Bacterial flora-improving agent, antibacterial agent, method for improving bacterial flora, and method for suppressing number of bacteria

Abstract

[Problem] To provide: a technique capable of reducing harmful bacteria without reducing beneficial bacteria; and a bacterial flora-improving agent and an antibacterial agent which use the same. According to the present invention, the number of harmful bacteria can be suppressed without suppressing the number of beneficial bacteria. Therefore, according to the present invention, bacterial flora can be effectively improved. Moreover, this antibacterial agent does not reduce beneficial bacteria, and thus the reduction of side effects such as diarrhea due to a reduction in the beneficial bacteria can be expected. [Solution] This bacterial flora-improving agent is characterized by containing an agaro-oligosaccharide as an active ingredient, wherein the active ingredient is used in one used amount among (a)-(c) below: (a) a used amount which does not suppress the number of bacteria belonging to the genus Bifidobacterium; (b) a used amount which does not suppress the number of bacteria belonging to the genus Lactobacillus; and (c) a used amount of at least 80 mg and less than 12,000 mg per day.

Description

Microbial flora improving agent, antibacterial agent, method for improving microbiota and method for suppressing bacterial count

[0001] The present invention relates to a bacterial flora improver, an antibacterial agent, a method for improving bacterial flora, and a method for suppressing bacterial count, which are characterized by using agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having 3,6-anhydro-L-galactose at the reducing end in amounts that do not suppress the bacterial count of Bifidobacterium and / or Lactobacillus.

[0002] Humans normally coexist with microorganisms that live in groups in specific areas of the body, such as the skin, nose, mouth, throat, large intestine, and vagina. The groups of microorganisms that exist in these specific environments are called "microbiota." The body's microbiota is deeply involved in the host's health, and it is known that maintaining a healthy microbiota leads to the maintenance and improvement of health.

[0003] Therefore, there is a demand for substances that improve the bacterial flora. For example, substances that are utilized by intestinal bacteria to improve the bacterial flora are called prebiotics, and known examples include fructooligosaccharides, galactooligosaccharides, and dietary fiber. Furthermore, Patent Document 1 discloses an agent for improving the intestinal flora containing soy isoflavone as an active ingredient.

[0004] Patent Publication No. 2023-080372

[0005] Many conventional prebiotics serve as substrates for specific beneficial bacteria such as bifidobacteria, promoting their growth or activating their metabolism to improve the microflora. However, there is a limit to the environmental carrying capacity of specific sites where microflora exist, such as the intestines or skin. If there is a competitive relationship between harmful and beneficial bacteria in the target microflora and harmful bacteria are already dominant, there is concern that the effectiveness of prebiotics, which serve as food for beneficial bacteria, in increasing the beneficial bacteria will be limited.

[0006] Therefore, the present inventors thought that by reducing harmful bacteria without reducing beneficial bacteria, even in situations where harmful bacteria are dominant, it would be possible to more effectively improve the bacterial flora. That is, an object of the present invention is to provide a technology that can reduce harmful bacteria without reducing beneficial bacteria, as well as a bacterial flora improver and an antibacterial agent that use the same.

[0007] Furthermore, there are many probiotic products (foods, supplements, feed, medicines, etc.) that contain representative beneficial bacteria such as Bifidobacterium and Lactobacillus. We believe that technology that can reduce harmful bacteria without reducing beneficial bacteria will contribute to improving the productivity, shelf life, and effectiveness of these products, as well as stabilizing and improving their manufacturing and quality.

[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 can suppress the bacterial count of harmful bacteria such as Ruminococcus and Fusobacterium at a certain concentration, without suppressing the bacterial count of typical beneficial bacteria such as Bifidobacterium and Lactobacillus. Based on this finding, the present inventors have completed the following inventions.

[0009] (1) A first aspect of the bacterial flora improver according to the present invention is characterized in that agarooligosaccharides are used as an active ingredient in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of Bifidobacterium bacteria, (b) an amount that does not suppress the number of Lactobacillus bacteria, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

[0010] (2) A first aspect of the antibacterial agent of the present invention is characterized in that agarooligosaccharides are used as an active ingredient in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of bacteria of the genus Bifidobacterium, (b) an amount that does not suppress the number of bacteria of the genus Lactobacillus, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

[0011] (3) In the present invention, the agarooligosaccharide may contain agarobiose.

[0012] (4) A second aspect of the bacterial flora improver of the present invention is characterized in that it contains 3,6-anhydro-L-galactose and / or an oligosaccharide having 3,6-anhydro-L-galactose at its reducing end as an active ingredient, and uses the active ingredient in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of Bifidobacterium bacteria, (b) an amount that does not suppress the number of Lactobacillus bacteria, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

[0013] (5) A second aspect of the antibacterial agent of the present invention is characterized in that the active ingredient is 3,6-anhydro-L-galactose and / or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end, and the active ingredient is used in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of Bifidobacterium bacteria, (b) an amount that does not suppress the number of Lactobacillus bacteria, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

[0014] (6) The method for improving bacterial flora according to the present invention (sometimes simply referred to as the "method for improving bacterial flora" in the present invention) comprises the step of using agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having ... 3,6-anhydro-L-galactose at the reducing end in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of bacteria of the genus Bifidobacterium; (b) an amount that does not suppress the number of bacteria of the genus Lactobacillus; or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

[0015] (7) The method for suppressing bacterial count according to the present invention (sometimes simply referred to as the "bacterial count suppression method" in the present invention) is a method for suppressing the bacterial count of bacteria other than those of the genus Bifidobacterium and / or Lactobacillus, and comprises a step of using agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having ... at their reducing ends in any of the following amounts (a) to (c): (a) an amount that does not suppress the bacterial count of the genus Bifidobacterium, (b) an amount that does not suppress the bacterial count of the genus Lactobacillus, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

[0016] (8) The use according to the present invention is the use of agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having agarooligosaccharides at the reducing end, for producing the bacterial flora improver or antibacterial agent according to the present invention.

[0017] The present invention may also be used outside of medical practice.

