JP2019043867A - Composition for promoting increase of Akkermansia muciniphila - Google Patents
Composition for promoting increase of Akkermansia muciniphila Download PDFInfo
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- JP2019043867A JP2019043867A JP2017166835A JP2017166835A JP2019043867A JP 2019043867 A JP2019043867 A JP 2019043867A JP 2017166835 A JP2017166835 A JP 2017166835A JP 2017166835 A JP2017166835 A JP 2017166835A JP 2019043867 A JP2019043867 A JP 2019043867A
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- JP
- Japan
- Prior art keywords
- akkermansia muciniphila
- composition
- promoting
- sgp
- neu5acα2
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
本発明は、シアリル糖ペプチドを含むアッカーマンシア・ムシニフィラ増加促進用組成物、及び該組成物を含む飲食品、医薬品、飼料に関する。 The present invention relates to a composition for promoting Akkermansia muciniphila increase containing a sialyl glycopeptide, and a food or drink, a pharmaceutical product and a feed containing the composition.
近年、腸内細菌が宿主の代謝調節機能に重要な役割を担っていること、そして腸内細菌叢の破綻により宿主の代謝機能に異変が生じ、様々な疾患の原因となることが明らかにされつつある。したがって、健全な腸内細菌叢を保つことが宿主の健康維持に重要である。宿主によい効果をもたらす腸内細菌としては、ビフィドバクテリウム属菌やある種のラクトバチルス属菌などが知られており、それらをプロバイオティクスとして食品とともに摂取する、または腸内でそれらの増殖を促進する食品をプレバイオティクスとして摂取することが推奨されている。 In recent years, it has been clarified that enteric bacteria play an important role in host's metabolic control function and that the breakdown of the enteric microbiota causes changes in the host's metabolic function and causes various diseases. It is getting worse. Therefore, maintaining a healthy intestinal flora is important for host health. Bifidobacterium bacteria and certain Lactobacillus bacteria are known as enteric bacteria that exert a good effect on the host, and they are taken together with food as probiotics, or those in the intestines. It is recommended to take food that promotes growth as prebiotics.
一方、最近の腸内細菌研究からは、宿主の健康に寄与する新たな腸内細菌が報告されてきている。アッカーマンシア・ムシニフィラ（Akkermansia muciniphila）は、ヒトの腸内細菌の１〜５％を占める細菌であり、宿主が消化管から分泌するムチンを分解・資化する特徴を有する（非特許文献１）。Clement K.らは、ヒト腸内におけるアッカーマンシア・ムシニフィラの量が、空腹時血糖値、血中インスリン値、およびウエスト周囲長と逆相関を示すことを報告している。すなわち、アッカーマンシア・ムシニフィラは、肥満、糖尿病、心疾患などの予防に効果的である可能性が示唆されている。また、マウスに高脂肪食を食べさせると肥満、腸管のバリア機能の低下、および脂肪組織の炎症マーカーが上昇、などの症状を呈するが、このとき盲腸内容物中のアッカーマンシア・ムシニフィラ量が低下することが報告されている。この高脂肪食のマウスに経口的に生きたアッカーマンシア・ムシニフィラを投与すると、脂肪量の増加、インスリン抵抗性、腸管バリア機能、脂肪組織の炎症などの代謝不全が改善された（非特許文献２）。 On the other hand, recent enteric bacteria research has reported new enteric bacteria that contribute to host health. Akkermansia muciniphila is a bacterium that accounts for 1 to 5% of human enteric bacteria, and has a feature of degrading and assimilating mucin secreted by the host from the digestive tract (Non-patent Document 1). Clement K. et al. Report that the amount of Akkermansia muciniphila in the human intestine is inversely correlated with fasting blood glucose levels, blood insulin levels, and waist circumference. That is, it has been suggested that Akkermansia muciniphila may be effective for the prevention of obesity, diabetes, heart disease and the like. In addition, when mice are fed a high-fat diet, they exhibit symptoms such as obesity, decreased intestinal barrier function, and increased inflammatory markers of adipose tissue, but at this time the amount of Akermansia muciniphila in the cecal contents decreases. It has been reported that. Oral administration of Akkermansia muciniphila to this high-fat diet improved amelioration of fat mass, insulin resistance, intestinal barrier function, and metabolic disorders such as inflammation of adipose tissue (Non-patent Document 2) ).
このように、腸管内でのアッカーマンシア・ムシニフィラ量が低下することは、腸の機能だけではなく全身の代謝機能を低下させることが明らかにされつつある。したがって現在では、腸内のアッカーマンシア・ムシニフィラを増加させるための様々な方法が検討されている。抗糖尿病薬として一般的に用いられているメトフォルミンの投与により、腸内のアッカーマンシア・ムシニフィラが増加することが報告され、アッカーマンシア・ムシニフィラ増加剤として期待されている。しかし、薬剤であることから既に糖尿病を発症している患者以外には適用することはできない。健康なヒト、または肥満気味なヒトなどが疾病予防を目的に食事として摂取できるアッカーマンシア・ムシニフィラ増加剤が期待されてきた。 Thus, it is becoming clear that the reduction of the amount of Akkermansia muciniphila in the intestinal tract reduces not only the function of the intestine but also the whole body metabolic function. Therefore, various methods for increasing Akkermansia muciniphila in the intestine are currently being considered. Administration of metformin, which is generally used as an anti-diabetic agent, is reported to increase Akkermansia muciniphila in the intestine, and is expected as an Akkermansia muciniphila increasing agent. However, because it is a drug, it can not be applied to patients who have already developed diabetes. Akkermansia muciniphila increasing agents have been expected that healthy humans or obese humans can consume as a diet for the purpose of disease prevention.
κ−カゼイングリコマクロペプチド（以下、ＧＭＰと略記する）は糖ペプチドの一種で、牛乳タンパク質の８０〜８５％を占めるカゼインのうちで唯一の糖タンパク質であるκ−カゼインの糖鎖を含むＣ末端ペプチドであり、κ−カゼインの１０６−１６９残基に相当し、分子量は、約７，０００である。また、ＧＭＰは、グルタミン酸残基を多く持つほか、糖鎖中にシアル酸残基を持つなど、カルボキシ基に富んでいる。
ＧＭＰはこれまでに、胃から出るホルモン（ＣＣＫ：満腹ホルモン）の分泌促進を介した食欲抑制作用（非特許文献３）、脂肪細胞への直接的な作用による脂肪蓄積抑制作用（非特許文献４）、ビフィズス菌の増殖促進作用（非特許文献５）、感染防御作用（特許文献１）、カルシウム吸収促進剤（特許文献２）などが報告されているが、アッカーマンシア・ムシニフィラに対する作用について記載や示唆のある文献等は知られていない。同様にＧＭＰ等を加水分解して得られるシアリル糖ペプチド（以下、ＳＧＰと略記する）も、アッカーマンシア・ムシニフィラに対する作用はなんら検討されていない。
κ-casein glycomacropeptide (hereinafter abbreviated as GMP) is a kind of glycopeptide and is a C-terminal containing a sugar chain of κ-casein, which is the only glycoprotein among casein which occupies 80 to 85% of milk protein. It is a peptide, corresponding to residues 106-169 of κ-casein, and has a molecular weight of about 7,000. Moreover, GMP has many glutamic acid residues and is rich in carboxy groups, such as having sialic acid residues in sugar chains.
