JP4726129B2 - Bifidobacterium growth-promoting peptide - Google Patents
Bifidobacterium growth-promoting peptide Download PDFInfo
- Publication number
- JP4726129B2 JP4726129B2 JP2006008738A JP2006008738A JP4726129B2 JP 4726129 B2 JP4726129 B2 JP 4726129B2 JP 2006008738 A JP2006008738 A JP 2006008738A JP 2006008738 A JP2006008738 A JP 2006008738A JP 4726129 B2 JP4726129 B2 JP 4726129B2
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- bifidobacteria
- growth
- actomyosin
- leu
- 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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Description
本発明は、食肉タンパク質等をパパイン等のプロテアーゼで分解することにより得られるビフィズス菌増殖促進効果を有する組成物、及び組成物中の主要有効成分であるペプチドおよびこのペプチドを有効成分として含有する食品、飼料、サプリメント、医薬品に関する。 The present invention relates to a composition having an effect of promoting growth of bifidobacteria obtained by degrading meat protein or the like with a protease such as papain, a peptide which is a main active ingredient in the composition, and a food containing the peptide as an active ingredient , Feed, supplements, pharmaceuticals.
ビフィズス菌（Bifidobacterium属の細菌）はヒトや動物の消化管内に棲息し、有益な働きをする微生物として注目されている。ビフィズス菌の宿主への有益な作用として、整腸作用を初め、免疫防御系改善作用、抗腫瘍作用などが報告されている。また、近年、プロバイオティクス（probiotics）という概念に対する関心が高まっている。Fuller（1989）〔非特許文献１〕は、プロバイオティクスを、「腸内微生物フローラのバランスを改善することによって宿主の健康に好影響を与える生菌体」とした。さらに、Havenaar（1992）〔非特許文献２〕は、この概念を拡張し、「ヒトや動物に投与した際に、微生物フローラの改善効果によって、消化器系、呼吸器系、泌尿器系等を対象に広く宿主に好影響を与える、生きた一種もしくは混合微生物」とした。ビフィズス菌は、プロバイオティクスとして用いられている代表的な細菌であり、生菌剤、サプリメント、食品等に広く産業的に利用されている。 Bifidobacteria (bacteria belonging to the genus Bifidobacterium) are attracting attention as microorganisms that live beneficially in the digestive tract of humans and animals. As beneficial effects of bifidobacteria on the host, an intestinal regulating action, an immune defense system improving action, an antitumor action and the like have been reported. In recent years, interest in the concept of probiotics has increased. Fuller (1989) [Non-Patent Document 1] described probiotics as “viable cells that positively affect the health of the host by improving the balance of the intestinal microbial flora”. In addition, Havenaar (1992) [Non-Patent Document 2] extends this concept to: “When administered to humans and animals, the effects of microbial flora improve the digestive system, respiratory system, urinary system, etc. It is a living species or mixed microorganism that has a positive impact on the host. Bifidobacteria are typical bacteria used as probiotics, and are widely used industrially in live bacteria, supplements, foods and the like.
また、ビフィズス菌等のプロバイオティクスとして用いられる細菌の増殖を促進させる物質も注目されている。このような物質は、プレバイオティクス（prebiotics）と呼ばれるようになっている。Gibson and Rooberfroid（1995）〔非特許文献３〕は、プレバイオティクスを、「腸内に生息する有用菌に選択的に働き、増殖を促進したり、その活性を高めることによって、宿主の健康に遊離に作用する物質」と定義している。プレバイオティクスとして、オリゴ糖（イヌリンやフルクトオリゴ糖など）のような難消化性糖類がよく知られており、すでに食品等において盛んに利用されている。 In addition, substances that promote the growth of bacteria used as probiotics such as bifidobacteria have attracted attention. Such materials have come to be called prebiotics. Gibson and Rooberfroid (1995) [Non-Patent Document 3] clarified that prebiotics can be used to improve the health of the host by selectively acting on useful bacteria that inhabit the intestines and promoting their growth or increasing their activity. It is defined as “a substance that acts freely”. As prebiotics, indigestible saccharides such as oligosaccharides (such as inulin and fructooligosaccharide) are well known and are already actively used in foods and the like.
一方、ビフィズス菌に対して増殖促進効果を示すペプチドも見出され、プレバイオティクスとしての可能性が注目されている。牛乳タンパク質の酵素分解物にはビフィズス菌増殖促進効果があるという報告が、これまでに比較的多くされてきたが、その多くはペプチドに結合する糖鎖部分の作用によることが明らかにされた。しかし、Liepleら（2002）〔非特許文献４〕は、人乳タンパク質のペプシン分解物にビフィズス菌増殖促進効果があることを見出し、この効果は糖鎖を含まないアミノ酸だけからなるペプチドによるものであることを示した。彼らは、分子量5,584および5,801Ｄａの２種のペプチドを同定し、これらがラクトフェリンの部分配列であることを報告した。さらに、これらのペプチドの配列情報をもとにし、prebiotic lactoferrin-derived peptide-I（PRELP-I）をデザイン・合成した。また、タンパク質分解物以外のビフィズス菌増殖促進ペプチドとして、Etohら（2000）〔非特許文献５〕は、ゴムの木の樹液から１０個のアミノ酸からなる特定配列のペプチドを発見した。 On the other hand, a peptide having a growth promoting effect on bifidobacteria has also been found, and its potential as a prebiotic has attracted attention. There have been a relatively large number of reports that enzymatic degradation products of milk proteins have a bifidobacteria growth-promoting effect, but it has been clarified that most of them are due to the action of a sugar chain moiety bound to a peptide. However, Lieple et al. (2002) [Non-Patent Document 4] found that a pepsin degradation product of human milk protein has a bifidobacteria growth-promoting effect, and this effect is due to a peptide consisting only of amino acids not containing sugar chains. It showed that there is. They identified two peptides with molecular weights of 5,584 and 5,801 Da and reported that these are partial sequences of lactoferrin. Furthermore, based on the sequence information of these peptides, prebiotic lactoferrin-derived peptide-I (PRELP-I) was designed and synthesized. As a bifidobacteria growth-promoting peptide other than protein degradation products, Etoh et al. (2000) [Non-Patent Document 5] discovered a peptide having a specific sequence consisting of 10 amino acids from the sap of a rubber tree.