[0018] According to the present invention, it is possible to suppress the number of harmful bacteria without suppressing the number of beneficial bacteria. It is believed that the specific reduction of harmful bacteria creates an environment in the bacterial flora in which beneficial bacteria can grow, resulting in an increase in the occupancy rate of beneficial bacteria. Therefore, according to the present invention, it is possible to effectively improve the bacterial flora. Furthermore, it can contribute to the production, quality improvement, and effectiveness improvement of probiotic products containing beneficial bacteria.

[0019] Furthermore, conventional oral antibacterial agents also exert antibacterial effects on beneficial intestinal bacteria, causing side effects such as diarrhea due to a decrease in beneficial bacteria.The present invention does not decrease beneficial bacteria, so it is expected that such side effects will be reduced.

[0020] Furthermore, the active ingredients of the present invention, agarooligosaccharides, 3,6-anhydro-L-galactose, and oligosaccharides having agarooligosaccharides at their reducing ends, are derived from agar, which has been consumed as a food since ancient times, and are therefore extremely safe. Therefore, according to the present invention, it is possible to improve the bacterial flora or suppress the bacterial count of specific bacteria without any safety concerns.

[0021] 1 is a bar graph showing the absorbance (OD660) of the culture medium in which Ruminococcus gnavus was cultured at varying concentrations of agarooligosaccharides in the medium. 2 is a bar graph showing the absorbance (OD660) of the culture medium in which Ruminococcus gnavus was cultured in the presence of agarooligosaccharides and disaccharides to octasaccharides. 3 is a bar graph showing the absorbance (OD660) of the culture medium in which Fusobacterium nucleatum was cultured at varying concentrations of agarooligosaccharides in the medium. 4 is a bar graph showing the absorbance (OD660) of the culture medium in which Fusobacterium nucleatum was cultured in the presence of agarooligosaccharides, disaccharides, or tetrasaccharides. 5 is a bar graph showing the absorbance (OD660) of the culture medium in which Bifidobacterium was cultured at varying concentrations of agarooligosaccharides in the medium. 1 is a bar graph showing the absorbance (OD660) of a culture medium in which Lactobacillus was cultured at varying concentrations of agarooligosaccharides. 2 is a bar graph showing the absorbance (OD660) of a culture medium in which Ruminococcus gnavus and Bifidobacterium longum were co-cultured in the presence of 0.1% by mass of agarooligosaccharides. 3 is a pie chart showing the number of bacteria in a culture medium inoculated with Ruminococcus gnavus and Bifidobacterium longum at a bacterial ratio of approximately 10:1 and co-cultured in the presence of 0.1% by mass of agarooligosaccharides.

[0022] The present invention is further described below. The present invention provides a bacterial flora-improving agent according to the first and second aspects and an antibacterial agent according to the first and second aspects. In the present invention, these agents may be collectively referred to as "the agent," or any one of these agents may be referred to as "the agent." The present invention also provides use of agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having 3,6-anhydro-L-galactose at the reducing end, for producing the agent.

[0023] The present invention also provides a method for improving bacterial flora and a method for suppressing bacterial count. The method for suppressing bacterial count is a method for suppressing the count of other bacteria without suppressing the count of Bifidobacterium and / or Lactobacillus. Here, "other bacteria" refers to bacteria other than Bifidobacterium and / or Lactobacillus.

[0024] A "microbial flora improver" refers to a composition that improves a bacterial flora. In the present invention, "improving a bacterial flora" refers to increasing the proportion or number of beneficial bacteria in the bacterial flora, or decreasing the proportion or number of harmful bacteria. Whether a bacterial flora has been improved can be confirmed, for example, by measuring the number of bacteria of one or more bacterial species reported as beneficial bacteria or one or more bacterial species reported as harmful bacteria in the bacterial flora.

[0025] The term "antibacterial agent" refers to a composition that has the activity of inhibiting the growth and proliferation of bacteria (antibacterial activity).

[0026] "Beneficial bacteria" refer to bacteria that can directly or indirectly provide some beneficial effect to humans or animals, such as bacteria that contribute to maintaining or improving health, the production or quality improvement of food, pharmaceuticals, cosmetics, etc., environmental conservation and improvement, and the promotion of crop growth. Specific examples of bacterial species include the Bifidobacterium genus (bifidobacteria) such as Biffidobacterium bifidum, B. breve, B. infantis (reclassified as B. longum subsp. infantis), B. longum, B. adolescentis, B. pseudolongum, B. thermophilum, B. lactis, B. animalis, and B. pseudocatenulatum; the Lactocaseibacillus genus such as Lacticaseibacillus casei and Lacticaseibacillus paracasei; the Lactiplantibacillus genus such as Lactiplantibacillus plantarum; and the Lactobacillus genus such as Lactobacillus delbrueckii, L. acidophilus, L. casei, L. paracasei, L. plantarum, L. helveticus, L. salivarius, L. rhamnosus, L. bulgaricus, L. fermentum, and L. reuteri.

[0027] "Harmful bacteria" refers to bacteria that can exert some kind of adverse effect on humans or animals, directly or indirectly. Examples include bacteria that have a negative impact on the host's health or the environment. Specific bacterial species include, for example, the genus Ruminococcus, such as Ruminococcus gnavus; the genus Fusobacterium, such as Fusobacterium mortiferum and F. nucleatum; the genus Blautia, such as Blautia wexlerae, B. luti, B. producta, and B. coccoides; the genus Enterococcus, such as Enterocloster bolteae and E. faecalis; the genus Treponema, such as Treponema brennaborense and T. denticola; the genus Streptococcus, such as Streptococcus parasanguinis and S. pneumoniae; Bacteroides acidifaciens, B. caccae, and B. Examples include Bacteroides such as B. acidifaciens and B. fragilis, Marvinbryantia formatexigens, Anaerotruncus colihominis, Tannerella forsythia, Porphyromonas gingivalis, Escherichia coli, Roseburia faecis, Faecalibacterium prausnitzii, Eubacterium rectale, Haemophlus influenzae, Moraxella catarrhalis, Rothia mucilaginosa, Mycoplasma salivarium, Helicobacter pylori, Actinobacillus actinomycetemcomitans, Gammaproteobacteria, and Erysipelotrichia.