Until now, GMP has an anorectic action through promoting secretion of a hormone (CCK: satiety hormone) that is emitted from the stomach (Non-patent Document 3), and an action to suppress fat accumulation by direct action on adipocytes (Non-patent document 4) ), Bifidobacterium growth-promoting action (Non-patent document 5), infection protection action (patent document 1), calcium absorption promoter (patent document 2), etc. have been reported, but their action on Akkermansia muciniphila There are no known documents with suggestions. Similarly, also for sialyl glycopeptide (hereinafter abbreviated as SGP) obtained by hydrolyzing GMP and the like, its action on Akkermansia muciniphila has not been examined at all.
本発明の課題は、従来にない新たなアッカーマンシア・ムシニフィラの増加促進用組成物、及び該組成物を含む食品、医薬品、飼料を提供することである。 An object of the present invention is to provide a novel composition for promoting the growth of Akkermansia muciniphila, and food, medicine and feed containing the composition.
本発明者らは、腸内のアッカーマンシア・ムシニフィラを増加させる素材を鋭意検討した結果、ＧＭＰの分解物であるＳＧＰにアッカーマンシア・ムシニフィラを増加させる作用があることを見出し、本発明を完成させるに至った。また、換言すればＳＧＰのこれまでに報告されていない新たな用途を見出したことに基づく発明である。即ち本発明は以下の構成を有する。
〔１〕スレオニン及び／又はセリン残基を有するペプチドに糖鎖が結合したシアリル糖ペプチドを含むアッカーマンシア・ムシニフィラ増加促進用組成物であって、
前記糖ペプチドの分子量が５００以上３，０００以下であり、
前記糖鎖が前記ペプチドのスレオニン又はセリン残基の水酸基に結合し、
前記糖鎖の構成糖がシアル酸、Ｎ−アセチルガラクトサミン及びガラクトースからなる群から選択される１つ以上であることを特徴とするアッカーマンシア・ムシニフィラ増加促進用組成物。
〔２〕前記シアリル糖ペプチドの糖鎖がシアル酸及びガラクト−Ｎ−ビオースからなるものである〔１〕に記載のアッカーマンシア・ムシニフィラ増加促進用組成物。
〔３〕前記シアル酸と前記ガラクト−Ｎ−ビオースのモル比が１：１〜２：１である〔２〕に記載のアッカーマンシア・ムシニフィラ増加促進用組成物。
〔４〕前記シアリル糖ペプチドのペプチド鎖のアミノ酸残基数が１以上１３以下である〔１〕〜〔３〕のいずれか１つに記載のアッカーマンシア・ムシニフィラ増加促進用組成物。
〔５〕スレオニン及び／又はセリン残基を有し当該残基の水酸基に糖鎖が結合したシアリル糖ペプチド、を含むアッカーマンシア・ムシニフィラ増加促進用組成物であって、
前記糖鎖が、以下の（１）〜（５）からなる群から選ばれる１つ以上であることを特徴とするアッカーマンシア・ムシニフィラ増加促進用組成物。
（１）Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1
（２）Galβ1-3(Neu5Acα2-6)GalNAcα1
（３）Neu5Acα2-3Galβ1-3GalNAcα1
（４）Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1
（５）Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1
〔６〕前記糖鎖が、前記（１）及び前記（２）〜（５）からなる群から選ばれる１つ以上である〔５〕に記載のアッカーマンシア・ムシニフィラ増加促進用組成物。
〔７〕シアリル糖ペプチドが乳由来であることを特徴とする〔１〕〜〔６〕のいずれか１つに記載のアッカーマンシア・ムシニフィラ増加促進用組成物。
〔８〕〔１〕〜〔７〕のいずれか１つに記載のアッカーマンシア・ムシニフィラ増加促進用組成物を含むアッカーマンシア・ムシニフィラ増加促進用飲食品。
〔９〕〔１〕〜〔７〕のいずれか１つに記載のアッカーマンシア・ムシニフィラ増加促進用組成物を含むアッカーマンシア・ムシニフィラ増加促進用医薬品。
〔１０〕〔１〕〜〔７〕のいずれか１つに記載のアッカーマンシア・ムシニフィラ増加促進用組成物を含むアッカーマンシア・ムシニフィラ増加促進用飼料。
The present inventors have intensively studied materials for increasing Akermansia muciniphila in the intestine and found that SGP, which is a degradation product of GMP, has an action to increase Akkermansia muciniphila, and complete the present invention It came to Also, in other words, it is an invention based on the finding of a new use that has not been reported so far in SGP. That is, the present invention has the following configuration.
[1] A composition for promoting Akkermansia muciniphila comprising a sialylglycopeptide in which a sugar chain is linked to a peptide having a threonine and / or a serine residue,
The molecular weight of the glycopeptide is 500 or more and 3,000 or less,
The sugar chain is bonded to the hydroxyl group of threonine or serine residue of the peptide,
A composition for promoting increase of Akkermansia muciniphila, wherein the constituent sugar of the sugar chain is one or more selected from the group consisting of sialic acid, N-acetylgalactosamine and galactose.
[2] The composition according to [1], wherein the sugar chain of the sialyl glycopeptide comprises sialic acid and galacto-N-biose.
[3] The composition for promoting increase of Akkermansia muciniphila according to [2], wherein the molar ratio of the sialic acid and the galacto-N-biose is 1: 1 to 2: 1.
[4] The composition for promoting an increase in Akkermansia muciniphila according to any one of [1] to [3], wherein the number of amino acid residues in the peptide chain of the sialyl glycopeptide is 1 or more and 13 or less.
[5] A composition for promoting the increase of Akkermansia muciniphila comprising a sialyl glycopeptide having a threonine and / or serine residue and having a sugar chain bonded to the hydroxyl group of the residue,
The composition for promoting Akkermansia muciniphila increase, characterized in that the sugar chain is one or more selected from the group consisting of the following (1) to (5):
(1) Neu5Acα2-3Galβ1-3 (Neu5Acα2-6) GalNAcα1
(2) Galβ1-3 (Neu5Acα2-6) GalNAcα1
(3) Neu5Acα2-3Galβ1-3GalNAcα1
(4) Neu5Acα2-3Galβ1-3 (O-Ac-Neu5Acα2-6) GalNAcα1
(5) Neu5Acα2-3Galβ1-3 (O-diAc-Neu5Acα2-6) GalNAcα1
[6] The composition for promoting an increase in Akkermansia muciniphila according to [5], wherein the sugar chain is one or more selected from the group consisting of (1) and (2) to (5).
[7] The composition according to any one of [1] to [6], wherein the sialyl glycopeptide is derived from milk.
[8] A food / drink product for promoting Akkermansia muciniphila comprising the composition for promoting Akkermansia muciniphila according to any one of [1] to [7].
[9] A pharmaceutical product for promoting Akkermansia muciniphila comprising the composition for promoting Akkermansia muciniphila according to any one of [1] to [7].
A diet for promoting Akkermansia muciniphila comprising the composition for promoting Akkermansia muciniphila according to any one of [10] [1] to [7].
本発明は、従来にない新たなアッカーマンシア・ムシニフィラの増加促進用組成物、及び該組成物を含むアッカーマンシア・ムシニフィラの増加促進用飲食品、医薬品、飼料を提供することが可能となった。 The present invention has made it possible to provide a novel composition for promoting the increase of Akkermansia muciniphila, and a food, a drug, and a feed for promoting the increase of Akkermansia muciniphila containing the composition.
