JP6666053B2 - IALD production promoter - Google Patents

IALD production promoter Download PDF

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JP6666053B2
JP6666053B2 JP2015071248A JP2015071248A JP6666053B2 JP 6666053 B2 JP6666053 B2 JP 6666053B2 JP 2015071248 A JP2015071248 A JP 2015071248A JP 2015071248 A JP2015071248 A JP 2015071248A JP 6666053 B2 JP6666053 B2 JP 6666053B2
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iald
bifidobacterium
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bacterium
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新井利信
瀬戸泰幸
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Megmilk Snow Brand Co Ltd
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Description

本発明は、ビフィドバクテリウム属の菌(Bifidobacterium属)、特にビフィドバクテリウム・ロンガム(Bifidobacterium longum)の菌を有効成分とする、IALD(Indole−3−aldehyde)の産生促進剤に関する。   TECHNICAL FIELD The present invention relates to an agent for promoting the production of IALD (Indol-3-aldehyde), comprising a bacterium belonging to the genus Bifidobacterium (genus Bifidobacterium), particularly a bacterium of Bifidobacterium longum as an active ingredient.

トリプトファンから分解合成されるIALDはAhRレセプターを介してIL−22の産生を促進することが知られている。IL−22は抗菌ペプチドやムチンの産生を促進するので、有益なサイトカインである。   It is known that IALD decomposed and synthesized from tryptophan promotes production of IL-22 via an AhR receptor. IL-22 is a beneficial cytokine because it promotes the production of antimicrobial peptides and mucins.

ラクトバチルス・ロイテリ(Lactobacillus reuteri)がトリプトファンからIALD(Indole−3−aldehyde)の合成を促進する事が報告されている(非特許文献1)。   It has been reported that Lactobacillus reuteri promotes the synthesis of IALD (indole-3-aldehyde) from tryptophan (Non-Patent Document 1).

芳香族炭化水素レセプター(AhR)活性化能を有する乳酸菌、ビフィドバクテリウム属の菌およびプロピオン酸菌からなる群が示されているが、IALDに関する記載も示唆もされていない(特許文献1)。   A group consisting of a lactic acid bacterium having the ability to activate an aromatic hydrocarbon receptor (AhR), a bacterium belonging to the genus Bifidobacterium and a propionic acid bacterium is shown, but neither description nor suggestion regarding IALD is given (Patent Document 1). .

再公表特許WO2012/033151Republished Patent WO2012 / 033151

Zelante T, Iannitti RG, Cunha C, De Luca A, Giovannini G, Pieraccini G, Zecchi R, D’Angelo C, Massi−Benedetti C, Fallarino F, Carvalho A, Puccetti P, Romani L. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin−22. Immunity. 2013 Aug 22;39(2):372−85.Zelante T, Iannitti RG, Cunha C, De Luca A, Giovannini G, Pierracini G, Zecchi R, D'Angelo C, Massi-Benedicti C, Fallarpo, Italy. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity. 2013 Aug 22; 39 (2): 372-85.

上記のようにIALDの産生促進によって、IL−22の産生が促進され、抗菌ペプチドやムチンを産生する事が確認されている。そこで、本発明は効率的にIALDの産生を促進し得る剤の提供を課題とする。   As described above, it has been confirmed that by promoting the production of IALD, the production of IL-22 is promoted, and that antibacterial peptides and mucin are produced. Therefore, an object of the present invention is to provide an agent capable of efficiently promoting the production of IALD.

本発明者らは、数多くの乳酸菌を試験に供し、ビフィドバクテリウム属の菌、特にビフィドバクテリウム・ロンガムの菌がIALDを産生する機能が非常に高いことを見出し、本発明を完成させるに至った。
すなわち、本発明は以下の構成からなる。
(1)ビフィドバクテリウム属の菌を有効成分とする、IALD産生促進剤。
(2)ビフィドバクテリウム属の菌がビフィドバクテリウム・ロンガムの菌である、上記(1)に記載のIALD産生促進剤。
(3)ビフィドバクテリウム属の菌がSBT−2928(FERM P−10657)である、上記(1)に記載のIALD産生促進剤。
The present inventors have conducted a number of lactic acid bacteria tests and found that bacteria of the genus Bifidobacterium, particularly bacteria of the genus Bifidobacterium longum, have a very high function of producing IALD, thereby completing the present invention. Reached.
That is, the present invention has the following configurations.
(1) An IALD production promoter comprising a Bifidobacterium bacterium as an active ingredient.
(2) The IALD production promoter according to (1), wherein the bacterium of the genus Bifidobacterium is a bacterium of Bifidobacterium longum.
(3) The IALD production promoter according to the above (1), wherein the bacterium belonging to the genus Bifidobacterium is SBT-2928 (FERM P-10657).

