Gum arabic seyal modified glycolytic enzyme, food and beverage composition, bifidobacterial growth promoter, method for producing oligosaccharides, method for promoting bifidobacterial growth, kit for detecting gum arabic seyal assimilation bacteria, and method for detecting gum arabic seyal assimilation bacteria

Abstract

To provide a gum arabic seyal modified glycolytic enzyme useful for assimilating modified sugars of seyal gum arabic.SOLUTION: A gum arabic seyal modified glycolytic enzyme exhibits endoglycosidase activity and comprises one of following amino acid sequences (a)-(d): (a) a specific amino acid sequence; (b) an amino acid sequence having at least 90% of sequence identity to the specific amino acid sequence; (c) a specific amino acid sequence, different from (a) and (b); and (d) an amino acid sequence having at least 90% of sequence identity to (c).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a seyal gum arabic-modifying glycolytic enzyme, a food and drink composition, a bifidobacterial growth promoter, a method for producing oligosaccharides, a method for promoting the growth of bifidobacterium, a kit for detecting seyal gum arabic assimilation bacteria, and a method for detecting seyal gum arabic assimilation bacteria. [Background technology]

[0002] Gum arabic is a dried secretion from wounds in the bark of the Acacia acacia tree, a member of the legume family native to the Nile region of Africa. Gum arabic is composed of a glycoprotein (arabinogalactan protein (AGP)) in which type II arabinogalactan (AG) and β-arabinooligosaccharide sugar chains are attached to peptide chains.

[0003] Gum arabic is divided into Senegal and seyal species. Senegal gum arabic is harvested from Acasia senegal. Senegal gum arabic has a higher protein content and is used as a thickener and emulsifier. Seyal gum arabic is harvested from Acasia seyal. Seyal gum arabic is used as a coating agent for confectionery and pharmaceuticals, as a base material for encapsulation, and as a gloss agent, taking advantage of its film-forming ability.

[0004] Non-Patent Document 1 compares gum arabic of seyal species with gum arabic of Senegal species. According to Non-Patent Document 1, gum arabic of seyal species had a higher L-arabinose content and a lower protein content than gum arabic of Senegal species. In addition, the presence of a sugar chain structure composed of β-L-arabinofuranose (hereinafter also referred to as "Araf") and β-L-arabinopyranose (hereinafter also referred to as "Arap") was shown in the modified sugar of gum arabic of seyal species. The detailed sugar chain structure of Senegal gum arabic has been analyzed, and Non-Patent Document 2 reports modification of the AGP side chain with α-D-galactopyranose (hereinafter also referred to as "Galp")-(1→3)-Araf and α-L-rhamnopyranose (hereinafter also referred to as "Rhap")-(1→4)-β-D-glucuronic acid (hereinafter also referred to as "GlcpA")-(1→6)-β-D-Galp-(1→6)-D-galactose (hereinafter also referred to as "Gal").

[0005] On the other hand, with regard to gum arabic from Seyal species, only a putative structure has been presented in which arabinose (hereinafter also referred to as "Ara") oligomers obtained from nuclear magnetic resonance (NMR) analysis are attached to the AGP side chain (see Non-Patent Document 3), and the detailed sugar chain structure is not known. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2019-17292 A [Non-patent literature]

[0007] [Non-Patent Document 1] Lizeth Lopez-Torrez and 4 others, “Acacia senegal vs. Acacia seyal gums-Part 1: Composition and structure of hyperbranched plant exudates”, Food Hydrocolloids, 2015, 51: 41-53 [Non-Patent Document 2] CATischer, et al., “The free reducing oligosaccharides of gum arabic: aids for structural assignments in the polysaccharide”, Carbohydrate Polymers, 2002, 47(2), 151-158 [Non-Patent Document 3] Shao-Ping Nie, 5 others, “The core carbohydrate structure of Acacia seyal var. seyal(Gum arabic)”, Food Hydrocolloids, 2013, 32(2), 221-227 [Non-Patent Document 4] Yuki Sasaki and 7 others, "Novel 3-O-α-D-galactosyl-α-L-arabinofuranosidase for the assimilation of gum arabic arabinogalactan protein in Bifidobacterium longum subsp.longum", Applied and Environmental Microbiology, 2021, 87, e02690-20 Summary of the Invention [Problem to be solved by the invention]

[0008] Patent Document 1 and Non-Patent Document 4 disclose 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase) that releases disaccharides (α-D-Galp-(1→3)-Araf) modified on the β1,6-galactan side chain of Senegal gum arabic. If modified glycolytic enzymes are available for Seyal gum arabic as well, it is expected that the detailed elucidation of the glycan structure will progress. Furthermore, such modified glycolytic enzymes can be used to utilize Seyal gum arabic as a prebiotic, a non-digestible food material that increases bacteria useful to humans.

[0009] The present invention has been made in view of the above circumstances, and aims to provide a seyal gum arabic modified glycolytic enzyme useful for assimilating modified sugars of seyal gum arabic.An object of the present invention is to provide a food composition, a bifidobacterial growth promoter and a method for promoting the growth of bifidobacterial in the intestine, which can promote the growth of bifidobacterium.An object of the present invention is to provide a method for producing oligosaccharides, which can produce target oligosaccharides efficiently with high selectivity.Another object of the present invention is to provide a kit for detecting seyal gum arabic assimilating bacteria and a method for detecting seyal gum arabic assimilating bacteria. [Means for solving the problem]

[0010] The seyal gum arabic modified glycolytic enzyme according to the first aspect of the present invention comprises: It has any one of the following amino acid sequences (a) to (d) and has endoglycosidase activity. (a) the amino acid sequence shown in SEQ ID NO:1 (b) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1 (c) the amino acid sequence shown in SEQ ID NO:2 (d) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2

[0011] The seyal gum arabic modified glycolytic enzyme according to the second aspect of the present invention comprises: It is encoded by DNA of (a) or (b) below. (a) DNA containing the nucleotide sequence shown in SEQ ID NO:3 (b) A DNA that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO:3 or a probe prepared from the complementary nucleotide sequence, and encodes a modified glycolytic enzyme from Seyal arabic gum having endoglycosidase activity.

[0012] The food and beverage composition according to the third aspect of the present invention is The present invention includes at least one of the above-mentioned gum arabic modified glycolytic enzyme of S. seyalii according to the first or second aspect of the present invention, a Bifidobacterium bacterium having a gene encoding the gum arabic modified glycolytic enzyme of S. seyalii, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium.

[0013] The food and beverage composition according to the third aspect of the present invention comprises: For promoting the decomposition of Seyal gum arabic.

[0014] The food and beverage composition according to the fourth aspect of the present invention is At least one of the gum arabic modified saccharidase according to the first or second aspect of the present invention, a Bifidobacterium bacterium having a gene encoding the gum arabic modified saccharidase, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium; A raw material containing gum arabic of seyal species; Includes.

