Agent and method for inhibiting the growth of Streptococcus anginosus

Abstract

The present invention provides a technology capable of effectively inhibiting the growth of Streptococcus anginosus. According to the present invention, the growth of Streptococcus anginosus can be inhibited in vivo. Furthermore, according to the present invention, by inhibiting the growth of Streptococcus anginosus in vivo, it is possible to contribute to the prevention or improvement of various diseases and unhealthy conditions in which this bacterium is involved in the onset or exacerbation of the disease, or in which an increase in this bacterium serves as an indicator of the onset or exacerbation of the disease. SOLUTION: A growth inhibitor for Streptococcus anginosus, which contains agarooligosaccharide as an active ingredient.

Description

[Technical Field]

[0001] The present invention relates to an agent and method for inhibiting the growth of Streptococcus anginosus. [Background technology]

[0002] Streptococcus anginosus is a Gram-positive, non-spore-forming, non-motile, facultative anaerobic coccus belonging to the genus Streptococcus. This bacterium is normally present in the body, such as the oral cavity, nasopharynx, gastrointestinal tract, and urogenital tract, and can occasionally become a pathogen causing oral and head and neck infections, central nervous system infections such as brain abscesses, bacteremia, infective endocarditis, intraperitoneal infections, pneumonia, empyema, skin and soft tissue infections, and osteomyelitis (Non-Patent Document 1).

[0003] It has also been reported that Streptococcus anginosus is a pathogen of aerobic vaginitis (Non-Patent Document 2) and a pathogen that promotes gastritis and gastric cancer (Non-Patent Document 3). Furthermore, in recent years, it has been shown that Streptococcus anginosus is found in significantly higher amounts in the feces of patients with pancreatic ductal carcinoma than in the feces of control subjects, making it useful as a marker for detecting pancreatic cancer (Non-Patent Documents 4 and 5). The present inventors have also reported that this bacterium is found in significantly higher amounts in the feces of patients with hepatocellular carcinoma than in healthy individuals (Non-Patent Document 6). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kameda General Hospital, Department of Infectious Diseases, Home > Microbiology Round, Posted: June 29, 2023, [online] [Searched October 23, 2024], Internet<URL:https: / / www.kameda.com / pr / infectious_disease / post_291.html> [Non-patent document 2] Zhi Tao et al., The Pathogenesis Of Streptococcus anginosus In Aerobic Vaginitis, Infection and Drug Resistance 2019:12 3745?3754 [Non-patent document 3] Kaili Fu et al., Streptococcus anginosus promotes gastric inflammation, atrophy, and tumorigenesis in mice, Cell 187(4), 2024 Feb 15, p.882-896, doi: 10.1016 / j.cell.2024.01.004. [Non-patent document 4] National Center for Global Health and Medicine, Home > Latest Information > Latest News > Discovery of Oral and Intestinal Bacterial Species Associated with Pancreatic Cancer and Prognosis: Possibility of New Tumor Markers for Early Detection of Pancreatic Cancer, April 19, 2022, Tokyo Medical University, National Center for Global Health and Medicine, European Molecular Biology Laboratory [online] [Retrieved October 23, 2024], Internet<URL:https: / / www.ncgm.go.jp / pressrelease / 2022 / 20220419.html> [Non-Patent Document 5] Nagata N, Nishijima S, Kojima Y et al, Metagenomic Identification of Microbial Signatures Predicting Pancreatic Cancer From a Multinational Study. Gastroenterology Vol.163 No.1, July 2022, p.222-238 [Non-patent document 6] Tadashi Fujii, Teiji Kuzuya, Nobuhiro Kondo, Kohei Funasaka, Eizaburo Ohno, Yoshiki Hirooka and Takumi Tochio. Altered intestinal Streptococcus anginosus and 5α-reductase gene levels in patients with hepatocellular carcinoma and elevated Bacteroides stercoris in atezolizumab / bevacizumab non-responders. Journal of Medical Microbiology 2024;73:001878. DOI 10.1099 / jmm.0.001878. Published 06 September 2024 Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, Streptococcus anginosus is a normal inhabitant of the body, but can also be considered a pathogen involved in the onset or worsening of various diseases and unhealthy conditions, such as purulent infections, aerobic vaginitis, gastritis, gastric cancer, etc. Therefore, the present inventors believed that if the proliferation of this bacterium could be inhibited in the body, it would be possible to contribute to the prevention or improvement of diseases and unhealthy conditions in which this bacterium is involved in the onset or worsening of these conditions.