本発明のビフィドバクテリウム属を有効成分とする、IALD産生促進剤によれば、効率的にIALDの産生を増加でき、これによってIL−22の産生を促進することにより、抗菌ペプチドやムチンの産生を増強する事ができる。   According to the IALD production promoter containing the Bifidobacterium genus of the present invention as an active ingredient, IALD production can be efficiently increased, thereby promoting the production of IL-22. Production can be enhanced.

各菌株のIALD産生能を比較したものである。It is a comparison of IALD-producing ability of each strain. 各菌株のIALD産生能を比較したものである。It is a comparison of IALD-producing ability of each strain.

本発明において、「IALD産生促進剤」とは、IALD(Indole−3−aldehyde)の産生を促進するための剤のことをいい、ビフィドバクテリウム属の菌を有効成分とする剤のことをいう。この剤は、有効成分であるビフィドバクテリウム属の菌のみからなる剤であってもよく、IALDの産生を抑制しないその他の物質を含む剤であってもよい。   In the present invention, the term "IALD production promoter" refers to an agent for promoting the production of IALD (indole-3-aldehyde), and refers to an agent containing a bacterium of the genus Bifidobacterium as an active ingredient. Say. This agent may be an agent comprising only a bacterium belonging to the genus Bifidobacterium, which is an active ingredient, or may be an agent containing another substance which does not suppress IALD production.

本発明において、「IALD」とは、インドール−3−アルデヒド(Indole−3−aldehyde)のことをいう。   In the present invention, "IALD" refers to indole-3-aldehyde.

本発明の「IALD産生促進剤」の有効成分であるビフィドバクテリウム属の菌として、ビフィドバクテリウム・アドレセンティス(Bifidobacterium adolescentis)、ビフィドバクテリウム・ロンガム(Bifidobacterium longum)、ビフィドバクテリウム・インファンティス(Bifidobacterium infantis)、ビフィドバクテリウム・シュードロンガム(Bifidobacterium pseudolongum)、ビフィドバクテリウム・ビフィダム(Bifidobacterium bifidum)、ビフィドバクテリウム・ブレイブ(Bifodobacterium breve)、ビフィドバクテリウム・シュードカテニュラタム(Bifidobacterium pseudocatenulatum)、ビフィドバクテリウム・カテニュラタム(Bifidobacterium catenulatum)、ビフィドバクテリウム・アングラタム(Bifidobacterium angulatum)またはビフィドバクテリウム・アニマリス(Bifidobacterium animalis)等の菌が挙げられ、これらを一種以上または二種以上組み合わせて含むものであっても良い。このうち、ビフィドバクテリウム・ロンガムの菌を有効成分とすることが好ましい。ビフィドバクテリウム・ロンガムの菌として、特にSBT−2928(FERM P−10657)を有効成分として含むことが好ましい。   Examples of Bifidobacterium genus bacteria that are the active ingredients of the "IALD production promoter" of the present invention include Bifidobacterium adressentis, Bifidobacterium longum, Bifidobacterium longum, and Bifidobacterium longum. -Bifidobacterium infantis, Bifidobacterium pseudolongum, Bifidobacterium bifidum, Bifidobacterium efibufib, and Bifidobacterium efibridobactivum. Catenulatum (Bifid but Bifidobacterium catenulatum, Bifidobacterium catenulatum, Bifidobacterium angulatum (Bifidobacterium angulatum) or Bifidobacterium animalis or Bifidium bacterium, and the like. These may be included in combination. Among them, it is preferable to use Bifidobacterium longum bacteria as an active ingredient. It is preferable to contain SBT-2928 (FERM P-10657) as an active ingredient, particularly as a bacterium of Bifidobacterium longum.

以下に、本発明の実施例をあげて本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。   Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the present invention is not limited thereto.

〔実施例1〕
1.1.菌体の準備
表1に示した乳酸菌11株及びビフィドバクテリウム属の菌12株を供試菌として使用した。乳酸菌はMRS液体培地にて、ビフィドバクテリウム属の菌はGAM液体培地にて37℃,16時間静置培養した。各菌体を遠心分離(1,912×g,4℃,10min)にて回収し、1mM MgSOを含有する100mM リン酸カリウム緩衝液(pH7、以下、緩衝液と呼ぶ)を用いて3回洗浄した。
[Example 1]
1.1. Preparation of bacterial cells 11 strains of lactic acid bacteria and 12 strains of the genus Bifidobacterium shown in Table 1 were used as test bacteria. Lactic acid bacteria were cultured in MRS liquid medium, and Bifidobacterium bacteria were cultured in GAM liquid medium at 37 ° C. for 16 hours. Each cell was collected by centrifugation (1,912 × g, 4 ° C., 10 min), and three times using 100 mM potassium phosphate buffer (pH 7, hereinafter referred to as buffer) containing 1 mM MgSO 4. Washed.

