Novel bifidobacterium and utilization of same

Abstract

To provide a novel bacterium belonging to Bifidobacterium longum subsp. infantis. Provided is a bacterium belonging to Bifidobacterium longum subsp. infantis, which has an ability to assimilate human breast milk oligosaccharides and xylooligosaccharides and does not have a drug resistance gene.

Description

Novel Bifidobacterium and its applications

[0001] The present invention relates to a bacterium of the genus Bifidobacterium, which is classified as Bifidobacterium longum subsp. infantis, and uses thereof.

[0002] The formation of the intestinal microbiota during infancy begins with the colonization of aerobic bacteria such as Enterobacteriaceae, and transitions to a bifidobacteria-dominated microbiota within a few days to several months (Non-Patent Document 1). Infant bifidobacteria metabolize human breast milk oligosaccharides to produce organic acids, lowering the pH in the intestine. Organic acids produced by bifidobacteria have been shown to be effective in preventing infectious diseases in mice (Non-Patent Document 2), and similar effects are expected in humans. Therefore, administering bifidobacteria as a probiotic during infancy to induce the early formation of a bifidobacteria flora is thought to be useful for promoting infant health.

[0003] For bifidobacteria to colonize and proliferate in the infant intestine, it is important that they be able to utilize human breast milk oligosaccharides (Non-Patent Document 3). It is known that the timing of bifidobacterial flora formation during infancy varies from individual to individual, and it has been observed in many infants that bifidobacteria with low utilization of human breast milk oligosaccharides are replaced by strains with high utilization of human breast milk oligosaccharides by the end of breastfeeding (Non-Patent Document 1). Therefore, bifidobacterial probiotics with high utilization of human breast milk oligosaccharides are effective for stably colonizing the intestine, efficiently metabolizing human breast milk oligosaccharides to produce organic acids, and exerting physiological effects.

[0004] After weaning, the diet contains a lot of plant-derived indigestible polysaccharides (e.g., xylans), and bifidobacterial strains that can utilize indigestible polysaccharides and are expected to grow in the intestine and produce high organic acids are thought to be promising candidates for probiotics.

[0005] Bifidobacterium longum subsp. infantis is one of the predominant bacteria in the infant intestine and is known to be able to utilize human breast milk oligosaccharides (e.g., Patent Documents 1 and 2). However, although approximately 250 types of human breast milk oligosaccharides have been identified, the diversity of Bifidobacterium longum subsp. infantis strains that can utilize individual breast milk oligosaccharides has not been fully investigated. Furthermore, no Bifidobacterium longum subsp. infantis bacteria have been known to date that can degrade the xylose backbone of xylooligosaccharides derived from xylans, the major indigestible polysaccharides found in the diet.

[0006] International Publication No. 2019 / 232284 Japanese Patent Application Laid-Open No. 2021-112166

[0007] Tsukuda, N. et al., ISME J, 2021, 15: 2574-2590Fukuda, S. et al., Nature, 2011, 469: 543-547Matsuki, T. et al., Nat Commun, 2016, 7: 11939

[0008] The present invention relates to providing a novel bacterium belonging to Bifidobacterium longum subsp. infantis that is capable of assimilating human breast milk oligosaccharides and xylooligosaccharides.

[0009] As a result of extensive research, the present inventors have isolated and identified a novel strain of Bifidobacterium longum subsp. infantis from a human infant, which can efficiently assimilate human breast milk oligosaccharides and also has the ability to assimilate xylooligosaccharides. Furthermore, the present inventors have found that this strain does not contain drug resistance genes.

[0010] That is, the present invention provides the following [1] to [9]. [1] A bacterium belonging to Bifidobacterium longum subsp. infantis that has the ability to assimilate human breast milk oligosaccharides and xylooligosaccharides and does not have a drug resistance gene. [2] The bacterium according to [1], which has the ability to assimilate at least one human breast milk oligosaccharide selected from the group consisting of fucosyllactoses, lacto-N-tetraose, 5- to 10-saccharide human neutral breast milk oligosaccharides, and sialyllactoses. [3] The bacterium according to [2], wherein the fucosyllactoses are 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose, the 5- to 10-saccharide human neutral breast milk oligosaccharides are lacto-N-fucopentaose I and lacto-N-difucohexaose I, and the sialyllactoses are 3'-sialyllactose and 6'-sialyllactose. [4] Bifidobacterium longum subsp. infantis YIT 13155 (NITE BP-04137) or a strain closely related thereto. [5] A food or drink containing the bacterium according to any one of [1] to [4]. [6] The food or drink according to [5], further comprising at least one selected from the group consisting of human breast milk oligosaccharides and xylooligosaccharides. [7] A pharmaceutical containing the bacterium according to any one of [1] to [4]. [8] The pharmaceutical according to [7], further comprising at least one selected from the group consisting of human breast milk oligosaccharides and xylooligosaccharides. [9] A primer pair consisting of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3, or a primer pair consisting of complementary sequences corresponding to said nucleotide sequences.

