JP2010246523A - New genus bifidobacterium microorganism and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new genus Bifidobacterium microorganism inhibiting the differentiation of Th17, inhibiting the production of IL-17, and utilizable for a food and drink, a medicine or the like. <P>SOLUTION: The genus Bifidobacterium microorganism containing 16SrRNA gene having a specific base sequence is regulated so that the amount of produced interleukin-17 in the supernatant, obtained by mixing mouse spleen cells, the heat-sterilized microorganisms, transforming growth factor beta and interleukin-6, and culturing the resultant mixture for 72 h, may be ≤30% of that obtained when the microorganism is not added. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel Bifidobacterium genus microorganism and use thereof.

Bifidobacterium (Bifidobacterium) microorganisms are microorganisms inhabiting the intestinal tract of all animals. The Bifidobacterium microorganisms are known to have physiological effects such as intestinal regulation and immunomodulation.

  It is known that the immunoregulatory action of Bifidobacterium microorganisms is performed by adjusting the balance between Th1 and Th2. However, the involvement of not only Th1 and Th2 but also Th17 has been clarified in immune regulation. However, among orally ingestible microorganisms, Th17 cannot be regulated, and instead, Th17 differentiation is promoted. There is a thing, and the knowledge regarding Th17 regulatory action of Bifidobacterium microorganisms is hardly seen.

  When differentiation of Th17 is promoted, interleukin-17 (IL-17) is produced, and IL-17 such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, type 1 diabetes, bronchial asthma and the like Diseases that have been suggested to be related can be caused. Therefore, if production of IL-17 can be suppressed, it is expected that it can be used for prevention and treatment of those diseases.

However, Bifidobacterium infantis is the only Bifidobacterium genus microorganism that can suppress Th17 differentiation and suppress IL-17 production so far (Non-Patent Documents). 1) The actual situation is that the use of these products for foods and drinks, medicines, etc. has not progressed.

Tanabe, S., et al .: Int. J. Mol. Med., 22: 181-185, 2008.

  Therefore, it has been desired to provide a novel microorganism belonging to the genus Bifidobacterium that can suppress the differentiation of Th17 and suppress the production of IL-17 and can be used for foods and drinks, pharmaceuticals, and the like.

  As a result of intensive studies to solve the above problems, the present inventors have found a novel Bifidobacterium having the above properties from the feces of infants and completed the present invention.

That is, the present invention is a microorganism belonging to the genus Bifidobacterium containing the base sequence of the 16S rRNA gene shown in SEQ ID NO: 1 with a homology of 97% or more.

  Moreover, this invention is the food / beverage products characterized by containing the said Bifidobacterium genus microorganisms.

  Furthermore, the present invention is a pharmaceutical product containing a Bifidobacterium microorganism.

  The Bifidobacterium microorganism of the present invention can suppress the differentiation of Th17 and suppress the production of IL-17.

  Therefore, the Bifidobacterium genus microorganism of the present invention becomes a food and drink, a pharmaceutical, and the like that can be used for the prevention and treatment of diseases that have been suggested to be associated with IL-17.

  In addition, the Bifidobacterium microorganism of the present invention promotes the production of interferon-γ and suppresses the production of interleukin-4, similarly to the above-mentioned conventional Bifidobacterium microorganism. And Th2 can be adjusted to the Th1 side.

  Therefore, the Bifidobacterium genus microorganism of the present invention becomes a food and drink, a pharmaceutical, and the like that can be used for the prevention and treatment of diseases associated with the balance of Th1 and Th2 being on the Th2 side. In this case, since the Bifidobacterium microorganism of the present invention can suppress Th17 differentiation and IL-17 production, the food and drink used for the conventional prevention and treatment of the above-mentioned diseases There are fewer side effects than pharmaceuticals.

