JP4647750B2 - Fraction containing high amount of milk basic cystatin and method for producing degradation product thereof - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、乳由来の塩基性タンパク質画分より乳塩基性シスタチン高含有画分を製造する方法に関する。
また、本発明は、得られた乳塩基性シスタチン高含有画分をプロテアーゼで分解することによって乳塩基性シスタチン高含有画分分解物を製造する方法に関する。
さらに、本発明は、これらの乳塩基性シスタチン高含有画分及び／又は乳塩基性シスタチン高含有画分分解物を配合し、必要に応じてカルシウム及び／又はビタミンを配合した骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患並びに歯周病の予防及び改善のための飲食品、医薬又は飼料に関する。
【０００２】
【従来の技術】
近年、高齢化に伴い、骨粗鬆症、骨折及び腰痛等の各種骨疾患が増加している。骨組織においては、絶えず骨形成と骨吸収が営まれており、若い時には骨形成と骨吸収のバランスがとれているが、加齢に伴い種々の原因でそのバランスが骨吸収に傾く。これが、長期間続くと骨組織が脆くなり、骨粗鬆症、骨折及び腰痛等の各種骨疾患を生じることになる。この骨吸収に傾くアンカップリングを防ぐことができれば、各種骨疾患を予防できると考えられている。
従来より、各種骨疾患の予防及び治療の方法として、1)食事によるカルシウムの補給、2)軽い運動、3)日光浴、4)薬物投与等が行われてきた。食事によるカルシウムの補給には、炭酸カルシウム及びリン酸カルシウム等のカルシウム塩や牛骨粉、卵殻及び魚骨粉等の天然カルシウムが用いられている。軽い運動については、軽いランニングや散歩等が良いとされているが、体が弱っていると軽い運動もやっかいなものとなり、ましてや寝たきりの老人になると殆ど運動することができなくなる。３番目の日光浴は、活性化ビタミンＤ3 の補給という点では良いとされているが、これだけでは不十分である。最後の薬物投与においては、１α−ヒドロキシビタミンＤ3 、カルシトニン製剤等が骨粗鬆症の治療及び改善に有効であることが知られている。なお、リウマチ等の骨関節疾患や歯周病も、最終的には骨吸収が起こることから、骨吸収を抑えることによりこれらの改善ができるものと考えられている。
【０００３】
本発明者らは、このような各種骨疾患及び骨関節疾患並びに歯周病の予防及び治療に有効な物質を得るために、乳清タンパク質中の骨芽細胞増殖因子及び骨吸収防止因子並びに骨強化作用を有する画分の探索を続けてきた。すなわち、乳、特に乳清タンパク質を分画し、破骨細胞の骨吸収を抑制する作用を有する画分を分画することを試みた。その結果、乳清タンパク質中の水溶性画分を逆浸透膜や電気透析膜等で処理して脱塩することにより得られるタンパク質及びペプチド混合物に骨強化作用があることを見出した（特開平4-183371号公報) 。そして、このタンパク質及びペプチド混合物の水溶液をエタノール処理、加熱処理、加塩処理、限外濾過膜処理等をして得られる画分に骨強化作用があることを見出した (特開平5-176715号公報、特開平5-320066号公報) 。また、本発明者らは、乳中に微量存在する塩基性タンパク質に骨芽細胞におけるコラーゲン合成促進作用及び骨吸収防止作用があることを見出した（特開平7-207509号公報) 。
【０００４】
一方、シスタチンは、システインプロテアーゼインヒビターとして、活性中心にＳＨ基を持つシステインプロテアーゼのタンパク質分解活性を阻害する物質であり、動物組織、細胞、血液及び尿中に見出されている。また、シスタチンの有用な作用として、ウイルスの増殖阻害作用が確認されている(Biochem. Biophys. Res. Commun., vol.127, p.1072, 1985) 。近年、高齢化の進行に伴い、破骨細胞の骨吸収に起因する骨粗鬆症が急増している。現在、破骨細胞の骨吸収を抑える医薬として、カルシトニン製剤が知られている。しかし、このカルシトニン製剤は、医薬品として使用されるホルモン製剤であり、食品素材として使用することができる安全な物質については、検討がなされていないのが現状である。なお、動物組織、細胞、血液及び尿からは、食品素材として使用することができるシスタチンを大量に得るには至っていない。
【０００５】
【発明が解決しようとする課題】
本発明者らは、骨吸収抑制作用を有する塩基性タンパク質について、骨吸収抑制作用を有する活性本体の分離精製を試み、分離精製した物質を同定したところ、この物質が乳塩基性シスタチンであることを確認した。また、この乳塩基性シスタチンが、乳以外に由来する他の種やタイプのシスタチンに比べて、特異的に破骨細胞の骨吸収を抑制する作用があることも見出した（特願平11-89946号）。
このような現状から、食品素材として使用することができる乳塩基性シスタチンを大量に、しかも安価に製造する方法が求められている。そこで、本発明では、乳由来の塩基性タンパク質画分から塩基性シスタチン高含有画分を製造する方法を提供することを課題とする。
また、乳塩基性シスタチン高含有画分のプロテア−ゼ分解物にも骨吸収抑制作用があることが判っているので、本発明では、乳塩基性シスタチン高含有画分分解物を製造する方法を提供することも課題とする。
さらに、本発明では、乳塩基性シスタチン高含有画分や乳塩基性シスタチン高含有画分分解物を配合した、骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患並びに歯周病の予防及び改善のための飲食品、医薬又は飼料を提供することを課題とする。
【０００６】
【課題を解決するための手段】
本発明は、乳由来の塩基性タンパク質画分を、必要に応じて、好ましくは80℃以上に加熱し、生じた沈殿を除去して上清を回収し、この上清にアルコールを好ましくは濃度70％となるように添加し、生じた沈殿を除去して乳塩基性シスタチン高含有画分を回収し、ヘパリン等の含硫酸基担体と接触させて担体に吸着しない画分を回収し、この画分を分画分子量10〜50kDa の限外濾過膜で処理して透過液を回収して、乳塩基性シスタチン高含有画分を製造することを特徴とする。
また、本発明は、上記の方法で製造された乳塩基性シスタチン高含有画分をさらにプロテアーゼで分解した乳塩基性シスタチン高含有画分分解物を製造することを特徴とする。
さらに、本発明は、上記の方法で製造された乳塩基性シスタチン高含有画分及び／又は乳塩基性シスタチン高含有画分分解物を配合し、さらに必要に応じてカルシウム及び／又はビタミンを配合した、骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患並びに歯周病の予防及び改善のための飲食品、医薬又は飼料を特徴とする。
【０００７】
【発明の実施の形態】
本発明では、乳から安全で、しかも大量に、乳塩基性シスタチン高含有画分を製造することができる。乳としては、生乳、粉乳、脱脂粉乳及び還元乳等が用いられる。
