes before and after weaning. They discovered that there are genes whose expression levels significantly increase in the gut microbiota after weaning, and among these genes, there is a gene of the Glycoside Hydrolase 59 (GH59) family of carbohydrate metabolizing enzymes derived from Bifidobacterium breve. Until now, the function of GH59 family enzymes possessed by microorganisms was unknown. As a result of analysis by the inventors, it was found that GH59 derived from B. breve has 6-O-glucuronyl-beta-galactosidase activity that cleaves the side chain ends of arabinogalactan protein (AGP), a proteoglycan universally distributed in plant tissues. The inventors concluded that this enzyme and B. breve containing this enzyme are useful for assisting the metabolism of dietary fiber in infants during the weaning period, and thus completed the present invention. Furthermore, based on the observation that the amount of the GH59 gene possessed by B. breve increases in the intestinal microbiota of infants after weaning, the inventors have also come to the realization that the degree of maturity of the intestinal microbiota of a target infant can be determined using the amount of this gene as an indicator.

[0007] In other words, the present invention is as follows: [1] Bifidobacterium breve possessing the following proteins (A), (B), or (C): (A) a protein containing the amino acid sequence of SEQ ID NO: 2; (B) a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 2, and having Glycoside Hydrolase 59 (GH59) activity; (C) a protein containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2, and having GH59 activity. [2] Bifidobacterium breve possessing the following DNA (a) or (b): (a) DNA containing the base sequence of SEQ ID NO: 1; (b) DNA encoding a protein that can hybridize under stringent conditions with DNA having a base sequence complementary to the base sequence of SEQ ID NO: 1, and having GH59 activity. [3] Bifidobacterium breve according to [1] or [2], selected from the group consisting of Bifidobacterium breve MCC01128 (NITE BP-04132), Bifidobacterium breve MCC10201 (NITE BP-04133), Bifidobacterium breve MCC10206 (NITE BP-04134), and Bifidobacterium breve MCC10258 (NITE BP-04135). [4] A composition for improving the metabolism of dietary fiber, comprising the bacterial cells of Bifidobacterium breve according to any one of [1] to [3], its culture, and / or a processed product thereof. [5] A carbohydrate-degrading composition having a higher-order structure, comprising the cells of Bifidobacterium breve described in any of [1] to [3], its culture, and / or a processed product thereof. [6] A food and beverage composition comprising the cells of Bifidobacterium breve described in any of [1] to [3], its culture, and / or a processed product thereof. [7] The food and beverage composition according to [6], further comprising a raw material containing arabinogalactan protein (AGP). [8] Bifidobacterium breve powder described in any of [1] to [3]. [9] A method for improving dietary fiber metabolism, comprising using the cells of Bifidobacterium breve described in any of [1] to [3], its culture, and / or a processed product thereof.A method for degrading carbohydrates having a higher-order structure, comprising using the cells of Bifidobacterium breve, its culture, and / or a processed product thereof as described in any of

[10] [1] to [3]. A method for producing oligosaccharides containing GlcA, comprising the step of reacting the cells of Bifidobacterium breve, its culture, and / or a processed product thereof as described in any of [1] to [3] with a raw material containing AGP.

[12] The following proteins (A), (B), or (C): (A) A protein containing the amino acid sequence of SEQ ID NO: 2; (B) A protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 2, and having GH59 activity; (C) A protein containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2, and having GH59 activity.

[13] DNA of the following (a) or (b): (a) DNA containing the base sequence of Sequence ID No. 1; (b) DNA that can hybridize under stringent conditions with DNA having a base sequence complementary to the base sequence of Sequence ID No. 1 and encodes a protein having GH59 activity.

[14] A composition for improving dietary fiber metabolism, comprising the protein described in

[12] , or the DNA described in

[13] or the protein encoded thereby.

[15] A food and beverage composition, comprising the protein described in

[12] , or the DNA described in

[13] or the protein encoded thereby.

[16] The food and beverage composition according to

[15] , further comprising a raw material containing arabinogalactan protein (AGP).

[17] A method for improving dietary fiber metabolism, comprising administering the protein described in

[12] , or the protein encoded by the DNA described in

[13] to a target.

[18] A method for decomposing carbohydrates having a higher-order structure, comprising using the protein described in

[12] , or the protein encoded by the DNA described in

[13] . A method for producing an oligosaccharide containing GlcA, comprising the step of reacting a protein described in

[19] or

[12] , or a DNA-encoded protein described in

[13] , with a raw material containing AGP.

[20] A method for determining the degree of maturity of the intestinal microbiota of an infant, wherein the abundance of the following DNA in the intestinal microbiota of the infant is used as an indicator: (a) DNA containing the base sequence of Sequence ID No. 1; (b) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of Sequence ID No. 1; (c) DNA containing a base sequence having 90% or more identity with the base sequence of Sequence ID No. 1.

[21] The method according to

[20] , comprising the steps of: calculating the relative abundance of the DNA in the total genes contained in the intestinal microbiota of the infant; and comparing the relative abundance with the relative abundance of the DNA in the intestinal microbiota of infants in general after weaning.

[0008] The present invention provides a composition that can assist in the metabolism of dietary fiber in infants during the weaning period. Such a composition can be ingested together with or added to weaning food containing dietary fiber, particularly AGP, and can help in the nutritional intake of infants during the weaning period. Furthermore, the present invention can be used to determine the degree of maturity of the intestinal microbiota of infants during the weaning period.

[0009] Graph showing changes in the relative abundance of enzyme genes in Japanese infant fecal metagenomic data before and after weaning. Schematic diagram of the gene cluster structure of four GH59 species derived from intestinal bacterial species. SDS-PAGE images of the purified recombinant proteins of four GH59 species derived from intestinal bacterial species. Figure showing the enzymatic activity of the recombinant proteins of four GH59 species derived from intestinal bacterial species. TLC images of the cleaved product after enzymatic treatment, and their structures. Figure showing the enzymatic activity of the recombinant protein of extracellularly localized Lr59A in the vegetable water-soluble fraction (TLC images of the cleaved product after enzymatic treatment). Oligosaccharide derived from gum arabic AGP (Rha-GlcA-Gal ２ A graph showing the growth of intestinal bacteria cultured in the presence of ) or galactose. Oligosaccharides derived from gum arabic AGP (Rha-GlcA-Gal ２ TLC image of residual sugars in the culture medium supernatant after culturing intestinal bacteria in the presence of (4MeGlcA-Gal). ２ A graph showing the growth of intestinal bacteria cultured in the presence of (4MeGlcA-Gal). ２TLC image of residual sugars in the culture medium supernatant after culturing intestinal bacteria in the presence of [unspecified substance].

[0010] This specification discloses the carbohydrate metabolic enzyme Glycoside Hydrolase 59 (GH59) possessed by Bifidobacterium breve (hereinafter also referred to as B. breve). The GH59 possessed by B. breve have a common amino acid sequence, and specifically, the following proteins (A), (B), or (C) are GH59 according to the present invention: (A) a protein containing the amino acid sequence of SEQ ID NO: 2; (B) a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 2, and having Glycoside Hydrolase 59 (GH59) activity; (C) a protein containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2, and having GH59 activity.

[0011] Here, the number of changes, i.e., deletions, substitutions, and / or additions, in the amino acid sequence shown in SEQ ID NO: 2 depends on the position or type of amino acid residue in the three-dimensional structure of the protein. The number of changes is not strictly limited, but may be 1 to 50 in SEQ ID NO: 2, 1 to 30 in another example, 1 to 15 in another example, 1 to 10 in another example, or 1 to 5 in another example. This is possible because amino acids can have a high degree of homology to each other, and mutations between these amino acids do not affect the activity of the protein or significantly alter the three-dimensional structure of the protein compared to the unmodified protein. Therefore, a protein containing changes in the amino acid sequence shown in SEQ ID NO: 2 may have an amino acid sequence with homology of 90% or more, 95% or more, 98% or more, or 99% or more of the entire amino acid sequence of SEQ ID NO: 2, as defined by the parameter "identity" when using the computer program BLAST, as long as GH59 activity is maintained or the three-dimensional structure of the protein is not significantly altered compared to a protein having the amino acid sequence shown in SEQ ID NO: 2.

[0012] Examples of deletions, substitutions, and / or additions of one or more amino acid residues include conservative mutations. A typical example of a conservative mutation is a conservative substitution. Conservative substitutions are, but are not limited to, substitutions between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid; between Ala, Leu, Ile, and Val when the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His, and Thr when the substitution site is a hydrophilic amino acid; between Gln and Asn when the substitution site is a polar amino acid; between Lys, Arg, and His when the substitution site is a basic amino acid; between Asp and Glu when the substitution site is an acidic amino acid; and between Ser and Thr when the substitution site is an amino acid with a hydroxyl group. Examples of conservative substitutions include substitutions of Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, and Ile to Leu or M Examples of substitutions include: substitution of et, Val or Phe; substitution of Leu to Ile, Met, Val or Phe; substitution of Lys to Asn, Glu, Gln, His or Arg; substitution of Met to Ile, Leu, Val or Phe; substitution of Phe to Trp, Tyr, Met, Ile or Leu; substitution of Ser to Thr or Ala; substitution of Thr to Ser or Ala; substitution of Trp to Phe or Tyr; substitution of Tyr to His, Phe or Trp; and substitution of Val to Met, Ile or Leu.

[0013] Non-conservative mutations are also examples of deletions, substitutions, and / or additions of one or more amino acid residues, provided that such mutations are compensated for by one or more other mutations at different positions in the amino acid sequence, so that GH59 activity is maintained or the three-dimensional structure of the protein is not significantly altered compared to the protein having the amino acid sequence shown in SEQ ID NO: 2.

[0014] The DNA encoding GH59 according to the present invention is either (a) or (b): (a) DNA containing the base sequence of SEQ ID NO: 1; (b) DNA that can hybridize under stringent conditions with DNA having a base sequence complementary to the base sequence of SEQ ID NO: 1 and that encodes a protein having GH59 activity.