本発明のＳＧＰを有効成分として含むアッカーマンシア・ムシニフィラの増加促進用組成物、及びＳＧＰを有効成分として含むアッカーマンシア・ムシニフィラの増加促進用飲食品、医薬品、飼料について以下に詳細に説明する。
本発明のアッカーマンシア・ムシニフィラの増加促進用組成物、及び該組成物を含む飲食品、医薬品、飼料について以下に詳細に説明する。
The composition for promoting the increase of Akkermansia muciniphila containing SGP of the present invention as an active ingredient, and the food, beverage, and medicine for promoting the increase of Akkermansia muciniphila containing SGP as an active ingredient will be described in detail below.
The composition for promoting the increase of Akkermansia muciniphila of the present invention, and the food, drink, medicine and feed containing the composition will be described in detail below.
（シアリル糖ペプチド：ＳＧＰ）
本発明のＳＧＰは、スレオニン及び／又はセリン残基を有するペプチドに糖鎖が結合した糖ペプチドであって、分子量が５００以上であり、糖鎖がスレオニン又はセリン残基の水酸基に結合し、糖鎖の構成糖がシアル酸、Ｎ−アセチルガラクトサミン、ガラクトースから選択される１つ以上であるものを用いることができる。
本発明のＳＧＰは、糖鎖がシアル酸、およびガラクト−Ｎ−ビオース(Galβ1-3GalNAc)から成るものが好ましく、シアル酸とガラクト−Ｎ−ビオースのモル比が１：１〜２：１であるものが更に好ましい。また、本発明のＳＰＧはペプチド鎖が１アミノ酸残基以上１３アミノ酸残基以下程度のものを用いることができる。さらに、本発明のＳＰＧはアセチル化したシアル酸を含むものも用いることができる。
(Sialyl glycopeptide: SGP)
The SGP of the present invention is a glycopeptide in which a sugar chain is linked to a peptide having a threonine and / or serine residue, and has a molecular weight of 500 or more, and the sugar chain is bonded to the hydroxyl group of threonine or serine residue, It is possible to use one in which the chain constituent sugar is one or more selected from sialic acid, N-acetylgalactosamine, galactose.
The SGP of the present invention is preferably one in which the sugar chain consists of sialic acid and galacto-N-biose (Galβ1-3GalNAc), and the molar ratio of sialic acid to galacto-N-biose is 1: 1 to 2: 1. Are more preferred. In addition, as the SPG of the present invention, one having a peptide chain of about 1 to 13 amino acid residues can be used. Furthermore, SPG of the present invention can also be used that contains acetylated sialic acid.
本願発明のアッカーマンシア・ムシニフィラ増加促進用組成物の一態様として、スレオニン及び／又はセリン残基を有し当該水酸基に糖鎖が結合したシアリル糖ペプチドであって、前記糖鎖が、以下の（１）〜（５）からなる群から選ばれる１つ以上であることを特徴とするシアリル糖ペプチドを含む組成物が挙げられる。
（１）Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1
（２）Galβ1-3(Neu5Acα2-6)GalNAcα1
（３）Neu5Acα2-3Galβ1-3GalNAcα1
（４）Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1
（５）Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1
One embodiment of the composition for promoting Akkermansia muciniphila increase of the present invention is a sialyl glycopeptide having a threonine and / or a serine residue and having a sugar chain bound to the hydroxyl group, wherein the sugar chain is 1) A composition comprising a sialylglycopeptide, characterized in that it is one or more selected from the group consisting of (1) to (5).
(1) Neu5Acα2-3Galβ1-3 (Neu5Acα2-6) GalNAcα1
(2) Galβ1-3 (Neu5Acα2-6) GalNAcα1
(3) Neu5Acα2-3Galβ1-3GalNAcα1
(4) Neu5Acα2-3Galβ1-3 (O-Ac-Neu5Acα2-6) GalNAcα1
(5) Neu5Acα2-3Galβ1-3 (O-diAc-Neu5Acα2-6) GalNAcα1
また、本願発明のアッカーマンシア・ムシニフィラ増加促進用組成物のもうひとつの態様として、前記糖鎖が、下記（１）及び下記（２）〜（５）からなる群から選ばれる１つ以上の組み合わせであることを特徴とするシアリル糖ペプチドを含む組成物が挙げられる。
（１）Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1
（２）Galβ1-3(Neu5Acα2-6)GalNAcα1
（３）Neu5Acα2-3Galβ1-3GalNAcα1
（４）Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1
（５）Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1
Moreover, as another embodiment of the composition for promoting Akkermansia muciniphila increase of the present invention, the sugar chain is one or more combinations selected from the group consisting of the following (1) and the following (2) to (5): And a composition comprising a sialyl glycopeptide characterized in that
(1) Neu5Acα2-3Galβ1-3 (Neu5Acα2-6) GalNAcα1
(2) Galβ1-3 (Neu5Acα2-6) GalNAcα1
(3) Neu5Acα2-3Galβ1-3GalNAcα1
(4) Neu5Acα2-3Galβ1-3 (O-Ac-Neu5Acα2-6) GalNAcα1
(5) Neu5Acα2-3Galβ1-3 (O-diAc-Neu5Acα2-6) GalNAcα1
本発明のＳＧＰは、ＧＭＰ等を酵素等で加水分解することにより得ることができる。
ＧＭＰはκ−カゼインの糖鎖を含むＣ末端ペプチドであり、牛乳の場合κ−カゼインの１０６−１６９残基に相当し、分子量は、約７，０００である。本発明のＳＧＰの原料となるＧＭＰは、牛乳以外にもヤギやヒツジなどの獣乳由来のホエイ中から得られるものであればどのようなものでも用いることができる。
また、本発明のＳＧＰは、微生物、植物、動物の臓器の抽出物を酵素などで加水分解することにより調製することもできる。さらに、化学的に合成されたもの、もしくは遺伝子組換え体で調製したものでもよい。
The SGP of the present invention can be obtained by hydrolyzing GMP or the like with an enzyme or the like.
GMP is a C-terminal peptide containing a sugar chain of κ-casein and corresponds to 106-169 residues of κ-casein in milk, and has a molecular weight of about 7,000. The GMP used as a raw material of SGP of the present invention may be any GMP other than milk as long as it can be obtained from whey derived from animal milk such as goats and sheep.
The SGP of the present invention can also be prepared by hydrolyzing the extract of a microorganism, a plant or an organ of an animal with an enzyme or the like. Furthermore, they may be chemically synthesized or those prepared by gene recombination.