また、本発明の乳塩基性シスタチン高含有画分分解物は、乳塩基性シスタチン高含有画分をトリプシン、キモトリプシン、ペプシン、パパイン、カリクレイン、カテプシン、サーモライシン、Ｖ８プロテアーゼ等のプロテアーゼで限定分解したペプチド混合物である。
さらに、本発明は、このような乳塩基性シスタチン高含有画分及び／又は乳塩基性シスタチン高含有画分分解物を飲食品、医薬、飼料等に配合したものである。すなわち、牛乳、乳飲料、ジュース、ゼリー、ビスケット、パン、麺、ソーセージ等の飲食品に配合すれば良く、錠剤や粉末、あるいは歯磨き剤やうがい薬等の医薬としても良く、飼料に配合しても良い。
【０００８】
なお、本発明の骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患並びに歯周病の予防及び改善のための飲食品、医薬又は飼料には、吸収性の良好なカルシウムを配合することが望ましい。このような吸収性の良好なカルシウムとしては、塩化カルシウム、炭酸カルシウム、乳酸カルシウム、卵殻、あるいは、乳由来のカルシウムを例示することができる。また、ビタミンＤやビタミンＫ等の骨形成に有効なビタミンを配合することが望ましい。これらのカルシウム及びビタミンは、乳塩基性シスタチンと作用機作が異なるため、骨形成に関して相乗的に効果がある。そして、乳塩基性シスタチンは、熱安定性が高く、食品加工上、極めて優れた性質を有するものである。
本発明においては、成人の場合、１日当たり１μg から100mg の乳塩基性シスタチン高含有画分及び／又は乳塩基性シスタチン高含有画分分解物を数回に分けて摂取すれば良い。このようにすることにより、骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患並びに歯周病を予防及び改善することができる。
次に、実施例を挙げて、本発明を詳しく説明する。
【０００９】
【実施例１】
スルホン化キトパール3,000gを充填し、脱イオン水で充分洗浄したカラムに脱脂乳10,000ｌを通液し、脱イオン水で充分カラムを洗浄した後、0.1 〜1.0M塩化ナトリウムの直線濃度勾配で溶出し、凍結乾燥して、乳由来の塩基性タンパク質画分の粉末を得た。この乳由来の塩基性タンパク質画分の粉末200gを濃度１％となるように酢酸緩衝液（pH５）で溶解してヘパリン固定化カラムに通液し、さらに酢酸緩衝液（pH５）20 lを通液して素通り画分40 lを得た。この画分を限外濾過膜処理（50kDa カット）して20倍に濃縮し、その際に得られた透過液を限外濾過膜処理（10kDa カット）して10倍に濃縮し、乳塩基性シスタチン高含有画分を得、これを凍結乾燥して乳塩基性シスタチン高含有画分の粉末60g を得た。なお、この画分中の乳塩基性シスタチン含量は1.8 ％であった。
【００１０】
【実施例２】
スルホン化キトパール3,000gを充填し、脱イオン水で充分洗浄したカラムに脱脂乳10,000ｌを通液し、脱イオン水で充分カラムを洗浄した後、0.1 〜1.0M塩化ナトリウムの直線濃度勾配で溶出し、凍結乾燥して、乳由来の塩基性タンパク質画分の粉末を得た。この乳由来の塩基性タンパク質画分の粉末２kgを５％濃度で溶解し、80℃、15分間加熱して遠心分離により上清を得た。この上清38kgを５℃に冷却した後、最終濃度40％となるようにエタノールを添加し、５℃で６時間後に遠心分離して沈殿を除去した。さらに、この上清に最終濃度70％となるようにエタノールを添加し、５℃で12時間後に遠心分離して乳塩基性シスタチン高含有画分240g（サンプルＡ）を得た。なお、この画分中の乳塩基性シスタチン含量は0.7 ％であった。
この乳塩基性シスタチン高含有画分200gを濃度１％となるように酢酸緩衝液（pH５）で溶解してヘパリン固定化カラムに通液し、さらに酢酸緩衝液（pH５）20 lを通液して素通り画分40 lを得た。この画分を限外濾過膜処理（50kDa カット）して20倍に濃縮し、その際に得られた透過液を限外濾過膜処理（10kDa カット）して10倍に濃縮し、乳塩基性シスタチン高含有画分を得、これを凍結乾燥して乳塩基性シスタチン高含有画分の粉末65g （サンプルＢ）を得た。なお、この画分中の乳塩基性シスタチン含量は2.2 ％であった。
【００１１】
【実施例３】
実施例２で得られた乳塩基性シスタチン高含有画分の粉末１g を水 100mlに懸濁し、最終濃度が１重量％となるようにパンクレアチンを加えて、37℃で５時間酵素処理した。そして、90℃で５分間加熱処理して酵素を失活させた後、凍結乾燥し、乳塩基性シスタチン高含有画分分解物0.91g （サンプルＣ）を得た。
【００１２】
【試験例１】
生後10〜20日齢のＩＣＲ系マウスの長管骨を摘出し、軟組織を除去した後、５％牛胎児血清を含むα−ＭＥＭ溶液中で長管骨を機械的に細切し、破骨細胞を含む全骨髄細胞を得た。この細胞について、約 200万細胞を５％牛胎児血清を含むα−ＭＥＭ溶液で象牙片の上にスポットした。数時間後、サンプルを添加した５％牛胎児血清を含むα−ＭＥＭ溶液を加えて、３日間培養し、破骨細胞の骨吸収活性を調べた。
骨吸収活性の評価は、培養後に象牙片上の細胞を剥がして、ヘマトキシリン染色し、PIASLA-555により画像解析して、骨吸収窩(pit) の数をカウントすることにより行った。
この結果、実施例２及び３で得られたサンプルＡ〜Ｃについて、それぞれ500ng/mlの濃度のサンプル溶液を調製し、骨吸収活性を調べた。すなわち、各サンプルを加えて細胞を培養した時の骨吸収窩(pit) の数をカウントし、サンプルを加えずに細胞を培養した時の骨吸収窩(pit) の数を100 ％として骨吸収活性を表し、表１に示した。
【００１３】
【表１】
─────────────────────
試験試料 骨吸収活性（％，±SD）
─────────────────────
サンプルＡ 81.3±3.9
サンプルＢ 70.4±5.6
サンプルＣ 61.5±3.2
─────────────────────
【００１４】
表１からみると、乳塩基性シスタチン高含有画分及び乳塩基性シスタチン高含有画分分解物に骨吸収活性を抑制する効果があることが判った。
但し、セタノール処理したサンプルＡは本発明方法であるサンプルＢ、Ｃほど骨吸収活性抑制効果はなかった。
【００１５】
【試験例２】
実施例２で得られたサンプルＢ及び実施例３で得られたサンプルＣについて、骨粗鬆症モデルラットを用いた動物実験を行った。
なお、ラットに投与した飼料の基本組成を表２に示す。
飼料中のカルシウム量とリン量は共に全ての群で飼料100g当たり300mg になるように、カルシウム：リン比を１：１とした。
【００１６】
【表２】
──────────────────────────
蔗糖 50.0 （重量％）
カゼイン 20.0
コーンスターチ 15.0
セルロース 5.0
トウモロコシ油 5.0
ビタミン混合（コリン含む） 1.0
ミネラル混合（注１） 4.0
──────────────────────────
1) 炭酸カルシウムをカルシウム源とした。
【００１７】
表２に示した基本組成の飼料に、サンプルＢ又はＣを添加し、以下の試験食を調製した。
試験食１：表２に示した基本組成の飼料＋サンプルＢ（0.01mg／100g）
試験食２：表２に示した基本組成の飼料＋サンプルＣ（0.01mg／100g）