[0011] The novel bacterium belonging to Bifidobacterium longum subsp. infantis according to the present invention has the ability to decompose and assimilate xylooligosaccharides derived from human breast milk oligosaccharides and xylans, which are abundant in the diet, mainly after weaning, and does not contain drug resistance genes that can be transmitted to other intestinal bacteria. Therefore, the bacterium of the present invention is expected to grow in the human intestine, have high organic acid production capabilities, and is also highly safe, making it suitable for use as a probiotic in pharmaceuticals, foods, beverages, and the like.

[0012] HPLC chromatogram of human breast milk oligosaccharide fraction derived from human breast milk. Diagram showing the distribution and phylogenetic tree of sugar utilization genes for each strain of Bifidobacterium longum subsp. infantis. Gray areas in the diagram indicate that the gene is present, and white areas indicate that the gene is not present. Diagram showing the residual sugars in the culture supernatant 48 hours after culture in a medium containing a human breast milk oligosaccharide fraction as a sugar source. Diagram showing the detection ability of primers specific to Bifidobacterium longum subsp. infantis Bi-17 or closely related strains.

[0013] The bacterium of the present invention is a bacterium belonging to Bifidobacterium longum subsp. infantis that has the ability to assimilate human breast milk oligosaccharides and xylooligosaccharides and does not have a drug resistance gene.

[0014] "Human milk oligosaccharides (HMOs)" are a general term for oligosaccharides contained in human breast milk, also known as human milk oligosaccharides. Examples of human breast milk oligosaccharides include fucosyllactoses in which fucose is bound to a lactose backbone, such as 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), and difucosyllactose (DFL); human neutral breast milk oligosaccharides consisting of 5 to 10 sugars in which fucose is bound to a lactose backbone, such as lacto-N-tetraose (LNT); lacto-N-fucopentaose I (LNFPI), and lacto-N-difucohexaose I (LNDFHI); and sialyllactoses in which sialic acid is bound to a lactose backbone, such as 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL).

[0015] The human breast milk oligosaccharides assimilated by the bacteria of the present invention include at least one of the above-mentioned human breast milk oligosaccharides, preferably at least one selected from the group consisting of fucosyllactoses, lacto-N-tetraose, human neutral breast milk oligosaccharides of 5 to 10 sugars, and sialyllactoses, more preferably at least one selected from the group consisting of fucosyllactoses, lacto-N-tetraose, at least one selected from the group consisting of human neutral breast milk oligosaccharides of 5 to 10 sugars, and at least one selected from the group consisting of sialyllactoses, and even more preferably 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose I, 3'-sialyllactose, and 6'-sialyllactose.

[0016] In the present invention, "having the ability to utilize human breast milk oligosaccharides" means having the ability to grow using human breast milk oligosaccharides as a sugar source. Specifically, for example, it means being able to grow in a medium containing human breast milk oligosaccharides as the sole sugar source. For example, the turbidity, e.g., OD , after 60 hours of culture in a medium containing only human breast milk oligosaccharides as a sugar source can be measured. ６００ The increase in turbidity is 0.015 or more. ６００ The increase is preferably 0.018 or more, more preferably 0.02 or more. Here, the human breast milk oligosaccharides used as the sugar source may be one of the above human breast milk oligosaccharides or a combination of two or more of them. When two or more of them are combined, the sugar source concentration refers to the total concentration of the two or more human breast milk oligosaccharides.

[0017] The culture medium used in testing human breast milk oligosaccharide assimilation can be a medium lacking a sugar source to which human breast milk oligosaccharides have been added. The composition of the sugar source-free medium can be ILS medium, Peptone-Yeast (PY) medium, or the like. The amount of human breast milk oligosaccharides added to the medium is preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass, even more preferably 0.1 to 1% by mass, and even more preferably 0.25% by mass. It is also desirable to culture the target bacteria belonging to Bifidobacterium longum subsp. infantis in a sugar source-free medium as a control and confirm the OD.

[0018] "Xylooligosaccharide (XOS)" refers to an oligosaccharide having a structure in which 2 to 10, preferably 2 to 5, xylose units are linked by β-1,4 bonds, and examples thereof include xylobiose, xylotriose, and xylotetraose. In vivo, xylooligosaccharides are produced by hydrolysis of xylans, the major indigestible polysaccharides contained in the diet, by xylanase from intestinal bacteria.

