JP6666053B2 - IALD production promoter - Google Patents
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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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- 238000004519 manufacturing process Methods 0.000 title claims description 24
- 239000004480 active ingredient Substances 0.000 claims description 11
- 241001608472 Bifidobacterium longum Species 0.000 claims description 10
- 229940009291 bifidobacterium longum Drugs 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 description 20
- 241000186000 Bifidobacterium Species 0.000 description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 7
- 102100030703 Interleukin-22 Human genes 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 108010074109 interleukin-22 Proteins 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 230000001737 promoting effect Effects 0.000 description 6
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 5
- OLNJUISKUQQNIM-UHFFFAOYSA-N indole-3-carbaldehyde Chemical compound C1=CC=C2C(C=O)=CNC2=C1 OLNJUISKUQQNIM-UHFFFAOYSA-N 0.000 description 4
- 239000004310 lactic acid Substances 0.000 description 4
- 235000014655 lactic acid Nutrition 0.000 description 4
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 241000186014 Bifidobacterium angulatum Species 0.000 description 2
- 241000186011 Bifidobacterium catenulatum Species 0.000 description 2
- 239000012901 Milli-Q water Substances 0.000 description 2
- 102000015728 Mucins Human genes 0.000 description 2
- 108010063954 Mucins Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000003984 Aryl Hydrocarbon Receptors Human genes 0.000 description 1
- 108090000448 Aryl Hydrocarbon Receptors Proteins 0.000 description 1
- 241001134770 Bifidobacterium animalis Species 0.000 description 1
- 241000186016 Bifidobacterium bifidum Species 0.000 description 1
- 241000186148 Bifidobacterium pseudolongum Species 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000186604 Lactobacillus reuteri Species 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 1
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940118852 bifidobacterium animalis Drugs 0.000 description 1
- 229940002008 bifidobacterium bifidum Drugs 0.000 description 1
- 229940004120 bifidobacterium infantis Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 229940001882 lactobacillus reuteri Drugs 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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). .
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.
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 ＭｇＳＯ４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)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015071248A JP6666053B2 (en) | 2015-03-31 | 2015-03-31 | IALD production promoter |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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