【０００１】
【発明の属する技術分野】
本発明は、酵母細胞壁画分を有効成分とするミネラル吸収促進剤及び該酵母細胞壁画分を有効成分とする骨粗鬆症の予防及び／又は改善剤に関する。
【０００２】
【従来の技術】
人類は加齢に伴い、体内カルシウム分布・濃度の調節能が変化することで骨組織における骨形成と骨吸収のバランスが崩れ始めるが、昨今、生活様式が機械化され身体への負担が軽減されているにも関わらず、足腰の衰えからくる疾患により病院を訪れる者の人数は増加の一途をたどっている。近年、老齢人口の増加に伴い、寝たきり老人や痴呆老人の治療及び介護が大きな社会問題となっているが、これらの大きな要因は、骨粗鬆症からくる腰の曲がりや痛み、それが原因として起こる骨折のしやすさと言われている。
【０００３】
骨は１８歳くらいまでに形成され、その後２０歳代から３０歳代にかけて強靭な骨形成が進むが、世代を問わず女性の社会進出が一層盛んとなっている現在では、女性のライフスタイルの乱れにより女性ホルモンの分泌に異常をきたし、その結果、生理不順や骨密度の低下等が誘引される。また、女性は妊娠や授乳により大量のカルシウムが胎児に供給されるため、骨に十分な量のカルシウムを貯えておく必要がある。女性は５０歳頃から閉経を迎え、それと同時に女性ホルモンの分泌が著しく減少することで、骨量および骨密度が急激に減少する。従来から、骨量や骨密度の減少を抑制するためには、運動による刺激、食品からの十分なカルシウム及びマグネシウムの摂取、腸管からのカルシウム吸収を促進する活性型ビタミンＤの補給等の、骨が減少する原因を改善していくことが重要と言われてきた。しかしながら、個々のライフスタイルや時間的な制約から常日頃から運動を継続すること、または、食事によりカルシウム、マグネシウム等のミネラル及び活性型ビタミンＤを効率良く摂取し続けることは困難である。
【０００４】
一方、従来、酵母又は酵母菌体構成成分を有効成分とする薬理用組成物に関する技術としては、特許公報に多くのものが開示されている。例えば、酵母を有効成分とする妊娠維持、流産の防止や胎児の発育促進に有用な組成物（特開平４−３０５５３０号公報）や、酵母菌体破砕液の遠心分離上清をプロテアーゼ処理し加熱後沈殿を除去することで得られる、酵母由来リボ核酸のミネラル吸収促進作用及びその製造方法（特開平６−２８７１４１号公報）や、ミネラル強化酵母を含有することを特徴とする高機能性食品（特開２０００−２５３８５１号公報）や、流動食中に酵母、特にミネラル成分、好ましくはその微量元素を高含量に含む酵母を配合した流動性等の物性に優れた流動食（特開２００１−４６０１６号公報）や、酒酵母クロムを２０〜５０質量％含有するミネラル含有組成物又は治療用組成物（特開２００２−３３２２３６号公報）や、亜鉛含有量の多い乾燥酵母と、塩化ナトリウム、塩化カリウム、硫酸マグネシウム、炭酸マグネシウム、卵殻カルシウムとを含む塩と、必要に応じてＬ−グルタミン酸塩とを含有させる食品添加用組成物（特開２００２−３３０７２７号公報）が開示されている。
【０００５】
また、ミネラル強化酵母を懸濁液中で熱水抽出して得られるミネラル分を含有する栄養補給用食品（特開２００２−３３５９１３号公報）や、ミネラルの供給素材として、好ましくは粒径４μｍ以下の粒子の割合が５０％以上となるよう、酵母の細胞を破砕したミネラル高含有酵母の噴霧乾燥物を含有することを特徴とする経口経管栄養組成物（特開２００２−３００８６１号公報）や、マンガン、銅又はモリブデンをミネラル成分とするミネラル酵母を含有する食品組成物（特開２００３−１９８号公報）が開示されている。しかしながら、これらの技術の手段はいずれも、酵母構成成分にミネラルの栄養源を求めたに留まり、ミネラル以外の酵母構成成分のミネラル吸収促進作用に関するものではない。また、酵母に含まれるミネラル分を濃縮した食品素材及びミネラル補給剤に関する技術が開示されているが（特開平９−２０５号公報）、ミネラル吸収促進または骨粗鬆症予防等の有効性については判定されておらず、実際に摂取した場合の効果の如何は不明である。
【０００６】
更に、酵母から抽出した酵母エキスに含まれるリボ核酸を摂取することでミネラル吸収促進作用を期待した技術が開示されているが（特開平５−２９２９１６号公報）、試験管内での擬似モデルによりミネラル吸収能を評価しているのみであり、摂取されたリボ核酸の生体内における動態、すなわち、胃や小腸における消化、分解及び吸収等の作用については全く考慮していないことから、ミネラル吸収促進作用の発現に重要となる消化管内におけるカルシウムの可溶化作用が十分に引き起こされ、ひいては、骨形成の促進が生じるかについては不明である。
【０００７】
一方、酵母細胞壁を有効成分として薬理活性組成物として利用する技術としては、生酵母を自己消化またはプロテアーゼ等の酵素処理により細胞内の可溶性エキスを除去したエキス抽出残渣の酵母細胞壁をミネラル吸収促進剤として、鉄分の吸収に利用する技術が開示されている（特開２００２−２５５８３２号公報）。しかしながら、該公報に開示されたものは、酵母細胞壁のラットへの投与により鉄のみかけの吸収率（％）が対照に比べ上昇したとする結果を示しているものの、試験群間の値に有意差を認めてはいない。また、鉄欠乏性貧血ラットに乾燥酵母、酵母細胞壁及び酵母エキスを投与することで、それらのヘモグロビン再生率（％）及びヘモグロビン増加（ｇ／Ｌ）を比較しているが、酵母細胞壁は対照に対しては有意な効果があるという結果であるものの、乾燥酵母との間には有意差を認めておらず、さらには、酵母細胞壁とともに酵母エキスも混合し投与している。したがって、該公報の記載からは、酵母細胞壁に特異な効果を確認することができないものである。
【０００８】
【特許文献１】
特開平４−３０５５３０号公報。
【特許文献２】
特開平５−２９２９１６号公報。
【特許文献３】
特開平６−２８７１４１号公報。
【特許文献４】
特開平９−２０５号公報。
【特許文献５】
特開２０００−２５３８５１号公報。
【特許文献６】
特開２００１−４６０１６号公報。
【特許文献７】
特開２００２−２５５８３２号公報。
【特許文献８】
特開２００２−３３２２３６号公報。
【特許文献９】
特開２００２−３３０７２７号公報。
【特許文献１０】
特開２００２−３３５９１３号公報。
【特許文献１１】
特開２００２−３００８６１号公報。
【特許文献１２】
特開２００３−１９８号公報。
【非特許文献１】
Ｂｉｏｓｃｉｅｎｃｅ， Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， ａｎｄ Ｂｉｏｃｈｅｍｉｓｔｒｙ， ｖｏｌ．６２， ｐ８３７， １９９８。
【０００９】
【発明が解決しようとする課題】
本発明の課題は、酵母細胞壁画分を有効成分とする安全な且つ効力の優れたミネラル吸収促進剤及び該酵母細胞壁画分を有効成分とする骨粗鬆症の予防及び／又は改善剤を提供することにある。
【００１０】
近年、食物繊維などの難消化性成分を摂取することで、消化管におけるミネラル吸収が亢進されるという報告がなされている（Ｋ． Ｓａｉｔｏ ｅｔ ａｌ．， Ｂｉｏｓｃｉｅｎｃｅ， Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， ａｎｄ Ｂｉｏｃｈｅｍｉｓｔｒｙ， ｖｏｌ．６２， ｐ８３７， １９９８）。摂取された難消化性成分は、大腸で腸内細菌により資化され発酵産物である短鎖脂肪酸へと変換されるが、この生成された短鎖脂肪酸は速やかに腸管から吸収され、大腸のエネルギー源となって大腸機能の正常化、活性化へ貢献すると言われている。かかる短鎖脂肪酸産生の促進素材として、ビフィズス菌や乳酸菌などの腸内細菌や、その成長促進因子であるオリゴ糖及び水可溶性食物繊維が挙げられるが、ビフィズス菌や乳酸菌などの腸内細菌を摂取しても、大腸へ達する前に胃酸の影響によりそのほとんどが死滅し、短鎖脂肪酸の高産生には寄与しないという問題があり、また、オリゴ糖を摂取すると、腸内常在細菌に資化されて短鎖脂肪酸を産生するものの、食物繊維のような膨潤性に乏しく、腸内細菌との接触性や消化管への物理的刺激に欠けることから、短鎖脂肪酸産生や腸管上皮細胞の代謝回転促進という観点から問題があった。
【００１１】
また、オリゴ糖は通常甘味を有していることから、食品に配合して使用する場合、その食品の本来の風味を損ねてしまうという弊害があった。更に、オリゴ糖や水可溶性食物繊維の摂取によっては、下痢や腹痛の発症といった副作用の表れる問題があった。したがって、食物繊維などの難消化性成分を摂取することで、消化管におけるミネラル吸収が亢進されるという報告はあるものの、その効果的な解決策が見い出されていないのが現状である。
現在、食生活や身体の生理的バランスから骨密度の減少、骨粗鬆症の問題が増大しており、それらを原因とする骨折など現代人が抱える特有の疾病に対する予防・治療法の開発が期待されている。特に、健康食品のような形での活性物質の摂取は、副作用が少なく簡便であることから、このような疾病の予防・治療法として関心が高まってきている。
【００１２】
本発明の課題は、これらのニーズに応えるもので、高齢者の骨量減少抑制及び骨粗鬆症の予防を始めとして、骨粗鬆症患者の症状改善、一般女性や妊婦の骨量及び骨密度の維持改善を目的として、ミネラルの吸収を効果的に促進させ、更には、骨密度の増加及び骨粗鬆症予防並びに症状の改善に有効な、副作用が少なく安全な、水への分散性が高く、より摂取しやすい素材としてのミネラル吸収促進剤及び骨粗鬆症の予防及び／又は改善剤、更には骨粗鬆症用薬理活性組成物を提供することにある。
【００１３】
【課題を解決するための手段】
本発明者は、上記課題を解決すべく、ミネラルの吸収を促進し、骨密度の増加や骨粗鬆症の予防及び／又は改善に有効であり、且つ飲食のような形で安全に摂取できる物質について、鋭意探索した結果、酵母菌体をアルカリ処理後水洗浄、物理的粉砕処理及び可溶性菌体成分の除去処理を行うことにより得られる酵母細胞壁画分が、安全な且つ効力の優れたミネラル吸収促進剤としての作用があり、また、酵母菌体をアルカリ処理後水洗浄、及び可溶性菌体成分の除去処理を行うことにより得られる酵母細胞壁画分が、骨粗鬆症の予防及び／又は改善剤として有効であることを見い出し、本発明を完成するに至った。
すなわち、本発明は、酵母菌体をアルカリ処理後水洗浄、物理的粉砕処理及び可溶性菌体成分の除去処理を行うことにより得られる酵母細胞壁画分を有効成分とするミネラル吸収促進剤、及び酵母菌体をアルカリ処理後水洗浄、及び可溶性菌体成分の除去処理を行うことにより得られる酵母細胞壁画分を有効成分とする骨粗鬆症の予防及び／又は改善剤からなるものである。本発明における物理的粉砕処理は、高圧ホモジナイザーや超音波発生器のような粉砕処理によって行われる。
