JP4476934B2 - Novel peptide and immunostimulant, functional food and method for producing functional food - Google Patents
Novel peptide and immunostimulant, functional food and method for producing functional food Download PDFInfo
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- JP4476934B2 JP4476934B2 JP2005505414A JP2005505414A JP4476934B2 JP 4476934 B2 JP4476934 B2 JP 4476934B2 JP 2005505414 A JP2005505414 A JP 2005505414A JP 2005505414 A JP2005505414 A JP 2005505414A JP 4476934 B2 JP4476934 B2 JP 4476934B2
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Description
【技術分野】
【０００１】
本発明は、免疫腑活作用を有する新規ペプチドおよび新規な免疫賦活剤、機能性食品ならびに機能性食品の製造方法に関する。
【背景技術】
【０００２】
日本は平均寿命８０年という世界有数の長寿国になったが、誰もが健康的な長寿をまっとうできるとは限らない。健康長寿の最大の敵は、がん・脳卒中・心臓病・肝臓病・腎臓病・糖尿病などの「生活習慣病」といわれる病気群である。これは、生活習慣に着目した生活習慣病という概念を新たに導入しようという狙いから、従来の加齢に注目した「成人病」にかわって、平成８年１２月に、旧厚生省「公衆衛生審議会」が提唱したものである。この「生活習慣病」の発症と進行には、生活習慣要因・遺伝要因・外部環境要因の３つの要因が関わっているが、生活習慣が基礎にある多くの病気は予防が可能であり、日常の食生活を改善することによって発病を予防するという一次予防の重要性が注目されている。
【０００３】
本来、食品の機能としては、生命活動を維持するための各種栄養成分の補給としての栄養機能(一次機能)と、おいしく感じて食べる嗜好感覚機能(二次機能)があるとされてきた。すなわち、おいしくて栄養源として優れているものが優れた食品とされていた。ところが、１９８０年代に、生体防御機能や病気の予防と回復などに関与する栄養成分が発見され、これまでの一次と二次機能に加えて重要な第三の機能として体の働きを調節する機能、すなわち免疫力の強化や調節、各種ホルモン分泌調整、老化の抑制など種々の生体調節機能を有する成分が含まれている食品の生理機能が注目されるようになった。
【０００４】
牛乳タンパク質は、主要な食品タンパク質の中でも、窒素や必須アミノ酸の優れた供給源となるばかりでなく、ミネラルやビタミンなどの栄養素の吸収を促進するもの、骨形成促進および骨吸収抑制作用を持つもの、血中コレステロールレベルの低下作用を持つもののように、生体の様々な機能を活発化させるような調節作用を発揮するものなど、本来経口的に摂取され、何らかのかたちで身体に働きかけるように設計されている。
【０００５】
１９７９年、Brantleらが牛乳カゼインの消化物がオピオイドアゴニスト活性を持つ旨報告をして以来、カゼイン消化物から種々の生物活性を有するペプチドが分離・同定されてきた(非特許文献１，２参照)。乳タンパク質から誘導される代表的なペプチドであるオピオイドペプチドは、体内での鎮痛作用ばかりでなく、腸管の蠕動運動や消化管機能の調節に関与する可能性が示唆されている(非特許文献３)。他にも、血圧降下作用を発揮するアンジオテンシンＩ変換酵素阻害ペプチド、マクロファージ貪食能の活性化作用や免疫促進作用をもつ免疫調節ペプチド、血小板凝集阻害ペプチド、回腸収縮ペプチドなどが知られている。それらのペプチドの中でもカルシウム吸収促進活性を持つカゼインホスホペプチドや血圧降下活性を持つαｓ１−カゼインの２３〜３４残基域のペプチドは、特定保健用食品としてすでに利用されるまでに至っており、牛乳カゼインの新たな利用分野が注目される一因となっている。
【０００６】
しかしながら、生体の免疫賦活化を誘導し、生体防御への寄与を狙った食品は未だ開発されていない。免疫機能の活性化を表す指標としては、様々なものがあるが、その中の１つとしてマクロファージ機能の誘導に関わる走化性が挙げられる。走化性因子による遊走は、免疫応答に重要なマクロファージの機能の１つであり、腸管内走化性因子により刺激されたマクロファージは腸管において感染部位へ遊走し病原体を殺傷すると考えられている。すなわち、腸管内の生体防御において、マクロファージの病原体への遊走は非常に重要な役割を担っていると言える。
【０００７】
マクロファージの走化性を誘導する物質はこれまでにも数多く報告されているが(非特許文献４)、ペプチド成分で走化性因子が同定されているものは、ｆＭＬＰを代表とする細菌由来のフォルミル化されたペプチドや、ヒト末梢血リンパ球から得られたペプチド(特許文献１)、ペプチドライブラリーから作られた化学合成ペプチド(非特許文献５)だけであり、食品由来のものとしては卵白から得られるオボアルブミンフラグメントが知られているにすぎない（特許文献２)。
【特許文献１】
【０００８】
特開平９−２２７５９２号 第２〜９頁
【特許文献２】
【０００９】
特開平９−１２４７０号 第２〜３頁
【非特許文献１】
【００１０】
ブラントル(V.Brantl)ら，「生理化学」(Hoppe-Seyler's Z. Physiol. Chem.），１９７９年，第１３６０号，ｐ.１２１１−１２１６
【非特許文献２】
【００１１】
エジーンズ(Agnes Henschen)ら，「生理化学」(Hoppe-Seyler's Z. Physiol. Chem.），１９７９年，第１３６０号，ｐ.１２１７−１２２４
【非特許文献３】
【００１２】
金丸義敬，「牛乳・乳成分の新しい機能(タンパク質)」，乳業技術，２０００年，第５０巻，ｐ.２２−３７
【非特許文献４】
【００１３】
義江修ら著、「ケモカインハンドブック」，２０００年，ｐ.１２
【非特許文献５】
【００１４】
インイン(Yingying Le)ら，「免疫学」(Journal of Immunology)，１９９８年，第１６３巻，ｐ.６７７７−６７８４
【００１５】
そこで、本発明者らは、腸管内で生体防御作用を発揮する食品を提供することを目的とし、乳タンパク成分の免疫賦活化能に注目して鋭意努力したところ、β−カゼインの消化物にマクロファージ遊走活性を見出し、本発明を完成するに至った。
【発明の開示】
【００１６】
本発明のペプチドは、配列番号１で示される次のアミノ酸配列からなる新規ペプチドである。
Tyr−Pro−Val−Glu−Pro
【００１７】
本発明の免疫賦活剤は、配列番号１で示される次のアミノ酸配列からなるペプチドを有効成分とし、本発明の用法は、配列番号１で示される次のアミノ酸配列からなるペプチドを免疫腑活剤として使用することを特徴としている。
Tyr−Pro−Val−Glu−Pro
【００１８】
また、本発明の機能性食品は、マクロファージ走化性誘導能を実質的に発揮する量の配列番号１で示される次のアミノ酸配列からなるペプチドを含有することを特徴としている。
Tyr−Pro−Val−Glu−Pro
【００１９】
さらに、本発明の機能性食品は、生体内の消化酵素がマクロファージ走行性誘導能を実質的に発揮する量の配列番号１で示される次のアミノ酸配列からなるペプチドを生じさせる程度に、食品中のβ−カゼイン１１８−１１９残基結合がアクチナーゼにより切断されていることを特徴としている。
Tyr−Pro−Val−Glu−Pro
【００２０】
本発明の機能性食品の製造方法は、マクロファージ走化性誘導能を実質的に発揮する量のペプチドが含まれるように、食品中のβ−カゼインをアクチナーゼにより消化する工程を有することを特徴としている。この場合において、配列番号１で示される次のアミノ酸配列からなるペプチドが含まれるようにするのがよい。
Tyr−Pro−Val−Glu−Pro
【図面の簡単な説明】
【００２２】
第１図は、ギムザ染色後のＰＶＰＦ膜を示す写真であって、同図ＡはＰＶＰＦ膜全体を、同図Ｂは顕微鏡観察された遊走細胞を示す。
第２図は、β−カゼインの各種酵素消化によるマクロファージ走化性の誘導を示す図である。
第３図は、β−カゼインの各種酵素消化後のＳＤＳポリアクリルアミド電気泳動図であって、同図Ａは酵素消化前の泳動図、同図Ｂは消化物(３ｋＤ以下)の泳動図である。
第４図は、各種酵素消化β−カゼインの分子量３ｋＤ以下の画分によるマクロファージ走化性の誘導を示す図である。
第５図は、各種酵素消化β−カゼインの分子量３ｋＤ以下の画分によるヒト抹消血単球の走化性の誘導を示す図であって、同図ＡはFACS CaliburTmによるモノサイトの純度を示す図、同図ＢはアクチナーゼＥ消化物により遊走した細胞を示す顕微鏡像、同図Ｃは酵素消化物により遊走した細胞数を示す図である。
第６図は、β−カゼインのアクチナーゼＥ消化により発現するマクロファージ走化性の経時的変化を示す図である。
第７図は、β−カゼイン消化物からの陰イオン交換クロマトグラフィーによるマクロファージ走化性因子の分画を示す図であって、上段は紫外吸収による図、下段は走化誘導された細胞数による図である。
第８図は、マクロファージ走化性ペプチドの高速液体クロマトグラフィーによる単離・精製を示す図であって、同図Ａ上段は３０％アセトニトリルによる溶出を示す図、同図Ａ下段はＱフラクションの溶出を示す図、同図ＢはＱ１、Ｑ２、Ｑ３フラクションによる走化性を示す図である。
第９図は、マクロファージ走化性ペプチドＱ１のチェッカーボード解析図である。
第１０図は、マクロファージ走化性ペプチドＱ１のカルシウム流動性を示す図であって、上段はＣａを取り込んだマクロファージのレーザー顕微鏡像、下段はマクロファージのＣａ取り込みの変化を示す図である。
第１１図は、マクロファージ走化性ペプチドＱ１の刺激によるマクロファージ内で流動するカルシウム濃度を示す図である。
【本発明を実施するための最良の形態】
【００２３】
本発明は、マクロファージの走化性能を誘導する新規ペプチドに関するもので、本発明に係る新規ペプチドは、配列番号１で示されるTyr−Pro−Val−Glu−Proのアミノ配列を有する(以下、該ペプチドを「β−カゾケモチド(β−casochemotide)」と称する。)。このペプチドは、β−カゼイン１１４〜１１８残基に相当するものであり、オピオイドペプチドとして単離同定されたβ−ネオカゾモルフィン(配列番号２：Tyr−Pro−Val−Glu−Pro−Phe、β−カゼインの１１４〜１１９残基に相当)のＣ末端(Phe)が欠損したものである。
【００２４】
β−カゾケモチドは、例えばβ−カゼインを、アクチナーゼＥ(販売名、科研製薬(株)製)に代表される各種アクチナーゼ(Actinase)やパパイン(Papain)などの蛋白消化酵素で処理することにより得られ、特にアクチナーゼで処理することによって高収率で当該ペプチドが得られる。この酵素処理は、特別な条件下で処理を行う必要はなく、通常の処理条件が選択される。例えば、下記表１に示すような至適ｐＨ、至適バッファー、至適温度にて行えるが、この条件下に制限されない。また、当該ペプチドは化学合成によっても得ることができる。
【００２５】