[0028] In the present invention, suppressing the number of bacteria and suppressing their proliferation are synonymous. Similarly, not suppressing the number of bacteria and not suppressing their proliferation are synonymous.

[0029] The phrase "suppressing the number of bacteria" includes not only a reduction in the number of bacteria but also a maintenance of the same level of the number of bacteria or an increase in the number of bacteria, but also a smaller increase compared to when the active ingredient of the present invention (agarooligosaccharide, 3,6-anhydro-L-galactose and / or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end) is not used.

[0030] Furthermore, "not suppressing the number of bacteria" includes cases where the number of bacteria is reduced, but is the same as (no significant difference from) the number of bacteria when the active ingredient of the present invention is not used, or cases where the degree of reduction is smaller than when the active ingredient of the present invention is not used.

[0031] Whether or not the number of specific bacteria is inhibited can be confirmed by a conventional method. For example, as shown in the examples described below, if a specific bacterium is isolated, the active ingredient of the present invention can be added to a culture medium for the bacterium, and the degree of proliferation can be confirmed by a turbidity method or the like, in comparison with a culture medium without the active ingredient.

[0032] Furthermore, for specimens in which multiple bacterial species have been cultured together or specimens reflecting bacterial flora (such as feces, cecal contents, intestinal lavage fluid, or oral scrapings), the number of bacteria in each specimen can be determined by quantitative PCR using primers specific to the bacteria in question. That is, by comparing the number of bacteria in the presence and absence of the active ingredient of the present invention using quantitative PCR, it is possible to determine whether the number of bacteria is suppressed for each specimen.

[0033] The present invention is characterized by using agarooligosaccharides, 3,6-anhydro-L-galactose and / or oligosaccharides having 3,6-anhydro-L-galactose at the reducing end as active ingredients.

[0034] 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, agarobiose (disaccharide), agarotetraose (tetrasaccharide), agarohexaose (hexasaccharide), agarooctaose (octasaccharide), and agarodecaose (decasaccharide). In the present invention, agarooligosaccharides contain at least one of these oligosaccharides, and may consist of one type or two or more types.

[0035] As shown in the Examples below, among agarooligosaccharides, agarobiose and agarotetraose exhibit particularly high growth inhibitory effects against harmful bacteria. Therefore, agarooligosaccharides preferably contain agarobiose. In this case, the agarooligosaccharide may consist solely of agarobiose, or may contain agarooligosaccharides other than agarobiose. In this case, the agarobiose content 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.

[0036] Agarooligosaccharides are oligosaccharides having 3,6-anhydro-L-galactose at the reducing end. Therefore, in the present invention, 3,6-anhydro-L-galactose or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end may be used as an active ingredient. 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.

[0037] 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.

[0038] 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. Acid decomposition can yield even-numbered sugars having 3,6-anhydro-L-galactopyranose at the reducing end.

[0039] Enzymatic degradation methods include degradation with α-agarase and degradation with β-agarase. As with acid degradation, α-agarase can be used to obtain an even-numbered sugar 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] An example of the sugar composition confirmed by HPLC for agarooligosaccharides prepared by decomposing agar with concentrated sulfuric acid is shown below (Shirai I, Sakai T, Shiba K, Uzuhashi Y, Karasawa K. Agaro-oligosaccharides prevent myostatin hyperexpression and myosin heavy chain protein degradation in C2C12 myotubes induced by tumor necrosis factor-α. CellBio. 2018;7(2):23-34.): Disaccharide (agarobiose): 41.8, Tetrasaccharide (agarotetraose): 41.0, Hexasaccharide (agarohexaose): 14.5, Octasaccharide (agarooctaose): 2.7.

[0044] 3,6-Anhydro-L-galactose can be prepared using commercially available reagents or by standard methods. Examples of such preparation methods include the method described in Japanese Patent No. 4007760. Specifically, 450 μL of a 100 mM aqueous solution of agarobiose was mixed with 50 μL of 10x phosphate-buffered saline and 50 μL of 10 units / μL β-galactosidase phosphate-buffered saline, and the mixture was 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 material. 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. 2The 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.

[0045] The active ingredient of the present invention can be used in a form that allows direct or indirect contact with the bacterial flora to be improved or the bacterial group whose bacterial count is to be suppressed, and a more specific mode of use can be appropriately determined depending on the subject of use and the purpose of use. For example, if the active ingredient is used to improve the bacterial flora carried by humans or animals or to suppress the bacterial count of bacteria carried by humans or animals, it can be orally ingested by humans or animals, or applied or sprayed to the site where the bacterial flora or bacteria are present, i.e., oral administration, enteral administration, transdermal administration, transmucosal administration, etc. can be exemplified as modes of use.

[0046] The active ingredient of the present invention can be used, for example, in the following amounts: (a) A usage amount that does not suppress the number of Bifidobacterium bacteria; A usage amount that exerts antibacterial activity without suppressing the number of Bifidobacterium bacteria; Or, A usage amount that suppresses the number of harmful bacteria without suppressing the number of Bifidobacterium bacteria; (b) A usage amount that does not suppress the number of Lactobacillus bacteria; A usage amount that exerts antibacterial activity without suppressing the number of Lactobacillus bacteria; Or, A usage amount that suppresses the number of harmful bacteria without suppressing the number of Lactobacillus bacteria; (c) A usage amount of 80 mg or more but less than 12,000 mg per day.

[0047] The amount used that does not suppress the number of Bifidobacterium bacteria can be appropriately set depending on the attributes and condition of the subject, the composition of the bacterial flora, the form and purpose of the product, etc. For example, when administered or ingested by humans or animals, the amount used (intake / administration) can be exemplified as a lower limit of 0.0125 mg / kg body weight or more, 0.025 mg / kg body weight or more, 0.05 mg / kg body weight or more, or 0.1 mg / kg body weight or more. On the other hand, the upper limit can be exemplified as 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, or 200 mg / kg body weight or less.