【００１４】
骨密度の増加や骨粗鬆症の予防及び／又は改善に有効な物質を探索する中で、ミネラルの吸収を促進し、且つ、飲食のような形で安全に摂取できる物質としては、食物繊維のようなものが考えられる。体に、摂取された食物繊維は消化管内において腸内細菌により分解を受け、発酵産物として酢酸、プロピオン酸及び酪酸などの短鎖脂肪酸が産生される。大腸内の短鎖脂肪酸産生に伴い、大腸の粘膜側に短鎖脂肪酸が豊富に存在することで大腸におけるミネラル吸収が亢進されることが明らかとなっている。この作用は、短鎖脂肪酸によるｐＨの低下及び浸透圧の上昇という物理的な影響から、大腸内にあるミネラルの可溶化率が上昇することで、大腸の粘膜側から管腔側へ通過するミネラル量が増加することが要因の一つと考えられている。したがって、より発酵性の高い食物繊維を摂取することが、大腸におけるミネラル吸収の促進効果を高める重要なポイントとなる（Ｃ． Ｄｅｍｉｇｎｅ ｅｔ ａｌ．， Ｊｏｕｒｎａｌ ｏｆ Ｎｕｔｒｉｔｉｏｎ， ｖｏｌ．１１９， ｐ１６２５， １９８９）。
【００１５】
本発明者らは、このような視点にたって、鋭意探索の結果、酵母の細胞内成分である酵母エキスを除去して得られる酵母細胞壁画分についての研究過程において、酵母細胞壁画分が水不溶性食物繊維を含有するにも関わらず、水への分散性、膨潤性に優れ、また摂取後の大腸における腸内細菌による資化性が高く、他の食物繊維素材に比べてより多くの短鎖脂肪酸を産生させる作用を持つことを見い出した。そこで、酵母細胞壁画分の薬理作用について種々検討を重ねたところ、酵母菌体をアルカリ処理後水洗浄し、更に可溶性菌体成分を除去するか、或いは可溶性菌体成分を除去した酵母菌体をアルカリ処理後水洗浄して得た酵母細胞壁画分を、高圧ホモジナイザーのような物理的粉砕処理を施して得られる酵母細胞壁画分が、消化管内における腸内細菌の短鎖脂肪酸産生の資化基質となる食物繊維含量を高め、しかも、自己消化による特有の異味異臭のない、摂取に適した酵母細胞壁画分となることを見い出し、本発明をなした。
【００１６】
すなわち具体的には本発明は、酵母菌体をアルカリ処理後水洗浄、物理的粉砕処理及び可溶性菌体成分の除去処理を行うことにより得られる酵母細胞壁画分を有効成分とするミネラル吸収促進剤（請求項１）や、物理的粉砕処理が高圧ホモジナイザー処理によって行われることを特徴とする請求項１記載のミネラル吸収促進剤（請求項２）や、溶性菌体成分の除去処理が、自己消化及び／又は酵素処理によって行われることを特徴とする請求項１又は２記載のミネラル吸収促進剤（請求項３）や、酵母菌体をアルカリ処理後水洗浄、及び可溶性菌体成分の除去処理を行うことにより得られる酵母細胞壁画分を有効成分とする骨粗鬆症の予防及び／又は改善剤（請求項４）や、酵母菌体をアルカリ処理後水洗浄、物理的粉砕処理及び可溶性菌体成分の除去処理を行うことにより得られる酵母細胞壁画分を有効成分とする骨粗鬆症の予防及び／又は改善剤（請求項５）や、物理的粉砕処理が高圧ホモジナイザー処理によって行われることを特徴とする請求項５記載の骨粗鬆症の予防及び／又は改善剤（請求項６）や、可溶性菌体成分の除去処理が、自己消化及び／又は酵素処理によって行われることを特徴とする請求項４〜６のいずれか記載の骨粗鬆症の予防及び／又は改善剤（請求項７）からなる。
【００１７】
【発明の実施の形態】
本発明は、酵母菌体をアルカリ処理後水洗浄し、更に可溶性菌体成分を除去するか、或いは可溶性菌体成分を除去した酵母菌体をアルカリ処理後水洗浄して得た酵母細胞壁画分を、高圧ホモジナイザーや超音波発生器のような物理的粉砕処理を施して得られる酵母細胞壁画分を有効成分とするミネラル吸収促進剤、及び、酵母菌体をアルカリ処理後水洗浄、及び可溶性菌体成分の除去処理を行うことにより得られる酵母細胞壁画分を有効成分とする骨粗鬆症の予防及び／又は改善剤からなるものである。
【００１８】
（原料酵母）
本発明の酵母細胞壁画分の原料となる酵母としては、分類学上酵母に属するもので可食性の酵母であれば特に制限はなく、例えば、ビール醸造工程の副生成物であるビール酵母の他、ワイン酵母、パン酵母、トルラ酵母、アルコール酵母、清酒用酵母などを挙げることができ、より具体的には、ビール酵母、パン酵母の属するサッカロマイセス属のサッカロマイセス・セレビッシェ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）或いはサッカロマイセス・パストリアヌス（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｐａｓｔｏｒｉａｎｕｓ）、その他サッカロマイセス・ルーキシ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｒｏｕｘｉｉ）、サッカロマイセス・カールスバーゲンシス（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃａｒｌｓｂｅｒｇｅｎｓｉｓ）、サッカロマイセス・ポンベ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ）、またメタノール資化性酵母であるキャンディダ属のキャンディダ・ウティリス（Ｃａｎｄｉｄａ ｕｔｉｌｉｓ）、キャンディダ・トロピカリス（Ｃａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓ）、キャンディダ・リポリティカ（Ｃａｎｄｉｄａ ｌｉｐｏｌｙｔｉｃａ）、キャンディダ・フレーベリ（Ｃａｎｄｉｄａ ｆｌａｖｅｒｉ）、キャンディダ・ボイジニィ（Ｃａｎｄｉｄａ ｂｏｉｄｉｎｉｉ）等を、さらにロドトルラ・ミニュータ（Ｒｈｏｄｏｔｒｕｒａ ｍｉｎｕｔａ）等を例示することができる。そして、これら酵母は、単独あるいは組み合わせて使用することができる。また、酵母としては生酵母を用いることが好ましいが、乾燥酵母等の生酵母以外の形態の酵母を用いる場合であっても、例えば水中等に懸濁して生酵母同様に処理することもできる。さらに、使用する酵母の形状や大きさに特に制限はなく、その大きさは１〜２０μｍの範囲のものが好ましい。
【００１９】
（酵母細胞壁画分の調製）
本発明において用いる酵母細胞壁画分の調製には、酵母菌体をアルカリ処理後水洗浄し、更に可溶性菌体成分を除去するか、或いは可溶性菌体成分を除去した酵母菌体をアルカリ処理後水洗浄することにより、酵母菌体から例えば蛋白質、アミノ酸、核酸などの水又は極性溶剤に可溶性の菌体成分のような可溶性菌体成分が除去された酵母細胞壁画分を調製することにより行う。酵母菌体から可溶性菌体成分を除去するには、通常酵素処理により酵母菌体を溶菌して可溶性菌体成分を菌体外に分離・除去することにより行われる。かかる酵素処理方法としては、酵母菌体の酵素を使用するいわゆる自己消化法や、外部からプロテアーゼ、ヌクレアーゼ、グルカナーゼ、エステラーゼなどの酵素を添加する酵素添加法や、それらを併用する方法などを例示することがでる。かかる酵素処理酵母菌体から、可溶性菌体成分を遠心分離などの除去処理を施すことによって酵母細胞壁画分を得ることができる。上記例示の酵素処理方法は、いずれも酵母菌体内成分を酵母エキスとして製造する際に用いる方法であることからして、製造コストの点を考慮すると、酵母細胞壁画分として、酵母エキス製造における副生成物である酵母エキス抽出残渣を利用することが有利である。
【００２０】
本発明で用いる酵母細胞壁画分の調製を、酵母エキス製造における副生成物である酵母エキス抽出残渣を利用して行う場合の該酵母エキスの製造方法としては、例えば、特開平８−５６６１１号公報に示される、加熱失活させた酵母の水懸濁液を酵素で分解処理した後、キトサンまたはキトサンとポリアクリル酸塩を添加して水不溶物の除去を行い、酵母エキス水溶液を得る酵母エキスの製造方法や、特開平９−５６３６１号公報に示される、高圧ホモジナイザー処理した酵母菌体懸濁液を中性〜弱アルカリ性に調整し、該酵母菌体懸濁液にエンド型プロテアーゼを含む酵素剤を添加し、自己消化させる酵母エキスの製造方法や、特開平１１−３３２５１１号公報に示される、原料となる酵母菌体を、加温処理した後に、核酸分解酵素の含まれる条件下で、かつ５´−ヌクレオチドを分解する酵素活性が抑制される条件下で菌体成分を消化し抽出する酵母エキスの製造法などが挙げられる。また、これらの技術以外にも、公知の酵母エキスの製造における酵母エキス抽出残渣を有効に用いることができる。
【００２１】
本発明で用いる酵母細胞壁画分の調製に際しては、酵母菌体又は酵母エキス抽出残渣を、生理活性効果の増大及び臭い等の物性の改善のために、アルカリ処理後、水洗浄処理を行う。例えば、酵母細胞壁画分として、酵母菌体又は酵母エキス抽出残渣を、アルコール処理及び／又はオゾン処理することなく、水洗浄又はアルカリ処理後水洗浄、若しくは水洗浄又はアルカリ処理後水洗浄に加え、本発明にしたがって、高圧ホモジナイザー処理等のような物理粉砕処理を行うことにより、より優れたミネラル吸収促進作用及び骨粗鬆症の予防や症状改善効果を奏するばかりでなく、自己消化による特有の異味異臭がなく、摂取に適した酵母細胞壁画分を得ることができる。かかるアルカリ処理後の水洗浄処理としては、酵母エキス抽出工程においてスラリー状の酵母菌体をアルカリ処理後水洗浄し、酵母菌体から得られた酵母エキス抽出残渣をさらにアルカリ処理し、その後水洗浄処理することが好ましいが、酵母菌体あるいは酵母エキス抽出残渣のいずれか一方に対してアルカリ処理後水洗浄を行ってもよい。
【００２２】
上記スラリー状の酵母菌体のアルカリ処理としては、例えば、固形分濃度を５〜２０重量％、好ましくは８〜１２重量％、より好ましくは約１０重量％に調整した酵母菌体スラリーに、そのｐＨが８〜１２、好ましくは９〜１０となるように水酸化ナトリウムを添加し、０〜２０℃、好ましくは０〜１０℃での攪拌処理を挙げることができる。また、かかるアルカリ処理後の水洗浄としては、通常の水洗浄方法を用いることができ、アルカリ処理後の菌体を遠心分離機等で脱水した後に行うことが洗浄効率の点からして好ましく、かかる洗浄工程は複数回行うこともできる。また、上記酵母エキス抽出残渣のアルカリ処理としては、例えば、固形分濃度を５〜２０重量％、好ましくは８〜１２重量％、より好ましくは約１０重量％に調整した酵母エキス抽出残渣スラリーに、そのｐＨが８〜１２、好ましくは９〜１０となるように水酸化ナトリウムを添加し、０〜７０℃、好ましくは０〜５０℃、より好ましくは１０〜３０℃での攪拌処理を挙げることができる。
【００２３】