[0019] In the present invention, "having the ability to utilize xylooligosaccharides" means having the ability to grow using xylooligosaccharides as a sugar source. Specifically, for example, it means being able to grow in a medium containing xylooligosaccharides as the sole sugar source. For example, the turbidity, e.g., OD , after 80 hours of culture in a medium containing only xylooligosaccharides as a sugar source can be measured. ６００ The increase in turbidity is 0.015 or more. ６００ The increase is preferably 0.018 or more, more preferably 0.02 or more.

[0020] The culture medium used in the test for xylooligosaccharide assimilation ability may be a medium from which a sugar source has been removed and to which xylooligosaccharides have been added. The composition of the medium from which a sugar source has been removed may be ILS medium, Peptone-Yeast (PY) medium, or the like. The amount of xylooligosaccharides added to the medium is preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass, even more preferably 0.1 to 1% by mass, and even more preferably 0.25% by mass. As a control, it is desirable to culture the target bacterium belonging to Bifidobacterium longum subsp. infantis in a medium from which a sugar source has been removed and confirm the OD.

[0021] Since xylooligosaccharides can be hydrolyzed by xylosidase, bacteria capable of assimilating xylooligosaccharides can be rephrased as bacteria having a xylosidase gene on their genome.

[0022] The term "xylosidase gene" refers to a gene (beta-xylosidase) encoding a xylosidase classified in glycoside hydrolase (GH) family 43. The "xylosidase gene" possessed on the genome of the bacterium belonging to Bifidobacterium longum subsp. infantis of the present invention specifically includes a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1, or a polynucleotide having 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more identity to said nucleotide sequence and encoding a protein having xylosidase activity. Here, the polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1 refers to the xylosidase gene possessed by Bi-17, which will be described later.

[0023] The identity of nucleotide sequences is determined by aligning the two nucleic acid sequences to be compared so that as many bases as possible match, and dividing the number of matched bases by the total number of bases, expressed as a percentage. Those skilled in the art can determine the identity of nucleotide sequences by appropriately setting the parameters of software such as BLAST, ClustalX, or Genetyx. Furthermore, xylosidase activity refers to the activity of using xylooligosaccharides as substrates to continuously produce xylose residues from the non-reducing ends of xylooligosaccharides (xylooligosaccharide hydrolysis activity).

[0024] The bacterium of the present invention does not have a drug resistance gene. A bacterium does not have a drug resistance gene when a gene that confers resistance to an antibacterial drug to the bacterium is not detected in the base sequence of the bacterium's genome. When the base sequence of the bacterium is compared with drug resistance genes listed in publicly known databases, the bacterium can be determined to have no drug resistance gene if no base sequence with 95% or more sequence identity to the drug resistance gene is found in the bacterium's genome sequence. Specifically, when the presence or absence of a drug resistance gene is searched for using the bacterium's genome sequence with ARG-ANNOT (https: / / ifr48.timone.univ-mrs.fr / blast / arg-annot_nt.html), the bacterium can be determined to have no drug resistance gene if no base sequence with 95% or more sequence identity to the drug resistance gene listed in the ARG-ANNOT database is found in the bacterium's genome sequence. Examples of such drug resistance genes include the macrolide antibiotic resistance gene (ermX), the aminoglycoside antibiotic resistance gene (rmtF), and the tetracycline resistance gene (tetR). The bacterium of the present invention does not have any of the drug resistance genes listed in the ARG-ANNOT database, including these genes.

[0025] As such, the bacterium of the present invention has the ability to decompose and assimilate human breast milk oligosaccharides and xylooligosaccharides and does not contain drug resistance genes, so it can grow well in the intestines of humans (preferably infants) and contribute to the supply of short-chain fatty acids such as acetic acid, lactic acid, and formic acid.Furthermore, there is a low risk of the drug resistance gene being transmitted to other intestinal bacteria, causing the emergence of drug-resistant bacteria, making it highly safe.In this specification, infants refer to children from 0 days to 2 years of age.

[0026] The bacterium of the present invention can be obtained by screening bacteria belonging to Bifidobacterium longum subsp. infantis present in humans (e.g., adults and infants, preferably infants) using as indicators the ability to assimilate human breast milk oligosaccharides, the ability to assimilate xylooligosaccharides or the presence of a xylosidase gene, and the absence of a drug resistance gene. Alternatively, the bacteria belonging to Bifidobacterium longum subsp. infantis of the present invention can be obtained by screening from bacteria produced by known breeding methods using any bacterium belonging to Bifidobacterium longum subsp. infantis, such as a bacterium belonging to Bifidobacterium longum subsp. infantis present in humans (e.g., adults or infants, preferably infants), as a parent strain, using the ability to assimilate human breast milk oligosaccharides, the ability to assimilate xylooligosaccharides or the presence of a xylosidase gene, and the absence of a drug resistance gene as indicators; by screening from bacteria produced by treating the parent strain with ultraviolet irradiation or a mutagen such as nitrosoguanidine (NTG) or ethylmethanesulfonate (EMS); or by screening from bacteria produced by modifying the genome of the parent strain by known site-directed mutagenesis methods, for example.

