JP2019017292A - Novel gum arabic assimilating bifidobacteria - Google Patents
Novel gum arabic assimilating bifidobacteria Download PDFInfo
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- JP2019017292A JP2019017292A JP2017138371A JP2017138371A JP2019017292A JP 2019017292 A JP2019017292 A JP 2019017292A JP 2017138371 A JP2017138371 A JP 2017138371A JP 2017138371 A JP2017138371 A JP 2017138371A JP 2019017292 A JP2019017292 A JP 2019017292A
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- bacterium
- bifidobacterium
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- gum arabic
- protein
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- 239000004299 sodium benzoate Substances 0.000 description 1
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- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
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- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
本発明は、新規ビフィドバクテリウム属細菌、新規アラビアガム分解酵素、及びそれらの用途に関する。 The present invention relates to a novel Bifidobacterium genus, a novel gum arabic degrading enzyme, and uses thereof.
アラビアガムは、アフリカ、ナイル地方原産のマメ科アカシア属アラビアゴムノキの樹皮の傷口からの分泌物を乾燥させたものである。日本では食品添加物指定されており、安全性も確認されている。乳化剤や安定剤として飲食品に広く用いられており、アイスクリームなどの菓子類や、ガムシロップが典型的な用途である。また医薬品の錠剤のコーティング剤や、絵具、インクなどの工業製品にも用いられている。 Gum arabic is dried from the cuts of the bark of the leguminous acacia genus Gum, native to the Nile region of Africa. In Japan, food additives are designated and safety has been confirmed. Widely used in food and drink as emulsifiers and stabilizers, confectionery such as ice cream and gum syrup are typical uses. It is also used in pharmaceutical tablets coatings, industrial products such as paints and inks.
アラビアガムは、ペプチド鎖にＩＩ型アラビノガラクタン（以下、「ＩＩ型ＡＧ」とも記載する）及びβ−アラビノオリゴ糖の糖鎖が付加した糖タンパク質（アラビノガラクタン・プロテイン（ＡＧＰ））で構成される（図１）。ＩＩ型ＡＧは、ＡＧＰのペプチド鎖に結合するβ１，３−ガラクタン主鎖に、β１，６−ガラクタン側鎖が付加した構造であり、その末端はアラビノースやラムノースやグルクロン酸などで修飾されている。ＩＩ型ＡＧの修飾糖のバリエーションは多岐にわたり、例えばアラビノースではフラノース型又はピラノース型がα結合又はβ結合で結合している。このような修飾糖の複雑性のために、アラビアガムは難消化性食物繊維の一つとされている。また、アラビアガムの他にも種々の野菜や果物にもＩＩ型ＡＧは含まれている。 Gum arabic is composed of a glycoprotein (arabinogalactan protein (AGP)) in which a sugar chain of type II arabinogalactan (hereinafter also referred to as “type II AG”) and β-arabino oligosaccharide is added to a peptide chain. (FIG. 1). Type II AG has a structure in which β1,6-galactan side chain is added to β1,3-galactan main chain that binds to the peptide chain of AGP, and its end is modified with arabinose, rhamnose, glucuronic acid, etc. . There are a wide variety of modified sugars of type II AG. For example, in arabinose, a furanose type or a pyranose type is linked by an α bond or a β bond. Due to the complexity of such modified sugars, gum arabic is considered one of the resistant fiber. In addition to gum arabic, type II AG is also contained in various vegetables and fruits.
これまでに、ビフィドバクテリウム・ロンガムの一部の菌とビフィドバクテリウム・アドレセンティスの一部の菌のみが、アラビアガムを利用して生育できる資化性を有することが報告されている（非特許文献２）。しかしながら、それらのビフィズス菌のアラビアガム分解のメカニズムは解明されていない。 So far, only some of Bifidobacterium longum and some of Bifidobacterium adrecentis have been reported to have assimilability that can be grown using gum arabic. (Non-patent document 2). However, the mechanism of the gum arabic degradation of these bifidobacteria has not been elucidated.
また、ヒト試験において、アラビアガムは腸内ビフィズス菌を増やす作用があることが報告されている（非特許文献１）。ＩＩ型ＡＧは、前述のとおり難消化性であるため、これを含有する植物を摂取した場合、通常は糖鎖構造が維持されて腸内に供給される。一般に腸内ビフィズス菌はオリゴ糖により増殖するが、腸内でＩＩ型ＡＧが何らかの分解作用を受けて、ビフィズス菌増殖に寄与するものと考えられている。 In human tests, gum arabic has been reported to have the effect of increasing intestinal bifidobacteria (Non-patent Document 1). Since type II AG is indigestible as described above, when a plant containing it is ingested, the sugar chain structure is usually maintained and supplied into the intestine. Intestinal bifidobacteria generally grow with oligosaccharides, but type II AG is considered to contribute to the growth of bifidobacteria by undergoing some degradation action in the intestine.
本発明者らはこれまでに、ＩＩ型ＡＧの分解に必須な酵素群の機能解析と、ビフィドバクテリウム・ロンガムＪＣＭ１２１７におけるＩＩ型ＡＧ分解代謝経路を明らかにした（非特許文献３）。しかしながら、前記ビフィドバクテリウム属細菌が有する糖質分解酵素（エキソ−β−１，３−ガラクタナーゼ）は、修飾糖が少ないカラマツ由来のＩＩ型ＡＧを分解するが、複雑な修飾糖を持つアラビアガム由来のＩＩ型ＡＧに対しては分解性を示さない。
アラビアガム由来のＩＩ型ＡＧは、その修飾糖を弱酸分解により除去すると、エキソ−β−１，３−ガラクタナーゼによる分解活性が１１．３倍に増加することも確認されている（非特許文献３）。このことから、アラビアガム資化性にはアラビアガムのＩＩ型ＡＧの修飾糖の分解・除去が大きく関与することが推測される。
The present inventors have so far clarified the functional analysis of enzyme groups essential for the degradation of type II AG and the type II AG degradation metabolic pathway in Bifidobacterium longum JCM1217 (Non-patent Document 3). However, the carbohydrase (exo-β-1,3-galactanase) possessed by the genus Bifidobacterium decomposes type II AG derived from larch with few modified sugars, but has complex modified sugars. Degradability is not shown for type II AG derived from gum arabic.
It has also been confirmed that type II AG derived from gum arabic increases the degradation activity by exo-β-1,3-galactanase by 11.3 times when the modified sugar is removed by weak acid degradation (non-patent document) 3). From this, it is speculated that degradation and removal of the modified sugar of type II AG of gum arabic is greatly involved in gum arabic utilization.
本発明は、アラビアガム資化性を有する新規なビフィドバクテリウム属細菌、及びアラビアガム由来のＩＩ型ＡＧの修飾糖を分解し得る酵素を提供することを課題とする。 An object of the present invention is to provide a novel bacterium belonging to the genus Bifidobacterium having an ability to assimilate gum arabic and an enzyme capable of degrading a modified sugar of type II AG derived from gum arabic.
本発明者らが鋭意研究を進めた結果、アラビアガム資化性を有する新規な５種のビフィドバクテリウム属細菌を見出した。また、かかる新規細菌が、アラビアガムから特定の二糖を遊離する分解活性を有する酵素を保持することを見出し、以下の本発明を完成させた。 As a result of diligent research conducted by the present inventors, five novel Bifidobacterium bacteria having arabic gum utilization were found. Moreover, it discovered that this novel bacterium hold | maintained the enzyme which has a decomposition activity which liberates specific disaccharide from gum arabic, and completed the following this invention.
本発明の第一の態様は、新規ビフィドバクテリウム属細菌であり、具体的には、ビフィドバクテリウム・ロンガムＭＣＣ０３００（ＮＩＴＥ ＢＰ−０２４９７）、ビフィドバクテリウム・ロンガムＭＣＣ１００４０（ＮＩＴＥ ＢＰ−０２４９８）、ビフィドバクテリウム・ロンガムＭＣＣ１００８５（ＮＩＴＥ ＢＰ−０２４９９）、ビフィドバクテリウム・ロンガムＭＣＣ１０１２７（ＮＩＴＥ ＢＰ−０２５００）、及びビフィドバクテリウム・ロンガムＭＣＣ１０１３０（ＮＩＴＥ ＢＰ−０２５０１）からなる群から選択されるビフィドバクテリウム属細菌である。 The first aspect of the present invention is a novel genus Bifidobacterium, specifically Bifidobacterium longum MCC0300 (NITE BP-02497), Bifidobacterium longum MCC10040 (NITE BP-02498) ), Bifidobacterium longum MCC10085 (NITE BP-02499), Bifidobacterium longum MCC10127 (NITE BP-02500), and Bifidobacterium longum MCC10130 (NITE BP-02501) Bifidobacterium.
本発明の第二の態様は、新規糖質分解酵素であり、具体的には以下の（Ａ）又は（Ｂ）のタンパク質である。
（Ａ）配列番号２の少なくとも第３７〜１１３６位に示されるアミノ酸配列を含むタンパク質
（Ｂ）配列番号２の少なくとも第３７〜１１３６位に示されるアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入、又は付加を含むアミノ酸配列を含み、かつ、エンドグリコシダーゼ活性を有するタンパク質
前記糖質分解酵素は、以下の（ａ）又は（ｂ）のＤＮＡでコードされるタンパク質でもある。
（ａ）配列番号１の少なくとも第１０９〜３４１１位に示される塩基配列を含むＤＮＡ
（ｂ）配列番号１の少なくとも第１０９〜３４１１位に示される塩基配列に相補的な塩基配列又は該相補配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、エンドグリコシダーゼ活性を有するタンパク質をコードするＤＮＡ
The second aspect of the present invention is a novel saccharide-degrading enzyme, specifically, the following protein (A) or (B).
(A) a protein comprising an amino acid sequence represented by at least positions 37 to 1136 of SEQ ID NO: 2 (B) substitution of one or several amino acids in the amino acid sequence represented by positions 37 to 1136 of SEQ ID NO: 2; A protein comprising an amino acid sequence containing a deletion, insertion, or addition and having endoglycosidase activity The glycolytic enzyme is also a protein encoded by the following DNA (a) or (b).
(A) DNA comprising a base sequence shown at least in positions 109 to 3411 of SEQ ID NO: 1
(B) hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown at least at positions 109 to 3411 of SEQ ID NO: 1 or a probe that can be prepared from the complementary sequence, and has endoglycosidase activity DNA encoding protein
本発明の第三の態様は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素（第二の態様のタンパク質）を含有する、飲食品組成物である。本態様の飲食品組成物は、好ましくはアラビアガム分解促進用である。 The third aspect of the present invention is the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the carbohydrase of the present invention (the protein of the second aspect). ) Containing the food and drink composition. The food / beverage product composition of this aspect is preferably for promoting gum arabic decomposition.
本発明の第四の態様は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素（第二の態様のタンパク質）を含有する、ビフィズス菌増殖促進助剤である。 The fourth aspect of the present invention is the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the carbohydrase of the present invention (the protein of the second aspect). ) Containing Bifidobacteria growth promoting aid.
本発明の第五の態様は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素（第二の態様のタンパク質）と、アラビアガムとを含有する、飲食品組成物である。本態様の飲食品組成物は、好ましくはビフィズス菌増殖促進用である。 The fifth aspect of the present invention is the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the saccharide-degrading enzyme of the present invention (the protein of the second aspect). ) And gum arabic. The food / beverage product composition of this aspect is preferably for promoting the growth of bifidobacteria.
本発明の第六の態様は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前
記細菌の菌体処理物、及び／又は本発明の糖質分解酵素（第二の態様のタンパク質）を、ＩＩ型アラビノガラクタンを含む原料に作用させる工程を含む、オリゴ糖の製造方法である。本態様において、前記オリゴ糖は、好ましくはＧａｌ−α１，３−Ａｒａ、Ａｒａｆ−β１，３−Ａｒａ、及び／又はＡｒａｐ−β１，３−Ａｒａである。
The sixth aspect of the present invention is the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the carbohydrase of the present invention (the protein of the second aspect). ) Is allowed to act on a raw material containing type II arabinogalactan. In this embodiment, the oligosaccharide is preferably Gal-α1,3-Ara, Araf-β1,3-Ara, and / or Arap-β1,3-Ara.
本発明の第七の態様は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素（第二の態様のタンパク質）を動物に投与することを含む、ビフィズス菌増殖促進方法である。 The seventh aspect of the present invention is the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the carbohydrase of the present invention (the protein of the second aspect). ) Is administered to an animal.
本発明の第八の態様は、配列番号１の塩基配列のうちの連続する２〜８塩基の配列を３’末端側に含む、１５〜３０塩基のＤＮＡからなるプライマー、及び配列番号１の塩基配列のうちの連続する２〜８塩基の配列に相補的な配列を３’末端側に含む、１５〜３０塩基のＤＮＡからなるプライマーからなるＰＣＲ用プライマーセットを含むキットである。本態様のキットは、好ましくはアラビアガム資化菌の検出用である。
本態様のキットを用いてＰＣＲを行う工程を含む、アラビアガム資化菌を検出する方法も提供される。
The eighth aspect of the present invention is a primer comprising 15 to 30 bases of DNA comprising the sequence of 2 to 8 bases in the base sequence of SEQ ID NO: 1 on the 3 ′ end side, and the base of SEQ ID NO: 1. A kit comprising a primer set for PCR comprising a primer comprising 15 to 30 bases of DNA comprising a sequence complementary to a sequence of 2 to 8 bases in the sequence on the 3 ′ end side. The kit of this embodiment is preferably for detection of gum arabic assimilating bacteria.