[0015] The food and drink composition according to the fourth aspect of the present invention comprises: It is used to promote the growth of bifidobacteria.

[0016] The food and beverage composition according to the fifth aspect of the present invention is The present invention includes a decomposition product of gum seyal arabic by at least one of the gum arabic modified saccharidase according to the first or second aspect of the present invention, a Bifidobacterium bacterium having a gene encoding the gum arabic modified saccharase, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium.

[0017] A bifidobacterium growth promoter according to a sixth aspect of the present invention comprises: The present invention includes at least one of the above-mentioned gum arabic modified glycolytic enzyme of S. seyalii according to the first or second aspect of the present invention, a Bifidobacterium bacterium having a gene encoding the gum arabic modified glycolytic enzyme of S. seyalii, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium.

[0018] A method for producing oligosaccharides according to a seventh aspect of the present invention comprises the steps of: The method includes a step of allowing at least one of the gum arabic modified saccharidase according to the first or second aspect of the present invention, a Bifidobacterium bacterium having a gene encoding the gum arabic modified saccharidase, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium to act on a raw material containing gum arabic.

[0019] The oligosaccharide is β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap- (1→3)-L-Ara or β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, This may also be the case.

[0020] A method for promoting the proliferation of bifidobacteria according to an eighth aspect of the present invention comprises: The method includes a step of administering to a non-human animal at least one of the seyal sp. gum arabic-modified glycolytic enzyme according to the first or second aspect of the present invention, a Bifidobacterium bacterium having a gene encoding the seyal sp. gum arabic-modified glycolytic enzyme, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium.

[0021] A kit for detecting gum arabic assimilation bacteria of S. seyal according to a ninth aspect of the present invention comprises: The present invention comprises a primer pair designed to include at least 10 consecutive bases contained in the DNA encoding the S. seyal gum arabic modified glycolytic enzyme according to the first or second aspect of the present invention in the base sequence of a PCR product.

[0022] A method for detecting gum arabic assimilation bacteria of S. seyal according to a tenth aspect of the present invention comprises: The method includes a step of carrying out PCR using the kit for detecting gum arabic bacteria of S. seyalii according to the ninth aspect of the present invention. Effect of the Invention

[0023] According to the present invention, there is provided a gum arabic seyal modified glycolytic enzyme useful for assimilating modified sugars of gum arabic seyal. According to the food composition, the bifidobacterium growth promoter and the method for promoting bifidobacterium growth of the present invention, it is possible to promote the growth of bifidobacteria in the intestine. According to the method for producing oligosaccharides of the present invention, it is possible to produce a target oligosaccharide efficiently with high selectivity. According to the present invention, there are also provided a kit for detecting gum arabic seyal assimilation bacteria and a method for detecting gum arabic seyal. [Brief description of the drawings]

[0024] [Figure 1] FIG. 2 is a diagram showing a chromatogram obtained by high performance anion exchange chromatography with pulsed amperometric analysis (HPAEC-PAD) in Test Example 1. [Diagram 2] FIG. 2 shows a chromatogram obtained by HPAEC-PAD in Test Example 2. [Diagram 3] FIG. 1 shows the results of a co-culture assimilation test of two types of bifidobacteria in Test Example 3. [Figure 4] FIG. 1 is a schematic diagram showing a thin layer chromatography (TLC) plate and the degradation of modified sugars of Seyal gum arabic in Test Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of".

[0026] Embodiment 1: Seyal gum arabic modified glycolytic enzyme The seyal gum arabic modified glycolytic enzyme according to the present embodiment has any one of the following amino acid sequences (a) to (d) and has endoglycosidase activity. (a) the amino acid sequence shown in SEQ ID NO:1 (b) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1 (c) the amino acid sequence shown in SEQ ID NO:2 (d) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2

[0027] The seyal gum arabic modified glycolytic enzyme has an endoglycosidase activity that selectively acts on seyal gum arabic modified sugars and releases β-arabino oligosaccharide trisaccharide, tetrasaccharide, and pentasaccharide. The seyal gum arabic modified glycolytic enzyme having the amino acid sequence shown in SEQ ID NO: 1 was identified in the BBCT_0495 gene (NCBI-Protein ID: BAR01463) of Bifidobacterium catenulatum JCM1194 strain (hereinafter referred to as "JCM1194"). The amino acid sequence identity between the amino acid sequence shown in SEQ ID NO: 1 and GAfase discovered from Bifidobacterium longum JCM7052 strain disclosed in the above non-patent document 4 is less than 28%.

[0028] Here, when two amino acid sequences are aligned to maximize their degree of identity, the ratio of the number of positions at which identical amino acids exist in both amino acid sequences to the total number of amino acids in the two sequences is called sequence identity.

[0029] The term "sequence identity of 90% or more" with respect to an amino acid sequence means at least 90% sequence identity. The amino acid sequence of the S. seyal gum arabic modified glycolytic enzyme may have sequence identity of 90% or more, preferably 92% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more to the amino acid sequence shown in SEQ ID NO: 1 or 2, so long as it has the above-mentioned endoglycosidase activity.

[0030] When the seyal gum arabic modified glycolytic enzyme according to the present embodiment has an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, the seyal gum arabic modified glycolytic enzyme may have an amino acid sequence for improving translation efficiency, an amino acid sequence for use in purification of the seyal gum arabic modified glycolytic enzyme, an amino acid sequence for improving expression efficiency of chaperone, etc., in addition to the amino acid sequence for improving translation efficiency, as long as the seyal gum arabic modified glycolytic enzyme has the above-mentioned endoglycosidase activity. Examples of the amino acid sequence for use in purification of the seyal gum arabic modified glycolytic enzyme include a histidine tag sequence (His-Tag), glutathione-S-transferase, and maltose binding protein. The amino acid sequence for improving translation efficiency, the amino acid sequence for use in purification, and the amino acid sequence for improving expression efficiency are added to at least one of the N-terminus and the C-terminus. The number of amino acids added to the N-terminus or the C-terminus is, for example, several, preferably 1 to 9.

[0031] The amino acid sequence shown in SEQ ID NO: 2 is the same as the amino acid sequence shown in SEQ ID NO: 1 except that the C-terminal cell wall anchoring region is removed, a His-Tag is added to the C-terminus, and a part of the N-terminal region is replaced with a SKIK sequence for solubilization. The seyal gum arabic modified glycolytic enzyme having the amino acid sequence shown in SEQ ID NO: 2 also has the above-mentioned endoglycosidase activity.

[0032] Even when the S. seyal gum arabic modified glycolytic enzyme of the present embodiment has an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, as described above, the S. seyal gum arabic modified glycolytic enzyme may have an amino acid sequence for improving translation efficiency, an amino acid sequence for improving expression efficiency of chaperone, etc., as long as it has the above-mentioned endoglycosidase activity.