試験食３：表２に示した基本組成の飼料（ただし、カルシウム源として、炭酸カルシウムに代えて乳由来のカルシウム（特開平4-306622号公報）を使用）＋サンプルＢ（0.01mg／100g）
【００１８】
動物は、40週齢のＳＤ系雌性ラットを用いた。骨粗鬆症モデルラットは、１週間予備飼育した後に卵巣摘出手術を施し、低カルシウム食で２ケ月間飼育することにより作成した。その時、疑似手術を施し、卵巣を摘出しないラットも作成した。そして、１試験群７匹で群分けし、各試験食を１ケ月間投与した。なお、擬似手術を施し、卵巣を摘出しないラットを用いた群（シャム群）及び対照群には、表２に示した基本組成の飼料を投与した。
試験食投与後、各試験群のラットの大腿骨を摘出し、骨塩量測定装置で骨塩量を測定し、破断特性測定装置で骨強度を測定した。
試験結果を表３及び表４に示す。
【００１９】
【表３】
────────────────────────
試験群 骨塩量 （mg，±SD）
────────────────────────
シャム群 120.2 ± 3.9
対照群 83.5 ± 4.9
試験食１投与群 104.7 ± 3.6
試験食２投与群 106.3 ± 4.1
試験食３投与群 111.3 ± 3.1
────────────────────────
【００２０】
表３に示したように、大腿骨の骨塩量は、対照群に比べて試験食投与群で統計的に有意に高い値を示した。このことから、サンプルＢ及びＣには骨吸収防止作用があることが判った。また、吸収性の良い乳由来のカルシウムを添加することにより、さらに作用が増強することが判った。
【００２１】
【表４】
──────────────────────
試験群 骨破断力（106dyn）
─────────────────────
シャム群 13.3 ± 3.6
対照群 6.7 ± 2.3
試験食１投与群 10.4 ± 2.9
試験食２投与群 10.9 ± 2.4
試験食３投与群 11.9 ± 3.1
──────────────────────
【００２２】
表４に示したように、骨破断力は、対照群に比べて試験食投与群で統計的に有意に高い値を示した。このことから、サンプルＢ及びＣには骨強化作用があることが判った。また、吸収性の良い乳由来のカルシウムを添加することにより、さらに作用が増強することが判った。
【００２３】
【試験例３】
表５に示した基本組成の水溶液に、サンプルＢ（0.01mg／100g）及びビタミンＤ（200IU ）を添加して混合し、容器に充填した後、加熱滅菌して飲料（試験品）を製造した。
なお、サンプルＢに代えてアルブミン（0.01mg／100g）を添加した飲料（対照品）も同様に製造した。
【００２４】
【表５】
──────────────────────
結晶ブドウ糖 15.0 （重量％）
カルシウム 0.5
水 74.5
──────────────────────
【００２５】
変形性関節症（関節の開裂の収縮）の患者16人を８人ずつ２群に分け、上記各飲料を１ヶ月間飲用してもらい、飲用前と飲用後の骨吸収の骨代謝マーカーである尿中デオキシピリジノリン量を測定した。また、問診により自覚症状を確認した。
試験結果を表６及び表７に示す。
【００２６】
【表６】
表６に示すように、カルシウム及びビタミンを配合した対照品投与群でもデオキシピリジノリン量が減少したものの、試験品投与群ではさらに大きく減少した。このことから、サンプルＢにより、骨の破壊による骨吸収が極めてよく抑制されていることが判った。
【００２８】
【表７】
表７に示すように、関節痛についても、各項目について痛みが軽減していることが判る。
【００３０】
【試験例４】
６週齢のゴールデンハムスターを１週間予備飼育した後、エーテル麻酔下でM1の歯頸部に滅菌した手術用縫合絹糸No４を５重に巻き付け、Keyes らの飼料（D #2000:Keyes,P.H. and Jordan:Archs. Oral.Biol,vol.9,pp.377-400,1964) で飼育することにより、歯周病を発病させた。ハムスター18匹を１試験群とし、サンプルＡ、Ｂ又はＣ10μｇを適当に希釈した試験液で、１日２回、口腔内を約10分間絶えず浸す処置を行った。なお、対照群は、蒸留水で処置を行った。処置開始４週間後に、2.5 ％グルタルアルデヒド溶液（pH7.4 ）を用いて約20分間固定灌流した後、下顎骨両側を摘出した。
歯槽骨減少量の評価は、2.5 ％グルタルアルデヒド溶液で固定した後、軟Ｘ線撮影を行った写真を画像解析装置（PIAS LA-555)で解析し、M1付近のエナメルセメント境と歯槽骨頂間の面積を計測して、歯槽骨減少量を評価した。
その試験結果を表８に示す。
【００３１】
【表８】
表８によると、対照群に比べて試験群では明らかに歯槽骨減少量が有意に低く、かつこの効果は濃度依存的であった。このことから、乳塩基性シスタチン高含有画分及びその分解物は、歯槽骨の減少を抑制し、歯周病予防に効果的であることが判る。
【００３３】
【実施例４】
表９に示した組成で各成分を混合し、容器に充填した後、加熱滅菌して、骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患の予防及び改善用の飲料を製造した。
【００３４】
【表９】
────────────────────────
混合異性化糖 15.00 （重量％）
果汁 10.00
クエン酸 0.50
サンプルＢ 0.01
香料 0.10
カルシウム 0.50
ビタミンＤ (200IU)
水 73.89
───────────────────────
【００３５】
【実施例５】
表１０に示した組成で各成分を混合し、加圧成型して、骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患の予防及び改善用の錠剤を製造した。
【００３６】
【表１０】
────────────────────────
含水結晶ブドウ糖 93.5（重量％）
サンプルＢ 0.1
カルシウム 5.0
ビタミンＤ （200IU)
シュガーエステル 1.0
香料 0.4
────────────────────────
【００３７】
【実施例６】
表１１に示した組成で各成分を混合し、容器に充填した後、加熱殺菌して、骨粗鬆症等の各種骨疾患及びリウマチ等の各種骨関節疾患の予防及び改善用のゼリーを製造した。
【００３８】
【表１１】
────────────────────
果糖 20.00 （重量％）
グラニュー糖 15.00
水飴 5.00
寒天 1.00
サンプルＣ 0.01
香料 0.10
カルシウム 0.10
水 58.79
────────────────────
【００３９】
【実施例７】
表１２に示した組成で各成分を混合し、乳化温度85℃で、骨粗鬆症等の各種骨疾患及びリウマチ等の各種骨関節疾患の予防及び改善用のチーズを製造した。
【００４０】
【表１２】
─────────────────────
ゴーダチーズ 43.0（重量％）
チェダーチーズ 43.5
クエン酸ナトリウム 2.0
サンプルＣ 0.1
乳由来のカルシウム 1.0
水 10.4
─────────────────────
【００４１】
【実施例８】