また、かかるアルカリ処理後の水洗浄としては、通常の水洗浄方法を用いることができ、アルカリ処理後の酵母エキス抽出残渣を遠心分離機等で脱水した後に行うことが洗浄効率の点からして好ましく、かかる洗浄工程は複数回行うこともできる。エタノール処理、オゾン処理、酸処理を行うことなく、このようなアルカリ処理後水洗浄処理により、異味異臭原因物質が簡便かつ低コストで除去することができ、単独で摂取する場合はもちろん、他の食品素材と混合使用する場合であっても、かかる食品素材の風味を損なうことがない無味無臭の酵母細胞壁画分を得ることができる。
【００２４】
本発明においては、酵素処理を速やかに行うとともに、より効力の優れた酵母細胞壁画分を得るために、酵素処理前や上記アルカリ処理前の酵母菌体に、物理粉砕処理（高圧ホモジナイザーなどにより細胞壁の物理的破壊を伴う前処理）を施す。物理粉砕処理として高圧ホモジナイザー（例えばＨｉｇｈ ｐｒｅｓｓｕｒｅ ｈｏｍｏｇｅｎｉｚｅｒ、ＡＰＶ製）や超音波発生器（例えばＵｌｔｒａｓｏｎｉｃｓ、Ｔｅｌｓｏｎｉｃｓ製）による粉砕処理等が挙げられる。このうち、高圧ホモジナイザーを用いる前処理は、例えば１００〜１０００ｋｇ／ｃｍ２の圧力下冷却しながら行うことが望ましい。さらには、酵母細胞壁画分の食物繊維含量を高めることを目的として、アルカリ処理後水洗浄後の酵母菌体に、高圧ホモジナイザーなどにより細胞壁の物理的破壊を伴う後処理を行うこともできる。この高圧ホモジナイザーを用いる後処理は、例えば１００〜１０００ｋｇ／ｃｍ２の圧力下冷却しながら行うことが望ましい。このような物理処理は、その効力の増大とともに、酵母菌体の分散性向上（食感の向上）や洗浄性の向上（異味異臭物質除去性の向上）を得る目的で、酵素処理後や上記アルカリ処理後の酵母菌体に施すことも出来る。
【００２５】
（薬理活性組成物）
本発明において酵母細胞壁画分を有効成分とするミネラル吸収促進剤及び骨粗鬆症の予防及び／又は改善剤は、薬理活性組成物として摂取することができる。
ここで、薬理活性組成物とは、酵母細胞壁画分を単独又は酵母細胞壁画分と他の成分若しくは素材との混合物からなり、酵母細胞壁画分の有する薬理作用の対象となる疾病に対する予防及び／又は症状改善用の製剤、並びに飲食品素材に配合した飲食品としての形態をとった飲食可能な組成物をいう。
本発明における、ミネラル吸収促進効果、骨密度上昇効果及び骨密度減少抑制効果を有する酵母細胞壁画分は、一般に消化管内でクロム、鉄等の３価のカチオンやカルシウム、マグネシウム、マンガン、モリブデン、セレン、鉄、銅、亜鉛等の２価のカチオンや、ナトリウム、カリウム等の１価のカチオンを始めとしたミネラルの可溶化率を向上させるために有効であるといわれている、短鎖脂肪酸の産生基質となりうる食物繊維などの難消化性成分を豊富に含有することが必要であるとともに、その難消化性成分は消化管内において腸内細菌により良好に発酵されることが好ましい。さらに、本発明に用いられる酵母細胞壁画分に含まれる食物繊維成分は６０％以上であることが好ましい。
【００２６】
本発明に用いられる酵母細胞壁画分に含有される食物繊維には、主にβ−１，３−グルカン、β−１，６−グルカン、及びα−１，６−マンナンから構成されるものであり、その中でも主体をなすβ−１，３−グルカンとβ−１，６−グルカンが豊富に含有されることで消化管内における高い発酵性が保証されるため、本発明における酵母細胞壁画分にはより多くのβ−グルカンが含有されることが好ましい。さらに、本発明に用いられる酵母細胞壁画分に含まれるβ−グルカンは、通常の酸加水分解法により酵母細胞壁画分を分解した後、高速液体クロマトグラフィーを用いた絶対検量線法により容易に測定することが可能であるが、該画分に含まれるβ−グルカン量は、４１％以上であることが好ましく、より好ましくは、４９％以上であることが好ましい。なお、上記用途剤としての効果を発揮するものであれば、酵母菌体をそのまま物理破砕処理することにより得られる酵母細胞壁画分でも構わない。
【００２７】
本発明の酵母細胞壁画分はミネラルの吸収促進剤、骨粗鬆症の予防、症状改善素材として、ヨーグルト、ドリンクヨーグルト、ジュース、牛乳、豆乳、酒類、コーヒー、紅茶、緑茶、煎茶、玉露、ウーロン茶、ウコン茶、プーアル茶、ジャスミン茶、スポーツ飲料、ミネラルウオーター等の各種飲料や、プリン、クッキー、パン、ケーキ、ドーナツ、ゼリーなどの洋菓子、煎餅、羊羹、大福、おはぎ、カステラなどの和菓子、冷菓、チューインガム等のパン・菓子類や、うどん、そば等の麺類や、かまぼこ、ハム、魚肉ソーセージ等の魚肉練り製品や、ハム、ソーセージ、ハンバーグ等の畜肉製品や、みそ、しょう油、ソース、ドレッシング、マヨネーズ、甘味料等の調味類や、たこ焼き、お好み焼き、焼きそば等の鉄板焼き食品や、豆腐、こんにゃく、その他漬物、佃煮、餃子、コロッケ、サンドイッチ、ピザ、ハンバーガー、サラダ等の各種総菜へ配合して飲食品として使用することで、より本発明の効果を発揮でき、特に、咀嚼力が減退するとともに、歯の状態が悪化した高齢者、食事内容に過敏な妊婦、体重増加の気になる女性のＱＯＬ（ｑｕａｌｉｔｙ ｏｆ ｌｉｆｅ）の改善に貢献することができる。
【００２８】
【実施例】
以下に、本発明を実施例により詳細に説明するが、本発明は以下の実施例によって限定されるものではない。なお、特にことわらない限り、実施例中に示された酵母菌体重量は全て実状態での重量（ドライウエイト）である。なお、これより以下、カルシウムは「Ｃａ」と表記する。
【００２９】
調製例１
ビール醸造工程より副生成物として得られる、発酵後ビール酵母スラリーの重量を精確に量った後、固形分が１０重量％になるように加水した。水酸化ナトリウムをｐＨ９となるまで添加し、１０℃で攪拌処理を行った後、遠心分離を行い、沈殿画分に加水して洗浄後、再度遠心分離を行った。固形分が１０重量％になるように加水した懸濁物を５０℃、１７時間の反応条件で自己消化させた後、遠心分離して、可溶性菌体成分を除去した自己消化残渣を酵母細胞壁画分とした。この酵母細胞壁画分のタンパク質含量は２３．７％、食物繊維含量（ＡＯＡＣ法）は６０．３％であった。
【００３０】
調製例２
ビール醸造工程より副生成物として得られる、発酵後ビール酵母スラリーの重量を精確に量った後、固形分が１０重量％になるように加水した。水酸化ナトリウムをｐＨ９となるまで添加し、１０℃で攪拌処理を行った後、遠心分離を行い、沈殿画分に加水して洗浄後、再度遠心分離を行った。固形分が１０重量％になるように加水した懸濁物を７００ｋｇ／ｃｍ２の圧力下冷却しながら高圧ホモジナイザー処理を行った後、懸濁物を５０℃、７時間の反応条件で自己消化させた後、さらにプロテアーゼを添加し５０℃で１８時間酵素反応を行った後、遠心分離を行い可溶性菌体成分を除去し、ここで得られる自己消化・酵素反応残渣を酵母細胞壁画分とした。この酵母細胞壁画分のタンパク質含量は２１．３％、食物繊維含量（ＡＯＡＣ法）は６２．７％であった。
【００３１】
調製例３
ビール醸造工程より副生成物として得られる、発酵後ビール酵母スラリーの重量を精確に量った後、固形分が１０重量％になるように加水した。水酸化ナトリウムをｐＨ９となるまで添加し、１０℃で攪拌処理を行った後、遠心分離を行い、沈殿画分に加水して洗浄後、再度遠心分離を行った。固形分が１０重量％になるように加水した懸濁物を７００ｋｇ／ｃｍ２の圧力下冷却しながら高圧ホモジナイザー処理を行った後、懸濁物を５０℃、７時間の反応条件で自己消化させた後、さらにプロテアーゼを添加し５０℃で１８時間酵素反応を行った後、遠心分離を行い可溶性菌体成分を除去した。この画分の固形分が１０重量％になるように加水して洗浄後に遠心分離を行う操作を２度繰り返し、ここで得られる沈殿画分を酵母細胞壁画分とした。この酵母細胞壁画分のタンパク質含量は１４．０％、食物繊維含量（ＡＯＡＣ法）は６７．３％であった。
【００３２】
調製例４
ビール醸造工程より副生成物として得られる、発酵後ビール酵母スラリーの重量を精確に量った後、固形分が１０重量％になるように加水した。水酸化ナトリウムをｐＨ９となるまで添加し、１０℃で攪拌処理を行った後、遠心分離を行い、沈殿画分に加水して洗浄後、再度遠心分離を行った。固形分が１０重量％になるように加水した懸濁物を７００ｋｇ／ｃｍ２の圧力下冷却しながら高圧ホモジナイザー処理を行った後、懸濁物を５０℃、７時間の反応条件で自己消化させた後、さらにプロテアーゼを添加し５０℃で１８時間酵素反応を行った後、遠心分離を行い可溶性菌体成分を除去した。得られた自己消化・酵素反応残渣に加水して洗浄後、再度遠心分離を行った後、固形分が１０重量％になるように加水した懸濁物を７００ｋｇ／ｃｍ２の圧力下冷却しながら高圧ホモジナイザー処理を行い酵母細胞壁画分とした。この酵母細胞壁画分の成分分析値を表１に示す。
【００３３】
【表１】
【００３４】
試験例１
調製例２で得られた酵母細胞壁画分（以下「本発明品１」という）の水中での膨潤能と、他の代表的な食物繊維素材の膨潤能との比較を行うため、消化管内を人工的に再現した環境下における水中沈定体積を測定した。サンプルとして本発明品１の他、市販のセルロース、小麦ふすま、コーンファイバー、ビートファイバー、及び酵母細胞壁（田辺製薬株式会社製「イムセルＢＦ」、以下「比較例品」という）を用い、これら各１ｇをそれぞれ１００ｍＬメジューム瓶にとり、１／１５Ｍリン酸緩衝液（Ｎａ２ＨＰＯ４を４．７ｇ、ＫＨ２ＰＯ４を４．５ｇとり、蒸留水を加え１Ｌに定容、ｐＨ６．８）を５０ｍＬ加えて攪拌した。超音波処理及び脱気処理を１分間行い、さらに超音波処理を３分間継続して行った後、１００ｍＬメスシリンダーに移し、上記緩衝液を加えて１００ｍＬに定容した。２４時間静置後、各サンプルの沈定体積（ｍＬ／ｇ）の測定を行った。結果を表２に示す。表２からもわかるように、本発明品１は他の代表的な食物繊維素材及び類似の酵母細胞壁素材である比較例品に比べて、水中での高い膨潤能を有することが判明した。以上のことから、骨密度上昇及び減少抑制に顕著な効果を発揮する本発明品は、水不溶性食物繊維を含有するにも関わらず、膨潤性、すなわち、水への分散性が極めて高いことが明らかとなり、より摂取しやすい素材としての薬理用組成物を提供されることが可能であることを確認した。
【００３５】
【表２】
【００３６】
試験例２