しかし、これらのビフィズス菌増殖促進効果を示すペプチドは、プレバイオティクスとして用いるには、なお、いくつかの問題が残されている。Liepleらのペプチドは、人乳由来のものであり、食品等の素材としては調製が困難であるし、合成ペプチドであるPRELP Iもコストの面から食品素材としての利用は困難であろう。また、Etohらの見出したペプチドは、食品由来のものでないため、安全性評価など検討すべき課題も残されている。さらに、これらのペプチドをヒトや動物が摂取した場合に、下部消化管内でビフィズス菌の増殖に寄与するためには、消化酵素（プロテアーゼ）の作用に対する抵抗性も求められるが、そのような検討は行われていない。 However, some problems still remain when these peptides showing the effect of promoting the growth of bifidobacteria are used as prebiotics. The peptide of Lieple et al. Is derived from human milk and is difficult to prepare as a material for foods, etc., and PRELP I, which is a synthetic peptide, may be difficult to use as a food material from the viewpoint of cost. In addition, since the peptide found by Etoh et al. Is not derived from food, there are still problems to be examined such as safety evaluation. Furthermore, when these peptides are ingested by humans or animals, resistance to the action of digestive enzymes (proteases) is also required to contribute to the growth of bifidobacteria in the lower digestive tract. Not done.
ところで、食肉タンパク質をプロテアーゼ分解することにより、生理活性ペプチドが生成することが報告されている〔特許文献１，２〕。このようなペプチドとして、血圧降下ペプチド、抗酸化ペプチドなどがある。食肉中には非常に多くの種類のタンパク質が存在するため、プロテアーゼ処理により様々なペプチドが生成することが期待される。しかし、これまでに、食肉タンパク質のプロテアーゼ分解物からビフィズス菌増殖促進ペプチドが見出された報告は全くない。 By the way, it has been reported that bioactive peptides are produced by protease degradation of meat protein [Patent Documents 1 and 2]. Examples of such peptides include blood pressure lowering peptides and antioxidant peptides. Since there are so many kinds of proteins in meat, it is expected that various peptides are generated by protease treatment. However, until now, there has been no report that a bifidobacteria growth-promoting peptide has been found from protease degradation products of meat proteins.
本発明の目的は、ヒトや動物の消化管内で有益な作用を示すビフィズス菌を増殖させるための有効な解決手段を得ることにある。具体的には、ビフィズス菌を増殖させることのできる食品・飼料あるいは医薬品のペプチド性素材を提供することである。このような素材は、摂取により消化管内の有用細菌であるビフィズス菌の増殖を促進し、宿主に有益な作用をするだけではなく、通常、食品中において増殖が緩慢であることが知られているビフィズス菌を、加工食品中において増殖を促進させる目的にも使用できる。 An object of the present invention is to obtain an effective solution for growing Bifidobacteria exhibiting beneficial effects in the digestive tract of humans and animals. Specifically, it is to provide a peptide material for food / feed or pharmaceuticals capable of growing Bifidobacteria. Such a material not only promotes the growth of bifidobacteria, a useful bacterium in the gastrointestinal tract when ingested, and has a beneficial effect on the host, but is generally known to be slow in food. Bifidobacteria can also be used for the purpose of promoting growth in processed foods.
上記の課題を解決すべく、本発明者らは、鋭意研究を進め、食肉タンパク質（アクトミオシン）をプロテアーゼ（パパイン等）で処理し、不要物を除去したペプチド含有画分に高いビフィズス菌増殖促進効果のあることを見出した。さらに、アクトミオシンのパパイン処理により得たペプチド含有画分からビフィズス菌増殖促進効果を有するペプチドを精製・同定して、本発明を完成するに至った。 In order to solve the above-mentioned problems, the present inventors proceeded with intensive studies and treated the meat protein (Actomyosin) with protease (papain, etc.) to remove the unnecessary substances and promote high growth of bifidobacteria. I found it effective. Furthermore, the present invention was completed by purifying and identifying a peptide having a bifidobacteria growth-promoting effect from a peptide-containing fraction obtained by papain treatment of actomyosin.