1.2.反応条件
反応は休止菌体系にて実施した。表1に示したように、菌体懸濁液を供試菌によってO.D.600=3.7〜7.9に調整して、次のA、Bの2つの反応液組成にて37℃で18時間、反応を行った。
反応液組成A:20mM トリプトファン
反応液組成B:20mM トリプトファン and 20mM α−ケトグルタル酸
1.2. Reaction conditions The reaction was performed in a quiescent bacterial system. As shown in Table 1, the cell suspension was subjected to O.D. D. The mixture was adjusted to 600 = 3.7 to 7.9 and reacted at 37 ° C. for 18 hours with the following two reaction liquid compositions of A and B.
Reaction solution composition A: 20 mM tryptophan Reaction solution composition B: 20 mM tryptophan and 20 mM α-ketoglutaric acid

1.3.反応産物の定量
反応液は遠心分離後(9,100×g,4℃,10min)、上清を回収し、フィルター(0.22μm)にて不溶物を除去したものをHPLC分析サンプルとした。分析はAgilent社の1200シリーズを使用した。溶離液A(95% Milli−Q water,5% Acetonitrile(ACN),0.05% Trifluoroacetic acid(TFA))で平衡化したODSカラム(Hydrosphere C18 粒子径(μm)S−5,細孔径 4.6X250,品番 HS12S05−2546WT,メーカー YMC)に分析サンプルを10μlインジェクトし、流速1ml/minとして溶離液B(5% Milli−Q water,95% ACN,0.05% TFA)の濃度勾配(%B(time):10(0)→50(16)→50(16)→100(16.1)→100(20)→10(20.1)→10(26))により行った。カラムオーブンは40℃とした。分光光度計にてIALDの吸収波長の試験を行った結果、IALDの検出は300nmにおける吸収を利用した。ここで、合成活性(Synthesizing activity)を、反応により生成したIALD合成量(Concentration. ppm)を供試菌のO.D.600の値で割った値と定義する(式1)。
1.3. Quantification of reaction product After the reaction solution was centrifuged (9,100 × g, 4 ° C., 10 min), the supernatant was recovered, and a filter (0.22 μm) from which insolubles were removed was used as an HPLC analysis sample. The analysis used Agilent 1200 series. 3. ODS column (Hydrosphere C18 particle size (μm) S-5, pore size) equilibrated with eluent A (95% Milli-Q water, 5% Acetonitrile (ACN), 0.05% Trifluoroacetic acid (TFA)). 6X250, product number HS12S05-2546WT, manufacturer YMC), inject 10 μl of the analysis sample, flow rate 1 ml / min, eluent B (5% Milli-Q water, 95% ACN, 0.05% TFA) concentration gradient (%) B (time): 10 (0) → 50 (16) → 50 (16) → 100 (16.1) → 100 (20) → 10 (20.1) → 10 (26)). The column oven was at 40 ° C. As a result of performing a test of the absorption wavelength of IALD with a spectrophotometer, the detection of IALD utilized absorption at 300 nm. Here, the synthesis activity (Synthesizing activity) is determined by the IALD synthesis amount (Concentration. Ppm) generated by the reaction. D. It is defined as a value divided by the value of 600 (Equation 1).

[式1]
[Equation 1]

結果を図1(反応液組成A)、図2(反応液組成B)に示した。
図1、図2より、α−ケトグルタル酸の有無に関わらず、ラクトバチルス属(Lactobacillus)と比較して、ビフィドバクテリウム属の菌(Bifidobacterium属)、特にビフィドバクテリウム・ロンガム(Bifidobacterium longum)のIALD産生能が優れていることが確認できた。また、その中でも特にSBT−2928(FERM P−10657)の効果が大きい事が確認できた。従って、これらのビフィドバクテリウム属の菌を有効成分として、IALD産生促進剤が提供できることが示された。
The results are shown in FIG. 1 (reaction solution composition A) and FIG. 2 (reaction solution composition B).
1 and 2, regardless of the presence or absence of α-ketoglutarate, compared to Lactobacillus, bacteria of the genus Bifidobacterium, in particular, Bifidobacterium longum. ) Was confirmed to be excellent in IALD-producing ability. In addition, it was confirmed that SBT-2928 (FERM P-10657) was particularly effective. Therefore, it was shown that an IALD production promoter could be provided using these Bifidobacterium bacteria as active ingredients.

本発明のビフィドバクテリウム属の有効成分とする、IALD産生促進剤によれば、効率的にIALDの産生を増加でき、これによってIL−22の産生を促進することにより、抗菌ペプチドやムチンの産生を増強する事ができる。   According to the IALD production promoter as an active ingredient of the genus Bifidobacterium of the present invention, the production of IALD can be efficiently increased, thereby promoting the production of IL-22. Production can be enhanced.

Claims (1)

ビフィドバクテリウム・ロンガムSBT−2928(FERM P−10657)を有効成分とするIALD産生促進剤。 An IALD production promoter comprising Bifidobacterium longum SBT-2928 (FERM P-10657) as an active ingredient.
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