WO2021166285A1 - 新規ビフィドバクテリウム属細菌及びそれを含む組成物 - Google Patents
新規ビフィドバクテリウム属細菌及びそれを含む組成物 Download PDFInfo
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- WO2021166285A1 WO2021166285A1 PCT/JP2020/030678 JP2020030678W WO2021166285A1 WO 2021166285 A1 WO2021166285 A1 WO 2021166285A1 JP 2020030678 W JP2020030678 W JP 2020030678W WO 2021166285 A1 WO2021166285 A1 WO 2021166285A1
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- sorbitol
- culture
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
- A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the present invention is a novel bifidobacteria and a composition containing the novel bifidobacteria, more specifically, a novel bifidobacteria having sorbitol assimilation ability isolated from the intestine of an athlete.
- the present invention relates to foods and drinks containing the genus bacteria and the novel bifidobacteria genus bacteria, probiotic compositions and the like.
- the health condition can be improved or improved by adjusting the intestinal environment.
- the intestinal flora composed of a large number of intestinal bacteria by analyzing fecal samples by a molecular biological method. Therefore, research from various viewpoints is being carried out in order to elucidate the relationship between the composition of the intestinal flora and the health condition.
- Non-Patent Document 1 As a result of analyzing the intestinal flora of 13 female marathon athletes, a certain percentage (6 out of 13 athletes) possessed Prevotella intestinal bacteria was found. Etc. have been reported.
- sugars such as glucose and sucrose
- sugars include sugar alcohols such as sorbitol and xylitol, and synthetic sweeteners such as aspartame.
- sugar alcohols are widely used as sweeteners, food additives, etc. because they are mainly plant-derived natural compounds, so they are highly safe, and because they are difficult to digest and absorb, they have low calories.
- Non-Patent Document 1 As a result of analyzing the intestinal bacterial flora of athletes, only the knowledge of what kind of microorganisms are present and at what ratio is obtained. , The individual microorganisms that make up the athlete's intestinal flora have not been investigated or isolated.
- oligosaccharides and polydextrose are difficult to be decomposed and absorbed in the digestive tract, and serve as a selective nutrient source for intestinal bacteria useful for improving health condition and promote their growth. It is used as prebiotics. Therefore, it is expected that sugar alcohols, which are widely used as constituents of foods and drinks, will be utilized as prebiotics.
- an object of the present invention is to isolate and provide a new lactic acid bacterium having a beneficial function from the intestinal flora of an athlete.
- Another object of the present invention is to obtain a new lactic acid bacterium having a sugar alcohol assimilation ability capable of growing using a sugar alcohol such as sorbitol and using the sugar alcohol as a prebiotic. To provide.
- Another object of the present invention is to provide a composition such as a food or drink or a probiotic composition containing the above-mentioned new lactic acid bacterium.
- the present inventors obtained 1000 or more samples from 500 or more athletes, analyzed the intestinal flora of the athletes, and diligently studied for the purpose of isolating a novel lactic acid bacterium useful as a new probiotic. , The present invention has been completed.
- the novel lactic acid bacterium of the present invention that solves the above-mentioned problems is Bifidobacterium longum AuB-001 strain (NITE BP-03095) of Bifidobacterium (Bifidobacterium genus bacterium).
- the Bifidobacterium longum AuB-001 strain (NITE BP-03095) according to the present invention is a novel Bifidobacterium genus bacterium having sorbitol assimilation ability. Since this novel bifidobacteria is a longum species, it can be used as a probiotic. In addition, since this novel Bifidobacterium bacterium has the ability to assimilate sorbitol, sorbitol contained in various foods and beverages and sorbitol naturally contained in fruits, etc. are prebiotics. Can be effectively used as. Furthermore, this novel Bifidobacterium genus bacterium has excellent acid resistance as compared with the reference strain (Bifidobacterium longum ATCC15707 strain).
- composition containing the novel Bifidobacterium genus bacterium of the present invention is a composition containing Bifidobacterium longum AuB-001 strain (NITE BP-03095).
- composition of the present invention contains Bifidobacterium longum AuB-001 strain (NITE BP-03095) and sorbitol. This results in a composition containing beneficial microorganisms and their nutrient sources, namely probiotics and prebiotics.
- composition of the present invention is a food or drink.
- the foods and drinks in the present invention naturally include supplements, health foods, functional foods, dietary supplements, foods for specified health use, and the like.
- composition of the present invention is a probiotic composition. This selects one aspect of the suitable composition.