すなわち本発明は、〔１〕アクトミオシンあるいはアクトミオシンを含む食肉タンパク質懸濁液をプロテアーゼで処理することにより得られるペプチド含有画分を有効成分とすることを特徴とするビフィズス菌増殖促進剤や、〔２〕プロテアーゼがパパインであることを特徴とする〔１〕記載のビフィズス菌増殖促進剤や、〔３〕ペプチド含有画分が、Glu-Leu-Metの配列で示されるペプチドを含有することを特徴とする〔１〕又は〔２〕記載のビフィズス菌増殖促進剤や、〔４〕Glu-Leu-Metの配列で示されるペプチド又はその塩を有効成分とするビフィズス菌増殖促進剤に関するものである。
また本発明は、〔５〕アクトミオシンあるいはアクトミオシンを含む食肉タンパク質懸濁液をプロテアーゼで処理することにより得られるペプチド含有画分を含有し、ビフィズス菌増殖促進効果を有することが表示されている食品、サプリメント、飼料、医薬品に関するものである。さらに本発明は、〔６〕Glu-Leu-Metの配列で示されるペプチド又はその塩に関するものである。
That is, the present invention provides [1] a Bifidobacterium growth promoter characterized by comprising as an active ingredient a peptide-containing fraction obtained by treating actomyosin or a meat protein suspension containing actomyosin with a protease, [2] The Bifidobacterium growth promoter according to [1], wherein the protease is papain, and [3] the peptide-containing fraction contains a peptide represented by the Glu-Leu-Met sequence. It relates to a bifidobacteria growth promoter according to [1] or [2], which is characterized, and [4] a bifidobacteria growth promoter comprising a peptide represented by the Glu-Leu-Met sequence or a salt thereof as an active ingredient .
The present invention also includes [5] actomyosin or a peptide-containing fraction obtained by treating a meat protein suspension containing actomyosin with a protease, and is shown to have a bifidobacteria growth promoting effect. It relates to food, supplements, feed, and medicines. Furthermore, the present invention relates to a peptide represented by [6] Glu-Leu-Met sequence or a salt thereof.
本発明の効果は、ヒトや動物の消化管内で有益な作用を示すビフィズス菌を増殖させ、ヒトや動物の健康維持や増進をはかるための問題に解決手段を与えることにある。すなわち、本発明により、ビフィズス菌増殖促進効果のある食品・飼料あるいは医薬品の素材を提供することができる。 The effect of the present invention is to provide a solution to the problem of growing bifidobacteria having a beneficial action in the digestive tract of humans and animals and maintaining and promoting the health of humans and animals. That is, according to the present invention, it is possible to provide a food / feed or pharmaceutical material having an effect of promoting the growth of bifidobacteria.
本発明のビフィズス菌増殖促進効果を有する組成物は、アクトミオシン分解物（ペプチド）を有効成分として含む。アクトミオシンは何に由来するものでもよいが、アクトミオシンは、動物の筋肉（骨格筋）に多く存在しており、これを抽出して用いることができる。用いる骨格筋は、入手のしやすさから、豚、牛、鶏等が適しているが、これらの畜種に限定されるものではない。また、魚類その他の海生生物等に由来するものでもよい。また、筋肉の部位や状態は、アクトミオシンが調製できるものであれば、特に限定されるものではない。アクトミオシンの調製方法は、特定のものを採用する必要はなく、組成物の用途（例えば、飼料等）によってはアクトミオシンを単離精製する必要はなく、単にアクトミオシンを含む食肉タンパク質懸濁液として調製しても構わない。 The composition having the effect of promoting the growth of Bifidobacterium of the present invention contains an actomyosin degradation product (peptide) as an active ingredient. Actomyosin may be derived from any source, but a large amount of actomyosin is present in animal muscles (skeletal muscles) and can be extracted and used. As the skeletal muscle to be used, pigs, cows, chickens and the like are suitable because of their availability, but they are not limited to these breeds. Further, it may be derived from fish or other marine organisms. The muscle site and state are not particularly limited as long as actomyosin can be prepared. It is not necessary to adopt a specific method for preparing actomyosin, and it is not necessary to isolate and purify actomyosin depending on the use of the composition (for example, feed etc.), but simply a meat protein suspension containing actomyosin. You may prepare as.
アクトミオシンの分解は、アクトミオシンの水懸濁液またはアクトミオシンを含む食肉タンパク質の水懸濁液にプロテアーゼを添加して行う。プロテアーゼは、アクトミオシンを適度に分解するものであれば、様々なものが使用可能である。例えば、フィチン、トリプシン、プロテイネースＫ、プロナーゼＥ、パパイン、ブロメライン、パンクレアチン、プロチン及びズブチリシン等が挙げられるが、これらに限定されるものではない。また、これらのプロテアーゼは単独で用いてもよいし２種以上を併用してもよい。パパインを用いた場合に、高い効果を有する組成物が得られるため、その使用が好ましい。 The degradation of actomyosin is performed by adding protease to an aqueous suspension of actomyosin or an aqueous suspension of meat protein containing actomyosin. Various proteases can be used as long as they appropriately decompose actomyosin. Examples include phytin, trypsin, proteinase K, pronase E, papain, bromelain, pancreatin, protin and subtilisin, but are not limited thereto. These proteases may be used alone or in combination of two or more. When papain is used, a composition having a high effect is obtained, and therefore its use is preferable.