[0015] Here, the nucleotide sequence of Sequence ID 1 may represent a nucleotide sequence encoding a protein having GH59 activity, which has the amino acid sequence shown in Sequence ID 2 using any synonymous amino acid codon according to the Standard Gene Code Table (see, for example, Lewin B., “Genes VIII”, 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). Therefore, a gene encoding a protein with GH activity may also be a gene having a mutant nucleotide sequence due to genetic code degeneracy.

[0016] The above "stringent conditions" may include conditions in which specific hybrids are formed, for example, hybrids with homology of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, as defined as the "identity" parameter when using the computer program BLAST, and nonspecific hybrids, for example, hybrids with lower homology than those listed above, are not formed. Examples of stringent conditions include washing once or twice, in other examples two or three times, at 60°C with a salt concentration of 1×SSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulfate), at 60°C, at a salt concentration of 0.1×SSC, 0.1% SDS, or at 65°C with a salt concentration of 0.1×SSC, 0.1% SDS. As DNA having a base sequence complementary to the base sequence of SEQ ID NO: 1, a portion of the sequence complementary to the sequence shown in SEQ ID NO: 1 may be used. Such probes can be prepared by PCR (polymerase chain reaction; see White TJ et al., The polymerase chain reaction, Trends Genet., 1989, 5:185-189) using oligonucleotides prepared based on the sequence shown in Sequence ID No. 1 as primers and a DNA fragment containing the nucleotide sequence that can be used as a probe as a template. The probe length is recommended to be greater than 50 bp, but can be appropriately selected depending on the hybridization conditions, and is usually between 100 bp and 1 kbp. For example, when using a DNA fragment with a length of approximately 300 bp as a probe, the washing conditions after hybridization may be, for example, 2×SSC, 0.1% SDS at 50°C, 60°C, or 65°C.

[0017] To assess the degree of identity of proteins or DNA, several calculation methods can be used, such as BLAST search, FASTA search, and the ClustalW method. BLAST (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov / BLAST / ) search is a heuristic search algorithm used by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx, which attribute significance to discoveries using the statistical methods of Karlin S. and Altschul SF ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci. USA, 1990, 87:2264-2268; "Applications and statistics for multiple high-scoring segments in molecular sequences". Proc. Natl. Acad. Sci. USA, 1993, 90:5873-5877). The computer program BLAST calculates three parameters: score, identity, and similarity. The FASTA search method is described by Pearson WR ("Rapid and sensitive sequence comparison with FASTP and FASTA", Methods Enzymol., 1990, 183:63-98).The ClustalW method is described by Thompson J.D. et al. (“CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, Nucleic Acids Res., 1994, 22:4673-4680).

[0018] In this specification, the term "identity" may mean "homology" between amino acid sequences or nucleotide sequences. The sequence identity between two sequences is calculated as the ratio of residues that match between the two sequences when the two sequences are aligned so as to obtain the maximum alignment. The "identity" between amino acid sequences specifically means, unless otherwise specified, the identity calculated by blastp using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). Also, the "identity" between nucleotide sequences specifically means, unless otherwise specified, the identity calculated by blastn using the default Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0019] This specification discloses B. breve that possesses the aforementioned GH59 protein or DNA encoding the same. Specifically, Bifidobacterium breve MCC01128, B. breve MCC10201, B. breve MCC10206, and B. breve MCC10258 are mentioned. However, other B. breve are also included in the B. breve of the present invention as long as they possess the aforementioned GH59 protein or DNA encoding the same.

[0020] Bifidobacterium breve MCC01128 was internationally deposited on July 10, 2024, with accession number NITE BP-04132 at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan, Room 122) under the Budapest Convention. Bifidobacterium breve MCC10201 was internationally deposited on July 10, 2024, with accession number NITE BP-04133 at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan, Room 122) under the Budapest Convention. Bifidobacterium breve MCC10206 was internationally deposited on July 10, 2024, with accession number NITE BP-04134 at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan, Room 122) under the Budapest Convention. Bifidobacterium breve MCC10258 was internationally deposited on July 10, 2024, with accession number NITE BP-04135 at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan, Room 122) under the Budapest Convention.

[0021] Furthermore, the bacteria identified by the bacterial names exemplified above are not limited to the actual strains deposited or registered with the designated institution under those bacterial names (hereinafter, for convenience of explanation, also referred to as "deposited strains"), but also include strains that are substantially equivalent thereto (also referred to as "derived strains" or "inducible strains"). In other words, it is not limited to the actual strains deposited with the depositary institution under the above accession number, but also includes strains that are substantially equivalent thereto. For each bacterium, "a strain substantially equivalent to the above-mentioned deposited strain" refers to a strain that belongs to the same species as the above-mentioned deposited strain, and furthermore has a similarity in genome sequence (Average Nucleotide Identity value) to the above-mentioned deposited strain of preferably 99.5% or more, more preferably 99.9% or more, and even more preferably 100% identity, and preferably has the same mycological properties as the above-mentioned deposited strain. Here, it is preferable that the strain substantially equivalent to the above-mentioned deposited strain possesses only genes derived from the deposited bacterium and does not have genes of foreign origin. For each bacterium, a strain substantially equivalent to the deposited strain may, for example, be a derivative strain with the deposited strain as the parent strain. Examples of derivative strains include strains bred from the deposited strain and strains that arose naturally from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutagenesis. Examples of mutagenesis 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. Examples of strains that arose naturally from the deposited strain include strains that arose naturally during the use of the deposited strain. Examples of such strains include mutant strains that arose naturally through the cultivation (e.g., subculturing) of the deposited strain. A derivative strain may be constructed by one modification, or by two or more modifications. Furthermore, a strain substantially equivalent to the above-mentioned deposited strain may be a bacterium determined to be the same bacterium by the Randomly Amplified Polymorphic DNA method or the Pulsed-field gel electrophoresis method (as described in Probiotics in food / Health and nutritional properties and guidelines for evaluation, page 43, 85).

[0022] This specification discloses a composition containing the aforementioned GH59 protein or the Bacillus breve cells carrying DNA encoding the same, its culture, and / or its cell-treated product. The cells may be live cells, dead cells, or a mixture of live and dead cells, but preferably contain live cells. The live cells may be live cells recovered from a culture or obtained from a commercial product. Also, the live cells may be in the form of a culture obtained by culturing the bacterium as it is, or in a form obtained by diluting or concentrating it. Further, as long as Bifidobacterium breve is alive, the live cells may be in the form of a bacterial powder produced by a method such as a liquid nitrogen freezing method, a spray drying method, or a freeze drying method, or may be in a form treated by an oil drop method.

[0023] As the culture of Bacillus breve, for example, the culture obtained by culturing may be used as it is, the culture may be diluted or concentrated and used, or the cells recovered from the culture may be used. Also, as the culture, a culture supernatant or a culture fraction may be used. When using the culture supernatant, for example, the supernatant of the culture solution when cultured in a GAM medium at 37°C for 16 h can be preferably used. As the cell-treated product of Bacillus breve, disruption, heating, drying, and their dilutions, dried products, or fractions can be used for the cells or the culture. The heat-treated product of the cells (heat-killed cells) can be obtained, for example, by treating the bacterium at 70 to 100°C for 10 to 40 minutes or at 90 to 150°C for 5 to 30 seconds. In addition, pressure may be applied during the heat treatment. During the heat treatment, the temperature does not necessarily have to be constant, as long as it is within the above temperature range for a predetermined time. The heat-killed cells of the bacterium may be used as they are after the heat treatment, or those treated by a treatment such as disruption, heat drying, freeze drying, or spray drying may be used. The culture and the cell-treated product of Bacillus breve cells usually refer to those containing the GH59 protein.

[0024] The method for culturing B. breve is not particularly limited, as long as the bacteria can grow. For example, the method commonly used for culturing B. breve can be used as is, or modified as appropriate. The culture temperature may be, for example, 25 to 50°C, and preferably 35 to 42°C. Cultivation can preferably be carried out under anaerobic conditions, for example, by aerating with an anaerobic gas such as carbon dioxide. Alternatively, cultivation can be carried out under microaerophilic conditions, such as liquid static culture. Cultivation can be continued, for example, until B. breve has grown to a desired extent.

[0025] The culture medium used for cultivation is not particularly limited, as long as it allows B. breve to grow. For example, the culture medium commonly used for culturing B. breve can be used as is or modified as appropriate. Specifically, as a carbon source, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysates, and molasses can be used depending on their assimilation properties. As a nitrogen source, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, as well as nitrates, can be used. Inorganic salts such as sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate can be used. Organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract may also be used. Commonly used culture media for *Breve* include reinforced Clostridial medium, MRS medium (de Man, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium), TOSP medium (TOS propionate medium), TOSP Mup medium (TOS propionate mupirocin medium), GAM (Gifu Anaerobic Medium) medium, and YCFA (Yeast Extract-casein Hydrolysate Acid) medium.

[0026] In the composition of the present invention, the content of the cells of B. breve is not particularly limited and is appropriately set according to the form of the composition. For example, as the cell mass of the bacterium, 1×10 ４ to 1×10 １３ cfu / g or 1×10 ４ to 1×10 １３ cfu / mL is preferably used, and 1×10 ５ to 1×10 １２ cfu / g or 1×10 ５ to 1×10 １２ cfu / mL is more preferably used, and 1×10 ６ to 1×10 １１ cfu / g or 1×10 ６ to 1×10 １１ cfu / mL is even more preferably used. In this specification, "cfu" represents colony forming unit (colony forming unit). When using dead cells, cfu / g or cfu / mL may be read as individual cells / g or individual cells / mL. When using the culture supernatant as the culture of B. breve, it is preferably 0.1 to 100% by mass of the whole composition, more preferably 1 to 90% by mass, and even more preferably 10 to 80% by mass. In the composition of the present invention, the content of the cells of B. breve relative to the whole composition is preferably 0.001% by mass or more and 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 when distributed as an oral composition.