- a composition containing a novel Bifidobacterium genus bacterium and a novel Bifidobacterium genus bacterium having the following excellent effects.
- the novel lactic acid bacterium of the present invention is Bifidobacterium longum AuB-001 strain (NITE BP-03095) having the ability to assimilate sorbitol.
- the novel Bifidobacterium bacterium of the present invention is isolated from the intestinal flora of an athlete (male, athletics, former Olympics representative from Japan).
- This novel Bifidobacterium genus bacterium that is, the Bifidobacterium longum AuB-001 strain, has been internationally deposited with the patented microorganism depository as follows. (1) Contract number: NITE BP-03095 (2) Original deposit date: December 25, 2019 (3) Depositary organization: Patent Microbial Deposit Center, Incorporated Administrative Agency Product Evaluation Technology Infrastructure Organization (Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture 292-0818) )
- the mycological properties of the Bifidobacterium longum AuB-001 strain isolated from the intestinal flora of athletes are shown in Tables 1 and 2 below.
- Each test shown in Table 1 below includes morphological observation using an optical microscope (Olympus Corporation product, model number: BX50F4) and the method of Barrow & Feltham et al. (Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edition, Cambridge: University Press). , 1993).
- the reaction test for each substrate shown in Table 2 was carried out using an anaerobic bacterium biochemical identification kit (Apikenki (API20A), a product of BioMérieux Japan Co., Ltd.).
- the Bifidobacterium longum AuB-001 strain can be grown by culturing.
- the culture temperature of the AuB-001 strain is preferably 20 ° C to 40 ° C, more preferably 25 ° C to 40 ° C, and particularly preferably 30 ° C to 40 ° C.
- the bacterium of the genus Bifidobacterium is an obligate anaerobic bacterium, it is preferable to culture it in an anaerobic environment, for example, it is preferable to culture it while aerating carbon dioxide gas or nitrogen gas.
- a medium generally used for culturing bifidobacteria can be used, and the medium is not particularly limited, but is limited to GAM bouillon “Nissui” (Nippon Suisan Kaisha Co., Ltd.) or TOS propionic acid agar medium. (Yakult Pharmaceutical Industry Co., Ltd.) and the like. Further, a saccharide such as sorbitol, which the AuB-001 strain has an assimilation ability, may be blended in this medium.
- the Bifidobacterium longum AuB-001 strain can be used in the state of the culture solution, the state in which the culture solution is concentrated or diluted, and the state of the cells recovered from the culture solution or the like. In addition, various additional treatments such as washing treatment, freeze-drying, L-drying, spray-drying and other drying treatments, and heat treatment may be applied to the culture solution and the recovered bacterial cells as long as the effects of the present invention are not lost. It is possible.
- the Bifidobacterium longum AuB-001 strain is preferably used as a live bacterium, but may be a dead bacterium, and a mixture of the live bacterium and the dead bacterium is used. You may.
- the Bifidobacterium longum AuB-001 strain according to the present invention can be used alone as a beneficial novel bifidobacteria, but can be used as various compositions obtained in combination with other components.
- can. As the composition containing the Bifidobacterium longum AuB-001 strain of the present invention, foods and drinks to be ingested orally, probiotic compositions, intestinal regulating compositions, quasi-drugs, pharmaceuticals and the like are preferably used. Can be mentioned.
- foods and drinks include supplements, health foods, functional foods, dietary supplements, foods for specified health use, and the like.
- the content of the AuB-001 strain in the composition varies depending on the use and mode of use of the composition, but as an example, it is preferably 1 ⁇ 10 6 to 1 ⁇ 10 12 cells / g, and 1 ⁇ 10 More preferably, it is 7 to 1 ⁇ 10 11 cells / g.
- Sorbitol can be contained in the composition containing the Bifidobacterium longum AuB-001 strain of the present invention. Since the AuB-001 strain has the ability to assimilate sorbitol, sorbitol, which is a constituent of the composition, serves as a selective nutrient source for the AuB-001 strain and can activate the growth and activity of the AuB-001 strain. ..
- the blending ratio of sorbitol in the composition is not particularly limited, but as an example, 0.01 to 20% by weight is preferable, 0.1 to 10% by weight is more preferable, and 0.5 to 5% by weight is particularly preferable. ..
- Food and drink are suitable for the use of the composition according to the present invention.
- supplement forms such as tablets and capsules, powders, granules and gels, beverages such as fermented milk and lactic acid bacteria beverages, soft drinks and sports beverages, dairy products such as yogurt and ice cream, candy and gum.