プロテアーゼを作用させる場合、温度、ｐＨ等を、それぞれのプロテアーゼの至適条件に設定すると、速やかに分解物を得ることができるが、プロテアーゼの添加量や反応時間によっても制御可能であるため、特定の条件に限定されるものではない。プロテアーゼによりアクトミオシンを分解した後に、溶液を９０〜１００℃に加熱して、プロテアーゼ活性を消失させる。 When proteases are allowed to act, if the temperature, pH, etc. are set to the optimum conditions for each protease, degradation products can be obtained quickly, but it can also be controlled by the amount of protease added and the reaction time. It is not limited to the above conditions. After degrading actomyosin with protease, the solution is heated to 90-100 ° C. to eliminate protease activity.
このように調製したアクトミオシンの分解物に、ビフィズス菌増殖促進効果を有するペプチドが存在し、その効果を大きく阻害する共存物はないので、そのままでも食品素材等に利用することができる。しかし、より有効性を高める必要がある場合は、活性成分（ペプチド）の濃縮や精製を行うことが望ましい。例えば、減圧濃縮機で濃縮し、チャンバー温度を９５℃以下に調整した噴霧乾燥機で乾燥し、得られた粉末を４０℃以下に冷却して粉末状製品を製造することができる。あるいは、凍結乾燥による濃縮や液体クロマトグラフィーによる分取等を行なってもよい。 The degradation product of actomyosin prepared as described above contains a peptide having a bifidobacteria growth-promoting effect, and there is no coexisting substance that greatly inhibits the effect, so that it can be used as it is as a food material. However, it is desirable to concentrate and purify the active ingredient (peptide) when it is necessary to increase its effectiveness. For example, it can be concentrated with a vacuum concentrator, dried with a spray dryer whose chamber temperature is adjusted to 95 ° C. or lower, and the resulting powder can be cooled to 40 ° C. or lower to produce a powder product. Alternatively, concentration by freeze drying, fractionation by liquid chromatography, or the like may be performed.
本発明者らは、アクトミオシンのプロテアーゼ（パパイン）分解物から、ビフィズス菌増殖促進効果を示すペプチドを発見した。すなわち、Glu-Leu-Metの配列を示すトリペプチドである。このペプチドは、アクトミオシンのパパイン分解物から後述の実施例に示すような操作により精製することも可能であるが、配列情報をもとに適当な化学合成法により得ることもできる。いずれの方法によって得たペプチドも同様のビフィズス菌増殖促進効果が認められる。 The present inventors have discovered a peptide exhibiting a bifidobacteria growth promoting effect from a protease (papain) degradation product of actomyosin. That is, it is a tripeptide showing the sequence of Glu-Leu-Met. This peptide can be purified from the papain degradation product of actomyosin by the procedure shown in the examples described later, but can also be obtained by an appropriate chemical synthesis method based on the sequence information. The same bifidobacteria growth-promoting effect is observed in the peptide obtained by any method.
以上に述べた有効成分（ペプチド）、あるいは有効成分を含む組成物は、通常、経口的にヒトあるいは動物に投与（摂取）される。また、本有効成分は、食肉を摂取した場合に、生体（消化管）内で消化酵素の作用により食肉タンパク質から生成するペプチドと類似の構造を示すものであり、多量の摂取によっても生体への悪影響はきわめて小さいものと判断できる。したがって、ヒトを含め動物への安全性の高い素材と言えよう。さらに、通常、食品中において増殖が緩慢であることが知られているビフィズス菌を、食品中において増殖を促進させる目的にも、以上に述べた有効成分（ペプチド）、あるいは有効成分を含む組成物は、使用できる。 The active ingredient (peptide) described above or a composition containing the active ingredient is usually administered (ingested) to a human or an animal orally. In addition, this active ingredient shows a structure similar to a peptide produced from meat protein by the action of digestive enzymes in the living body (digestive tract) when meat is ingested, and even when ingested in large quantities, It can be judged that the adverse effect is extremely small. Therefore, it can be said that the material is highly safe for animals including humans. Furthermore, the above-mentioned active ingredient (peptide) or a composition containing the active ingredient is also used for the purpose of promoting the growth of Bifidobacterium, which is known to be slow in food, in food. Can be used.
従って、本発明の組成物は、ビフィズス菌増殖促進剤として、また、ビフィズス菌増殖促進効果を有する食品、サプリメント、飼料、医薬品として広く利用できる。
本発明の有効成分（ペプチド）を経口投与（摂取）する場合、本発明の効果が損なわれない範囲で、上記の各製品に慣用される他の成分を含んでもよい。このような添加剤の例としては、賦形剤（ラクトース、スターチ等）、結合剤（シロップ、ゼラチン等）、香料、崩壊剤等が挙げられる。また、水溶性食物繊維、不溶性食物繊維、オリゴ糖等のビフィズス菌の増殖に有用な成分を添加してもよい。さらに、ビタミンＡ、ビタミンＢ1、ビタミンＢ2、ビタミンＢ6、ビタミンＢ12、ビタミンＣ、ビタミンＤ、ビタミンＥ、ナイアシン（ニコチン酸）、パントテン酸、葉酸等のビタミン類、リジン、スレオニン、トリプトファン等の必須アミノ酸類や、カルシウム、マグネシウム、鉄、亜鉛、銅等のミネラル類及び、例えば、α−リノレン酸、ＥＰＡ、ＤＨＡ、カゼインホスホペプチド（ＣＰＰ）、カゼインカルシウムペプチド（ＣＣＰ）等の人の健康に寄与する物質類、その他の食品や食品添加物として認可されている有用物質の１種又は２種以上が使用できる。
あるいは適当な食品等に添加・加工（混合、加熱等）してもよい。
Therefore, the composition of the present invention can be widely used as a bifidobacteria growth promoter and as a food, supplement, feed or pharmaceutical product having a bifidobacteria growth promoting effect.