調製例３で得られた酵母細胞壁画分（以下「本発明品２」という）の、健常ラットを用いた消化管からのＣａ吸収及び骨密度に与える影響についての実験を行った。供試動物として、Ｗｉｓｔａｒ系雄性ラット（７週齢、１５０〜１７０ｇ）を用い、試験飼料投与前の１週間、表３に示す対照群の組成飼料で予備飼育を行い、実験環境への馴化を行った後、これらラットを各群８匹ずつに区分けして使用した。供試飼料（被検サンプル）としては、表３に組成が示されている調製例３により調製された本発明品２群、食物繊維成分を配合しない対照群及び酵母細胞壁画分と等量の酵母細胞壁（イムセルＢＦ）を含む比較例品群を用い、供試飼料は自由摂取にてラットに与え、２週間飼育した。なお、本発明品２又は比較例品のタンパク質含量及び脂質含量を測定することで、飼料中のタンパク質含量及び脂質含量が均等になるようカゼイン及び大豆油の配合量を補正した。供試飼料の投与２週間後、糞便、右側大腿骨及び腹部大動脈血の採取を行い、糞便排泄量、糞便中Ｃａ排泄量及び血清中Ｃａ濃度の測定を行った。採取した糞便は直ちに凍結乾燥を行った後、乾燥重量を測定し、採取した血液は直ちに遠心分離を行い血清を分取した。また、大腿骨については、骨密度及び骨塩量をｐＱＣＴ法にて測定した。
【００３７】
骨は大きく分けて皮質骨と海綿骨に分かれるが、代謝回転は内部にある海綿骨のほうが速く、中心部の海綿骨がもろくなることで骨全体の強度も低下するため、海綿骨の骨密度を高めることが骨粗鬆症予防に重要なポイントの一つとなることから、総骨密度、海綿骨骨密度、全骨塩量及び海綿骨骨塩量を指標として効果を判定した。結果を表４に示す。本発明で得られた本発明品の摂取により、糞便中へのＣａ排泄量が有意に減少したことから、Ｃａの体内への吸収率が向上したことが判明した。さらに、総骨密度、海綿骨骨密度、全骨塩量及び海綿骨骨塩量の比較においては、本発明品２群が他の２群に比べ有意に高い値となった。以上のことから、本発明品２には骨密度上昇効果のあることが明らかとなり、これは、本発明品を始めとした食物繊維成分を摂取しない場合、もしくは、比較例品を摂取した場合に比べて顕著に高い効果であった。
【００３８】
【表３】
【００３９】
【表４】
【００４０】
試験例３
調製例４で得られた酵母細胞壁画分（以下「本発明品３」という）の健常ラットを用いた消化管からのＣａ吸収及び骨密度に与える影響についての実験を行った。供試動物として、Ｗｉｓｔａｒ系雄性ラット（７週齢、１５０〜１７０ｇ）を用い、試験飼料投与前の１週間、表５に示す対照群の組成飼料で予備飼育を行い、実験環境への馴化を行った後、これらラットを各群８匹ずつに区分けして使用した。供試飼料（被検サンプル）としては、表５に組成が示されている調製例４により調製された本発明品群、食物繊維分を配合しない対照群及び酵母細胞壁画分と等量の酵母細胞壁（イムセルＢＦ）を含む比較例品群を用い、供試飼料は自由摂取にてラットに与え、２週間飼育した。なお、本発明品３又は比較例品のタンパク質含量及び脂質含量を測定することで、飼料中のタンパク質含量及び脂質含量が均等になるようカゼイン及び大豆油の配合量を補正した。供試飼料の投与２週間後、糞便、右側大腿骨及び腹部大動脈血の採取を行い、糞便排泄量、糞便中Ｃａ排泄量及び血清中Ｃａ濃度の測定を行った。採取した糞便は直ちに凍結乾燥を行った後、乾燥重量を測定し、採取した血液は直ちに遠心分離を行い血清を分取した。また、大腿骨については、骨密度及び骨塩量をｐＱＣＴ法にて測定した。
【００４１】
なお、効果の判定は、試験例２と同様に、総骨密度、海綿骨骨密度、全骨塩量及び海綿骨骨塩量を効果の指標とした。結果を表６に示す。本発明で得られた本発明品３の摂取により、糞便中へのＣａ排泄量が有意に減少したことから、Ｃａの体内への吸収率が向上したことが判明した。さらに、海綿骨骨塩量の比較においては、本発明品３群が比較例品群に比べ有意に高い値となった。また、海綿骨骨密度について本発明品群と比較例品群とを比較したところ、ｐ値が５％水準では有意でなかったものの、ｐ＝０．０７と限りなく有意に近い結果にて本発明品３の比較例品に対する高い効果が判明した。以上のことから、本発明品３には骨組織増強効果のあることが明らかとなり、これは、本発明品３を始めとした食物繊維成分を摂取しない場合、もしくは、比較例品を摂取した場合に比べて顕著に高い効果であった。
【００４２】
【表５】
【００４３】
【表６】
【００４４】
試験例４
調製例２で得られた酵母細胞壁画分（以下「本発明品１」という）の、食餌中のＣａ量の低減により作製した骨密度減少モデルラット（骨粗鬆症モデル）を用いた骨密度に与える影響についての実験を行った。供試動物として、ＳＤ系雄性ラット（３週齢、４５〜５５ｇ）を用い、表７に示すＬｏｗ Ｃａ飼料（０．００３％ Ｃａ）にて２週間飼育することで骨密度減少モデルを作製した後、これらラットを各群８匹ずつに区分けした。引き続きラットへ供試する飼料として、本発明品１配合飼料及び対照飼料に含まれる食物繊維含量が均等になるよう、代表的な食物繊維素材であるセルロース（旭化成株式会社製、アビセル）を比較例品（以下「比較例品２」）として配合した対照飼料を設定した。供試飼料として表８に組成を示した、Ｎｏｒｍａｌ Ｃａ含有比較例品２配合飼料（０．５％ Ｃａ）、Ｎｏｒｍａｌ Ｃａ含有１０％本発明品１配合飼料（０．５％ Ｃａ）、Ｈｉｇｈ Ｃａ含有比較例品２配合飼料（１．２％ Ｃａ）、およびＨｉｇｈ Ｃａ含有１０％本発明品１配合飼料（１．２％ Ｃａ）をそれぞれ２週間自由摂取させた。各飼料中のＣａ含量は、飼料組成であるＡＩＮ−９３Ｇを基本としたミネラル混合組成物中のＣａ含量を調整することで補正した。供試飼料の投与２週間後、右側大腿骨及び腹部大動脈血の採取を行った。採取した血液は直ちに遠心分離を行い血清を分取した後、血清中Ｃａ濃度の測定を行った。また、大腿骨については、骨密度及び骨塩量をｐＱＣＴ法にて測定した。
【００４５】
骨は大きく分けて皮質骨と海綿骨に分かれるが、食餌中のＣａ量の低減によって骨密度が減少する場合、まず初めに皮質骨から骨吸収作用が進行することで骨密度の低下が起こることから、総骨密度及び皮質骨骨密度を指標として効果を判定した。結果を表９及び表１０に示す。Ｎｏｒｍａｌ Ｃａ飼料では本発明品１の配合による骨密度への影響に有意な差は観察されなかったが、Ｈｉｇｈ Ｃａ飼料においては本発明品１の摂取により対照と比較し総骨密度および皮質骨骨密度の減少が有意に抑制された。以上のことから、本発明品１には食餌中のＣａ量の低減によって生じる骨密度の減少によって生じる骨粗鬆症様の症状を抑制する効果のあることが明らかとなった。この効果は、食物繊維素材である比較例品２を摂取した場合に比べて顕著に高いものであった。
【００４６】
【表７】
【００４７】
【表８】
【００４８】
【表９】
【００４９】
【表１０】
【００５０】
試験例５
卵巣摘出手術を行うことでエストロゲン欠乏により発症する閉経性骨粗鬆症モデルラットを用い、調製例２で得られた酵母細胞壁画分（以下「本発明品１」という）摂取が骨密度に及ぼす影響を評価する実験を行った。
Ｗｉｓｔａｒ系雌性ラット（８週齢、１８０〜２１０ｇ）を固形飼料（ＣＥ−２、日本クレア製）にて１週間馴化後、ラットを各群１０匹ずつに区分けした。表１１に示す、Ｈｉｇｈ Ｃａ含有１０％本発明品１配合飼料（２．５％ Ｃａ）及び本発明品を配合しないＨｉｇｈ Ｃａ飼料（２．５％ Ｃａ）を対照飼料として１週間投与した後、両側卵巣摘出術を行った。引き続き、同一の試験飼料をそれぞれ３週間自由摂取させた後、右側大腿骨及び腹部大動脈血の採取を行った。採取した血液は直ちに遠心分離を行い血清を分取した後、血清中Ｃａ濃度の測定を行った。また、大腿骨については、骨密度をｐＱＣＴ法にて測定した。
【００５１】
エストロゲン欠乏により発症する閉経性骨粗鬆症モデルの場合、中心部の海綿骨からもろくなることで骨全体の強度が低下するため、海綿骨の骨密度を高めることが骨粗鬆症予防に重要なポイントの一つとなることから、総骨密度及び海綿骨骨密度を指標として効果を判定した。なお、本発明品１無配合飼料を摂取する偽手術群を別途設定することで卵巣摘出ラットの骨密度低下を確認した。結果を表１２に示す。本発明品１の摂取により対照と比較し海綿骨骨密度の減少が有意に抑制された。以上のことから、本発明品の摂取により、Ｃａ吸収もしくは骨形成が助長され骨粗鬆症の症状改善もしくは予防に貢献することが明らかとなった。
【００５２】
【表１１】
【００５３】
【表１２】
【００５４】
試験例６
本発明における酵母細胞壁画分は摂取された後、胃内における消化酵素や物理的作用の影響をほとんど受けることなく下部消化管に到達し、そこで腸内細菌の発酵基質となることで酢酸、プロピオン酸及び酪酸といった短鎖脂肪酸に変換されるが、該短鎖脂肪酸の作用によって消化管内が酸性域に保たれることで消化管粘膜側に存在するカルシウムを始めとしたミネラルの可溶化率が高まり、よって、ミネラル吸収が促進され、ひいては、骨密度上昇効果及び骨密度減少抑制効果が発揮される。本発明における酵母細胞壁画分は、短鎖脂肪酸の産生基質となりうる食物繊維などの難消化性成分を豊富に含有するが、その食物繊維成分が豊富に含有されることを調べるため、調製例２で得られた酵母細胞壁画分（以下「本発明品１」という）、調製例３で得られた酵母細胞壁画分（以下「本発明品２」という）、調製例４で得られた酵母細胞壁画分（以下「本発明品３」という）及び類似の酵母細胞壁（田辺製薬株式会社製「イムセルＢＦ」、以下「比較例品」という）に存在する食物繊維含量について、ＡＯＡＣ公定のプロスキー法により測定を行った。その結果（表１３）、本発明品１、本発明品２及び本発明品３には６０％以上の食物繊維が含まれていることが判明した。一方、比較例品に含まれるグルコース量は５４．５％であることが分かり、十分なミネラル吸収促進、骨密度の上昇及び骨密度減少抑制効果の発現には、６０％以上の食物繊維の存在が必要であることを確認した。
【００５５】
【表１３】
【００５６】
試験例７
本発明における酵母細胞壁画分には、短鎖脂肪酸の産生基質となりうる食物繊維などの難消化性成分を豊富に含有ことが試験例６から明らかとなったが、本発明品中にある食物繊維の中でも主体をなすβ−１，３−グルカン及びβ−１，６−グルカンが豊富に含有されることを調べるため、調製例３で得られた酵母細胞壁画分（以下「本発明品２」という）、調製例４で得られた酵母細胞壁画分（以下「本発明品３」という）及び類似の酵母細胞壁（田辺製薬株式会社製「イムセルＢＦ」、以下「比較例品」という）に存在するβ−グルカン由来のグルコース含量について、通常の酸加水分解法により本発明品又は比較例品を分解した後、高速液体クロマトグラフィーを用いた絶対検量線法により測定を行った。
【００５７】