[0027] In the Examples below, Bifidobacterium longum subsp. infantis Bi-15, Bi-16, Bi-17, and Bi-18 were selected as the bacteria of the present invention. The bacteria of the present invention are not limited to these strains, as long as they belong to Bifidobacterium longum subsp. infantis and have the ability to assimilate human breast milk oligosaccharides and xylooligosaccharides and do not have a drug resistance gene, and also include strains that are biologically and genetically closely related to these strains. Furthermore, the bacteria of the present invention may be naturally occurring strains, or mutants or genetically modified strains of naturally occurring strains, as long as they belong to Bifidobacterium longum subsp. infantis and have the ability to assimilate human breast milk oligosaccharides and xylooligosaccharides and do not have a drug resistance gene.

[0028] Bi-17 refers to Bifidobacterium longum subsp. infantis YIT 13155, and was deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on July 24, 2024 (NITE BP-04137). The bacterium of the present invention is preferably Bi-17 or a closely related strain thereof, due to its high ability to assimilate human breast milk oligosaccharides.

[0029] Here, closely related strains refer to strains whose ANI (Average Nucleotide Identity) values ​​using draft genomes are 98% or higher, preferably 99% or higher, more preferably 99.9% or higher, and even more preferably 99.99% or higher. The Bifidobacterium longum subsp. infantis Bi-15, Bi-16, Bi-17, and Bi-18 strains selected in the Examples described below have the lowest ANI value of 99.997% and the highest ANI value of 99.9992%, and can be said to be closely related strains.

[0030] Primers specific to Bifidobacterium longum subsp. infantis Bi-17 and related strains include an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 (5'-AAATATAGCGCTCGCCGACA-3') or a sequence complementary to said sequence, or an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 (5'-AGCCTACTGCCGCTTGATTT-3') or a sequence complementary to said sequence. Bi-17 and related strains can be specifically detected or quantified using a primer pair consisting of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3, or a primer pair consisting of a complementary sequence corresponding to said nucleotide sequence. For example, by using this primer pair and performing a PCR reaction using DNA contained in a sample (e.g., a human fecal sample) as a template, Bi-17 and related strains in the sample can be rapidly and easily detected or quantified.

[0031] The base sequences shown in SEQ ID NOs: 2 and 3 and the complementary sequences corresponding to said base sequences consist of base sequences in which one or two bases have been deleted, substituted, added or inserted, or base sequences which have 90% or more, preferably 95% or more, and more preferably 98% or more identity to the base sequences of SEQ ID NOs: 2 and 3 or the complementary sequences corresponding to said base sequences, and oligonucleotides which have the same function as primers as oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 2 and 3 or the complementary sequences corresponding to said base sequences are treated as equivalent to oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 2 and 3.

[0032] The above oligonucleotides can be synthesized by methods known in the art as oligonucleotide synthesis methods, such as the phosphotriethyl method and the phosphodiester method, using a commonly used automatic DNA synthesizer.

[0033] The form in which the bacteria of the present invention are used is not particularly limited, and they may be freeze-dried or used as a culture containing these bacteria, but in either form, it is preferable that the bacteria are in a live state.

[0034] The bacterium of the present invention can also be used in the form of conventional pharmaceutical preparations by mixing it with a solid or liquid non-toxic pharmaceutical carrier. Examples of such preparations include solid preparations such as tablets, granules, powders, and capsules; liquid preparations such as solutions, suspensions, and emulsions; and lyophilized preparations. These preparations can be prepared by conventional pharmaceutical techniques. Examples of such non-toxic pharmaceutical carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and physiological saline. Conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonicity agents, and excipients can also be added as needed.

[0035] To promote the growth of the bacterium of the present invention, at least one selected from the group consisting of human breast milk oligosaccharides and xylooligosaccharides that can be assimilated by the bacterium of the present invention can also be added to the pharmaceutical preparation. Examples of human breast milk oligosaccharides include fucosyllactoses such as 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose; human neutral breast milk oligosaccharides consisting of 5 to 10 sugars such as lacto-N-tetraose; lacto-N-fucopentaose I and lacto-N-difucohexaose I; and sialyllactoses such as 3'-sialyllactose and 6'-sialyllactose. Commercially available human breast milk oligosaccharides can be used. For example, 2'-fucosyllactose can be obtained from Advanced Protein Technologies, Inc., 3-fucosyllactose from Advanced Protein Technologies, Inc., difucosyllactose from OligoTech, Inc., lacto-N-fucopentaose I from Dextra Laboratories, Inc., lacto-N-difucohexaose I from Iso Sep, Inc., and lacto-N-tetraose from Iso Sep. Human breast milk oligosaccharides can also be prepared by known methods, such as isolation from breast milk, enzymatic production, or chemical synthesis. Examples of xylooligosaccharides include xylobiose, xylotriose, and xylotetraose. Commercially available xylooligosaccharides can be used, and are available from Bussan Food Science Co., Ltd., etc. Xylooligosaccharides can also be prepared by known methods, such as by treating biomass raw materials containing xylans with xylanase. The above-mentioned human breast milk oligosaccharides and xylooligosaccharides can be used alone or in combination of two or more.