このものは、マクロファージ走化性を誘導する作用を有し、免疫賦活剤として応用できる。β−カゾケモチドは、牛乳等中のβ−カゼインを利用して得られ、牛乳アレルギーを引き起こすアレルゲン中に該ペプチドと同じ配列を有するものはないので、その安全性は高いと考えられる。
【００２６】
本発明の機能性食品には、保健機能食品（特定保健用食品、栄養機能食品）や生理機能を標榜した食品が含まれ、マクロファージ走化性誘導能を実質的に発揮する量のペプチドを含有する。該機能性食品は、例えば、β−カゼインの蛋白消化物から単離されあるいは合成された本発明に係るペプチドを、目的とする食品に添加して得られる。また、β−カゼインの酵素消化物をそのまま食品中に添加してもよく、食品の製造工程中にて、マクロファージ走化性誘導能を実質的に発揮する量のペプチドが含まれるように、蛋白消化酵素を用いてβ−カゼインを消化させてもよい。例えば、原乳に酵素処理を行って乳飲料を製造する、酵素処理を行った原乳を原材料としてチーズ、ヨーグルトなどの乳製品を製造することが例示される。用いられる蛋白消化酵素は目的とする消化物(ペプチド)を産生する限り特に限定されるものではないが、好ましくはアクチナーゼである。
【００２７】
また、食品添加物として認められているアクチナーゼＡＳ(例えば科研製薬(株)製)などで分解して、食品中に含まれるβ−カゼイン中の１１８−１１９残基結合を切断しておいてもよい。そして、この食品が摂取されると、例えばペプシンやトリプシンなどの常在性の消化酵素によってさらにβ−カゼインの１１３−１１４残基結合が切断され、本発明のペプチドが体内にて産生される。このように、食品中のβ−カゼイン１１３−１１４残基結合をあらかじめ切断しておき、体内の消化酵素によってマクロファージ走化性誘導能を実質的に発揮する量のペプチドを生じさせてもよい。
【００２８】
本発明の機能性食品には、上記した乳製品以外にも種々の食品が考えられ、その種類等は限定されない。消化酵素による処理工程を加えることを考慮するならば、本発明は、β−カゼインを多く含む食品やβ−カゼインを多く含む食材を原材料にした食品に好適である。
【００２９】
β−カゼインの含有量はマクロファージ走化性誘導能を実質的に発揮する量とされるが、その目安は１回摂取量中０.０１〜１０ｍｇ程度、好ましくは０.１〜５ｍｇ程度である。
【実施例１】
【００３０】
次に、本発明について以下の実施例に基づいてさらに詳細に説明する。
１．ペプチドの調整
(１)β−カゼインの酵素分解
β−カゼイン１０mg(シグマ社製)を、Amharらの方法(J. Dairy Science,81,3131−3138(1998))の方法に従い、表１に示す至適バッファー１０ｍｌで溶解し、アクチナーゼＥ０.５ｍｇを加えて５％ＣＯ２、至適温度で２４時間反応させた。反応後、直ちに沸騰水中で１０分間加熱して酵素を失活させた後、水冷し、試験試料とした。なお、参考例として、同じβ−カゼインを他の６種のプロテアーゼ(α−キモトリプシン、ペプシン、サーモリシン、トリプシン、プロテアーゼＫ、パパイン)で処理したものを用いた。
【００３１】
【表１】
（２)限外濾過
上記(１)で得られた酵素消化物をCENTRIPREP３(ミリポア社製)で限外濾過(３０００ｒｐｍ、４℃)して、分子量３０００以下の画分を得た。
【００３３】
(３)Ｃ18逆相担体による分画
上記(２)で得られた分子量３０００以下のβ‐カゼイン酵素消化物のうちアクチナーゼＥによる消化物を、Ｃ18逆相担体であるSep-PakTMPlusＣ18カートリッジ(ウォーターズ社製)に吸着させた後に、イオン交換水、３０v/v％アセトニトリル、６０v/v％アセトニトリル、９０v/v％アセトニトリルをそれぞれ１０ｍｌ通し、各溶液による溶出画分を得た。その後、遠心エバポレーターによってアセトニトリルを除去した後、凍結乾燥し、−２０℃で保存した。
【００３４】
(４)イオン交換クロマトグラフィーによる活性成分の分画
上記(３)によって得られた３０v/v％アセトニトリル溶出画分を移動相Ａ(１ｍｌ)に溶解させ、Bio Logic chromatography system(バイオラド ラボラトリー社製)により以下の条件でイオン交換クロマトグラフィーに供した。
【００３５】
＜陽イオン交換クロマトグラフィー条件＞
カラム：HiTrapTM SP HP(アマシャム ファルマシア バイオテックＡＢ社製)
溶媒：移動相Ａ(５０ｍＭリン酸緩衝液ｐＨ６.０)
移動相Ｂ(１ＭＮａＣｌを含む５０ｍＭリン酸緩衝液ｐＨ６.０)
溶出：移動相Ａで８分間保持した後、５分間で移動相Ｂを５０％まで直線的
に上昇させ、その後４分間で移動相Ｂを１００％まで上昇させた。
流速：５.０ｍｌ/min
分取：１.０ｍｌ/fraction
温度：室温
検出：２１４ｎｍ
【００３６】
＜陰イオン交換クロマトグラフィー条件＞
カラム：HiTrapTM Q HP(アマシャム ファルマシア バイオテックＡＢ社製)
溶媒：移動相Ａ(２０ｍＭトリス−塩酸緩衝液(pH８.２))
移動相Ｂ(１ＭＮａＣｌを含む２０ｍＭトリス−塩酸緩衝液(ｐＨ８.
２))
溶出：移動相Ａで８分間保持した後、５分間で移動相Ｂを５０％まで直線的
に上昇させ、その後４分間で移動相Ｂを１００％まで上昇させた。
流速：５.０ｍｌ/min
分取：１.０ｍｌ/fraction
温度：室温
検出：２１４ｎｍ
【００３７】
(５)高速液体クロマトグラフィーによる活性ペプチドの分析および単離・精製
上記(４)で得られた活性画分(陰イオンクロマトグラフィ溶出フラクションＮｏ.４２〜４６)をイオン交換水に溶解し、逆相(reversed-phase、以下ＲＰ)モードでの高速液体クロマトグラフィー(High-performance liquid chromatography、以下ＨＰＬＣ)により分析および分取を行った。
【００３８】
＜ＲＰ−ＨＰＬＣ条件＞
カラム：Mightysil RP-18 GP(５μm、４.６×１５０ｍｍ、関東化学社製)
溶媒：移動相Ａ(０.０５v/v％ＴＦＡを含む３０％アセトニトリル)
移動相Ｂ(０.０５v/v％ＴＦＡを含む６０％アセトニトリル)
溶出：３０分間で移動相Ａ１００％から移動相Ｂ１００％に直線的に上昇さ
せた後、移動相Ｂで１０分間保持した。
流速：０.５ｍｌ/min
温度：４０℃
検出：２１４ｎｍ
【００３９】
本操作で得られたピークについて分取して、上記(３)の方法に準じＣ18逆相担体であるSep−pakTMPlus Ｃ18カートリッジに吸着させた後に、イオン交換水、３０％アセトニトリルをそれぞれ１０ｍｌ通し、３０％アセトニトリルの溶出画分を回収する事でＴＦＡを除去した。さらに、遠心エバポレーターによってアセトニトリルを除去した後、凍結乾燥し、−２０℃で保存した。
【００４０】
２．電気泳動分析
上記ペプチドの調整(１)および(２)において得た各フラクション１００μｌにＳＤＳ化試薬を１００μｌ加え、８５℃で２分加熱することによりＳＤＳ化した。ＳＤＳ化したサンプルは１レーンあたり１０μｌをアプライし、１２５Ｖの定電圧により泳動した。なお、ゲルは４.７５％濃縮ゲル、２０％分離ゲルを用い、分子量マーカーとしてペプチド分子量マーカー「第一」(第一化学薬品(株)製)およびprecision prestained standard(バイオラド ラボラトリー社製)を用いた。泳動後のゲルはＰＶＤＦ膜であるImmobilon-PSQ(ミリポア社製)にブロッティング装置SEMI-PHORTM(ヘファー理化学機器社製)を用いてエレクトロブロッティングした。すなわち、Western buffer(２０ｍＭ Tris base、 ２００ｍＭGlycin/dH２O)で浸した２枚の濾紙をブロッティング装置に敷き、完全に気泡を抜いた。その上に、１００％メタノールで５分間振盪した後Western bufferで浸したＰＶＤＦ膜を載せ、その上に分離ゲルを載せ、Western bufferで浸した２枚の濾紙をゲルの上にかぶせて完全に気泡を抜いた後、８０ｍＡで２時間ブロッティングを行った。タンパク質が転写されたＰＶＤＦ膜はQuick−CBB(和光純薬工業(株)製)により５分間振盪して染色した後、５０％メタノールで振盪することによってバックグラウンドを脱色した。
【００４１】
３．マクロファージ走化性試験
(１)マクロファージの調製
マウス由来マクロファージＪ７７４−１株(東北大学医学部加齢研究所付属医用細胞資源センターより譲渡)を、ＲＰＭＩ−１６４０培地(１０％ＦＣＳ含有)(シグマ社製)で培養し、１０継代目まで実験に供した。
【００４２】
(２)マクロファージ走化性の測定
ｉ)マクロファージＪ７７４−１をＲＰＭＩ−１６４０培地(１０％ＦＣＳ含有)(シグマ社製)で培養し、アッセイ時にはＲＰＭＩ−１６４０完全培地(１％ＢＳＡ含有)で洗浄後、１×１０６cells/ｍｌに調製した。
ii)マクロファージ走化性の測定は、本発明者らが先に報告した方法(Int. J. Food Microbiol.,77,29-38,(2002))に従い行った。すなわち、試料をＲＰＭＩ−１６４０完全培地で２倍希釈し、４８−well microchemotaxis chamber(ニューロ プローブ社製)の下部wellに２８μｌづつ入れた(３連)。この上に２５×８０mm、５.０μｍのＰＶＰＦ膜(ニューロ プローブ社製)を載せた後、ゴムのガスケット、蓋を順に載せてねじで止め、上部のwellにマクロファージ懸濁液を５０μｌづつ分注した。５％ＣＯ２、３７℃で２時間培養後、ＰＶＰＦ膜の表側、つまり細胞を播いた側をＰＢＳ(オキソイド社製)で洗浄し、フィルターワイパー(ニューロ プローブ社製)で細胞をぬぐいとり、フィールド染色液(武藤化学薬品社製)によりＰＶＰＦ膜をギムザ染色した。顕微鏡下(倍率２００倍)で遊走したマクロファージ数を試料当たり１０視野計数し、平均遊走数(high power field；ＨＰＦ)を算出した。
【００４３】
４．ヒト末梢血モノサイトの走化性の測定
(１)ヒト末梢血からのモノサイトの分離
ヘパリン採血した血液１００ｍｌを、HISTOPAQUE−1077(シグマ ダイアグノスティックス社製)を１５ｍｌ分注した５０ｍｌのチューブに２５ｍｌずつ重層し、遠心分離(１５００ｒｐｍ、３０min、２０℃)した。単核球層を回収した後、ＰＢＳを多量に加えて遠心洗浄(１５００ｒｐｍ、５min、４℃)し、同様の操作を３回行った後に生細胞数を計数した。次いで５ｍＭ ＥＤＴＡ(フロー ラボラトリー社製)、０.５％ＢＳＡ(シグマ社製)を含むＰＢＳで１／２０に希釈したビオチン標識マウス抗ヒト−ＣＤ１１ｂ抗体(ファルミンゲン社製)を１００μｌ加え、４℃で３０分間反応させた。ＰＢＳ(５ｍＭ ＥＤＴＡ、０.５％ＢＳＡ含有)を１.５ｍｌを加え遠心洗浄して上清を除去した後に、ＰＢＳ(５ｍＭ、０.５％ＢＳＡ含有)９０μｌ、MACS streptavidin microbeads(Miltenyi Boitec GmbH社製)を１０μｌ加え４℃で１５分間反応させた。ＰＢＳ(５ｍＭ ＥＤＴＡ、０.５％ＢＳＡ含有)で洗浄した後、ＰＢＳ（５ｍＭ ＥＤＴＡ、０.５％ＢＳＡ含有）５００μｌに懸濁し、magnetic cell separation system （以下、「MACS」と称する）（Miltenyi Biotec GmbH社製）によりMiniMACSカラム（ＲＳ+、Miltenyi Biotec GmbH社製）に吸着した細胞（モノサイト）を分離した。分離したモノサイトは生細胞数を計数した後、上記３.マクロファージ走化性試験(２)の方法に準じモノサイト走化性を測定した。
【００４４】
(２)ヒト末梢血モノサイトの純度検定