[0033] The seyal gum arabic modified glycolytic enzyme according to the present embodiment may be encoded by the following DNA (a) or (b): (a) DNA containing the nucleotide sequence shown in SEQ ID NO:3 (b) A DNA that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO:3 or a probe prepared from the complementary nucleotide sequence, and encodes a modified glycolytic enzyme from Seyal arabic gum having endoglycosidase activity. In addition, the DNA containing the base sequence shown in SEQ ID NO:3 encodes the amino acid sequence shown in SEQ ID NO:2.

[0034] "Stringent conditions" are conditions under which specific hybrids are formed and non-specific hybrids are not formed. Examples of stringent conditions include conditions under which DNAs with high homology, for example, DNAs with a homology of 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97%, and particularly preferably 99% or more, hybridize with each other, and DNAs with lower homology do not hybridize with each other. Examples of stringent conditions include hybridization at 42°C and washing at 42°C with a buffer containing 1×SSC (Saline Sodium Citrate Buffer) and 0.1% SDS (Sodium Dodecyl Sulfate), or hybridization at 65°C and washing at 65°C with a buffer containing 0.1×SSC and 0.1% SDS. In addition, there are various factors that affect the stringency of hybridization other than the above temperature conditions, and a person skilled in the art can achieve stringency equivalent to the hybridization stringency exemplified above by appropriately combining various factors.

[0035] The probe used for hybridization may be a part of the complementary sequence of the base sequence shown in SEQ ID NO: 3. For example, when a DNA fragment having a length of about 300 base pairs is used as the probe, washing conditions for hybridization include washing at 50° C. with a buffer containing 2×SSC and 0.1% SDS.

[0036] The conditions under which the seyal gum arabic modified glycolytic enzyme of the present embodiment exerts its endoglycosidase activity are not particularly limited. Conditions under which the seyal gum arabic modified glycolytic enzyme is used include, for example, a temperature of preferably 35 to 55°C, more preferably 45 to 50°C, and a pH of preferably 4.0 to 7.0, more preferably 5.0 to 6.0.

[0037] The endoglycosidase activity can be confirmed, for example, by the following method: A sugar chain having an α1,3 structure between Gal and L-Araf is reacted with the seyal gum arabic modified glycolytic enzyme, and the generated trisaccharide, tetrasaccharide or pentasaccharide is detected by a chromatography method such as HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection), and the enzyme activity is calculated from the amount of the detected trisaccharide, tetrasaccharide or pentasaccharide.

[0038] The gum arabic modified glycolytic enzyme according to the present embodiment can be produced by purifying it from JCM1194 or the like by a known method. It may also be produced by using a genetic engineering technique to produce a recombinant protein in an appropriate host bacterium. In this case, the entire gene sequence including the coding region of the signal peptide of the gene encoding the gum arabic modified glycolytic enzyme may be introduced into a host and expressed, and the mature protein of the gum arabic modified glycolytic enzyme of the seyal species may be obtained through processing in the host, or the gene sequence excluding the coding region of the signal peptide of the gene encoding the gum arabic modified glycolytic enzyme of the seyal species may be expressed in the host.

[0039] The seyal gum arabic modified glycolytic enzyme according to the present embodiment recognizes the glycan present in the seyal gum arabic modified glycan and cleaves the α1,3 structure between L-Araf and Gal located at the reducing end of the glycan, thereby releasing oligosaccharides (β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara and β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara). Such a β-arabino oligosaccharide trisaccharide-, tetrasaccharide- and pentasaccharide-releasing Gum arabic modified glycolytic enzyme has not been found so far, and the Gum arabic modified glycolytic enzyme is a novel enzyme. In the description of the sugar bond in this specification, for example, "1→2" indicates that the 1st carbon of one sugar is bonded to the 2nd carbon of another sugar.

[0040] Embodiment 2: Food and drink composition The food and drink composition according to the present embodiment includes at least one of the seyal gum arabic-modified glycolytic enzyme according to the above-mentioned embodiment 1, a Bifidobacterium bacterium having a gene encoding the seyal gum arabic-modified glycolytic enzyme, a culture of the Bifidobacterium bacterium, and a processed cell product of the Bifidobacterium bacterium (hereinafter collectively referred to as "seyal gum arabic-modified glycolytic enzyme, etc."). An example of the Bifidobacterium bacterium is JCM1194. As the Bifidobacterium bacterium, a culture obtained after culture may be used as it is, or may be used after dilution or concentration, or a cell recovered from the culture may be used. In addition, the Bifidobacterium bacterium may be a live bacterium or a dead bacterium, or may include both live and dead bacteria. Examples of treated bacterial cells include immobilized bacterial cells obtained by immobilizing bacterial cells with acrylamide, carrageenan, or the like, cell disruptants obtained by partially or completely disrupting the cell walls and cell membranes of Bifidobacterium bacterial cells by standard methods such as ultrasonication or homogenization, the supernatant obtained by centrifugation (water-soluble fraction), a fraction obtained by partially purifying the supernatant by ammonium sulfate treatment, or the like, and a concentrate obtained by concentrating the supernatant.

[0041] The food and beverage composition may be produced by adding at least one of the seyal gum arabic-modified glycolytic enzymes to a known food or beverage, or may be produced as a new food and beverage composition by mixing at least one of the seyal gum arabic-modified glycolytic enzymes into the raw materials of the food and beverage.

[0042] The food and beverage compositions of this embodiment may be in any form, such as liquid, paste, solid, powder, etc., and include, for example, wheat flour products, instant foods, processed agricultural products, processed marine products, processed livestock products, milk and dairy products, oils and fats, basic seasonings, complex seasonings, foods, frozen foods, confectioneries, beverages, etc.

[0043] The intake amount of the food and beverage composition according to the present embodiment can be appropriately selected. For example, the intake amount of the gum arabic modified glycolytic enzyme of Seyal species per kg of body weight per day is 1 μg / kg / day, 10 μg / kg / day, or 100 μg / kg / day. For example, the intake or administration amount of the Bifidobacterium genus bacteria per kg of body weight per day is 1×10 6 ~1×10 12 CFU / kg / day, 1×10 7 ~1×10 11 CFU / kg / day or 1 x 10 8 ~1×10 10 When a culture of a Bifidobacterium bacterium or a treated cell product of the Bifidobacterium bacterium is used, the intake or administration amount thereof may be the above-mentioned intake or administration amount when converted into the intake or administration amount of the Bifidobacterium bacterium.