[0036] The bacteria of the present invention can be used not only as formulations as described above, but also by being incorporated into foods and beverages. When incorporated into foods and beverages, they may be contained as is or together with various nutritional components. Specifically, when incorporating the bacteria of the present invention into foods and beverages, additives that can be used in foods and beverages may be appropriately used, and the bacteria may be formed into edible forms, i.e., granules, particles, tablets, capsules, pastes, etc., using conventional means. They may also be added to various foods, such as processed meat foods such as ham and sausage, processed seafood foods such as kamaboko and chikuwa, bread, confectionery, butter, powdered milk, or beverages such as water, fruit juice, milk, soft drinks, and tea drinks. Foods and beverages also include animal feed.

[0037] To promote the growth of the bacterium of the present invention, the food or drink may contain one or more selected from the group consisting of human breast milk oligosaccharides and xylooligosaccharides that can be assimilated by the bacterium of the present invention. Specific examples of human breast milk oligosaccharides and xylooligosaccharides are the same as those for the pharmaceutical products described above.

[0038] Furthermore, suitable foods and beverages include fermented milk foods and beverages, such as fermented soy milk, fermented fruit juice, and fermented vegetable juice, which contain the bacteria of the present invention in a viable state. Fermented milk foods and beverages are particularly preferred. Fermented milk foods and beverages can be produced according to conventional methods. For example, when producing fermented milk, the bacteria of the present invention are inoculated and cultured in a sterilized milk medium, either alone or together with other microorganisms, and then homogenized to obtain a fermented milk base. A separately prepared syrup solution is then added and mixed, homogenized using a homogenizer, or the like, and a flavor is added to produce the final product. The fermented milk foods and beverages obtained in this manner can be in any form, such as a plain type (without syrup (sweetener)), soft type, fruit-flavored type, solid, or liquid.

[0039] Such fermented milk foods and drinks can contain optional ingredients such as sweeteners such as syrup, emulsifiers, and thickeners (stabilizers). Sugars such as glucose, sucrose, fructose, high-fructose corn syrup, high-fructose corn syrup, palatinose, trehalose, lactose, xylose, galactooligosaccharides (GOS), xylooligosaccharides (XOS), arabinoxylooligosaccharides (AXOS), xylan, arabinoxylan, arabinooligosaccharides (AOS), arabinan, maltose, honey, and molasses; sugars such as sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, and reduced maltose syrup. The sweetener may contain high-intensity sweeteners such as alcohol, aspartame, thaumatin, sucralose, acesulfame K, stevia, etc.; emulsifiers such as glycerin fatty acid esters, polyglycerin fatty acid esters, sorbitan fatty acid esters, lecithin, etc.; and thickeners (stabilizers) such as agar, gelatin, carrageenan, guar gum, xanthan gum, pectin, locust bean gum, gellan gum, carboxymethylcellulose, soybean polysaccharides, propylene glycol alginate, etc. Other ingredients that can be added include vitamins such as vitamin A, B vitamins, vitamin C, and vitamin E; minerals such as calcium, magnesium, zinc, iron, and manganese; acidulants such as citric acid, lactic acid, acetic acid, malic acid, tartaric acid, and gluconic acid; milk fats such as cream, butter, and sour cream; flavors such as yogurt, berry, orange, quince, perilla, citrus, apple, mint, grape, apricot, pear, custard cream, peach, melon, banana, tropical, herbal, black tea, and coffee; herbal extracts; and brown sugar extracts.