[0027] This specification also provides a composition containing the aforementioned GH59 protein. When incorporating it into the composition, it can be incorporated in any manner as long as the protein does not impair the GH59 activity. The content of the GH59 protein in the composition of the present invention is not particularly limited and is appropriately set according to the form of the composition. For example, it can be 1×10 －７ to 100% by mass of the whole composition, 1×10 －６ to 95% by mass, or 1×10 －５It may be up to 90% by mass. These may be within the range of content typically used when distributed as an oral composition.

[0028] In this specification, GH59 activity refers to 6-O-β-glucuronyl-β-galactosidase. The GH59 possessed by B. breve has 6-O-β-glucuronyl-β-galactosidase activity and can act on and decompose carbohydrates having higher-order structures. Carbohydrates having higher-order structures refer to so-called complex carbohydrates, which are a general term for complexes of carbohydrates with other biomolecules. Examples of such complex carbohydrates include glycoproteins. The target on which the GH59 possessed by B. breve acts through its 6-O-β-glucuronyl-β-galactosidase activity may also be dietary fiber. Dietary fiber refers to carbohydrates that cannot be digested by human digestive enzymes, and as dietary fiber, those that dissolve in water, i.e., water-soluble dietary fiber, are preferred. In this invention, B. As a carbohydrate or dietary fiber having a higher-order structure on which GH59 possessed by B. breve acts, arabinogalactan protein (AGP), which is a glycoprotein and a water-soluble dietary fiber, is particularly preferred. GH59 acts on the side chain ends of AGP and can cleave the side chains. GalcA-β1,6-Gal and GalcA-β1,6-Gal2 are preferred as such side chains. However, in the present invention, the carbohydrate or dietary fiber having a higher-order structure on which GH59 possessed by B. breve acts is not limited to AGP, and may be other carbohydrates having GalcA-β1,6-Gal or GalcA-β1,6-Gal2 in the side chain.

[0029] From the above, compositions containing B. breve cells possessing the aforementioned GH59 protein or DNA encoding it, their cultures, and / or processed cell products, and compositions containing GH59 protein, can each be used as compositions for improving dietary fiber metabolism, and can also be used as food and beverage compositions. Furthermore, a method for improving dietary fiber metabolism can be used by using one or more selected from GH59 protein, B. breve cells possessing the GH59 protein or DNA encoding it, their cultures, and / or processed cell products, and the compositions of the present invention described above. Specifically, dietary fiber metabolism in a subject can be improved by administering the bacterial cells or compositions according to the present invention to that subject.

[0030] Furthermore, by reacting the GH59 protein, the cells of B. breve possessing the GH59 protein or DNA encoding it, its culture, and / or its cell treatment, and the aforementioned composition of the present invention with a carbohydrate having a higher-order structure, the carbohydrate can be degraded by an enzymatic reaction mediated by the GH59 protein. Also, by reacting the GH59 protein, the cells of B. breve possessing the GH59 protein or DNA encoding it, its culture, and / or its cell treatment, and the aforementioned composition of the present invention with a raw material containing AGP, an oligosaccharide containing glucuronic acid (GlcA) can be produced by an enzymatic reaction mediated by the GH59 protein. Note that the oligosaccharide containing GlcA is an oligosaccharide residue present in the side chain of AGP and is cleaved by the GH59 protein. As oligosaccharides containing GlucA, oligosaccharides containing GlucA and galactose (Gal) are preferred, oligosaccharides consisting of GlucA and Gal are more preferred, and GlucA-β1,6-Gal and GlucA-β1,6-Gal ２Oligosaccharides selected from the above are even more preferred. Here, the higher-order carbohydrate is preferably water-soluble, for example, AGP is a preferred example. AGP is a glycoprotein unique to plants and is universally found in angiosperms, gymnosperms, mosses, algae, and fungi. Raw materials containing AGP include these plants and their processed products. It may also be an additive in processed foods, for example, gum arabic, which is used as a stabilizer or emulsifier, is also a preferred raw material containing AGP. The conditions for the enzymatic reaction with GH59 protein are not particularly limited, but it is preferably carried out at a temperature of 15 to 50°C, and more preferably at a temperature of 35 to 40°C. The pH of the reaction system is preferably 4 to 7, and more preferably 5 to 6. The reaction time can be 5 hours or more, preferably 10 hours or more, more preferably 15 hours or more, and there is no particular upper limit, but it may be 48 hours or less, and preferably 24 hours or less.

[0031] The target of administration (ingestion) of the composition of the present invention is not particularly limited to animals, but is usually a mammal, and is preferably a human. Furthermore, although the target is not particularly limited, a healthy person is preferred. Here, a healthy person means a person who is not suffering from disease or illness, and is the target of administration (ingestion) for non-therapeutic purposes. Furthermore, the composition of the present invention may also be administered (ingested) for therapeutic purposes, in which case the target would be a person with a condition in which the metabolism of dietary fiber is reduced. Furthermore, although the target of administration (ingestion) of the composition of the present invention is not particularly limited, an infant in the weaning period is preferred. The target may also be an adult.

[0032] Furthermore, "administering to a subject" may be synonymous with "allowing the subject to ingest." Ingestion may be voluntary (ad libido) or compulsory (forced ingestion). Specifically, administration may involve, for example, supplying to a subject by incorporating GH59 protein, or B. breve cells possessing GH59 protein or DNA encoding it, their cultures, and / or processed products thereof, into food, beverages, animal feed, or pharmaceuticals, thereby allowing the subject to ingest them ad libido.

[0033] The timing and duration of ingestion (administration) of the composition of the present invention are not particularly limited and can be appropriately selected depending on the condition of the recipient.

[0034] Another aspect of the present invention is the use of GH59 protein or B. breve containing it in the production of a composition for improving the metabolism of dietary fiber. Another aspect of the present invention is the use of GH59 protein or B. breve containing it in the improvement of the metabolism of dietary fiber. Another aspect of the present invention is GH59 protein or B. breve containing it, used for improving the metabolism of dietary fiber. Another aspect of the present invention is the use of GH59 protein or B. breve containing it in the production of a composition for the breakdown of carbohydrates having a higher-order structure. Another aspect of the present invention is the use of GH59 protein or B. breve containing it in the breakdown of carbohydrates having a higher-order structure. Another aspect of the present invention is GH59 protein or B. breve containing it, used for the breakdown of carbohydrates having a higher-order structure.

[0035] The composition of the present invention may be in the form of food or pharmaceuticals, or it may be included as an additive in food or pharmaceuticals. The route of intake (administration) of the composition of the present invention may be either oral or parenteral, but it is usually oral. Parenteral intake (administration) may include transdermal, intravenous, rectal, and inhalation.

[0036] The amount of the composition of the present invention to be ingested (administered) is appropriately selected depending on the age (months), sex, condition, and other conditions of the person receiving the intake (administration). For example, the amount of B. breve in the composition of the present invention is preferably 10 as the bacterial cell content. ６ ~10 １２ cfu / day, more preferably 10 ７ ~10 １１ cfu / day, particularly preferably 10 ８ ~10 １０ The amount can also be adjusted to be suitable for intake within the range of cfu / day. Furthermore, the composition of the present invention contains B. breve in a total amount, preferably 10 ６ ~10 １２ cfu / day, more preferably 10 ８ ~10１１ cfu / day, particularly preferably 10 ９ ~10 １０ It is preferable to use the product in an intake of cfu / day. Regardless of the amount or duration of intake (administration), the composition of the present invention can be taken (administered) once a day or in multiple divided doses.

[0037] When the composition of the present invention is intended for oral intake, it is preferable to provide it in the form of a food or beverage. In this case, the food or beverage is usually used for non-therapeutic purposes. As long as the food or beverage does not impair the effects of the present invention and can be taken orally, its form and properties are not particularly limited, and it can be manufactured using ordinary methods with raw materials commonly used in food and beverages, except for containing GH59 protein or B. breve containing it.

[0038] Food and beverages include, regardless of form, such as liquid, paste, gel, solid, or powder, for example: tablets; liquid foods (nutritional foods for tube feeding); wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken batter, and breadcrumbs; instant noodles, cup noodles, retort and prepared foods, canned prepared foods, microwaveable foods, instant soups and stews, instant miso soup and clear soups, canned soups, freeze-dried foods, and other instant foods; canned agricultural products, canned fruits, and jasmine. Processed agricultural products such as marmalades, pickles, boiled beans, dried agricultural products, and cereals (grain processed products); processed marine products such as canned seafood, fish ham and sausages, processed seafood products, seafood delicacies, and tsukudani (simmered seafood); processed livestock products such as canned and paste meats, and meat ham and sausages; dairy and dairy products such as processed milk, milk beverages, yogurt (fermented milk), lactic acid bacteria beverages, cheese, ice cream, cream, and other dairy products; fats and oils such as butter, margarine, and vegetable oil; soy sauce, miso, and sausages. Basic seasonings such as vinegars, tomato-based seasonings, mirin, and vinegars; compound seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soup bases, spices, and other compound seasonings; frozen foods such as raw frozen foods, semi-cooked frozen foods, and cooked frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice sweets, bean sweets, dessert sweets, jelly, and other confectionery. Examples include confectionery; carbonated drinks, natural fruit juices, fruit juice drinks, fruit juice-containing soft drinks, fruit pulp drinks, fruit juice drinks with fruit pulp, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic beverages, and other beverages for enjoyment; baby food, furikake (rice seasoning), nori (rice seasoning for ochazuke), and other commercially available foods; nutritional compositions such as prepared milk (including powdered milk and liquid milk); enteral nutrition foods; and functional foods (foods for specified health uses, foods with nutritional function claims).

[0039] Of these, nutritional compositions are preferred. In the present invention, "nutritional composition" is not particularly limited as a form of food or beverage, but is preferably prepared milk, liquid food, etc., and more preferably prepared milk. The target of consumption is infants, toddlers, children, or adults, but is preferably infants and toddlers, and more preferably toddlers during the weaning period. Prepared milk includes prepared powdered milk and prepared liquid milk. Prepared powdered milk is defined in the Ministerial Ordinance on Standards for Ingredients of Milk and Dairy Products (Milk and Dairy Products Ordinance) as "a powder made by processing raw milk, milk, special milk, or food manufactured using these as raw materials, or using them as the main raw materials, with nutrients necessary for infants added." Prepared liquid milk is defined in the same ordinance as "a liquid made by processing raw milk, milk, special milk, or food manufactured using these as raw materials, or using them as the main raw materials, with nutrients necessary for infants added." Furthermore, prepared milk is a blend of various nutrients such as proteins, carbohydrates, lipids (oils and fats), vitamins, and minerals, and also includes those processed into powder or liquid form. Furthermore, prepared milk includes "infant formula," "infant liquid milk," and "infant formula for pregnant and lactating women" as special dietary foods stipulated in the Health Promotion Act, and also includes forms such as infant formula, nutritional powder for adults, and nutritional powder for the elderly. Follow-up milk is also preferably included.