When the active ingredient (peptide) of the present invention is orally administered (taken), other ingredients commonly used in the above products may be included as long as the effects of the present invention are not impaired. Examples of such additives include excipients (such as lactose and starch), binders (such as syrup and gelatin), fragrances, and disintegrants. In addition, components useful for the growth of bifidobacteria such as water-soluble dietary fiber, insoluble dietary fiber, and oligosaccharide may be added. In addition, vitamin A, vitamin B 1 , vitamin B 2 , vitamin B 6 , vitamin B 12 , vitamin C, vitamin D, vitamin E, niacin (nicotinic acid), pantothenic acid, folic acid and other vitamins, lysine, threonine, tryptophan Essential amino acids such as, minerals such as calcium, magnesium, iron, zinc and copper, and humans such as α-linolenic acid, EPA, DHA, casein phosphopeptide (CPP), casein calcium peptide (CCP), etc. Substances that contribute to health, and one or more useful substances approved as other foods and food additives can be used.
Or you may add and process (mixing, heating, etc.) to suitable foodstuffs.
実施例
以下、本発明を実施例で説明する。以下の実施例は、本発明を説明するためにあげた例であり、これにより本発明を限定するものではない。
Examples Hereinafter, the present invention will be described with reference to Examples. The following examples are given to illustrate the present invention and are not intended to limit the present invention.
（アクトミオシン分解物の調製方法）
アクトミオシンは、豚骨格筋から調製した。すなわち、挽き肉にした豚骨格筋（大腿二頭筋）に緩衝液（０．６Ｍ ＫＣｌ／０．０４Ｍ ＮａＨＣＯ3／０．０１ｍ Ｎａ2ＣＯ3）を加え、混合・遠心分離を繰り返した後、透析による脱塩と凍結乾燥により、アクトミオシン標品を得た。蒸留水１０ｍｌにアクトミオシン１００ｍｇを入れ、十分に懸濁させた。これに、５種類のプロテアーゼ（パパイン、フィチン、トリプシン、プロテイネースＫ、プロナーゼＥ）のうちの１種を１０ｍｇ添加し、３７℃で３時間反応させた。反応終了後、９５℃で１０分間加熱し、プロテアーゼを失活させた。遠心分離により不溶物を除去した上澄液をアクトミオシン分解物溶液とした。
(Method for preparing actomyosin degradation product)
Actomyosin was prepared from porcine skeletal muscle. That is, a buffer solution (0.6 M KCl / 0.04 M NaHCO 3 /0.01 m Na 2 CO 3 ) is added to minced porcine skeletal muscle (biceps femoris), dialysis is performed after repeated mixing and centrifugation. Actomyosin preparations were obtained by desalting and freeze-drying. 100 mg of actomyosin was added to 10 ml of distilled water and sufficiently suspended. To this, 10 mg of one of 5 types of proteases (papain, phytin, trypsin, proteinase K, pronase E) was added and reacted at 37 ° C. for 3 hours. After completion of the reaction, the protease was inactivated by heating at 95 ° C. for 10 minutes. The supernatant from which insolubles were removed by centrifugation was used as an actomyosin degradation product solution.
（ビフィズス菌増殖促進効果の測定方法）
ビフィズス菌に対するアクトミオシン分解物あるいはペプチドの増殖促進効果は、以下の方法により測定した。まず、１１０℃で１０分間加熱殺菌した１０％還元脱脂乳（以下、脱脂乳）５ｍｌに、測定試料（アクトミオシン分解物溶液あるいはペプチド溶液）を５０μｌ添加した。これに、増殖促進効果の指標菌として用いたビフィズス菌の培養液（ＭＲＳ液体培地で前培養したもの）１０μｌを接種した。なお、ビフィズス菌にはヒト腸管由来の７菌株（Bifidobacterium bifidum JCM1254、B. breve JCM1192、B. longum JCM1217、B. catenulatum JCM1194、B. angulatum JCM7096、B. pseudocatenulum JCM1200、B. adolescentis JCM7042）を用いて検討したが、脱脂乳中においてペプチドの増殖促進効果が最も顕著にあらわれたB. bifidum JCM1254を最終的な指標菌として用いることとした。ビフィズス菌接種後の脱脂乳は、３７℃で４８時間培養した後に、ｐＨを測定した。ビフィズス菌は増殖に伴い、乳酸や酢酸を生成し、培地pHを低下させることが知られている。このため、培地ｐＨの低下程度から、ビフィズス菌の増殖程度を判定することができる。
(Measurement method of bifidobacteria growth promotion effect)
The growth promoting effect of actomyosin degradation products or peptides against Bifidobacteria was measured by the following method. First, 50 μl of a measurement sample (Actomyosin degradation product solution or peptide solution) was added to 5 ml of 10% reduced skim milk (hereinafter, skim milk) sterilized by heating at 110 ° C. for 10 minutes. This was inoculated with 10 μl of a bifidobacteria culture solution (pre-cultured in MRS liquid medium) used as an indicator for the growth promoting effect. In addition, 7 strains derived from human intestinal tract (Bifidobacterium bifidum JCM1254, B. breve JCM1192, B. longum JCM1217, B. catenulatum JCM1194, B. angulatum JCM7096, B. pseudocatenulum JCM1200, B. adolescentis JCM7042) are used as Bifidobacterium. As a final indicator, B. bifidum JCM1254, which showed the most prominent effect of peptide growth in skim milk, was examined. The skim milk after inoculation with bifidobacteria was cultured at 37 ° C. for 48 hours, and then the pH was measured. It is known that bifidobacteria produce lactic acid and acetic acid as they grow and lower the pH of the medium. For this reason, the degree of growth of bifidobacteria can be determined from the degree of decrease in medium pH.