表１３に示した組成で各成分を混合し、骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患の予防及び改善用のイヌ飼育用飼料（ドッグフード）を製造した。
【００４２】
【表１３】
────────────────────
大豆粕 12.0 （重量％）
脱脂粉乳 14.0
大豆油 4.0
コーン油 2.0
パーム油 27.9
トウモロコシ澱粉 15.0
小麦粉 9.0
ふすま 2.0
ビタミン混合物 9.0
ミネラル混合物 2.0
セルロース 3.0
サンプルＢ 0.1
────────────────────
【００４３】
【実施例９】
表１４に示した組成で各成分を混合し、クリームを調製した。そして、このクリームを容器に充填して、歯周病予防及び改善用の歯磨き剤を製造した。
【００４４】
【表１４】
──────────────────────────
グリセリン 70.49 （重量％）
二酸化ケイ素 20.00
キサンタンガム 1.00
ミントフレーバー 1.00
二酸化チタン 0.70
フッ化ナトリウム 0.30
蒸留水 6.50
サンプルＣ 0.01
──────────────────────────
【００４５】
【発明の効果】
本発明で得られる乳塩基性シスタチン高含有画分やこれをプロテアーゼで分解した乳塩基性シスタチン高含有画分分解物は、骨形成促進及び骨吸収抑制作用を有しているので、これらを飲食品、医薬及び飼料等に配合することによって、骨粗鬆症等の各種骨疾患及びリウマチ等の骨関節疾患並びに歯周病の予防及び改善に有効に利用し得る。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a milk basic cystatin-rich fraction from a milk-derived basic protein fraction.
The present invention also relates to a method for producing a milk basic cystatin-rich fraction degradation product by decomposing the obtained milk basic cystatin-rich fraction with a protease.
Furthermore, the present invention provides various kinds of bones such as osteoporosis in which these milk basic cystatin-rich fractions and / or milk basic cystatin-rich fractions are blended, and calcium and / or vitamins are blended as necessary. The present invention relates to foods and beverages, medicines or feeds for the prevention and improvement of diseases and bone and joint diseases such as rheumatism and periodontal diseases.
[0002]
[Prior art]
In recent years, various bone diseases such as osteoporosis, broken bones, and back pain are increasing with aging. In bone tissue, bone formation and bone resorption are constantly carried out. When young, bone formation and bone resorption are balanced, but the balance tends to bone resorption due to various causes with aging. If this continues for a long period of time, the bone tissue becomes brittle, and various bone diseases such as osteoporosis, fractures, and back pain occur. It is considered that various bone diseases can be prevented if this uncoupling that is inclined to bone resorption can be prevented.
Conventionally, as methods for preventing and treating various bone diseases, 1) calcium supplementation with meals, 2) light exercise, 3) sunbathing, 4) drug administration, and the like have been performed. For calcium supplementation by meals, calcium salts such as calcium carbonate and calcium phosphate and natural calcium such as cow bone meal, eggshell and fish bone meal are used. For light exercise, light running and walking are said to be good, but if the body is weak, light exercise becomes troublesome, and even if you are a bedridden elderly person, you can hardly exercise. A third sun bath is considered good in terms of supplementing activated vitamin D 3 , but this alone is not sufficient. In the last drug administration, it is known that 1α-hydroxyvitamin D 3 , calcitonin preparation and the like are effective for the treatment and improvement of osteoporosis. It should be noted that bone joint diseases such as rheumatism and periodontal diseases are also considered to be able to be improved by suppressing bone resorption because bone resorption eventually occurs.