上記(１)に従い分離したヒト末梢血モノサイトの純度はFACS CaliburTM flow cytometer(FACS；ベクトン ディキンソン社製)により検定した。すなわち、MACSにより分離したモノサイトに、FACS washing buffer(２％ＦＣＳ、０.０２％ＮａＮ3/ＰＢＳ)を１ｍｌ加えピペッティングにより洗浄後、遠心分離(３２００ｒｐｍ、３min、４℃)により上澄液を除去した。この操作を３回繰り返した後、ビオチン標識マウス抗ヒトＣＤ１１ｂＭＡＣ−１抗体(ファーミンゲン社製)を１０μｌ加え遮光して４℃で１５分間反応させた。これにFACS washing buffer(２％ＦＣＳ、０.０２％ＮａＮ3/ＰＢＳ)を１ｍｌ加えピペッティングにより洗浄後、遠心分離(３２００ｒｐｍ、３min、４℃)により上澄液を除去した後、R-Phycoerythrin標識ストレプトアビジン(ターゴ社製)を１０μｌを加え、遮光して４℃で１５分間反応させた。これにFACS washing bufferを１ｍｌ加え洗浄後、遠心分離(３２００ｒｐｍ、３min、４℃)により上澄液を除去し、Sheath液(５００μｌ)を加えよく懸濁させた。これを２０μmのナイロンメッシュに通した後に、FACSによりヒト末梢血モノサイトの純度を検定した。
【００４５】
(３)ヒト末梢血モノサイトの走化性の測定
ヒト末梢血モノサイトの走化性は、試験試料として限外濾過(上記１.ペプチド調整(２))で得た画分をＲＰＭＩ−１６４０完全培地(１％ＢＳＡ含有)で１ｍｇ／ｍｌの濃度となるように希釈したものを用い、４８−well microchemotaxis chamberを用いて、上記３.マクロファージ走化性試験(２)に従い測定した。
【００４６】
５．β−カゼインのアクチナーゼＥによる分解物におけるマクロファージ走化性誘導の経時的変化
１０ｍＭのＣａＣｌ2を含むトリス−塩酸緩衝液にβ−カゼインを溶解し、アクチナーゼＥを加えて５％ＣＯ２、３７℃で１、３、６、１２、２４時間反応後、CENTRIPREP３により限外濾過(３０００ｒｐｍ、４℃)を行い分子量３０００以下の画分を得た。これらの画分に対するマクロファージ走化性を、４８−well microchemotaxis chamberを用いて、上記３.マクロファージ走化性試験(２)に従い測定した。
【００４７】
６．高速原子衝突質量分析法(FAB-MS)によるペプチドの分子量測定
溶媒にはイオン交換水、およびマトリックスにはグリセロールを選択した。上記１.ペプチド調整(５)において得た画分はPOSITIVEモードおよび低分解能の各種条件下で、高速原子衝突質量分析法(fast atom bombardment-mass spectrometry、以下「FAB-MS」と略す)により、試料の分子イオンピーク(Ｍ＋)の検出測定を行った。測定機種は、高性能二重収束質量分析計 ＪＭＳ−７００(日本電子株式会社製)を用いた。
【００４８】
７．プロテインシーケンサーによるペプチドのアミノ酸配列分析
上記１.ペプチド調整(５)において得た画分を、ＰＶＤＦ膜に疎水的に結合させた。分離カラムには、ＰＴＨ-Ｃ18カラム(２.１×２２０mm；パーキン エルマー社製)を用い、プロテインシーケンサー(model４９０ Procise；パーキン エルマー、アプライド バイオシステム社製)により、アミノ酸配列分析を行った。また、溶媒Ａ３(３.５％テトラヒドロフラン水溶液(パーキン エルマー社製))、溶媒Ｂ(アセトニトリルおよびイソプロピルアルコール混合液(パーキン エルマー社製))などを含む１４０Ｃマイクログラジェント送液システム (１４０Ｃ microgradient delivery system；アプライド バイオシステム社製) に必要な関連試薬は、アプライド バイオシステム社指定品を使用した。
【００４９】
８．活性画分によるチェッカーボード解析
マクロファージ走化性因子が、chemokinetic factor(活性化物質により細胞の運動がアトランダムに激しくなる、方向性なし)であるのか、もしくはchemotactic factor(方向性がある)であるのかを確認するために第９図に示すような方法でチェッカーボード解析を行った。すなわち、Chemotaxis chamber下部のwellに上記１.ペプチド調整(５)において得た活性ペプチドを様々な濃度に希釈したものを入れ、上部のwellに走化性因子の希釈液で作った細胞懸濁液を入れた。次いで、上記３.マクロファージ走化性試験(２)に準じてマクロファージの走化性を測定した。
【００５０】
９．レーザー顕微鏡によるマクロファージ細胞内Ｃａ2+流動性(Ｃａ flux)の測定
マクロファージ懸濁液(１×１０６cells/ｍｌ)を１００μｌ、Non-Coat ３５ｍＭDish(松浪硝子工業(株)製)の中央に入れ、ＲＰＭＩ−１６４０(１０％ＦＣＳ含有)を加えてドロップを作り、これを５％ＣＯ２、３７℃で２４時間インキュベートする。マクロファージがDishの底面に接着している事を確認し５％ＦＣＳを含むＲＰＭＩ−１６４０で２回洗浄後、再び５％ＦＣＳを含むＲＰＭＩ−１６４０でドロップを作る。次に、ジメチルスルホシキド(ＤＭＳＯ、和光純薬工業(株)製)で溶解したFluo-３AM(０.８ｍＭ)を５μｌずつ添加し、５％ＣＯ２、３７℃で１時間インキュベート後、ＲＰＭＩ−１６４０完全培地で２回洗浄し、ＲＰＭＩ−１６４０完全培地で再度ドロップを作った。Ｃａ fluxの測定にはレーザー顕微鏡を用い、Fluo-３AM強度が安定したところで、次の１０.Ｃａイオン濃度の測定法と同様に調製したイオノマイシン、ＥＧＴＡ、ｆＭＬＰまたは上記１.ペプチド調整(５)において得た活性画分を５μｌ入れて輝度の変化をグラフ化した。
【００５１】
１０．マクロファージ中のＣａイオン濃度の測定
(１)マクロファージへの蛍光色素の取り込み
マクロファージ懸濁液(１×１０６cells/ml)１ｍｌに、０.８ｍＭのFluo-３AM(Molecular Probes社製)を５μｌ添加し、３０分間３７℃の恒温槽でインキュベーションした。その後遠心分離(１５００ｒｐｍ、５min、４℃)し、上澄液を除去後５％ＦＣＳを含むＲＰＭＩ−１６４０を１ｍｌ加えて再懸濁した。
【００５２】
(２)ネガティブ試料とポジティブ試料の測定
ネガティブ試料の測定；インキュベーションした細胞懸濁液をFACSにセットし、１０秒ほど取り込みを行った後、いったん取り外して、ＲＰＭＩ−１６４０で溶解した６ｍＭのＥＧＴＡ(和光純薬工業(株)製)を５００μｌ添加し、すばやくセットし直した。この作業の間、データ取り込みは続けたままにした。
ポジティブ試料の測定；インキュベーションした細胞懸濁液をFACSにセットし、１０秒ほ ど取り込みを行った後、いったん取り外して、ＤＭＳＯで溶解した７００μＭのイオノマイシン(シグマ社製)を１０μｌ添加し、すばやくセットし直した。この作業の間、データ取り込みは続けたままにした。
【００５３】
(３)サンプルデータの測定
上記１.ペプチド調整(５)において得た活性画分を２×１０-1Ｍ、２×１０-2Ｍ、２×１０-3Ｍに段階希釈し、ｆＭＬＰは２×１０-3Ｍ、２×１０-4Ｍ、２×１０-5Ｍに段階希釈した。インキュベーションした細胞懸濁液をFACSにセットし、１０秒ほど取り込みを行った後、いったん取り外して、各濃度のサンプルを１０μｌ添加し、すばやくセットし直した。この作業の間、データ取り込みは続けたままにした。
【００５４】
１１．試験結果
(１)酵素分解により生じたペプチドに対するマクロファージ走化性
β−カゼインを７種類の酵素(α−キモトリプシン，ペプシン，サーモリシン，トリプシン，プロテアーゼＫ，アクチナーゼＥ，パパイン)で分解して生じたペプチドに対するマクロファージ走化性を、４８−well microchemotaxis chamberを用いて測定した。ギムザ染色法でＰＶＰＦ膜を染色したところ、コントロールである未分解のβ−カゼインのみの場合と比べて、プロテアーゼＫ、アクチナーゼＥおよびパパインによる分解物がかなり濃く染色された(第１図)。
【００５５】
これを光学顕微鏡を用いて倍率２００倍で一視野あたりの遊走細胞数をカウントしたところ、コントロールと比べて、プロテアーゼＫ、アクチナーゼＥおよびパパインによる分解物においてそれぞれ、約６.８倍、４.３倍、４.０倍多く遊走し、有意な差が認められた(第２図)。なお、第２図中、Ｃ、Ｓはそれぞれ酵素未消化物、酵素消化物を示し、＊印は５％の有意水準で、＊＊印は１％の有意水準で有意差があったことを意味する。また、結果はＨＰＦ１０視野中の平均細胞数である。
【００５６】
(２)限外濾過による各種酵素分解生成ペプチドのマクロファージ走化性
(ｉ)β−カゼインを各種酵素で分解したときに生成するペプチドを、CENTRIPREP3を用いて限外濾過し、分子量３０００以下の画分のみを回収した。この低分子ペプチドと限外濾過前のペプチドをSDS-PAGEに供した結果をそれぞれ第３図Ｂ、第３図Ａに示す。各図１、１０は分子量マーカー、２はα−キモトリプシン消化物、３はペプシン消化物、４はサーモリシン消化物、５はトリプシン消化物、６はプロテアーゼＫ消化物、７はアクチナーゼ消化物、８はパパイン消化物、９は未消化のβ−カゼインについて示す。α−キモトリプシンとトリプシンによる分解生成ペプチドのバンドは、限外濾過後に極端に減少していた。また、アクチナーゼＥによる分解生成ペプチドのバンドは限外濾過の前後のどちらも検出されなかった。
【００５７】
(ii)各種酵素分解ペプチドの分子量３０００以下の画分について、マクロファージ走化性を測定したところ、コントロールと比べて、プロテアーゼＫ、アクチナーゼＥおよびパパインによる分解物においてそれぞれ約５.５倍、５.８倍、５.１倍のＨＰＦ値を示し、遊走マクロファージ数に有意な差が認められた(第４図)。なお、第４図中、＊印は５％の有意水準で有意差があったことを意味する。また、結果はＨＰＦ１０視野中の平均細胞数である。
【００５８】
(３)ヒト末梢血由来モノサイトのβ−カゼイン由来ペプチドに対する走化性
上記(１)(２)に示したように酵素分解物ペプチド成分による走化性の誘導がマウス由来のマクロファージに対して認められた。次に、ヒト由来のモノサイトに対する走化性を、上記４.ヒト末梢血モノサイトの走化性の測定法に従って検討した。HISTPAQUEにより末梢血から分離された単核球のうち、モノサイトは２２.６％であり、さらにMagnetic Cell Separation System(ＭＡＣＳ)による磁気細胞分離法により分離したモノサイトは、FACS CaliburTMflow cytometerによる解析で約９７.５％であることを確認した(第５図Ａ)。ギムザ染色法によりＰＶＰＦ膜を染色し、光学顕微鏡により倍率２００倍で遊走したモノサイト(第５図Ｂ：アクチナーゼＥによる場合を示す)をカウントしたところ、コントロールである未分解のβ−カゼインのみの場合と比べて、パパインとアクチナーゼＥによる分解物に有意差が認められたが、パパインによる分解物がコントロールの約１.９倍のモノサイトを遊走させたのに対して、アクチナーゼＥによる分解物はコントロールの約３.３倍と、最も強い走化性が認められた（第５図Ｃ）。なお、第５図Ｃ中、＊印は５％の有意水準で有意差があったことを意味する。また、結果はＨＰＦ１０視野中の平均細胞数である。
【００５９】
(４)アクチナーゼＥによる分解ペプチドにおけるマクロファージ走化性誘導の経時的変化
上記(１)〜(３)の結果から、マウス由来のマクロファージとヒト由来のモノサイトの両方に対して走化性ペプチドを誘導する効果的な酵素は、アクチナーゼＥであることが判明した。そこで、アクチナーゼＥによる分解ペプチドにおける走化性誘導の経時的変化を調べた。その結果、第６図に示すように、３時間以上で遊走マクロファージ数に有意差が認められ、３時間と６時間では約１.６倍、１２時間では約２.５倍、２４時間では約３.５倍のマクロファージが遊走し、走化活性は経時的に上昇する傾向を示した。これらの結果から、β−カゼインの分解にはアクチナーゼＥが好ましく、３７℃で２４時間反応させるのがよいことが見出された。以下においては、この条件で得られた消化物について試験を行った。