（ＳＧＰの製造方法）
本発明のＳＧＰの製造方法についてその一態様を示し説明する。
本発明のＳＧＰは、例えばＧＭＰを加水分解することにより得られるため、まず、ＧＭＰの製造方法の一態様について説明する。
ＧＭＰを含む組成物は、特許第２６７３８２８号、または特開平６−２５２９７号公報に記載の方法により調製することができる。すなわち、ＧＭＰを含有する乳質原料物質、例えばチーズホエー、ホエー蛋白質濃縮物、除蛋白質チーズホエー等を、まずｐＨ４未満に調整した後、分画分子量１０，０００〜５０，０００の膜を用い、限外濾過処理をして透過液を得、好ましくは再度、該透過膜をｐＨ４以上に調整した後、分画分子量５０，０００以下の膜を用いて脱塩し濃縮することによりＧＭＰを調整する方法が挙げられる。また、乳質ホエーをｐＨ６．０以上に調整し４０〜７９℃で加熱処理したものを膜処理に付して乳清たんぱく質を除去し、ＧＭＰを濃縮液側に回収し、得られた濃縮液をｐＨ５．５以下にした後、再び膜処理に供してＧＭＰを透過液側に回収することによりＧＭＰ含有量の高い組成物を調製する方法を挙げることができる。
得られた組成物中のＧＭＰ量は、例えば、特許文献２の実施例に示されるようなウレア−ＳＤＳ電気泳動法により分析することができる。
次に、このようにして得られるＧＭＰをエンド型プロテアーゼまたはエキソ型プロテアーゼで処理する工程と、前記工程で得られた処理物を分画分子量５００以上３，０００以下の限外ろ過に供する処理工程を経ることにより本発明のＳＧＰを製造することができる。
糖ペプチド製造に用いるプロテアーゼの種類は、ペプチド結合を加水分解し、上記（シアリル糖ペプチド：ＳＧＰ）で記載したような分子量と糖鎖を有する糖ペプチドを得ることができるものであれば特に限定されるものではなく、１種類、又は複数のエンド型プロテアーゼ、エキソ型プロテアーゼを用いることができる。好ましくは、食品や医薬品の製造に使用可能な酵素がよく、アクチナーゼＥ（科研ファルマ）、アクチナーゼＡＳ（科研ファルマ）、ヌクレイシン（ＨＢＩ）、オリエンターゼＡＹ（ＨＢＩ）、スミチームＦＰ（新日本化学工業）、スミチームＳＰＰ−Ｇ（新日本化学工業）、プロテアーゼＡ（天野エンザイム）、ペプチダーゼＲ（天野エンザイム）などを単独、あるいは組み合わせて使用することができる。
(Method of manufacturing SGP)
One aspect of the method for producing SGP of the present invention will be shown and described.
Since the SGP of the present invention is obtained, for example, by hydrolyzing GMP, first, one aspect of the method for producing GMP will be described.
The composition containing GMP can be prepared by the method described in Japanese Patent No. 2673828 or JP-A No. 6-25297. That is, first, GMP-containing milk material substances such as cheese whey, whey protein concentrate, deproteinized cheese whey etc. are first adjusted to a pH of less than 4, and then a membrane with a molecular weight cut off of 10,000 to 50,000 is used. Method of adjusting GMP by obtaining the permeated liquid by ultrafiltration, preferably again adjusting the permeable membrane to a pH of 4 or more, desalting using a membrane having a molecular weight cut off of 50,000 or less, and concentrating Can be mentioned. Also, after adjusting the whey whey to pH 6.0 or higher and subjecting it to a heat treatment at 40 to 79 ° C, it is subjected to membrane treatment to remove whey protein and recover GMP to the concentrate side, and the obtained concentrate is obtained After the pH is adjusted to 5.5 or less, a method of preparing a composition having a high GMP content can be mentioned by subjecting it to membrane treatment again to recover GMP to the permeate side.
The amount of GMP in the obtained composition can be analyzed by, for example, urea-SDS electrophoresis as shown in the example of Patent Document 2.
Next, a step of treating the thus obtained GMP with endo type protease or exo type protease, and a step of subjecting the treated product obtained in the above step to ultrafiltration with a molecular weight cut off of 500 or more and 3,000 or less The SGP of the present invention can be produced by
The type of protease used for glycopeptide production is not particularly limited as long as it can hydrolyze peptide bonds to obtain a glycopeptide having a molecular weight and a sugar chain as described in (Sialyl glycopeptide: SGP). Instead, one or more endo-type proteases and exo-type proteases can be used. Preferably, enzymes that can be used for the production of foods and pharmaceuticals are good, such as Actinase E (Kampan Pharma), Actinase AS (Kampin Pharma), Nuclein (HBI), Orientase AY (HBI), Sumi Team FP (New Nippon Chemical Industries) Sumiteam SPP-G (Shin Nippon Chemical Co., Ltd.), Protease A (Amano Enzyme), Peptidase R (Amano Enzyme), etc. can be used alone or in combination.
（ＳＧＰのアッカーマンシア・ムシニフィラの増加促進作用の評価）
本発明のＳＧＰによるアッカーマンシア・ムシニフィラの増加促進作用は、ＳＧＰを投与した場合と非投与の場合における腸内または糞便中のアッカーマンシア・ムシニフィラの増加を比較し、非投与よりも投与の方が増加した場合に本発明の増加促進作用があると評価することができる。増加は増殖と同義で用いられる。アッカーマンシア・ムシニフィラの増加・増殖は菌の増加・増殖の評価に通常用いられる方法であればいずれでもよく、例えば、以下の実施例に記載した方法を挙げることができる。
(Evaluation of SGP's increase promotion action of Akkermansia muciniphila)
The increase-promoting action of Akkermansia muciniphila by the SGP of the present invention is compared with the increase of Akkermansia muciniphila in the intestine or feces with and without administration of SGP, and administration is more effective than no administration. When it is increased, it can be evaluated that there is an increase promoting effect of the present invention. An increase is used synonymously with proliferation. The increase and growth of Akkermansia muciniphila may be any method generally used for evaluating the increase and growth of bacteria, and examples thereof include the methods described in the following examples.
（ＳＧＰを含む飲食品、医薬品、飼料）
本発明の上記製造方法により得られたＳＧＰ（あるいはＳＧＰを含む組成物）は、そのまま飲食品の原材料として用いることができ、ＳＧＰを含む組成物を添加すること以外は、各食品の定法により製造すれば良い。
したがって、本発明の有効量のＳＧＰはどのような飲食品に配合しても良く、飲食品の製造工程中に原料に添加しても良い。飲食品の例としては、チーズ、発酵乳、乳製品乳酸菌飲料、乳酸菌飲料、バター、マーガリンなどの乳製品、乳飲料、果汁飲料、清涼飲料などの飲料、ゼリー、キャンディー、プリン、マヨネーズなどの卵加工品、バターケーキなどの菓子・パン類、さらには、各種粉乳の他、乳幼児食品、栄養組成物などを挙げることができるが特に限定されるものではない。
このようにして製造された有効量のＳＧＰを含む飲食品は、アッカーマンシア・ムシニフィラの増加促進用飲食品として提供される。
(Food and drink including SGP, medicine, feed)
SGP (or a composition containing SGP) obtained by the above-mentioned production method of the present invention can be used as it is as a raw material for food and drink, and it is produced by the usual method of each food except that the composition containing SGP is added. Just do it.
Therefore, the effective amount of SGP of the present invention may be added to any food or drink and may be added to the raw material during the process of manufacturing the food or drink. Examples of food and drink include cheese, fermented milk, dairy lactic acid bacteria beverage, lactic acid bacteria beverage, dairy products such as butter and margarine, beverages such as milk beverages, fruit juice beverages and soft drinks, eggs such as jelly, candy, pudding and mayonnaise Examples of the processed products, confectionery / breads such as butter cakes, and various powdered milks, infant foods, nutritional compositions and the like can be mentioned, but are not particularly limited.
The food and drink containing the effective amount of SGP thus produced are provided as a food for promoting the increase of Akkermansia muciniphila.
本発明の上記製造方法により得られたＳＧＰ（あるいはＳＧＰを含む組成物）は、そのまま医薬品の原材料として用いることができ、ＳＧＰを含む組成物を添加すること以外は、錠剤、カプセル、粉末、シロップ等の定法により製造すれば良い。
したがって、本発明のＳＧＰを有効成分として含む医薬品の製剤化に際しては、製剤上許可されている賦型剤、安定剤、矯味剤などを適宜混合して製剤化するほか、ＳＧＰをそのまま乾燥して粉末剤、散剤として用いることもできる。また、アッカーマンシア・ムシニフィラの増加促進作用を妨げない範囲で、賦型剤、結合剤、崩壊剤、滑沢剤、矯味矯臭剤、懸濁剤、コーティング剤、その他の任意の薬剤を混合して製剤化することもできる。剤形としては、錠剤、カプセル剤、顆粒剤、散剤、粉剤、シロップ剤などが可能である。
このようにして製造された有効量のＳＧＰを含む医薬品は、アッカーマンシア・ムシニフィラの増加促進用医薬品として提供される。
SGP (or a composition containing SGP) obtained by the above-mentioned production method of the present invention can be used as it is as a raw material for pharmaceuticals, and except that a composition containing SGP is added, tablets, capsules, powders, syrups It may be manufactured by a standard method such as.