[0044] The content of the S. seyal gum arabic modified glycolytic enzyme in the food and beverage composition according to the present embodiment can be appropriately selected based on the intake amount. The content of the S. seyal gum arabic modified glycolytic enzyme in the food and beverage composition is, for example, 1 μg / g, 10 μg / g, or 100 μg / g. The content of the Bifidobacterium bacteria in the food and beverage composition according to the present embodiment can be appropriately selected based on the intake amount, and is, for example, 1×106 ~1×10 12 CFU / g, 1×10 7 ~1×10 11 CFU / g or 1×10 8 ~1×10 10 The content of the culture of the Bifidobacterium bacteria or the treated bacterial cell product in the food or drink composition may be such that, when converted into the content of the Bifidobacterium bacteria, it satisfies the above-mentioned content.

[0045] In addition, CFU is an abbreviation for colony forming units. When the bacteria is dead, CFU can be replaced with individual cells.

[0046] The above-mentioned gum arabic modified glycolytic enzyme of S. seyale can cleave and release oligosaccharides from AGP of S. seyale gum arabic by endoglycosidase activity. The oligosaccharides and the remaining sugar chains of the degraded modified sugars are easily degraded by another hydrolase (exoglycosidase, etc.). Therefore, the food and beverage composition according to the present embodiment may be used for promoting the decomposition of gum arabic, particularly for promoting the decomposition of S. seyale gum arabic.

[0047] In another aspect of the present embodiment, there is provided a use of the above-mentioned gum arabic modified saccharase etc. in the manufacture of a food and drink composition for promoting the decomposition of gum arabic of seyal species. In another aspect of the present embodiment, there is provided a use of the above-mentioned gum arabic modified saccharase etc. in promoting the decomposition of gum arabic of seyal species. In another aspect of the present embodiment, there is provided a method for promoting the decomposition of gum arabic of seyal species, comprising administering the above-mentioned gum arabic modified saccharase etc. to an animal. The animal here may be a human or a non-human animal.

[0048] In another aspect of the present embodiment, there is provided a food and drink composition comprising a decomposition product of gum seyal arabic by the gum seyal arabic modified glycolytic enzyme or the like. The decomposition product comprises at least the above-mentioned β-arabino oligosaccharide trisaccharide, tetrasaccharide and pentasaccharide. The food and drink composition may be used for promoting the growth of bifidobacteria. In another aspect of the present embodiment, there is provided a food and drink composition comprising the gum seyal arabic modified glycolytic enzyme or the like and a raw material comprising gum seyal arabic. The raw material is not particularly limited as long as it contains gum seyal arabic. In particular, gum seyal arabic is known as a prebiotic dietary fiber and is preferred in that its safety in food and drink applications is recognized.

[0049] The gum arabic modified saccharidase can promote the proliferation of bifidobacteria in the intestine through the decomposition of gum arabic, and therefore the food and drink composition containing a raw material containing gum arabic and the gum arabic modified saccharidase may be used for promoting the proliferation of bifidobacteria.

[0050] In another aspect of the present embodiment, there is provided a use of the gum arabic modified seyal etc. and a raw material containing gum arabic seyal in the manufacture of a food and drink composition for promoting the proliferation of bifidobacteria. In another aspect of the present embodiment, there is provided a use of the gum arabic modified seyal etc. and a raw material containing gum arabic seyal in promoting the proliferation of bifidobacteria. In another aspect of the present embodiment, there is provided a method for promoting the proliferation of bifidobacteria, comprising a step of administering the gum arabic modified seyal etc. and a raw material containing gum arabic seyal to an animal. The animal here may be a human or a non-human animal.

[0051] The food and drink composition according to the present embodiment may be provided or sold as a food and drink labeled with uses (including health uses) for which the decomposition of gum arabic, the proliferation of bifidobacteria, or oligosaccharides are effective, such as prevention of disease, reduction of risk of disease, alleviation of symptoms of disease, and treatment of disease. The act of "labeling" includes all acts for informing consumers of the above uses. Any expression that can recall or infer the above uses falls under the category of "labeling", regardless of the purpose of the labeling, the content of the labeling, the object to be displayed, the medium, etc.

[0052] It is preferable that the "labeling" be done in an expression that allows consumers to directly recognize the above-mentioned uses. Specifically, this includes the acts of transferring, delivering, displaying for the purpose of transferring or delivering, or importing food and beverage products or product packaging on which the above-mentioned uses are written, displaying or distributing advertisements, price lists, or transaction documents related to the products and writing the above-mentioned uses in information containing the above-mentioned uses and providing them by electromagnetic means (such as the Internet), etc.

[0053] Examples of "labeling" include labeling as health food, functional food, enteral nutritional food, special purpose food, health functional food, food for specified health uses, nutrient functional food, functional food, medical drug, etc. Among these, in particular, labeling approved by the Consumer Affairs Agency, for example, labeling approved under the system related to food for specified health uses, nutrient functional food, or food with functional labeling, or a similar system, can be mentioned. Specifically, labeling as food for specified health uses, labeling as food for conditional specified health uses, labeling that affects the structure or function of the body, labeling that reduces the risk of disease, labeling of functionality based on scientific evidence, etc. can be mentioned, and more specifically, labeling as food for specified health uses (especially labeling of health uses) and similar labeling are typical examples.

[0054] Embodiment 3: Bifidobacteria growth promoter The bifidobacterium proliferation promoter according to the present embodiment contains the Seyal sp. gum arabic-modified glycolytic enzyme according to the above-mentioned first embodiment as an active ingredient.

[0055] Indigestible food materials that increase bacteria useful to humans, such as bifidobacteria, are attracting attention as prebiotics. Currently, nine types of oligosaccharides are recognized as foods for specified health uses that have the effect of increasing bifidobacteria. It has also been revealed that lacto-N-biose (β-D-Gal-1,3-D-GlcNAc (LNB)) is a growth factor that selectively increases bifidobacteria in infants. The above-mentioned oligosaccharides that are excised and released from seyal gum arabic by the seyal gum arabic-modified glycolytic enzyme are trisaccharides, tetrasaccharides, and pentasaccharides that have structures different from known prebiotic oligosaccharides. The above-mentioned oligosaccharides can be decomposed into monosaccharides by exoglycosidases (β-L-arabinopyranosidase, β-L-arabinofuranosidase) contained in bifidobacteria, preferably bifidobacteria that have seyal gum arabic-modified glycolytic enzyme, and assimilated. Therefore, these oligosaccharides can be used as highly selective prebiotic oligosaccharides that can selectively grow bifidobacteria. Furthermore, the remaining sugar chains after these oligosaccharides are easily decomposed by other hydrolases (exoglycosidases, etc.), and can be assimilated by bifidobacteria. Therefore, the bifidobacteria growth promoter promotes the growth and proliferation of bifidobacteria by its endoglycosidase activity.