There is also provided a method for detecting gum arabic assimilating bacteria comprising the step of performing PCR using the kit of this embodiment.
本発明により、アラビアガム資化性を有する新規ビフィドバクテリウム属細菌が提供される。また、本発明により、アラビアガム由来のＩＩ型ＡＧの修飾糖を分解することができる新規糖質分解酵素が提供される。前記ビフィドバクテリウム属細菌や前記糖質分解酵素は、飲食品組成物や剤に含有させることにより、アラビアガムの分解用途、オリゴ糖の製造、又はビフィズス菌増殖促進用途に利用することができる。 INDUSTRIAL APPLICABILITY According to the present invention, a novel Bifidobacterium genus bacterium having gum arabic utilization is provided. The present invention also provides a novel saccharide-degrading enzyme capable of degrading a modified sugar of type II AG derived from gum arabic. The Bifidobacterium genus bacteria and the saccharide-degrading enzyme can be used for the application of degrading gum arabic, the production of oligosaccharides, or the application of promoting the growth of bifidobacteria by containing them in food and beverage compositions or agents. .
次に、本発明の実施形態について説明する。ただし、本発明は以下の実施形態に限定されず、本発明の範囲内で自由に変更することができる。 Next, an embodiment of the present invention will be described. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention.
＜ビフィドバクテリウム属細菌＞
本発明の第一の態様は、新規ビフィドバクテリウム属細菌である。具体的には、ビフィドバクテリウム・ロンガムＭＣＣ０３００（ＮＩＴＥ ＢＰ−０２４９７）、ビフィドバクテリウム・ロンガムＭＣＣ１００４０（ＮＩＴＥ ＢＰ−０２４９８）、ビフィドバクテリウム・ロンガムＭＣＣ１００８５（ＮＩＴＥ ＢＰ−０２４９９）、ビフィドバクテリウム・ロンガムＭＣＣ１０１２７（ＮＩＴＥ ＢＰ−０２５００）、又はビフィドバクテリウム・ロンガムＭＣＣ１０１３０（ＮＩＴＥ ＢＰ−０２５０１）である。以下、上
記５種類の新規ビフィドバクテリウム属細菌を包括して「本発明のビフィドバクテリウム属細菌」と記載することがある。
<Bifidobacterium genus>
The first aspect of the present invention is a novel genus Bifidobacterium. Specifically, Bifidobacterium longum MCC0300 (NITE BP-02497), Bifidobacterium longum MCC10040 (NITE BP-02498), Bifidobacterium longum MCC10085 (NITE BP-02499), Bifidobacteria Um longum MCC10127 (NITE BP-02500) or Bifidobacterium longum MCC10130 (NITE BP-02501). Hereinafter, the above five kinds of novel Bifidobacterium bacteria may be collectively referred to as “the Bifidobacterium bacteria of the present invention”.
本発明のビフィドバクテリウム属細菌は、アラビアガムを糖源として生育が可能であり、すなわちアラビアガム資化性を有する。本細菌は、アラビアガムのＩＩ型ＡＧの修飾糖から特定の二糖を遊離させることができる、後述の糖質分解酵素を保持するため、アラビアガムのＩＩ型ＡＧの修飾糖を分解し、その結果アラビアガムを資化することができるものである。 The genus Bifidobacterium of the present invention can grow using gum arabic as a sugar source, that is, has gum arabic utilization. This bacterium can release a specific disaccharide from the modified sugar of type II AG of gum arabic. In order to retain the below-mentioned saccharide-degrading enzyme, the modified sugar of type II AG of gum arabic is decomposed. As a result, gum arabic can be assimilated.
細菌がアラビアガム資化性を有するか否かは、例えば以下の方法により確認することができる。
すなわち、供試菌を、糖源をアラビアガムのみに変更したＭＲＳ（de Man-Rogosa-Sharpe）液体培地１０ｍＬに、１０８ＣＦＵ（Colony Forming Unit）を接種し、嫌気条件下で３７℃で２４時間培養したときの濁度（ＯＤ６００）が、０．３以上である場合、当該菌はアラビアガム資化性を有すると判断する。
Whether or not bacteria have gum arabic assimilation can be confirmed, for example, by the following method.
That is, 10 8 CFU (Colony Forming Unit) was inoculated into 10 mL of MRS (de Man-Rogosa-Sharpe) liquid medium in which the sugar source was changed to only gum arabic, and the test bacteria were incubated at 37 ° C. under anaerobic conditions. If the turbidity (OD 600 ) when cultivated for a period of time is 0.3 or more, the bacterium is judged to have assimilation ability for gum arabic.
本発明のビフィドバクテリウム属細菌の遺伝学的性質を調べるため、１６ＳｒＲＮＡ遺伝子塩基配列を常法により同定した。さらに、各ビフィドバクテリウム属細菌の１６ＳｒＲＮＡ遺伝子塩基配列について、米国立バイオテクノロジー情報センター（以下、「ＮＣＢＩ」とも記載する）のデータベースにて、ＢＬＡＳＴ（Basic Local Alignment Search
Tool）解析により前記塩基配列の相同性検索を行った。
その結果、本発明のビフィドバクテリウム属細菌５種は、表１に示す通りビフィドバクテリウム・ロンガムＫＣＴＣ ３１２８Ｔと前記塩基配列において９９％以上の相同性があり、ビフィドバクテリウム・ロンガムに属するビフィドバクテリウム属細菌であることが確認された。
本発明のビフィドバクテリウム属細菌５種は、平成２９年６月２２日に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（郵便番号：292-0818、住所：千葉県木更津市かずさ鎌足2-5-8 122号室）に、ブダペスト条約に基づく国際寄託がなされ、表１に示す受託番号がそれぞれ付与されている。
In order to investigate the genetic properties of the Bifidobacterium of the present invention, the 16S rRNA gene base sequence was identified by a conventional method. Furthermore, the BLAST (Basic Local Alignment Search) database for the 16S rRNA gene base sequence of each genus Bifidobacterium is obtained from the database of the National Center for Biotechnology Information (hereinafter also referred to as “NCBI”).
Tool) The homology search of the base sequence was performed by analysis.
As a result, the five Bifidobacterium bacteria of the present invention have 99% or more homology with Bifidobacterium longum KCTC 3128T in the base sequence as shown in Table 1, and Bifidobacterium longum has It was confirmed that the bacterium belongs to the genus Bifidobacterium.
The five species of the genus Bifidobacterium of the present invention were incorporated on June 22, 2017 by the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Kazusa Kamashita, Kisarazu City, Chiba Prefecture) 2-5-8 Room 122) has been deposited internationally based on the Budapest Treaty and given the deposit numbers shown in Table 1.
本発明のビフィドバクテリウム属細菌は、上記寄託菌に制限されず、同寄託菌と実質的に同等の細菌であってもよい。実質的に同等の細菌とは、本発明のビフィドバクテリウム属細菌と同種属の細菌であって、上記寄託菌と同程度の高いアラビアガム資化性を有する細菌をいう。また、実質的に同等の細菌は、１６ＳｒＲＮＡ遺伝子の塩基配列が、上記寄託菌の１６ＳｒＲＮＡ遺伝子の塩基配列と９８％以上、好ましくは９９％以上、より好ましくは１００％の相同性を有し、かつ、好ましくは上記寄託菌と同一の菌学的性質を有する。さらに、本発明のビフィドバクテリウム属細菌は、本発明の効果が損なわれない限り、寄託菌、又はそれと実質的に同等の細菌から、変異処理、遺伝子組換え、自然変異株の選択等によって育種された変異株であってもよい。 The bacterium belonging to the genus Bifidobacterium of the present invention is not limited to the above-mentioned deposited bacterium, and may be a bacterium substantially equivalent to the deposited bacterium. The substantially equivalent bacterium refers to a bacterium belonging to the same genus as the Bifidobacterium genus bacterium of the present invention and having a high gum arabic assimilation ability similar to that of the deposited bacterium. In addition, the substantially equivalent bacterium has a homology of 98% or more, preferably 99% or more, more preferably 100%, with the base sequence of the 16S rRNA gene of the 16S rRNA gene of the deposited bacterium, and Preferably, it has the same bacteriological properties as the deposited bacteria. Further, the Bifidobacterium genus bacterium of the present invention can be selected from a deposited bacterium or a bacterium substantially equivalent thereto by mutation treatment, gene recombination, selection of a natural mutant, etc., as long as the effects of the present invention are not impaired. It may be a bred mutant.
本発明のビフィドバクテリウム属細菌又は当該ビフィドバクテリウム属細菌の培養物は、常法により当該ビフィドバクテリウム属細菌を培養することにより容易に取得できる。培養する方法は、ビフィドバクテリウム属細菌が増殖できる限り特に限定されず、当該細菌の性質に応じた適当な条件下で培養を行うことができる。例えば、培養温度は２５〜５０℃でよく、３５〜４２℃であることが好ましい。また培養は嫌気条件下で行うことが好ましく、例えば、炭酸ガス等の嫌気ガスを通気しながら培養することができる。また、液体静置培養等の微好気条件下で培養してもよい。 The Bifidobacterium bacterium of the present invention or the culture of the Bifidobacterium bacterium can be easily obtained by culturing the Bifidobacterium bacterium by a conventional method. The culture method is not particularly limited as long as Bifidobacterium can grow, and culture can be performed under appropriate conditions according to the nature of the bacteria. For example, the culture temperature may be 25 to 50 ° C, and preferably 35 to 42 ° C. Moreover, it is preferable to perform culture | cultivation on anaerobic conditions, for example, it can culture | cultivate, ventilating anaerobic gas, such as a carbon dioxide gas. Moreover, you may culture | cultivate on microaerobic conditions, such as liquid stationary culture.
本発明のビフィドバクテリウム属細菌を培養する培地としては、特に限定されず、ビフィドバクテリウム属細菌の培養に通常用いられる培地を用いることができる。すなわち、炭素源としては、アラビアガムの他に、例えば、グルコース、ガラクトース、ラクトース、アラビノース、マンノース、スクロース、デンプン、デンプン加水分解物、廃糖蜜等の糖類を資化性に応じて使用できる。窒素源としては、例えば、アンモニア、硫酸アンモニウム、塩化アンモニウム、硝酸アンモニウムなどのアンモニウム塩類や硝酸塩類を使用できる。また、無機塩類としては、例えば、塩化ナトリウム、塩化カリウム、リン酸カリウム、硫酸マグネシウム、塩化カルシウム、硝酸カルシウム、塩化マンガン、硫酸第一鉄等を用いることができる。また、ペプトン、大豆粉、脱脂大豆粕、肉エキス、酵母エキス等の有機成分を用いてもよい。 The medium for culturing the Bifidobacterium of the present invention is not particularly limited, and a medium usually used for culturing the Bifidobacterium can be used. That is, as the carbon source, for example, sugars such as glucose, galactose, lactose, arabinose, mannose, sucrose, starch, starch hydrolyzate, and molasses can be used depending on the assimilation property in addition to gum arabic. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic components such as peptone, soybean powder, defatted soybean meal, meat extract, yeast extract and the like may also be used.
本発明のビフィドバクテリウム属細菌は、後述の飲食品組成物、ビフィズス菌増殖促進助剤（間接的にビフィズス菌の生育及び増殖を促進させる薬剤）、オリゴ糖の製造方法、又はビフィズス菌増殖方法の態様を含め、細菌そのもの、培養物、又は菌体処理物の形態で用いることができる。すなわち、培養した後、得られた培養物をそのまま用いてもよく、希釈又は濃縮して用いてもよく、培養物から回収した菌体を用いてもよい。また、本発明において用いられるビフィドバクテリウム属細菌は、生菌であっても死菌であってもよく、生菌と死菌との両方を含むものでもよい。さらに、ビフィドバクテリウム属細菌の菌体処理物であってもよく、菌体処理物としては、例えば、菌体をアクリルアミドやカラギーナン等で固定化した固定化菌体、ビフィドバクテリウム属細菌の菌体の細胞壁及び細胞膜が超音波処理やホモジナイザー処理等の常法により一部又は完全に破砕された細胞破砕物、その遠心分離上清（水溶性画分）、その上清を硫安処理等で部分精製した画分、又は前記上清を濃縮したもの等が挙げられる。 The Bifidobacterium genus bacterium of the present invention includes a food / beverage product composition, a bifidobacteria growth promoting aid (an agent that indirectly promotes the growth and proliferation of bifidobacteria), a method for producing an oligosaccharide, or a bifidobacteria growth described below. It can be used in the form of a bacterium itself, a culture, or a treated microbial cell, including a method embodiment. That is, after culturing, the obtained culture may be used as it is, diluted or concentrated, or cells recovered from the culture may be used. Moreover, the Bifidobacterium genus bacteria used in the present invention may be live or dead, and may include both live and dead. Furthermore, it may be a cell-treated product of Bifidobacterium genus. Examples of the cell-treated product include immobilized cells obtained by immobilizing cells with acrylamide, carrageenan, etc., Bifidobacterium bacteria Cell walls and cell membranes of microbial cells in which cells are partially or completely crushed by conventional methods such as ultrasonic treatment and homogenizer treatment, centrifuged supernatant (water-soluble fraction), and the supernatant treated with ammonium sulfate, etc. Or a fraction obtained by partially purifying the supernatant.