[0048] <Method for Calculating Fecal Concentration> Alternatively, as shown in the Examples below, agarooligosaccharides do not inhibit the number of Bifidobacterium bacteria at a concentration of less than 0.3% by mass in a medium. Meanwhile, the average adult fecal volume is generally 100-500 g per day (Reference 1), of which approximately 80% is water (Reference 2), resulting in 80-400 mL / day of water excretion. If the agarooligosaccharide concentration in this fecal water (fecal concentration) is less than 0.3% by mass, it is considered that the number of Bifidobacterium bacteria in the intestine will not be inhibited, which is 80-400 g / day x 0.3% by mass = 240-1200 mg / day. Therefore, an example of a daily dose (intake / administration) of the active ingredient of the present invention that does not inhibit the number of Bifidobacterium bacteria is less than 1200 mg per adult. *Reference material 1: Tsurumi Clinic, Home > Announcements / Columns > Asking Dr. Tsurumi about "Having a bowel movement once every two days will make you sick" [online] [searched November 29, 2023], Internet<https: / / www.tsurumiclinic.com / news / 2020 / 06 / 20200624-225.htmL> *Reference 2: Taiho Pharmaceutical, Understanding the intestinal environment from feces! > Feces are an important message from the body [online] [searched November 30, 2023], Internet<https: / / www.taiho.co.jp / kenko / otayori / chounai04.htmL>

[0049] <Calculation of the Tolerance Per Bacterial Count> Alternatively, as shown in the Examples below, when 1.5 mg (0.3% by mass) of agarooligosaccharides was added to approximately 500 μL of the main culture medium, the turbidity of Bifidobacterium was 0.14 (1.96 × 10^8 CFU / mL of viable bacteria according to the McFarland turbidimetric method), and adding less than this amount did not inhibit the Bifidobacterium bacterial count. In other words, the tolerance of Bifidobacterium to agarooligosaccharides (the amount that does not inhibit their growth) can be considered to be less than 1.5 mg per 0.98 × 10^8 viable bacteria. Meanwhile, the average adult stool volume is 100–500 g per day (Reference 1), and the bacterial count in feces is approximately 1.011 billion / g (Reference 3), meaning the daily bacterial count is approximately 1.0–5.0 × 10^13. Since the proportion of Bifidobacterium in the intestinal bacteria of Japanese people is said to be approximately 0.18% (Reference 4), the number of Bifidobacteria excreted per day is approximately 1.0-5.0 x 10^13 x 0.18% = approximately 180-900 x 10^8. If this number of Bifidobacteria excreted is considered to be the intestinal population, the agarooligosaccharide tolerance of Bifidobacterium in the intestine is approximately 180-900 x 10^8 / 0.98 x 10^8 x 1.5 mg = 276-918 mg per day. Therefore, for the active ingredient of the present invention, an example of a daily dosage (intake / administration) that does not suppress the number of Bifidobacterium bacteria is less than 918 mg per adult. *Reference 3: Hirokazu Tsuji, Development and application of the intestinal flora analysis system Yakult Intestinal Flora-SCAN (YIF-SCAN): A quantitative perspective on human intestinal flora, Chemistry and Biology, Vol. 56, No. 5, pp. 371-375, 2018. *Reference 4: Suguru Nishijima et al., The gut microbiome of healthy Japanese and its microbial and functional uniqueness, DNA Research, Volume 23, Issue 2, April 2016, Pages 125-133, https: / / doi.org / 10.1093 / dnares / dsw002

[0050] The dosage that does not suppress the number of Lactobacillus bacteria can be appropriately determined depending on the attributes and condition of the subject, the composition of the bacterial flora, the form and purpose of the product, etc. For example, when administered or ingested to humans or animals, the dosage (intake / administration) for an adult per day can be exemplified as follows: lower limit: 0.0125 mg / kg body weight or more, 0.025 mg / kg body weight or more, 0.05 mg / kg body weight or more, 0.1 mg / kg body weight or more, etc. Meanwhile, upper limit: 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, etc.

[0051] Alternatively, as shown in the Examples below, agarooligosaccharides do not inhibit the number of Lactobacillus bacteria at a concentration of less than 3% by mass in the culture medium. Applying this value to the above-mentioned method for calculating fecal concentration results in 80-400 g / day x 3% by mass = 2,400-12,000 mg / day. Therefore, an example of the amount of use (intake / administration) of the active ingredient of the present invention that does not inhibit the number of Bifidobacterium bacteria is less than 12,000 mg per adult per day.

[0052] On the other hand, as shown in the examples below, agarooligosaccharides suppress the number of harmful bacteria when their concentration in the culture medium is at least 0.1% by mass. Applying this value to the above-mentioned method for calculating fecal concentration results in 80-400 g / day x 0.1% by mass = 80-400 mg / day. That is, for the active ingredient of the present invention, the daily dose (intake / administration) required to exert antibacterial activity without suppressing the number of Bifidobacterium bacteria can be exemplified as 80 mg or more and less than 1200 mg per adult. Furthermore, the daily dose (intake / administration) required to exert antibacterial activity without suppressing the number of Lactobacillus bacteria can be exemplified as 80 mg or more and less than 12,000 mg per adult.

[0053] The content of the active ingredient in the product can also be appropriately set based on the above-mentioned usage amount guidelines and depending on the form and purpose 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.

[0054] The active ingredient of the present invention may be used as it is in the form of a pharmaceutical product, a quasi-drug, a reagent, other medicine, food or drink, a supplement, etc., or may be used as a raw material for these products by blending it with other ingredients. These products can be produced by methods known to those skilled in the art using the active ingredient as a raw material.

[0055] 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.

[0056] <Test Method> (1) Preparation of Agar-oligosaccharides 50 g of agar ("Ultra Agar AX-30" Ina Food Industry Co., Ltd.) was added to 1,000 g of purified water and heated to dissolve, after which 2 g of concentrated sulfuric acid was added and stirred at 90°C for 3 hours. After adjusting the pH to 3.5 with sodium hydroxide, the mixture was treated with activated carbon and filtered through filter paper to recover the filtrate. This was further filtered through a filter with a pore size of 0.1 μm, and the filtrate was recovered and powdered by vacuum freeze-drying to obtain agaro-oligosaccharide powder.