[0003]
In order to obtain substances effective for the prevention and treatment of such various bone diseases and osteoarticular diseases and periodontal diseases, the present inventors have developed osteoblast growth factors and bone resorption prevention factors in whey proteins and bones. We have continued to search for fractions that have a reinforcing effect. That is, it tried to fractionate milk, especially whey protein, and fraction which has the effect | action which suppresses the bone resorption of an osteoclast. As a result, it was found that the protein and peptide mixture obtained by treating and desalting the water-soluble fraction in whey protein with a reverse osmosis membrane, an electrodialysis membrane or the like has a bone strengthening action (Japanese Patent Laid-Open No. 4). -183371). And, it was found that a fraction obtained by subjecting an aqueous solution of the protein and peptide mixture to ethanol treatment, heat treatment, salting treatment, ultrafiltration membrane treatment, etc. has a bone strengthening action (Japanese Patent Laid-Open No. 5-177715). JP-A-5-320066). Further, the present inventors have found that a basic protein present in a trace amount in milk has an effect of promoting collagen synthesis in osteoblasts and an effect of preventing bone resorption (Japanese Patent Laid-Open No. 7-207509).
[0004]
On the other hand, cystatin, as a cysteine protease inhibitor, is a substance that inhibits the proteolytic activity of cysteine protease having an SH group at the active center, and is found in animal tissues, cells, blood and urine. In addition, as a useful action of cystatin, an inhibitory action on virus growth has been confirmed (Biochem. Biophys. Res. Commun., Vol. 127, p. 1072, 1985). In recent years, with the progress of aging, osteoporosis caused by osteoclastic bone resorption has increased rapidly. Currently, calcitonin preparations are known as drugs that suppress bone resorption of osteoclasts. However, this calcitonin preparation is a hormonal preparation used as a pharmaceutical product, and the present situation is that a safe substance that can be used as a food material has not been studied. In addition, cystatin which can be used as a food material has not been obtained in large quantities from animal tissues, cells, blood and urine.
[0005]
[Problems to be solved by the invention]
The inventors of the present invention have tried to isolate and purify an active substance having a bone resorption inhibitory action on a basic protein having a bone resorption inhibitory action, and identified the separated and purified substance. As a result, the substance is milk basic cystatin. It was confirmed. It was also found that this milk basic cystatin has an action of specifically inhibiting osteoclast bone resorption compared to other species and types of cystatin derived from non-milk (Japanese Patent Application No. 11- 89946).
Under such circumstances, there is a demand for a method for producing a large amount of milk basic cystatin that can be used as a food material at low cost. Therefore, an object of the present invention is to provide a method for producing a fraction containing a high amount of basic cystatin from a milk-derived basic protein fraction.
In addition, since it is known that the protease degradation product of the milk basic cystatin-rich fraction also has a bone resorption inhibitory action, the present invention provides a method for producing a milk basic cystatin-rich fraction degradation product. Providing is also an issue.
Furthermore, in the present invention, various bone diseases such as osteoporosis, osteoarthritis diseases such as rheumatism, and periodontal disease can be prevented and improved by blending a fraction containing milk basic cystatin and a fraction containing a fraction containing milk basic cystatin. It is an object of the present invention to provide food, beverage, medicine, or feed for food.
[0006]
[Means for Solving the Problems]
In the present invention, the milk-derived basic protein fraction is preferably heated to 80 ° C. or higher as necessary, the resulting precipitate is removed and the supernatant is recovered, and alcohol is preferably added to the supernatant. It is added to 70%, the resulting precipitate is removed and a fraction containing a high amount of milk basic cystatin is recovered, and a fraction not adsorbed on the carrier is recovered by contacting with a sulfate-containing carrier such as heparin. The fraction is treated with an ultrafiltration membrane having a fractional molecular weight of 10 to 50 kDa, and the permeate is collected to produce a fraction containing a high amount of milk basic cystatin.
In addition, the present invention is characterized by producing a milk basic cystatin-rich fraction-decomposed product obtained by further decomposing a milk basic cystatin-rich fraction produced by the above-described method with a protease.
Furthermore, the present invention contains a milk basic cystatin-rich fraction and / or a milk basic cystatin-rich fraction decomposed product produced by the above method, and calcium and / or vitamins if necessary. It is characterized by foods, beverages, medicines or feeds for the prevention and improvement of various bone diseases such as osteoporosis and bone and joint diseases such as rheumatism and periodontal disease.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, a fraction containing a high amount of milk basic cystatin can be produced in a safe and large amount from milk. As milk, raw milk, powdered milk, skim milk powder, reduced milk and the like are used.
Further, the fraction containing a high amount of milk basic cystatin according to the present invention is a peptide obtained by limited decomposition of a milk basic cystatin high content fraction with a protease such as trypsin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, or V8 protease. It is a mixture.
Furthermore, this invention mix | blends such a milk basic cystatin high content fraction and / or a milk basic cystatin high content fraction decomposition product in food-drinks, a pharmaceutical, feed. In other words, it can be added to foods and drinks such as milk, milk drinks, juice, jelly, biscuits, bread, noodles, sausages, etc., and it can also be used as pharmaceuticals such as tablets and powders, toothpaste and mouthwash, etc. Also good.
[0008]
In addition, various bone diseases such as osteoporosis of the present invention, osteoarthritis diseases such as rheumatism, and food and drink, medicine or feed for the prevention and improvement of periodontal disease, it is desirable to mix calcium with good absorbability . Examples of such calcium having good absorbability include calcium chloride, calcium carbonate, calcium lactate, eggshell, or calcium derived from milk. Moreover, it is desirable to mix vitamins effective for bone formation such as vitamin D and vitamin K. These calcium and vitamins have a synergistic effect on bone formation due to the different mechanism of action from milk basic cystatins. Milk basic cystatin has high heat stability and has extremely excellent properties in food processing.