[0006] Furthermore, as mentioned above, Streptococcus anginosus is abundant in the feces of patients with pancreatic ductal carcinoma and hepatocellular carcinoma, and this bacterium can be considered an indicator of these cancers. Based on this, the present inventors believed that if the number of this bacterium in the body could be suppressed, it could contribute to the prevention and improvement of pancreatic cancer and hepatocellular carcinoma.

[0007] That is, an object of the present invention is to provide a technique that can effectively inhibit the growth of Streptococcus anginosus. Another object of the present invention is to provide a technique that can contribute to the prevention or improvement of various diseases and unhealthy conditions in which Streptococcus anginosus is involved in the onset or exacerbation of the disease, or in which an increase in the bacterium serves as an indicator of the onset or exacerbation of the disease. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors have found that agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having 3,6-anhydro-L-galactose at the reducing end (hereinafter, these may be collectively referred to as the "active ingredient" or any one or more of these may be referred to as the "active ingredient") can inhibit the growth of Streptococcus anginosus and reduce its presence in bacterial flora. Based on this finding, the present inventors have completed the following inventions.

[0009] (1) A first aspect of the growth inhibitor for Streptococcus anginosus according to the present invention (sometimes referred to as "the agent") contains agarooligosaccharide as an active ingredient.

[0010] (2) The agent may be used to prevent or ameliorate aerobic vaginitis. That is, the present invention also provides an agent for preventing or ameliorating aerobic vaginitis, which contains, as an active ingredient, one or more selected from agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having agarooligosaccharides at their reducing ends.

[0011] (3) The agent may be used to prevent or ameliorate gastritis or gastric cancer. That is, the present invention also provides an agent for preventing or ameliorating gastritis or gastric cancer, which contains as an active ingredient one or more selected from agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having agarooligosaccharides at their reducing ends.

[0012] (4) The agent may be used for preventing or ameliorating pancreatic cancer. That is, the present invention also provides an agent for preventing or ameliorating pancreatic cancer, which comprises, as an active ingredient, one or more selected from agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having agarooligosaccharides at their reducing ends.

[0013] (5) In the present invention, the agarooligosaccharide may contain agarobiose.

[0014] (6) A second embodiment of the present agent contains 3,6-anhydro-L-galactose or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end as an active ingredient.

[0015] (7) The method of the present invention for inhibiting the growth of Streptococcus anginosus (sometimes referred to as "the method") comprises the step of administering to a human or animal one or more selected from agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having agarooligosaccharides at their reducing ends.

[0016] (8) The present invention may also provide a method for preventing or ameliorating aerobic vaginitis by inhibiting the growth of Streptococcus anginosus in a living body. That is, the present invention also provides a method for preventing or ameliorating aerobic vaginitis, comprising the step of administering to a human or animal one or more selected from agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having agarooligosaccharides at their reducing ends.

[0017] (9) The present invention may also provide a method for preventing or ameliorating gastritis or gastric cancer by inhibiting the growth of Streptococcus anginosus in a living body. That is, the present invention also provides a method for preventing or ameliorating gastritis or gastric cancer, comprising the step of administering to a human or animal one or more selected from agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having agarooligosaccharides at their reducing ends.

[0018] (10) The present invention may also provide a method for preventing or ameliorating pancreatic cancer by inhibiting the growth of Streptococcus anginosus in a living body. That is, the present invention also provides a method for preventing or ameliorating pancreatic cancer, comprising the step of administering to a human or animal one or more selected from agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having agarooligosaccharides at their reducing ends.

[0019] The present invention may be practiced outside of medical practice. [Effects of the Invention]

[0020] According to the present invention, the growth of Streptococcus anginosus can be inhibited in vivo. Furthermore, by inhibiting the growth of Streptococcus anginosus in vivo, the present invention can contribute to the prevention or improvement of various diseases and unhealthy conditions in which this bacterium is involved in the onset or exacerbation of the disease, or in which an increase in this bacterium serves as an indicator of the onset or exacerbation of the disease.