[0040] Furthermore, functional foods and beverages are preferably formulated as various supplements, as this makes it easier for consumers to understand the amount of active ingredients they are taking. Additionally, supplements are preferably consumed with meals or added to meals, from the perspective of promoting the metabolism of dietary fiber. Meals to which supplements are added typically contain carbohydrates with a higher-order structure, such as AGP. These meals may be regular meals for adults, or baby food or toddler food for infants. When formulated as supplements, they can be solid preparations such as powders, granules, tablets, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions; which may be enterically coated or otherwise treated. In such formulations, the drug can be formulated together with, for example, sugars such as lactulose, maltitol, and lactitol, and other sugars such as dextrin and starch; proteins such as gelatin, soy protein, and corn protein; amino acids such as alanine, glutamine, and isoleucine; polysaccharides such as cellulose and gum arabic; and oils and fats such as soybean oil and neutral fatty acid triglycerides. The formulation can also be carried out in accordance with the description of components, carriers, and methods related to the formulation of pharmaceuticals described later.

[0041] Furthermore, it can also be used as animal feed as a form of food or beverage. Examples of animal feed include pet food, livestock feed, and fish feed. The form of the feed is not particularly limited, and in addition to GH59 protein or B. breve containing it, it may contain, for example, grains such as corn, wheat, barley, rye, and milo; vegetable oils such as soybean oil cake, rapeseed oil cake, coconut oil cake, and linseed oil cake; brans such as wheat bran, wheat bran, rice bran, and defatted rice bran; processed food products such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skim milk powder, whey, yellow grease, and taro; yeasts such as Torula yeast and brewer's yeast; mineral feeds such as tricalcium phosphate and calcium carbonate; oils and fats; single amino acids; sugars, etc. While there are no particular restrictions on the form of the aforementioned food and beverage, beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit juices, and lactic acid bacteria drinks (including concentrated stocks and powders for preparation of these beverages) are particularly preferred from the viewpoint of efficiently ingesting the aforementioned compound.

[0042] When the composition of the present invention is in the form of food or beverage (including animal feed), it can be provided and sold as food or beverage labeled with its use in improving the metabolism of dietary fiber. Furthermore, GH59 protein or B. breve containing it can be used in the manufacture of such food or beverage products.

[0043] Such "display" acts include all acts that make consumers aware of the aforementioned use, and any expression that can evoke or infer the aforementioned use falls under the "display" acts of this invention, regardless of the purpose of the display, the content of the display, or the object or medium on which it is displayed. Furthermore, it is preferable that the "display" is made in an expression that allows consumers to directly recognize the above use. Specifically, examples include acts of transferring, delivering, displaying for transfer or delivery, or importing food and beverage products or product packaging on which the above use is described; displaying or distributing advertisements, price lists, or transaction documents related to products on which the above use is described; or providing information containing these on an electromagnetic (Internet, etc.) basis on which the above use is described.

[0044] On the other hand, the content of the display is preferably a display approved by the government or other administrative body (for example, a display approved based on various systems established by the government and made in accordance with such approval). Furthermore, it is preferable to attach such display content to packaging, containers, catalogs, brochures, point-of-purchase (POP) displays and other promotional materials used at sales sites, and other documents.

[0045] Furthermore, "labeling" also includes labels for health foods, functional foods, enteral nutrition foods, foods for special dietary uses, health functional foods, foods for specified health uses, nutrient function foods, foods with functional claims, quasi-drugs, etc. In particular, labels approved by the Consumer Affairs Agency include, for example, labels approved under the systems for foods for specified health uses, foods with nutrient function, or foods with functional claims, or similar systems. Specifically, these include labels for foods for specified health uses, labels for conditionally specified health uses, labels that indicate an effect on the structure or function of the body, labels that indicate a reduction in disease risk, and labels that indicate functionality based on scientific evidence. More specifically, typical examples include labels for foods for specified health uses (especially labels indicating health uses) and similar labels as defined in the Cabinet Office Ordinance concerning the permission of special use labeling, etc., as stipulated in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009).

[0046] Examples of such labeling include claims related to improving the metabolism of dietary fiber or the breakdown of carbohydrates with higher-order structures. More specifically, these may include claims such as "improves the metabolism of dietary fiber," "aids in the absorption of nutrients from vegetables," "breaks down dietary fiber and provides a nutrient source for intestinal bacteria," "promotes the growth of intestinal bacteria," and "breaks down dietary fiber contained in vegetables in baby food."

[0047] The compositions of the present invention may also be in the form of pharmaceuticals. In this case, the compositions of the present invention may be used for therapeutic purposes, specifically for treating or preventing conditions in which dietary fiber metabolism is impaired.

[0048] The route of administration of the drug may be either oral or parenteral, but oral administration is preferred. Parenteral administration methods include transdermal, intravenous, rectal, and inhalation. The drug can be formulated into any desired dosage form depending on the method of administration. For example, for oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. It can also be made into an enteric-coated preparation by enteric coating, etc. For parenteral administration, it can be formulated into suppositories, ointments, injections, etc. In formulation, in addition to the GH59 protein or B. breve containing it, ingredients such as excipients, pH adjusters, colorants, and flavorings commonly used in formulation can be used. It is also possible to use other pharmacoactive ingredients or ingredients that have known or future-discovered effects on improving the metabolism of dietary fiber in combination. Furthermore, formulation can be carried out by any known method depending on the dosage form. During formulation, carriers commonly used in formulation may be added as appropriate. Examples of such carriers include excipients, binders, disintegrants, lubricants, stabilizers, and flavoring / odorizing agents.

[0049] 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, hydroxypropyl methylcellulose, carboxymethylcellulose, and carboxymethylcellulose 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.

[0050] Examples of binders include, in addition to the above-mentioned excipients, gelatin; polyvinylpyrrolidone; macrogol, and the like.

[0051] Examples of disintegrants include, in addition to the above-mentioned excipients, chemically modified starches or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone.

[0052] Examples of lubricants include talc; stearic acid; metal stearate salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as beegum and gayl wax; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylate 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; and starch derivatives.

[0053] Examples of stabilizers include para-hydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.

[0054] Examples of flavoring and odor-masking agents include sweeteners, acidulants, and flavorings. For liquid formulations intended for oral administration, the carrier used may be a solvent such as water.

[0055] The timing of taking the pharmaceutical product of the present invention is not particularly limited, for example, before meals, after meals, between meals, or before going to bed.

[0056] This specification also discloses a method for determining the degree of maturity of the intestinal microbiota of infants. This method determines the extent to which the intestinal microbiota is approaching a mature state after weaning, based on the fact that the expression level of the GH59 gene derived from B. breve increases in the intestinal microbiota of infants after weaning compared to before weaning. In this method, the expression level of the GH59 gene in the intestinal microbiota of the target infant, i.e., the abundance of the following (a), (b), or (c) DNA is used as an indicator: (a) DNA containing the base sequence of Sequence ID No. 1; (b) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of Sequence ID No. 1; (c) DNA containing a base sequence having 90% or more identity with the base sequence of Sequence ID No. 1. Specifically, as shown in the test example described below, the method is performed by the steps of calculating the relative abundance of the DNA in the total genes contained in the intestinal microbiota of the target infant, and comparing the relative abundance with the relative abundance of the DNA in the intestinal microbiota of infants in general after weaning. The relative abundance of the aforementioned DNA in the total genes contained in the gut microbiota of a target infant can be measured from the infant's feces using conventional methods. For example, it can be measured using homology searches of DNA base sequences with well-known software, detecting the sequence by creating primers specific to the sequence, or searching based on three-dimensional structure. The relative abundance of the aforementioned DNA in the gut microbiota of infants in general after weaning can be calculated, for example, based on the gut microbiota metagenomic sequences of weaned infants obtained from public databases. The target infants are preferably those in the weaning period.

[0057] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0058] <Test Example 1> Comparative Analysis of Genomic Data of Gut Microbiota Before and After Weaning by a Person Skilled in the Art. Metagenomic sequence data obtained from the gut microbiota contained in the feces of Japanese infants was classified and analyzed using a method specialized for carbohydrate metabolism-related enzymes (Glycoside Hydrolase family classification) to search for enzyme genes whose abundance fluctuates before and after weaning. The bioinformatics platform METAnnotatorX2 (https: / / christianmilani86.wixsite.com / bioinformatics / copia-di-megannotator) was used for functional profiling of the reads from which the metagenomic sequences were obtained. Specifically, filtering was performed to remove sequences with a length of less than 50 bp and sequences with an average quality value < 25 from the raw data in fastq format. Subsequently, the human genome (GRCh38) was processed using bowtie2 (https: / / bowtie-bio.sourceforge.net / bowtie2 / index.shtml) with the options --no-hd --no-sq --no-unal to remove host-derived sequences from the mapped reads. The filtered sequences were used as input for performing a sequence homology search (rapsearch, rapsearch v2.24; https: / / omics.informatics.indiana.edu / mg / RAPSearch / ) against a carbohydrate-active enzyme (CAZY) database. The threshold was e-value < 10. -8The relative abundance of genes according to the CAZY classification was calculated based on these results. The threshold for considering the abundance to have changed before and after weaning was set as a p-value < 0.05 for the change in the relative abundance of each gene before and after weaning by t-test, and an increase of more than twofold after weaning. As a result of the analysis, 18 genes were found, and in particular, GH59, a gene whose function is unknown in prokaryotes, was found to have a significantly increased abundance in the gut microbiota after weaning compared to before weaning (Figure 1). Next, the bacterial species from which the GH59 gene originates was investigated from the sequence data. When a phasic search (phasic > 70%, coverage > 60%) was performed on known GH59 sequences (531 sequences, 340 unique sequences) using rapsearch, it was confirmed that sequences originating from B. breve accounted for the majority.