- beverages such as fermented milk and lactic acid bacteria beverages, soft drinks and sports beverages
- dairy products such as yogurt and ice cream, candy and gum.
- chocolate and other confectionery, bread, porridge, cereals, noodles, jelly, soup, seasonings and other food and drink compositions can be combined in this composition. be.
- These food and drink compositions can be produced by adding, for example, the Bifidobacterium longum AuB-001 strain in powder form or suspension form to the raw material of food and drink and mixing them.
- the Bifidobacterium longum AuB-001 strain it is also possible to add the Bifidobacterium longum AuB-001 strain to a fermented raw material such as milk and cultivate or ferment it to produce a fermented food.
- the use of the composition according to the present invention it is also preferable to use it as a probiotic composition for adjusting the intestinal environment.
- the form of the probiotic composition can be the same as that of the above-mentioned food and drink.
- the prebiotic component include, but are not limited to, oligosaccharides such as galactooligosaccharide and fructooligosaccharide, and dietary fiber such as polydextrose and inulin, in addition to the above-mentioned sorbitol. Can be selected and incorporated into the composition.
- it is also possible to select one or more probiotics such as Bifidobacterium longum strain, Bifidobacterium strain or Lactobacillus strain other than AuB-001 strain and blend them in the composition. ..
- Example 1 Isolation of lactic acid bacteria Fecal samples were collected from a large number of athletes and lactic acid bacteria were isolated. Specifically, it was carried out by the following method. The stool sample was suspended in sterilized PBS, and the suspension was further diluted with PBS to prepare a serial diluent, which was applied to TOS propionic acid agar medium (a product of Yakult Pharmaceutical Co., Ltd.). This plate medium was anaerobically cultured in an anaerobic jar at 37 ° C. for 2 to 3 days using an oxygen scavenger / carbon dioxide generator (Mitsubishi Gas Chemical Company, Inc. product, Aneropack (registered trademark) / Kenki).
- TOS propionic acid agar medium a product of Yakult Pharmaceutical Co., Ltd.
- This plate medium was anaerobically cultured in an anaerobic jar at 37 ° C. for 2 to 3 days using an oxygen scavenger / carbon dioxide generator (Mitsubishi Gas Chemical Company
- Bacterial isolates colonized on a plate medium were caught and inoculated into a liquid medium (GAM bouillon “Nissui", manufactured by Nissui Pharmaceutical Co., Ltd.) having the medium composition shown in Table 3 below, and the temperature was 37 ° C. under anaerobic conditions. Was cultured in. Then, as described in detail in Example 2 below, the DNA of the isolate is extracted from each culture medium, and the extracted DNA is subjected to ribosomal RNA gene (rDNA) base sequence analysis to estimate the bacterial species of the isolate. bottom.
- rDNA ribosomal RNA gene
- the culture medium of the isolate presumed to be of the genus Bifidobacterium was anaerobic in the GAM bouillon "Nissui" liquid medium shown in Table 3 after single colony isolation using TOS propionic acid agar medium for pure culture. Under the conditions, the cells were cultured at 37 ° C., made into a 20% glycerol stock, and stored at ⁇ 80 ° C.
- Example 2 Identification of isolates (1)
- 16S ribosomal RNA gene (rDNA) nucleotide sequence analysis was performed in order to estimate the bacterial species of the colonized bacterial isolate.
- the DNA of the isolate was extracted from the culture medium, and the 16S rRNA gene was amplified by PCR on the extracted DNA.
- the sequences of the primer sets used for PCR were "27F: 5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO: 1)" and "1492R: 5'-GGTTTACCTTGTCGACTT-3' (SEQ ID NO: 2)".
- the nucleotide sequence of each PCR product was determined, and the nucleotide sequence of the 16S rRNA gene region of each isolate was obtained.
- the sequence of this 16S rRNA gene region was searched for homology by the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) on the NCBI international nucleotide sequence database (Genbank).
- the nucleotide sequence of the 16S rRNA gene region of a bacterial isolate isolated from a fecal sample of a male athlete was changed to "Bifidobacterium longum subsp. Suillum strain Su 851 16S ribosomal. It was found to show as high a homology as 99.3% with "RNA, partial sequence”. Therefore, the strain of this isolate was presumed to be Bifidobacterium longum and named AuB-001 strain.
- the nucleotide sequence of the determined 16S rRNA gene region (SEQ ID NO: 3) of this isolate (AuB-001 strain) is shown in Table 4 below.
- Example 3 3. Identification of isolates (2)
- the AuB-001 strain presumed to be Bifidobacterium longum was subjected to ANI (Average nucleicide identity) analysis.