＜糖質分解酵素＞
本発明の第二の態様は、新規糖質分解酵素であり、具体的には、配列番号２の少なくとも第３７〜１１３６位に示されるアミノ酸配列を含むタンパク質である。
<Glycolytic enzyme>
The second aspect of the present invention is a novel saccharide-degrading enzyme, specifically, a protein comprising the amino acid sequence shown at least at positions 37 to 1136 of SEQ ID NO: 2.
また、本発明の糖質分解酵素は、後述するエンドグリコシダーゼ活性が損なわれない限り、配列番号２の少なくとも第３７〜１１３６位に示されるアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入、又は付加の変異が加えられたアミノ酸配列であってもよい。
ここで、１個又は数個とは、アミノ酸残基のタンパク質の立体構造における位置や種類によっても異なるが、好ましくは１〜３０個、より好ましくは１〜２０個、さらに好ましくは１〜１０個、特に好ましくは１〜５個である。
かかる変異としては、置換が好ましく、アミノ酸の種類を変更しない範囲での置換が望ましい。すなわち、前記配列におけるアミノ酸と、同アミノ酸を置換するアミノ酸は構造上の分類で類似種類のアミノ酸であることが望ましい。具体的には前記配列のアミノ酸が酸性アミノ酸又は酸性アミノ酸アミドである場合はアスパラギン酸、グルタミン酸、アスパラギン、又はグルタミンのうちのいずれかとの置換、塩基性アミノ酸である場合はリジン、ヒスチジン、又はアルギニンのうちのいずれかとの置換、芳香族アミノ酸である場合
はフェニルアラニン、チロシン、又はトリプトファンのうちのいずれかとの置換、脂肪族アミノ酸又はオキシアミノ酸である場合は、グリシン、アラニン、バリン、ロイシン、イソロイシン、セリン又はスレオニンのうちのいずれかとの置換、及び前記以外のアミノ酸（システイン、メチオニン、プロリン等）の場合はエンドグリコシダーゼ活性を損なわない範囲で任意に置換することが望ましい。なお、本明細書において、アミノ酸はＬ体である。
Further, the carbohydrase of the present invention is a substitution or deletion of one or several amino acids in the amino acid sequence shown at least at positions 37 to 1136 of SEQ ID NO: 2 unless the endoglycosidase activity described later is impaired. An amino acid sequence to which an insertion or addition mutation is added may also be used.
Here, one or several amino acid residues vary depending on the position and type of protein in the three-dimensional structure of the amino acid residue, but preferably 1-30, more preferably 1-20, and even more preferably 1-10. Particularly preferably, 1 to 5.
As such mutation, substitution is preferable, and substitution within a range that does not change the type of amino acid is desirable. That is, it is desirable that the amino acid in the sequence and the amino acid that substitutes the same amino acid are amino acids of similar types in structural classification. Specifically, when the amino acid of the sequence is an acidic amino acid or acidic amino acid amide, it is substituted with any of aspartic acid, glutamic acid, asparagine, or glutamine, and when it is a basic amino acid, lysine, histidine, or arginine. Substitution with any of these, substitution with any of phenylalanine, tyrosine, or tryptophan if it is an aromatic amino acid, glycine, alanine, valine, leucine, isoleucine, serine if it is an aliphatic amino acid or oxyamino acid Alternatively, in the case of substitution with any of threonine and amino acids other than those described above (cysteine, methionine, proline, etc.), it is desirable to arbitrarily substitute as long as endoglycosidase activity is not impaired. In addition, in this specification, an amino acid is L-form.
本発明の糖質分解酵素は、配列番号１の少なくとも第１０９〜３４１１位に示される塩基配列を含むＤＮＡでコードされるタンパク質でもある。 The saccharide-degrading enzyme of the present invention is also a protein encoded by DNA comprising a base sequence shown at least at positions 109 to 3411 of SEQ ID NO: 1.
また、本発明の糖質分解酵素は、後述するエンドグリコシダーゼ活性が損なわれない限り、配列番号１の少なくとも第１０９〜３４１１位に示される塩基配列に相補的な塩基配列又は該相補配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズするＤＮＡでコードされるタンパク質であってもよい。
前記ストリンジェントな条件とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。一例を示せば、相同性が高いDNA同士、例えば８０％以上、好ましくは９０％以上、より好ましくは９５％以上、より好ましくは９７％、特に好ましくは９９％以上の相同性を有するＤＮＡ同士がハイブリダイズし、それより相同性が低いＤＮＡ同士がハイブリダイズしない条件、例えば、４２℃でのハイブリダイゼーション、及び１×ＳＳＣ（Saline Sodium Citrate Buffer）、０．１％のＳＤＳ（Sodium Dodecyl Sulfate）を含む緩衝液による４２℃での洗浄処理を挙げることができ、６５℃でのハイブリダイゼーション、及び０．１×ＳＳＣ、０．１％のＳＤＳを含む緩衝液による６５℃での洗浄処理をより好ましくは挙げることができる。なお、ハイブリダイゼーションのストリンジェンシーに影響を与える要素としては、上記温度条件以外に種々の要素があり、当業者であれば、種々の要素を適宜組み合わせて、上記例示したハイブリダイゼーションのストリンジェンシーと同等のストリンジェンシーを実現することが可能である。
ハイブリダイゼーションに用いるプローブは、配列番号１の少なくとも第１０９〜３４１１位に示される塩基配列の相補配列の一部であってもよい。例えば、プローブとして、３００ｂｐ程度の長さのＤＮＡ断片を用いる場合には、ハイブリダイゼーションの洗いの条件は、２×ＳＳＣ、０．１％のＳＤＳを含む緩衝液による５０℃での洗浄処理が挙げられる。
In addition, the saccharide-degrading enzyme of the present invention is prepared from a base sequence complementary to the base sequence shown at least at positions 109 to 3411 of SEQ ID NO: 1 or the complementary sequence so long as endoglycosidase activity described later is not impaired. It may be a protein encoded by DNA that hybridizes with the resulting probe under stringent conditions.
The stringent condition refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, DNAs having high homology, for example, DNAs having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97%, particularly preferably 99% or more. Conditions under which hybridization occurs and DNAs with lower homology do not hybridize, for example, hybridization at 42 ° C., 1 × SSC (Saline Sodium Citrate Buffer), 0.1% SDS (Sodium Dodecyl Sulfate) And washing treatment at 42 ° C. with a buffer solution containing, more preferably, hybridization at 65 ° C. and washing treatment at 65 ° C. with a buffer solution containing 0.1 × SSC, 0.1% SDS. Can be mentioned. In addition, there are various factors other than the above temperature conditions as factors affecting the stringency of hybridization, and those skilled in the art will be able to combine various components as appropriate to achieve the same stringency of hybridization as exemplified above. Stringency can be realized.
The probe used for hybridization may be a part of a complementary sequence of the base sequence shown at least at positions 109 to 3411 of SEQ ID NO: 1. For example, when a DNA fragment having a length of about 300 bp is used as a probe, the washing conditions for hybridization include a washing treatment at 50 ° C. with a buffer containing 2 × SSC and 0.1% SDS. It is done.
本発明の糖質分解酵素は、本発明のビフィドバクテリウム属細菌が共通してその遺伝子を保有する酵素である。
本発明者らは、本発明のビフィドバクテリウム属細菌のゲノム配列について、遺伝子予測ソフトであるProdigal（v2.50）（http://prodigal.ornl.gov/）を用いて遺伝子領域を推定した。そして、予測された遺伝子のアミノ酸配列について、ＮＣＢＩのタンパク質データベースnrを用いて相同性検索を行い、遺伝子の機能予測を行ったところ、本発明のビフィドバクテリウム属細菌に共通して保存された、アラビアガム分解代謝を担う遺伝子クラスターを見出した。さらに、前記遺伝子クラスター内には、糖質加水分解酵素ドメインを有するタンパク質が存在することが見出された。すなわち、本発明の糖質分解酵素である、ＩＩ型ＡＧエンド１，３−α−Ｌ−アラビノフラノシダーゼ（以下、「ＥＡＦａｓｅ」とも記載する）である。
配列番号１は、ビフィドバクテリウム・ロンガムのＥＡＦａｓｅ遺伝子の塩基配列であり、配列番号２はそのアミノ酸配列である。
The saccharide-degrading enzyme of the present invention is an enzyme commonly possessed by the Bifidobacterium bacterium of the present invention.
The present inventors estimated the gene region of the genome sequence of the genus Bifidobacterium of the present invention using Prodigal (v2.50) (http://prodigal.ornl.gov/), which is a gene prediction software. did. The amino acid sequence of the predicted gene was subjected to homology search using the NCBI protein database nr, and the function of the gene was predicted. As a result, it was conserved in common with the Bifidobacterium of the present invention. A gene cluster responsible for the degradation metabolism of gum arabic was found. Furthermore, it was found that a protein having a carbohydrate hydrolase domain exists in the gene cluster. That is, it is a type II AG endo 1,3-α-L-arabinofuranosidase (hereinafter also referred to as “EAFase”), which is a saccharide-degrading enzyme of the present invention.
SEQ ID NO: 1 is the base sequence of the EAFase gene of Bifidobacterium longum, and SEQ ID NO: 2 is its amino acid sequence.
InterPro（https://www.ebi.ac.uk/interpro/）を用いてドメイン検索を行ったところ、ＥＡＦａｓｅは、Ｎ末端側（配列番号２の第１〜３６位を含む）にシグナルペプチド（ＳＰ）を有することが予測された。また、Ｃ末端側に膜貫通領域（ＴＭ）を有し、菌体表
層に局在化する酵素であることが予測された（図２）。これにより、本発明のビフィドバクテリウム属細菌を細菌そのもの、培養物、又は菌体処理物の形態で用いる場合においても、後述のようにＥＡＦａｓｅの活性を発揮することができる。
さらに、Protein Homology/analogY Recognition Engine V 2.0（http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index）を用いて糖質加水分解酵素ドメインの構造予測を行ったところ、既存酵素との類似性が低いことが分かった。そのため、本発明の糖質分解酵素であるＥＡＦａｓｅは、新規の糖質加水分解酵素ファミリー（ＧＨファミリー）に属すると考えられる。
When domain search was performed using InterPro (https://www.ebi.ac.uk/interpro/), EAFase was found to have a signal peptide (including positions 1-36 of SEQ ID NO: 2) on the N-terminal side (including SEQ ID NO: 2). SP) was predicted. Moreover, it was estimated that it is an enzyme which has a transmembrane region (TM) on the C-terminal side and is localized on the surface of the cell (FIG. 2). Thereby, even when using the Bifidobacterium genus bacteria of this invention with the form of bacteria itself, a culture, or a microbial cell processed material, the activity of EAFase can be exhibited as mentioned later.
Furthermore, the structure of carbohydrate hydrolase domain using Protein Homology / analogY Recognition Engine V 2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) As a result of the prediction, it was found that the similarity with existing enzymes was low. Therefore, EAFase, which is the saccharide-degrading enzyme of the present invention, is considered to belong to a novel saccharide-hydrolyzing enzyme family (GH family).
ＥＡＦａｓｅのアミノ酸配列について、ＮＣＢＩのタンパク質データベースnrを用いて相同性検索を行ったところ、１つのタンパク質を除き、ホモログは見つからなかった。ヒットしたタンパク質（ビフィドバクテリウム・ロンガムのhypothetical protein（WP_077381861.1））は、nrデータベースにアミノ酸配列が登録されているものの、機能については明らかにされていない。かかる登録アミノ酸配列は、遺伝子予測ソフトなどを用いて予測された遺伝子の塩基配列をアミノ酸配列に変換しただけのものと推測される。
したがって、ＥＡＦａｓｅは、配列及び機能が新たに見出された新規の糖質分解酵素である。また、ＥＡＦａｓｅは、ビフィドバクテリウム・ロンガム以外の細菌の既知のタンパク質中には見出されず、かつビフィドバクテリウム・ロンガムのうち特定の菌種のみが保有する酵素である。
When the homology search was performed for the amino acid sequence of EAFase using NCBI protein database nr, no homolog was found except for one protein. Although the amino acid sequence of the hit protein (Bifidobacterium longum hypothetical protein (WP_077381861.1)) is registered in the nr database, its function has not been clarified. Such a registered amino acid sequence is presumed to be the one obtained by converting the base sequence of a gene predicted using gene prediction software or the like into an amino acid sequence.
Therefore, EAFase is a novel saccharide-degrading enzyme whose sequence and function are newly found. EAFase is not found in known proteins of bacteria other than Bifidobacterium longum, and is an enzyme possessed only by a specific bacterial species among Bifidobacterium longum.