It is a phylogenetic tree showing the relationship between a known Bifidobacterium microorganism and the Bifidobacterium microorganism of the present invention. It is drawing which shows Th17 inhibitory activity of Bifidobacterium sp. CFB1 strain | stump | stock (in FIG. 2,-is an unstimulated control and + is a negative object).

  The novel Bifidobacterium microorganism of the present invention can be obtained from infant feces, intestinal tract and the like, for example, by the following method.

  First, fresh feces immediately after excretion of feces of healthy infants are put in an anaerobic pack (Anerogas Pack: manufactured by Mitsubishi Gas Chemical) and transported at a temperature at which the feces are not frozen, for example, refrigerated below 7 ° C. . The transported stool is serially diluted with anaerobic serial dilution method (Tomochitsu Mitsuoka, “World of Enterobacteriaceae”, 2nd edition, 53-65, Sobunsha, 1984), and part of this is BS agar medium or BL It is smeared on an agar medium (both manufactured by Eiken Chemical Co., Ltd.) and cultured anaerobically at 37 ° C. for 48 hours by the steel wool method. The colony obtained after culturing is picked and confirmed for purification under the same conditions as the separation medium. The purified microorganism is stored frozen at -85 ° C.

  Next, the 16S rRNA gene region is amplified by colony PCR using the following 27F and 75R primers, for example, and the 27S, 75R, 520F, 520R, 930F, and 800R. Using the primers of 1100F and 1100R, the entire 16S rRNA gene base sequence is determined by ABI PRISM 3100 DNA Sequencer (manufactured by Applied Biosystem). Among the base sequences determined here, those having a base length of about 1300 bp and having a homology of 97% or more with the 16S rRNA gene shown in SEQ ID NO: 1 were selected, and thereafter, regular property tests (Tai Uchimura and Sanae Okada) , "Lactic acid bacteria experiment manual", supervised by Michio Kosaki, first edition, 29-72, Asakura Shoten, 1992), etc., and the novel Bifidobacterium of the present invention is obtained as having the following taxonomic properties.

<PCR primer for 16S rDNA gene amplification and nucleotide sequence determination>
27F: 5′-AGAGTTTGATCCCTGCTCAG-3 ′
75R: 5′-CCCGGGATCCAAGCTTACCGGTTACCTTGTAC
GACTT-3 '
520F: 5′-CAGGAGTGCCAGCAGCCCGCGG-3 ′
520R: 5′-ACCGCCGCTGCTGGC-3 ′
930F: 5′-GCACAAGCGGTGGAGCATGTGG-3 ′
800R: 5′-CAGGACTACCAGGGTATCTAAT-3 ′
1100F: 5′-CAGGAGCAACGAGCCGCAACCC-3 ′
1100R: 5'-AGGGTTGCCGCTCGTTG-3 '