- ANI analysis is performed by dividing the entire genome sequence of the strain to be investigated and the existing strain into fragments of about 1000 bases on a computer, and calculating the similarity of each fragment of both genome sequences by a sequence search algorithm.
- the 70% concordance in the DNA-DNA hybrid formation test is said to correspond to 95% in the ANI value. Therefore, if the ANI value is 95% or more, it is determined to be the same species, and if the ANI value is less than 95%, it is determined to be a different species.
- the entire genome sequence of the AuB-001 strain was determined by a sequencer, and a base sequence of 2,548,718 bp was obtained.
- the ANI analysis was performed programmatically on the ANItools Online (http://ani.mypathogen.cn/) website. The results are shown in Table 5 below.
- the AuB-001 strain showed an ANI value of 95% or more with respect to a plurality of strains of Bifidobacterium longum species. Therefore, the strain of the AuB-001 strain of the present invention was identified as Bifidobacterium longum. Moreover, since the ANI value with the closely related strain was less than 100%, it was clarified that the AuB-001 strain is a novel strain of Bifidobacterium longum. This Bifidobacterium longum AuB-001 strain was deposited as NITE BP-03095 at the Patent Microorganisms Depositary Center.
- Example 4 4. Examination of saccharification ability
- a reaction test was conducted using each saccharide as a substrate. The test was conducted using an anaerobic bacterium biochemical identification kit (Apicenki (API20A), a product of Biomeryu Japan Co., Ltd.), and as a comparative control, the same applies to the Bifidobacterium longum ATCC15707 strain, which is a reference strain of Bifidobacterium longum. Was tested. The results are shown in Table 6 below.
- the AuB-001 strain according to the present invention is the reference strain B. Unlike the longum ATCC15707 strain, it was revealed that it has the ability to assimilate sorbitol, which is a sugar alcohol.
- Bifidobacterium longum which has the ability to assimilate sorbitol, from the intestinal flora of athletes supports the diversity and functionality of the intestinal flora of athletes.
- Example 5 Analysis of organic acid production
- Organic acids keep the intestinal acidity, promote intestinal peristalsis and water secretion from the intestinal tract, and have the effects of preventing infection, suppressing the production of spoilage products, and improving bowel movements and bowel movements. Has an action. Therefore, it is known that the intestinal environment is adjusted and contributes to health by retaining the bacteria that produce organic acids in the intestine. Therefore, in order to investigate the organic acid-producing ability of the Bifidobacterium longum AuB-001 strain, the amount of organic acid produced in the culture solution was analyzed.
- culture solution A containing no sorbitol and (2) culture solution B containing sorbitol were used as culture solutions, and the amount of organic acid produced in each culture solution was measured.
- a Nissui liquid medium containing sorbitol at a concentration of 0.5% was used. The test was conducted as follows.
- Each culture solution was filtered through a membrane filter having a pore size of 0.20 ⁇ m, and the concentration of each organic acid was measured with a high performance liquid chromatograph device (manufactured by Shimadzu Corporation, organic acid analysis system) using the filtrate as a sample solution. ..
- the detailed measurement conditions are as follows.
- a reference strain Bifidobacterium longum ATCC15707 strain
- AuB-001 strain a reference strain
- colonies of the ATCC15707 strain grown on TOS propionic acid agar medium at 37 ° C. for 72 hours by anaerobic culture were sterilized. Suspended in saline, Macfarland turbidity No. Prepared to 0.5.
- the culture solution B. longum ATCC15707 strain
- the test was carried out in the same manner as described above, and the concentration of the organic acid was measured. The results are shown in Tables 8 and 9 and FIGS. 1 and 2 below.
- the total number of bacteria in the culture solution A of the AuB-001 strain after anaerobic culture using the culture solution A was 1.5 ⁇ 10 10 (cells / mL), and the total number of bacteria in the culture solution A of the ATCC15707 strain was 1.5 ⁇ 10 10 (cells / mL). total bacterial count was 8.9 ⁇ 10 9 (cells / mL ).
- the total number of bacteria in the culture solution B of the AuB-001 strain after anaerobic culture using the culture solution B (Table 7) was 1.2 ⁇ 10 10 (cells / mL), and the culture solution B of the ATCC15707 strain. total bacterial count in the medium was 4.9 ⁇ 10 9 (cells / mL ).