[0057] (2) Preparation of disaccharides to octasaccharides The agarooligosaccharides prepared by test method (1) were subjected to recycle size exclusion chromatography, and fractions containing disaccharides, tetrasaccharides, hexasaccharides, and octasaccharides were separated. Recycle size exclusion chromatography was performed under the following conditions. <Conditions for recycle size exclusion chromatography> System: LaboACE LC-7080 Plus (Japan Analytical Industry Co., Ltd.) Column: JAIGEL-W252 / W253 (Japan Analytical Industry Co., Ltd.) Mobile phase: Aqueous solution containing 0.005% (v / v) acetic acid and 10% (v / v) ethanol Flow rate: 3.5 mL / min

[0058] The composition of each fraction was confirmed using high-performance liquid chromatography (Prominence® HPLC system (Shimadzu Corporation)). The HPLC 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).

[0059] Each fraction was dried to obtain agarobiose, agarotetraose, agarohexaose, and agarooctaose. In this example, agarobiose, agarotetraose, agarohexaose, and agarooctaose may be referred to as "disaccharide," "tetrasaccharide," "hexasaccharide," and "octasaccharide," respectively.

[0060] (3) Bacterial strains The bacterial strains used were (A) to (F) shown in Table 1. In Table 1, "JCM" is the abbreviation for the Microbial Materials Development Laboratory, BioResource Research Center, RIKEN. All strains were cultured anaerobically using the anaerobic culture kit "Anelopack" (Mitsubishi Gas Chemical).

[0061] (4) Culture medium For the strains (a) to (d), the culture medium was 1 L of Brain-Heart Infusion medium (*1) supplemented with 5 g of yeast extract, 5 g of K2HPO4, 8 g of glucose, 0.5 g of L-cysteine ​​hydrochloride, 1 g of Tween 80, 0.005 g of hemin, 0.002 g of vitamin K1, 0.001 g of resazurin sodium salt, 0.025 g of acetic acid, and 0.01 g of MgSO2·7H2O. *1 Composition of Brain-Heart Infusion medium (per liter): Brain-heart infusion, 5g / L yeast extract, 5g K2HPO4, 8g glucose, 0.5g L-cysteine ​​hydrochloride, 1g Tween 80, 0.005g hemin, 0.002g vitamin K1, 1mg resazurin, 50mL salt solution (*2). *2 Composition of salt solution (per liter): 5g sodium acetate, 2g ammonium citrate, 0.2g MgSO2·7H2O, 0.05g MnSO4·H2O, pH 6.8.

[0062] For strains (E) and (F), the culture medium used was Merck-Millipore MRS medium (*3). *3 MRS medium composition (g / L): 10.0 g casein-derived peptone, 8.0 g meat extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g K2HPO4, 1.0 g Tween 80, 2.0 g ammonium citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, pH 5.7 (25°C).

[0063] (5) Statistical analysis The results of bacterial counts measured by the turbidimetric method and the results of quantitative PCR were analyzed using the statistical analysis software GraphPad Prism version 9.5.1 (GraphPad Software) by the Kruskal-Wallis test and unpaired t-test, respectively. In this analysis, a P value of <0.05 was considered significant and is indicated by an asterisk.

[0064] Example 1: Effect on Ruminococcus gnavus. The growth of Ruminococcus gnavus cultured in the presence of agarooligosaccharides was examined. While Ruminococcus gnavus is found in extremely low abundance in the intestines of healthy individuals, its abundance has been reported to be elevated in the intestines of patients with inflammatory bowel disease (*4), moyamoya disease, non-moyamoya intracranial aortic disease (*5), heart failure (*6), spondyloarthritis (*7), coronary artery disease (*8), and atherosclerotic cardiovascular disease (*9), as well as in individuals with high body fat (*10). It has also been reported to produce inflammatory polysaccharides and induce the secretion of inflammatory cytokines from host dendritic cells, causing Crohn's disease (*11), making it an undesirable bacterium, at least in humans. ※4. Hall AB, Yassour M, Sauk J, Garner A, Jiang X, Arthur T, et al. A novel Ruminococcus gnavus clade enriched in inflammatory bowel disease patients. Genome Med. 2017;9(1):103; doi: 10.1186 / s13073-017-0490-5. ※5. Yohei Mineharu et al., Increased abundance of Ruminococcus gnavus in gut microbiota is associated with moyamoya disease and non-moyamoya intracranial large artery disease, Sci Rep. 2022 Nov 24;12(1):20244. doi: 10.1038 / s41598-022-24496-9. *6. Simadibrata, Daniel M. MD et al., S178?A Systematic Review of the Gut Microbiota Profile in Patients With Heart Failure, The American Journal of Gastroenterology 117(10S):p e129, October 2022. | DOI: 10.14309 / 01.ajg.0000857352.83593.b6 ※7. Maxime Breban et al., Faecal microbiota study reveals specific dysbiosis in spondyloarthritis, Ann Rheum Dis. 2017 Sep;76(9):1614-1622. doi: 10.1136 / annrheumdis-2016-211064. Epub 2017 Jun 12. ※8. Takumi Toya et al., Coronary artery disease is associated with an altered gut microbiome composition, PLoS One. 2020 Jan 29;15(1):e0227147. doi: 10.1371 / journal.pone.0227147. eCollection 2020. ※9. I C L van den Munckhof 1 et al., Role of gut microbiota in chronic low-grade inflammation as potential driver for atherosclerotic cardiovascular disease: a systematic review of human studies, Obes Rev. 2018 Dec;19(12):1719-1734. doi: 10.1111 / obr.12750. Epub 2018 Aug 24. ※10. Louise Grahnemo et al., Cross-sectional associations between the gut microbe Ruminococcus gnavus and features of the metabolic syndrome, Lancet Diabetes Endocrinol. 2022 Jul;10(7):481-483. doi: 10.1016 / S2213-8587(22)00113-9. ※11. Henke MT, Kenny DJ, Cassilly CD, Vlamakis H, Xavier RJ, Clardy J.Ruminococcus gnavus, a member of the human gut microbiome associated with Crohn's disease, produces an inflammatory polysaccharide. Proceedings of the National Academy of Sciences. 2019;116(26):12672-7.