本発明の糖質分解酵素であるＥＡＦａｓｅは、糖鎖内のＬ−Ａｒａｆと３位で結合したＡｒａｐ又はＧａｌｐの２位及び３位の構造を認識し、前記Ｌ−Ａｒａｆのα１，３構造を切断する、エンドグリコシダーゼ活性を有する。認識部位がＧａｌ−ＡｒａとＡｒａ−Ａｒａの二種あるのは、β−Ａｒａｐとα−Ｇａｌｐとが類似する構造であるからと考えられる。なお、β−Ａｒａｆに対しても、弱いながら認識することができる。
なお、本明細書において「Ａｒａ」はアラビノース、「Ｇａｌ」はガラクトースをそれぞれ表し、これらの後ろの「ｐ」はピラノース環構造、「ｆ」はフラノース環構造をそれぞれ表す。
EAFase, which is a saccharide-degrading enzyme of the present invention, recognizes the structures at positions 2 and 3 of Arap or Galp bonded to L-Araf in the sugar chain at the 3-position, and the α1,3 structure of the L-Araf is recognized. Has endoglycosidase activity to cleave. It is thought that there are two types of recognition sites, Gal-Ara and Ara-Ara, because β-Arap and α-Galp have similar structures. Note that β-Araf can be recognized although it is weak.
In this specification, “Ara” represents arabinose, “Gal” represents galactose, “p” behind them represents a pyranose ring structure, and “f” represents a furanose ring structure.
図３を参照して説明すると、ＥＡＦａｓｅは、アラビアガムのＩＩ型ＡＧ中の糖側鎖においては、６，４分岐を持つＧａｌに付加された網掛け部分のＧａｌｐ−α１，３−Ａｒａｆ−α１，３−を認識し、Ｌ−Ａｒａｆ−α１，３構造を切断して、Ｇａｌｐ−α１，３−Ａｒａｆ（以下、「ＧＡ」とも記載する）を遊離する（図３（ａ）、（ｂ））。なお、アラビアガムのＩＩ型ＡＧ中の糖側鎖のバリエーションはこれに限らず多岐にわたって存在するが、ＥＡＦａｓｅは他に、６，４分岐を持つＧａｌに付加されたＡｒａｐ−β１，３−Ａｒａｆ−α１，３−を認識し、Ｌ−Ａｒａｆ−α１，３構造を切断して、Ａｒａｐ−β１，３−Ａｒａｆ（以下、「ＡＡ」とも記載する）を遊離することもできる。
なお、アラビアガムのＩＩ型ＡＧには、ＧＡとＡＡとが７：１（モル比）で含まれていることが知られている（C.A. Tischer et al., Carbohydr. Poly. 47: 151-158 (2002)）。ＥＡＦａｓｅは、これらの二糖をアラビアガムから切断し、遊離させることができる酵素であるともいえる。これまでにＧＡやＡＡを遊離させる酵素は知られていないため、ＥＡＦａｓｅは新規な機能を有する酵素である。
Referring to FIG. 3, EAFase is a shaded portion of Galp-α1,3-Araf-α1 added to Gal having 6,4 branches in the sugar side chain in type II AG of gum arabic. , 3- is recognized, and the L-Araf-α1,3 structure is cleaved to release Galp-α1,3-Araf (hereinafter also referred to as “GA”) (FIGS. 3A and 3B). ). In addition, the variation of sugar side chain in type II AG of gum arabic is not limited to this, but EAFase is also Arap-β1,3-Araf- added to Gal having 6, 4 branches. It is also possible to recognize α1,3- and cleave the L-Araf-α1,3 structure to release Arap-β1,3-Araf (hereinafter also referred to as “AA”).
It is known that type II AG of gum arabic contains GA and AA in a ratio of 7: 1 (molar ratio) (CA Tischer et al., Carbohydr. Poly. 47: 151-158). (2002)). It can be said that EAFase is an enzyme that can cleave these disaccharides from gum arabic and release them. Since no enzyme that releases GA or AA has been known so far, EAFase is an enzyme having a novel function.
また、ＥＡＦａｓｅは、カラマツのＩＩ型ＡＧ中の糖側鎖の一例においては、Ａｒａｐ−β１，３−Ａｒａｆ−α１，３−を認識し、Ｌ−Ａｒａｆ−α１，３構造を切断して、ＡＡを遊離する（図３（ｃ））。ＥＡＦａｓｅは、イネのＩＩ型ＡＧ中の糖側鎖の一例においては、Ａｒａｆ−β１，３−Ａｒａｆ−α１，３−を認識し、Ｌ−Ａｒａｆ−α１，３構造を切断して、Ａｒａｆ−β１，３−Ａｒａｆを遊離する（図３（ｄ））。 In addition, EAFase recognizes Arap-β1,3-Araf-α1,3- and cleaves L-Araf-α1,3 structure in an example of sugar side chain in type II AG of larch, Is released (FIG. 3 (c)). In one example of sugar side chains in rice type II AG, EAFase recognizes Araf-β1,3-Araf-α1,3-, cleaves L-Araf-α1,3 structure, and Araf-β1 , 3-Araf is released (FIG. 3 (d)).
本発明の糖質分解酵素がエンドグリコシダーゼ活性を発揮する条件は、特に限定されな
いが、例えば温度は３５〜５５℃が好ましく、４５〜５０℃がより好ましい。また、ｐＨは４．０〜７．０が好ましく、５．０〜６．０がより好ましい。
The conditions under which the saccharide-degrading enzyme of the present invention exhibits endoglycosidase activity is not particularly limited, but for example, the temperature is preferably 35 to 55 ° C, more preferably 45 to 50 ° C. Moreover, 4.0-7.0 are preferable and, as for pH, 5.0-6.0 are more preferable.
ここで、エンドグリコシダーゼ活性は、例えば以下の方法により確認することができる。
すなわち、Ｌ−Ａｒａｆと３位で結合したＡｒａｐ又はＧａｌｐの構造を有する糖鎖に本発明の糖質分解酵素を反応させて、ＨＰＡＥＣ−ＰＡＤ（High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection）等のクロマトグラフィー法により生成した二糖を検出し、その量から酵素活性を算出すればよい。
Here, endoglycosidase activity can be confirmed, for example, by the following method.
That is, a sugar chain having the structure of Arap or Galp bonded to L-Araf at the 3-position is reacted with the carbohydrase of the present invention to perform chromatography such as HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection). What is necessary is just to detect the disaccharide produced | generated by the chromatography method and to calculate an enzyme activity from the quantity.
本発明の糖質分解酵素は、本発明のビフィドバクテリウム属細菌から公知の方法により精製して製造することができる。
また、遺伝子工学的手法を用いて適当な宿主細菌に組換えタンパク質を産生させることにより製造してもよい。このとき、ＥＡＦａｓｅ遺伝子のシグナルペプチドのコード領域を含む全遺伝子配列を宿主に導入して発現させ、宿主内でのプロセッシングを経て本発明の糖質分解酵素の成熟タンパク質を取得してもよいし、ＥＡＦａｓｅ遺伝子のシグナルペプチドのコード領域を除いた遺伝子配列を宿主に発現させてもよい。
The saccharide-degrading enzyme of the present invention can be purified and produced from the Bifidobacterium genus bacterium of the present invention by a known method.
Moreover, you may manufacture by making a suitable host bacterium produce a recombinant protein using a genetic engineering technique. At this time, the entire gene sequence including the coding region of the signal peptide of the EAFase gene may be introduced into the host for expression, and the mature protein of the glycolytic enzyme of the present invention may be obtained through processing in the host. A gene sequence excluding the coding region of the signal peptide of the EAFase gene may be expressed in the host.
＜ビフィズス菌増殖促進助剤＞
本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素は、ビフィズス菌増殖促進助剤の有効成分として利用することができる。
前述の通り、本発明のビフィドバクテリウム属細菌は本発明の糖質分解酵素を保持し、本発明の糖質分解酵素はエンドグリコシダーゼ活性によりＩＩ型ＡＧの糖鎖からオリゴ糖を切り出し遊離することができる。
<Bifidobacteria growth promoter>
The Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the saccharide-degrading enzyme of the present invention can be used as an active ingredient of a Bifidobacterium growth promoting aid. it can.
As described above, the Bifidobacterium genus bacterium of the present invention retains the saccharide-degrading enzyme of the present invention, and the saccharide-degrading enzyme of the present invention cleaves and releases oligosaccharides from the sugar chains of type II AG by endoglycosidase activity. be able to.
近年、ビフィズス菌に代表されるヒトにとって有用な細菌（プロバイオティクス）を増やす難消化性の食品素材が、プレバイオティクスとして注目されている。現在、９種類のオリゴ糖がビフィズス菌を増やす効果がある特定保健用食品として認められている。また、ラクト−Ｎ−ビオース（Ｇａｌ−１，３−ＧｌｃＮＡｃ（ＬＮＢ））が，乳幼児のビフィズス菌を選択的に増やす増殖因子であることが明らかにされている(T. Katayama, Biosci. Biotechnol. Biochem. 80:621-632 (2016)、M. Kitaoka., Adv. Nutr. 3:422S-429S (2012))。
アラビアガムやカラマツ等のＩＩ型ＡＧを含む原料からＥＡＦａｓｅによって切り出され遊離するオリゴ糖（Ｇａｌ−α１，３−Ａｒａ、Ａｒａｐ−β１，３−Ａｒａ、Ａｒａｆ−β１，３−Ａｒａ等）は、既知のプレバイオティクスオリゴ糖とは異なる構造を有する二糖である。これらの二糖は、ビフィズス菌、好ましくはＥＡＦａｓｅを保有するビフィズス菌の菌体内で該菌が保有するエキソグリコシダーゼ（α−ガラクトシダーゼ、β−Ｌ−アラビノピラノシダーゼ、又はβ−Ｌ−アラビノフラノシダーゼ）により単糖に分解され資化され得る。したがって、これらの二糖は、ビフィズス菌、特にビフィドバクテリウム・ロンガムやビフィドバクテリウム・アドレセンティスを選択的に増殖させ得る高選択性プレバイオティクスオリゴ糖として利用し得る。
また、これらの二糖が切り出された残余の糖鎖を含むＩＩ型ＡＧは、別の加水分解酵素（エキソグリコシダーゼ等）による分解を受けやすくなるため、ビフィズス菌に資化され得る。
したがって、本発明のビフィズス菌増殖促進助剤は、そのエンドグリコシダーゼ活性によって、間接的にビフィズス菌の生育及び増殖を促進させる作用を有する。
In recent years, indigestible food materials that increase useful bacteria (probiotics) represented by bifidobacteria have attracted attention as prebiotics. At present, nine types of oligosaccharides are recognized as foods for specified health use that have the effect of increasing bifidobacteria. In addition, it has been clarified that lacto-N-biose (Gal-1,3-GlcNAc (LNB)) is a growth factor that selectively increases bifidobacteria in infants (T. Katayama, Biosci. Biotechnol. Biochem. 80: 621-632 (2016), M. Kitaoka., Adv. Nutr. 3: 422S-429S (2012)).
Oligosaccharides (Gal-α1,3-Ara, Arap-β1,3-Ara, Araf-β1,3-Ara, etc.) that are cut out from a raw material containing type II AG such as gum arabic and larch and released by EAFase are known. It is a disaccharide having a different structure from the prebiotic oligosaccharide. These disaccharides are bisglycosides, preferably exoglycosidase (α-galactosidase, β-L-arabinopyranosidase, or β-L-arabino) possessed by Bifidobacterium containing EAFase. Furanosidase) can be decomposed into monosaccharides and utilized. Therefore, these disaccharides can be used as highly selective prebiotic oligosaccharides capable of selectively growing Bifidobacteria, particularly Bifidobacterium longum and Bifidobacterium adrecentis.
In addition, type II AG containing the remaining sugar chain from which these disaccharides are cut out is susceptible to degradation by another hydrolase (such as exoglycosidase), and thus can be assimilated by bifidobacteria.
Therefore, the bifidobacteria growth promoting aid of the present invention has an action of indirectly promoting the growth and proliferation of bifidobacteria by its endoglycosidase activity.
本発明のビフィズス菌増殖促進助剤は、経口投与及び非経口投与のいずれでもよく、投与方法に応じて、適宜所望の剤形に製剤化することができる。例えば、経口投与の場合、
散剤、顆粒剤、錠剤、カプセル剤等の固形製剤；溶液剤、シロップ剤、懸濁剤、乳剤等の液剤等に製剤化することができる。また、非経口投与の場合、座剤、軟膏剤又は点眼剤等に製剤化することができる。
The bifidobacteria growth promoting aid of the present invention may be either oral or parenteral, and can be appropriately formulated into a desired dosage form according to the administration method. For example, in the case of oral administration,
It can be formulated into solid preparations such as powders, granules, tablets and capsules; liquids such as solutions, syrups, suspensions and emulsions. In the case of parenteral administration, it can be formulated into a suppository, an ointment, an eye drop or the like.
また、製剤化に際しては、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素の他に、通常製剤化に用いられている賦形剤、ｐＨ調整剤、着色剤、矯味剤等の成分を用いることができる。
加えて、製剤化は剤形に応じて適宜公知の方法により実施できる。製剤化に際しては、適宜、製剤担体を配合して製剤化してもよい。
In addition, in addition to the Bifidobacterium bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the saccharide-degrading enzyme of the present invention, the formulation is usually used for formulation. Ingredients such as excipients, pH adjusters, colorants, and flavoring agents can be used.