In the present invention, in the case of an adult, 1 μg to 100 mg of a milk basic cystatin-rich fraction and / or a milk basic cystatin-rich fraction degradation product may be taken in several portions per day. By doing in this way, various bone diseases, such as osteoporosis, bone joint diseases, such as rheumatism, and periodontal disease can be prevented and improved.
Next, an example is given and the present invention is explained in detail.
[0009]
[Example 1]
The column was packed with 3,000 g of sulfonated chitopearl, and 10,000 l of skimmed milk was passed through a column that had been thoroughly washed with deionized water. After thoroughly washing the column with deionized water, elution was performed with a linear concentration gradient of 0.1 to 1.0 M sodium chloride. And lyophilized to obtain a milk-derived basic protein fraction powder. 200 g of this milk-derived basic protein fraction powder is dissolved in acetate buffer (pH 5) to a concentration of 1%, passed through a heparin-immobilized column, and further passed through 20 l of acetate buffer (pH 5). A liquid fraction of 40 l was obtained. This fraction was concentrated 20 times by ultrafiltration membrane treatment (50 kDa cut), and the permeate obtained at that time was concentrated 10 times by ultrafiltration membrane treatment (10 kDa cut). A fraction containing a high content of cystatin was obtained, and this was freeze-dried to obtain 60 g of a powder containing a high content of milk basic cystatin. The milk basic cystatin content in this fraction was 1.8%.
[0010]
[Example 2]
The column was packed with 3,000 g of sulfonated chitopearl, and 10,000 l of skimmed milk was passed through a column that had been thoroughly washed with deionized water. After thoroughly washing the column with deionized water, elution was performed with a linear concentration gradient of 0.1 to 1.0 M sodium chloride. And lyophilized to obtain a milk-derived basic protein fraction powder. A 2 kg powder of this milk-derived basic protein fraction was dissolved at a concentration of 5%, heated at 80 ° C. for 15 minutes, and centrifuged to obtain a supernatant. After cooling 38 kg of the supernatant to 5 ° C., ethanol was added to a final concentration of 40%, and the mixture was centrifuged at 5 ° C. for 6 hours to remove precipitates. Further, ethanol was added to the supernatant to a final concentration of 70%, and the mixture was centrifuged at 5 ° C. for 12 hours and then centrifuged to obtain a milk basic cystatin-rich fraction 240 g (sample A). The milk basic cystatin content in this fraction was 0.7%.
200 g of this milk basic cystatin-rich fraction is dissolved in an acetate buffer solution (pH 5) to a concentration of 1%, passed through a heparin-immobilized column, and further passed through 20 l of acetate buffer solution (pH 5). As a result, a passing fraction of 40 l was obtained. This fraction was concentrated 20 times by ultrafiltration membrane treatment (50 kDa cut), and the permeate obtained at that time was concentrated 10 times by ultrafiltration membrane treatment (10 kDa cut). A fraction containing a high content of cystatin was obtained, and this was freeze-dried to obtain 65 g of a powder containing a high content of milk basic cystatin (sample B). The milk basic cystatin content in this fraction was 2.2%.
[0011]
[Example 3]
1 g of the powder of the milk basic cystatin-rich fraction obtained in Example 2 was suspended in 100 ml of water, pancreatin was added to a final concentration of 1% by weight, and the enzyme treatment was performed at 37 ° C. for 5 hours. Then, the enzyme was inactivated by heat treatment at 90 ° C. for 5 minutes, and then freeze-dried to obtain 0.91 g (sample C) of a degradation product containing a high amount of milk basic cystatin.
[0012]
[Test Example 1]
After removing the long bones of ICR mice 10 to 20 days old and removing soft tissues, the long bones were mechanically cut in an α-MEM solution containing 5% fetal calf serum, and osteoclasts were obtained. Whole bone marrow cells containing cells were obtained. About 2 million cells were spotted on ivory pieces with an α-MEM solution containing 5% fetal calf serum. Several hours later, an α-MEM solution containing 5% fetal calf serum to which the sample was added was added, and the mixture was cultured for 3 days, and the bone resorption activity of osteoclasts was examined.
Evaluation of bone resorption activity was performed by peeling cells on ivory pieces after culture, staining with hematoxylin, image analysis with PIASLA-555, and counting the number of bone resorption pits.
As a result, a sample solution having a concentration of 500 ng / ml was prepared for each of the samples A to C obtained in Examples 2 and 3, and the bone resorption activity was examined. That is, the number of bone resorption pits when cells were cultured with each sample added, and the number of bone resorption pits when cells were cultured without adding samples was taken as 100%. The activity is shown in Table 1.
[0013]
[Table 1]
─────────────────────
Test sample Bone resorption activity (%, ± SD)
─────────────────────
Sample A 81.3 ± 3.9
Sample B 70.4 ± 5.6
Sample C 61.5 ± 3.2
─────────────────────
[0014]
From Table 1, it was found that the milk basic cystatin-rich fraction and the milk basic cystatin-rich fraction degradation product have an effect of suppressing bone resorption activity.
However, the sample A treated with cetanol was not as effective in suppressing bone resorption activity as the samples B and C of the present invention.
[0015]
[Test Example 2]
For the sample B obtained in Example 2 and the sample C obtained in Example 3, an animal experiment using an osteoporosis model rat was performed.
Table 2 shows the basic composition of the feed administered to rats.
The calcium: phosphorus ratio was 1: 1 so that the amount of calcium and phosphorus in the feed was 300 mg per 100 g of feed in all groups.
[0016]
[Table 2]
──────────────────────────
Sucrose 50.0 (wt%)
Casein 20.0
Cornstarch 15.0
Cellulose 5.0
Corn oil 5.0
Vitamin mix (including choline) 1.0
Mineral mixing (Note 1) 4.0
──────────────────────────
1) Calcium carbonate was used as the calcium source.
[0017]
Sample B or C was added to the feed having the basic composition shown in Table 2 to prepare the following test meals.