(アクトミオシン分解物のビフィズス菌増殖促進効果)
豚骨格筋アクトミオシンのプロテアーゼ分解物５種のビフィズス菌（B. bifidum JCM1254）増殖促進効果を測定した。図１に示した結果のように、アクトミオシンのパパイン分解物に強い活性（脱脂乳のｐＨ低下＝ビフィズス菌増殖促進効果）が見られた。
(Actomyosin degradation product promotes the growth of bifidobacteria)
The growth promoting effect of 5 types of Bifidobacteria (B. bifidum JCM1254) protease degradation products of pig skeletal muscle actomyosin was measured. As shown in the results of FIG. 1, a strong activity was observed on the papain degradation product of actomyosin (pH reduction of skim milk = bifidobacteria growth promoting effect).
（ビフィズス菌増殖促進ペプチドの精製方法）
目的ペプチドの精製は、ゲル濾過クロマトグラフィーと逆相高速液体クロマトグラフィー（ＨＰＬＣ）の組み合わせにより実施した。ゲル濾過クロマトグラフィーは、カラムにセファデックスＧ２５Ｆｉｎｅ（２６ｍｍ×１００ｍｍ）を用い、溶出液には蒸留水を使用した。流速は２ｍｌ／ｍｉｎとし、溶出液中のペプチドの検出は、２１５ｎｍの吸光度の測定により行った。アクトミオシン分解物溶液１ｍｌをカラムに負荷し、溶出液を２分間隔（４ｍｌずつ）で分画採取した。各画分は凍結乾燥させた後に、蒸留水に再溶解させ、ビフィズス菌増殖促進効果を判定した。
(Method for purifying bifidobacteria growth-promoting peptide)
The target peptide was purified by a combination of gel filtration chromatography and reverse phase high performance liquid chromatography (HPLC). In the gel filtration chromatography, Sephadex G25 Fine (26 mm × 100 mm) was used for the column, and distilled water was used for the eluent. The flow rate was 2 ml / min, and the peptide in the eluate was detected by measuring the absorbance at 215 nm. 1 ml of the actomyosin degradation product solution was loaded onto the column, and the eluate was fractionated at intervals of 2 minutes (4 ml each). Each fraction was freeze-dried and then redissolved in distilled water to determine the bifidobacteria growth promoting effect.
逆相ＨＰＬＣは、カラムにCAPCELL PAK C18 UG-120 4.6 x 150mmを用い、溶出液Ａ（０．１％トリフルオロ酢酸を含む蒸留水）から溶出液Ｂ（０．１％トリフルオロ酢酸を含むアセトニトリル）への直線濃度勾配系で溶出を行った。流速１ｍｌ／ｍｉｎで、溶出開始後１０分目までは溶出液Ａを流し、１０分目から濃度勾配を開始し、溶出時間４０分目でＢ液濃度が２０％に達するようにした。ＨＰＬＣ装置には、島津製作所製高速液体クロマトグラフＬＣ−ＶＰシステムを利用した。溶出液中のペプチドの検出は、２１５ｎｍの吸光度の測定により行った。分画採取した溶出液（各フラクション）は、凍結乾燥させた後に、蒸留水に再溶解させ、ビフィズス菌増殖促進効果を判定した。 Reversed-phase HPLC uses CAPCELL PAK C18 UG-120 4.6 x 150 mm as the column, and eluent A (distilled water containing 0.1% trifluoroacetic acid) to eluent B (acetonitrile containing 0.1% trifluoroacetic acid). Elution was performed with a linear concentration gradient system. At a flow rate of 1 ml / min, eluent A was allowed to flow until 10 minutes after the start of elution, a concentration gradient was started from 10 minutes, and the concentration of solution B reached 20% at the elution time of 40 minutes. For the HPLC apparatus, a high performance liquid chromatograph LC-VP system manufactured by Shimadzu Corporation was used. Detection of the peptide in the eluate was performed by measuring absorbance at 215 nm. The fractionated eluate (each fraction) was freeze-dried and then redissolved in distilled water to determine the effect of promoting bifidobacteria growth.
（ペプチドの構造決定方法）
精製したペプチドのアミノ酸配列は、自動エドマン法（気相法）によるＮ末端アミノ酸分析により決定した。自動分析装置としてアプライドバイオシステムズ社製プロテインシークエンサーモデル４７０Ａを使用した。また、ペプチドの分子量測定には、島津製作所製質量分析装置QP8000αを使用した。
(Method for determining peptide structure)
The amino acid sequence of the purified peptide was determined by N-terminal amino acid analysis by the automated Edman method (gas phase method). A protein sequencer model 470A manufactured by Applied Biosystems was used as an automatic analyzer. In addition, a mass spectrometer QP8000α manufactured by Shimadzu Corporation was used for measuring the molecular weight of the peptide.