Therefore, when formulating a pharmaceutical containing the SGP of the present invention as an active ingredient, it is possible to appropriately mix and formulate an excipient, stabilizer, flavoring agent, etc. approved on the formulation, and dry the SGP as it is. It can also be used as a powder or powder. In addition, an excipient, a binder, a disintegrant, a lubricant, a flavoring agent, a suspending agent, a coating agent, and any other drug may be mixed within a range that does not interfere with the increase promoting action of Akkermansia muciniphila. It can also be formulated. As the dosage form, tablets, capsules, granules, powders, powders, syrups and the like can be used.
The pharmaceutical preparation containing an effective amount of SGP thus produced is provided as a pharmaceutical for promoting the increase of Akkermansia muciniphila.
本発明の上記製造方法により得られたＳＧＰ（あるいはＳＧＰを含む組成物）は、そのまま飼料の原材料として用いることができ、ＳＧＰを含む組成物を添加すること以外は、飼料の定法により製造すれば良い。
したがって、本発明の有効量のＳＧＰは前記飲食品と同様にどのような飼料に配合しても良く、飼料の製造工程中に原料に添加しても良い。
このようにして製造された有効量のＳＧＰを含む飼料は、アッカーマンシア・ムシニフィラの増加促進用飼料として提供される。
SGP (or a composition containing SGP) obtained by the above-mentioned production method of the present invention can be used as it is as a raw material for feed, and it can be produced according to the standard method of feed except for adding a composition containing SGP. good.
Therefore, the effective amount of SGP of the present invention may be added to any feed as the food and drink, and may be added to the raw material during the feed production process.
The feed containing the effective amount of SGP thus produced is provided as a feed for promoting the growth of Akkermansia muciniphila.
（ＳＧＰの摂取量）
本発明のＳＧＰを飲食品、医薬品、飼料などの素材又はそれら素材の加工品に配合させてアッカーマンシア・ムシニフィラの増加促進用組成物を製造する場合、配合割合は特に限定されず、製造の容易性や好ましい一日投与量にあわせて適宜調節すればよい。
一日投与量は、投与対象の症状、年齢などを考慮してそれぞれ個別に決定されるが、成人ヒトの場合、ＳＧＰを1日当り０．１ｇ以上を摂取すればよく、１ｇ以上が好ましく、１０ｇ以上がさらに好ましい。
(Intake of SGP)
When the composition of the present invention for promoting the increase of Akkermansia muciniphila is prepared by incorporating the SGP of the present invention into raw materials such as food and drink, pharmaceuticals, feeds, or processed products of those raw materials, the mixing ratio is not particularly limited, It may be appropriately adjusted according to the nature and the preferable daily dose.
Although the daily dose is determined individually in consideration of the condition to be administered, age, etc., in the case of an adult human, SGP may be taken at least 0.1 g per day, preferably 1 g or more, 10 g The above is more preferable.
以下、本発明の実施例を詳細に説明するが、本発明はこれらに限定されるものではない。 Examples of the present invention will be described in detail below, but the present invention is not limited thereto.
〔実施例１〕本発明のＳＧＰの調製方法
１．ＧＭＰの製造方法
ホエイタンパク質濃縮物（サンラクトＮ−２、太陽化学製）１ｋｇを５０℃の水５０Ｌに溶解し、濃塩酸によりｐＨ３．５に調整した。これを、分画分子量２０，０００の限外ろ過膜（ＧＲ６１ＰＰ、ＤＤＳ製）を用い、５０℃、圧力０．４ＭＰａ、平均透過液流速５２．４Ｌ／ｍ２・ｈにて限外ろ過を行なった。透過液量が４０Ｌに達した時点で濃縮液に５０℃の水４０Ｌを加え、連続して限外ろ過を行なった。以上の様にして連続運転を行い、透過液を１６０Ｌ得た。
得られた透過液に２５％苛性ソーダを加え、ｐＨ７．０とし、再度同じ条件、同じ限外ろ過膜で濃縮液が５Ｌになるまで限外ろ過を行い、脱塩濃縮した。続いて５０℃の水を加え、濃縮液量を常に１０Ｌに保ちながら、これまでと同じ条件、同じ限外ろ過膜でダイアフィルトレーションを行い、さらに脱塩した。このダイアフィルトレーションにより透過液量が８０Ｌに達した時点で濃縮液に水を加えるのをやめ、濃縮液量が２Ｌになるまで限外ろ過にて濃縮し、この濃縮液を乾燥し、ＧＭＰ５４ｇを得た。
Example 1 Method of Preparing SGP of the Present Invention
1. Production method of GMP 1 kg of whey protein concentrate (Sunlacto N-2, manufactured by Solar Chemical Co., Ltd.) was dissolved in 50 L of water at 50 ° C., and adjusted to pH 3.5 with concentrated hydrochloric acid. This is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cut off of 20,000 (GR 61 PP, manufactured by DDS) at 50 ° C., a pressure of 0.4 MPa, and an average permeate flow rate of 52.4 L / m 2 · h The When the permeate volume reached 40 L, 40 L of water at 50 ° C. was added to the concentrate and ultrafiltration was performed continuously. Continuous operation was performed as described above to obtain 160 L of permeate.
To the obtained permeated liquid, 25% caustic soda was added to adjust to pH 7.0, and under the same conditions, under the same ultrafiltration membrane, ultrafiltration was performed until the concentrated liquid reached 5 L, and it was desalted and concentrated. Subsequently, water at 50 ° C. was added, and diafiltration was performed with the same ultrafiltration membrane under the same conditions as the above, while keeping the concentrated liquid volume always at 10 L, and further desalted. Water is not added to the concentrate when the permeate volume reaches 80 L by this diafiltration, concentrated by ultrafiltration until the concentrate volume becomes 2 L, and the concentrate is dried, GMP 54 g I got
２．ＳＧＰの製造方法
上記１．で得られたＧＭＰを用い、３０Ｋｇの５％ＧＭＰ溶液を調製した。これをエンド型プロテアーゼ（アクチナーゼＡＳ、科研ファルマ製）を添加して５０℃で６時間反応させた。これを、分画分子量１，０００の限外ろ過膜を用いて限外ろ過を行なった。濃縮液に水を加えて連続して限外ろ過を行なうことでダイアフィルトレーションを行ない、５倍の加水濃縮を行なった後に濃縮液画分を得た。この濃縮液を乾燥し、ＳＧＰ（１）２７１．７ｇを得た。
３．２種類の酵素を組合わせたＳＧＰの製造方法
上記１．で得られたＧＭＰを用い、３０Ｋｇの５％ＧＭＰ溶液を調製した。これをエンド型プロテアーゼ（オリエンターゼＡＹ、ＨＢＩ製）を添加して５０℃で４時間反応させた後、エキソ型プロテアーゼ（ペプチダーゼＲ、天野エンザイム製）を添加して３７℃で２時間反応した。これを、分画分子量１，０００の限外ろ過膜を用いて限外ろ過を行なった。濃縮液に水を加えて連続して限外ろ過を行なうことでダイアフィルトレーションを行ない、５倍の加水濃縮を行なった後に濃縮液画分を得た。この濃縮液を乾燥し、ＳＧＰ（２）を３１２．６ｇ得た。
2. Method for producing SGP A 30 kg 5% GMP solution was prepared using the GMP obtained above. This was added with endo type protease (Actinase AS, manufactured by Kaken Pharma) and reacted at 50 ° C. for 6 hours. This was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cut off of 1,000. Water was added to the concentrated solution and diafiltration was performed by continuous ultrafiltration, and after concentrated by 5 times of hydrolysis, a concentrated solution fraction was obtained. The concentrate was dried to obtain 271.7 g of SGP (1).