In addition, formulation can be appropriately performed by a known method according to the dosage form. Upon formulation, a formulation carrier may be appropriately blended to formulate.
本発明のビフィズス菌増殖促進助剤の摂取量又は投与量は、剤形に合わせて適宜選択することができるが、例えば、体重１ｋｇあたりの１日の本発明のビフィドバクテリウム属細菌の摂取量又は投与量として、１×１０６〜１×１０１２ＣＦＵ／ｋｇ／日が好ましく、１×１０７〜１×１０１１ＣＦＵ／ｋｇ／日がより好ましく、１×１０８〜１×１０１０ＣＦＵ／ｋｇ／日がさらに好ましい。本発明のビフィドバクテリウム属細菌の培養物や前記細菌の菌体処理物を用いる場合のその摂取量又は投与量は、本発明のビフィドバクテリウム属細菌の摂取量又は投与量に換算された場合に上記摂取量又は投与量となることが好ましい。また、例えば、体重１ｋｇあたりの１日の本発明の糖質分解酵素の摂取量又は投与量として、１μｇ／ｋｇ／日が好ましく、１０μｇ／ｋｇ／日がより好ましく、１００μｇ／ｋｇ／日がさらに好ましい。 The intake or dose of the bifidobacteria growth promoting aid of the present invention can be appropriately selected according to the dosage form. For example, the intake of the Bifidobacterium of the present invention per day per kg of body weight The amount or dose is preferably 1 × 10 6 to 1 × 10 12 CFU / kg / day, more preferably 1 × 10 7 to 1 × 10 11 CFU / kg / day, and more preferably 1 × 10 8 to 1 × 10 10. More preferred is CFU / kg / day. The intake or dose of the Bifidobacterium bacterium culture of the present invention or the processed bacterial cell product is converted to the intake or dose of the Bifidobacterium bacterium of the present invention. The above intake or dose is preferable. In addition, for example, the intake or dose of the saccharide-degrading enzyme of the present invention per 1 kg of body weight per day is preferably 1 μg / kg / day, more preferably 10 μg / kg / day, further 100 μg / kg / day. preferable.
また、本発明のビフィズス菌増殖促進助剤中の本発明のビフィドバクテリウム属細菌の含有量は、前記摂取量に基づいて適宜選択することができるが、例えば、１×１０６〜１×１０１２ＣＦＵ／ｇ、好ましくは１×１０７〜１×１０１１ＣＦＵ／ｇ、より好ましくは１×１０８〜１×１０１０ＣＦＵ／ｇとすることができる。本発明のビフィドバクテリウム属細菌の培養物や前記細菌の菌体処理物を用いる場合のその含有量は、本発明のビフィドバクテリウム属細菌の含有量に換算された場合に上記含有量となることが好ましい。また、本発明のビフィズス菌増殖促進助剤中の本発明の糖質分解酵素の含有量は、例えば、１μｇ／ｇ、好ましくは１０μｇ／ｇ、より好ましくは１００μｇ／ｇとすることができる。 Further, the content of the Bifidobacterium genus bacterium of the present invention in the Bifidobacterium growth-promoting assistant of the present invention can be appropriately selected based on the above intake, but for example, 1 × 10 6 to 1 × 10 12 CFU / g, preferably 1 × 10 7 to 1 × 10 11 CFU / g, more preferably 1 × 10 8 to 1 × 10 10 CFU / g. The content of the culture of the Bifidobacterium genus bacterium of the present invention or the processed product of the bacterium is the above content when converted to the content of the Bifidobacterium bacterium of the present invention. It is preferable that In addition, the content of the saccharide-degrading enzyme of the present invention in the Bifidobacterium growth promoting aid of the present invention can be, for example, 1 μg / g, preferably 10 μg / g, more preferably 100 μg / g.
なお、前記単位のうちＣＦＵは、colony forming unitsの略であり、コロニー形成単位である。該細菌が死菌の場合、ＣＦＵは個細胞（cells）と置き換えることができる。 Of the above units, CFU is an abbreviation for colony forming units and is a colony forming unit. If the bacterium is dead, the CFU can be replaced with cells.
また、前記製剤担体としては、剤形に応じて、各種有機又は無機の担体を用いることができる。固形製剤の場合の担体としては、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯味矯臭剤等が挙げられる。 In addition, as the preparation carrier, various organic or inorganic carriers can be used depending on the dosage form. Examples of the carrier in the case of a solid preparation include excipients, binders, disintegrants, lubricants, stabilizers, and flavoring agents.
賦形剤としては、例えば、乳糖、白糖、ブドウ糖、マンニット、ソルビット等の糖誘導体；トウモロコシデンプン、馬鈴薯デンプン、α−デンプン、デキストリン、カルボキシメチルデンプン等のデンプン誘導体；結晶セルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム等のセルロース誘導体；アラビアガム；デキストラン；プルラン；軽質無水珪酸、合成珪酸アルミニウム、メタ珪酸アルミン酸マグネシウム等の珪酸塩誘導体；リン酸カルシウム等のリン酸塩誘導体；炭酸カルシウム等の炭酸塩誘導体；硫酸カルシウム等の硫酸塩誘導体等が挙げられる。 Examples of the excipient include sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbit; starch derivatives such as corn starch, potato starch, α-starch, dextrin and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Cellulose derivatives such as hydroxypropylmethylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate and magnesium magnesium aluminosilicate; phosphate derivatives such as calcium phosphate; And carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate and the like.
結合剤としては、例えば、上記賦形剤の他、ゼラチン；ポリビニルピロリドン；マクロゴール等が挙げられる。 Examples of the binder include gelatin, polyvinyl pyrrolidone, macrogol and the like in addition to the above excipients.
崩壊剤としては、例えば、上記賦形剤の他、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドン等の化学修飾されたデンプン又はセルロース誘導体等が挙げられる。 Examples of the disintegrant include, in addition to the above excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.
滑沢剤としては、例えば、タルク；ステアリン酸；ステアリン酸カルシウム、ステアリン酸マグネシウム等のステアリン酸金属塩；コロイドシリカ；ピーガム、ゲイロウ等のワックス類；硼酸；グリコール；フマル酸、アジピン酸等のカルボン酸類；安息香酸ナトリウム等のカルボン酸ナトリウム塩；硫酸ナトリウム等の硫酸塩類；ロイシン；ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウム等のラウリル硫酸塩；無水珪酸、珪酸水和物等の珪酸類；デンプン誘導体等が挙げられる。 As the lubricant, for example, talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and geirow; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid Carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic acid hydrate; starch derivatives and the like It is done.
安定剤としては、例えば、メチルパラベン、プロピルパラベン等のパラオキシ安息香酸エステル類；クロロブタノール、ベンジルアルコール、フェニルエチルアルコール等のアルコール類；塩化ベンザルコニウム；無水酢酸；ソルビン酸等が挙げられる。 Examples of the stabilizer include paraoxybenzoates such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like.
矯味矯臭剤としては、例えば、甘味料、酸味料、香料等が挙げられる。
なお、経口投与用の液剤の場合に使用する担体としては、水等の溶剤、矯味矯臭剤等が挙げられる。
Examples of the flavoring agent include sweeteners, acidulants, and fragrances.
In addition, as a carrier used in the case of a liquid for oral administration, a solvent such as water, a flavoring agent and the like can be mentioned.
本発明のこの態様の別の側面は、ビフィズス菌増殖促進助剤の製造における、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素の使用であるということもできる。
また、この態様の別の側面は、ビフィズス菌増殖促進における本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素の使用であるということもできる。
Another aspect of this embodiment of the present invention is a bifidobacteria genus bacterium of the present invention, a culture of the bacterium, a treated product of the bacterium, and / or the present invention in the production of a Bifidobacterium growth promoting aid. It can also be said that this is the use of carbohydrase.
In addition, another aspect of this embodiment is the use of the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the saccharide-degrading enzyme of the present invention in promoting the growth of bifidobacteria. It can also be used.
この態様の別の側面は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素を動物に投与することを含む、ビフィズス菌増殖促進方法であるともいえる。ここで、動物は、特に限定されないが、通常はヒトである。 Another aspect of this embodiment comprises administering to the animal the Bifidobacterium bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the glycolytic enzyme of the present invention. It can be said that this is a method for promoting the growth of bifidobacteria. Here, the animal is not particularly limited, but is usually a human.
＜飲食品組成物＞
本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素は、飲食品組成物に含有させることができる。
かかる飲食品組成物は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素を公知の飲食品に添加することによって製造してもよいし、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素を飲食品の原料中に混合して新たな飲食品組成物として製造することもできる。
<Food and beverage composition>
The Bifidobacterium bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the saccharide-degrading enzyme of the present invention can be contained in a food or drink composition.
Such a food / beverage product composition is obtained by adding the Bifidobacterium genus bacterium of the present invention, a culture of the bacterium, a treated product of the bacterium, and / or a saccharide-degrading enzyme of the present invention to a known food / beverage product. The Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the saccharide-degrading enzyme of the present invention are mixed in the raw material of the food and drink And it can also manufacture as a new food-drinks composition.
また、本発明飲食品組成物は、液状、ペースト状、固体、粉末等の形態を問わず、錠菓、流動食、飼料（ペット用を含む）等のほか、例えば、小麦粉製品、即席食品、農産加工品、水産加工品、畜産加工品、乳・乳製品、油脂類、基礎調味料、複合調味料・食品類、冷凍食品、菓子類、飲料、これら以外の市販品等が挙げられる。 Moreover, this invention food-drinks composition is liquid, paste-form, solid, powder etc. regardless of forms, tablet confectionery, liquid food, feed (including pet use) etc., for example, flour products, instant foods, Examples include processed agricultural products, processed fishery products, processed livestock products, milk / dairy products, fats and oils, basic seasonings, compound seasonings / foods, frozen foods, confectionery, beverages, and other commercial products.
本発明の飲食品組成物の摂取量は、適宜選択することができるが、例えば、体重１ｋｇあたりの１日の本発明のビフィドバクテリウム属細菌の摂取量として、１×１０６〜１×１０１２ＣＦＵ／ｋｇ／日が好ましく、１×１０７〜１×１０１１ＣＦＵ／ｋｇ／日がより好ましく、１×１０８〜１×１０１０ＣＦＵ／ｋｇ／日がさらに好ましい。本発明のビフィドバクテリウム属細菌の培養物や前記細菌の菌体処理物を用いる場合のその摂取量は
、本発明のビフィドバクテリウム属細菌の摂取量に換算された場合に上記摂取量又は投与量となることが好ましい。また、例えば、体重１ｋｇあたりの１日の本発明の糖質分解酵素の摂取量として、１μｇ／ｋｇ／日が好ましく、１０μｇ／ｋｇ／日がより好ましく、１００μｇ／ｋｇ／日がさらに好ましい。
The intake of the food / beverage product composition of the present invention can be selected as appropriate. For example, the intake of the Bifidobacterium of the present invention per day per kg of body weight is 1 × 10 6 to 1 ×. 10 12 CFU / kg / day is preferable, 1 × 10 7 to 1 × 10 11 CFU / kg / day is more preferable, and 1 × 10 8 to 1 × 10 10 CFU / kg / day is more preferable. The amount of intake in the case of using the Bifidobacterium bacterium culture of the present invention or the bacterial cell processed product is converted into the amount of the Bifidobacterium genus of the present invention. Or it becomes preferable that it becomes a dosage. Further, for example, the daily intake amount of the glycolytic enzyme of the present invention per kg of body weight is preferably 1 μg / kg / day, more preferably 10 μg / kg / day, and further preferably 100 μg / kg / day.
また、本発明の飲食品組成物中の本発明のビフィドバクテリウム属細菌の含有量は、前記摂取量に基づいて適宜選択することができるが、例えば、１×１０６〜１×１０１２ＣＦＵ／ｇ、好ましくは１×１０７〜１×１０１１ＣＦＵ／ｇ、より好ましくは１×１０８〜１×１０１０ＣＦＵ／ｇとすることができる。本発明のビフィドバクテリウム属細菌の培養物や前記細菌の菌体処理物を用いる場合のその含有量は、本発明のビフィドバクテリウム属細菌の含有量に換算された場合に上記含有量となることが好ましい。また、本発明の飲食品組成物中の本発明の糖質分解酵素の含有量は、例えば、１μｇ／ｇ、好ましくは１０μｇ／ｇ、より好ましくは１００μｇ／ｇとすることができる。 Moreover, although content of the Bifidobacterium genus bacteria of this invention in the food-drinks composition of this invention can be suitably selected based on the said intake, for example, 1 * 10 < 6 > -1 * 10 < 12 >. CFU / g, preferably 1 × 10 7 to 1 × 10 11 CFU / g, more preferably 1 × 10 8 to 1 × 10 10 CFU / g. The content of the culture of the Bifidobacterium genus bacterium of the present invention or the processed product of the bacterium is the above content when converted to the content of the Bifidobacterium bacterium of the present invention. It is preferable that Moreover, content of the saccharide-degrading enzyme of this invention in the food-drinks composition of this invention can be 1 microgram / g, for example, Preferably it is 10 microgram / g, More preferably, it can be 100 microgram / g.