- the AuB-001 strain in the culture solution A (without sorbitol addition), has the same organic acid producing ability as the reference strain ATCC15707, and all of them contain lactic acid, acetic acid and formic acid. It was found to be produced in large quantities. It was also found that the AuB-001 strain had a high production ability of lactic acid, formic acid and acetic acid even in the culture solution B to which sorbitol was added. From this, it was clarified that the AuB-001 strain can proliferate actively in the presence of sorbitol and produce an organic acid.
- Example 6 Analysis of saccharifying ability and organic acid production
- a carbohydrate-free PY medium supplemented with glucose or sorbitol as a carbohydrate source was added instead of the glucose-containing culture medium used in Example 5.
- the AuB-001 strain and the reference strain (Bifidobacterium longum ATCC15707 strain) were used to analyze the saccharifying ability, that is, the proliferative ability and the amount of organic acid produced in each culture medium.
- the composition of the PY medium used in this example is shown in Tables 10 and 11 below.
- the PY medium was obtained by heating and dissolving the solution 1 shown in Table 10, then adding the solution 2 and autoclaving at 121 ° C. for 15 minutes. After quenching this, the culture medium to which D (-)-sorbitol was added so as to have a concentration of 0.5% by weight was defined as "PY medium + 0.5% sorbitol", and D (+)-glucose was 0.5% by mass.
- the culture medium added to a concentration was defined as "PY medium + 0.5% glucose”.
- the resazurin solution (* 1) in Table 10 is a solution prepared by dissolving 25 mg in 100 mL of distilled water
- the salt solution (* 2) is a solution prepared from the components shown in Table 11, and a hemin solution (* 1) is used.
- 50 mg of hemin was dissolved in 1N NaOH and messed up to 100 mL with distilled water, and 0.15 mL of vitamin K1 was dissolved in 30 mL of 95% ethanol for Vitamin K1 solution (* 4). I used the one.
- the test was conducted as follows. Colonies of AuB-001 strain grown on TOS propionic acid agar medium (Yakult Yakuhin Kogyo Co., Ltd.) by anaerobic culture at 37 ° C. for 72 hours were taken and suspended in sterile physiological saline to obtain McFarland turbidity No. Prepared to 0.5. 0.1 mL of this AuB-001 strain bacterial solution was inoculated into 10 mL of each culture solution of "PY medium + 0.5% sorbitol" and "PY medium + 0.5% glucose", and sterilized and pressurized at 37 ° C. for 20 hours. It was anaerobically cultured using a test tube.
- each culture part from each culture was serially diluted stock solution to 10 4 times, was determined total number of bacteria each culture per 1mL counts the number of bacteria using a hemocytometer. Further, each of the remaining culture solutions was filtered through a membrane filter having a pore size of 0.20 ⁇ m, and the concentration of each organic acid was measured using the filtrate as a sample solution under the same measurement method and conditions as in Example 5 described above.
- the reference strain Bifidobacterium longum ATCC15707 strain was used, and the colonies of the ATCC15707 strain grown on TOS propionic acid agar medium at 37 ° C. for 72 hours by anaerobic culture were suspended in sterile physiological saline. Turbidity, Macfarland Turbidity No. Prepared to 0.5. About the culture solution (B. longum ATCC15707 strain) in which 0.1 mL of the bacterial solution of this reference strain was inoculated into 10 mL of each culture solution and anaerobically cultured at 37 ° C. for 20 hours, and the culture solution (Blank) not inoculated with the bacteria. was also tested in the same manner, and the total number of bacteria and the concentration of each organic acid per 1 mL of each culture solution were measured. The results are shown in Tables 12 and 13 and FIGS. 3 and 4 below.
- the AUB-001 strain was vigorously grown to 4.8 ⁇ 10 9 cells / mL, a large amount of lactic acid and acetic acid while produced, the ATCC15707 strain is the reference strain, the total number of bacteria did not proliferate and 5.6 ⁇ 10 6 cells / mL, it was shown that hardly produce lactic acid and acetic acid.
- the AuB-001 strain can proliferate using sorbitol even though it is a bifidobacteria longum species, and can produce lactic acid and acetic acid as final metabolites.
- Example 7 Examination of acid resistance
- Bifidobacterium spp. are vulnerable to a low pH environment, so when ingested orally, they are easily killed by the influence of gastric juice, and there is a problem that it is difficult to reach the intestine alive. Therefore, the acid resistance of the Bifidobacterium longum AuB-001 strain according to the present invention was investigated using a test solution adjusted to pH 2.0 that imitated gastric juice.