[0056] The bifidobacteria growth promoter may be administered orally or parenterally, and may be formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, it may be a solid preparation such as a powder, granule, tablet, or capsule; or a liquid preparation such as a solution, syrup, suspension, or emulsion. In addition, in the case of parenteral administration, it may be a suppository, ointment, or the like.

[0057] In addition, in the formulation, in addition to the above-mentioned gum arabic modified glycolytic enzyme, excipients, pH adjusters, colorants, flavoring agents, and other components that are usually used in formulations can be used. Formulation can be carried out by a known method as appropriate depending on the dosage form. When formulating, a formulation carrier may be appropriately blended to form the formulation.

[0058] The intake or administration amount of the bifidobacterium growth promoter can be appropriately selected according to the dosage form. For example, the intake or administration amount of the gum arabic modified seyal species per kg of body weight per day is 1 μg / kg / day, 10 μg / kg / day, or 100 μg / kg / day. For example, the intake or administration amount of the bifidobacterium per kg of body weight per day is 1×10 6 ~1×10 12 CFU / kg / day, 1×10 7 ~1×10 11 CFU / kg / day or 1 x 10 8 ~1×10 10 When a culture of a Bifidobacterium bacterium or a treated cell product of the Bifidobacterium bacterium is used, the intake or administration amount thereof may be the above-mentioned intake or administration amount when converted into the intake or administration amount of the Bifidobacterium bacterium.

[0059] The content of the gum arabic modified glycolytic enzyme of S. seyal in the bifidobacterium growth promoter is, for example, 1 μg / g, 10 μg / g, or 100 μg / g. The content of the bifidobacterium bacteria in the food and beverage composition according to the present embodiment can be appropriately selected based on the intake amount. For example, the content of the bifidobacterium bacteria in the food and beverage composition according to the present embodiment can be selected from 1×10 6 ~1×10 12 CFU / g, 1×10 7 ~1×10 11 CFU / g or 1×10 8 ~1×10 10 The content of the culture product of the Bifidobacterium bacteria or the treated bacterial cell product in the food or drink composition is preferably the above-mentioned content when converted into the content of the Bifidobacterium bacteria.

[0060] As the pharmaceutical carrier, various organic or inorganic carriers can be used depending on the dosage form. Examples of carriers in the case of solid preparations include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, etc.

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

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

[0063] Examples of disintegrants include, in addition to the above-mentioned excipients, croscarmellose sodium, sodium carboxymethyl starch, chemically modified starches such as crosslinked polyvinylpyrrolidone, cellulose derivatives, and the like.

[0064] Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and geranium stearate; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; starch derivatives, and the like.

[0065] Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.

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

[0067] In another aspect of the present embodiment, there is provided a use of the above-mentioned gum arabic modified saccharolytic enzyme or the like in the production of a bifidobacterium growth promoter. In another aspect, there is provided a use of the above-mentioned gum arabic modified saccharolytic enzyme or the like in promoting the growth of bifidobacterium.

[0068] Another aspect of the present embodiment may be a method for promoting the growth of bifidobacteria, comprising administering the gum arabic modified glycolytic enzyme or the like to an animal, which may be a human or a non-human animal.

[0069] Embodiment 4: Method for producing oligosaccharides The method for producing oligosaccharides according to the present embodiment includes a step of allowing the gum arabic modified glycolytic enzyme or the like to act on a raw material containing gum arabic, as described above. As described above, the gum arabic modified glycolytic enzyme can cleave and release oligosaccharides from the sugar chains of gum arabic by endoglycosidase activity, thereby producing oligosaccharides. The oligosaccharides produced by this production method are not particularly limited, but are usually oligosaccharides having an arabinose reducing end, such as β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, or β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara.

[0070] The conditions for allowing the seyal gum arabic-modified glycolytic enzyme or the like to act on a raw material containing seyal gum arabic are not particularly limited, but are usually conditions under which the seyal gum arabic-modified glycolytic enzyme can exert its endoglycosidase activity.

[0071] The amount of the gum arabic modified seyal enzyme acting on the raw material can be, for example, 1 μg / g, 10 μg / g, or 100 μg / g relative to 1 g of the raw material. The amount of the Bifidobacterium bacteria acting on the raw material can be appropriately determined depending on the type of raw material and reaction conditions. For example, the amount of the Bifidobacterium bacteria acting on the raw material can be 1×10 6 ~1×10 12 CFU / g, 1×10 7 ~1×10 11 CFU / g or 1×10 8 ~1×10 10 The amount of the culture of Bifidobacterium or treated bacterial cells to be acted on the raw material is preferably the above amount when converted into the amount of the Bifidobacterium.

[0072] The raw materials are as described above, and may be appropriately selected depending on the type of the target oligosaccharide. For example, when gum arabic of Seyal species is used as the raw material, β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, and β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara can be selectively produced.

[0073] The produced oligosaccharides can be used as highly selective prebiotic oligosaccharides that can selectively grow bifidobacteria, especially Bifidobacterium catenulatum, Bifidobacterium pseudcatenulatum, and Bifidobacterium adolescentis.

[0074] It was known that oligosaccharides could be prepared by chemical methods (weak acid decomposition treatment). However, chemical methods have the disadvantage that reaction conditions such as time and temperature must be strictly controlled so as not to decompose the product into monosaccharides, and that a large amount of impurities is contained. The method for producing oligosaccharides according to the present embodiment is an enzymatic method, and therefore can produce the desired oligosaccharides efficiently with high selectivity, making it very useful.

[0075] Embodiment 5: Kit for detecting Seyal gum arabic assimilation bacteria The kit for detecting S. seyal gum arabic assimilation bacteria according to the present embodiment includes a primer pair designed to include at least 10 consecutive bases, for example, 10 to 20 bases or 10 to 30 bases, contained in the DNA encoding the S. seyal gum arabic modified glycolytic enzyme in the base sequence of a PCR product. The DNA is, for example, the base sequence (SEQ ID NO: 4) of the BBCT_0495 gene (GH39) of JCM1194.

[0076] The primer pair may be designed based on SEQ ID NO: 4 as oligonucleotides so as to satisfy the following requirements: the primer pair can be amplified under normal PCR conditions; the base sequence does not generate a PCR product in any other region of the base sequence encoding the seyal gum arabic modified glycolytic enzyme; the PCR product has a length of 80 to 550 base pairs so that it can be used in quantitative PCR; and the Tm value is within the range of 55 to 65°C.

[0077] The PCR primer pair included in the kit for detecting gum arabic seyal species according to the present embodiment can selectively amplify a part of DNA encoding the gum arabic seyal species modified glycolytic enzyme by performing PCR using nucleic acid in a test sample as a template. The DNA amplified by PCR is a sequence specific to the gum arabic seyal species modified glycolytic enzyme and is distinguished from other gene sequences. As described above, the gum arabic seyal species modified glycolytic enzyme can decompose modified sugars of gum arabic seyal species, so it can be said that bacteria possessing the gum arabic seyal species modified glycolytic enzyme have the ability to assimilate gum arabic seyal species. Therefore, the kit for detecting gum arabic seyal species assimilation bacteria can be used to detect gum arabic seyal species assimilation bacteria.