<Taxonomic properties>
(1) Form of microorganism Type: Aspergillus Cell size: Length 0.2-0.3 μm × Width 1.0-1.3 μm
Cell edge: round Cell chain: short chain / single cell spore: no spore formation (2) colony morphology ^*
Diameter: 1mm
Color tone: Brown Shape: Circular Raised state: Half-lens shape Perimeter: Full edge Surface shape, etc .: Smooth Transparency: Opaque Consistency: Butter-like * When microorganisms are cultured in BL medium at 37 ° C for 48 hours under anaerobic conditions Form.
(3) Utilization saccharide fermentability test of carbon source ^**
D-xylose +
D-ribose +
D-Mannose weak
D-cellobiose +
D-trehalose-
Merezitose −
Dextrin weak
Starch −
Inulin weak
Mannitol −
Sorbitol +
Arabinose +
Glucose +
Fructose +
Galactose +
Sucrose +
Maltose +
Lactose +
Melibiose +
Raffinose +
Salicin +
Glycerol −
Rhamnose −
No saccharide −
** + is assimilated,-is not assimilated.
(4) Enzyme activity test ^***
Alkaline phosphatase −
Esterase (C4) −
Esterase lipase (C8) +
Lipase (C14) −
Leucine allylamidase +
Valine allylamidase −
Cystine allylamidase −
Trypsin −
α-chymotrypsin −
Acid phosphatase +
Naphthol-AS-BI-
-Phosphohydrolase α-galactosidase +
β-galactosidase +
β-glucuronidase −
α-Glucosidase +
β-glucosidase +
N-acetyl-β-glucosaminidase −
α-mannosidase −
α-fucosidase −
*** API ZYM (bioMerieux Japan) was used. + Is assimilable and-is not assimilable.
(5) Culture conditions ABCM liquid medium and BL agar medium are suitable. An anaerobic culture is required, and it grows by either anaerobic system by Anero Pack (Mitsubishi Gas Chemical) or carbon dioxide replacement by steel wool method. Without anaerobic culture similar to these, it will not grow at all. In the case of the ABCM liquid medium, the OD value becomes 1.2 or more by culturing at 37 ° C. for 24 hours, and in the case of the BL agar medium, colonies are formed by culturing at 37 ° C. for 48 hours.
(6) Growth conditions, etc. Growth temperature test 15 ° C −
37 ° C +
45 ° C-
Aerobic test-
Anaerobic test ＋
Initial pH test pH 4.5-
pH 7.2 +
pH 8.0 +
Gas production test −
(7) G + C content 56 ± 2 mol%
(8) 16S rRNA gene base sequence homology Reference strains of known relatives belonging to the genus Bifidobacterium are Bifidobacterium catenulatum JCM1194 strain (94%: 1,316 / 1) , 388 bp), Bifidobacterium angulatum JCM7096 strain (94%: 1,310 / 1,389 bp), Bifidobacterium dentium JCM1195 strain (94%: 1,321 / 1,403 bp). Generally, if the homology is less than 97%, it is considered as a different species, but the homology was 94% even in the three closest strains. Accordingly, if the homology is 97% or more, preferably 98% or more, more preferably 99% or more with respect to the sequence shown in SEQ ID NO: 1 in the sequence listing, it is included in the Bifidobacterium microorganism of the present invention.
(9) DNA-DNA homology DNA-DNA hybridization is performed by a microplate method using fluorescence detection (Ezaki, T., et al .: Int. J. Syst. Bacteriol., 39: 224-229, 1989). As a result of DNA-DNA hybridization with the reference strain of the above-mentioned related species, Bifidobacterium catenuratum JCM1194 strain 16%, Bifidobacterium anguratam JCM7096 strain 23%, Bifidobacterium denthium JCM1195 27% homology with the strain. Accordingly, if the DNA-DNA homology is 60%, it is included in the Bifidobacterium microorganism of the present invention.
(10) Phylogenetic analysis A phylogenetic tree was created with the analysis software CLUSTAL W. FIG. 1 is a phylogenetic tree showing the relationship between known Bifidobacterium microorganisms and the Bifidobacterium microorganism of the present invention. The numerical value shown at the branch point in the figure is the bootstrap confidence value, and the symbol in parentheses represents the accession number.
(11) IL-17 production inhibitory action After culturing a mixture of mouse spleen cells, heat-sterilized Bifidobacterium microorganisms, transforming growth factor beta (TGF-β) and IL-6 for 72 hours, The production amount of IL-17 in the liquid is 30% or less, preferably 20% or less when the microorganism is not added.
(12) Th1 / Th2 regulating action A mixture of mouse spleen cells and heat-sterilized Bifidobacterium microorganisms was cultured for 72 hours, and then interferon-γ (IFN-γ) in the supernatant was The ratio of the production amount to the production amount of interleukin-4 (IL-4) is 2 times or more, preferably 10 times or more that when the microorganism is not added.