[0040] In the production of fermented milk foods and drinks, microorganisms other than the bacterium of the present invention can also be used in combination. Examples of such microorganisms include bacteria of the genus Lacticaseibacillus, such as Lacticaseibacillus paracasei, Lactobacillus casei, and the like. casei), Lactobacillus acidophilus (L. acidophilus), Lactobacillus plantarum (L. plantarum), Lactobacillus buchneri (L. buchneri), Lactobacillus gallinarum (L. gallinarum), Lactobacillus amylovorus (L. amylovorus), Lactobacillus brevis (L. brevis), Lactobacillus rhamnosus (L. rhamnosus), Lactobacillus kefir (L. kefir), Lactobacillus Lactobacillus paracasei (L. paracasei), Lactobacillus crispatus (L. crispatus), Lactobacillus zeae (L. zeae), Lactobacillus helveticus (L. helveticus), Lactobacillus salivarius (L. salivarius), Lactobacillus gasseri (L. gasseri), Lactobacillus fermentum (L. fermentum), Lactobacillus reuteri (L. reuteri), Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus), Lactobacillus delbrueckii subsp. Lactobacillus bacteria such as L. delbrueckii subsp. delbrueckii and L. johnsonii, Streptococcus bacteria such as Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis. Lactococcus bacteria such as Lactococcus lactis subsp. cremoris, Enterococcus faecalis,Examples of suitable bacteria include Enterococcus bacteria such as Enterococcus faecium, Bacillus bacteria such as Bacillus subtilis, and yeasts belonging to the genera Saccharomyces, Torulaspora, and Candida, such as Saccharomyces cerevisiae, Torulaspora delbrueckii, and Candida kefir. Fermented milk foods and beverages are preferably produced using the bacteria of the present invention in combination with one or more bacteria selected from the group consisting of Lacticaseibacillus, Lactobacillus, Streptococcus, and Lactococcus, as these bacteria are highly palatable and easy to eat and drink.

[0041] Although there is no strict limit to the dosage when using the bacterium of the present invention, a suitable dosage is 10 viable cells per day. ５ cfu ~ 10 １３ cfu, especially 10 ８ cfu ~ 10 １２ The subjects for which the bacterium of the present invention is used are not particularly limited, but it is particularly suitable for use in infants and young children.

[0042] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples in any way.

[0043] Reference Example 1: Purification and Analysis of Human Milk Oligosaccharides. Four volumes of chloroform / methanol (2:1 V / V) were added to human breast milk, and the mixture was thoroughly stirred and centrifuged (3,000 rpm, 30 minutes). The supernatant was collected, and chloroform / methanol was added to the lower layer again, followed by the same procedure. The solvent was removed from the two supernatants using a rotary evaporator. The dried sugar fraction was dissolved in 70% ethanol and allowed to stand overnight at 4°C. The precipitated lactose was removed by filtration, and the solution was collected and concentrated. The concentrated solution was subjected to a gel filtration column to remove the lactose fraction and void fraction (acidic oligosaccharides). The ABEE method was used to analyze human milk oligosaccharides (HMOs). First, the sample was treated with chloroform / methanol to remove proteins and lipids. The treated sample was mixed with ABEE reagent and reacted at 80°C for 45 minutes. After diethyl ether extraction to remove the ABEE reagent, quantification was performed by HPLC under the following measurement conditions. Instrument: Shimadzu Prominence (Shimadzu Corporation) Solvent: 100 mM ammonium acetate buffer (pH 4.5): acetonitrile = 87:13 Column: L-column 2 (Chemicals Evaluation and Research Institute, Japan) Analysis time: 55 min Flow rate: 1 mL / min (0-32.5 min) → 2 mL / min (33-55 min) Temperature: 40°C Internal standard: 1 mM arabinose

[0044] The HPLC chromatogram is shown in Figure 1. An oligosaccharide fraction containing 6.1 mM 2'-fucosyllactose (2'-FL), 13.4 mM 3-fucosyllactose (3-FL), 4.1 mM difucosyllactose (DFL), 25.0 mM lacto-N-fucopentaose I (LNFPI), and 4.1 mM lacto-N-difucohexaose I (LNDFHI) was obtained.

[0045] Example 1 Selection of Bifidobacterium bacteria capable of assimilating breast milk oligosaccharides and xylooligosaccharides <Method> (1) Strains used: 26 strains of Bifidobacterium longum subsp. infantis (hereinafter referred to as Bi-01 to Bi-26) isolated from human infants and Bifidobacterium infantis YIT 12734 as the type strain. T (ATCC15697, hereafter YIT 12734 T ) was used.

[0046] (2) Analysis of carbohydrate utilization: 2 μL of a bacterial suspension prepared by suspending colonies cultured on GAM agar medium for 2 days in physiological saline was added to 40 μL of ILS medium prepared so that the final concentration of each of the following carbohydrates was 0.25% by mass, and the mixture was overlaid with 50 μL of mineral oil and cultured at 37° C. under anaerobic conditions for 100 hours. OD600 was measured every 30 minutes using a microplate reader (BioTek). 2'-Fucosyllactose (2'-FL, Advanced Protein Technologies) 3-Fucosyllactose (3-FL, Advanced Protein Technologies) 3'-Sialyllactose (3'-SL, Sigma-Aldrich) 6'-Sialyllactose (6'-SL, Sigma-Aldrich) Difucosyllactose (DFL, OligoTech) Lacto-N-fucopentaose I (LNFPI, Dextra Laboratories) Lacto-N-difucohexaose I (LNDFHI, Iso Sep) Lacto-N-tetraose (LNT, Iso Sep) Xylooligosaccharides (XOS, Bussan Food Science Co., Ltd.)