【００６０】
(５)β−カゼイン由来ペプチドの各分画ステップにおける回収率とマクロファージ走化性試験
β−カゼイン由来ペプチドを分画するために、アクチナーゼＥによる処理物(３７℃、２４時間反応物)について、限外濾過、Ｃ18逆相クロマトグラフィーを行った。それぞれの分画後の回収率を表２にタンパク含量(％)で示し、各画分の走化性も測定した(表２)。その結果、限外濾過による活性の低下は見られず、またその後に行ったＣ18逆相クロマトグラフィーから得られた画分では３０％アセトニトリル、６０％アセトニトリル溶出画分にマクロファージ走化性因子が存在する事が分かった。しかしながら、６０％アセトニトリル溶出画分は収量が１.８％と非常に低く、それよりもアセトニトリル濃度が低い３０％アセトニトリル溶出画分を用いるのが好ましいと判断された。
【００６１】
【表２】
(６)陽イオン交換クロマトグラフィーによる活性ペプチド成分の分画とマクロファージ走化性試験
Ｃ18逆相クロマトグラフィーにより得られた３０％アセトニトリル溶出画分を陽イオン交換クロマトグラフィーに供した。その結果、リン酸塩緩衝液(ｐＨ６.０)による陽イオン交換クロマトグラフィーではほとんどすべてがカラム素通り成分であり、それぞれのフラクションについてマクロファージ走化性を48-well microchemotaxis chamberを用いて測定したところ、カラム素通り画分に走化性が認められた(図示せず)。
【００６４】
(７)陰イオン交換クロマトグラフィーによる活性ペプチド成分の分画とマクロファージ走化性試験
Ｃ18逆相クロマトグラフィーにより得られた３０％アセトニトリル溶出画分を陰イオン交換クロマトグラフィーに供した。その結果、トリス−塩酸緩衝溶液(ｐＨ８.２)による陰イオン交換クロマトグラフィーではカラム吸着画分の２つピークとカラム素通り画分とに分離した(第７図上段)。それぞれのフラクションについてマクロファージ走化性を48-well microchemotaxis chamberを用いて測定したところ、陰イオン交換クロマトグラフィーの素通り画分と吸着画分の最初のピークには有意な活性が認められなかったのに対し、吸着画分の２つ目のピーク：フラクションナンバー(F.N.)４２〜４６に走化性が認められた(第７図下段)。以下、このフラクションナンバー４２〜４６の画分をＱ画分と称する。
【００６５】
(８)高速液体クロマトグラフィー(ＨＰＬＣ)による活性成分の分析および単離・精製
Ｃ18逆相クロマトグラフィーで得られた３０％アセトニトリル溶出画分とＱ画分をＲＰ−ＨＰＬＣに供した。その結果、３０％アセトニトリル溶出画分からマルチなピークが検出された(第８図Ａ上段)。一方、Ｑ画分からは主に３成分(溶出時間の早い順にＱ１，Ｑ２およびＱ３)が検出され、その割合はＱ１が９０.７％、Ｑ２とＱ３が４.０%であった(第８図Ａ下段)。それぞれの画分を分取、単離後、走化性の測定を行った結果、Ｑ２とＱ３に関しては収量が少なかったために測定には至らなかったが、Ｑ１はＱ画分とほぼ同程度の活性が認められた(第８図Ｂ)。
【００６６】
(９)高速原子衝突質量分析法(FAB-MS)によるペプチドの分子量測定
高速液体クロマトグラフィーにより分取したＱ１、Ｑ２、Ｑ３の分子量を測定したところ、それぞれ６０４、５２３、７５１であった。
【００６７】
(１０)Ｑ１、Ｑ２、Ｑ３のアミノ酸配列分析
高速液体クロマトグラフィーにより分取したＱ１、Ｑ２、Ｑ３についてプロテインシーケンサーによってアミノ酸分析を行った。これにより、
Ｑ１：Tyr−Pro−Val−Glu−・・・
Ｑ２：Glu−Met−・・・
Ｑ３：Tyr−Pro−Ｘ−Glu−・・・(Ｘ＝読解不可能)
という配列が得られた。
【００６８】
さらに、Ｎ末端アミノ酸配列と分子量から、β−カゼインの配列と照らし合わせたところ、Ｑ１、Ｑ２、Ｑ３は、以下に示すように、β−カゼインの１０８〜１１９残基由来のペンタ、テトラ、ヘキサペプチドであることが同定された。この３つのペプチドの等電点はいずれも３〜４の値を示し、そのため陽イオン交換カラムには吸着しなかったと考えられる。
Ｑ１：Tyr−Pro−Val−Glu−Pro(１１４〜１１８残基 Ｍ.Ｗ.＝６０３.６：配列番号１で示される)
Ｑ２：Glu−Met−Pro−Phe(１０８〜１１１残基 Ｍ.Ｗ.＝５２２.６：配列番号３で示される))
Ｑ３：Tyr−Pro−Val−Glu−Pro−Phe(１１４〜１１９残基 Ｍ.Ｗ.＝５２２.６：配列番号２で示される)
【００６９】
なお、β−カゼインは不均一性を示すことが知られており、１１７残基目がGlnであるものと１１７残基目がGluであるものとがあるが、得られたＱ１、Ｑ３は、１１７残基目がGluであるβ−ペプチド由来のものであった。また、収量の多かったＱ１について、その活性を測定したところ、ＨＰＬＣ分取後においてもマクロファージ走化性活性を失わなかった。走化性活性(誘導能)の発現にはタンパク質の高次構造が深く関与すると考えられており、ＨＰＬＣ分取すれば高圧によりタンパク質の高次構造が破壊されるため走化性誘導能を失うという見解もあるが、得られたペプチドは５つのアミノ酸からなるペンタペプチド(ＹＰＶＥＰ)であって、立体構造が変化しにくかったために活性を失わなかったものと考えられる。
【００７０】
(１１)β−カゼイン由来マクロファージ走化性ペプチドのチェッカーボード解析
Ｑ１による活性が、化学運動性によるものか、走化性によるものかを確認するためにチェッカーボード解析を行った。その結果、上部wellのペプチド濃度が下部wellよりも高いときは、ほとんどマクロファージの遊走が認められなかった。これに対し、下部wellのペプチド濃度が上部wellよりも高いときには、１ＨＰＦあたり１９から１２０もの多くのマクロファージが遊走していることが確認できた(第９図)。このことから、このペプチドはマクロファージに対して走化性を誘導することが確認された。
【００７１】
(１２)マクロファージの細胞内Ｃａ2+流動性(Ｃａ flux)
まず初めに、Ｃａインジケーター(fluo-３AM)を用いて、レーザー顕微鏡により細胞内Ｃａ2+流動性を測定した。強力な走化性ペプチドとして知られているｆＭＬＰが、Ｑ１のコントロールとして用いられた。最近になって、そのレセプターが７回膜貫通型であったことが明らかにされたためである。マクロファージにおけるその活性化機構は、レセプターに走化性因子が結合すると、イノシトールリン酸の代謝を介してＣａイオンが貯蔵器官から放出され、シグナリングによってＭａｃＩのような接着因子が活性化されて、遊走を開始すると考えられる。そして、近年、ケモカインに代表される走化性因子は、Ｇプロテイン関連の７回膜貫通型レセプターを介し活性を発現させることが明らかにされた。この測定から、走化性ペプチドＱ１には、濃度依存的にＣａ fluxが認められ、１×１０-3Ｍ濃度で顕著なピークが得られた(第１０図下段左図)。一方、コントロールとして用いたｆＭＬＰはまったくピークが認められなかった(同図下段右図)。そこで、実際に細胞質内Ｃａ濃度をフローサイトメトリーにより測定したところ、計算式から細胞内Ｃａ濃度は、Ｑ１で刺激した細胞の細胞内Ｃａ濃度のみが高く約６２０ｎＭで、ｆＭＬＰは約１７０ｎＭとコントロールと同程度であり、レーザー顕微鏡によるデータを支持するものであり(第１１図)、β−カゾケモチドは、ｆＭＬＰとは異なるレセプターを介してマクロファージに作用するものと考えられる。
【００７２】
以上の実験結果より、アクチナーゼＥによる分解生成ペプチド群のうち、分子量３０００以下の画分が、マウス由来マクロファージとヒト末梢血モノサイトの両者に対して強い走化性を示し、未分解β−カゼインと比べるとマクロファージに対して約５.８倍、ヒトモノサイトに対して約３.３倍の細胞数が遊走した。しかし、このペプチド群はSDS-PAGEで検出することができなかった。これは、アクチナーゼＥが、各種の基質に対して強力な加水分解力を有しているため、タンパク質の各種のペプチド結合に作用し、その結果タンパク分解産物としてペプチド成分にとどまらず多量の遊離アミノ酸を産出するという特性を持ち、上記実施例においては、β−カゼインがアクチナーゼＥによって非常に低分子のペプチドかアミノ酸レベルにまで分解され、ゲルを通過してしまったためと考えられる。
【００７３】
マクロファージの細胞内Ｃａ2+流動性(Ｃａ flux)に与える影響について、強力な走化性ペプチドとして知られているｆＭＬＰをコントロールとして測定したところ、本発明に係るペプチドは、ｆＭＬＰとは異なる流動性が認められた。α−カゼインがｆＭＬＰと同じレセプターを認識する可能性を示唆する報告もあるが、その走化性ペプチド配列に関する知見や、ｆＭＬＰと異なるレセプターを介して走化性を発現するという報告もなく、本発明のペプチドは、マクロファージの走化性を亢進する新規な誘導因子であると言える。
【００７４】
また、本発明のβ−カゾケモチドは、５つのアミノ酸からなるペンタペプチド(ＹＰＶＥＰ)であるので、立体構造が変化しにくく、高圧が負荷されるＨＰＬＣ分取によっても走化性誘導能を失うことなく容易に分取できるものである。
【００７５】
β−ペプチドの１１３残基(Lys)−１１４残基(Tyr)の結合がペプシンおよびトリプシンで切断され、１１９（Phe）−１２０(Thr)の結合がキモトリプシンで切断されることが明らかにされている(Yunden Jinsmaaら，Peputides，20,957−962(1997))。したがって、本発明のペプチド、β−カゾケモチドは、それよりも短いのでこれ以上の酵素分解を受けないものと推定され、経口投与に十分利用しうるものである。
【００７６】
また、古くから大きな関心事である牛乳アレルギーを引き起こすアレルゲン中にこのペプチドと同じ配列を持つものはない。各種の走化性因子を用いてラットに皮下注射をしたところ、ｆＭＬＰではすぐに炎症反応を起こして、好中球が多く集まってきたのに対して、β−カゼインではそれほど急激な反応が見られなかったことも知られており、本発明のペプチドは生体にとって有害な物質ではないと考えられる。
【００７７】
このように当該ペプチドは、マクロファージの走行性を誘導し、免疫賦活機能を発揮する新規ペプチドである。これを食品成分として有効に利用するためには、該ペプチドを食品中に添加したり、食品中のβ−カゼインをアクチナーゼＥなどの消化酵素により消化させるのがよい。また、あらかじめβ−カゼインの１１８−１１９残基結合を、例えば食品添加物として厚生労働省から認可されているアクチナーゼＡＳ(例えば科研製薬(株)製)で分解しておき、摂取したこれらの分解物の消化によって該ペプチドを生成させることもできる。
【産業上の利用可能性】
【００７８】
本発明は、マクロファージ走化性を亢進する新たな新規ペプチドを提供する。このペプチドは、乳製品を始めとする食品に含まれているβ−カゼインをアクチナーゼＥなどのタンパク消化酵素により分解することで得られるので、非常に安全性の高いものである。そして、当該ペプチドを直接利用しあるいは消化管内で当該ペプチドを生じやすいようにあらかじめ食品を酵素処理することで、食品由来成分の免疫賦活化機能を利用した新たな機能性食品が提供される。【Technical field】
[0001]
The present invention relates to a novel peptide having an immunostimulatory action, a novel immunostimulator, a functional food, and a method for producing a functional food.