[0021] Furthermore, the agarooligosaccharides used as active ingredients in the present invention are oligosaccharides derived from agar, which has been consumed as a food since ancient times, and are extremely safe. Therefore, according to the present invention, the growth of Streptococcus anginosus can be inhibited without concerns about safety or side effects. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a bar graph showing the turbidity (OD660) of the culture medium in which Streptococcus anginosus was cultured in the presence of sucrose (Suc) or agarooligosaccharides (AOS). In the figure, the plots show the measured values ​​for each sample. [Figure 2]This table shows the 20 microbial strains that make up the Human Resident Microbial DNA Cocktail (product name "DNA-Mock-003", lot 240101ND, NBRC). This table was taken from the product data sheet (National Institute of Technology and Evaluation, HOME > Biotechnology > Microorganisms and Industrial Use > Microbiome > NBRC Human Resident Microbial Cocktail, [online] [searched July 29, 2024], Internet). <URL: https: / / www.nite.go.jp / nbrc / industry / microbiome / cocktail20220113.html><URL: https: / / www.nite.go.jp / data / 000152907.pdf> ). [Figure 3] 1 is a bar graph showing the turbidity (OD660) of the culture medium obtained by co-cultivating Streptococcus anginosus and a human resident bacterial DNA cocktail in the absence (Sample 1) or presence (Sample 2) of agarooligosaccharides. In the figure, the plots show the measured values ​​for each sample. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be described in detail below.

[0024] Agarooligosaccharides are even-numbered oligosaccharides consisting of repeating units of agarobiose, a disaccharide composed of D-galactose and 3,6-anhydro-L-galactose. Examples of agarooligosaccharides include the smallest unit, the disaccharide agarobiose, the tetrasaccharide agarotetraose, the hexasaccharide agarohexaose, the octasaccharide agarooctaose, and the decasaccharide agarodecaose. In the present invention, agarooligosaccharides contain at least one of these oligosaccharides, and may consist of one type or two or more types. For example, agarooligosaccharides may consist solely of agarobiose, or may contain agarooligosaccharides other than agarobiose. In this case, the content of agarobiose in the agarooligosaccharide may be, for example, 1 to 100% by mass, 10 to 100% by mass, 20 to 100% by mass, 30 to 100% by mass, 40 to 100% by mass, or 50 to 100% by mass.

[0025] Agarooligosaccharides are oligosaccharides having 3,6-anhydro-L-galactose at the reducing end. Therefore, the active ingredient of the present invention may be 3,6-anhydro-L-galactose or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end. In this case, the number of sugars in the oligosaccharide may be, for example, 2 to 8 sugars, 2 to 10 sugars, or 2 to 12 sugars.

[0026] Agaroligosaccharides can be commercially available agarooligosaccharides (agar oligosaccharides), or can be produced by conventional methods. A typical method for producing agarooligosaccharides is, for example, a method of hydrolyzing agar. Hydrolysis can be performed using either an acid or an enzyme.

[0027] Examples of acid decomposition methods include those using solid acids as described in Japanese Patent No. 4796697, mineral acids such as sulfuric acid and hydrochloric acid, and organic acids such as acetic acid and citric acid, but any method can be used. Acid decomposition can produce an even-numbered sugar having 3,6-anhydro-L-galactopyranose at the reducing end.

[0028] Enzymatic degradation methods include degradation with α-agarase and degradation with β-agarase. As with acid degradation, α-agarase can be used to obtain even-numbered sugars having 3,6-anhydro-L-galactopyranose at the reducing end. Degradation with α-agarase can be carried out, for example, by the method described in Japanese Patent Application Publication No. H2-65789.

[0029] The agar hydrolysate may be used as agarooligosaccharides directly, or may be purified or pH-adjusted before use. Purification methods include filtration using filter paper or activated carbon. The agarooligosaccharide solution obtained by hydrolysis may be used in liquid form, or, if necessary, may be powdered by vacuum freeze-drying or other methods.

[0030] Agar is a dehydrated and dried mucilage extracted from red seaweeds such as Gelidium and Gracilaria, and contains the polysaccharides agarose and agaropectin as its main components. In addition to agar, substances containing agarose and agaropectin can also be used as raw materials for producing agarooligosaccharides. Specific examples of such substances include solutions obtained by hot water extraction of red algae from the Gelidaceae, Gracilaria, and Gracilaria families, which are the raw materials for agar. Examples of red algae from the Gelidaceae family include Acanthus nigricans, Acanthus nigricans, Acanthus nigricans, Acanthus obscurus, and Acanthus japonica. Examples of red algae from the Gracilaria family include Gracilaria gracilaria and Gracilaria sieboldii. Examples of red algae from the Gracilaria family include Acanthus gistus and Acanthus sieboldii. These red algae can be used alone or in combination of two or more.