[0047] (3) Comparative genome analysis and phylogenetic analysis To construct the pangenome of Bifidobacterium infantis, the genome sequences of a total of 27 strains used were annotated using Prokka. The output GFF3 file was submitted to Roary, and a pangenome was created by classifying genes with sequence identity of 95% or more as identical genes. The number of core genes possessed by all strains was 1,338, and the number of accessory genes possessed only by some strains was 3,500. The pangenome sequence was submitted to eggNOGmapper (http: / / eggnog-mapper.embl.de / ) for KO annotation. A phylogenetic tree was created using UPGMA using MEGA-6, using SNP sequence information from the core genome. ARG-ANNOT (version ARG-ANNOT NT V6) (https: / / ifr48.timone.univ-mrs.fr / blast / arg-annot_nt.html) was used to search for drug resistance genes, and PHASTER (https: / / phaster.ca / ) was used to search for phage sequences. A drug resistance gene or phage sequence was determined to be present when a nucleotide sequence with 95% or more sequence identity to a drug resistance gene or phage sequence listed in the database was found.

[0048] <Results> (1) Comparative genome analysis of strains Comparative genome analysis was performed on 26 strains (Bi-01 to Bi-26). The total base count of the 26 strains was 2.61±0.05 Mbp, the number of CDS (Coding Sequences) was 2,247±85, and the number of phage was 2.61±0.05 (Table 1).

[0049]

[0050] (i) Distribution of antibiotic resistance genes Of the 26 strains used (Bi-01 to Bi-26), 14 strains carried the ermX gene, a resistance gene for macrolide antibiotics (Table 1). In addition, some of the strains carrying ermX also carried the aminoglycoside antibiotic resistance gene (rmtF) and the tetracycline resistance gene (tetR) (Table 1). No drug resistance genes other than the three genes listed in Table 1 were detected in any of the strains. Furthermore, no drug resistance genes were detected in the reference strain.

[0051] (ii) The distribution of genes involved in HMO utilization is shown in Figure 2. All 26 strains used (Bi-01 to Bi-26) possessed a gene encoding the ABC transporter for fucosyllactose (FL). All strains also possessed a gene involved in the uptake of lacto-N-tetraose (LNT). However, it was thought that there existed a strain (LNT_A) that directly incorporated LNT as a tetrasaccharide into the bacteria, and a strain (LNT_B) that decomposed LNT into lacto-N-biose (LNB) and lactose outside the bacteria using lacto-N-biosidase (GH136). The gene region predicted to be involved in the utilization of long-chain fucosylated HMOs (approximately 5-7 sugars) (the region corresponding to the type strain's Gene IDs: Blon_2331 to Blon_2361) contained genes encoding five glycolytic enzymes (β-galactosidase (Gh2), α-L-fucosidase (GH95), α-L-fucosidase (GH29), sialidase (GH33), and β-hexosaminidase (GH20)) and seven substrate-binding proteins (SBPs) of ABC transporters. The genes encoding the five glycolytic enzymes were conserved among all strains, including the type strain, but the number of genes encoding substrate-binding proteins varied among the strains. Other genes were present in all strains, while some were present only in some strains. The gene region predicted to be involved in sialyloligosaccharide utilization (the gene region corresponding to Gene ID: Blon_0642 to Blon_0651 of the reference strain) was conserved in all strains except for one strain (Bi-04).

[0052] (iii) The results of the utilization of plant-derived polysaccharides and the distribution of genes involved in the utilization are shown in Figure 2. Of the 26 strains used (Bi-01 to Bi-26), 12 strains possessed the GH43 gene, which is predicted to be involved in the utilization of plant-derived polysaccharides. Of these, seven strains possessed GH43_subfamily 11, which has been reported as a xylosidase (Saito, Y. et al., Appl Environ Microbiol, 2020. 86(24)), six strains possessed GH43_subfamily 34, which has been reported as an arabinofuranosidase (Sakka, M. et al., Enzyme Microb Technol, 2019. 124: 23-31), and four strains possessed GH43_subfamily 22, which has been reported as an arabinofuranosidase (Sakka, M. et al., Enzyme Microb Technol, 2019. 124: 23-31). On the other hand, the type strain did not possess GH43_subfamily 11, which has been reported as a xylosidase.

[0053] (2) Sugar utilization (i) The results of HMO utilization are shown in Figure 3. In a medium containing the neutral oligosaccharide fraction purified from breast milk in Reference Example 1 as the sole sugar source, all 26 strains used (Bi-01 to Bi-26) and all of the type strains showed growth. After 48 hours of culture, LNDFHI was found to remain in the culture medium of Bi-06, but no remaining sugars were found in the culture medium of the other strains.