3.2 Method for Producing SGP Combining Two or More Kinds of Enzymes Above. A 30 kg 5% GMP solution was prepared using the GMP obtained above. This was added to endo-type protease (Orientase AY, manufactured by HBI) and allowed to react at 50 ° C. for 4 hours, then exo-type protease (Peptidase R, manufactured by Amano Enzyme) was added, and reacted at 37 ° C. for 2 hours. This was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cut off of 1,000. Water was added to the concentrated solution and diafiltration was performed by continuous ultrafiltration, and after concentrated by 5 times of hydrolysis, a concentrated solution fraction was obtained. The concentrate was dried to obtain 312.6 g of SGP (2).
〔実施例２〕ＳＧＰのシアリダーゼ処理
実施例１で調製したＳＧＰ（１）を２ｍＭ塩化カルシウムを含む１００ｍＭ酢酸緩衝液（ｐＨ５．０）で２０ｍｇ／ｍＬに調製し、それにシアリダーゼ（Clostridium perfringens由来：Ｓｉｇｍａ，Ｎ２８７６−２５ＵＮ）を１０Ｕ／ｍＬとなるように加え、３７℃で２０時間反応させた。酵素添加なしのブランクは、酵素を添加せずに同条件で反応させた。反応後は５分間ボイルすることで、反応を停止させた。得られた反応液は、サイズ排除クロマトグラフィー（ＳＥＣ）に供した。ＳＥＣ分析には、２本のＴＳＫｇｅｌ Ｇ３０００ＰＷ（東ソー）を装着したＬ−２０００（ＨＩＴＡＣＨＩ）システムを用い、ＵＶ２１４ｎｍの吸収を検出した。移動相として０．１％トリフルオロ酢酸を含む４０％アセトニトリル溶液を用い、流速０．３ｍｌ／ｍｉｎで１２０分間室温でアイソクラチック（isocratic）に溶出した。
ＳＥＣ分析の結果を図１に示す。７０分付近に大きなピークが認められたが、このピークは、酵素反応に用いた酢酸緩衝液である。
ＳＧＰの主要なピークは５０分から６０分の間に認められたが、これらはシアリダーゼ処理することで全体的に低分子側にシフトした。このことから、調製したＳＧＰ（１）の主体は、シアル酸を含有する化合物であることが明らかとなった。
Example 2 Sialidase Treatment of SGP SGP (1) prepared in Example 1 was prepared at 20 mg / mL in 100 mM acetate buffer (pH 5.0) containing 2 mM calcium chloride, and sialidase (from Clostridium perfringens origin: Sigma) , N 2876-25UN) to 10 U / mL, and allowed to react at 37 ° C. for 20 hours. Blanks without addition of enzyme were reacted under the same conditions without addition of enzyme. After the reaction, the reaction was stopped by boiling for 5 minutes. The resulting reaction solution was subjected to size exclusion chromatography (SEC). For SEC analysis, UV-214 nm absorbance was detected using an L-2000 (HITACHI) system equipped with two TSKgel G3000PW (Tosoh). Using a 40% acetonitrile solution containing 0.1% trifluoroacetic acid as a mobile phase, it was eluted in an isocratic at room temperature for 120 minutes at a flow rate of 0.3 ml / min.
The results of the SEC analysis are shown in FIG. A large peak was observed around 70 minutes, which is the acetate buffer used for the enzyme reaction.
The major peaks of SGP were observed between 50 and 60 minutes, but they were totally shifted to the low molecular side by sialidase treatment. From this, it became clear that the main component of the prepared SGP (1) is a sialic acid-containing compound.
〔実施例３〕ＳＧＰのＬＣ／ＭＳを用いた構造解析
ＬＣ／ＭＳ分析には、高速液体クロマトグラフィー（ＨＰＬＣ）ＰａｒａｄｉｇｍＭＳ２（Michrom Bioresources）に接続したイオントラップ型質量分析計ＬＴＱ Ｖｅｌｏｓ（Thermofisher scientific）を用い（ＬＣ−ＥＳＩ−ＩＴ ＭＳ）、分離カラムとしてＩｎｅｒｔＳｕｓｔａｉｎ Ｃ１８（φ０．２ｍｍ×１５０ｍｍ，ジーエルサイエンス）を装着した。移動相には、０．１％のギ酸を含んだ２％アセトニトリル溶液（Ａ液）、および０．１％ギ酸を含んだ９０％アセトニトリル溶液（Ｂ液）を用いた。各ＳＧＰを５０％アセトニトリル−０．１％ギ酸溶液で１００μｇ／ｍＬに調製し、２５μＬをＨＰＬＣに導入した。移動相は２μＬ／ｍｉｎで通液し、試料導入後０分から９０分の間にＢ液の割合を０％から４５％まで直線的に増加させた。質量分析計は、ＭＳ測定（ｍ／ｚ４００−２０００，ポジティブモード）とＭＳ／ＭＳ測定を行った。
ＭＳ／ＭＳ測定は、ＭＳのうち強度の高い順に５つのイオンを自動的にプレカーサーイオンとして選択し、コリジョンエネルギー３５Ｖで破壊して生じたプロダクトイオンのｍ／ｚを観測した。プレカーサーイオンとプロダクトイオンのｍ／ｚの差が２９２であるものをＮｅｕ５Ａｃ、３３４をＮ，Ｏ−ジアセチルノイラミン酸（O-Ac-Neu5Ac）、３７６をＮ，Ｏ，Ｏ−トリアセチルノイラミン酸（O,O-diAc-Neu5Ac）、２０３をＧａｌＮＡｃ、１６２をＧａｌと帰属し、プロダクトイオンのパターンから糖鎖構造を決定した。糖鎖が完全に解離したと思われるイオンのｍ／ｚをペプチドにプロトンが付加したイオンに由来すると推定し、そのｍ／ｚに相当するペプチド配列をＧＭＰの配列中から探索することで、糖ペプチドの構造を推定した。
実施例１で調製したＳＧＰ（１）について、ＬＣ／ＭＳ分析で得られたＭＳスペクトルおよびＭＳ／ＭＳスペクトルから、ＳＧＰに含まれる糖ペプチドの糖鎖構造を推定した。
表１に、ＬＣ／ＭＳ分析で検出した糖ペプチドの糖鎖構造を示す。その結果、検出したいずれの糖ペプチドもシアル酸を含む糖鎖が結合したペプチドであった。検出した多くの糖ペプチドがシアル酸を２分子結合したNeu5Acα1-3Galβ1-3(Neu5Acα1-6)GalNAcの糖鎖構造（Glycan type 3, 4, 5, 6, 7）を有していたが、シアル酸が１分子であるNeu5Acα1-3Galβ1-3GalNAc（Glycan type 1）やGalβ1-3(Neu5Acα1-6)GalNAc（Glycan type 2）も検出された。さらに、シアル酸のＯ−アセチル体であるＮ，Ｏ−ジアセチルノイラミン酸（O-Ac-Neu5Ac、Glycantype 4）、およびＮ，Ｏ，Ｏ−トリアセチルノイラミン酸（O- diAc-Neu5Ac、Glycan type 5）も検出された。、また、一つのペプチド鎖に2本の糖鎖が結合した糖ペプチド（Glycan type 6, 7）も検出された。
ＭＳ／ＭＳスペクトル解析から検出したペプチド鎖は、いずれもウシのグリコマクロペプチドに含まれるペプチド鎖であった。検出した全ての糖ペプチドにはセリンまたはスレオニン残基が1分子以上存在し、ペプチド鎖長は最も短いもので１残基、最も長いもので１３残基であった。また、検出したペプチド鎖の分子量は、７４５から２９２８の範囲であった。
[Example 3] Structural analysis of SGP using LC / MS For LC / MS analysis, ion trap mass spectrometer LTQ Velos (Thermofisher scientific) connected to high performance liquid chromatography (HPLC) Paradigm MS 2 (Michrom Bioresources) Using (LC-ESI-IT MS), InertSustain C18 (φ 0.2 mm × 150 mm, GL Science) was installed as a separation column. As the mobile phase, a 2% acetonitrile solution (solution A) containing 0.1% formic acid and a 90% acetonitrile solution (solution B) containing 0.1% formic acid were used. Each SGP was adjusted to 100 μg / mL with 50% acetonitrile-0.1% formic acid solution, and 25 μL was introduced to HPLC. The mobile phase was passed at 2 μL / min, and the proportion of solution B was linearly increased from 0% to 45% between 0 and 90 minutes after sample introduction. The mass spectrometer performed MS measurement (m / z 400-2000, positive mode) and MS / MS measurement.