The novel Bifidobacterium genus microorganism of the present invention is one of the above taxonomic properties, at least one or more selected from the group consisting of the following (a) to (f), preferably all It can be distinguished from Bifidobacterium microorganisms.
(I) Mouse spleen cells, heat-sterilized Bifidobacterium microorganisms, a mixture of TGF-β and IL-6 were cultured for 72 hours, and then the amount of IL-17 produced in the supernatant was 30% or less without addition of (ii) G + C content 56 ± 2 mol%
(C) No growth without anaerobic culture (d) No β-glucuronidase activity, which is a detrimental enzyme activity in the digestive tract (e) No spore formation, takes the form of a rod-shaped cell (f) Mouse spleen cells, A mixture of heat-sterilized microorganisms belonging to the genus Bifidobacterium is cultured for 72 hours, and then the amount of interferon-γ (IFN-γ) produced in the supernatant and interleukin-4 (IL-4) The production ratio is more than twice that when the microorganism is not added.

  An example of a novel Bifidobacterium genus microorganism having the above 16S rDNA base sequence and taxonomic properties is Bifidobacterium sp. CFB1 strain isolated by the present inventors from infant feces. This Bifidobacterium sp. CFB1 strain was established on March 17, 2009 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1-chome, Tsukuba City, Ibaraki 305-8586, Japan). ) (Deposit number FERM P-21789).

  The aforementioned Bifidobacterium genus microorganisms of the present invention can be used for various foods and beverages, pharmaceuticals and the like containing the same as other known Bifidobacterium microorganisms.

  Examples of the food and drink include sweets such as candy, gum, cookies, rice crackers, beverages such as soft drinks, carbonated beverages, alcoholic beverages, foods such as noodles and bread, fermented milk products such as yogurt and soy milk yogurt, etc. Fermented dairy products are particularly preferable. Moreover, these food-drinks can be manufactured in accordance with a conventional method.

  Examples of the above-mentioned pharmaceuticals include normal tablets, powders, capsules and the like, which are viable cells, dried cells, or a pulverized product thereof alone or mixed with a normal pharmaceutical carrier. Moreover, these pharmaceutical products can be manufactured according to a conventional method.

  In these foods and beverages, since the Bifidobacterium microorganism of the present invention can suppress Th17 differentiation and IL-17 production, multiple sclerosis, rheumatoid arthritis, inflammatory bowel It can be used for the prevention and treatment of diseases, psoriasis, type 1 diabetes, bronchial asthma and other diseases that are suggested to be associated with IL-17.

  In addition, the Bifidobacterium microorganism of the present invention not only suppresses Th17 differentiation and IL-17 production, but also promotes interferon-γ production and interleukin-4 production. Therefore, the balance between Th1 and Th2 can be adjusted to the Th1 side. Therefore, the said food-drinks and a pharmaceutical can be utilized also for the prevention and treatment of the disease relevant to the balance of Th1 and Th2 becoming Th2 side, such as allergic diseases, such as hay fever and atopic dermatitis. In addition, in this case, since the Bifidobacterium microorganism of the present invention can suppress Th17 differentiation and IL-17 production, the food and drink used for the prevention and treatment of the conventional diseases described above. There are fewer side effects than pharmaceuticals.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these Examples at all.

Example 1
Isolation of Bifidobacterium sp. CFB1 strain:
Fresh feces immediately after excretion of a healthy infant (male: 1 year and 6 months) were placed in an anaerobic pack (Anerogas Pack: manufactured by Mitsubishi Gas Chemical) and transported by refrigeration at 7 ° C or lower. The transported stool is serially diluted with anaerobic serial dilution method (Tomochitsu Mitsuoka, “World of Enteric Fungi”, 2nd Edition, 53-65, Sobunsha, 1984), and a portion of this is diluted with BL agar medium (both Eiken Chemical Co., Ltd.) and anaerobic culture at 37 ° C. for 48 hours by the steel wool method.

  The colonies obtained after the culture were picked and confirmed for purification under the same conditions as the separation medium. The purified microorganism was stored frozen at -85 ° C. The entire base sequence of the 16S rDNA of the preserved microorganism was sequenced using the following primers and determined by BLAST search (DDBJ website: http://www.ddbj.nig.ac.jp).