その結果（表１４）、本発明品２及び本発明品３には４９％以上のグルコースが含まれていることが判明した。一方、比較例品に含まれるグルコース量は４０．１％であることが分かり、十分なミネラル吸収促進、骨密度の上昇及び骨密度減少抑制効果の発現には、４１％以上のグルコース、すなわち、グルコースを構成糖とする多糖であるβ−グルカンの存在が重要であることを確認した。また、腸内細菌細胞の資化されうる構成多糖を成すグルコース及びマンノースの合計含有量の比較においては、本発明品２及び本発明品３には７５％以上の多糖類が含まれていることが判明した。一方、比較例品に含まれる多糖類の総量には６１．８％であることが分かり、十分なミネラル吸収促進、骨密度の上昇及び骨密度減少抑制効果の発現には、６３％以上の多糖成分、すなわち、グルコース及びマンノースを構成糖とする多糖成分の存在が重要であることを確認した。
【００５８】
【表１４】
【００５９】
試験例８
本発明における酵母細胞壁画分は、摂取され消化管に到達した後、腸内細菌の発酵基質となることで、酢酸、プロピオン酸及び酪酸といった短鎖脂肪酸に変換され、それらの該短鎖脂肪酸の作用によって消化管内に存在するカルシウムを始めとしたミネラルの可溶化率が高まることで、ミネラルの吸収促進が発現され、ひいては、骨密度上昇効果及び骨密度減少抑制効果がもたらされる。本発明における酵母細胞壁画分が、腸内細菌によりどの程度発酵され短鎖脂肪酸が産生されるかについて調べるため、調製例３で得られた酵母細胞壁画分（以下「本発明品２」という）、調製例４で得られた酵母細胞壁画分（以下「本発明品３」という）及び類似の酵母細胞壁（田辺製薬株式会社製「イムセルＢＦ」、以下「比較例品」という）を正常ラットに投与し、摂取後の盲腸内に産生される短鎖脂肪酸量を測定する実験を行った。供試動物として、Ｗｉｓｔａｒ系雄性ラット（７週齢、１５０〜１７０ｇ）を用い、試験飼料投与前の１週間、試験例２の表３に示す対照群の組成飼料で予備飼育を行い、実験環境への馴化を行った後、ラットを各群８匹ずつに区分けして使用した。
【００６０】
試験群としては、試験例２の表３に組成が示されている調製例３により調製された本発明品２群、試験例３の表５に組成が示されている調製例４により調製された本発明品３群、及び酵母細胞壁画分と等量の比較例品を含む比較例品群を設定し、供試飼料は自由摂取にてラットに与え、２週間飼育した。なお、食物繊維素材を含有しない飼料を投与する対照群も設定した。本発明品２、本発明品３又は比較例品のタンパク質含量及び脂質含量を測定することで、飼料中のタンパク質含量及び脂質含量が均等になるようカゼイン及び大豆油の配合量を補正した。供試飼料の投与２週間後に解剖を行い、盲腸内にある発酵産物を採取した後、直ちに加水し十分に分散させ、遠心分離及びろ過することで短鎖脂肪酸の抽出を行い、この抽出液を盲腸内容物抽出液として分取した。該抽出液に存在する短鎖脂肪酸含量について、高速液体クロマトグラフィーを用いた絶対検量線法により測定を行った。その結果（表１５）、本発明品２又は本発明品３を摂取することで対照及び比較例品に比べて有意に高い短鎖脂肪酸産生が観察された。また、この効果は、比較例品を摂取した場合に比べて顕著に高いものであった。以上のことから、本発明品の摂取により、Ｃａ吸収もしくは骨形成が促進され骨粗鬆症の症状改善もしくは予防に有効であることが明らかとなった。
【００６１】
【表１５】
【００６２】
【発明の効果】
本発明のミネラル吸収促進剤により、高齢者の骨量減少抑制及び骨粗鬆症の予防を始めとして、骨粗鬆症患者の症状改善、一般女性や妊婦の骨量及び骨密度の維持改善を目的として、Ｃａ等のミネラルの吸収を効果的に促進させ、更には、骨密度の増加及び骨粗鬆症の予防ならびに症状の改善に有効な、副作用が少なく安全な、薬理活性組成物を提供することができる。本発明の有効成分は、水への分散性が高く、より摂取しやすい素材として、種々の薬理活性組成物としての投与形態或いはこれを含有してなる飲食品として摂取することができる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a mineral absorption promoter containing a yeast cell wall fraction as an active ingredient and an agent for preventing and / or improving osteoporosis containing the yeast cell wall fraction as an active ingredient.
[0002]
[Prior art]
As humans age, the balance between bone formation and bone resorption in bone tissue starts to change due to changes in the ability to regulate calcium distribution and concentration in the body, but recently the lifestyle has been mechanized and the burden on the body has been reduced. Despite this, the number of people visiting the hospital is steadily increasing due to diseases caused by weakness in the legs. In recent years, the treatment and care of bedridden and demented elderly people has become a major social problem with the increase in the aging population. It is said to be easy.
[0003]
Bone is formed by the age of about 18 years, and tough bone formation progresses from the 20s to the 30s. However, women's social advancement is becoming more active in all generations. The disturbance causes abnormal secretion of female hormones, which results in irregular menstruation and a decrease in bone density. In addition, a large amount of calcium is supplied to the fetus during pregnancy and lactation, so it is necessary to store a sufficient amount of calcium in bones. Women undergo menopause around the age of 50, and at the same time, the secretion of female hormones is significantly reduced, resulting in a sharp decrease in bone mass and bone density. Conventionally, in order to suppress a decrease in bone mass and bone density, exercise such as stimulation by exercise, intake of sufficient calcium and magnesium from food, supplementation of active vitamin D which promotes absorption of calcium from the intestinal tract, etc. It has been said that it is important to improve the cause of the decrease. However, due to individual lifestyles and time constraints, it is difficult to continue exercising on a regular basis, or to efficiently consume minerals such as calcium and magnesium and active vitamin D through meals.
[0004]
On the other hand, conventionally, many techniques related to pharmacological compositions containing yeast or a yeast cell component as an active ingredient are disclosed in patent gazettes. For example, a composition useful for maintaining pregnancy, preventing miscarriage and promoting fetal growth, which contains yeast as an active ingredient (JP-A-4-305530), or a centrifuged supernatant of a yeast cell lysate is treated with protease and heated. A mineral-enhancing action of yeast-derived ribonucleic acid and a method for producing the same (JP-A-6-287141) obtained by removing post-sedimentation, and a highly functional food containing mineral-enriched yeast ( Japanese Patent Application Laid-Open (JP-A) No. 2001-46016), and a liquid food having excellent physical properties such as fluidity in which yeast, especially a mineral component, preferably a yeast containing a trace element thereof is contained in a high content in a liquid food (JP-A-2001-46016). Japanese Patent Application Laid-Open No. 2002-332236), a mineral-containing composition or a therapeutic composition containing 20 to 50% by mass of sake yeast chromium (Japanese Patent Application Laid-Open No. 2002-332236), and a dried yeast having a high zinc content. And a salt containing sodium chloride, potassium chloride, magnesium sulfate, magnesium carbonate, and eggshell calcium, and if necessary, L-glutamate, a food additive composition (JP-A-2002-330727) is disclosed. Have been.