In MS / MS measurement, five ions among MSs in descending order of intensity were automatically selected as precursor ions, and m / z of product ions generated by destruction with collision energy of 35 V was observed. Neu5Ac, 334, N, O-Diacetylneuraminic acid (O-Ac-Neu5Ac), 376, N, O, O-Triacetylneuraminic acid in which the m / z difference between the precursor ion and the product ion is 292 (O, O-diAc-Neu5Ac), 203 was assigned to GalNAc, 162 to Gal, and the sugar chain structure was determined from the product ion pattern. It is assumed that m / z of the ion which is thought to be completely dissociated from the sugar chain is derived from the ion obtained by adding a proton to the peptide, and the peptide sequence corresponding to the m / z is searched from among the GMP sequences. The structure of the peptide was deduced.
For SGP (1) prepared in Example 1, the sugar chain structure of the glycopeptide contained in SGP was estimated from the MS spectrum and the MS / MS spectrum obtained by LC / MS analysis.
Table 1 shows sugar chain structures of glycopeptides detected by LC / MS analysis. As a result, all of the detected glycopeptides were peptides to which a sugar chain containing sialic acid was bound. Many glycopeptides detected had the sugar chain structure (Glycan type 3, 4, 5, 6, 7) of Neu5Acα1-3Galβ1-3 (Neu5Acα1-6) GalNAc in which two molecules of sialic acid were linked, but One molecule of acid, Neu5Acα1-3Galβ1-3GalNAc (Glycan type 1) and Galβ1-3 (Neu5Acα1-6) GalNAc (Glycan type 2), was also detected. Furthermore, N, O-diacetylneuraminic acid (O-Ac-Neu5Ac, Glycantype 4), which is an O-acetyl form of sialic acid, and N, O, O-triacetylneuraminic acid (O-diAc-Neu5Ac, Glycan type 5) was also detected. Also, a glycopeptide (Glycan type 6, 7) in which two sugar chains were linked to one peptide chain was also detected.
The peptide chains detected by MS / MS spectral analysis were all peptide chains contained in bovine glycomacropeptide. In all the glycopeptides detected, at least one molecule of serine or threonine residue was present, and the shortest peptide chain length was one residue and the longest was 13 residues. Also, the molecular weight of the detected peptide chain was in the range of 745 to 2928.
〔実施例４〕ＳＧＰの構成糖と存在比
ＳＧＰは糖鎖とペプチド鎖から成るが、アッカーマンシア・ムシニフィラの増加作用にはＳＧＰ中の糖鎖の構成糖とその存在比率が重要と考えられる。
実施例３で示したように、ＳＧＰの糖鎖は、シアル酸、およびガラクト−Ｎ−ビオース（Galb1-3GalNAc）から成ることが明らかとなった。よって、次にＳＧＰ中のシアル酸とガラクト-Ｎ-ビオースのそれぞれの量を測定し、これらの存在比を調べた。
実施例１で調製したＳＧＰ中のシアル酸量は、以下に記載した方法で測定した。すなわち、シアル酸（Ｎ−アセチルノイラミン酸）量を測定するために、ねじ口試験管に１００μＬのサンプル溶液、８００μＬの水、および１００μＬの１Ｎ硫酸溶液を添加し、ブロックヒーターを用いて８０℃で４５分間、加熱した。冷却後、等量の１００ｍＭの水酸化ナトリウムで中和した後、０．４５μｍのフィルターで不溶物を除去した。得られた反応液中の糖含量は、Carbopak PA1カラムを装着したDIONEX ICS-5000DPシステムを用いて測定した。
実施例１で調製したＳＧＰからガラクト−Ｎ−ビオースを遊離させるために、まず実施例２に記載した方法でＳＧＰをシアリダーゼ処理した。シアリダーゼ処理後のＳＧＰは、引き続きＯ−グルカナーゼ（エンド−a−Ｎ−アセチルガラクトサミニダーゼ）処理することで、ＳＧＰからガラクト−Ｎ−ビオースを遊離させた。遊離したガラクト−Ｎ−ビオースは、Carbopak PA1カラムを装着したDIONEX ICS-5000DPシステムを用いて測定した。
シアル酸（Ｎ−アセチルノイラミン酸）とガラクト−Ｎ−ビオースのモル比は、実施例１で調製したＳＧＰ（１）が１．８６：１、ＳＧＰ（２）が１．６７：１であった
Example 4 Constituent Sugars and Abundance Ratio of SGP Although SGP is composed of a sugar chain and a peptide chain, it is considered that the constituent sugars of sugar chains in SGP and the proportion thereof are important for the increase action of Akkermansia muciniphila.
As shown in Example 3, it was revealed that the sugar chain of SGP consists of sialic acid and galacto-N-biose (Galb1-3GalNAc). Therefore, next, the amount of each of sialic acid and galacto-N-biose in SGP was measured, and their abundance ratio was examined.
The amount of sialic acid in SGP prepared in Example 1 was measured by the method described below. That is, in order to measure the amount of sialic acid (N-acetylneuraminic acid), 100 μL of sample solution, 800 μL of water, and 100 μL of 1 N sulfuric acid solution are added to a screw test tube, and 80 ° C. using a block heater. Heat for 45 minutes. After cooling, after neutralization with an equal volume of 100 mM sodium hydroxide, insolubles were removed with a 0.45 μm filter. The sugar content in the resulting reaction solution was measured using a DIONEX ICS-5000 DP system equipped with a Carbopak PA1 column.
In order to release galacto-N-biose from SGP prepared in Example 1, SGP was first treated with sialidase by the method described in Example 2. After sialidase treatment, SGP was subsequently treated with O-glucanase (endo-a-N-acetylgalactosaminidase) to release galacto-N-biose from SGP. Released galacto-N-biose was measured using a DIONEX ICS-5000 DP system equipped with a Carbopak PA1 column.