[0031] The sugar composition of agarooligosaccharides can be confirmed by liquid chromatography, including high performance liquid chromatography, as shown in the Examples below. This allows agarooligosaccharides with a desired number of sugars, such as agarobiose only, agarotetraose only, or agarohexaose only, to be fractionated and used after adjusting the sugar composition of the agarooligosaccharides.

[0032] 3,6-Anhydro-L-galactose can be prepared using commercially available reagents or by standard methods. Examples of such methods include the method described in Japanese Patent No. 4007760. Specifically, 50 μL of 10x phosphate-buffered saline and 50 μL of 10 units / μL β-galactosidase phosphate-buffered saline solution were added to 450 μL of a 100 mM aqueous solution of agarobiose, mixed, and allowed to react at 37°C for 1 hour. 5 mL of a 1:1 mixture of 1-butanol and ethanol was added to the reaction mixture, and the mixture was centrifuged to precipitate insoluble matter. The resulting supernatant was subjected to column chromatography using a silica gel column, and the mixture was compressed at 0.3 kg / cm using a compressor with a 5:5:1 mixture of 1-butanol, ethanol, and water as the eluent. 2 The mixture is pressurized to 100°C and separated. By separating the fractions so that each fraction is 7 mL, a liquid containing highly purified 3,6-anhydro-L-galactose can be obtained, for example, in fractions 14 to 17. These fractions can be collected and evaporated to dryness under reduced pressure to obtain 3,6-anhydro-L-galactose.

[0033] "Suppressing the growth" of Streptococcus anginosus includes not only reducing the number of bacteria of this bacterium (reducing the proportion of bacteria in the bacterial flora), but also maintaining the number of bacteria at a similar level or increasing the number of bacteria but to a smaller extent than when the active ingredient of the present invention is not used. Furthermore, suppressing growth and suppressing the number of bacteria are synonymous.

[0034] Whether or not the growth of Streptococcus anginosus can be inhibited can be confirmed by a conventional method. For example, if the bacterium is isolated, the active ingredient can be added to the culture medium, and the number of bacteria can be confirmed by a turbidity method or the like, in comparison with a culture medium without the active ingredient.

[0035] Furthermore, when Streptococcus anginosus is not isolated (e.g., feces, saliva, vaginal secretions, nasal secretions, cocktail bacterial solutions containing multiple bacterial species, human intestinal models, etc.), a method can be used in which bacterial 16S rDNA is amplified by polymerase chain reaction (PCR) using the total genomic DNA of bacteria extracted from the sample as a template, and the amplified product is decoded by next-generation sequencing (NGS). The bacterial species and abundance are identified based on a 16S database, and the abundance ratio of this bacterium is determined and compared between samples administered with and without the active ingredient of the present invention. Alternatively, quantitative PCR can be performed on the total genomic DNA of the bacteria using primers specific to Streptococcus anginosus. When determining the number of this bacterium by PCR, because the amount of genes purified from the sample (purification efficiency) is not constant, it is preferable to calculate the proportion of this bacterium (abundance ratio, occupancy rate) in the total number of bacteria or the amount of template DNA and make a judgment based on that proportion.

[0036] When quantifying Streptococcus anginosus by PCR, specific primers can be designed based on a partial sequence of genomic DNA (a conserved region of this bacterium) that is conserved in this bacterium but not in closely related species, identified based on published sequence information. For example, the genome of Streptococcus anginosus DSM 20563 is publicly available under GenBank accession number AFIM01000064.1.

[0037] For example, Non-Patent Document 6 identifies a partial sequence of the DNA sequence (SEQ ID NO: 1) of the N-acetylneuraminic acid lyase (nanA) gene possessed by this bacterium as a conserved region. Then, as primers specific to this bacterium, a forward primer (SEQ ID NO: 2) corresponding to positions 425-446 in SEQ ID NO: 1 and a reverse primer (SEQ ID NO: 3) corresponding to positions 526-548 in SEQ ID NO: 1 are used (Non-Patent Document 6; METHODS, Primer design). That is, Non-Patent Document 6 quantifies Streptococcus anginosus by quantifying the partial sequence of the nanA gene. In the present invention, this bacterium can also be quantified using such specific primers.