Test meal 1: feed of the basic composition shown in Table 2 + sample B (0.01 mg / 100 g)
Test meal 2: Feed with the basic composition shown in Table 2 + Sample C (0.01 mg / 100 g)
Test meal 3: Feed having the basic composition shown in Table 2 (however, calcium derived from milk (Japanese Patent Laid-Open No. 4-306622) is used instead of calcium carbonate as a calcium source) + sample B (0.01 mg / 100 g)
[0018]
The animal used was a 40-week-old SD female rat. An osteoporosis model rat was prepared by pre-breding for 1 week, then performing an oophorectomy and breeding on a low calcium diet for 2 months. At that time, a sham operation was performed to create a rat that did not remove the ovaries. Then, each group was divided into 7 groups per test group, and each test meal was administered for 1 month. In addition, the feed of the basic composition shown in Table 2 was administered to the group (sham group) and the control group using rats that had undergone sham surgery and did not remove ovaries.
After administration of the test meal, the femurs of the rats of each test group were removed, the bone mineral content was measured with a bone mineral content measurement device, and the bone strength was measured with a fracture characteristic measurement device.
The test results are shown in Tables 3 and 4.
[0019]
[Table 3]
────────────────────────
Test group Bone mineral content (mg, ± SD)
────────────────────────
Siamese group 120.2 ± 3.9
Control group 83.5 ± 4.9
Test meal 1 administration group 104.7 ± 3.6
Test meal 2 administration group 106.3 ± 4.1
Test meal 3 administration group 111.3 ± 3.1
────────────────────────
[0020]
As shown in Table 3, the bone mineral content of the femur was statistically significantly higher in the test food administration group than in the control group. From this, it was found that Samples B and C have a bone resorption preventing action. It was also found that the action was further enhanced by adding calcium derived from milk having good absorbability.
[0021]
[Table 4]
──────────────────────
Test group Bone breaking force (10 6 dyn)
─────────────────────
Siamese group 13.3 ± 3.6
Control group 6.7 ± 2.3
Test meal 1 administration group 10.4 ± 2.9
Test meal 2 administration group 10.9 ± 2.4
Test meal 3 administration group 11.9 ± 3.1
──────────────────────
[0022]
As shown in Table 4, the bone breaking force was statistically significantly higher in the test meal administration group than in the control group. From this, it was found that Samples B and C have a bone strengthening action. It was also found that the action was further enhanced by adding calcium derived from milk having good absorbability.
[0023]
[Test Example 3]
Sample B (0.01 mg / 100 g) and vitamin D (200 IU) were added to the aqueous solution having the basic composition shown in Table 5, mixed, filled into a container, and then heat sterilized to produce a beverage (test product). .
In addition, it replaced with the sample B and the drink (control product) which added albumin (0.01 mg / 100g) was manufactured similarly.
[0024]
[Table 5]
──────────────────────
Crystalline glucose 15.0 (wt%)
Calcium 0.5
Water 74.5
──────────────────────
[0025]
It is a bone metabolism marker for bone resorption before and after drinking, with 16 patients with osteoarthritis (joint cleavage contraction) divided into 2 groups of 8 and each drink given above for 1 month The amount of deoxypyridinoline in urine was measured. In addition, subjective symptoms were confirmed through an interview.
The test results are shown in Tables 6 and 7.
[0026]
[Table 6]
As shown in Table 6, although the amount of deoxypyridinoline was reduced in the control product administration group containing calcium and vitamin, it was further greatly reduced in the test product administration group. From this, it was found that sample B significantly suppressed bone resorption due to bone destruction.
[0028]
[Table 7]
As shown in Table 7, it can be seen that also the joint pain is reduced for each item.
[0030]
[Test Example 4]
After pre-feeding a 6-week-old golden hamster for 1 week, sterilized surgical suture silk No4 was wrapped around the M1 tooth neck under ether anesthesia five times, and the feed from Keyes et al. (D # 2000: Keyes, PH and Periodontal disease was caused by rearing in Jordan: Archs. Oral. Biol, vol. 9, pp. 377-400, 1964). Eight hamsters were used as one test group, and a treatment in which the oral cavity was continuously immersed for about 10 minutes twice a day with a test solution in which 10 μg of sample A, B or C was appropriately diluted was performed. The control group was treated with distilled water. Four weeks after the start of the treatment, fixed perfusion was performed for about 20 minutes using a 2.5% glutaraldehyde solution (pH 7.4), and then both sides of the mandible were removed.
The amount of alveolar bone loss was evaluated by fixing with a 2.5% glutaraldehyde solution and then analyzing the soft X-ray photograph using an image analyzer (PIAS LA-555). The enamel cement boundary near M1 and the alveolar crest Was measured to evaluate the amount of alveolar bone loss.
The test results are shown in Table 8.
[0031]
[Table 8]
According to Table 8, the amount of alveolar bone loss was clearly lower in the test group than in the control group, and this effect was concentration dependent. From this, it can be seen that the milk-basic cystatin-rich fraction and the degradation product thereof are effective in preventing periodontal disease by suppressing the decrease in alveolar bone.
[0033]
[Example 4]
Each component was mixed with the composition shown in Table 9, filled into a container, and then heat sterilized to produce beverages for prevention and improvement of various bone diseases such as osteoporosis and osteoarthritis diseases such as rheumatism.
[0034]
[Table 9]
────────────────────────
Mixed isomerized sugar 15.00 (wt%)
Fruit juice 10.00
Citric acid 0.50
Sample B 0.01
Fragrance 0.10
Calcium 0.50
Vitamin D (200IU)
Wed 73.89
───────────────────────
[0035]
[Example 5]
Each component was mixed with the composition shown in Table 10, and it pressure-molded, and manufactured the tablet for prevention and improvement of various bone diseases, such as osteoporosis, and osteoarthritis diseases, such as rheumatism.
[0036]
[Table 10]
────────────────────────
Water-containing crystal glucose 93.5 (wt%)
Sample B 0.1
Calcium 5.0
Vitamin D (200IU)
Sugar Ester 1.0
Fragrance 0.4
────────────────────────
[0037]
[Example 6]
Each component was mixed with the composition shown in Table 11, filled into a container, and then heat sterilized to produce a jelly for prevention and improvement of various bone diseases such as osteoporosis and various bone and joint diseases such as rheumatism.