[0078] Test samples to which the kit for detecting gum arabic bacteria assimilation of seyal species can be applied include, but are not limited to, mixed cultures of lactic acid bacteria, foods and drinks containing lactic acid bacteria such as yogurt and cheese, laboratory animals such as mice and rats, human feces, intestinal fluids, etc. Methods for extracting nucleic acid from these samples include, for example, a method of physically destroying cells with enzymes such as lysozyme, beads, etc., or a method using a commercially available nucleic acid extraction kit.

[0079] The kit for detecting S. seyal gum arabic assimilation bacteria may comprise, in addition to the above primer pair, for example, PCR reagents (thermostable DNA polymerase, dNTPs), a buffer solution, an instruction manual, etc. Furthermore, the kit for detecting S. seyal gum arabic assimilation bacteria may comprise reagents necessary for detecting S. seyal gum arabic assimilation bacteria, reagents necessary for detecting bacteria of other strains, species, or genera, etc.

[0080] The kit for detecting gum seyal arabic assimilation bacteria can easily test whether or not intestinal bifidobacteria grow by ingestion of gum seyal arabic, and thus gum seyal arabic can be provided as an appropriate supplement to gum seyal arabic responders whose intestinal bifidobacteria grow by ingestion of gum seyal arabic.

[0081] In another aspect of the present embodiment, there is provided a method for detecting gum arabic bacteria of S. seyal, comprising a step of carrying out PCR using a kit for detecting gum arabic bacteria of S. seyal.

[0082] In addition, the term "detection" as used herein includes not only a qualitative detection for examining the presence or absence of gum arabic bacteria of S. seyal but also a quantitative detection (measurement) for quantitatively examining the amount of gum arabic bacteria of S. seyal.

[0083] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0084] [Example 1: Preparation of recombinant enzyme] A plasmid (pET23d_SKIK_BBCT_0495Cdel93) containing the BBCT_0495 gene, which was totally synthesized using the genome information of JCM1194, was transformed using E. coli BL21(DE3)pLysS. The base sequence of the plasmid is shown in SEQ ID NO:5. In this plasmid, the BBCT_0495 gene has been modified so as to encode a recombinant enzyme in which the C-terminal cell wall anchoring region has been removed, a His-Tag has been added to the C-terminus, and a part of the N-terminal region has been replaced with a SKIK sequence for solubilization. The amino acid sequence of the recombinant enzyme is shown in SEQ ID NO:2.

[0085] 2.5 μl of plasmid was added to 50 μl of competent cells that had been melted on ice, and the mixture was left to stand on ice for 20 minutes. The mixture was incubated at 42°C for 45 seconds, and then left to stand on ice for 2 minutes. 0.9 ml of SOC medium was placed in a sterilized 15 ml tube, and the entire amount of competent cells was added. The mixture was cultured with shaking at 37°C for 1 hour, and 50 μl of the mixture was applied to an LB agar plate (ampicillin + chloramphenicol). The LB agar plate was incubated overnight at 37°C, and the resulting colonies were cultured in 3 ml of LB medium containing ampicillin and chloramphenicol to prepare glycerol stocks.

[0086] The glycerol stock was inoculated into 3 ml of LB medium containing ampicillin and chloramphenicol to obtain an OD 600 The mixture was cultured at 37°C with shaking until the pH reached 0.5. 30 µl of IPTG was added, and the mixture was cultured at 15°C overnight.

[0087] 600μl of Bug Buster Extraction Reagent (Millipore) was added to 3ml of the obtained bacterial cells, and the mixture was suspended and left to stand at room temperature for 10 minutes. 10μl of 10-fold diluted turbonuclease was added and left to stand at room temperature for 5 minutes. The mixture was centrifuged (12000rpm) at 4℃ for 10 minutes, and the supernatant (hereinafter referred to as "BBCT_0495 BugBuster treatment") was collected.

[0088] The crude enzyme contained in the supernatant was purified via His-Tag attached to the C-terminus. 3 ml of equilibration buffer (50 mM sodium phosphate, 300 mM sodium chloride (pH 7.0)) was applied to a column containing 1 ml of Metal Affinity Resin. 1.2 ml of equilibration buffer was added to 600 μl of crude enzyme and applied to the column. Furthermore, 5 ml of equilibration buffer was applied to the column. An appropriate amount of equilibration buffer was mixed with elution buffer (0 mM sodium phosphate, 300 mM sodium chloride, 100 mM imidazole (pH 7.0)) to prepare elution solutions with each imidazole concentration (0 mM, 5 mM, 10 mM, 25 mM, 50 mM). The column was transferred to another test tube, and 5 ml of elution solution with 0 mM imidazole was applied. Thereafter, 5 ml of elution solution was applied in the order of 5 mM, 10 mM, 25 mM, and 50 mM, and the eluted fraction was collected. Each time the eluate was passed through the column, the column was transferred to a different test tube.

[0089] The 25 mM and 50 mM elution fractions were desalted and concentrated using a VIVASPIN TURBO (30 kDa cutoff, Sartorius Japan).

[0090] (result) When the obtained sample was examined for protein by SDS-PAGE, a band was detected at the expected molecular weight of about 105 kDa.

[0091] [Test Example 1: Functional analysis of recombinant enzymes] (Purification of Seyal gum arabic and recovery of free oligosaccharides) 148.4 g of gum arabic (Fiber Gum TAN, Acasia seyal 100%, Nexira) was dissolved in 520 ml of Elix water. 1800 ml of ethanol and 400 μl of 6M HCl were added, and the mixture was left at low temperature for 2 hours, and then filtered by suction through a glass filter. The precipitate fraction remaining on the filter was dissolved in 520 ml of Elix water, and 1800 ml of ethanol and 400 μl of 6M HCl were added, and the mixture was filtered by suction through a glass filter. About 200 ml of 80% ethanol was added to the precipitate fraction remaining on the filter, and the mixture was stirred on the filter. The mixture was filtered by suction through a glass filter, and washed again with 80% ethanol in the same manner. Next, about 100 ml of acetone was added to the precipitate fraction remaining on the filter, and the mixture was mixed on the filter, and then filtered by suction. The precipitate fraction on the filter was collected, placed on a glass dish, and dried in a desiccator. After drying, the mixture was dissolved in about 500 ml of Elix water, and freeze-dried. The freeze-dried purified Gum Acacia seyalii was used as Gum Acacia seyalii below.