- AuB-001 strain was used at 37 ° C. using TOS propionic acid agar medium (Yakult Yakuhin Kogyo Co., Ltd.) and an oxygen scavenger / carbon dioxide generator (Mitsubishi Gas Chemical Co., Ltd. product, Aneropouch (registered trademark) / Kenki). It was anaerobically cultured for 24 hours. The fresh culture strains were suspended in sterile physiological saline to prepare a bacterial solution so that the number of bacteria per 1mL becomes 10 8 cells / mL.
- the AuB-001 strain was superior to the reference strain ATCC15707 in acid resistance. Specifically, when contacted with artificial gastric juice of pH 2.0 for 30 minutes, the AuB-001 strain survived 2.3 ⁇ 10 3 CFU / mL, but the number of surviving bacteria of the ATCC15707 strain was 3.3 CFU. It was / mL.
- the Bifidobacterium longum AuB-001 strain according to the present invention has a sorbitol assimilation ability capable of actively proliferating using sorbitol and producing lactic acid and acetic acid as final metabolites. It was found that it has excellent acid resistance as well as having. Therefore, it was shown that the AuB-001 strain has different properties from the known Bifidobacterium longum species including the reference strain.
- novel Bifidobacterium longum AuB-001 strain (NITE BP-03095) of the present invention and the composition containing the same are bifidobacteria useful as new probiotics and improve the intestinal environment. It is effectively used for foods (including supplements, health foods, functional foods, foods for specified health uses, etc.) and is widely useful in the medical field.
- SEQ ID NO: 1 Forward primer for PCR amplification of 16S rRNA gene region
- SEQ ID NO: 2 Reverse primer for PCR amplification of 16S rRNA gene region
- SEQ ID NO: 3 New Bifidobacterium longum AuB-001 strain Base sequence of 16S rRNA gene region
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Abstract
Description
(1)プロバイオティクスとして使用され得る新たなビフィドバクテリウム属細菌であり、食品や飲料、医薬品等の組成物に配合して使用することができる。
(2)様々な食品や飲料に広く配合され、天然果実等にも多く含まれるソルビトールの資化能を有するビフィドバクテリウム属細菌であるため、ソルビトールをプレバイオティクスとして有効活用することができる。
(3)基準株(Bifidobacterium longum ATCC15707株)と比べて耐酸性に優れるため、経口摂取した場合でも生きた状態で腸内に到達され易い。
(1)受託番号:NITE BP-03095
(2)原寄託日:2019年12月25日
(3)寄託機関:独立行政法人製品評価技術基盤機構 特許微生物寄託センター(〒292-0818 千葉県木更津市かずさ鎌足2-5-8 122号室)
1.乳酸菌の単離
多数のアスリートから糞便サンプルを採集し、乳酸菌の単離を行った。具体的には、次の方法にて行った。糞便サンプルを滅菌したPBSに懸濁し、この懸濁液をさらにPBSで希釈して系列希釈液を調製し、TOSプロピオン酸寒天培地(ヤクルト薬品工業株式会社製品)に塗布した。この平板培地を嫌気ジャー内で脱酸素・炭酸ガス発生剤(三菱ガス化学株式会社製品、アネロパック(登録商標)・ケンキ)を用いて37℃で2~3日間嫌気培養した。平板培地上でコロニー形成した細菌分離株を釣菌し、以下表3に示す培地組成の液体培地(GAMブイヨン「ニッスイ」、日水製薬株式会社製品)に接種し、嫌気性条件下で37℃にて培養した。そして、以下実施例2で詳述するように、各培養液から分離株のDNAをそれぞれ抽出し、抽出されたDNAについてリボソームRNA遺伝子(rDNA)塩基配列解析を行い、分離株の菌種を推定した。ビフィドバクテリウム属と推定された分離株の培養液は、純粋培養のためにTOSプロピオン酸寒天培地を用いて単一集落分離した後、表3に示すGAMブイヨン「ニッスイ」液体培地において嫌気性条件下で37℃にて培養し、20%グリセロールストックにして-80℃で保管した。