[0065] (1) Cultivation in the Presence of Agaroo-oligosaccharides. Ruminococcus gnavus was inoculated into the culture medium and then statically cultured anaerobically at 37°C for 37 hours. This was used as the seed broth. Agaroo-oligosaccharides were added to a 20% (w / w) glucose aqueous solution to final concentrations of 0, 0.1, and 0.2% by mass to prepare a sugar solution. 470 μL of the culture medium was dispensed into 96-deep-well plates (AxyGen Scientific, CA, USA), and 2.5 μL of the sugar solution was dispensed into each well. 25 μL of the seed broth was then inoculated into each well and cultured under the same conditions for 23 hours (final concentrations of agaro-oligosaccharides in the culture medium were 0, 0.1, and 0.2% by mass).

[0066] (2) Measurement of bacterial count by turbidity method. 20 μL of the culture medium from the main culture was sampled and diluted 10-fold by adding 180 μL of water. The absorbance (OD660) of the diluted culture medium was measured using a microplate reader (Wako SUNRISE Rainbow) (N=8). The results are shown in Figure 1.

[0067] As shown in Figure 1, the absorbance (OD660) was 1.27 at an agarooligosaccharide concentration of 0% by mass, but 0.77 and 0.09 at 0.1% and 0.2% by mass, respectively, both of which were lower than the 0% concentration. In other words, agarooligosaccharides suppressed the bacterial count of Ruminococcus gnavus at concentrations of 0.1% and 0.2% by mass. These results demonstrate that agarooligosaccharides can suppress the growth of Ruminococcus bacteria at both 0.1% and 0.2% by mass concentrations in the medium.

[0068] (3) Culture in the Presence of Disaccharides to Octasaccharides Ruminococcus gnavus was cultured by the method described in Example 1(1) except that agarooligosaccharides were replaced with disaccharides, tetrasaccharides, hexasaccharides, or octasaccharides, and the number of bacteria was measured by the turbidity method described in Example 1(2) (N=2). The concentration of disaccharides to octasaccharides in the medium was 0.1% by mass. The results are shown in Figure 2.

[0069] As shown in Figure 2, the absorbance (OD660) was 0.03, 0.04, 0.02, and 0.06 when the medium contained 0.1% by mass of a disaccharide, tetrasaccharide, hexasaccharide, or octasaccharide, respectively. These values ​​were significantly lower than the 1.37 value obtained when the medium contained no disaccharide, tetrasaccharide, hexasaccharide, or octasaccharide (glucose only). These results demonstrate that agarooligosaccharides, agarobiose, agarotetraose, agarohexaose, and agarooctaose, at a concentration of 0.1% by mass in the medium, can inhibit the growth of Ruminococcus. Furthermore, because all of these oligosaccharides contain 3,6-anhydro-L-galactose at their reducing ends, it was also demonstrated that 3,6-anhydro-L-galactose or oligosaccharides containing 3,6-anhydro-L-galactose at their reducing ends can inhibit the growth of Ruminococcus.

[0070] Example 2: Effect on Fusobacterium nucleatum The growth rate of Fusobacterium nucleatum cultured in the presence of agarooligosaccharides was examined. Fusobacterium nucleatum is a pathogenic bacterium that is known to cause periodontal disease and has also been shown to be involved in the progression of colon cancer (*12, *13). *12. Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res. 2012;22(2):299-306; doi: 10.1101 / gr.126516.111. *13. Wu J, Li Q, Fu X. Fusobacterium nucleatum contributes to the carcinogenesis of colorectal cancer by inducing inflammation and suppressing host immunity. Translational oncology. 2019;12(6):846-51.

[0071] (1) Cultivation in the Presence of Agarooligosaccharides Ruminococcus gnavus was cultured in place of Fusobacterium nucleatum by the method described in Example 1(1), and the absorbance of the culture solution was measured by the turbidity method described in Example 1(2) (N=8). However, the concentration of agarooligosaccharides in the medium was 0.2% by mass, and the main culture time was 9 hours. The results are shown in Figure 3.

[0072] As shown in Figure 3, the absorbance (OD660) was 0.96 when the agarooligosaccharide concentration in the medium was 0% by mass, but was significantly lower at 0.40 when the agarooligosaccharide concentration was 0.2% by mass. In other words, agarooligosaccharides suppressed the number of Fusobacterium nucleatum bacteria at a concentration of 0.2% by mass. This result demonstrated that agarooligosaccharides can suppress the growth of Fusobacterium at a concentration of 0.2% by mass in the medium.

[0073] (2) Cultivation in the Presence of Disaccharides and Tetrasaccharides: Ruminococcus gnavus was replaced with Fusobacterium nucleatum, and agarooligosaccharides were replaced with disaccharides or tetrasaccharides. Fusobacterium nucleatum was cultured by the method described in Example 1(1), and the number of bacteria was measured by the turbidity method described in Example 1(2) (N=2). The concentration of disaccharides and tetrasaccharides in the medium was 0.1% by mass. The results are shown in Figure 4.

[0074] As shown in Figure 4, the absorbance (OD660) was 0.04 and 0.27 when the medium contained 0.1% by mass of a disaccharide or 0.2% by mass of a tetrasaccharide, respectively. Both values ​​were significantly lower than the 1.23 value obtained when the medium contained no disaccharide or tetrasaccharide (glucose only). In particular, the disaccharide had the lowest absorbance. These results demonstrated that agarooligosaccharides, agarobiose, and agarotetraose, at a concentration of 0.1% by mass in the medium, can inhibit the growth of Fusobacterium. Furthermore, because all of these oligosaccharides contain 3,6-anhydro-L-galactose at their reducing ends, it was also demonstrated that 3,6-anhydro-L-galactose or oligosaccharides containing it at their reducing ends can inhibit the growth of Fusobacterium.