[0059] <Test Example 2> Screening of GH59-containing bacterial strains A Genbank ID list was obtained for known sequences (531 sequences, 340 unique sequences; http: / / www.cazy.org / IMG / cazy_data / GH59.txt) registered in the CAZY database as GH59, and the sequences listed were obtained from Genbank (https: / / www.ncbi.nlm.nih.gov / protein / ). Targeting the genome sequences of Bifidobacterium bacteria, homology searches (rapsearch v2.24) were performed using the obtained sequences as a database (comparison target), with a threshold of 70% or more sequence homology and 60% or more sequence coverage for any of the sequences. As a result, the following Bifidobacterium breve was found to possess GH59 and to be a candidate probiotic bacterium capable of metabolizing carbohydrates using AGP. The sequence numbers listed next to the bacteria below refer to the amino acid sequence of GH59 possessed by the bacterium and the nucleic acid sequence encoding it. Bifidobacterium breve MCC01128 (SEQ ID NO: 4, SEQ ID NO: 3) Bifidobacterium breve MCC10201 (SEQ ID NO: 6, SEQ ID NO: 5) Bifidobacterium breve MCC10206 (SEQ ID NO: 8, SEQ ID NO: 7) Bifidobacterium breve MCC10258 (SEQ ID NO: 10, SEQ ID NO: 9) In addition, the amino acid sequence common to GH59 possessed by Bifidobacterium breve is shown as SEQ ID NO: 2, and the nucleic acid sequence encoding it is shown as SEQ ID NO: 1.

[0060] <Test Example 3> Functional Analysis of GH59 Derived from Intestinal Bacteria Species (1) Preparation of Recombinant GH59 (1-1) Genetic Information of Target Sequences and Plasmid Construction Functional analysis was performed on four types of GH59 derived from Bifidobacterium breve MCC01128, Lacticaseibacillus rhamnosus JCM1136, and Ruminococcus bicirculans strain 80 / 3. These four types of GH59 are referred to as Bb59 (BBM1128_RS10615, WP_021649418.1), Lr59A (Q777_RS12345, WP_012807330.1), Lr59B (Q777_RS12295, WP_019728571.1), and Rb59 (RBI_I00669, CCO04392.1), respectively (Figure 2). From the predicted domain structures of each GH59, Lr59A and Rb59 were predicted to have a signal peptide sequence at the N-terminus and a transmembrane domain at the C-terminus, and to be enzymes localized on the bacterial surface.

[0061] For the expression plasmids, the His6-tag was designed to be attached to the N-terminus only for Bb59, and to the C-terminus for the other three species. The Bb59 gene was amplified by PCR using the region excluding the N-terminus 1-8aa (the nucleotide sequence region corresponding to the amino acid sequence 9-913 aa), the Lr59A gene by PCR using the internal region excluding the signal peptide sequence (the nucleotide sequence region corresponding to the amino acid sequence 40-1463aa), and the full-length Lr59B gene by PCR using the primers shown in Table 1 (SEQ ID NOs. 11-18, in order). Since R. bicirculans 80 / 3 cells were unavailable, the Rb59 gene was amplified by codon optimization targeting the region excluding the signal peptide sequence (1-23aa) and the transmembrane domain (863-885aa) (the nucleotide sequence region corresponding to the amino acid sequence 24-862aa), designing a complementary sequence at the end, and artificially synthesizing it in two fragments using Eurofins.

[0062]

[0063] (1-2) Genome extraction L. rhamnosus JCM1136 TFor genome extraction from the cells, the Wizard® Genomic DNA (Deoxyribonucleic acid) Purification Kit (Promega) was used. Before using the kit, the cells were suspended in 10 mg / mL lysozyme solution and incubated at 37 °C for 60 minutes. Genome extraction from B. breve MCC01128 was performed by phenol-chloroform extraction. Specifically, the cells (1 mL of culture medium) were suspended in 150 μL of 0.5 mg / mL lysozyme-containing TE buffer and incubated at 37 °C for 3 hours. 30 μL of 5M NaCl was added, vortexed, and after spin-down, 15 μL of 10% SDS was added, mixed once by inversion, spun down, and incubated at room temperature. After 30 minutes, 100 μL of phenol-chloroform solution was added, mixed 30 times by inversion, and the supernatant was collected after centrifugation (5,800 × g, 4 °C, 5 min). 150 μL of supernatant was mixed with 375 μL of ethanol, then inverted 10 times. After spinning down, the mixture was allowed to stand overnight at -30 °C. The mixture was then centrifuged (12,000 × g, 4 °C, 10 min), the supernatant was discarded, 750 μL of 70% ethanol was added, and the mixture was centrifuged again (12,000 × g, 4 °C, 5 min) to remove the supernatant. After completely evaporating the ethanol using a speed backer, 200 μL of sterile water was added and the mixture was dissolved.

[0064] (1-3) Plasmid preparation (1-3-1) Cloning of Bb59 Using the genomic DNA of B. breve MCC01128 as a template, a solution containing genomic DNA (final concentration 4.0 ng / μL) and a primer set (each final concentration 0.3 μM) was prepared, and PCR was performed as described in Table 2. For the vector side, PCR was performed using the Nde I, Xho I treated pET23b plasmid as a template, as described in Table 3.

[0065]

[0066]

[0067] (1-3-2) Cloning of Lr59A and Lr59B genes. Lr59A is L. rhamnosus JCM1136 T Using the genomic DNA as a template, a solution containing genomic DNA (final concentration 3.3 ng / μL) and the above-mentioned primer set (each with a final concentration of 0.3 μM) was prepared, and the reactions described in Table 4 were carried out.

[0068]

[0069] Each PCR product was confirmed by agarose electrophoresis, and DNA extraction from the gel was performed using the Wizard® SV Gel and PCR Clean-Up System (Promega). DNA concentration was measured using a NanoPhotometer N60 (Implen) by measuring the absorbance at 260 nm (A). 260 This was calculated by measuring ( ).

[0070] (1-3-3) In-Fusion Cloning Reaction The In-Fusion Snap Assembly Master Kit (Takara Bio) was used to ligate the DNA fragments. For Bb59, approximately 10 ng each of PCR-amplified vector DNA and insert DNA was added. For Lr59A and Lr59B, the above-mentioned insert DNA (3.3 ng and 8.3 ng for Lr59A and Lr59B, respectively) and 5 ng each of Nde I and Xho I-treated pET23b plasmid were added, and the reaction was carried out by incubation in 2.5 μL of 1 × In-Fusion solution at 50 °C for 15 minutes.

[0071] (1-3-4) E. coli transformation and plasmid extraction Competent E. coli cells were prepared using the Mix & Go E. coli Transformation Buffer Set (ZYMO RESEARCH), aliquoted in 100 μL portions, and stored at -80 °C. E. coli DH5α [F - ,Φ80, lacZΔM15, Δ(lacZYA-argF), U169 recA1, endA1 hsdR17(r K - , mK +) , phoA supE44 λ - Thaw [thi-1, gyrA96, relA1] on ice, add 2.5 μL of In-Fusion reaction solution, vortex for 1 second, let stand on ice for 3 minutes, and incubate at 42 °C for 45 seconds. Then, immediately vortex for 1 second, let stand on ice for several minutes, add 400 μL of Luria-Bertani (LB) broth, Miller (DIFCO) liquid medium, and perform recovery culture at 37 °C for 1 hour. Spread LB agar (LBamp medium) to which ampicillin (Amp) was added to a final concentration of 100 μg / mL and culture at 37 °C until colonies formed. After single isolation of the colonies obtained from the agar medium, inoculate a single colony in liquid LBamp medium and culture overnight with shaking (180 rpm) in an aerobic environment at 37 °C. Plasmid extraction from E. coli was performed using the Wizard® Plus SV Minipreps DNA Purification System (Promega). DNA sequencing analysis of plasmids and specific regions of the genome was outsourced to Eurofins Genomics K.K.

[0072] (1-4) Expression and Purification of Recombinant GH59 (1-4-1) Expression and Purification of Recombinant Bb59 A Bb59 expression plasmid, whose sequence from start codon to stop codon had been confirmed by sequence analysis, was transformed into E. coli BL21 (DE3) and cultured on LB amp agar. Then, several colonies were combined and inoculated into 5 mL of LB amp liquid medium and cultured overnight with shaking (180 rpm) under aerobic conditions at 37 °C. Subsequently, recombinant protein expression was performed on the pre-cultured cells using the Overnight Express Autoinduction System (Novagen), an induction method that does not require IPTG induction. Specifically, following the kit protocol, 900 μL of pre-cultured bacteria were inoculated into approximately 90 mL of liquid medium, which was created by mixing 1.8 mL, 4.5 mL, and 90 μL of OnEx Solution 1, 2, and 3, respectively, into 84 mL of LB amp liquid medium. The culture was then rotated (120 rpm) for 24 hours under aerobic conditions at 25 °C. After culturing, the bacteria were divided into three 30 mL portions by centrifugation (12,000 × g, 4 °C, 10 mins) and stored at -30 °C until use. For the extraction procedure, the bacteria (60 mL of culture solution) stored at -30 °C were thawed on ice and then suspended in 3 mL of Lysis buffer [50 mM HEPES {4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid}, 300 mM NaCl, 10 mM imidazole, pH 8.0]. Subsequently, the cells were sonicated and disrupted using a micro-sonic homogenizer Q-125 (QSONICA) under conditions of a pulse interval of 1 sec on / 2 sec off and an amplitude of 30% for 15 minutes in ice. After cell disruption, the supernatant was collected by centrifugation (15,000 × g, 4 °C, 10 min). Due to viscosity, lysis buffer was added to a total volume of 10 mL, and the solution was subjected to affinity column purification.