[0038] [SEQ ID NO: 1] nanA gene (length 645 bases) derived from Streptococcus anginosus DSM 20563, Accession No.: GenBank: AFIM01000064.1 (genome): 39668-40312 (corresponding portion of nanA gene) tcatttatat ttgttaaaaa tgttctccat gataggcaat tgcaaaaatc ctaaggctgc aaagccgaca aatacaaaaa gagtaaagcc tagcagcaac acaaatcctg aactagagag gaaaaagatg attgtagcta ataaggctat tatcaaaaag aaccacggga aatgtaaact taccatgatc agtcccgttt gtaatagatc ttttaccgtc atttcaaact tagctgccaa aggatagaca cataaaaata gcaaactagc aaaaatgatg acgccgatac aggttgcttt taacatttga aatggcattg tttcatgctt ccaaaaaagc aggaggtcaa atagactgat acctacaatg ctgagttcta gcaaaccaag tttgaaaccg accttccaat ttttcctaaa tgcctgcaca taggttgctg ttacttttat gcgtcgcgaa tctctgatga caaacaaagt ttcatacaag ctaattttgg caataccaat cgtaacaagt ggtagacaag acaaaagaaa gatgaggttc acagtcacca aatctaatat tttctcacat attctcataa agaaattatc tgtatcaaat atacttcgta ttaactgcga acctctcttt tccat

[0039] <Primers for amplifying the conserved region of the nanA gene of this bacterium> [SEQ ID NO: 2] Forward primer: 5'-CGCAGTTGGCAGGTGTAGCTCT-3' [SEQ ID NO: 3] Reverse primer: 5'-CCAAGTGCTGCAAAGGTTTGAAT-3'

[0040] As mentioned above, Streptococcus anginosus has been reported to be a pathogen of aerobic vaginitis (Non-Patent Document 2). In Non-Patent Document 2, comprehensive analysis of the microbiota was conducted using vaginal secretions from women with aerobic vaginitis, bacterial vaginosis, and healthy women. The results showed that Streptococcus anginosus was almost absent in both bacterial vaginosis and healthy women, whereas this bacterium accounted for the largest proportion of women with aerobic vaginitis (Figure 6). Furthermore, when co-cultured with vaginal epithelial cells and an LDH assay was performed, this bacterium induced lysis of the epithelial cells (Figures 8 and 9).

[0041] Therefore, if the proliferation of Streptococcus anginosus in the body can be suppressed, the number of pathogens in people who may suffer from aerobic vaginitis or in patients with aerobic vaginitis can be reduced, which is thought to contribute to the prevention or amelioration of aerobic vaginitis. In other words, the active ingredient of the present invention can be used to prevent or ameliorate aerobic vaginitis.

[0042] Furthermore, as mentioned above, Streptococcus anginosus has been reported to be a pathogen that promotes gastritis and gastric cancer (Non-Patent Document 3). In Non-Patent Document 3, the presence of this bacterium in the gastric mucosa at each stage of gastric cancer development was examined, and it was found that the presence was greater in atrophic gastritis and intestinal metaplasia than in superficial gastritis, and was highest in the gastric cancer stage (Figure 1). Mice were orally administered this bacterium once every three days and infected, and acute gastritis developed in two weeks (Figure 1) and chronic gastritis in three months (Figure 2). Gastric cancer development progresses from atrophy to metaplasia and dysplasia, and gastritis is a major risk factor. Mice infected with this bacterium showed atrophy of gastric parietal cells 9 to 12 months after infection, and metaplasia and low-grade dysplasia of the gastric mucosa 12 months after infection. The bacterium was present at high densities in the areas of metaplasia and dysplasia (Figure 3). Furthermore, the expression of tight junction proteins CLDN18 and OCLN was reduced in the gastric tissue of mice infected with this bacterium, and the barrier function was impaired (Figure 4). 3) Metaplasia and decreased expression of tight junction proteins were also observed in germ-free mice infected with this bacterium, indicating that this bacterium alone can promote gastric cancer formation (Figure 4). Tumor weight significantly increased in both mice subcutaneously implanted with mouse gastric cancer cell line YTN16 and injected with this bacterium at the same site, and in mice injected with YTN16 into the gastric serosa and orally administered with this bacterium (Figure 5). Infection of a mouse model of MNU-induced gastric cancer with this bacterium also increased the number, size, and degree of dysplasia of tumors, promoting cancer progression (Figure 5). It is thought that the surface protein TMPC of this bacterium binds to the ANXA2 receptor on gastric epithelial cells, allowing this bacterium to form colonies in the gastric mucosa (Figure 6), and that this promotes cancer formation through activation of the MAPK signaling pathway (Figure 7).