[Background]
[0002]
Japan has become one of the world's longest-lived countries with an average life expectancy of 80 years, but not everyone can have a healthy longevity. The greatest enemy of healthy longevity is a group of diseases called “lifestyle-related diseases” such as cancer, stroke, heart disease, liver disease, kidney disease, and diabetes. In December 1996, the Ministry of Health and Welfare “Public Health Deliberation” replaced the “adult disease” that focused on conventional aging, with the aim of introducing a new concept of lifestyle-related disease that focused on lifestyle. It was advocated by the Association. The development and progression of this “lifestyle-related disease” involves three factors: lifestyle factors, genetic factors, and external environmental factors. Many diseases that are based on lifestyle habits can be prevented in daily life. The importance of primary prevention, which is to prevent disease by improving the dietary habits of children, has attracted attention.
[0003]
Originally, food functions have been considered to have a nutritional function (primary function) as a supplement of various nutritional components to maintain life activity and a taste sensory function (secondary function) to eat deliciously. That is, what was delicious and excellent as a nutrient source was regarded as an excellent food. However, in the 1980s, nutrients involved in biological defense functions and disease prevention and recovery were discovered, and functions that regulate the body's function as an important third function in addition to the primary and secondary functions so far That is, attention has been paid to the physiological functions of foods containing components having various biological regulation functions such as enhancement and regulation of immunity, regulation of various hormone secretions, suppression of aging, and the like.
[0004]
Milk protein is not only an excellent source of nitrogen and essential amino acids among major food proteins, but also promotes the absorption of nutrients such as minerals and vitamins, and promotes bone formation and suppresses bone resorption It is designed to act on the body in some way by ingesting it orally, such as those that act to regulate various functions of the body, such as those that lower blood cholesterol levels. ing.
[0005]
Since 1979, Brantle et al. Reported that casein digests of milk have opioid agonist activity, peptides having various biological activities have been isolated and identified from casein digests (see Non-Patent
[0006]
However, foods that induce immune activation of the living body and aim at contribution to biological defense have not been developed yet. There are various indexes indicating the activation of immune function, and one of them is chemotaxis related to the induction of macrophage function. Migration by chemotactic factors is one of the functions of macrophages important for immune responses, and macrophages stimulated by intestinal chemotactic factors are thought to migrate to the site of infection in the intestine and kill pathogens. That is, it can be said that migration of macrophages to pathogens plays a very important role in the defense of the intestinal tract.
[0007]
Many substances that induce the chemotaxis of macrophages have been reported so far (Non-patent Document 4), but those in which chemotactic factors have been identified as peptide components are derived from bacteria such as fMLP. Only formylated peptides, peptides obtained from human peripheral blood lymphocytes (Patent Document 1), and chemically synthesized peptides made from peptide libraries (Non-Patent Document 5), food-derived egg whites Only the ovalbumin fragment obtained from is known (Patent Document 2).
[Patent Document 1]
[0008]
JP-A-9-227592, pages 2-9
[Patent Document 2]
[0009]
JP-A-9-12470, pages 2-3
[Non-Patent Document 1]
[0010]
V.Brantl et al., “Hoppe-Seyler's Z. Physiol. Chem., 1979, 1360, p. 1211-1216.
[Non-Patent Document 2]
[0011]
Agnes Henschen et al., “Hoppe-Seyler's Z. Physiol. Chem., 1979, No. 1360, pp. 1217-1224.
[Non-Patent Document 3]
[0012]
Yoshitaka Kanamaru, “New functions of milk and milk components (protein)”, Dairy Technology, 2000, 50, p.22-37
[Non-Patent Document 4]
[0013]
Osamu Yoshie et al., “Chemokine Handbook”, 2000, p.12
[Non-Patent Document 5]
[0014]
Yingying Le et al., “Journal of Immunology”, 1998, 163, p. 6777-6784.
[0015]
Accordingly, the present inventors have sought to provide a food that exerts a biodefense action in the intestinal tract and have made extensive efforts focusing on the immunostimulatory ability of the milk protein component. Macrophage migration activity was found and the present invention was completed.
DISCLOSURE OF THE INVENTION
[0016]
The peptide of the present invention is a novel peptide consisting of the following amino acid sequence represented by SEQ ID NO: 1.
Tyr-Pro-Val-Glu-Pro
[0017]
The immunostimulant of the present invention has the following amino acid sequence represented by SEQ ID NO: 1. Consist of A peptide is an active ingredient, and the usage of the present invention is the following amino acid sequence represented by SEQ ID NO: 1. Consist of It is characterized by using a peptide as an immunostimulant.
Tyr-Pro-Val-Glu-Pro
[0018]
In addition, the functional food of the present invention is the following amino acid sequence represented by SEQ ID NO: 1 in an amount that substantially exhibits the ability to induce macrophage chemotaxis. Consist of It is characterized by containing a peptide.
Tyr-Pro-Val-Glu-Pro
[0019]
Furthermore, the functional food of the present invention is such that the digestive enzyme in the body produces a peptide consisting of the following amino acid sequence represented by SEQ ID NO: 1 in an amount that substantially exhibits the ability to induce macrophage running. Β-casein 118-119 residues of Actinase It is characterized by being cut by.
Tyr-Pro-Val-Glu-Pro
[0020]
In the method for producing a functional food of the present invention, β-casein in food is added so as to contain an amount of the peptide that substantially exhibits the ability to induce macrophage chemotaxis. Actinase It has the process of digesting by. In this case, the next amino acid sequence represented by SEQ ID NO: 1 Consist of Peptides should be included.
Tyr-Pro-Val-Glu-Pro
[Brief description of the drawings]
[0022]
FIG. 1 is a photograph showing a PVPF membrane after Giemsa staining. FIG. 1A shows the whole PVPF membrane, and FIG. 1B shows migratory cells observed under a microscope.
FIG. 2 is a diagram showing the induction of macrophage chemotaxis by various enzyme digests of β-casein.
FIG. 3 is an SDS polyacrylamide electrophoretic diagram after various enzyme digestion of β-casein. FIG. 3A is an electrophoretic diagram before enzyme digestion, and FIG. 3B is an electrophoretic diagram of a digest (3 kD or less). .
FIG. 4 shows the induction of macrophage chemotaxis by fractions of various enzyme-digested β-casein having a molecular weight of 3 kD or less.
FIG. 5 is a graph showing the induction of chemotaxis of human peripheral blood monocytes by fractions having a molecular weight of 3 kD or less of various enzyme digested β-caseins, and FIG. 5A shows the purity of monosites by FACS CaliburTm. The figure and the same figure B are the microscope images which show the cell which migrated by the actinase E digested material, and the same figure C is a figure which shows the number of the cells which migrated by the enzyme digested material.
FIG. 6 is a graph showing changes over time in macrophage chemotaxis expressed by actinase E digestion of β-casein.
FIG. 7 is a diagram showing fractionation of macrophage chemotactic factors from β-casein digests by anion exchange chromatography, with the upper graph by ultraviolet absorption and the lower graph by the number of cells induced to chemotaxis. FIG.
FIG. 8 shows the isolation and purification of macrophage chemotactic peptides by high-performance liquid chromatography, where the upper part of FIG. A shows the elution with 30% acetonitrile, and the lower part of FIG. A shows the elution of the Q fraction. FIG. 4B is a diagram showing chemotaxis by Q1, Q2 and Q3 fractions.
FIG. 9 is a checkerboard analysis diagram of macrophage chemotactic peptide Q1.
FIG. 10 is a diagram showing the calcium fluidity of macrophage chemotactic peptide Q1, wherein the upper stage shows a laser microscopic image of macrophages that have incorporated Ca, and the lower stage shows changes in Ca uptake of macrophages.
FIG. 11 is a diagram showing the concentration of calcium flowing in macrophages by stimulation with macrophage chemotactic peptide Q1.
[Best Mode for Carrying Out the Invention]
[0023]
The present invention relates to a novel peptide that induces macrophage chemotaxis performance, and the novel peptide according to the present invention has an amino sequence of Tyr-Pro-Val-Glu-Pro represented by SEQ ID NO: 1 (hereinafter referred to as the said peptide). The peptide is referred to as “β-casochemotide”.) This peptide corresponds to β-casein 114 to 118 residues, and β-neocasomorphin (SEQ ID NO: 2: Tyr-Pro-Val-Glu-Pro-Phe, β, isolated and identified as an opioid peptide). -C-terminal (Phe) of casein (corresponding to residues 114 to 119) is deleted.
[0024]
β-casochemotide is obtained, for example, by treating β-casein with protein digestion enzymes such as various actinases represented by actinase E (trade name, manufactured by Kaken Pharmaceutical Co., Ltd.) and papain. In particular, the peptide can be obtained in high yield by treatment with actinase. This enzyme treatment does not require treatment under special conditions, and normal treatment conditions are selected. For example, it can be performed at the optimum pH, optimum buffer, and optimum temperature as shown in Table 1 below, but is not limited to these conditions. The peptide can also be obtained by chemical synthesis.