[0005]
Further, as a nutritional supplement food containing a mineral component obtained by hot-water extraction of a mineral-enriched yeast in a suspension (Japanese Patent Application Laid-Open No. 2002-335913), or a mineral supply material, preferably a particle size of 4 μm or less Oral tube feeding composition comprising a spray-dried product of a mineral-rich yeast obtained by crushing yeast cells so that the percentage of particles becomes 50% or more (Japanese Patent Application Laid-Open No. 2002-300861), Discloses a food composition containing a mineral yeast containing manganese, manganese, copper or molybdenum as a mineral component (JP-A-2003-198). However, all of these techniques merely seek mineral nutrients from the yeast components, and do not relate to the mineral absorption promoting action of yeast components other than minerals. In addition, although a technique relating to a food material in which the mineral content contained in yeast is concentrated and a mineral supplement is disclosed (JP-A-9-205), the effectiveness of promoting mineral absorption or preventing osteoporosis has been determined. No, it is unclear what effect it would actually take.
[0006]
Furthermore, a technology has been disclosed which is expected to promote mineral absorption by ingesting ribonucleic acid contained in a yeast extract extracted from yeast (Japanese Patent Application Laid-Open No. 5-292916). It only evaluates the absorption ability and does not consider the in vivo kinetics of ingested ribonucleic acid, that is, the effects of digestion, decomposition and absorption in the stomach and small intestine. It is not known whether the solubilization of calcium in the gastrointestinal tract, which is important for the expression of osteoporosis, is sufficiently induced, and thus promotion of bone formation occurs.
[0007]
On the other hand, as a technique for utilizing yeast cell wall as a pharmacologically active composition as an active ingredient, the yeast cell wall of extract extraction residue obtained by removing live soluble extract from cells by autolysis or enzymatic treatment with protease is used as a mineral absorption promoter. For example, Japanese Patent Application Laid-Open No. 2002-255832 discloses a technique used for absorbing iron. However, although the results disclosed in the publication indicate that administration of yeast cell walls to rats increased the apparent absorption rate (%) of iron as compared with the control, the results showed that the apparent absorption rate (%) was significantly different between the test groups. I do not admit the difference. Also, administration of dry yeast, yeast cell wall and yeast extract to iron-deficient anemic rats compared their hemoglobin regeneration rate (%) and hemoglobin increase (g / L). Although there is a significant effect on the yeast, no significant difference was observed between the yeast and the dried yeast. Furthermore, the yeast extract was mixed with the yeast cell wall and administered. Therefore, it is not possible to confirm the effect specific to the yeast cell wall from the description in this publication.
[0008]
[Patent Document 1]
JP-A-4-305530.
[Patent Document 2]
JP-A-5-292916.
[Patent Document 3]
JP-A-6-287141.
[Patent Document 4]
JP-A-9-205.
[Patent Document 5]
JP-A-2000-253851.
[Patent Document 6]
JP-A-2001-46016.
[Patent Document 7]
JP-A-2002-255832.
[Patent Document 8]
JP-A-2002-332236.
[Patent Document 9]
JP-A-2002-330727.
[Patent Document 10]
JP-A-2002-335913.
[Patent Document 11]
JP-A-2002-300861.
[Patent Document 12]
JP-A-2003-198.
[Non-patent document 1]
Bioscience, Biotechnology, and Biochemistry, vol. 62, p837, 1998.
[0009]
[Problems to be solved by the invention]
An object of the present invention is to provide a safe and highly effective mineral absorption promoter comprising a yeast cell wall fraction as an active ingredient and an agent for preventing and / or improving osteoporosis comprising the yeast cell wall fraction as an active ingredient. is there.