[0043] Therefore, if the proliferation of Streptococcus anginosus in the body can be suppressed, the number of pathogens in people who may be susceptible to gastritis or gastric cancer or in patients with such diseases can be reduced, which is thought to contribute to the prevention or amelioration of gastritis or gastric cancer. In other words, the active ingredient of the present invention can be used to prevent or ameliorate gastritis or gastric cancer.

[0044] Furthermore, as mentioned above, it has been reported that Streptococcus anginosus can be an indicator of pancreatic cancer (Non-Patent Documents 4 and 5). In Non-Patent Document 5, saliva and fecal samples from 47 Japanese patients with pancreatic cancer and 235 controls were analyzed using shotgun metagenomic sequencing. The authors identified 18 oral bacteria and 30 intestinal bacteria (including Streptococcus anginosus) that were increased or decreased in patients with pancreatic cancer (Figure 1D). Among these, increases in the intestinal bacteria V atypica, V parvula, S anginosus, and S oralis, and decreases in F prausnitzii, were observed in cohort studies of Spanish patients with pancreatic cancer (57 patients, 50 controls) and German patients with pancreatic cancer (44 patients, 32 controls), similar to those observed in Japanese patients (Figure 4B). Furthermore, the presence of S anginosus was significantly increased in patients with intraductal papillary mucinous neoplasms (IPMN), a risk factor for pancreatic cancer, compared with controls (Figure 3B).

[0045] Therefore, if the proliferation of Streptococcus anginosus in the body can be suppressed, the number of indicator microorganisms for pancreatic cancer in people who may be susceptible to pancreatic cancer or in patients with pancreatic cancer can be reduced, which is thought to contribute to the prevention or amelioration of pancreatic cancer. In other words, the active ingredient of the present invention can be used to prevent or ameliorate pancreatic cancer.

[0046] The active ingredient of the present invention can be used, for example, in a form to be administered to humans or animals. More specific modes of use can be appropriately determined depending on the recipient, application site, purpose of use, etc. For example, the administration method and route may be any as long as the active ingredient reaches the site where Streptococcus anginosus inhabits. Specific examples include oral ingestion, nasal drip, placement under the tongue (sublingually) or between the gums and cheek, and insertion into the rectum (transrectally) or vagina (transvaginally).

[0047] The dosage of the active ingredient can also be appropriately determined depending on the recipient and site of administration, the form of the product, the intended use, etc. Specific examples of dosages include, for example, 0.01 mg / kg body weight or more, 0.1 mg / kg body weight or more, 1 mg / kg body weight or more, 5 mg / kg body weight or more, 10 mg / kg body weight or more, 1000 mg / kg body weight or less, 800 mg / kg body weight or less, 600 mg / kg body weight or less, 400 mg / kg body weight or less, 200 mg / kg body weight or less, 100 mg / kg body weight or less, and 10 mg / kg body weight or less per day for an adult.

[0048] The active ingredient may be used as it is in the form of food and drink, supplements, pharmaceuticals, quasi-drugs, feed, etc., or may be used by blending it with other ingredients as an ingredient in food and drink, supplements, pharmaceuticals, quasi-drugs, feed, etc. These products can be produced by methods known to those skilled in the art using the active ingredient as a raw material.

[0049] The content of the active ingredient in the product can also be appropriately set depending on the form and use of the product. Specific examples of the content include 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 35% by mass or less, 30% by mass or less, 10% by mass or less, and 5% by mass or less.

[0050] The present invention will be described below based on examples, but the technical scope of the present invention is not limited to the features shown in these examples. [Example]

[0051] Example 1: Preparation of agarooligosaccharides 50 g of agar ("Ultra Agar AX-30" manufactured by Ina Food Industry Co., Ltd.) was added to 1000 g of purified water and heated to dissolve. 2 g of concentrated sulfuric acid was then added and stirred at 90°C for 3 hours. The pH was adjusted to 3.5 with sodium hydroxide, and the mixture was treated with activated carbon. The filtrate was then filtered through a filter paper and collected. This was then further filtered through a 0.1 μm pore filter, and the collected filtrate was then powdered by vacuum freeze-drying to obtain agarooligosaccharide powder.