The molar ratio of sialic acid (N-acetylneuraminic acid) to galacto-N-biose is 1.86: 1 for SGP (1) and 1.67: 1 for SGP (2) prepared in Example 1. The
〔試験例１〕
マウスに通常餌（コントロール）、実施例１で調製したＳＧＰ（１）を１０％添加した餌を３週間摂取させた。摂取期間終了後に盲腸内容物を採取し、ＤＮＡを抽出した。得られたＤＮＡから総菌量とアッカーマンシア・ムシニフィラ量を測定するために、リボゾームＲＮＡ遺伝子をターゲットとした定量的ＰＣＲを行なった。ヒトにおける難消化性成分の主な発酵部位は大腸でありアッカーマンシア・ムシニフィラも大腸に存在するが、マウスでの主な発酵部位は盲腸であるため、この試験では盲腸内容物について測定した。したがって、実験結果は盲腸内容物中の総菌量、及びアッカーマンシア・ムシニフィラ量を示すが、これらの結果が盲腸での作用に限定するわけではなく、ヒトでの主な発酵部位である大腸における大腸内容物または糞便中の総菌量、及びアッカーマンシア・ムシニフィラ量に置き換えることができる。
総菌量はＣｏｎｔｒｏｌ群に比べてＳＧＰを１０％添加した餌を摂取したＳＧＰ群で有意に増加した（図２）。
また、アッカーマンシア・ムシニフィラ量は、Ｃｏｎｔｒｏｌ群に比べて、ＳＧＰ群で顕著に増加した（図３）。ＳＧＰ群は、Ｃｏｎｔｒｏｌ群よりも約１，０００倍多いアッカーマンシア・ムシニフィラが検出された。
これらの結果から、ＳＧＰは腸内のアッカーマンシア・ムシニフィラが増加させる作用を有していることが明らかとなった。
[Test Example 1]
The mice were fed a normal diet (control) and a diet to which 10% of SGP (1) prepared in Example 1 was added for 3 weeks. Cecal contents were collected after the intake period and DNA was extracted. In order to measure the total amount of bacteria and the amount of Akkermansia muciniphila from the obtained DNA, quantitative PCR targeting ribosomal RNA gene was performed. The main fermentation site of the indigestible component in humans is the large intestine and Akkermansia muciniphila is also present in the large intestine, while the main fermentation site in mice is the cecum, so in this test the cecal content was determined. Therefore, although the experimental results show the total amount of bacteria in the cecal contents and the amount of Akkermansia muciniphila, these results are not limited to the action in the cecum, but in the large intestine which is the main fermentation site in humans. It can be replaced by the total amount of bacteria in the contents of the large intestine or feces, and the amount of Akkermansia muciniphila.
The total amount of bacteria was significantly increased in the SGP group fed a diet containing 10% SGP compared to the Control group (FIG. 2).
Also, the amount of Akkermansia muciniphila increased significantly in the SGP group as compared to the Control group (FIG. 3). In the SGP group, about 1,000 times more Akermansia muciniphila was detected than in the Control group.
From these results, it became clear that SGP has an action to increase Akkermansia muciniphila in the intestine.
〔実施例５〕サプリメントの製造
実施例１で得られたＳＧＰ粉末３０ｇに、ビタミンＣとクエン酸の等量混合物４０ｇ、グラニュー糖１００ｇ、コーンスターチと乳糖の等量混合物６０ｇを加えて混合した。混合物をスティック状袋に詰め、本発明のアッカーマンシア・ムシニフィラ増加促進用サプリメントを製造した。
Example 5 Preparation of Supplement To 30 g of the SGP powder obtained in Example 1, 40 g of an equivalent mixture of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of an equivalent mixture of corn starch and lactose were added and mixed. The mixture was packed in a stick-like bag to produce an Akkermansia muciniphila increasing supplement of the present invention.
〔実施例６〕飲料の製造
表２に示した配合により原料を混合し、容器に充填した後、加熱殺菌して、本発明のアッカーマンシア・ムシニフィラ増加促進用飲料を製造した。
Example 6 Preparation of Beverage The ingredients were mixed according to the formulation shown in Table 2 and filled in a container, which was then heat sterilized to produce an Akkermansia muciniphila increase promoting beverage of the present invention.
〔実施例７〕医薬品（カプセル剤）の製造
表３に示した配合により原料を混合し、造粒により顆粒状とした後、空カプセルに１０ｍｇずつ充填して、本発明のアッカーマンシア・ムシニフィラ増加促進用医薬品を含むカプセル剤を製造した。
[Example 7] Preparation of pharmaceutical (capsule) The raw materials were mixed according to the formulation shown in Table 3 and granulated to form granules, and then 10 mg each was filled into empty capsules to increase Akkermansia muciniphila of the present invention. A capsule containing a drug for promotion was manufactured.
本発明によれば、ＳＧＰを有効成分とする新たなアッカーマンシア・ムシニフィラ増加促進用組成物、及びＳＧＰを有効成分とするアッカーマンシア・ムシニフィラの増加促進用飲食品、医薬品、飼料を提供することが可能となった。 According to the present invention, it is possible to provide a new composition for promoting Akkermansia muciniphila comprising SGP as an active ingredient, and a food, beverage, medicine, feed for the promotion of Akkermansia muciniphila comprising SGP as an active ingredient. It has become possible.
Claims (10)
前記糖ペプチドの分子量が５００以上３，０００以下であり、
前記糖鎖が前記ペプチドのスレオニン又はセリン残基の水酸基に結合し、
前記糖鎖の構成糖がシアル酸、Ｎ−アセチルガラクトサミン及びガラクトースからなる群から選択される１つ以上であることを特徴とするアッカーマンシア・ムシニフィラ増加促進用組成物。 What is claimed is: 1. A composition for increasing Akkermansia muciniphila comprising a sialylglycopeptide in which a threonine and / or a peptide having a serine residue is linked to a sugar chain,
The molecular weight of the glycopeptide is 500 or more and 3,000 or less,
The sugar chain is bonded to the hydroxyl group of threonine or serine residue of the peptide,
A composition for promoting increase of Akkermansia muciniphila, wherein the constituent sugar of the sugar chain is one or more selected from the group consisting of sialic acid, N-acetylgalactosamine and galactose.
前記糖鎖が、以下の（１）〜（５）からなる群から選ばれる１つ以上であることを特徴とするアッカーマンシア・ムシニフィラ増加促進用組成物。
（１）Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1
（２）Galβ1-3(Neu5Acα2-6)GalNAcα1
（３）Neu5Acα2-3Galβ1-3GalNAcα1
（４）Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1
（５）Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1 It is a composition containing an threonine and / or serine residue and a sialyl glycopeptide in which a sugar chain is bonded to a hydroxyl group of the residue, and the composition for promoting increase of Akkermansia muciniphila,
The composition for promoting Akkermansia muciniphila increase, characterized in that the sugar chain is one or more selected from the group consisting of the following (1) to (5):
(1) Neu5Acα2-3Galβ1-3 (Neu5Acα2-6) GalNAcα1
(2) Galβ1-3 (Neu5Acα2-6) GalNAcα1
(3) Neu5Acα2-3Galβ1-3GalNAcα1
(4) Neu5Acα2-3Galβ1-3 (O-Ac-Neu5Acα2-6) GalNAcα1
(5) Neu5Acα2-3Galβ1-3 (O-diAc-Neu5Acα2-6) GalNAcα1
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