前述の通り、本発明のビフィドバクテリウム属細菌は本発明の糖質分解酵素を保持し、本発明の糖質分解酵素はエンドグリコシダーゼ活性によりアラビアガムのＩＩ型ＡＧの糖鎖からオリゴ糖（ＧＡ及びＡＡ）を切り出し遊離することができる。かかる切り出しにより分解を受けた修飾糖の残余の糖鎖は、別の加水分解酵素（エキソグリコシダーゼ等）による分解を受けやすくなる。
そのため、本発明の飲食品組成物は、アラビアガム分解促進の用途に好ましく供することができる。
As described above, the bacterium belonging to the genus Bifidobacterium of the present invention retains the saccharide-degrading enzyme of the present invention, and the saccharide-degrading enzyme of the present invention has oligosaccharide (from the sugar chain of type II AG of gum arabic by endoglycosidase activity. GA and AA) can be excised and released. The remaining sugar chain of the modified sugar that has been degraded by such excision is likely to be degraded by another hydrolase (such as exoglycosidase).
Therefore, the food-drinks composition of this invention can be preferably provided for the use of gum arabic decomposition promotion.
本発明のこの態様の別の側面は、アラビアガム分解促進用飲食品組成物の製造における、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素の使用であるということもできる。
また、この態様の別の側面は、アラビアガム分解促進における本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素の使用であるということもできる。
この態様の別の側面は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素を動物に投与することを含む、アラビアガム分解促進方法であるともいえる。ここで、動物は、特に限定されないが、通常はヒトである。
Another aspect of this embodiment of the present invention is to provide a Bifidobacterium bacterium of the present invention, a culture of the bacterium, a treated product of the bacterium, and / or Or it can be said that it is use of the saccharide-degrading enzyme of this invention.
In addition, another aspect of this embodiment is the use of the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated bacterial body of the bacterium, and / or the carbohydrase of the present invention in promoting gum arabic degradation. It can also be used.
Another aspect of this embodiment comprises administering to the animal the Bifidobacterium bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the glycolytic enzyme of the present invention. It can also be said that this is a method for promoting gum arabic decomposition. Here, the animal is not particularly limited, but is usually a human.
本発明の飲食品組成物は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素に加えて、ＩＩ型ＡＧを含む原料をさらに含有させることができる。
ここで、前記原料はＩＩ型ＡＧを含むものであれば特に限定されず、例えばアラビアガム、カラマツ、イネ、ダイコン、サツマイモ、小麦、リンゴ、ブドウ、エンドウ、トウモロコシ、ニンジン等が挙げられる。特に、アラビアガムは、プレバイオティクス食物繊維として知られており、飲食用途での安全性も認められているため、好ましい。
前述の本発明のビフィズス菌増殖促進助剤で説明した通り、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素は、ＩＩ型ＡＧの分解を介して、腸内のビフィズス菌の増殖を促進させることができる。
そのため、ＩＩ型ＡＧを含む原料を、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素とともに含有する飲食品組成物は、ビフィズス菌増殖促進の用途に好ましく供することができる。
In addition to the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the saccharide-degrading enzyme of the present invention, the food and drink composition of the present invention is a type II AG. The raw material containing can be further contained.
Here, the raw material is not particularly limited as long as it contains type II AG, and examples thereof include gum arabic, larch, rice, radish, sweet potato, wheat, apple, grape, pea, corn, carrot and the like. In particular, gum arabic is known as a prebiotic dietary fiber and is preferred because it is recognized as safe for food and drink.
As explained in the aforementioned Bifidobacterium growth promoting aid of the present invention, the Bifidobacterium genus of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or the carbohydrase of the present invention Can promote the growth of Bifidobacteria in the intestine through degradation of type II AG.
Therefore, a food / beverage composition containing a raw material containing type II AG together with the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the processed bacterial cell of the bacterium, and / or the saccharide-degrading enzyme of the present invention. The product can be preferably used for the purpose of promoting the growth of bifidobacteria.
本発明のこの態様の別の側面は、ビフィズス菌増殖促進用飲食品組成物の製造における、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及
び／又は本発明の糖質分解酵素と、ＩＩ型ＡＧを含む原料との使用であるということもできる。
また、この態様の別の側面は、ビフィズス菌増殖促進における本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素と、ＩＩ型ＡＧを含む原料との使用であるということもできる。
この態様の別の側面は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素と、ＩＩ型ＡＧを含む原料とを動物に投与することを含む、ビフィズス菌増殖促進方法であるともいえる。ここで、動物は、特に限定されないが、通常はヒトである。
Another aspect of this embodiment of the present invention is to provide a Bifidobacterium growth-promoting food or drink composition, wherein the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated product of the bacterium, and / or Or it can also be said that it is use of the saccharide-degrading enzyme of this invention, and the raw material containing II type AG.
In addition, another aspect of this embodiment is the bifidobacteria genus bacteria of the present invention in promoting the growth of bifidobacteria, the culture of the bacteria, the processed bacterial body of the bacteria, and / or the carbohydrase of the present invention. It can also be said that it is the use with the raw material containing II type AG.
Another aspect of this embodiment is a raw material comprising the Bifidobacterium bacterium of the present invention, a culture of the bacterium, a treated product of the bacterium, and / or a saccharide-degrading enzyme of the present invention, and type II AG It can also be said that this is a method for promoting the growth of bifidobacteria comprising administering to the animal. Here, the animal is not particularly limited, but is usually a human.
また、本発明で定義される飲食品組成物は、アラビアガムの分解、ビフィズス菌の増殖、又はオリゴ糖が有効に作用する疾患の予防、疾患のリスク低減、疾患の症状緩和、及び／又は疾患の治療等の用途（保健用途を含む）が表示された飲食品として提供・販売されることが可能である。
「表示」行為には、需要者に対して前記用途を知らしめるための全ての行為が含まれ、前記用途を想起・類推させ得るような表現であれば、表示の目的、表示の内容、表示する対象物・媒体等の如何に拘わらず、全て本発明の「表示」行為に該当する。
In addition, the food and drink composition defined in the present invention includes the degradation of gum arabic, the growth of bifidobacteria, or the prevention of diseases in which oligosaccharides effectively act, the risk reduction of diseases, the alleviation of disease symptoms, and / or the diseases It can be provided / sold as a food / beverage product displaying the use (including health use) of the treatment.
The “display” act includes all acts for informing the consumer of the use, and if the expression can remind the user of the use, the purpose of the display, the content of the display, the display Regardless of the target object / medium, etc., all fall under the “display” act of the present invention.
また、「表示」は、需要者が上記用途を直接的に認識できるような表現により行われることが好ましい。具体的には、飲食品に係る商品又は商品の包装に前記用途を記載したものを譲渡し、引き渡し、譲渡若しくは引き渡しのために展示し、輸入する行為、商品に関する広告、価格表若しくは取引書類に上記用途を記載して展示し、若しくは頒布し、又はこれらを内容とする情報に上記用途を記載して電磁気的（インターネット等）方法により提供する行為等が挙げられる。 Moreover, it is preferable that the “display” is performed by an expression that allows the consumer to directly recognize the above-described use. Specifically, it is the act of transferring, displaying, importing, displaying, or importing products that are related to food or drinks or products that describe the use, on advertisements, price lists, or transaction documents. For example, an act of describing and displaying the above uses or distributing them, or describing the above uses in information including the contents and providing them by an electromagnetic (Internet or the like) method can be given.
一方、表示内容としては、行政等によって認可された表示（例えば、行政が定める各種制度に基づいて認可を受け、そのような認可に基づいた態様で行う表示等）であることが好ましい。また、そのような表示内容を、包装、容器、カタログ、パンフレット、ＰＯＰ等の販売現場における宣伝材、その他の書類等へ付することが好ましい。 On the other hand, the display content is preferably a display approved by the government or the like (for example, a display that is approved based on various systems determined by the government and performed in a mode based on such approval). Moreover, it is preferable to attach such display contents to advertising materials at sales sites such as packaging, containers, catalogs, pamphlets, POPs, and other documents.
また、「表示」には、健康食品、機能性食品、経腸栄養食品、特別用途食品、保健機能食品、特定保健用食品、栄養機能食品、機能性表示食品、医薬用部外品等としての表示も挙げられる。この中でも特に、消費者庁によって認可される表示、例えば、特定保健用食品、栄養機能食品、若しくは機能性表示食品に係る制度、又はこれらに類似する制度にて認可される表示等が挙げられる。具体的には、特定保健用食品としての表示、条件付き特定保健用食品としての表示、身体の構造や機能に影響を与える旨の表示、疾病リスク減少表示、科学的根拠に基づいた機能性の表示等を挙げることができ、より具体的には、健康増進法に規定する特別用途表示の許可等に関する内閣府令（平成二十一年八月三十一日内閣府令第五十七号）に定められた特定保健用食品としての表示（特に保健の用途の表示）及びこれに類する表示が典型的な例である。 In addition, “labeling” includes health food, functional food, enteral nutrition food, special purpose food, health functional food, food for specified health use, nutrition functional food, functional label food, quasi-drug, etc. A display is also included. Among these, in particular, indications approved by the Consumer Affairs Agency, for example, indications approved in systems related to foods for specified health use, functional nutritional foods, functional indication foods, or similar systems, etc. can be mentioned. Specifically, labeling as a food for specified health use, labeling as a conditionally specified food for specified health use, labeling that affects the structure and function of the body, labeling for reducing the risk of disease, and functionality based on scientific evidence Labeling, etc., and more specifically, Cabinet Office Ordinance concerning permission for special purpose labeling provided for in the Health Promotion Act (Cabinet Office Ordinance No. 57, August 31, 2000) The labeling as food for specified health (particularly the labeling of health use) and the like are the typical examples.
＜オリゴ糖の製造方法＞
本発明のオリゴ糖の製造方法は、本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素を、ＩＩ型ＡＧを含む原料に作用させる工程を含む。
前述の通り、本発明のビフィドバクテリウム属細菌は本発明の糖質分解酵素を保持し、本発明の糖質分解酵素はエンドグリコシダーゼ活性によりＩＩ型ＡＧの糖鎖からオリゴ糖を切り出し遊離することができるため、オリゴ糖の製造が実現される。
本発明の製造方法により製造されるオリゴ糖は、特に限定されないが、通常はアラビノースの還元末端を有するオリゴ糖であり、例えばＧａｌ−１，３−ＡｒａもしくはＧａｌ
−１，３−Ａｒａ−Ｘ、又はＡｒａ−１，３−ＡｒａもしくはＡｒａ−１，３−Ａｒａ−Ｘである（Ｘは任意の単糖又は二糖〜五糖のオリゴ糖を表す）。
<Method for producing oligosaccharide>
The oligosaccharide production method of the present invention comprises a Bifidobacterium genus bacterium of the present invention, a culture of the bacterium, a treated product of the bacterium, and / or a saccharide-degrading enzyme of the present invention, and a type II AG. A step of acting on the raw material.
As described above, the Bifidobacterium genus bacterium of the present invention retains the saccharide-degrading enzyme of the present invention, and the saccharide-degrading enzyme of the present invention cleaves and releases oligosaccharides from the sugar chains of type II AG by endoglycosidase activity. Production of oligosaccharides is realized.
The oligosaccharide produced by the production method of the present invention is not particularly limited, but is usually an oligosaccharide having a reducing end of arabinose, such as Gal-1,3-Ara or Gal.
-1,3-Ara-X, or Ara-1,3-Ara or Ara-1,3-Ara-X (X represents any monosaccharide or disaccharide to pentasaccharide oligosaccharide).
本発明のビフィドバクテリウム属細菌、前記細菌の培養物、前記細菌の菌体処理物、及び／又は本発明の糖質分解酵素を、ＩＩ型ＡＧを含む原料に作用させる工程を行う条件は、特に限定されないが、通常は本発明の糖質分解酵素がそのエンドグリコシダーゼ活性を発揮できる条件で行われる。なお、かかる条件は、前述の本発明の糖質分解酵素の説明に準じる。 Conditions for performing the step of allowing the Bifidobacterium genus bacterium of the present invention, the culture of the bacterium, the treated bacterial body of the bacterium, and / or the saccharide-degrading enzyme of the present invention to act on a raw material containing type II AG are as follows: Although not particularly limited, it is usually carried out under conditions that allow the carbohydrase of the present invention to exert its endoglycosidase activity. Such conditions are in accordance with the description of the saccharide-degrading enzyme of the present invention described above.
原料に作用させる本発明のビフィドバクテリウム属細菌の量は、原料の種類や反応条件によって適宜定めることができるが、例えば、原料１ｇに対する量として、１×１０６〜１×１０１２ＣＦＵ／ｇ、好ましくは１×１０７〜１×１０１１ＣＦＵ／ｇ、より好ましくは１×１０８〜１×１０１０ＣＦＵ／ｇとすることができる。本発明のビフィドバクテリウム属細菌の培養物や前記細菌の菌体処理物を用いる場合の量は、本発明のビフィドバクテリウム属細菌の量に換算された場合に上記量となることが好ましい。
原料に作用させる本発明の糖質分解酵素の量は、例えば、原料１ｇに対する量として、１μｇ／ｇ、好ましくは１０μｇ／ｇ、より好ましくは１００μｇ／ｇとすることができる。
The amount of the genus Bifidobacterium of the present invention to act on the raw material can be appropriately determined depending on the type of raw material and reaction conditions. For example, the amount per 1 g of the raw material is 1 × 10 6 to 1 × 10 12 CFU / g, preferably 1 × 10 7 to 1 × 10 11 CFU / g, more preferably 1 × 10 8 to 1 × 10 10 CFU / g. The amount in the case of using the culture of Bifidobacterium of the present invention or the treated product of the bacterium may be the above amount when converted to the amount of the Bifidobacterium of the present invention. preferable.