[0052] The composition of the prepared agarooligosaccharides was measured using high-performance liquid chromatography (Prominence® HPLC system (Shimadzu Corporation)). Measurement conditions were as follows: two columns (TSKgel® α-2500, Tosoh Corporation) connected in series, elution with HO as solvent, a flow rate of 0.3 mL / min, and a temperature of 60°C, and detection was by RI (differential refractive index). The results were as follows. In this example, a composition containing the following disaccharides to decasaccharides is referred to as "agarooligosaccharides (AOS)." Disaccharide (agarobiose): 31.5% by mass Tetrasaccharide (agarotetraose): 30.1% by mass Hexasaccharide (agarohexaose): 21.2% by mass Octasaccharide (agarooctaose): 11.6% by mass Decasaccharide (agarodecaose): 5.6% by mass

[0053] Example 2: Growth inhibitory effect of Streptococcus anginosus: Evaluation by turbidity (1)Culture conditions etc. The culture medium used was RF medium*, a modified version of Brain-Heart Infusion medium (Thermo Scientific). Anaerobic culture was performed using the Anaeropack anaerobic culture kit (Mitsubishi Gas Chemical) by static culture at 37°C. *Composition of RF medium: 1L Brain-Heart Infusion medium, 5g yeast extract, 5g K2HPO4, 8g glucose, 0.5g L-cysteine ​​hydrochloride, 1g Tween 80, 0.005g hemin, 0.002g vitamin K1, 0.001g resazurin sodium, 0.025g acetate, 0.01g MgSO2·7H2O, pH 6.8.

[0054] (2) Cultivation in the presence of agarooligosaccharides Streptococcus anginosus JCM 12993 (Microbial Materials Development Laboratory, BioResource Research Center, RIKEN) (sometimes referred to as the "SA strain") was anaerobically cultured in RF medium for 23.5 hours, and this was used as the seed culture medium. Two types of medium were prepared as main culture media: RF medium supplemented with sucrose to a final concentration of 0.5% by mass (Sample 1), and RF medium supplemented with agarooligosaccharides to a final concentration of 0.5% by mass (Sample 2). 0.4 mL of the main culture medium was dispensed into deep well plates (AxyGen Scientific, CA, USA), and 20 μL of a 10-fold diluted seed culture medium was inoculated into each well and cultured anaerobically for 44 hours (main culture). Hereinafter, the culture solutions obtained by main culture using Sample 1 and Sample 2 are referred to as Sample 1 and Sample 2, respectively.

[0055] (3) Measurement of bacterial count by turbidity method After the main culture, 20 μL of the culture medium was sampled and diluted 10-fold by adding 180 μL of water. The light transmittance of the diluted culture medium at a wavelength of 660 nm was measured using a microplate reader (Wako SUNRISE Rainbow) to calculate the turbidity (OD660) (primary measurement value). The RF medium was also diluted 10-fold and measured in the same way to calculate the turbidity (diluted medium turbidity). The diluted medium turbidity was subtracted from the primary measurement value, and the result was multiplied by 10 to obtain the turbidity of the culture medium. The turbidity was calculated as the average value of eight samples for each sample. Statistical analysis between groups was performed using the Mann-Whitney test using medical statistical analysis software GraphPad Prism (GraphPad Software), with a P value of <0.05 indicating a significant difference. The results are shown in Figure 1.

[0056] As shown in Figure 1, the turbidity (OD660) of the culture solution was 2.09 for Sample 1 (main culture in a sucrose-containing medium), while it was 0.10 for Sample 2 (main culture in a medium containing 0.5% AOS), which was significantly lower than Sample 1. In other words, the turbidity of the Streptococcus anginosus culture solution decreased in the presence of agarooligosaccharides. These results demonstrated that agarooligosaccharides can inhibit the growth of Streptococcus anginosus.

[0057] Example 3: Growth inhibitory effect of Streptococcus anginosus: Evaluation based on abundance ratio (1) Cultivation in the presence of agarooligosaccharides Culture conditions were as described in Example 2(1). The SA strain was anaerobically cultured in RF medium for 23.5 hours, and this was used as the SA seed mother liquor. A human commensal bacterial DNA cocktail (product name "DNA-Mock-003", lot 240101ND, National Biotechnology Center (NBRC), National Institute of Technology and Evaluation) (sometimes referred to as the "cocktail strain") was anaerobically cultured in RF medium for 23.5 hours, and this was used as the cocktail seed mother liquor. The cocktail strains were a mixture of 20 strains shown in Figure 2 from the microbial strains held by the NBRC, with each strain containing an equal number of copies of genomic DNA. The 20 strains are species known to inhabit the human intestine, oral cavity, skin, etc. Shotgun sequencing of the cocktail strains confirmed that each strain was detected at a detection rate of approximately 5% (Tourlousse, DM, Narita, K., Miura, T. et al. Characterization and demonstration of mock communities as control reagents for accurate human microbiome community measurements. Microbiology Spectrum, 10(2): e01915-21.).