[0010]
In recent years, it has been reported that ingestion of indigestible components such as dietary fiber enhances mineral absorption in the digestive tract (K. Saito et al., Bioscience, Biotechnology, and Biochemistry, vol. 62, p837, 1998). Ingested indigestible components are assimilated by intestinal bacteria in the large intestine and are converted into short-chain fatty acids, which are fermentation products.The generated short-chain fatty acids are quickly absorbed from the intestinal tract, and the energy of the large intestine is reduced. It is said to contribute to the normalization and activation of colon function as a source. Intestinal bacteria such as bifidobacteria and lactic acid bacteria, and oligosaccharides and water-soluble dietary fibers that are growth promoters thereof are mentioned as materials for promoting the production of short-chain fatty acids, and ingesting intestinal bacteria such as bifidobacteria and lactic acid bacteria. However, there is a problem that almost all of them are killed by the effect of stomach acid before reaching the large intestine, and do not contribute to high production of short-chain fatty acids. Although it produces short-chain fatty acids, it lacks swelling like dietary fiber, lacks contact with intestinal bacteria and lacks physical irritation to the digestive tract, resulting in short-chain fatty acid production and metabolism of intestinal epithelial cells. There was a problem from the viewpoint of promoting rotation.
[0011]
In addition, since oligosaccharides usually have a sweet taste, when used in a food, the oligosaccharide has an adverse effect of impairing the original flavor of the food. Furthermore, there is a problem that side effects such as diarrhea and abdominal pain appear depending on the intake of oligosaccharides and water-soluble dietary fiber. Therefore, although there are reports that ingestion of indigestible components such as dietary fiber enhances mineral absorption in the digestive tract, at present, no effective solution has been found.
At present, the problem of bone mineral density and osteoporosis is increasing due to the dietary balance and physiological balance of the body, and the development of prevention and treatment methods for modern diseases such as bone fractures caused by them is expected. I have. In particular, ingestion of active substances in the form of health foods has been increasing in interest as a method for preventing and treating such diseases, because it has few side effects and is simple.
[0012]
The object of the present invention is to meet these needs, and aims at improving the symptoms of osteoporosis patients, including maintaining bone mass and bone density of general women and pregnant women, including suppressing bone loss and preventing osteoporosis in the elderly. As a material that effectively promotes the absorption of minerals, and is effective in increasing bone density and preventing osteoporosis and improving symptoms, with fewer side effects, safe dispersibility in water, and easier intake. And an agent for preventing and / or improving osteoporosis, and a pharmacologically active composition for osteoporosis.
[0013]
[Means for Solving the Problems]
The present inventor, in order to solve the above problems, promotes the absorption of minerals, is effective in preventing and / or improving bone density and osteoporosis, and a substance that can be safely taken in the form of food and drink, As a result of diligent search, the yeast cell wall fraction obtained by subjecting yeast cells to alkali treatment and then washing with water, physical pulverization and removal of soluble cell components is a safe and effective mineral absorption promoter. The yeast cell wall fraction obtained by subjecting yeast cells to an alkali treatment followed by water washing and removal of soluble bacterial components is effective as a preventive and / or ameliorating agent for osteoporosis. This led to the completion of the present invention.
That is, the present invention provides a mineral absorption promoter containing a yeast cell wall fraction as an active ingredient, which is obtained by subjecting yeast cells to an alkali treatment followed by washing with water, physical pulverization treatment, and removal of soluble cell components, and yeast. It comprises a preventive and / or ameliorating agent for osteoporosis containing a yeast cell wall fraction obtained by subjecting cells to an alkali treatment, followed by washing with water and a treatment for removing soluble cell components. The physical pulverization in the present invention is performed by a pulverization using a high-pressure homogenizer or an ultrasonic generator.
[0014]
In the search for a substance that is effective in increasing bone density and preventing and / or improving osteoporosis, substances that promote mineral absorption and that can be safely taken in the form of food and drink, such as dietary fiber, Things are conceivable. Ingested dietary fiber is degraded by intestinal bacteria in the digestive tract, and short-chain fatty acids such as acetic acid, propionic acid and butyric acid are produced as fermentation products. With the production of short-chain fatty acids in the large intestine, it has been clarified that the abundance of short-chain fatty acids on the mucosal side of the large intestine enhances mineral absorption in the large intestine. This effect is due to the physical effect of lowering pH and osmotic pressure caused by short-chain fatty acids, and increasing the solubilization rate of minerals in the large intestine. It is considered that the increase is one of the factors. Therefore, ingestion of dietary fiber with higher fermentability is an important point for enhancing the effect of promoting mineral absorption in the large intestine (C. Demign et al., Journal of Nutrition, vol. 119, p1625, 1989).
[0015]
The present inventors have taken this point of view, and as a result of intensive search, in the course of research on the yeast cell wall fraction obtained by removing the yeast extract, which is an intracellular component of yeast, the yeast cell wall fraction was found to be water-insoluble. Despite containing dietary fiber, it is excellent in dispersibility and swelling in water, and is highly assimilated by intestinal bacteria in the large intestine after ingestion, and has more short chains compared to other dietary fiber materials It has been found to have the action of producing fatty acids. Therefore, when the pharmacological action of the yeast cell wall fraction was repeatedly examined, the yeast cells were alkali-treated and washed with water to further remove the soluble cell components, or the yeast cells from which the soluble cell components were removed were used. The yeast cell wall fraction obtained by washing with water after alkali treatment is subjected to a physical pulverization treatment such as a high-pressure homogenizer, and the yeast cell wall fraction is used as a substrate for intestinal bacterial short-chain fatty acid production in the digestive tract. The present inventors have found that a yeast cell wall fraction suitable for ingestion, which has an increased dietary fiber content and has no peculiar off-flavor due to autolysis, is suitable for ingestion.
[0016]
That is, specifically, the present invention relates to a mineral absorption promoter comprising a yeast cell wall fraction obtained by performing an alkali treatment on a yeast cell, washing with water, a physical pulverization treatment and a treatment for removing a soluble bacterial component as an active ingredient. (1) The physical absorption treatment is performed by a high-pressure homogenizer treatment, and the mineral absorption enhancer (Claim 2) according to (1) or the treatment for removing soluble bacterial components is autolyzed. And / or enzymatic treatment, wherein the mineral absorption enhancer according to claim 1 or claim 2 (claim 3), the yeast cells are alkali-treated and then washed with water and the soluble cell components are removed. A preventive and / or ameliorating agent for osteoporosis containing a yeast cell wall fraction obtained as an active ingredient (claim 4); And an agent for preventing and / or ameliorating osteoporosis containing a yeast cell wall fraction as an active ingredient obtained by performing the removal treatment of the yeast (claim 5), and the physical pulverization treatment is performed by a high-pressure homogenizer treatment. The agent for preventing and / or ameliorating osteoporosis according to claim 5 (claim 6) and the treatment for removing soluble bacterial components are carried out by autolysis and / or enzymatic treatment. An agent for preventing and / or improving osteoporosis according to any one of claims (claim 7).
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention provides a yeast cell wall fraction obtained by subjecting yeast cells to an alkali treatment followed by washing with water and further removing soluble cell components, or a yeast cell from which the soluble cell components have been removed is subjected to alkali treatment and then washed with water. A mineral absorption enhancer containing a yeast cell wall fraction obtained by subjecting the yeast cell wall fraction obtained by a physical pulverization treatment such as a high-pressure homogenizer or an ultrasonic generator to an active ingredient; It comprises a preventive and / or ameliorating agent for osteoporosis containing a yeast cell wall fraction obtained by performing a body component removal treatment as an active ingredient.
[0018]
(Raw yeast)
The yeast used as the raw material of the yeast cell wall fraction of the present invention is not particularly limited as long as it belongs to the taxonomic yeast and is an edible yeast, and includes, for example, beer yeast which is a by-product of the brewing process. , Wine yeast, baker's yeast, torrla yeast, alcoholic yeast, yeast for sake, etc., and more specifically, brewer's yeast, Saccharomyces cerevisiae of the genus Saccharomyces to which baker's yeast belongs, or Saccharomyces cerevisiae. (Saccharomyces pastorianus), other Saccharomyces rouxii, Saccharomyces curlsbergensis (Saccharomyces carlsbergensis), Saccharomyces Saccharomyces pombe, and Candida utilis of the genus Candida, which is a methanol-assimilating yeast, Candida tropicalis, Candida lipoditica, and Candida tropicalis. -Friedberg (Candida flaveri), Candida boidini (Candida boidini) and the like, and furthermore, Rhodotorula minuta (Rodotorura minuta) and the like can be exemplified. These yeasts can be used alone or in combination. It is preferable to use live yeast as the yeast, but even when yeast other than live yeast such as dried yeast is used, it can be suspended in water or the like and treated in the same manner as live yeast. Furthermore, the shape and size of the yeast used are not particularly limited, and the size is preferably in the range of 1 to 20 μm.
[0019]
(Preparation of yeast cell wall fraction)
In preparing the yeast cell wall fraction used in the present invention, the yeast cells are alkali-treated and then washed with water to further remove the soluble cell components, or the yeast cells from which the soluble cell components have been removed are subjected to alkali treatment and then subjected to water treatment. Washing is carried out by preparing a yeast cell wall fraction from which yeast cells have been removed from soluble yeast components such as proteins, amino acids, nucleic acids, etc., which are soluble in water or polar solvents. The removal of soluble bacterial components from yeast cells is usually carried out by lysing yeast cells by enzyme treatment and separating and removing the soluble bacterial components outside the cells. Examples of such an enzyme treatment method include a so-called autolysis method using an enzyme of a yeast cell, an enzyme addition method in which an enzyme such as a protease, a nuclease, a glucanase, and an esterase are added from the outside, and a method in which they are used in combination. I can do it. A yeast cell wall fraction can be obtained by subjecting soluble enzyme components to removal treatment such as centrifugation from the enzyme-treated yeast cells. Since all of the above-described enzyme treatment methods are methods used when producing a yeast intracellular component as a yeast extract, considering the production cost, the yeast cell wall fraction is used as a secondary in yeast extract production. It is advantageous to use the yeast extract extract residue that is the product.