The amount of the saccharide-degrading enzyme of the present invention that acts on the raw material can be, for example, 1 μg / g, preferably 10 μg / g, and more preferably 100 μg / g, based on 1 g of the raw material.
本発明の製造方法において、原料はＩＩ型ＡＧを含むものであれば特に限定されず、例えばアラビアガム、カラマツ、イネ、ダイコン、サツマイモ、小麦、リンゴ、ブドウ、エンドウ、トウモロコシ、ニンジン等が挙げられ、目的とするオリゴ糖の種類によって適宜選択すればよい。例えば、アラビアガムを原料としたときは、Ｇａｌ−α１，３−Ａｒａ（ＧＡ）、及びＡｒａｐ−β１，３−Ａｒａ（ＡＡ）を選択的に製造することができる。また、カラマツを原料としたときは、Ａｒａｐ−β１，３−Ａｒａ（ＡＡ）を選択的に製造することができる。また、イネを原料にしたときは、Ａｒａｐ−β１，３−Ａｒａｆ、及びＡｒａｆ−β１，３−Ａｒａを製造することができる。 In the production method of the present invention, the raw material is not particularly limited as long as it contains type II AG, and examples thereof include gum arabic, larch, rice, radish, sweet potato, wheat, apple, grape, pea, corn, carrot and the like. The selection may be made as appropriate depending on the type of the target oligosaccharide. For example, when gum arabic is used as a raw material, Gal-α1,3-Ara (GA) and Arap-β1,3-Ara (AA) can be selectively produced. Moreover, when larch is used as a raw material, Arap-β1,3-Ara (AA) can be selectively produced. Moreover, when rice is used as a raw material, Arap-β1,3-Araf and Araf-β1,3-Ara can be produced.
製造されたオリゴ糖は、ビフィズス菌、特にビフィドバクテリウム・ロンガムやビフィドバクテリウム・アドレセンティスを選択的に増殖させ得る高選択性プレバイオティクスオリゴ糖として利用し得る。 The produced oligosaccharide can be used as a highly selective prebiotic oligosaccharide capable of selectively proliferating Bifidobacteria, particularly Bifidobacterium longum or Bifidobacterium adrecentis.
従来、ＧＡやＡＡといったＩＩ型ＡＧ由来のオリゴ糖は、化学的手法（弱酸分解処理）で調製できることは知られていた。しかしながら、かかる手法では、単糖にまで分解しないように時間や温度等の反応条件を厳密にコントロールする必要があるうえ、不純物が多く含まれる点で、オリゴ糖の製造方法としては満足なものではなかった。
本発明の製造方法は、酵素学的手法であるため、高い選択性で効率的に目的のオリゴ糖を製造することができるため、非常に有用である。
Conventionally, it has been known that oligosaccharides derived from type II AG such as GA and AA can be prepared by a chemical method (weak acid decomposition treatment). However, this method requires strict control of reaction conditions such as time and temperature so that it does not decompose into monosaccharides, and is not satisfactory as a method for producing oligosaccharides because it contains many impurities. There wasn't.
Since the production method of the present invention is an enzymological method, the target oligosaccharide can be efficiently produced with high selectivity, and thus is very useful.
＜アラビアガム資化菌の検出用キット＞
本発明はさらに、配列番号１の塩基配列のうちの連続する２〜８塩基の配列を３’末端側に含む、１５〜３０塩基のＤＮＡからなるプライマー、及び配列番号１の塩基配列のうちの連続する２〜８塩基の配列に相補的な配列を３’末端側に含む、１５〜３０塩基のＤＮＡからなるプライマーからなるＰＣＲ用プライマーセットを含むキットを提供する。
<Kit for detection of gum arabic assimilation bacteria>
The present invention further includes a primer comprising 15 to 30 bases of DNA comprising a sequence of 2 to 8 bases of the base sequence of SEQ ID NO: 1 on the 3 ′ end side, and a base sequence of SEQ ID NO: 1 Provided is a kit comprising a primer set for PCR comprising a primer comprising 15 to 30 bases of DNA comprising a sequence complementary to a continuous 2 to 8 bases sequence on the 3 ′ end side.
ＰＣＲ用プライマーセットは、通常のＰＣＲ条件で増幅可能であること、本発明の糖質分解酵素をコードするＤＮＡ配列の他の領域では増幅断片が生じない配列であること、増幅断片が定量的ＰＣＲにも適用可能なように８０〜５５０ｂｐの長さになるようにすること、かつ、Ｔｍ値が５５〜６５℃の範囲内になること、を満たすようにオリゴヌクレオチドを配列番号１に基づいて設計すればよい。
例えば、配列番号１の塩基配列の第１８１７〜１８２１位の配列を３’末端側に含む２０塩基程度のＤＮＡをフォワードプライマーとして、配列番号１の塩基配列の第１９３５〜１９３９位の配列に相補的な配列を３’末端側に含む２０塩基程度のＤＮＡをリバースプライマーとして、プライマーセットを構成することができる。
The PCR primer set can be amplified under normal PCR conditions, is a sequence that does not produce an amplified fragment in other regions of the DNA sequence encoding the glycolytic enzyme of the present invention, and the amplified fragment is quantitative PCR The oligonucleotide is designed on the basis of SEQ ID NO: 1 so that the length is 80 to 550 bp so that it can be applied, and the Tm value is within the range of 55 to 65 ° C. do it.
For example, a DNA of about 20 bases containing the sequence of positions 1817 to 1821 of the base sequence of SEQ ID NO: 1 on the 3 ′ end side is used as a forward primer and complementary to the sequences of positions 1935 to 1939 of the base sequence of SEQ ID NO: 1. A primer set can be constructed using a reverse primer of about 20 bases of DNA containing such a sequence on the 3 ′ end side.
本発明のキットに含まれるＰＣＲプライマーセットは、被験試料中の核酸を鋳型としてＰＣＲを行うことにより、本発明の糖質分解酵素をコードするＤＮＡ配列の一部を選択的に増幅し得る。また、かかるＰＣＲにより増幅されるＤＮＡ配列は、本発明の糖質分解酵素に固有の配列であり、他の遺伝子配列と区別される。
前述の通り、本発明の糖質分解酵素はアラビアガムの修飾糖を分解することができるため、該酵素を保有する細菌はアラビアガム資化性を有するといえる。したがって、本発明のキットは、アラビアガム資化菌の検出の用途に好ましく供することができる。
本発明により、さらに、本発明のキットを用いてＰＣＲを行う工程を含む、アラビアガム資化菌を検出する方法も提供される。
The PCR primer set included in the kit of the present invention can selectively amplify a part of the DNA sequence encoding the carbohydrase of the present invention by performing PCR using the nucleic acid in the test sample as a template. Moreover, the DNA sequence amplified by such PCR is a sequence unique to the carbohydrase of the present invention, and is distinguished from other gene sequences.
As described above, since the saccharide-degrading enzyme of the present invention can degrade the modified sugar of gum arabic, it can be said that the bacterium having the enzyme has assimilation ability of gum arabic. Therefore, the kit of the present invention can be preferably used for the detection of gum arabic assimilating bacteria.
The present invention further provides a method for detecting gum arabic assimilating bacteria, which comprises the step of performing PCR using the kit of the present invention.
なお、ここで「検出」とは、アラビアガム資化菌の存在の有無を調べる定性的な検出の意味の他に、アラビアガム資化菌の存在量を定量的に調べるような定量的な検出（測定）の意味も含む。 Here, “detection” means not only qualitative detection of the presence or absence of gum arabic assimilating bacteria but also quantitative detection such as quantitatively examining the amount of gum arabic assimilating bacteria. Also includes the meaning of (measurement).
本発明のキットを適用し得る被験試料としては、特に限定されないが、例えば、乳酸菌の混合培養物やヨーグルトやチーズなどの乳酸菌を含む飲食品、マウスやラットなどの実験動物やヒトの糞便・腸液などを挙げることができる。
これらの試料から核酸を抽出する方法は、例えば、Ｌｙｓｏｚｙｍｅなどの酵素やビーズなどで物理的に細胞を破壊して抽出する方法や、市販の核酸抽出キットを使用する方法等が挙げられ、特に限定されない。
The test sample to which the kit of the present invention can be applied is not particularly limited. For example, a mixed culture of lactic acid bacteria, foods and drinks containing lactic acid bacteria such as yogurt and cheese, laboratory animals such as mice and rats, and human feces and intestinal fluids And so on.
Methods for extracting nucleic acids from these samples include, for example, methods of physically destroying cells with enzymes such as Lysozyme and beads, methods of using commercially available nucleic acid extraction kits, etc. Not.
本発明のキットには、前記ＰＣＲプライマーセットの他に、例えば、ＰＣＲ用試薬（耐熱性ＤＮＡポリメラーゼ、ｄＮＴＰ）、緩衝液、説明書等を含めてもよい。また、アラビアガム資化菌の検出に必要な試薬や、他の株・種・属の細菌の検出に必要な試薬等も含めてもよい。 In addition to the PCR primer set, the kit of the present invention may contain, for example, a PCR reagent (heat-resistant DNA polymerase, dNTP), a buffer, instructions, and the like. Further, reagents necessary for detecting gum arabic assimilating bacteria, reagents necessary for detecting bacteria of other strains, species, and genera may be included.
以下に実施例を用いて本発明を説明するが、本発明はこれら実施例に限定されるものではない。 The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
［試験例１］アラビアガム資化性試験
表２に記載の各供試菌を、糖源をアラビアガムのみに変更したＭＲＳ液体培地（表３）２００μＬに、１ｖ/ｖ％（約１×１０６ＣＦＵ）ずつ接種し、嫌気条件下で３７℃にて培養した。培養４８時間後に濁度（ＯＤ６００）を測定し、菌を接種しなかった培地を同様に培養したコントロールの濁度を差し引いた値を以下の基準に照らし、資化性の有無を判定した。
ＯＤ６００＜０．１５：資化性なし、ＯＤ６００≧０．１５：資化性あり
[Test Example 1] Gum arabic assimilation test Each test bacterium described in Table 2 was added to 200 μL of MRS liquid medium (Table 3) in which the sugar source was changed to only gum arabic, and 1 v / v% (about 1 × 10%). 6 CFU) and inoculated at 37 ° C. under anaerobic conditions. The turbidity (OD 600 ) was measured 48 hours after the culture, and the value obtained by subtracting the turbidity of the control in which the medium not inoculated with the bacteria was similarly cultured was determined according to the following criteria to determine the presence or absence of assimilability.
OD 600 <0.15: no assimilability, OD 600 ≧ 0.15: assimilability
結果を表２に示す。全１１３種の供試菌のうち、Bifidobacterium longum ＭＣＣ０３００、ＭＣＣ１００４０、ＭＣＣ１００８５、ＭＣＣ１０１２７、及びＭＣＣ１０１３０の５種のみが、アラビアガム資化性を有することが認められた。 The results are shown in Table 2. Of the 113 test bacteria in total, only 5 kinds of Bifidobacterium longum MCC0300, MCC10040, MCC10085, MCC10127, and MCC10130 were found to have arabic gum utilization.
［試験例２］アラビアガム資化性ビフィドバクテリウム属細菌を反応させたアラビアガムからの遊離糖の解析
ビフィドバクテリウム・ロンガムＪＣＭ１２１７及びビフィドバクテリウム・ロンガムＭＣＣ０３００を、試験例１と同様に糖源をアラビアガムのみに変更したＭＲＳ液体培地で、嫌気条件下で３７℃にて４８時間培養した。培養後に集菌し、５ｍＭ酢酸バッファー（ｐＨ５．０）で洗菌し、冷凍した。冷凍菌体１０ｍｇ（洗菌菌体重量）を、表４に記載の２％（20μｇ／μＬ）アラビアガム溶液を含む混合溶液２ｍＬに加え、３７℃で３日間反応させた。
[Test Example 2] Analysis of free sugar from gum arabic reacted with gum arabic-assimilating Bifidobacterium genus Bifidobacterium longum JCM1217 and Bifidobacterium longum MCC0300 were the same as in Test Example 1. In an MRS liquid medium in which the sugar source was changed to only gum arabic, the cells were cultured at 37 ° C. for 48 hours under anaerobic conditions. The cells were collected after culturing, washed with 5 mM acetate buffer (pH 5.0), and frozen. 10 mg of frozen cells (bacterial cell weight) was added to 2 mL of a mixed solution containing 2% (20 μg / μL) gum arabic solution described in Table 4 and reacted at 37 ° C. for 3 days.
反応後の菌液上清をＨＰＡＥＣ−ＰＡＤで解析した。測定条件を以下に記す。
クロマト機材：ICS-3000, Dionex Corp.