[0073] Purification was performed by Ni-NTA (Nickel-nitrilotriacetic acid) affinity chromatography, anion exchange chromatography, and gel filtration chromatography. Ni-NTA affinity chromatography was performed using Ni-NTA agarose (Qiagen). The procedure was carried out according to the column's instruction manual. Specifically, 1 mL of resin was packed into the column vessel, equilibrated with lysis buffer (50 mM HEPES, 300 mM NaCl, 10 mM imidazole, pH 8.0), and then 10 mL of the supernatant of the cell disruption solution was passed through the column three times to bind the protein to the column. Subsequently, the column was washed by adding 10 mL of wash buffer (50 mM HEPES, 300 mM NaCl, 20 mM imidazole, pH 8.0). After washing the column three times, 3 mL of Elution buffer (50 mM HEPES, 300 mM NaCl, 500 mM imidazole, pH 8.0) was added to the column to elute the protein. Similarly, 3 mL of Elution buffer was added again, and elution was performed a total of two times. The resulting recombinant enzyme solution was desalted and concentrated using Amicon Ultra 10K (Merck Millipore) and replaced with 20 mM Tris-HCl buffer (pH 8.0). Furthermore, it was subjected to gradient elution with NaCl using AKTA pure 25 (Cytiva) on a MonoQ 5 / 50 GL anion exchange column (GE Healthcare) [10 mM sodium phosphate (pH 8.0), 0-0.5 M NaCl, 20 column volume (20 mL)]. Subsequently, the purified fraction was collected, desalted and concentrated using Amicon Ultra 10K (4 mL), and replaced with 20 mM Tris-HCl buffer (pH 8.0). Then, gel filtration chromatography was performed using Superdex 200 increase 10 / 30 GL (GE Healthcare). Elution was performed using 20 mM Tris-HCl buffer (pH 8.0). The collected fraction was concentrated using Amicon Ultra 10K (4 mL).

[0074] (1-4-2) The Lr59A expression plasmid, whose sequence from start codon to stop codon has been confirmed by recombinant Lr59A expression and purification sequence analysis, was used in Escherichia coli BL21 (DE3) ΔlacZ / pRARE2[F - , ompT, hsdSB (rB - , mB - After transforming [ΔlacZ, gal dcm (DE3), argU (AGA, AGG) proL (CCC)] using the same procedure as above, several colonies were combined and inoculated into 5 mL of LB medium supplemented with a final concentration of 100 μg / mL Amp and 10 μg / mL Cm, and incubated overnight with shaking (180 rpm) under aerobic conditions at 37 °C. 1.25 mL of the pre-culture solution was inoculated into 125 mL of LB liquid medium supplemented with the same concentrations of Amp and Cm, and OD was used. 600 The cells were cultured in aerobic conditions at 37 °C at 120 rpm until the concentration reached 0.5. Then, IPTG (Isopropyl β-d-thiogalactopyranoside) was added to a final concentration of 1 mM, and the cells were cultured in aerobic conditions at 18 °C at 120 rpm for 35 hours. After culturing, the cells were dispensed into 30 mL aliquots, and four aliquots were collected by centrifugation (8,000 × g, 4 °C, 10 mins). The cells were stored at -80 °C until use. For extraction, the cells were suspended in 5 mL of Lysis buffer (50 mM sodium phosphate buffer, 300 mM NaCl, pH 7.4), and then sonication was performed on ice in a manner almost identical to that used for Bb59.

[0075] Affinity purification was performed using TALON metal affinity resin (Clontech). 2 mL of resin was packed into a column vessel and equilibrated with Lysis buffer (50 mM sodium phosphate buffer, 300 mM NaCl, pH 7.4). 5 mL of the supernatant from the cell lysate was diluted to 30 mL by adding 25 mL of Lysis buffer and applied to the column to bind the protein. After washing with 30 mL of Lysis buffer, elution was performed by adding 10 mL each of buffers (pH 7.4) containing 5, 10, 20, 30, 40, 50, and 100 mM imidazole. The eluted fractions containing the target protein (20 mM and 30 mM imidazole eluted fractions) were desalted and concentrated using Amicon Ultra 50K (Merck Millipore) (15 mL) and replaced with 20 mM Tris-HCl buffer (pH 8.0).

[0076] (1-4-3) The Lr59B expression plasmid, whose sequence from start codon to stop codon has been confirmed by recombinant Lr59B expression and purification sequence analysis, was used in Escherichia coli BL21 (DE3) ΔlacZ / pRARE2 [F - , ompT, hsdSB (rB - , mB - After transforming [ΔlacZ, gal dcm (DE3), argU (AGA, AGG) proL (CCC)] using the same procedure as above, several colonies were combined and inoculated into 5 mL of LB medium supplemented with a final concentration of 100 μg / mL Amp and 10 μg / mL Cm, and incubated overnight with shaking (180 rpm) under aerobic conditions at 37 °C. 1 mL of the pre-culture solution was inoculated into 100 mL of LB liquid medium supplemented with the same concentrations of Amp and Cm, and OD was performed. 600The cells were cultured in aerobic conditions at 25 °C for 9.5 hours at 120 rpm until the concentration reached 0.3. Then, IPTG was added to a final concentration of 1 mM, and the cells were cultured in aerobic conditions at 18 °C for 39 hours at 120 rpm. After culturing, the cells were dispensed into 25 mL aliquots, and four aliquots were collected by centrifugation (8,000 × g, 4 °C, 10 mins). The aliquots were stored at -80 °C until use. For extraction, the cells were suspended in 5 mL of Lysis buffer (50 mM sodium phosphate buffer, 300 mM NaCl, pH 7.4), and subsequent sonication of the cells in ice was performed in much the same manner as for Bb59.

[0077] Affinity purification was performed using TALON metal affinity resin (Clontech) following the same procedure as for Lr59A. After purification, the eluted fractions containing the target protein (30 mM and 50 mM imidazole eluted fractions) were desalted and concentrated using Amicon Ultra 10K (Merck Millipore) (4 mL), and the solvent was replaced with 20 mM Tris-HCl buffer (pH 8.0).

[0078] (1-4-4) For the expression of recombinant Rb59 and the ligation of the purified total synthesized DNA with the vector DNA, the In-Fusion Snap Assembly Master Kit (Takara Bio) was used. 25 ng each of the two total synthesized DNA fragments and 3 ng of Nde I, Xho I-treated pET23b plasmid were added, and the reaction was carried out by incubation in 5 μL of 1 × In-Fusion solution at 50 °C for 15 minutes. Transformation of E. coli DH5α and plasmid extraction were performed using the same procedure as above. The Rb59 expression plasmid, whose sequence from start codon to stop codon had been confirmed by sequencing analysis, was transformed into E. coli BL21 (DE3) using the same procedure as above, and recombinant protein expression was performed using the Overnight Express Autoinduction System (Novagen), similar to Bb59. Specifically, 50 μL of pre-cultured bacterial cells were inoculated into a total of 5 mL of liquid medium prepared by mixing 105 μL, 368 μL, and 5.25 μL of OnEx Solution 1, 2, and 3, respectively, into LB amp liquid medium, and the culture was incubated with shaking (180 rpm) at 37°C for 18 hours. Extraction was performed using BugBuster protein extraction reagent (Novagen). After suspension in Bugbuster, the solution was incubated at room temperature for 10 minutes, then centrifuged at 15,000 rpm at room temperature for 10 minutes, and the supernatant was collected.

[0079] Subsequently, the supernatant was purified by Ni-NTA (Nickel-nitrilotriacetic acid) affinity chromatography, anion exchange chromatography, and gel filtration. Ni-NTA affinity chromatography was performed using Ni-NTA spin columns (Qiagen) according to the protocol. Specifically, 600 μL of lysis buffer was added to the Ni-NTA spin columns and equilibrated by centrifugation (300 × g, 4 °C, 5 min). Then, twice the amount of lysis buffer was added to the Bugbuster supernatant and passed through the column twice (300 × g, 4 °C, 5 min) to bind the protein to the column. After that, 600 μL of wash buffer (50 mM HEPES, 300 mM NaCl, 20 mM imidazole, pH 8.0) was added and the column was washed (900 × g, 4 °C, 2 min). After washing the column three times, elution buffer (50 mM HEPES, 300 mM NaCl, 500 mM imidazole, pH 8.0) was added to the column to elute the protein (900 × g, 4 °C, 2 min). Elution was repeated twice. The resulting recombinant enzyme solution was desalted and concentrated using Amicon Ultra 10K (Merck Millipore), and the solvent was replaced with 20 mM Tris-HCl buffer (pH 8.0). Furthermore, the solution was subjected to gradient elution with NaCl using AKTA pure 25 (Cytiva) on a MonoQ 5 / 50 GL anion exchange column (GE Healthcare) [10 mM sodium phosphate (pH 8.0), 0-0.5 M NaCl, 20 column volume (20 mL)]. Subsequently, the purified fraction was collected, concentrated using Amicon Ultra 10K, and the solvent was replaced with 20 mM Tris-HCl buffer (pH 8.0). Subsequently, gel filtration chromatography was performed using Superdex 200 increase 10 / 30 GL (GE Healthcare). Elution was performed using 20 mM Tris-HCl buffer (pH 8.0).The recovered fraction was replaced with 50 mM sodium acetate buffer (pH 5.5) using Amicon Ultra 10K (4 mL).

[0080] (1-5) Quantitative analysis of four recombinant GH59s and confirmation by SDS-PAGE. The concentrations of the purified proteins were calculated by measuring the absorbance at 280 nm using a NanoPhotometer N60 micro-spectrophotometer and referring to the predicted extinction coefficients of each enzyme based on the amino acid sequence (Table 5) using the ExPASy PlotParam tool (https: / / web.expasy.org / protparam / ).

[0081]

[0082] The purified proteins were identified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). A separation gel with an 8.75% acrylamide concentration was used. The Unstained Protein Marker, Broad Range (New England Biolabs) was used as the marker. Quick-CBB (Coomassie brilliant blue, Wako) was used for gel staining, and destaining was performed with MilliQ water (Figure 3).