2.分離株の同定(1)
実施例1において、コロニー形成した細菌分離株の菌種を推定するために、16SリボソームRNA遺伝子(rDNA)塩基配列解析を行った。まず、培養液から分離株のDNAを抽出し、抽出されたDNAについて、PCRにて16S rRNA遺伝子を増幅した。PCRに用いたプライマーセットの配列は、「27F:5’-AGAGTTTGATCCTGGCTCAG-3’(配列番号1)」及び「1492R:5’-GGTTACCTTGTTACGACTT-3’(配列番号2)」とした。各PCR産物の塩基配列決定を行い、各分離株の16S rRNA遺伝子領域の塩基配列を得た。この16SrRNA遺伝子領域の配列について、NCBIの国際塩基配列データベース(Genbank)上で、BLASTプログラム(https://blast.ncbi.nlm.nih.gov/Blast.cgi)による相同性検索を行った。
3.分離株の同定(2)
実施例2において、ビフィドバクテリウム・ロンガムと推定されたAuB-001株について、ANI(Average nucleotide identity)解析を行った。ANI解析とは、調査対象株及び既存株の全ゲノム配列をコンピュータ上で約1000塩基程度の断片に分け、両者のゲノム配列の各断片の類似度を配列検索アルゴリズムで計算することにより行われるものであり、DNA-DNAハイブリッド形成試験における70%の一致度は、ANI値では95%に相当するとされている。それゆえ、ANI値が95%以上であれば同種と判定され、ANI値が95%未満であれば別種と判定される。AuB-001株の全ゲノム配列をシーケンサーにより決定し、2,548,718bpの塩基配列を得た。ANI解析はANItools Online(http://ani.mypathogen.cn/)ウェブサイトのプログラムにより行った。結果を以下表5に示す。
4.糖資化能の検討
ビフィドバクテリウム・ロンガム(Bifidobacterium longum)種に同定されたAuB-001株の糖資化能を調べるため、各糖類を基質とした反応試験を行った。試験は嫌気性菌生化学的同定キット(アピケンキ(API20A)、ビオメリュー・ジャパン株式会社製品)を用いて行い、比較対照として、ビフィドバクテリウム・ロンガムの基準株であるBifidobacterium longum ATCC15707株についても同様の試験を行った。結果を以下表6に示す。
5.有機酸産生量の分析
有機酸は腸管内を酸性に保ち、腸の蠕動運動や腸管からの水の分泌を促進するほか、感染防御、腐敗産物の生産抑制、便性・便通の改善効果を有する作用を有する。そのため、有機酸を産生する菌を腸内に保有することにより、腸内環境が整い、健康に寄与することが知られている。そこで、ビフィドバクテリウム・ロンガム(Bifidobacterium longum)AuB-001株の有機酸産生能を調べるため、培養液中における有機酸産生量を分析した。なお、培養液として、(1)ソルビトールを含まない培養液A、(2)ソルビトールを含有する培養液B、の2種類の培養液を用い、各培養液中における有機酸産生量を測定した。(1)ソルビトールを含まない培養液Aとしては、上記表3に示したGAMブイヨン「ニッスイ」液体培地を用い、(2)ソルビトールを含有する培養液Bとしては、下記表7に示すGAMブイヨン「ニッスイ」液体培地にソルビトールを0.5%となるように配合したものを使用した。試験は次のようにして行った。TOSプロピオン酸寒天培地(ヤクルト薬品工業株式会社)で37℃、72時間、嫌気培養により生育させたAuB-001株のコロニーを取り、滅菌生理食塩水に懸濁してマックファーランド濁度No.0.5に調製した。この菌液0.1mLを各培養液5mLにそれぞれ接種し、37℃で20時間、脱酸素・炭酸ガス発生剤(三菱ガス化学株式会社製品、アネロパウチ(登録商標)・ケンキ)を用いて嫌気培養した。各培養液を孔径0.20μmのメンブレンフィルターでろ過し、ろ液を試料液として、高速液体クロマトグラフ装置(株式会社島津製作所製品、有機酸分析システム)にて、各有機酸の濃度を測定した。詳細な測定条件は次の通りである。
・カラム:Shim-pack SCR-102(H)、300mm×8mm ID、2本直列で使用
・ガードカラム:Shim-pack SCR-102(H)、50mm×6mm ID
・溶離液:5mmol/L p-トルエンスルホン酸
・反応液:5mmol/L p-トルエンスルホン酸、100μmol/L EDTA、20mmol/L Bis-Tris
・流速:0.8mL/min
・オーブン温度:45℃
・検出器:電気伝導度検出器 CDD-10A
6.糖資化能及び有機酸産生量の分析
次に、実施例5で用いたグルコースを含有する培養液に替えて、炭水化物を含有しないPY培地にグルコース又はソルビトールを炭水化物源としてそれぞれ添加した培養液を用い、AuB-001株及び基準株(Bifidobacterium longum ATCC15707株)の各培養液中における糖資化能、すなわち、増殖能と有機酸産生量を分析した。
7.耐酸性の検討
一般的にビフィドバクテリウム属細菌は低いpH環境に弱いため、経口摂取した場合には胃液の影響を受けて死滅しやすく、生きたまま腸に到達し難いという問題がある。そこで、胃液を模したpH2.0に調整した試験液を用い、本発明に係るビフィドバクテリウム・ロンガム(Bifidobacterium longum)AuB-001株の耐酸性について調べた。
配列番号2:16SrRNA遺伝子領域のPCR増幅のためのリバースプライマー
配列番号3:新規ビフィドバクテリウム・ロンガム(Bifidobacterium longum)AuB-001株の16SrRNA遺伝子領域の塩基配列
Claims (5)
- ビフィドバクテリウム・ロンガム(Bifidobacterium longum)AuB-001株(NITE BP-03095)。
- ビフィドバクテリウム・ロンガム(Bifidobacterium longum)AuB-001株(NITE BP-03095)を含有することを特徴とする組成物。
- ビフィドバクテリウム・ロンガム(Bifidobacterium longum)AuB-001株(NITE BP-03095)及びソルビトールを含有することを特徴とする組成物。