豚骨格筋アクトミオシンのパパイン分解物から、ビフィズス菌増殖促進効果を示すペプチドの精製を、ゲル濾過クロマトグラフィーと２回の逆相ＨＰＬＣ（１ｓｔ，２ｎｄ ＨＰＬＣ ｒｕｎ）により行った。まず、アクトミオシンのパパイン分解物をゲル濾過クロマトグラフィーにより分画した結果、溶出時間１２〜１４ｍｉｎの画分に最も強いビフィズス菌増殖効果が認められた。次にこの画分を逆相ＨＰＬＣに供し、２分間隔で溶出液を採取した（１ｓｔ ＨＰＬＣ ｒｕｎ）。各画分の活性を測定した結果、溶出時間２８〜３０分の画分に最も高い活性が認められた。この画分を再度、同条件の逆相ＨＰＬＣに供した（２ｎｄ ＨＰＬＣ ｒｕｎ）。溶出時間２４〜３３分の溶出液を、出現したピークに注目し、９画分に分けて採取した。各画分の活性を測定した結果、溶出時間２９．５〜３０．０分の画分に最も高い活性が認められた。この画分を逆相ＨＰＬＣに供した結果、１本のピークであることが確認されたため、精製を終了させた。 From the papain degradation product of porcine skeletal muscle actomyosin, a peptide exhibiting the effect of promoting the growth of bifidobacteria was purified by gel filtration chromatography and two reverse phase HPLC (1st, 2nd HPLC run). First, the papain degradation product of actomyosin was fractionated by gel filtration chromatography. As a result, the strongest bifidobacteria growth effect was observed in the fraction with an elution time of 12 to 14 min. Next, this fraction was subjected to reverse phase HPLC, and the eluate was collected at 2-minute intervals (1st HPLC run). As a result of measuring the activity of each fraction, the highest activity was observed in the fraction having an elution time of 28 to 30 minutes. This fraction was again subjected to reverse phase HPLC under the same conditions (2nd HPLC run). The eluate having an elution time of 24 to 33 minutes was collected in 9 fractions, paying attention to the peak that appeared. As a result of measuring the activity of each fraction, the highest activity was observed in the fraction having an elution time of 29.5 to 30.0 minutes. As a result of subjecting this fraction to reverse phase HPLC, it was confirmed that it was a single peak, and thus purification was terminated.
上述のように精製して得たペプチドのアミノ酸配列をプロテインシークエンサーにより解析した結果、Glu-Leu-Metであった。この配列から算出した分子量（391.52）は、質量分析装置による測定値（392.56）から算出した値（392.56 - 1.00 = 391.56）とよく一致していた。以上の結果から、精製したビフィズス菌増殖促進ペプチドの構造（アミノ酸配列）は、Glu-Leu-Metであると結論した。このペプチドのアミノ酸配列は、豚骨格筋のミオシン重鎖の配列中に認められるものであり、アクトミオシンをパパインで分解した際に、ミオシン重鎖から生成したものと考えられる。 As a result of analyzing the amino acid sequence of the peptide obtained by purification as described above using a protein sequencer, it was Glu-Leu-Met. The molecular weight (391.52) calculated from this sequence was in good agreement with the value (392.56-1.00 = 391.56) calculated from the measured value (392.56) by the mass spectrometer. From the above results, it was concluded that the structure (amino acid sequence) of the purified bifidobacteria growth-promoting peptide was Glu-Leu-Met. The amino acid sequence of this peptide is found in the myosin heavy chain sequence of porcine skeletal muscle, and is considered to have been generated from the myosin heavy chain when actomyosin was degraded with papain.
アクトミオシンのパパイン分解物中より発見したビフィズス菌増殖促進ペプチド（Glu Leu-Met）の配列情報をもとに、合成ペプチドを調製した。合成ペプチドの調製には、アプライドバイオシステムズ社製ペプチド合成装置モデル４３０Ａを使用した。合成ペプチドは、逆相ＨＰＬＣにより、純度９０％以上になるように精製した。
合成したトリペプチド（Glu-Leu-Met）がビフィズス菌増殖促進効果を示すことを確認すると共に、この活性が単に構成アミノ酸や構成部分ペプチド（ジペプチド）に起因するものではないことを示した。図２は、合成したトリペプチド（Glu-Leu-Met）と部分配列ジペプチド（Glu-Leu, Leu-Met）、そして構成アミノ酸（Glu, Leu, Met, ３種アミノ酸混合）のビフィズス菌増殖促進効果を測定した結果を示したグラフである。それぞれのペプチドとアミノ酸は、脱脂乳中の濃度が０．１ｍＭになるように添加した。Glu-Leu-Metの部分配列であるジペプチド（Glu-Leu, Leu-Met）と構成アミノ酸（Glu, Leu, Met, 3種アミノ酸混合）は、いずれもGlu-Leu-Metより弱い活性しか示さなかった。このことから、Glu-Leu-Metのビフィズス菌増殖促進効果は、構成アミノ酸が単なる窒素源として利用されることにより発現するのではなく、トリペプチド配列に重要な意義があることが確認された。
A synthetic peptide was prepared based on the sequence information of the bifidobacteria growth-promoting peptide (Glu Leu-Met) discovered from the papain degradation product of actomyosin. A peptide synthesizer model 430A manufactured by Applied Biosystems was used for the preparation of the synthetic peptide. The synthetic peptide was purified to a purity of 90% or more by reverse phase HPLC.