[0058] The SA seed mother liquor and cocktail seed mother liquor were each diluted 10-fold and mixed in equal amounts to prepare a mixed seed mother liquor. Two types of medium were prepared as main culture media: RF medium (Sample 1) and RF medium supplemented with agarooligosaccharides to a final concentration of 0.5% by mass (Sample 2). Each main culture medium was dispensed into deep well plates (AxyGen Scientific, CA, USA) at 0.4 mL per well, after which 20 μL of the mixed seed mother liquor was inoculated and cultured anaerobically for 23.5 hours (main culture). Hereafter, the culture solutions obtained by main culture using Sample 1 and Sample 2 are referred to as Sample 1 and Sample 2, respectively.

[0059] (2) Measurement of bacterial count by turbidity method The turbidity (OD660) of the culture solution after the main culture was measured by the method described in Example 2(3). The average turbidity was calculated by averaging four specimens for each sample. The results are shown in Figure 3. As shown in Figure 3, the turbidity (OD660) of the culture solution was 3.19 for Sample 1 (main culture in RF medium) and 1.86 for Sample 2 (main culture in AOS-containing medium), and there was no significant difference between the two (ns). In other words, there was no significant difference in the total number of bacteria in the presence of agarooligosaccharides compared to their absence.

[0060] (3) Comprehensive analysis of bacterial flora After the main culture, the culture medium was diluted 10-fold, and a 0.5 mL aliquot was incubated at 70°C for 10 minutes. The samples were then disrupted using zirconia beads at 4,300 rpm for 2 minutes in a FastPrep FP100A device (MP Biomedicals). The mixture was centrifuged at 15,000 rpm for 1 minute, and the supernatant was collected and used as total bacterial DNA. Using total bacterial DNA as a template, PCR was performed using the universal primers SEQ ID NOS: 4 and 5 to amplify the V3-V4 region of bacterial 16S rDNA (Takahashi S, et al., (2014) Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing. PLoS ONE 9(8): e105592. Published: August 21, 2014). Forward primer (Pro341F): 5'-CCTACGGGNBGCASCAG-3' (SEQ ID NO: 4) Reverse primer (Pro805R): 5'-GACTACNVGGGTATCTAATCC-3' (SEQ ID NO: 5)

[0061] The PCR-amplified products were then sequenced by next-generation sequencing (NGS). NGS was performed using the Illumina MiSeq platform (Illumina) and MiSeq Reagent Kit ver. 3 (Illumina) using a paired-end method (2 × 300 bp). The sequences were analyzed using the EzBioCloud 16S database and the 16S Microbiome Pipeline (EzBioCloud 16S-based MTP app, https: / / www.EZbiocloud.net) to identify species and determine their abundance (occupancy). The abundance was calculated as the percentage of the number of reads for each bacterial species relative to the total number of reads. This NGS analysis was performed by Seibu Giken Co., Ltd.

[0062] As a result, the abundance ratio of Streptococcus anginosus was 14.69% in Sample 1 (main culture in RF medium), while it was 0.00% in Sample 2 (main culture in AOS-containing medium). In other words, in the presence of agarooligosaccharides, the abundance ratio of Streptococcus anginosus decreased to almost 0. These results demonstrate that agarooligosaccharides can inhibit the growth of Streptococcus anginosus even in a bacterial flora environment where a considerable amount of multiple other bacterial species are present.

Claims

1. A growth inhibitor of Streptococcus anginosus, containing agarooligosaccharide as the active ingredient.

2. The agent according to claim 1, which is used to prevent or improve aerobic vaginitis.

3. 2. The agent according to claim 1, which is used to prevent or ameliorate gastritis or gastric cancer.

4. The agent according to claim 1, wherein the agarooligosaccharide is an agarooligosaccharide containing agarobiose.

5. A method for inhibiting the growth of Streptococcus anginosus in a living body (excluding medical procedures), comprising the step of administering agarooligosaccharides to a human or animal.

6. 6. The method according to claim 5, which is a method for preventing or ameliorating aerobic vaginitis.

7. The method according to claim 5, which is a method for preventing or ameliorating gastritis or gastric cancer.

8. An agent according to any one of claims 1 to 4 selected from food, beverages, supplements, pharmaceuticals, quasi-drugs and feed.

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