[0075] Example 3: Effects on Bifidobacterium The growth rate of Bifidobacterium (bifidobacteria) cultured in the presence of agarooligosaccharides was examined. Bifidobacteria are known to contribute to the health of the host, including preventing infectious diseases and modulating the immune system (*14, *15). *14. Lim HJ, Shin HS. Antimicrobial and Immunomodulatory Effects of Bifidobacterium Strains: A Review. J Microbiol Biotechnol. 2020;30(12):1793-800; doi: 10.4014 / jmb.2007.07046. *15. Valdes L, Salazar N, Gonzalez S, Arboleya S, Rios-Covian D, Genoves S, et al. Selection of potential probiotic bifidobacteria and prebiotics for elderly by using in vitro faecal batch cultures. European Food Research and Technology. 2017;243:157-65.

[0076] Ruminococcus gnavus was cultured using the method described in Example 1(1) instead of Bifidobacterium longum or Bifidobacterium adolescentis, and the absorbance of the culture solution was measured using the turbidity method described in Example 1(2) (N=4). However, the concentrations of agarooligosaccharides in the medium were 0% by mass, 0.1% by mass, 0.2% by mass, and 0.3% by mass. The results are shown in Figure 5.

[0077] 5, the absorbance (OD660) of Bifidobacterium longum was 0.51 at an agarooligosaccharide concentration of 0% by mass, while it was 0.53 and 0.52 at 0.1% and 0.2% by mass, respectively, which was equivalent to that at 0% by mass. At 0.3% by mass, it was 0.06, which tended to be smaller than that at 0% by mass, but there was no significant difference.

[0078] The absorbance (OD660) of Bifidobacterium adolescentis was 0.64 at 0% agarooligosaccharide concentration, while it was 0.65 and 0.49 at 0.1% and 0.2% agarooligosaccharide concentrations, respectively, which were equivalent to 0% agarooligosaccharide concentration. At 0.3% agarooligosaccharide concentration, it was 0.14, which was smaller than that at 0% agarooligosaccharide concentration.

[0079] That is, agarooligosaccharides did not inhibit the bacterial counts of Bifidobacterium longum and Bifidobacterium adolescentis at concentrations of less than 0.3% by mass. This result revealed that agarooligosaccharides do not inhibit the growth of bifidobacteria at concentrations of less than 0.3% by mass in the medium.

[0080] Example 4: Effect on Lactobacillus The growth rate of Lactobacillus cultured in the presence of agarooligosaccharides was examined. Like bifidobacteria, Lactobacillus is a beneficial bacterium known to contribute to the health of the host, including preventing infectious diseases (*16, *17). *16. Barrons R, Tassone D. Use of Lactobacillus probiotics for bacterial genitourinary infections in women: a review. Clin Ther. 2008;30(3):453-68; doi: 10.1016 / j.clinthera.2008.03.013. *17. Reid G, Burton J. Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect. 2002;4(3):319-24; doi: 10.1016 / s1286-4579(02)01544-7.

[0081] Ruminococcus gnavus was cultured using the method described in Example 1(1) instead of Lactobacillus plantarum or Lactobacillus casei, and the absorbance of the culture solution was measured by the turbidity method described in Example 1(2) (N=4). The concentrations of agarooligosaccharides in the medium were 0%, 1%, 2%, 3%, and 5% by mass. The results are shown in Figure 6.

[0082] 6, the absorbance (OD660) of Lactobacillus plantarum was 2.49 at 0% agarooligosaccharide concentration, 2.01 at 1% and 2% by mass, and 1.86 at 3% and 5% by mass, respectively, which were equivalent to the 0% agarooligosaccharide concentration. The absorbance (OD660) was 1.59 at 3% by mass and 1.40 at 5% by mass, which were smaller than the 0% agarooligosaccharide concentration.

[0083] The absorbance (OD660) of Lactobacillus casei was 2.74 at 0% agarooligosaccharide concentration, 2.05 at 1% and 2% by mass, and 1.81 at 3% and 5% by mass, respectively, which were equivalent to the 0% agarooligosaccharide concentration. The absorbance (OD660) was 1.72 at 3% by mass and 1.43 at 5% by mass, which were smaller than the 0% agarooligosaccharide concentration.

[0084] That is, agarooligosaccharides did not inhibit the bacterial counts of Lactobacillus plantarum and Lactobacillus casei at concentrations of less than 3% by mass. These results demonstrate that agarooligosaccharides do not inhibit the growth of Lactobacillus bacteria at concentrations of less than 3% by mass in the medium.

[0085] Example 5: Examination of bacterial counts in mixed culture Since it was revealed that agarooligosaccharides inhibit the growth of Ruminococcus but do not inhibit the growth of Bifidobacteria, the bacterial counts of these bacteria were determined when they were mixed and cultured in the presence of 0.1% by mass of agarooligosaccharides.

[0086] (1) Mixed culture in the presence of agarooligosaccharides Ruminococcus gnavus and Bifidobacterium longum were inoculated into culture media and then subjected to static anaerobism at 37°C for 37 hours to prepare Ruminococcus gnavus seed liquor (OD660 = 1.42, gnavus seed liquor) and Bifidobacterium longum seed liquor (OD660 = 2.63, longum seed liquor). These seed liquors were inoculated simultaneously at the following ratios and cultured according to the method described in Example 1 (1). The total number of bacteria was measured using the turbidity method described in Example 1 (2) (N = 8). The concentration of agarooligosaccharides in the medium was 0.1% by mass. The results are shown in Figure 7. (Ki) Gnavas species mother liquor: Longum species mother liquor = 20:0 (μL) (H) Gnavas species mother liquor: Longum species mother liquor = 20:1 (μL) (I) Gnavas species mother liquor: Longum species mother liquor = 20:10 (μL) (Ko) Gnavas species mother liquor: Longum species mother liquor = 0:1 (μL)

[0087] As shown in Figure 7, the absorbance (OD660) of (K) inoculated with only Bifidobacterium longum seed mother liquor was 0.03, while (K) and (Ke) inoculated with Bifidobacterium longum seed mother liquor were 0.48 and 0.52, respectively, which was significantly higher than (K). (K) inoculated with only Bifidobacterium longum seed mother liquor was 0.45, which tended to be higher than (K). In other words, when Ruminococcus gnavus was mixed with Bifidobacterium longum and cultured, the inhibitory effect on the total bacterial count was significantly reduced.