[0083] (2) Measurement of enzyme activity using recombinant GH59 (2-1) Preparation of substrate Senegalese gum arabic was purchased from Sigma-Aldrich (St. Louis, MO, USA). The precipitate fraction was collected by ethanol precipitation beforehand, and endogenous monosaccharides, disaccharides, and oligosaccharides were removed before use. In addition, for Senegalese gum arabic, recombinant enzyme treatment was performed to trim side-chain modified sugars and was also used as a substrate. To separate the polysaccharide fraction after enzyme treatment, the precipitate was collected by ethanol precipitation. Specifically, trimmed Senegalese gum arabic was prepared by reacting 2% Senegalese gum arabic with 1.75 μg / mL of GH39 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase), 0.66 μg / mL of GH39 3-O-β-L-arabinopyranosyl-α-L-arabinofuranosidase (AAfase), and 1.06 μg / mL of GH43_22 / GH43_34 α-L-arabinofuranosidase (BlArafE) in 50 mM sodium acetate buffer (pH 6.0) at 37 °C for 16 hours. After the reaction, the enzymes were inactivated by incubation at 100 °C for 5 minutes to stop the reaction. Subsequently, ethanol was added to achieve a final concentration of 80%, and the mixture was vortexed for several minutes before being allowed to stand at 4°C for 16 hours. After centrifugation at 12,000 × g for 10 minutes at 4°C, the precipitate was collected, the ethanol was evaporated, and the precipitate was dissolved in Elix water. The recombinant enzymes used were GAfase (Y. Sasaki et al., Appl. Environ. Microbiol., 87, e02690-02620 (2021)), AAfase (Y. Sasaki et al., Microbiome Research Reports, 2 (2023)), and BlArafE (Y. Sasaki et al., Appl. Environ. Microbiol., aem. 02187-02121 (2022)), which were expressed and purified according to previously reported methods.

[0084] (2-2) Enzymatic reaction using polysaccharide substrate Senegalese gum arabic or side-chain trimmed Senegalese gum arabic with a final concentration of 1.6% was reacted with various recombinant GH59 (Bb59, Lr59A, Lr59B, Rb59) at 1.25 nM concentrations in 50 mM sodium acetate buffer (pH 5.5) at 37 °C for 16 hours, and the reaction solution was analyzed by TLC (Thin-layer chromatography). In the TLC analysis, 1 μL of each sample was spotted onto a TLC aluminum plate silica gel 60 (Merck) and developed with the developing solvent (n-butanol:acetic acid:water = 2:1:1). After development, a diphenylamine / aniline / phosphoric acid solution (100 mL acetone, 1 g diphenylamine, 1 mL aniline, 10 mL phosphoric acid) was sprayed and heated in an oven until the sugars changed color. The results are shown in Figure 4. In TLC, among the multiple water-soluble polysaccharide spots present before the enzymatic reaction, the AGP spot became fainter, and spots of AGP degradation products, which were not present before the reaction, appeared. This indicates that AGP was degraded and an AGP degradation product (GlcA-β1,6-Gal2) was produced. From these results, it was confirmed that AGP, one of the components of plant cell walls, is degraded and sugars are released.

[0085] (2-3) Extraction of water-soluble fraction derived from vegetables: The vegetables were cut and ground in a mixer while adding water until the total volume was approximately 2L. The mixture was heated in a water bath for 2 hours, stirring occasionally, and then centrifuged at 8,000 rpm for 7 minutes at 4°C to collect the supernatant. The mixture was filtered using a coffee filter, and the filtrate was collected, after which three times the amount of ethanol was added. After being suspended for a while, it was left to stand overnight at 4°C, and then centrifuged at 3,000 rpm for 10 minutes at 4°C to collect the precipitate fraction. The centrifugation was repeated three times, and only the precipitate fraction was collected. The ethanol was completely evaporated, and the mixture was frozen at -80°C and freeze-dried.

[0086] (2-4) Activity measurement of Lr59A on water-soluble fraction derived from vegetables The water-soluble fraction derived from vegetables obtained was again precipitated in 80% ethanol, and the precipitated fraction was collected. The water-soluble vegetable fraction with a final concentration of 1.6% was incubated with 2.5 nM Lr59A in 50 mM sodium acetate buffer (pH 5.5) at 37 °C for 16 hours, and the reaction solution was analyzed by TLC using the method described above. The results are shown in Figure 5. In TLC, among the multiple water-soluble polysaccharide spots that were present before the enzymatic reaction, the AGP spot became fainter, and spots of AGP degradation products that were not present before the reaction appeared. This indicates that AGP was degraded and AGP degradation products (GlcA-β1,6-Gal) were produced. From these results, it was confirmed that AGP, one of the components of plant cell walls, is degraded and sugars are released.

[0087] <Test Example 4> Assimilation test using B. breve with or without the Bb59 gene (1-1) Preparation of MRSCS medium The medium used was Difco® Lactobacilli de Man, Rogasa and Sharpe (MRS) Broth (BD biosciences, San Jose, USA) medium (MRSCS medium) containing 0.34% (w / v) sodium ascorbate and 0.02% (w / v) cysteine. MRSCS medium was prepared with the composition shown in Table 6. Culturing was performed under anaerobic conditions using AnaeroPack (MITSUBISHI GAS CHEMICAL).

[0088]

[0089] (1-2) Culture test on Rha-GlcA-Gal2 oligosaccharide derived from gum arabic AGP (1-2-1) Preparation of sugar source An assimilation test was performed using Rha-GlcA-Gal2 oligosaccharide (Rha-α1,4-GlcA-β1,6-Gal-β1,6-Gal), which is a side chain component of gum arabic AGP and a substrate for Bb59. Previous reports have shown that when B. longum JCM7052 is cultured on gum arabic, the Rha-GlcA-Gal2 oligosaccharide is not utilized by B. longum JCM7052 and remains in the culture medium (Y. Sasaki et al., Appl. Environ. Microbiol., aem. 02187-02121 (2022)). Therefore, Rha-GlcA-Gal2 oligosaccharide was prepared from the supernatant after culturing B. longum JCM7052 on gum arabic. Specifically, B. longum JCM7052 was cultured anaerobically at 37 °C for 48 hours in MRSCS medium containing a final concentration of 5% Senegalese gum arabic. The supernatant was collected by centrifugation (8000 × g, 10 minutes, room temperature), and ethanol was added to the supernatant to create an 85% ethanol environment, and ethanol precipitation was performed. After suspending with ethanol, the mixture was allowed to stand overnight at 4°C and then centrifuged again (8000 × g, 10 minutes, room temperature). Although the target Rha-GlcA-Gal2 is an oligosaccharide, it precipitated at this stage (likely due to the presence of glucuronic acid, an acidic sugar). Therefore, the precipitated fraction was collected, the pH was adjusted again to approximately 2, and ethanol precipitation was performed in a 70% ethanol environment. At this stage, the oligosaccharide migrated to the supernatant, so centrifugation was performed under the same conditions, and the supernatant was collected. The supernatant was dried using a speed bag, and the target sugar was recovered.

[0090] (1-2-2) Culture test and evaluation of sugar utilization capacity B. breve MCC01128 (Bb59-containing strain) and B. breve JCM1192 T(Bb59-non-containing strain) was cultured overnight in liquid MRSCS at 37 °C under anaerobic conditions, and then diluted with sterile water to an OD600 (Optical density at 600 nm) of 1.0. Pre-cultured cells were added to 500 μL of MRSCS medium in an amount of 5 μL (initial OD600 = 0.01), and cultured for 48 hours at 37 °C under anaerobic conditions in MRSCS medium containing either 1% galactose or 0.08% Rha-GlcA-Gal2 oligosaccharide as the sole carbon source. The culture was performed in three parallel cultures, and a sugar-free medium was also used for comparison. The OD600 of each medium without bacteria was used as the growth rate at 0h, and the OD600 at each observation point was subtracted from the OD600 at 0h for evaluation. OD600 measurements were performed by adding 100 μL of culture medium to a 96-well half-well plate (Corning, C3596) and using an absorbance microplate reader, Multiskan GO Advanced (Themo Fisher Scientific). For residual sugar analysis after culturing, the culture medium was centrifuged at 15,000 rpm for 10 minutes at 4 °C, and the supernatant was collected and analyzed by TLC. Supernatant analysis was performed in a single series. For the TLC analysis method, 1 μL of each sample was spotted onto a TLC aluminum plate silica gel 60 (Merck) and developed with the developing solvent (n-butanol:acetic acid:water = 2:1:1). After development, a diphenylamine / aniline / phosphoric acid solution (100 mL acetone, 1 g diphenylamine, 1 mL aniline, 10 mL phosphoric acid) was sprayed, and the plate was heated in an oven until the sugars changed color. The culture curves for each bacterium are shown in Figure 6, and the TLC results are shown in Figure 7. In TLC, the AGP spots became fainter in the culture medium after culturing, and spots of AGP degradation products, which were not present before culturing, appeared. This indicates that AGP was degraded and AGP degradation products were generated. From this, it was confirmed that B. breve MCC01128 has the ability to degrade AGP and generate AGP degradation products (GlcA-β1,6-Gal).

[0091] (1-3) Culture test on 4MeGlcA-Gal2 oligosaccharide derived from vegetable AGP (1-3-1) Preparation of sugar source Hot water extraction of water-soluble fractions was performed from radish and cabbage. 1.6 kg of radish (including water content) was finely chopped, 0.1 NaCl was added to make a total liquid volume of approximately 2 L, and it was pulverized in a mixer. It was heated at 100 °C for 60 minutes with occasional stirring, and the supernatant was collected using a Buchner funnel. The supernatant was then filtered through a 0.2 μm filter, and the tranquilizer was concentrated using an evaporator. The concentrated water-soluble fraction was centrifuged (12,000 × g, 4°C, 10 minutes), and the supernatant was collected. Three times the volume of ethanol was added to the collected supernatant, and after stirring at room temperature for 2 hours using a stirrer bar, it was left to stand at 4°C for 4 days. The precipitate fraction was collected by centrifugation (8,000 × g, 4°C, 15 minutes), and the collected precipitate fraction was washed twice with 80% ethanol. After centrifugation again (8,000 × g, 4°C, 15 minutes), the precipitate fraction was dissolved in Elix water, and then dialyzed with tap water using a dialysis membrane for 3 days at 4°C. The fraction after dialyzed was freeze-dried. The water-soluble fraction from cabbage was extracted using almost the same procedure.