<PCR primer for 16S rDNA gene amplification and nucleotide sequence determination>
27F: 5′-AGAGTTTGATCCCTGCTCAG-3 ′
75R: 5′-CCCGGGATCCAAGCTTACCGGTTACCTTGTAC
GACTT-3 '
520F: 5′-CAGGAGTGCCAGCAGCCCGCGG-3 ′
520R: 5′-ACCGCCGCTGCTGGC-3 ′
930F: 5′-GCACAAGCGGTGGAGCATGTGG-3 ′
800R: 5′-CAGGACTACCAGGGTATCTAAT-3 ′
1100F: 5′-CAGGAGCAACGAGCCGCAACCC-3 ′
1100R: 5'-AGGGTTGCCGCTCGTTG-3 '

  The sequence (1,394 bp) of 16S rDNA of Bifidobacterium sp. Strain CFB1 determined above is shown in SEQ ID NO: 1 in the sequence listing.

Example 2
Mouse spleen cell stimulation test (1):
The Bifidobacterium sp. CFB1 strain obtained in Example 1 was anaerobically cultured at 37 ° C. for 24 hours using ABCM liquid medium (Eiken Chemical Co., Ltd.). These cultures were harvested by centrifugation at 3000 × g, 10 minutes, 4 ° C. and washed 3 times with distilled water. The cells were sterilized by heat treatment at 100 ° C. for 50 minutes, lyophilized and stored.

Spleen cells of 6-week-old female BALB / c mice were treated with 10% (V / V) heat-inactivated fetal calf serum, 1 ml of RPMI-1640 medium supplemented with 10 μM 2-mercaptoethanol, 10 mM HEPES, penicillin and streptomycin. was suspended in the spleen cell suspension was appropriately diluted 10 ⁷ cells / ml of culture medium, TGF-beta of 2 ng, 20 ng of IL-6 and heat sterilization was Bifidobacterium sp CFB1 strain (heat sterilization The previous number of bacteria (10 ⁷ CFU) was added to the medium, and cultured at 37 ° C. in the presence of 5% carbon dioxide for 72 hours.

  After the culture, the IL-17 concentration in the supernatant was measured by a microbead method using a Bio-Plex Suspension Array System (BioRad Laboratories) to determine the fluorescence intensity value. TGF-β, IL-6 and Bifidobacterium sp. CFB1 strain were not added to the unstimulated control (−), TGF-β and IL-6 were added, but Bifidobacterium sp. CFB1 strain was not added. Was used as a negative control (+). The test results are shown in FIG.

  As a result of the above test, the production amount of IL-17 of Bifidobacterium sp. CFB1 strain was 17.4% of the negative control.

  From this, it was shown that the Bifidobacterium sp. CFB1 strain suppresses Th17 differentiation and suppresses IL-17 production.

Example 3
Yogurt production:
10% by mass skimmed milk was sterilized at 110 ° C. for 20 minutes and dispensed into test tubes in 10 ml portions. The test tube was kept anaerobic by rapid cooling in ice, and Lactobacillus delbrueckii subsp. Bulgaricus B5b strain (foundation) previously cultured in MRS liquid medium (Oxoid). in Japan dairy technical Association (Yubinbango102-0073 Chiyoda-ku, tokyo Kudankita 1-14-19) sale shares) of 10 ⁸ level of bacterial solution of 0.5% by weight of the amount and ABCM liquid medium (Eiken Chemical Co., Ltd.) The cultured Bifidobacterium sp. CFB1 strain at 10 ⁸ level was inoculated in an amount of 2% by mass. Next, this was cultured at 37 ° C. for 16 hours to produce yogurt. The viable cell count of this yogurt was 8.2 × 10 ⁸ CFU / ml for Lactobacillus delbruecki subspecies Bulgaricus B5b and 5.6 × 10 ⁷ CFU for Bifidobacterium sp. CFB1. / Ml. Moreover, the pH of this yogurt fell moderately and the flavor was also good.