- 前記組成物は飲食品であることを特徴とする請求項2又は3に記載の組成物。
- 前記組成物がプロバイオティクス組成物であることを特徴とする請求項2~4のいずれか1項に記載の組成物。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH1175830A (ja) * | 1997-09-16 | 1999-03-23 | Snow Brand Milk Prod Co Ltd | 新規ビフィズス菌及びその利用 |
WO2003048343A1 (fr) * | 2001-12-07 | 2003-06-12 | Morinaga Milk Industry Co., Ltd. | Bifidobacterium longum |
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Publication number | Priority date | Publication date | Assignee | Title |
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TW357191B (en) * | 1996-06-04 | 1999-05-01 | Food Industry Development Res Institute | Bifidobacteria strains with acid, bile salt and oxygen tolerance and their culture method the invention relates to bifidobacteria strains with acid, bile salt and oxygen tolerance and their culture method |
JP5286424B2 (ja) * | 2010-03-26 | 2013-09-11 | 森永乳業株式会社 | 生菌数の測定方法 |
ITMI20110793A1 (it) * | 2011-05-09 | 2012-11-10 | Probiotical Spa | Ceppi di batteri probiotici e composizione sinbiotica contenente gli stessi destinata alla alimentazione dei neonati. |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH1175830A (ja) * | 1997-09-16 | 1999-03-23 | Snow Brand Milk Prod Co Ltd | 新規ビフィズス菌及びその利用 |
WO2003048343A1 (fr) * | 2001-12-07 | 2003-06-12 | Morinaga Milk Industry Co., Ltd. | Bifidobacterium longum |
Non-Patent Citations (4)
Title |
---|
ANONYMOUS: "Pre-sale of supplements for research on intestinal bacteria of AuB athl etes led by former soccer representative Keita Suzuki", 2 January 2020 (2020-01-02), XP055849906, Retrieved from the Internet <URL:https://web.archive.org/web/20200102160038/https://www.makuake.com/project/aub> [retrieved on 20200930] * |
MATSUO KAORI, YOSHIDA NOBORU,TAMARI YUTARO,HAMADA HIROKI,UENO KOJI,OYANAGI ERI ,YANO HIROMI,ONODERA SHO: "Correlation between physical characteristics and composition of gut microbiota in marathon runners", JOURNAL OF KAWASAKI UNIVERSITY OF MEDICAL WELFARE, vol. 29, no. 1, 1 January 2019 (2019-01-01), JP, pages 99 - 105, XP055849914, ISSN: 0917-4605, DOI: 10.15112/00014619 * |
NORITOSHI TAKAHASHI , JIN-ZHONG XIAO , KAZUHIRO MIYAJI , TOMOKO YAESHIIMA , AKINORI HIRAMATSU , KEIJI IWATSUKI , SADAYUKI KOKUBO ,: "Selection of acid tolerant bifidobacteria and evidence for a low-pH-inducible acid tolerance response in bifidobacterium longum", JOURNAL OF DAIRY RESEARCH, vol. 71, no. 3, 200400801, pages 340 - 345, DOI: 10.1017/S0022029904000251 * |
TOMOHIKO FUJISAWA: "Current taxonomy of lactobacillus, enterococcus, lactococcus, bifidobacterium and related organisms", JOURNAL OF INTESTINAL MICROBIOLOGY, vol. 22, no. 4, 6 November 2008 (2008-11-06), JP, pages 241 - 252, XP055849896, ISSN: 1343-0882, DOI: 10.11209/jim.22.241 * |
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