カラム：CarboPac PA-1
移動相：A (0.1 M NaOH) 、B (0.5 M sodium acetate and 0.1 M NaOH)
プログラム: 0-5 min, 100% eluent A (0.1 M NaOH); 5-30 min, 0%-100% eluent B (0
.5 M sodium acetate and 0.1 M NaOH); and 30-35 min, 100% eluent B.
流速：１．０ｍＬ／ｍｉｎ
温度：３０℃
検出器：ＰＡＤ
結果を図４に示す。アラビアガム資化性を有するビフィドバクテリウム・ロンガムＭＣＣ０３００は、アラビアガムからＧａｌ−α１，３−Ａｒａを遊離させたことが認められた。一方、ビフィドバクテリウム・ロンガムＪＣＭ１２１７では、二糖の遊離は認められなかった。
The supernatant of the bacterial solution after the reaction was analyzed by HPAEC-PAD. The measurement conditions are described below.
Chromatographic equipment: ICS-3000, Dionex Corp.
Column: CarboPac PA-1
Mobile phase: A (0.1 M NaOH), B (0.5 M sodium acetate and 0.1 M NaOH)
Program: 0-5 min, 100% eluent A (0.1 M NaOH); 5-30 min, 0% -100% eluent B (0
.5 M sodium acetate and 0.1 M NaOH); and 30-35 min, 100% eluent B.
Flow rate: 1.0 mL / min
Temperature: 30 ° C
Detector: PAD
The results are shown in FIG. It was confirmed that Bifidobacterium longum MCC0300 having arabic gum assimilation property released Gal-α1,3-Ara from gum arabic. On the other hand, in Bifidobacterium longum JCM1217, no disaccharide release was observed.
［試験例３］組み換えＥＡＦａｓｅを用いた解析
（１）組み換えＥＡＦａｓｅの作製
ＥＡＦａｓｅ遺伝子のシグナルペプチドをコードする部位を除いた配列（配列番号１の第１０９〜３４１１位の配列）を有するＤＮＡをＰＣＲで増幅し、定法によりｐＥＴ２３ｄベクターに組み込み、取得したプラスミドをｐＥＴ２３ｄ＿ＥＡＦａｓｅと名付けた。
定法により大腸菌ＢＬ２１（ＤＥ３）をｐＥＴ２３ｄ＿ＥＡＦａｓｅで形質転換し、Overnight_Express Autoinduction System（メルク株式会社製）を用いて酵素誘導産生した。BugBuster protein extraction reagent (Novagen)を用いて菌体内酵素を抽出した後、タンパク質のＣ末端に付加したHis-tagを利用して酵素精製を行い、組み換えＥＡＦａｓｅタンパク質を得た。
[Test Example 3] Analysis using recombinant EAFase (1) Preparation of recombinant EAFase DNA having a sequence excluding the site encoding the signal peptide of EAFase gene (positions 109 to 3411 of SEQ ID NO: 1) was obtained by PCR. The amplified plasmid was incorporated into a pET23d vector by a conventional method, and the obtained plasmid was named pET23d_EAFase.
Escherichia coli BL21 (DE3) was transformed with pET23d_EAFase by a conventional method, and enzyme-induced production was performed using Overnight_Express Autoinduction System (Merck Co., Ltd.). After extracting the intracellular enzyme using BugBuster protein extraction reagent (Novagen), enzyme purification was performed using His-tag added to the C-terminus of the protein to obtain a recombinant EAFase protein.
（２）組み換えＥＡＦａｓｅを反応させたアラビアガム又はカラマツＩＩ型ＡＧからの遊離糖の解析
（１）で取得した組み換えＥＡＦａｓｅの溶液（０．１７０μｇ／μＬ）１μＬを、１Ｍ酢酸バッファー（ｐＨ５．０）１０μＬ及び滅菌水１６９μＬ中で、アラビアガム又はカラマツＩＩ型ＡＧ４００μｇと３７℃にて４８時間反応させた。回収した反応液の上清を試験例２と同様にＨＰＡＥＣ−ＰＡＤで解析した。
結果を図５に示す。アラビアガムからは、主にＧａｌ−α１，３−Ａｒａ（ＧＡ）が、カラマツＩＩ型ＡＧからはＡｒａｐ−β１，３−Ａｒａ（ＡＡ）が、それぞれ遊離した。この結果は、アラビアガム由来ＩＩ型ＡＧの修飾糖のＧＡとＡＡのモル比が７：１であることや、カラマツＩＩ型ＡＧではＧＡの存在が報告されていないことと一致する。
(2) Analysis of free sugar from gum arabic or larch type II AG reacted with recombinant EAFase 1 μL of recombinant EAFase solution (0.170 μg / μL) obtained in (1) was added to 1M acetate buffer (pH 5.0) In 10 μL and 169 μL of sterilized water, the reaction was carried out at 37 ° C. for 48 hours with 400 μg of gum arabic or larch type II AG. The supernatant of the collected reaction solution was analyzed by HPAEC-PAD in the same manner as in Test Example 2.
The results are shown in FIG. Gal-α1,3-Ara (GA) was released mainly from gum arabic, and Arap-β1,3-Ara (AA) was released from larch type II AG. This result is consistent with the fact that the molar ratio of GA to AA in the modified sugar of gum arabic type II AG is 7: 1, and the presence of GA is not reported in larch type II AG.
（３）組み換えＥＡＦａｓｅのアラビアガム又はカラマツＩＩ型ＡＧに対する基質分解率の解析
表５に示す組成で、（１）で取得した組み換えＥＡＦａｓｅをアラビアガム又はカラマツＩＩ型ＡＧに３７℃にて４８時間反応させた。回収した反応液のうち１００μＬにエタノール４００μＬを加え、１〜２時間４℃で冷却した。４℃、１５０００ｒｐｍで２０分間遠心分離し、得られた上清についてSpeedVac遠心濃縮システム（ThermoFisher社）を使用して遠心濃縮した。濃縮後、濃縮物を滅菌水で１ｍＬに調整し、フェノール硫酸法で遊離糖の量を測定した。標準試料としては、ガラクトースの０、２０、４０、６０、８０及び１００μｇ/ｍＬ水溶液を用いた。
(3) Analysis of substrate degradation rate of recombinant EAFase to gum arabic or larch type II AG With the composition shown in Table 5, the recombinant EAFase obtained in (1) was reacted with gum arabic or larch type II AG at 37 ° C. for 48 hours. I let you. 400 μL of ethanol was added to 100 μL of the collected reaction solution, and cooled at 4 ° C. for 1-2 hours. Centrifugation was performed at 4 ° C. and 15000 rpm for 20 minutes, and the resulting supernatant was subjected to centrifugal concentration using a SpeedVac centrifugal concentration system (ThermoFisher). After concentration, the concentrate was adjusted to 1 mL with sterilized water, and the amount of free sugar was measured by the phenol-sulfuric acid method. As standard samples, 0, 20, 40, 60, 80 and 100 μg / mL aqueous solutions of galactose were used.
表６にガラクトース標準試料のＯＤ４９０値を、表７に測定試料のＯＤ４９０値及び換算糖濃度を、それぞれ示す。当初基質濃度と換算糖濃度とを用いて、数式（１）及び（２）の通り基質分解率を算出した。ＥＡＦａｓｅのアラビアガムに対する基質分解率は１４．３％と非常に高く、カラマツＩＩ型ＡＧに対する基質分解率は０．６０４％であった。 Table 6 shows the OD 490 value of the galactose standard sample, and Table 7 shows the OD 490 value and converted sugar concentration of the measurement sample. Using the initial substrate concentration and the converted sugar concentration, the substrate degradation rate was calculated as in equations (1) and (2). The substrate degradation rate of EAFase for gum arabic was very high at 14.3%, and the substrate degradation rate for larch type II AG was 0.604%.
（４）組み換えＥＡＦａｓｅの基質特異性の解析
（１）で取得した組み換えＥＡＦａｓｅの溶液（０．１７０μｇ／μＬ）１μＬを、１Ｍ酢酸バッファー（ｐＨ５．０）２μＬ及び滅菌水３２μＬ中で、オリゴ糖１μｇを基質として３７℃にて１２時間反応させた。回収した反応液の上清を試験例２と同様にＨＰＡＥＣ−ＰＡＤで解析した。なお、基質としたオリゴ糖は、Arap-β1,3-Araf-α1,3-Galp-β1,6-Galp-β1,6-Gal又はAraf-β1,3-Araf-α1,3-Galp-β1,6-Galであり、これらはそれ
ぞれ、カラマツＩＩ型ＡＧからexo-β1,3-galactanaseを用いた加水分解反応により取得したもの、及び穂ばらみ期のイネから（K. Kawaguchi et al., Plant. J. 9: 777-785 (1996)）抽出したものである。
結果を図６に示す。Arap-β1,3-Araf-α1,3-Galp-β1,6- Galp-β1,6-GalからはArap-β1,3-Araが、Araf-β1,3-Araf-α1,3- Galp-β1,6-GalからはAraf-β1,3-Araが、それぞれ遊離することが確認された。Araf-β1,3-Araf-α1,3-Galp-β1,6-Galについては、反応開始９６時間後もAraf-β1,3-Araf-α1,3-Galp-β1,6-Galのピークが残っていたため、ＥＡＦａｓｅは、Arap-β1,3-Araf-α1,3-Galp-β1,6-Galp-β1,6-Galには強く作用するが、Araf-β1,3-Araf-α1,3-Galp-β1,6-Galに対する作用は弱いものと推測される。
(4) Analysis of substrate specificity of recombinant EAFase 1 μL of recombinant EAFase solution (0.170 μg / μL) obtained in (1) was added in 1 μg of oligosaccharide in 2 μL of 1M acetate buffer (pH 5.0) and 32 μL of sterilized water. Was allowed to react at 37 ° C. for 12 hours. The supernatant of the collected reaction solution was analyzed by HPAEC-PAD in the same manner as in Test Example 2. The oligosaccharide used as the substrate was Arap-β1,3-Araf-α1,3-Galp-β1,6-Galp-β1,6-Gal or Araf-β1,3-Araf-α1,3-Galp-β1 These were obtained from hydrolysis of larch type II AG using exo-β1,3-galactanase and from the rice at the booting stage (K. Kawaguchi et al., Plant. J. 9: 777-785 (1996)).
The results are shown in FIG. From Arap-β1,3-Araf-α1,3-Galp-β1,6-Galp-β1,6-Gal, Arap-β1,3-Ara becomes Araf-β1,3-Araf-α1,3-Galp- It was confirmed that Araf-β1,3-Ara was released from β1,6-Gal, respectively. For Araf-β1,3-Araf-α1,3-Galp-β1,6-Gal, the peak of Araf-β1,3-Araf-α1,3-Galp-β1,6-Gal is still present 96 hours after the start of the reaction. EAFase acts strongly on Arap-β1,3-Araf-α1,3-Galp-β1,6-Galp-β1,6-Gal, but Araf-β1,3-Araf-α1,3 The effect on -Galp-β1,6-Gal is assumed to be weak.
Claims (14)
（Ａ）配列番号２の少なくとも第３７〜１１３６位に示されるアミノ酸配列を含むタンパク質
（Ｂ）配列番号２の少なくとも第３７〜１１３６位に示されるアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入、又は付加を含むアミノ酸配列を含み、かつ、エンドグリコシダーゼ活性を有するタンパク質 The following protein (A) or (B).
(A) a protein comprising an amino acid sequence represented by at least positions 37 to 1136 of SEQ ID NO: 2 (B) substitution of one or several amino acids in the amino acid sequence represented by positions 37 to 1136 of SEQ ID NO: 2; A protein comprising an amino acid sequence containing a deletion, insertion, or addition and having endoglycosidase activity
（ａ）配列番号１の少なくとも第１０９〜３４１１位に示される塩基配列を含むＤＮＡ
（ｂ）配列番号１の少なくとも第１０９〜３４１１位に示される塩基配列に相補的な塩基配列又は該相補配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、エンドグリコシダーゼ活性を有するタンパク質をコードするＤＮＡ A protein encoded by the following DNA (a) or (b):
(A) DNA comprising a base sequence shown at least in positions 109 to 3411 of SEQ ID NO: 1
(B) hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown at least at positions 109 to 3411 of SEQ ID NO: 1 or a probe that can be prepared from the complementary sequence, and has endoglycosidase activity DNA encoding protein
ＩＩ型アラビノガラクタンを含む原料とを含有する、飲食品組成物。 The Bifidobacterium genus bacterium according to claim 1, the culture of the bacterium, a treated product of the bacterium, and / or the protein according to claim 2 or 3,
The food-drinks composition containing the raw material containing II type arabinogalactan.
配列番号１の塩基配列のうちの連続する２〜８塩基の配列に相補的な配列を３’末端側に含む、１５〜３０塩基のＤＮＡからなるプライマー
からなるＰＣＲ用プライマーセットを含むキット。 A primer comprising 15 to 30 bases of DNA comprising a sequence of 2 to 8 bases of the base sequence of SEQ ID NO: 1 on the 3 ′ end side, and 2 to 8 of the base sequence of SEQ ID NO: 1 A kit comprising a primer set for PCR comprising a primer comprising 15 to 30 bases of DNA comprising a sequence complementary to the base sequence on the 3 ′ end side.
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