[0025]
This has an effect of inducing macrophage chemotaxis and can be applied as an immunostimulator. β-casochemotide is obtained using β-casein in milk and the like, and since there is no allergen that causes milk allergy having the same sequence as the peptide, its safety is considered high.
[0026]
The functional foods of the present invention include health functional foods (foods for specified health use, nutritional functional foods) and foods with physiological functions, and contain an amount of peptide that substantially exhibits macrophage chemotaxis inducing ability. To do. The functional food can be obtained, for example, by adding a peptide according to the present invention isolated or synthesized from a protein digest of β-casein to a target food. In addition, an enzyme digest of β-casein may be added as it is to the food, and the protein is contained so that the amount of peptide that substantially exhibits the ability to induce macrophage chemotaxis is included in the food production process. You may digest (beta) -casein using a digestive enzyme. For example, the raw milk is subjected to an enzyme treatment to produce a milk beverage, and the raw milk subjected to the enzyme treatment is used as a raw material to produce a dairy product such as cheese or yogurt. The protein digestive enzyme to be used is not particularly limited as long as the target digest (peptide) is produced, but is preferably actinase.
[0027]
Alternatively, it may be decomposed with actinase AS (for example, manufactured by Kaken Pharmaceutical Co., Ltd.) that is recognized as a food additive to cleave the 118-119 residue bond in β-casein contained in food. Good. When this food is ingested, the 113-114 residue bond of β-casein is further cleaved by a resident digestive enzyme such as pepsin or trypsin, and the peptide of the present invention is produced in the body. Thus, the β-casein 113-114 residue bond in food may be cleaved in advance, and a peptide having an amount that substantially exhibits the ability to induce macrophage chemotaxis may be generated by digestive enzymes in the body.
[0028]
In addition to the dairy products described above, various foods can be considered as the functional food of the present invention, and the types thereof are not limited. Considering the addition of a digestive enzyme treatment step, the present invention is suitable for foods that are rich in β-casein and foods that are made from foods that are rich in β-casein.
[0029]
The β-casein content is considered to be an amount that substantially exhibits the ability to induce macrophage chemotaxis, but the standard is about 0.01 to 10 mg, preferably about 0.1 to 5 mg in a single intake. .
[Example 1]
[0030]
Next, the present invention will be described in more detail based on the following examples.
1. Peptide preparation
(1) Enzymatic degradation of β-casein
10 mg of β-casein (manufactured by Sigma) was dissolved in 10 ml of the optimum buffer shown in Table 1 according to the method of Amhar et al. (J. Dairy Science, 81, 3131-3138 (1998)) to obtain 0.5 mg of
[0031]
[Table 1]
(2) Ultrafiltration
The enzyme digest obtained in (1) above was ultrafiltered (3000 rpm, 4 ° C.) with CENTRIPREP3 (Millipore) to obtain a fraction having a molecular weight of 3000 or less.
[0033]
(3) C 18 Fractionation with reversed phase carrier
Among the β-casein enzyme digests having a molecular weight of 3000 or less obtained in (2) above, digests with actinase E are obtained as C 18 Sep-Pak as a reverse phase carrier TM PlusC 18 After adsorbing to a cartridge (manufactured by Waters), 10 ml each of ion-exchanged water, 30 v / v% acetonitrile, 60 v / v% acetonitrile, and 90 v / v% acetonitrile were passed through to obtain elution fractions from each solution. Then, after removing acetonitrile with a centrifugal evaporator, it lyophilized | freeze-dried and preserve | saved at -20 degreeC.
[0034]
(4) Fractionation of active ingredients by ion exchange chromatography
The 30 v / v% acetonitrile elution fraction obtained by (3) above was dissolved in mobile phase A (1 ml) and subjected to ion exchange chromatography under the following conditions using a Bio Logic chromatography system (manufactured by BioRad Laboratory).
[0035]
<Cation exchange chromatography conditions>
Column: HiTrap TM SP HP (Amersham Pharmacia Biotech AB)
Solvent: mobile phase A (50 mM phosphate buffer pH 6.0)
Mobile phase B (50 mM phosphate buffer pH 6.0 containing 1 M NaCl)
Elution: Hold for 8 minutes in mobile phase A, then linearize mobile phase B to 50% in 5 minutes
The mobile phase B was raised to 100% in 4 minutes.
Flow rate: 5.0 ml / min
Preparative: 1.0ml / fraction
Temperature: room temperature
Detection: 214nm
[0036]
<Anion exchange chromatography conditions>
Column: HiTrap TM Q HP (Amersham Pharmacia Biotech AB)
Solvent: Mobile phase A (20 mM Tris-HCl buffer (pH 8.2))
Mobile phase B (20 mM Tris-HCl buffer (
2))
Elution: Hold for 8 minutes in mobile phase A, then linearize mobile phase B to 50% in 5 minutes
The mobile phase B was raised to 100% in 4 minutes.
Flow rate: 5.0 ml / min
Preparative: 1.0ml / fraction
Temperature: room temperature
Detection: 214nm
[0037]
(5) Analysis, isolation and purification of active peptides by high performance liquid chromatography
The active fraction (anion chromatography elution fractions No. 42 to 46) obtained in (4) above is dissolved in ion-exchanged water and subjected to high performance liquid chromatography (High-Resolution) in reversed-phase (hereinafter referred to as RP) mode. Analysis and fractionation were performed by -performance liquid chromatography (hereinafter HPLC).
[0038]
<RP-HPLC conditions>
Column: Mightysil RP-18 GP (5 μm, 4.6 × 150 mm, manufactured by Kanto Chemical Co., Inc.)
Solvent: Mobile phase A (30% acetonitrile with 0.05 v / v% TFA)
Mobile phase B (60% acetonitrile containing 0.05 v / v% TFA)
Elution: linear increase from 100% mobile phase A to 100% mobile phase B in 30 minutes
And then held in mobile phase B for 10 minutes.
Flow rate: 0.5 ml / min
Temperature: 40 ° C
Detection: 214nm
[0039]
The peak obtained by this operation is fractionated, and C according to the method of (3) above. 18 Sep-pak as a reverse phase carrier TM Plus C 18 After adsorbing to the cartridge, 10 ml each of ion-exchanged water and 30% acetonitrile was passed, and TFA was removed by collecting the fraction eluted with 30% acetonitrile. Furthermore, after removing acetonitrile with a centrifugal evaporator, it lyophilized | freeze-dried and preserve | saved at -20 degreeC.
[0040]
2. Electrophoretic analysis
100 μl of an SDS reagent was added to 100 μl of each of the fractions obtained in the preparations (1) and (2) of the above peptide and heated at 85 ° C. for 2 minutes to form SDS. The SDS-modified sample was applied with 10 μl per lane and electrophoresed at a constant voltage of 125V. The gel used was 4.75% concentrated gel and 20% separation gel, and peptide molecular weight marker “Daiichi” (Daiichi Chemical Co., Ltd.) and precision prestained standard (BioRad Laboratories) were used as molecular weight markers. It was. The gel after electrophoresis is a PVDF membrane Immobilon-PSQ (Millipore) and blotting device SEMI-PHOR TM Electroblotting was performed using (Hefer Riken Chemical Co., Ltd.). That is, Western buffer (20 mM Tris base, 200 mM Glycin / dH 2 Two pieces of filter paper soaked in O) were laid on a blotting apparatus, and air bubbles were completely removed. A PVDF membrane soaked in 100% methanol for 5 minutes and then soaked in Western buffer was placed on it, a separation gel was placed on top of it, and two filter papers soaked in Western buffer were placed on the gel and completely bubbled. Then, blotting was performed at 80 mA for 2 hours. The PVDF membrane to which the protein was transferred was stained with Quick-CBB (manufactured by Wako Pure Chemical Industries, Ltd.) for 5 minutes, and then the background was decolorized by shaking with 50% methanol.
[0041]
3. Macrophage chemotaxis test
(1) Preparation of macrophages
Mouse-derived macrophage J774-1 strain (transferred from Tohoku University Medical Research Center for Aging Medicine) was cultured in RPMI-1640 medium (containing 10% FCS) (manufactured by Sigma) and experimented until the 10th passage. Provided.
[0042]
(2) Measurement of macrophage chemotaxis
i) Macrophage J774-1 was cultured in RPMI-1640 medium (containing 10% FCS) (manufactured by Sigma), washed with RPMI-1640 complete medium (containing 1% BSA) at the time of assay, and 1 × 10 6 Prepared to cells / ml.
ii) Macrophage chemotaxis was measured according to the method previously reported by the present inventors (Int. J. Food Microbiol., 77, 29-38, (2002)). That is, the sample was diluted 2-fold with RPMI-1640 complete medium and placed in 28 μl each in the lower well of a 48-well microchemotaxis chamber (manufactured by Neuroprobe) (triplicate). A 25 × 80 mm, 5.0 μm PVPF membrane (manufactured by Neuroprobe) was placed on top of this, then a rubber gasket and a lid were placed in order and fixed with screws, and 50 μl of macrophage suspension was dispensed into the upper well. did. 5% CO 2 After culturing at 37 ° C. for 2 hours, the front side of the PVPF membrane, that is, the side on which the cells were seeded, was washed with PBS (Oxoid), wiped with a filter wiper (Neuro Probe), and field staining solution (Muto) The PVPF membrane was Giemsa-stained by Chemicals). The number of macrophages that migrated under a microscope (200 times magnification) was counted in 10 fields per sample, and the average number of migration (high power field; HPF) was calculated.
[0043]
4). Measurement of chemotaxis of human peripheral blood monosites
(1) Separation of monosite from human peripheral blood
100 ml of heparin collected blood was layered on a 50 ml tube into which 15 ml of HISTOPAQUE-1077 (manufactured by Sigma Diagnostics) was dispensed, and centrifuged (1500 rpm, 30 min, 20 ° C.). After recovering the mononuclear cell layer, a large amount of PBS was added to perform centrifugal washing (1500 rpm, 5 min, 4 ° C.), and the number of viable cells was counted after performing the same operation three times. Next, 100 μl of biotin-labeled mouse anti-human-CD11b antibody (manufactured by Pharmingen) diluted to 1/20 with PBS containing 5 mM EDTA (manufactured by Flor Laboratories) and 0.5% BSA (manufactured by Sigma) was added at 4 ° C. The reaction was allowed for 30 minutes. After adding 1.5 ml of PBS (containing 5 mM EDTA and 0.5% BSA) and washing by centrifugation and removing the supernatant, 90 μl of PBS (containing 5 mM and 0.5% BSA), MACS streptavidin microbeads (Miltenyi Boitec GmbH) 10 μl) was added and reacted at 4 ° C. for 15 minutes. After washing with PBS (containing 5 mM EDTA and 0.5% BSA), it was suspended in 500 μl of PBS (containing 5 mM EDTA and 0.5% BSA), and magnetic cell separation system (hereinafter referred to as “MACS”) (Miltenyi Biotec The cells (monosite) adsorbed on the MiniMACS column (RS +, manufactured by Miltenyi Biotec GmbH) were separated by GmbH. After the number of viable cells was counted for the separated monosites, the monosite chemotaxis was measured according to the method of 3. Macrophage chemotaxis test (2) above.