In addition to confirming that the synthesized tripeptide (Glu-Leu-Met) has a bifidobacteria growth-promoting effect, it was shown that this activity is not simply caused by a constituent amino acid or a constituent peptide (dipeptide). Fig. 2 shows the bifidobacteria growth-promoting effect of the synthesized tripeptide (Glu-Leu-Met), partial sequence dipeptide (Glu-Leu, Leu-Met), and constituent amino acids (Glu, Leu, Met, mixed with three amino acids) It is the graph which showed the result of having measured. Each peptide and amino acid were added so that the concentration in skim milk would be 0.1 mM. The dipeptide (Glu-Leu, Leu-Met), which is a partial sequence of Glu-Leu-Met, and the constituent amino acids (Glu, Leu, Met, mixed with 3 amino acids) both show weaker activity than Glu-Leu-Met. It was. From this, it was confirmed that the growth-promoting effect of Glu-Leu-Met on bifidobacteria is not expressed by using the constituent amino acid as a mere nitrogen source, but has an important significance in the tripeptide sequence.
Glu-Leu-Metのビフィズス菌増殖促進効果を、ビフィズス菌２６菌株（B. bifidum ５株、B. breve ５株、B. adolescentis ７株、B. infantis ５株、B. longum ３株、B. catenulatum １株）を用いて検討した。その結果、２６株中１１株（B. bifidum ３株、B. breve ３株、B. adolescentis １株、B. infantis １株、B. longum ３株）に対して効果が認められた。 The growth-promoting effect of Glu-Leu-Met on Bifidobacterium 26 strains (B. bifidum 5 strains, B. breve 5 strains, B. adolescentis 7 strains, B. infantis 5 strains, B. longum 3 strains, B. longum strains) catenulatum 1 strain). As a result, 11 strains out of 26 (3 B. bifidum, 3 B. breve, 1 B. adolescentis, 1 B. infantis, 3 B. longum) were observed.
Glu-Leu-Metの病原細菌の増殖に対する影響を検討した。７種の病原細菌（Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfuringens, Bacillus cereus）を接種した液体培地に、Glu-Leu-Metを０．１ｍＭになるように添加した場合、Glu-Leu-Met無添加のものと比べて、いずれの病原細菌の増殖も影響（増殖促進および増殖抑制）は見られなかった。この結果から、Glu-Leu-Metの微生物増殖促進効果は、ビフィズス菌に対して選択性の高い作用と見られる。 The effect of Glu-Leu-Met on the growth of pathogenic bacteria was examined. When Glu-Leu-Met is added to a concentration of 0.1 mM to a liquid medium inoculated with seven pathogenic bacteria (Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfuringens, Bacillus cereus) The growth of any pathogenic bacterium was not affected (proliferation promotion and growth inhibition) compared to those without Glu-Leu-Met. From these results, it can be seen that the effect of Glu-Leu-Met on promoting microbial growth is a highly selective effect on bifidobacteria.
ペプチドを経口摂取（投与）し、下部消化管内でビフィズス菌増殖促進効果があらわれるためには、消化酵素（プロテアーゼ）による分解が行われずに消化管を通過することが必須である。合成ペプチド（Glu-Leu-Met）溶液（１ｍｇ／ｍｌ）に、代表的な消化酵素であるペプシンやパンクレアチンを添加（１ｍｇ／ｍｌ）し、３７℃で２時間反応させた結果、いずれの酵素によってもGlu-Leu-Metの分解は全く認められなかった（高速液体クロマトグラフ質量分析装置により確認）。この結果から、Glu-Leu-Metは、消化酵素耐性が高く、ヒトや動物が経口摂取した場合、分解されないままで下部消化管に到達し、ビフィズス菌の増殖に寄与することが期待できる。 In order to ingest (administer) a peptide orally and have a bifidobacteria growth promoting effect in the lower gastrointestinal tract, it is essential that the peptide pass through the gastrointestinal tract without being decomposed by a digestive enzyme (protease). As a result of adding typical digestive enzymes pepsin and pancreatin (1 mg / ml) to a synthetic peptide (Glu-Leu-Met) solution (1 mg / ml) and reacting at 37 ° C. for 2 hours, which enzyme Also showed no degradation of Glu-Leu-Met (confirmed by high performance liquid chromatograph mass spectrometer). From this result, Glu-Leu-Met is highly resistant to digestive enzymes and can be expected to contribute to the growth of bifidobacteria by reaching the lower gastrointestinal tract without being decomposed when ingested by humans or animals.
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| JP2003102427A (en) * | 2001-09-27 | 2003-04-08 | Prima Meat Packers Ltd | Physiologically functional food material or food and drink having stress stomach ulcer preventing function |
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| JPH05336954A (en) * | 1992-06-05 | 1993-12-21 | Eisai Co Ltd | Method for culturing microorganism and medium for microorganism |
| JPH05344865A (en) * | 1992-06-16 | 1993-12-27 | Yukie Murai | Healthy food |
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