[0092] After recovering the water-soluble fraction, the starch fraction contained in the recovered water-soluble fraction was removed by amylase and glucoamylase treatment. Specifically, after recovering the water-soluble fraction from the vegetables as described above, 1 g of the water-soluble fraction of radish was incubated with 600 U of amylase (from A. oryzae, Sigma) or 30-60 U of amyloglucosidase (from A. nigar, Sigma) in 100 mL of 50 mM sodium acetate buffer at 37 °C for 4 hours. Then, the mixture was heated at 100 °C for 5 minutes to inactivate the enzymes. Ethanol was added to the reaction mixture to create an 80% ethanol environment, and the mixture was suspended and allowed to stand overnight at 4 °C to perform ethanol precipitation. Then, the mixture was centrifuged (8,000 × g, 15 min, 4 °C) and the precipitate was recovered. The recovered precipitate fraction was then washed with 80% ethanol, centrifuged again, suspended in Elix water, and freeze-dried to obtain the starch-removed fraction. The same procedure was used to remove starch from the water-soluble fraction of cabbage.

[0093] (1-3-2) Culture Test and Evaluation of Sugar Utilization Ability According to H. Ichinose et al. (Applied and Environmental Microbiology, May 2006, Vol. 72, Issue 5), Clostridium thermocellum and other Clostridium species can act on AGP in radish to cleave 4MeGlcA-Gal2. Based on this, the inventors confirmed that when B. longum JCM1217 was cultured in water-soluble fractions of radish and cabbage, 4MeGlcA-Gal2, which is a substrate for Bb59, accumulated. Therefore, an assimilation test of B. breve was performed using the supernatant after culturing B. longum JCM1217. First, as the sole carbon source, 8 μL of pre-cultured B. longum JCM1217 was inoculated into 800 μL of MRSCS medium containing starch-removed radish water-soluble fractions and cabbage water-soluble fractions with final concentrations of 4% and 2%, respectively, and cultured at 37 °C under anaerobic conditions for 57 hours. After culturing, sufficient accumulation of 4MeGlcA-Gal2 was confirmed by TLC, and then the medium was centrifuged at 15,000 rpm for 10 minutes at room temperature, and the supernatant was collected. Subsequently, several μL of 1.25N NaOH was added to neutralize the medium, and then the medium was sterilized using a 0.2 μm pore size filter (SY13PL-S, PES). A culture test using B. breve was performed in a total of 140 μL of culture medium, consisting of 70 μL of B. longum JCM1217 culture medium obtained by the method described above and 70 μL of fresh 2×MRS medium (the final concentrations of the starch-removed radish water-soluble fraction and cabbage water-soluble fraction after B. longum culture were 2% and 1%, respectively). The B. breve strains MCC01128 and JCM1192 used in the test were pre-cultured in liquid MRSCS medium. The pre-cultured cells were centrifuged at 3000×g for 10 minutes, and the cells were suspended in sugar-free MRSCS to an OD600 of 1.0. 7 μL of this suspension was added to the main culture medium (initial OD600 = 0.05). The main culture was carried out at 37 °C for 86 hours under anaerobic conditions, and the OD600 was measured after 24, 48, and 86 hours. The culture test was performed in three consecutive cultures, with the sugar-free medium used for comparison.The OD600 of each culture medium without added bacteria was used as the growth rate at 0h, and the evaluation was performed by subtracting the 0h OD600 from the OD600 at each observation time. OD600 measurement was performed by adding 30 μL of diluted culture medium to a half-well 96-well plate (Corning, C3596) and using an absorbance microplate reader Multiskan GO Advance (Themo Fisher Scientific). For residual sugar analysis after culturing, the culture medium after 24 and 48 hours was centrifuged at 15,000 rpm for 10 minutes at 4°C, the supernatant was collected, and TLC analysis was performed using the procedure described above. One representative sample from three supernatant analyses is described. The culture curve for B. breve MCC01128 is shown in Figure 8, and the TLC results are shown in Figure 9. In TLC, the 4MeGlcA-Gal2 spot became fainter in the culture medium after culturing. This indicates that 4MeGlcA-Gal2 was degraded. This confirms that B. breve MCC01128 has the ability to decompose 4MeGlcA-Gal2 and utilize AGP.

[0094] <Test Example 5> Determining the maturity of an infant's gut microbiota using the amount of GH59 gene present in the gut microbiota of an infant during weaning The maturity of the subject infant's gut microbiota is determined by comparing the amount of GH59 gene present in the gut microbiota of an infant during weaning with the amount of GH59 gene present in the subject infant's gut microbiota. The GH59 gene used to calculate the relative abundance is (a) DNA containing the base sequence of Sequence ID No. 1, (b) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of Sequence ID No. 1, or (c) DNA containing a base sequence having 90% or more identity with the base sequence of Sequence ID No. 1. The amount of GH59 gene present in the gut microbiota of an infant during weaning is calculated as follows. Using the gut microbiota metagenomic sequence (PRJEB6456) of infants during weaning, obtained from a public database, the resulting metagenomic sequence is imported into METAnnatorX2. Filtering is then performed on the raw data in fastq format, removing sequences with an average quality value < 25, thereby eliminating sequences with low average reliability for bases (i.e., sequences unnecessary for analysis). Subsequently, host-derived sequences are also removed from the reads mapped to the human genome, as they are also unnecessary for analysis. The filtered sequences are used as input for a phasic sequence search (rapsearch v2.24; https: / / omics.informatics.indiana.edu / mg / RAPSearch / ) against the CAZY database. The threshold is e-value < 10. -8 The relative abundance of the gene is calculated using the CAZY classification. The amount of the GH59 gene in the infant gut microbiota of the target infant is calculated using a standard method with the infant's feces. In this way, it is possible to judge the maturity of the infant's gut environment by using the relative abundance of GH59 as a standard.

Claims

1. Bifidobacterium breve possessing any of the following proteins: (A), (B), or (C): (A) a protein containing the amino acid sequence of SEQ ID NO: 2; (B) a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 2, and which has Glycoside Hydrolase 59 (GH59) activity; (C) a protein containing an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 2, and which has GH59 activity.

2. Bifidobacterium breve possessing either (a) or (b) the following DNA: (a) DNA containing the nucleotide sequence of Sequence ID No. 1; (b) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 1 and that encodes a protein having GH59 activity.

3. Bifidobacterium breve according to claim 1 or 2, selected from the group consisting of Bifidobacterium breve MCC01128 (NITE BP-04132), Bifidobacterium breve MCC10201 (NITE BP-04133), Bifidobacterium breve MCC10206 (NITE BP-04134), and Bifidobacterium breve MCC10258 (NITE BP-04135).

4. A composition for improving the metabolism of dietary fiber, comprising the cells of Bifidobacterium breve described in claim 1 or 2, a culture thereof, and / or a processed product thereof.

5. A carbohydrate-degrading composition having a higher-order structure, comprising the cells of Bifidobacterium breve described in claim 1 or 2, a culture thereof, and / or a processed product thereof.

6. A food and beverage composition comprising the cells of Bifidobacterium breve described in claim 1 or 2, a culture thereof, and / or a processed product thereof.

7. The food and beverage composition according to claim 6, further comprising an ingredient containing arabinogalactan protein (AGP).

8. Bifidobacterium breve powder according to claim 1 or 2.

9. A method for improving dietary fiber metabolism, comprising using the cells of Bifidobacterium breve described in claim 1 or 2, a culture thereof, and / or a processed product thereof.

10. A method for degrading carbohydrates having a higher-order structure, comprising using the cells of Bifidobacterium breve described in claim 1 or 2, a culture thereof, and / or a processed product thereof.

11. A method for producing oligosaccharides containing glucuronic acid (GlcA), comprising the step of reacting the cells of Bifidobacterium breve described in claim 1 or 2, a culture thereof, and / or a processed product thereof, with a raw material containing AGP.

12. The following proteins (A), (B), or (C): (A) a protein containing the amino acid sequence of SEQ ID NO: 2; (B) a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 2, and which has Glycoside Hydrolase 59 (GH59) activity; (C) a protein containing an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2, and which has GH59 activity.

13. DNA of the following (a) or (b): (a) DNA containing the base sequence of SEQ ID NO: 1; (b) DNA that can hybridize under stringent conditions with DNA having a base sequence complementary to the base sequence of SEQ ID NO: 1 and that encodes a protein having GH59 activity.

14. A composition for improving dietary fiber metabolism, comprising the protein described in claim 12, or the DNA described in claim 13 or a protein encoded thereby.

15. A food or beverage composition comprising the protein described in claim 12, or the DNA described in claim 13 or a protein encoded therefrom.

16. The food and beverage composition according to claim 15, further comprising an ingredient containing arabinogalactan protein (AGP).

17. A method for improving dietary fiber metabolism, comprising using the protein described in claim 12, or the DNA-encoded protein described in claim 13.

18. A method for degrading a carbohydrate having a higher-order structure, comprising using the protein described in claim 12 or the DNA-encoded protein described in claim 13.

19. A method for producing an oligosaccharide containing glucuronic acid (GlcA), comprising the step of reacting a raw material containing AGP with the protein described in claim 12 or the DNA-encoded protein described in claim 13.

20. A method for determining the maturity of the intestinal microbiota of an infant, wherein the amount of any of the following DNAs in the intestinal microbiota of the infant is used as an indicator: (a) DNA containing the base sequence of Sequence ID No. 1; (b) DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence of Sequence ID No. 1; (c) DNA containing a base sequence having 90% or more identity with the base sequence of Sequence ID No.

1.

21. The method according to claim 20, comprising the steps of: calculating the relative abundance of the DNA in the total genes contained in the gut microbiota of a target infant; and comparing the relative abundance with the relative abundance of the DNA in the gut microbiota of infants in general after weaning.

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