[0038]
[Table 11]
────────────────────
Fructose 20.00 (wt%)
Granulated sugar 15.00
Minamata 5.00
Agar 1.00
Sample C 0.01
Fragrance 0.10
Calcium 0.10
Wed 58.79
────────────────────
[0039]
[Example 7]
Each component was mixed with the composition shown in Table 12, and the cheese for prevention and improvement of various bone diseases, such as osteoporosis, and various bone joint diseases, such as rheumatism, was manufactured at the emulsification temperature of 85 degreeC.
[0040]
[Table 12]
─────────────────────
Gouda cheese 43.0 (wt%)
Cheddar cheese 43.5
Sodium citrate 2.0
Sample C 0.1
Calcium from milk 1.0
Wed 10.4
─────────────────────
[0041]
[Example 8]
Each component was mixed with the composition shown in Table 13 to produce a dog breeding feed for preventing and ameliorating various bone diseases such as osteoporosis and osteoarthritis diseases such as rheumatism.
[0042]
[Table 13]
────────────────────
Soybean cake 12.0 (wt%)
Nonfat dry milk 14.0
Soybean oil 4.0
Corn oil 2.0
Palm oil 27.9
Corn starch 15.0
Flour 9.0
Bran 2.0
Vitamin mixture 9.0
Mineral mixture 2.0
Cellulose 3.0
Sample B 0.1
────────────────────
[0043]
[Example 9]
Each component was mixed with the composition shown in Table 14 to prepare a cream. And this cream was filled in the container, and the dentifrice for periodontal disease prevention and improvement was manufactured.
[0044]
[Table 14]
──────────────────────────
Glycerin 70.49 (wt%)
Silicon dioxide 20.00
Xanthan gum 1.00
Mint flavor 1.00
Titanium dioxide 0.70
Sodium fluoride 0.30
Distilled water 6.50
Sample C 0.01
──────────────────────────
[0045]
【The invention's effect】
The milk basic cystatin-rich fraction obtained by the present invention and the milk basic cystatin-rich fraction degradation product obtained by decomposing this with a protease have a bone formation promoting and bone resorption inhibiting action. It can be effectively used for the prevention and improvement of various bone diseases such as osteoporosis, osteoarthritis diseases such as rheumatism, and periodontal disease by blending with products, medicines and feeds.
Claims (3)
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000184349A JP4647750B2 (en) | 2000-06-20 | 2000-06-20 | Fraction containing high amount of milk basic cystatin and method for producing degradation product thereof |
| US09/876,267 US6649590B2 (en) | 2000-06-09 | 2001-06-06 | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| NZ515948A NZ515948A (en) | 2000-06-09 | 2001-06-07 | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| CA2349980A CA2349980C (en) | 2000-06-09 | 2001-06-07 | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| NZ512182A NZ512182A (en) | 2000-06-09 | 2001-06-07 | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| EP01112980.6A EP1161881B1 (en) | 2000-06-09 | 2001-06-08 | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| AU51853/01A AU784087B2 (en) | 2000-06-09 | 2001-06-08 | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| EP05017629A EP1602284A1 (en) | 2000-06-09 | 2001-06-08 | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000184349A JP4647750B2 (en) | 2000-06-20 | 2000-06-20 | Fraction containing high amount of milk basic cystatin and method for producing degradation product thereof |
| US09/876,267 US6649590B2 (en) | 2000-06-09 | 2001-06-06 | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2002000193A JP2002000193A (en) | 2002-01-08 |
| JP4647750B2 true JP4647750B2 (en) | 2011-03-09 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2008214237A (en) * | 2007-03-02 | 2008-09-18 | Snow Brand Milk Prod Co Ltd | Rankl production inhibitor |
| JP5736533B2 (en) * | 2007-07-19 | 2015-06-17 | 学校法人北里研究所 | Peptide pet food material with anti-stress action and palatability improvement effect |
| WO2011037957A2 (en) * | 2009-09-24 | 2011-03-31 | U.S. Foods & Pharmaceuticals, Inc. | Compositions and methods for bone repair, maintenance and regeneration |
| MY167580A (en) * | 2012-07-31 | 2018-09-20 | Megmilk Snow Brand Co Ltd | Novel fermented milk product and method for producing the same |
| BR112015002053A2 (en) * | 2012-07-31 | 2017-07-04 | Megmilk Snow Brand Co Ltd | cheese, methods of preventing bone disease, and of producing cheese |
| US20150343031A1 (en) * | 2012-07-31 | 2015-12-03 | Megmilk Snow Brand Co., Ltd. | Beverage, and method of producing the same |
| CA2879995C (en) * | 2012-07-31 | 2019-09-03 | Megmilk Snow Brand Co., Ltd. | Novel powdered milk product and method for producing the same |
| CA2879959C (en) * | 2012-07-31 | 2017-03-14 | Megmilk Snow Brand Co., Ltd. | Novel protein material |
| JP2016152809A (en) * | 2016-04-05 | 2016-08-25 | 雪印メグミルク株式会社 | Beverage and manufacturing method therefor |
| JP6562958B2 (en) * | 2017-02-28 | 2019-08-21 | 雪印メグミルク株式会社 | Fermented milk and method for producing the same |
| JP6357265B2 (en) * | 2017-06-07 | 2018-07-11 | 雪印メグミルク株式会社 | Protein material for bone disease prevention or treatment |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09191858A (en) * | 1996-01-23 | 1997-07-29 | Snow Brand Milk Prod Co Ltd | Basic protein composition, basic peptide composition and use thereof |
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| JP4592127B2 (en) * | 1999-03-30 | 2010-12-01 | 雪印乳業株式会社 | Bone resorption inhibitor |
| JP2001346519A (en) * | 2000-06-09 | 2001-12-18 | Snow Brand Milk Prod Co Ltd | Method for producing fraction containing high content of milk basic cystatin and decomposed product thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09191858A (en) * | 1996-01-23 | 1997-07-29 | Snow Brand Milk Prod Co Ltd | Basic protein composition, basic peptide composition and use thereof |
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