[0088] (2) Measurement of the number of each bacterium by quantitative PCR. Since the seed broth of (H) was as described above, the ratio of the number of bacteria at the start of the mixed culture was Ruminococcus gnavus to Bifidobacterium longum = approximately 10:1. Therefore, the number of each bacterium in the culture broth of (H) after the mixed culture was measured by quantitative PCR. As an indicator of the number of Ruminococcus gnavus bacteria, the DNA copy number of the gyrB gene (the gene encoding the β subunit of DNA gyrase) of Ruminococcus gnavus was measured. As an indicator of the number of Bifidobacterium longum bacteria, the DNA copy number of the 16S rRNA gene of Bifidobacterium was measured.

[0089] Specifically, approximately 480 μL of the remaining culture medium (H) after absorbance measurement was centrifuged at 4,000 rpm for 10 minutes, and the precipitate (bacterial cells) was collected and suspended in 0.5 mL of water. The suspension was then incubated at 70°C for 10 minutes, followed by disruption with zirconia beads at 4,300 rpm for 10 minutes using a FastPrep FP100A (MP Biomedicals) to obtain a disrupted solution. DNA was extracted from the disrupted solution using a Magtration System 12GC and a GC series MagDEA DNA 200 (Precision System Science), and the resulting supernatant was centrifuged at 15,000 rpm for 1 minute. The precipitate was collected and used as template DNA. The following primers were used: <For amplification of the gyrB gene of Ruminococcus gnavus> Forward primer: 5'-GGAGCAGACCAGATCCAAAT -3' (SEQ ID NO: 1) Reverse primer: 5'-CCAATATACATTCCCGGTCTTT -3' (SEQ ID NO: 2) <For amplification of the 16S ribosomal RNA gene of Bifidobacterium> Forward primer: 5'-GATTCTGGCTCAGGATGAACGC -3' (SEQ ID NO: 1) Reverse primer: 5'-CTGATAAGGACGCGACCCCAT -3' (SEQ ID NO: 2)

[0090] PCR reactions and detection were performed using a "QuantStudio 3" (Thermo Fisher Scientific). The reaction solution was prepared using the "PowerTrack SYBR Green Master Mix" (Thermo Fisher Scientific) reagent according to the accompanying instructions. The reaction conditions were an initial 2 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C, 15 seconds at 55°C, and 15 seconds at 72°C, followed by 1 minute at 72°C. After the reaction, melting curve analysis was performed to confirm the specificity of the amplification reaction using each primer. Since bacteria generally possess an average of four copies of the 16S rRNA gene, the DNA copy number of the 16S rRNA gene divided by four was used to determine the bacterial count of Bifidobacterium longum. The DNA copy number of the gyrB gene was used directly to determine the bacterial count of Ruminococcus gnavus. The percentage of each bacterial count, with the total bacterial count set at 100%, was calculated (N = 8). The results are shown in Figure 8.

[0091] As shown in Figure 8, the number of Ruminococcus gnavus bacteria was approximately 4%, while the number of Bifidobacterium longum bacteria was approximately 96%, with most of the bacteria being Bifidobacterium longum. That is, the ratio of Ruminococcus gnavus to Bifidobacterium longum at the start of the mixed culture was approximately 10:1, but after culture in the presence of 0.1% by mass of agarooligosaccharides, the ratio became approximately 4:96. These results demonstrate that agarooligosaccharides at a concentration of 0.1% by mass specifically inhibit the growth of Ruminococcus genus bacteria, thereby improving the occupancy rate of Bifidobacterium.

Claims

1. A bacterial flora improver, characterized in that agarooligosaccharides are used as an active ingredient in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of Bifidobacterium bacteria, (b) an amount that does not suppress the number of Lactobacillus bacteria, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

2. An antibacterial agent containing agarooligosaccharide as an active ingredient, wherein the active ingredient is used in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of Bifidobacterium bacteria, (b) an amount that does not suppress the number of Lactobacillus bacteria, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

3. The agent according to claim 1 or 2, wherein the agarooligosaccharide is an agarooligosaccharide containing agarobiose.

4. A bacterial flora improver, comprising 3,6-anhydro-L-galactose and / or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end as an active ingredient, wherein the active ingredient is used in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of Bifidobacterium bacteria, (b) an amount that does not suppress the number of Lactobacillus bacteria, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

5. An antibacterial agent comprising 3,6-anhydro-L-galactose and / or an oligosaccharide having 3,6-anhydro-L-galactose at the reducing end as an active ingredient, wherein the active ingredient is used in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of bacteria of the genus Bifidobacterium, (b) an amount that does not suppress the number of bacteria of the genus Lactobacillus, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

6. A method for improving bacterial flora (excluding medical procedures), which comprises a step of using agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having agarooligosaccharides at the reducing end in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of Bifidobacterium bacteria, (b) an amount that does not suppress the number of Lactobacillus bacteria, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

7. A method (excluding medical procedures) for suppressing the number of bacteria of the genus Bifidobacterium and / or Lactobacillus without suppressing the number of bacteria of other bacteria, which comprises a step of using agarooligosaccharides, 3,6-anhydro-L-galactose, and / or oligosaccharides having agarooligosaccharides at the reducing end in any of the following amounts (a) to (c): (a) an amount that does not suppress the number of bacteria of the genus Bifidobacterium, (b) an amount that does not suppress the number of bacteria of the genus Lactobacillus, or (c) an amount that is 80 mg or more but less than 12,000 mg per day.

8. Use of agarooligosaccharide, 3,6-anhydro-L-galactose and / or an oligosaccharide having ... at the reducing end, for producing the agent according to any one of claims 1 to 5.

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