Example 4
Mouse spleen cell stimulation test (2):
Spleen cells of 6-week-old female BALB / c mice were treated with 10% (V / V) heat-inactivated fetal calf serum, 1 ml of RPMI-1640 medium supplemented with 10 μM 2-mercaptoethanol, 10 mM HEPES, penicillin and streptomycin. was suspended in the spleen cell suspension was appropriately diluted 10 ⁷ cells / ml of culture medium, example 2 bi sterilized by heating in the same manner as Bifidobacterium sp CFB1 strain (a few bacteria before heat sterilization 10 ⁷ CFU) was added to the medium, and cultured at 37 ° C. in the presence of 5% carbon dioxide for 72 hours.

  After culturing, the IFN-γ and IL-4 concentrations in the supernatant were measured by the microbead method using a Bio-Plex Suspension Array System (BioRad Laboratories) to determine the fluorescence intensity value. did. An unstimulated control (-) was obtained by adding no Bifidobacterium sp. CFB1 strain. The test results are shown in Table 1.

  As a result of the above test, the addition of Bifidobacterium sp. CFB1 strain increased the IFN-γ concentration in the supernatant, but decreased the IL-4 concentration. Since IFN-γ is a typical cytokine produced by Th1 cells and IL-4 is produced by Th2 cells, the IFN-γ / IL-4 ratio was calculated and used as an index of Th1 / Th2 balance. By adding Bifidobacterium sp. CFB1 strain, the Th1 / Th2 balance increased from 165 to 2,832.

  From this, it was found that Bifidobacterium sp. CFB1 strain has the ability to adjust the Th1 / Th2 balance to the Th1 side.

  Since the Bifidobacterium microorganism of the present invention suppresses Th17 differentiation and suppresses IL-17 production, it can be used for the prevention and treatment of diseases that have been suggested to be associated with IL-17. It can be used for food and drinks and medicines that can be used.

Claims (10)

A microorganism belonging to the genus Bifidobacterium containing the base sequence of the 16S rRNA gene shown in SEQ ID NO: 1 in the sequence listing with a homology of 97% or more. The microorganism of the genus Bifidobacterium according to claim 1, which has one or more taxonomic properties selected from the group consisting of the following (a) to (f).
(I) Mouse spleen cells, heat-sterilized Bifidobacterium microorganisms, a mixture of transforming growth factor beta and interleukin-6 were cultured for 72 hours, and then interleukin-17 in the supernatant. Is less than 30% when the microorganism is not added (b) G + C content is 56 ± 2 mol%
(C) No growth without anaerobic culture (d) No β-glucuronidase activity, which is a detrimental enzyme activity in the digestive tract (e) No spore formation, takes the form of a rod-shaped cell (f) Mouse spleen cells, A mixture of heat-sterilized microorganisms belonging to the genus Bifidobacterium is cultured for 72 hours, and then the ratio of the production of interferon-γ and the production of interleukin-4 in the supernatant is added to the microorganism. More than twice The Bifidobacterium microorganism according to claim 1 or 2, wherein the Bifidobacterium microorganism is Bifidobacterium sp. CFB1 strain (deposit number FERM P-21789).   A food or drink comprising the Bifidobacterium microorganism according to any one of claims 1 to 3.   The food or drink according to claim 4, which suppresses the production of interleukin-17.   The food or drink according to claim 4 or 5, which promotes production of interferon-γ and suppresses production of interleukin-4.   The food or drink according to any one of claims 4 to 6, which is a fermented milk product.   A pharmaceutical comprising the Bifidobacterium microorganism according to any one of claims 1 to 3.   The pharmaceutical product according to claim 8, which suppresses the production of interleukin-17.   The pharmaceutical product according to claim 8 or 9, which promotes production of interferon-γ and suppresses production of interleukin-4.

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