[0054] (ii) Growth results for each HMO molecular species medium are shown in Table 2, along with the presence or absence of genes involved in HMO utilization in the genome. All 26 strains (Bi-01 to Bi-26) grew in media containing 2'-FL, 3-FL, DFL, LNT, LNFPI, and LNDFHI, each of which is a neutral HMO, as the sole sugar source. All strains except Bi-22 and Bi-23 grew in media containing LNFP as the sole sugar source. Two strains (Bi-08 and Bi-04) did not grow in media containing 3'-SL, an acidic HMO. Three strains (Bi-08, Bi-04, and Bi-01) did not grow in media containing 6'-SL as the sugar source, and some strains showed weak growth.

[0055] (iii) The results of plant-derived polysaccharide utilization, along with the presence or absence of genes involved in xylooligosaccharide utilization in the genome, are shown in Table 2. In a medium containing xylooligosaccharide, a plant-derived polysaccharide, as the sole sugar source, 7 of the 26 strains used (Bi-01 to Bi-26) showed growth.

[0056]

[0057] As is clear from the above results, the 26 strains of Bifidobacterium longum subsp. infantis isolated from infants varied in their utilization of HMOs and xylooligosaccharides, and the presence or absence of drug resistance genes varied. Among these, Bi-15, Bi-16, Bi-17, and Bi-18 exhibited high utilization of carbohydrates, including HMOs, but did not possess drug resistance genes. These strains exhibited ANI values ​​ranging from 99.997% (lowest) to 99.9992% (highest), indicating that they are closely related. These strains can utilize a variety of HMOs in the intestine during lactation and can also utilize xylooligosaccharides, a plant-derived polysaccharide ingested after weaning, as a substrate. Therefore, these strains are expected to produce organic acids and exert physiological effects in the intestine of infants and young children regardless of age, and because they do not possess drug resistance genes, they are safer in that they do not transmit drug resistance genes to other intestinal bacteria. In particular, Bi-16 and Bi-17 have higher HMO utilization, and Bi-17 has superior growth in HMO medium.

[0058] Example 2 Primers specific to Bi-17 and closely related strains <Method> (1) Strains used Bifidobacterium longum subsp. infantis Bi-01 to Bi05, Bi-15 to Bi18 obtained in Example 1, the type strain, and other Bifidobacterium longum subsp. infantis strains were used.

[0059] (2) PCR The PCR reaction solution used TB Green® Premix Ex Taq® II (Takara Bio Inc.) as the enzyme, with a primer concentration of 0.4 μM. The TB Green Premix EX Taq II and ROX Reference Dye II concentrations were prepared according to the manufacturer's instructions. The reaction solution was subjected to PCR using an Applied Biosystems® 7500 real-time PCR system, with initial denaturation at 95°C for 30 seconds followed by 40 cycles of 95°C for 5 seconds and then 60°C for 34 seconds. To confirm the length of the amplified fragments, the amplified products were electrophoresed on a 1.5% agarose gel in TAE buffer at 85 V for 60 minutes.

[0060] (3) Results The results are shown in Figure 4. By using a primer consisting of the base sequence shown in SEQ ID NO: 2 and a primer consisting of the base sequence shown in SEQ ID NO: 3, Bi-17 and related strains could be specifically detected.

Claims

1. A bacterium belonging to Bifidobacterium longum subsp. infantis that has the ability to assimilate human breast milk oligosaccharides and xylooligosaccharides and does not have drug resistance genes.

2. The bacterium described in claim 1, which has the ability to assimilate at least one human breast milk oligosaccharide selected from the group consisting of fucosyllactoses, lacto-N-tetraose, human neutral breast milk oligosaccharides of 5 to 10 sugars, and sialyllactoses.

3. The bacterium described in claim 2, wherein the fucosyllactoses are 2'-fucosyllactose, 3-fucosyllactose and difucosyllactose, the 5-10 sugar human neutral breast milk oligosaccharides are lacto-N-fucopentaose I and lacto-N-difucohexaose I, and the sialyllactoses are 3'-sialyllactose and 6'-sialyllactose.

4. Bifidobacterium longum subsp. infantis YIT 13155 (NITE BP-04137) or a strain closely related thereto.

5. A food or drink containing the bacterium described in any one of claims 1 to 4.

6. The food or drink according to claim 5, further comprising at least one selected from the group consisting of human breast milk oligosaccharides and xylooligosaccharides.

7. A pharmaceutical containing the bacterium according to any one of claims 1 to 4.

8. The pharmaceutical product according to claim 7, further comprising at least one selected from the group consisting of human breast milk oligosaccharides and xylooligosaccharides.

9. A primer pair consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 2 and an oligonucleotide having the base sequence shown in SEQ ID NO: 3, or a primer pair consisting of complementary sequences corresponding to the base sequences.

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