[0044]
(2) Purity test of human peripheral blood monosite
Purity of human peripheral blood monosites isolated according to (1) above is FACS Calibur TM The assay was performed using a flow cytometer (FACS; manufactured by Becton Dickinson). That is, FACS washing buffer (2% FCS, 0.02% NaN) was added to the monosite separated by MACS. Three After adding 1 ml of / PBS) and washing by pipetting, the supernatant was removed by centrifugation (3200 rpm, 3 min, 4 ° C.). After repeating this operation three times, 10 μl of biotin-labeled mouse anti-human CD11bMAC-1 antibody (manufactured by Pharmingen) was added and allowed to react at 4 ° C. for 15 minutes. FACS washing buffer (2% FCS, 0.02% NaN Three After adding 1 ml of / PBS) and washing by pipetting, the supernatant was removed by centrifugation (3200 rpm, 3 min, 4 ° C.), and 10 μl of R-Phycoerythrin-labeled streptavidin (manufactured by Targo) was added and protected from light. The reaction was performed at 4 ° C. for 15 minutes. To this was added 1 ml of FACS washing buffer, and after washing, the supernatant was removed by centrifugation (3200 rpm, 3 min, 4 ° C.), and a Sheath solution (500 μl) was added to suspend well. After passing this through a 20 μm nylon mesh, the purity of the human peripheral blood monosite was assayed by FACS.
[0045]
(3) Measurement of chemotaxis of human peripheral blood monosite
The chemotaxis of human peripheral blood monosites was determined by examining the fraction obtained by ultrafiltration (1. Peptide preparation (2) above) as a test sample using RPMI-1640 complete medium (containing 1% BSA) at a concentration of 1 mg / ml. It was measured according to the above-mentioned 3. Macrophage chemotaxis test (2) using a 48-well microchemotaxis chamber.
[0046]
5). Time course of induction of macrophage chemotaxis in degradation product of β-casein by actinase E
10 mM CaCl 2 Β-casein is dissolved in a Tris-HCl buffer solution containing A, and actinase E is added to add 5% CO. 2 After reaction at 37 ° C. for 1, 3, 6, 12, 24 hours, ultrafiltration (3000 rpm, 4 ° C.) was performed with CENTRIPREP3 to obtain a fraction having a molecular weight of 3000 or less. Macrophage chemotaxis to these fractions was measured using a 48-well microchemotaxis chamber according to 3. Macrophage chemotaxis test (2) above.
[0047]
6). Molecular weight measurement of peptides by fast atom collision mass spectrometry (FAB-MS)
Ion exchange water was selected as the solvent, and glycerol was selected as the matrix. The fraction obtained in 1. Peptide preparation (5) above was obtained by fast atom bombardment-mass spectrometry (hereinafter abbreviated as “FAB-MS”) under various conditions of POSITIVE mode and low resolution. Sample molecular ion peak (M + ) Was detected. As a measurement model, a high performance double convergence mass spectrometer JMS-700 (manufactured by JEOL Ltd.) was used.
[0048]
7). Amino acid sequence analysis of peptides by protein sequencer
The fraction obtained in the above 1. Preparation of peptide (5) was hydrophobically bound to the PVDF membrane. For the separation column, PTH-C 18 Amino acid sequence analysis was performed with a protein sequencer (model 490 Procise; Perkin Elmer, Applied Biosystems) using a column (2.1 × 220 mm; manufactured by Perkin Elmer). In addition, a 140C microgradient delivery system containing a solvent A3 (3.5% tetrahydrofuran aqueous solution (Perkin Elmer)), a solvent B (acetonitrile and isopropyl alcohol mixed solution (Perkin Elmer)), and the like. The relevant reagents required for Applied Biosystems) were those specified by Applied Biosystems.
[0049]
8). Checkerboard analysis with active fractions
To confirm whether the macrophage chemotactic factor is a chemokinetic factor (activator activates cell movement at random, non-directional) or chemotactic factor (directional). The checkerboard analysis was performed by the method shown in FIG. That is, the lower part of the Chemotaxis chamber is filled with various concentrations of the active peptide obtained in 1. Preparation of peptides (5) above, and the upper part of the cell suspension is made of a chemotactic factor dilution. Put. Subsequently, the chemotaxis of macrophages was measured according to the above 3. Macrophage chemotaxis test (2).
[0050]
9. Macrophage intracellular Ca by laser microscopy 2+ Measurement of fluidity (Ca flux)
Macrophage suspension (1 × 10 6 cells / ml) in the center of Non-Coat 35 mM Dish (manufactured by Matsunami Glass Industry Co., Ltd.), and RPMI-1640 (containing 10% FCS) is added to make a drop. 2 Incubate at 37 ° C. for 24 hours. After confirming that the macrophages adhere to the bottom of the dish, wash twice with RPMI-1640 containing 5% FCS, and again make a drop with RPMI-1640 containing 5% FCS. Next, 5 μl of Fluo-3AM (0.8 mM) dissolved in dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and 5% CO 2 was added. 2 After incubating at 37 ° C. for 1 hour, the plate was washed twice with RPMI-1640 complete medium, and a drop was made again with RPMI-1640 complete medium. The Ca flux was measured using a laser microscope, and when the Fluo-3AM intensity was stabilized, the following 10. In the preparation of ionomycin, EGTA, fMLP or the above-mentioned 1. Peptide preparation (5) 5 μl of the obtained active fraction was added and the change in luminance was graphed.
[0051]
10. Measurement of Ca ion concentration in macrophages
(1) Incorporation of fluorescent dyes into macrophages
Macrophage suspension (1 × 10 6 5 μl of 0.8 mM Fluo-3AM (Molecular Probes) was added to 1 ml of cells / ml) and incubated for 30 minutes in a 37 ° C. thermostat. Thereafter, the mixture was centrifuged (1500 rpm, 5 min, 4 ° C.), and after removing the supernatant, 1 ml of RPMI-1640 containing 5% FCS was added and resuspended.
[0052]
(2) Measurement of negative and positive samples
Measurement of negative sample: The incubated cell suspension was set in FACS, taken up for about 10 seconds, then removed and 6 mM EGTA (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in RPMI-1640 was removed. 500 μl was added and reset quickly. Data acquisition continued during this work.
Measurement of positive sample: Incubated cell suspension was set in FACS, taken up for about 10 seconds, then removed, 10 μl of 700 μM ionomycin (Sigma) dissolved in DMSO was added, and set quickly. Reworked. Data acquisition continued during this work.
[0053]
(3) Measurement of sample data
The active fraction obtained in the above 1. Preparation of peptide (5) is 2 × 10 -1 M, 2 × 10 -2 M, 2 × 10 -3 Serial dilution to M, fMLP 2 × 10 -3 M, 2 × 10 -Four M, 2 × 10 -Five Serial dilution to M. The incubated cell suspension was set in FACS and taken up for about 10 seconds, then removed and 10 μl of each concentration of sample was added and quickly set again. Data acquisition continued during this work.
[0054]
11. Test results
(1) Macrophage chemotaxis to peptides generated by enzymatic degradation
Measurement of macrophage chemotaxis against peptides produced by degradation of β-casein with seven types of enzymes (α-chymotrypsin, pepsin, thermolysin, trypsin, protease K, actinase E, papain) using a 48-well microchemotaxis chamber did. When the PVPF membrane was stained by the Giemsa staining method, degradation products by protease K, actinase E, and papain were stained much darker than in the case of only undegraded β-casein as a control (FIG. 1).
[0055]
When the number of migrating cells per visual field was counted at 200 times magnification using an optical microscope, the degradation products of protease K, actinase E and papain were about 6.8 times and 4.3 times, respectively, compared with the control. A significant difference was observed (Fig. 2). In FIG. 2, C and S indicate the undigested enzyme and the digested enzyme, respectively. * Indicates a significant level of 5% and ** indicates a significant difference of 1%. means. The result is the average number of cells in the HPF10 visual field.
[0056]
(2) Macrophage chemotaxis of various enzymatic degradation peptides by ultrafiltration
(i) The peptide produced when β-casein was decomposed with various enzymes was ultrafiltered using CENTRIPREP3, and only the fraction having a molecular weight of 3000 or less was recovered. The results of subjecting this low molecular weight peptide and the peptide before ultrafiltration to SDS-PAGE are shown in FIG. 3B and FIG. 3A, respectively. 1 and 10 are molecular weight markers, 2 is an α-chymotrypsin digest, 3 is a pepsin digest, 4 is a thermolysin digest, 5 is a trypsin digest, 6 is a protease K digest, 7 is an actinase digest, 8 is Papain digest, 9 indicates undigested β-casein. The bands of peptides formed by degradation by α-chymotrypsin and trypsin were extremely reduced after ultrafiltration. Moreover, the band of the degradation product peptide by actinase E was not detected before and after the ultrafiltration.
[0057]
(ii) Macrophage chemotaxis was measured for fractions having molecular weights of 3000 or less of various enzyme-degrading peptides. As compared with the control, the degradation products of protease K, actinase E and papain were about 5.5 times and 5. The HPF values were 8 times and 5.1 times, and a significant difference was observed in the number of migratory macrophages (FIG. 4). In FIG. 4, an asterisk (*) means that there was a significant difference at a significance level of 5%. The result is the average number of cells in the HPF10 visual field.
[0058]
(3) Chemotaxis of human peripheral blood-derived monosites to β-casein-derived peptides
As shown in the above (1) and (2), induction of chemotaxis by the enzyme degradation product peptide component was observed for mouse-derived macrophages. Next, chemotaxis to human-derived monosites was examined according to the above-described 4. Method for measuring chemotaxis of human peripheral blood monosites. Of the mononuclear cells separated from peripheral blood by HISTPAQUE, 21.6% of monosites were obtained, and monosites separated by magnetic cell separation using the Magnetic Cell Separation System (MACS) were analyzed by FACS CaliburTM flow cytometer. It was confirmed that it was about 97.5% (FIG. 5A). Monosite (Fig. 5B: showing the case of actinase E) stained with a Giemsa staining method and stained with a PVPF membrane at a magnification of 200 times with an optical microscope. As a result, only undegraded β-casein as a control was counted. Compared with the case, a significant difference was observed in the degradation product by papain and actinase E, but the degradation product by papain migrated about 1.9 times the monosite of the control, whereas the degradation product by actinase E. Was about 3.3 times the control, and the strongest chemotaxis was observed (FIG. 5C). In FIG. 5C, the symbol * indicates that there was a significant difference at a significance level of 5%. The result is the average number of cells in the HPF10 visual field.
[0059]
(4) Time-dependent changes in macrophage chemotaxis induction by degradation peptides by actinase E
From the results of (1) to (3) above, it was found that an effective enzyme that induces chemotactic peptides for both mouse-derived macrophages and human-derived monosites is actinase E. Therefore, the time course change of chemotaxis induction in the degradation peptide by actinase E was examined. As a result, as shown in FIG. 6, there was a significant difference in the number of migratory macrophages after 3 hours, about 1.6 times at 3 hours and 6 hours, about 2.5 times at 12 hours, and about 24 times at 24 hours. 3.5-fold macrophages migrated and the chemotaxis activity tended to increase over time. From these results, it was found that actinase E is preferable for the degradation of β-casein and it is preferable to react at 37 ° C. for 24 hours. In the following, the digested material obtained under these conditions was tested.
[0060]
(5) Recovery rate and macrophage chemotaxis test in each fractionation step of β-casein-derived peptide
In order to fractionate β-casein-derived peptides, a product treated with actinase E (reaction product at 37 ° C. for 24 hours) was subjected to ultrafiltration, C 18 Reverse phase chromatography was performed. The recovery after each fractionation is shown in Table 2 in terms of protein content (%), and the chemotaxis of each fraction was also measured (Table 2). As a result, no decrease in activity due to ultrafiltration was observed, and C 18 It was found that the macrophage chemotactic factor was present in the fraction eluted from 30% acetonitrile and 60% acetonitrile in the fraction obtained from reverse phase chromatography. However, the 60% acetonitrile elution fraction had a very low yield of 1.8%, and it was judged preferable to use the 30% acetonitrile elution fraction having a lower acetonitrile concentration.