[0092] (Reactivity of recombinant enzymes to Seyal gum arabic) Enzyme reaction was carried out using the recombinant enzyme prepared in Test Example 1 with gum arabic of Seyal as a substrate, and the product was analyzed by HPAEC-PAD. The reaction composition was 200 μl of substrate (final concentration 1%), 20 μl of 1M acetate buffer (pH 6.0) (final concentration 50 mM), 20 μl of recombinant enzyme (final concentration 7.01 nM), and 160 μl of water, totaling 400 μl. Water was added to the control sample instead of the recombinant enzyme. After incubation at 37°C for 16 hours, the reaction was stopped in a boiling bath for 3 minutes.

[0093] The conditions for HPAEC-PAD were as follows: Detector: Pulsed amperometric detector Column: CarboPack PA-1 Flow: 1.00mL / min Temperature: 30℃ Capture time: 60 minutes Sample volume: 25 μl Eluent: A: 100mM NaOH, B: 100mM NaOH containing 500mM CH3COONa Methods: 0-5 min: A100% 5~30 minutes: A100%→0%, B0%→100% 30~35 minutes: B100% 35~45 minutes: A100%

[0094] (result) The chromatogram obtained by HPAEC-PAD is shown in Figure 1. Peaks corresponding to three free oligosaccharides were confirmed. Hereinafter, the recombinant enzyme having endoglycosidase activity is also referred to as "endo-AAfase."

[0095] [Test Example 2: Purification and structural determination of free oligosaccharides] (Enzyme reaction) To analyze the structure of free oligosaccharides by NMR, the recombinant enzyme was reacted with gum arabic of Seyal species in a methanol environment to add a methyl group to the reducing end of the free oligosaccharides. The composition of the reaction solution is shown in Table 1.

[0096] [Table 1]

[0097] After incubating the reaction solution at 37°C for 5 days, ethanol was added to the reaction solution little by little (total amount of ethanol added was 1875 ml, final concentration 80%) and stirred for 1 hour. The reaction solution was placed in a refrigerator and centrifuged at 8000 rpm for 10 minutes. The supernatant was collected, concentrated with an evaporator, and then dissolved in 1000 ml of Elix water.

[0098] (Activated carbon column purification) As the activated carbon column, Autoprep (trademark) FiberS6 (6 ml syringe) and Autoprep (trademark) FiberS@Liq (connected type) were used. 400 ml of 1 L of sample was passed through column A, which was made up of five connected columns, and 600 ml was passed through column B, which was made up of ten connected columns. Column A was equilibrated with 50 ml of methanol and 50 ml of water, and then the sample was passed through. It was then washed with 600 ml of water, and 200 ml of 10% methanol, 200 ml of 25% methanol, 200 ml of 50% methanol, and 550 ml of 100% methanol were passed through in this order. Column B was equilibrated with 300 ml of methanol and 300 ml of water, and then the sample was passed through. It was then washed with 600 ml of water, and 200 ml of 10% methanol, 200 ml of 30% methanol, 200 ml of 60% methanol, and 12,000 ml of 100% methanol were passed through in this order.

[0099] The 50% and 100% methanol fractions from column A, and the 60% and 100% methanol fractions from column B were concentrated and then purified by gradient elution of water and acetonitrile on a PBr column (10 mm 1D x 250 mm, Nacalai Tesque). Each purified fraction was concentrated and freeze-dried. The freeze-dried sample was dissolved in 100 μl of sterile water and measured by HPAEC-PAD and high-resolution ESI-TOF MS.

[0100] 1 H, 13 The structures of the glycosyltransferases were identified by NMR analysis, including C and heteronuclear multiple bond correlation (HMQC). The freeze-dried samples were dissolved in heavy water (D2O). 1 H and 13 C NMR spectrum and 2D spectrum ( 1 H- 1H COSY, TOCSY, HMQC, HMBC) were measured in D2O at room temperature using a JEOL ECX 400 spectrometer (400 MHz). ESI-TOF mass spectra were recorded on a JEOL AccuTOF JMS-T700LCK with CF3CO2Na as the internal standard. A3-Me and A5-Me were acetylated and analyzed in the same manner, since some of the proton peaks were close to each other and difficult to assign. Double Quantum Filtered-COrrelation SpectroscopY (DQF-COSY) was also performed for the structure analysis of A5-Me.

[0101] (result) The chromatogram obtained by HPAEC-PAD is shown in Figure 2. Almost a single peak was obtained for each free oligosaccharide. The sugar structure was estimated by ESI-TOF MS, and A3-Me (Calcd for C 16 H 28 NaO 13 [M+Na] + 451.1428 found 451.1447), A4-Me(Calcd for C 21 H 36 NaO 17 [M+Na] + 583.1850 found 583.1827) and A5-Me (Calcd for C 26 H 44 NaO 21 [M+Na] + 715.2273 found 715.2294), the calculated values ​​calculated from the predicted structure were in agreement with the measured values.

[0102] In the results of A3-Me, the cross peaks of β-ArafH1 / β-ArafH4, β-ArapH1 / β-ArapH5, and α-ArafH1 / α-ArafH4 were observed in the Heteronuclear Multiple Bond Correlation (HMBC) spectrum, and the furan ring structure and pyran ring structure were identified, and they were found to be β-Araf, β-Arap, and α-Araf, respectively. Furthermore, the cross peaks of α-ArafH1 / OMe, β-ArapH1 / α-ArafC3, and β-ArafH1 / β-ArapC2 were observed, and the bond positions were clarified, and the structure of β-L-Araf-(1→2)-β-L-Arap-(1→3)-α-L-Araf-OMe shown in formula (1) was clarified. In addition, the analysis of acetylated A3-Me eliminated the overlap of the signals of β-ArapC2 and C3, and each peak could be assigned. A4-Me and A5-Me were also analyzed in the same manner, and their structures were revealed to be β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-α-L-Araf-OMe shown in formula (2) and β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-α-L-Araf-OMe shown in formula (3), respectively.

[0103] [ka]

[0104] [ka]

[0105] [ka]

[0106] [Test Example 3: Co-culture assimilation test of two types of bifidobacteria using gum arabic of Seyal species] Bifidobacterium longum JCM1217 (hereinafter referred to as "JCM1217") and JCM1194 were used. First, preculture was performed as follows. L-cysteine ​​hydrochloride monohydrate was added to MRS medium (MRS: water = 2.75 g: 50 ml) to a final concentration of 0.04%. 10 ml of the medium was dispensed into test tubes and autoclaved (121°C, 20 minutes). The medium was inoculated, and the test tubes and Anaeropack were placed in a jar to create anaerobic conditions, and static culture was performed at 37°C for 18 hours.

[0107] The main culture was carried out in MRS medium containing gum arabic (final concentration 1%) as the only carbon source. The composition of the medium used for the main culture is shown below.