[0061]
[Table 2]
(6) Fractionation of active peptide components and macrophage chemotaxis test by cation exchange chromatography
C 18 The fraction eluted with 30% acetonitrile obtained by reverse phase chromatography was subjected to cation exchange chromatography. As a result, almost all cation exchange chromatography using phosphate buffer (pH 6.0) was a column pass-through component, and macrophage chemotaxis was measured for each fraction using a 48-well microchemotaxis chamber. Chemotaxis was observed in the column passage fraction (not shown).
[0064]
(7) Fractionation of active peptide components by anion exchange chromatography and macrophage chemotaxis test
C 18 The fraction eluted with 30% acetonitrile obtained by reverse phase chromatography was subjected to anion exchange chromatography. As a result, in anion exchange chromatography using a Tris-hydrochloric acid buffer solution (pH 8.2), it was separated into two peaks of the column adsorption fraction and a column pass-through fraction (the upper part of FIG. 7). When macrophage chemotaxis was measured for each fraction using a 48-well microchemotaxis chamber, no significant activity was observed in the first peak of the anion exchange chromatography flow-through and adsorbed fractions. On the other hand, chemotaxis was observed in the second peak of the adsorbed fraction: fraction numbers (FN) 42 to 46 (lower part of FIG. 7). Hereinafter, the fractions of fraction numbers 42 to 46 are referred to as Q fraction.
[0065]
(8) Analysis, isolation and purification of active ingredients by high performance liquid chromatography (HPLC)
C 18 The 30% acetonitrile elution fraction and Q fraction obtained by reverse phase chromatography were subjected to RP-HPLC. As a result, a multi-peak was detected from the fraction eluted with 30% acetonitrile (the upper part of FIG. 8A). On the other hand, three components (Q1, Q2 and Q3 in the order of the elution time) were detected mainly from the Q fraction, and the ratio was 90.7% for Q1 and 4.0% for Q2 and Q3 (No. 8 (Figure A lower). After fractionation and isolation of each fraction, chemotaxis was measured. As a result, Q2 and Q3 were not measured because of low yield, but Q1 was almost the same as Q fraction. Activity was observed (FIG. 8B).
[0066]
(9) Molecular weight measurement of peptides by fast atom collision mass spectrometry (FAB-MS)
The molecular weights of Q1, Q2, and Q3 collected by high performance liquid chromatography were measured and found to be 604, 523, and 751, respectively.
[0067]
(10) Amino acid sequence analysis of Q1, Q2, Q3
Amino acid analysis was performed on Q1, Q2, and Q3 separated by high performance liquid chromatography using a protein sequencer. This
Q1: Tyr-Pro-Val-Glu -...
Q2: Glu-Met -...
Q3: Tyr-Pro-X-Glu -... (X = Unreadable)
The following sequence was obtained.
[0068]
Furthermore, when compared with the β-casein sequence from the N-terminal amino acid sequence and molecular weight, Q1, Q2, and Q3 are penta-, tetra-, and hexa-derived from 108-119 residues of β-casein as shown below. It was identified as a peptide. The isoelectric points of these three peptides all showed values of 3 to 4, and it is considered that they were not adsorbed on the cation exchange column.
Q1: Tyr-Pro-Val-Glu-Pro (114 to 118 residues MW = 603.6: represented by SEQ ID NO: 1)
Q2: Glu-Met-Pro-Phe (108 to 111 residues MW = 522.6: indicated by SEQ ID NO: 3))
Q3: Tyr-Pro-Val-Glu-Pro-Phe (residues 114 to 119, MW = 522.6: represented by SEQ ID NO: 2)
[0069]
Note that β-casein is known to exhibit heterogeneity, and there are those in which the 117th residue is Gln and the 117th residue is Glu. The obtained Q1 and Q3 are: It was derived from β-peptide whose 117th residue is Glu. Further, when the activity of Q1 having a high yield was measured, macrophage chemotactic activity was not lost even after HPLC fractionation. It is thought that the higher order structure of the protein is deeply involved in the expression of the chemotactic activity (inducibility), and if the HPLC fractionation, the higher order structure of the protein is destroyed by high pressure, so that the chemotaxis induction ability is lost However, it is considered that the obtained peptide was a pentapeptide (YPVEP) consisting of 5 amino acids, and the activity was not lost because the steric structure was difficult to change.
[0070]
(11) Checkerboard analysis of β-casein-derived macrophage chemotactic peptide
In order to confirm whether the activity by Q1 is due to chemical motility or chemotaxis, a checkerboard analysis was performed. As a result, when the peptide concentration in the upper well was higher than that in the lower well, macrophage migration was hardly observed. In contrast, when the peptide concentration in the lower well was higher than that in the upper well, it was confirmed that as many as 19 to 120 macrophages migrated per HPF (FIG. 9). From this, it was confirmed that this peptide induces chemotaxis to macrophages.
[0071]
(12) Intracellular Ca of macrophages 2+ Flowability (Ca flux)
First, intracellular Ca using a Ca indicator (fluo-3AM) with a laser microscope. 2+ The fluidity was measured. FMLP, known as a potent chemotactic peptide, was used as a control for Q1. This is because it was recently revealed that the receptor was a seven-transmembrane type. The activation mechanism in macrophages is that when a chemotactic factor binds to a receptor, Ca ions are released from the storage organ through metabolism of inositol phosphate, and an adhesion factor such as MacI is activated by signaling to migrate. Is considered to start. In recent years, chemotactic factors such as chemokines have been shown to express activity via G protein-related seven-transmembrane receptors. From this measurement, Ca flux was observed in the chemotactic peptide Q1 in a concentration-dependent manner. -3 A prominent peak was obtained at the M concentration (the lower left figure of FIG. 10). On the other hand, no peak was observed in the fMLP used as a control (the lower right diagram in the figure). Therefore, when the cytoplasmic Ca concentration was actually measured by flow cytometry, the intracellular Ca concentration of the cells stimulated with Q1 was high, about 620 nM, and fMLP was about 170 nM. It is comparable and supports the data obtained by a laser microscope (FIG. 11), and β-casochemotide is considered to act on macrophages via a receptor different from fMLP.
[0072]
From the above experimental results, in the peptide group produced by degradation by actinase E, a fraction having a molecular weight of 3000 or less showed strong chemotaxis to both mouse-derived macrophages and human peripheral blood monosites, and undegraded β-casein. Compared with, the number of cells migrated about 5.8 times that of macrophages and about 3.3 times that of human monosites. However, this peptide group could not be detected by SDS-PAGE. This is because actinase E has a strong hydrolyzing power with respect to various substrates, and thus acts on various peptide bonds of proteins. As a result, not only peptide components but also a large amount of free amino acids as proteolytic products It is considered that β-casein was degraded to a very low molecular peptide or amino acid level by actinase E and passed through the gel in the above examples.
[0073]
Macrophage intracellular Ca 2+ When the influence on fluidity (Ca flux) was measured using fMLP, which is known as a potent chemotactic peptide, as a control, the peptide according to the present invention showed fluidity different from that of fMLP. Although there is a report suggesting the possibility that α-casein recognizes the same receptor as fMLP, there is no knowledge about its chemotactic peptide sequence and no report that chemotaxis is expressed through a receptor different from fMLP. The peptide of the invention can be said to be a novel inducer that enhances the chemotaxis of macrophages.
[0074]
Moreover, since β-casochemotide of the present invention is a pentapeptide (YPVEP) consisting of five amino acids, the three-dimensional structure is hardly changed, and the chemotaxis inducing ability is not lost even by HPLC fractionation under high pressure. It can be easily sorted.
[0075]
It has been clarified that the bond of 113 residues (Lys) -114 residues (Tyr) of β-peptide is cleaved by pepsin and trypsin, and the bond of 119 (Phe) -120 (Thr) is cleaved by chymotrypsin. (Yunden Jinsmaa et al., Peputides, 20,957-962 (1997)). Therefore, since the peptide of the present invention, β-casochemotide, is shorter than that, it is presumed that the peptide will not be subjected to further enzymatic degradation and can be sufficiently used for oral administration.
[0076]
In addition, none of the allergens that cause milk allergy, a major concern since ancient times, have the same sequence as this peptide. When rats were injected subcutaneously with various chemotactic factors, fMLP immediately caused an inflammatory reaction and many neutrophils were collected, whereas β-casein showed a very rapid reaction. It is also known that the peptide of the present invention is not harmful to the living body.
[0077]
Thus, the peptide is a novel peptide that induces macrophage mobility and exerts an immunostimulatory function. In order to effectively use this as a food ingredient, it is preferable to add the peptide into food or to digest β-casein in food with a digestive enzyme such as actinase E. In addition, the 118-119 residue bond of β-casein is decomposed in advance with, for example, actinase AS (for example, manufactured by Kaken Pharmaceutical Co., Ltd.) approved by the Ministry of Health, Labor and Welfare as a food additive, and these decomposition products ingested The peptide can also be produced by digestion of.
[Industrial applicability]
[0078]
The present invention provides a novel novel peptide that enhances macrophage chemotaxis. This peptide is very safe because it is obtained by decomposing β-casein contained in foods such as dairy products with a protein digestion enzyme such as actinase E. And the novel functional food using the immunostimulatory function of a food-derived component is provided by using the peptide directly or subjecting the food to an enzyme treatment in advance so as to easily generate the peptide in the digestive tract.
Claims (4)
Tyr−Pro−Val−Glu−ProA novel peptide comprising the following amino acid sequence represented by SEQ ID NO: 1.
Tyr-Pro-Val-Glu-Pro
Tyr−Pro−Val−Glu−ProThe immunostimulant which uses the peptide which consists of the following amino acid sequence shown by sequence number 1 as an active ingredient.
Tyr-Pro-Val-Glu-Pro
Tyr−Pro−Val−Glu−ProA functional food containing a peptide comprising the following amino acid sequence represented by SEQ ID NO: 1 in an amount that substantially exhibits the ability to induce macrophage chemotaxis.
Tyr-Pro-Val-Glu-Pro
Tyr−Pro−Val−Glu−ProFunctional food having a step of treating β-casein in food with actinase so as to include a peptide consisting of the following amino acid sequence represented by SEQ ID NO: 1 in an amount that substantially exhibits macrophage chemotaxis induction ability Manufacturing method.
Tyr-Pro-Val-Glu-Pro
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| JP2003109082 | 2003-04-14 | ||
| PCT/JP2004/005280 WO2004092206A1 (en) | 2003-04-14 | 2004-04-13 | Novel peptide, immunopotentiator and functional food and process for producing functional food |
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| WO2015021530A1 (en) | 2013-08-12 | 2015-02-19 | Mansel Griffiths | Antiviral methods and compositions comprising probiotic bacterial molecules |
| KR20240046640A (en) * | 2017-03-16 | 2024-04-09 | 마이크로신테시스 인크. | Probiotic molecules for reducing pathogen virulence |
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| JPH0789873B2 (en) * | 1987-12-16 | 1995-10-04 | 雪印乳業株式会社 | Process for producing peptide mixture with high leucine content |
| JPH08269090A (en) * | 1995-03-28 | 1996-10-15 | Snow Brand Milk Prod Co Ltd | New peptide |
| AU6919700A (en) * | 1999-08-17 | 2001-03-13 | Regen Therapeutics Plc | Use of colostrinin, constituent peptides thereof, and analogs thereof for inducing cytokines |
| AU4239302A (en) * | 2001-06-28 | 2003-01-02 | Pfizer Products Inc. | Benzoic acid substituted benzopyrans for the treatment of atherosclerosis |
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