[0108] [Table 2]

[0109] 5 ml of 2xMRS and 5 ml of 2% seyal gum arabic as a sugar source were dispensed into test tubes and autoclaved (121°C, 20 min). 100 μl of the pre-cultured cells were inoculated into the medium for main culture. The inoculated test tubes and Anaeropack were placed in a jar, and the tubes were made anaerobic and cultured at 37°C. The absorbance (600 nm) was measured 6, 9.5, 12, 24, and 32 hours after the start of culture. 1 ml of the medium was collected 32 hours after the start of culture, centrifuged at 15,000 rpm for 10 minutes, and 1 μl of the supernatant was spotted on a TLC and developed.

[0110] For TLC, samples and standards were spotted on a plate (TLC aluminum sheet, silicagel60) and air-dried. An appropriate amount of developing solvent (n-propanol:ethanol:water = 7:1:2) was poured onto the developing layer, and the sheet was immersed in it to develop. When the developing solvent rose to the top, the sheet was removed and air-dried in a fume hood. A color developer was sprayed on the plate, air-dried again, and heated at 130°C for about 1 minute. The color developer was orcinol sulfuric acid, a 10:1 mixture of 10% sulfuric acid containing 1% FeCl3 and 6% orcinol.

[0111] (result) Figure 3 shows the change in absorbance of the medium over time. It was confirmed that JCM1194, which has the BBCT_0495 gene, grew well, while JCM1217, which does not have the BBCT_0495 gene, grew only slightly. Furthermore, the highest absorbance was observed when co-cultured.

[0112] Figure 4 shows the plate after TLC development. Accumulation of S4 (α-L-Rhap-(1→4)-β-D-GlcpA-(1→6)-β-D-Galp-(1→6)-D-Gal) was confirmed in the remaining sugars after cultivation. This indicates that the modified β-arabinooligosaccharides of gum arabic of Seyal species were removed by the action of endo-AAfase, and the AGP decomposition enzyme group contained in JCM1217 acted.

[0113] [Test Example 4: Investigation of strains carrying the BBCT_0495 gene] Using the amino acid sequence of BBCT_0495 shown in SEQ ID NO: 1 as the query sequence, the complete genome or homologous sequences on the contig were searched for using tblastn from the National Center for Biotechnology Information's BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) as the search target database as the base sequence. Among the strains registered in the NCBI database, those detected with both identity and coverage of 90% or more in the tblastn search results were calculated as gene-carrying strains.

[0114] (result) 5 of 58 strains (8.6%) of Bifidobacterium catenulatum, 19 of 284 strains (6.7%) of Bifidobacterium pseudocatenulatum, and 17 of 199 strains (8.5%) of Bifidobacterium adolescentis had the BBCT_0495 gene. In Bifidobacterium adolescentis, a strain with a moderately similar sequence was detected, with an identity of 77% and a coverage of 73%, and including these, the number of strains was 37 of 199 strains (18.6%).

[0115] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. The above-described embodiments are for the purpose of explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not the embodiments. Various modifications made within the scope of the claims and the scope of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0116] The present invention is useful in producing prebiotic-containing foods.

Claims

1. The present invention relates to a method for producing an endoglycosidase comprising the steps of: Seyal gum arabic modified glycolytic enzyme. (a) the amino acid sequence shown in SEQ ID NO:1 (b) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1; (c) the amino acid sequence shown in SEQ ID NO:2 (d) an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:2

2. A modified glycolytic enzyme from Seyal arabic gum, encoded by the following DNA (a) or (b): (a) DNA containing the base sequence shown in SEQ ID NO:3 (b) a DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or a probe prepared from the complementary base sequence, and that encodes a modified glycolytic enzyme from Seyal arabic gum having endoglycosidase activity.

3. The present invention comprises at least one of the gum arabic modified glycolytic enzyme of claim 1 or 2, a Bifidobacterium bacterium having a gene encoding the gum arabic modified glycolytic enzyme of claim 1, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium. Food and drink composition.

4. For promoting the decomposition of gum arabic from Seyal species. The food and beverage composition according to claim 3.

5. At least one of the gum arabic modified glycolytic enzyme of claim 1 or 2, a Bifidobacterium bacterium having a gene encoding the gum arabic modified glycolytic enzyme of claim 1 or 2, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium. A raw material containing gum arabic of seyal species; A food or beverage composition comprising:

6. For promoting the growth of bifidobacteria, The food and beverage composition according to claim 5.

7. The present invention relates to a method for producing a gum arabic seyal according to claim 1, a method for producing a gum arabic seyal according to claim 2, a method for producing a gum arabic seyal according to claim 3, a method for producing a gum arabic seyal according to claim 4, a method for producing a gum arabic seyal according to claim 5, a method for producing a gum arabic seyal according to claim 6, a method for producing a gum arabic seyal according to claim 7, a method for producing a gum arabic seyal according to claim 8, a method for producing a gum arabic seyal according to claim 9, a method for producing a gum arabic seyal according to claim 10, a method for producing a gum arabic seyal according to claim 11, a method for producing a gum arabic seyal according to claim 12, a method for producing a gum arabic seyal according to claim 13, a method for producing a gum arabic seyal according to claim 14, a method for producing a gum arabic seyal according to claim 15, a method for producing a gum arabic seyal according to claim 16, Food and drink composition.

8. The present invention comprises at least one of the gum arabic modified glycolytic enzyme of claim 1 or 2, a Bifidobacterium bacterium having a gene encoding the gum arabic modified glycolytic enzyme of claim 1, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium. Bifidobacteria growth promoter.

9. The method includes a step of allowing at least one of the seyal gum arabic modified glycolytic enzyme according to claim 1 or 2, a Bifidobacterium bacterium having a gene encoding the seyal gum arabic modified glycolytic enzyme, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium to act on a raw material containing seyal gum arabic. A method for producing oligosaccharides.

10. The oligosaccharide is β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap- (1→3)-L-Ara or β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Arap-(1→3)-L-Ara, A method for producing the oligosaccharide according to claim 9.

11. The method includes administering at least one of the gum arabic modified glycolytic enzyme of claim 1 or 2, a Bifidobacterium bacterium having a gene encoding the gum arabic modified glycolytic enzyme, a culture of the Bifidobacterium bacterium, and a treated cell product of the Bifidobacterium bacterium to a non-human animal. A method for promoting the growth of bifidobacteria.

12. A primer pair designed to include at least 10 consecutive bases contained in the DNA encoding the seyal gum arabic modified glycolytic enzyme according to claim 1 or 2 in the base sequence of a PCR product. A kit for detecting gum arabic-utilizing bacteria of Seyal species.

13. A step of performing PCR using the kit for detecting gum arabic assimilation bacteria of seyal species according to claim 12, A method for detecting gum arabic-utilizing bacteria of Seyal species.

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