[0020]
When the yeast cell wall fraction used in the present invention is prepared using a yeast extract extraction residue that is a by-product in the production of yeast extract, a method for producing the yeast extract is disclosed in, for example, JP-A-8-56611. After decomposing the aqueous suspension of the heat-inactivated yeast shown in the above with an enzyme, the yeast extract is obtained by adding chitosan or chitosan and polyacrylate to remove water-insoluble substances, thereby obtaining an aqueous yeast extract solution. And a yeast cell suspension treated with a high-pressure homogenizer as described in JP-A-9-56361, which is adjusted to neutral to weakly alkaline, and an enzyme containing endo-type protease in the yeast cell suspension. A method for producing a yeast extract to be added and auto-digesting, and a method for heating a yeast cell as a raw material as disclosed in Japanese Patent Application Laid-Open No. 11-332511, which contains a nuclease. That under the conditions, and 5'-nucleotides decomposing enzyme activity like the preparation of the yeast extract to extract and digest the bacterial component under the conditions suppressed. In addition to these techniques, yeast extract extraction residues in the production of known yeast extracts can be used effectively.
[0021]
In the preparation of the yeast cell wall fraction used in the present invention, the yeast cell or yeast extract extraction residue is subjected to an alkali treatment and then a water washing treatment in order to increase the physiological activity effect and improve physical properties such as odor. For example, as a yeast cell wall fraction, a yeast cell or a yeast extract extraction residue, without alcohol treatment and / or ozone treatment, added to water washing or water washing after alkali treatment, or water washing or water washing after alkali treatment, According to the present invention, by performing a physical pulverization treatment such as a high-pressure homogenizer treatment, not only a more excellent effect of promoting mineral absorption and an effect of preventing or improving osteoporosis, but also a unique off-flavor due to autolysis is eliminated. Thus, a yeast cell wall fraction suitable for ingestion can be obtained. As the water washing treatment after the alkali treatment, the yeast cells in a slurry state are alkali-treated and washed with water in the yeast extract extraction step, and the yeast extract extraction residue obtained from the yeast cells is further alkali-treated and then washed with water. Although it is preferable to carry out the treatment, any one of the yeast cells and the yeast extract extraction residue may be subjected to alkali treatment followed by water washing.
[0022]
As the alkali treatment of the slurry yeast cells, for example, a yeast cell slurry adjusted to a solid content concentration of 5 to 20% by weight, preferably 8 to 12% by weight, more preferably about 10% by weight, Sodium hydroxide is added so that the pH becomes 8 to 12, preferably 9 to 10, and a stirring treatment at 0 to 20 ° C, preferably 0 to 10 ° C can be mentioned. Further, as the water washing after the alkali treatment, a normal water washing method can be used, and it is preferable to perform the washing after dehydrating the cells after the alkali treatment with a centrifugal separator or the like from the viewpoint of washing efficiency, Such a washing step can be performed a plurality of times. As the alkali treatment of the yeast extract extraction residue, for example, a yeast extract extraction residue slurry adjusted to a solid content concentration of 5 to 20% by weight, preferably 8 to 12% by weight, more preferably about 10% by weight, Sodium hydroxide is added so that the pH becomes 8 to 12, preferably 9 to 10, and stirring at 0 to 70 ° C, preferably 0 to 50 ° C, more preferably 10 to 30 ° C. it can.
[0023]
In addition, as the water washing after the alkali treatment, a normal water washing method can be used.From the viewpoint of washing efficiency, the washing is performed after dehydrating the yeast extract extraction residue after the alkali treatment with a centrifuge or the like. Preferably, such a washing step can be performed a plurality of times. Without performing ethanol treatment, ozone treatment, and acid treatment, such an alkali treatment and water washing treatment can remove the off-flavor and off-odor causing substances easily and at low cost. Even when used in combination with a food material, a tasteless and odorless yeast cell wall fraction that does not impair the flavor of the food material can be obtained.
[0024]
In the present invention, in order to perform the enzyme treatment promptly and obtain a yeast cell wall fraction with superior efficacy, the yeast cells before the enzyme treatment or before the alkali treatment are subjected to physical pulverization treatment (cell wall treatment using a high-pressure homogenizer or the like). Pretreatment with physical destruction of Examples of the physical pulverization include pulverization using a high-pressure homogenizer (for example, High pressure homogenizer, manufactured by APV) and an ultrasonic generator (for example, manufactured by Ultrasonics or Telsonics). Among them, the pretreatment using the high-pressure homogenizer is, for example, 100 to 1000 kg / cm. 2 It is desirable to carry out while cooling under a pressure of. Furthermore, for the purpose of increasing the dietary fiber content of the yeast cell wall fraction, post-treatment involving physical destruction of the cell wall can be performed on the yeast cells that have been washed with water after alkali treatment, using a high-pressure homogenizer or the like. The post-treatment using this high-pressure homogenizer is, for example, 100 to 1000 kg / cm. 2 It is desirable to carry out while cooling under a pressure of. Such physical treatment is carried out after the enzymatic treatment or the above-mentioned treatment for the purpose of obtaining the enhancement of the dispersibility of yeast cells (improvement of texture) and the improvement of washability (improvement of removal of off-flavor and off-odor substances) together with the increase of the efficacy. It can also be applied to yeast cells after alkali treatment.
[0025]
(Pharmacologically active composition)
In the present invention, the mineral absorption promoter and the osteoporosis prevention and / or amelioration agent containing the yeast cell wall fraction as an active ingredient can be ingested as a pharmacologically active composition.
Here, the pharmacologically active composition comprises the yeast cell wall fraction alone or a mixture of the yeast cell wall fraction and other components or materials, and prevents and / or prevents diseases targeted for pharmacological action having the yeast cell wall fraction. Or a edible composition in the form of a food or beverage compounded with a preparation for improving symptoms and a food or drink material.
In the present invention, the yeast cell wall fraction having a mineral absorption promoting effect, a bone density increasing effect, and a bone density decrease suppressing effect is generally used in the digestive tract to contain trivalent cations such as chromium and iron, calcium, magnesium, manganese, molybdenum, and selenium. Production of short-chain fatty acids, which is said to be effective for improving the solubilization rate of minerals such as divalent cations such as iron, copper and zinc, and monovalent cations such as sodium and potassium It is necessary to contain abundant indigestible components such as dietary fiber which can serve as a substrate, and it is preferable that the indigestible components be fermented well by intestinal bacteria in the digestive tract. Further, the dietary fiber component contained in the yeast cell wall fraction used in the present invention is preferably at least 60%.
[0026]
The dietary fiber contained in the yeast cell wall fraction used in the present invention is mainly composed of β-1,3-glucan, β-1,6-glucan, and α-1,6-mannan. Among them, the high content of β-1,3-glucan and β-1,6-glucan, which are the main components, ensures high fermentability in the digestive tract. Preferably contains more β-glucan. Further, β-glucan contained in the yeast cell wall fraction used in the present invention is easily measured by an absolute calibration curve method using high performance liquid chromatography after decomposing the yeast cell wall fraction by a usual acid hydrolysis method. However, the amount of β-glucan contained in the fraction is preferably 41% or more, more preferably 49% or more. In addition, a yeast cell wall fraction obtained by subjecting yeast cells to physical crushing as it is may be used as long as it exhibits the effect as the above-mentioned use agent.
[0027]
The yeast cell wall fraction of the present invention is a mineral absorption promoter, a material for preventing osteoporosis and improving symptoms, as yogurt, drink yogurt, juice, milk, soy milk, liquor, coffee, black tea, green tea, green tea, gyokuro, oolong tea, turmeric tea , Pour tea, jasmine tea, sports drinks, various drinks such as mineral water, western confectionery such as pudding, cookies, bread, cake, donut, jelly, etc., Japanese confectionery such as rice crackers, yokan, daifuku, hayagi, castella etc. Bread and confectionery, noodles such as udon, buckwheat, etc., fish paste products such as kamaboko, ham, fish sausage, meat products such as ham, sausage, hamburger steak, miso, soy sauce, sauce, dressing, mayonnaise, sweetener Teppanyaki food such as takoyaki, okonomiyaki, fried noodles, tofu, Garlic, other pickles, boiled soy sauce, gyoza, croquettes, sandwiches, pizza, hamburgers, salads, etc., can be used as foods and beverages to exert the effect of the present invention, especially the chewing power It can contribute to improvement of quality of life (QOL) of elderly people whose teeth have deteriorated, whose teeth have deteriorated, pregnant women who are sensitive to dietary contents, and women who are worried about weight gain.
[0028]
【Example】
Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples. Unless otherwise specified, all yeast cell weights shown in the examples are actual weights (dry weights). Hereinafter, calcium is referred to as “Ca”.
[0029]
Preparation Example 1
After accurately weighing the fermented brewer's yeast slurry obtained as a by-product from the beer brewing process, water was added so that the solid content was 10% by weight. Sodium hydroxide was added until the pH became 9, and the mixture was stirred at 10 ° C., centrifuged, washed by adding water to the precipitate fraction, and then centrifuged again. The suspension hydrolyzed to a solid content of 10% by weight was autolyzed under the reaction conditions of 50 ° C. and 17 hours, and then centrifuged to remove the autolysed residue from which soluble bacterial components were removed. Minutes. The protein content of the yeast cell wall fraction was 23.7%, and the dietary fiber content (AOAC method) was 60.3%.
[0030]
Preparation Example 2
After accurately weighing the fermented brewer's yeast slurry obtained as a by-product from the beer brewing process, water was added so that the solid content was 10% by weight. Sodium hydroxide was added until the pH became 9, and the mixture was stirred at 10 ° C., centrifuged, washed by adding water to the precipitate fraction, and then centrifuged again. A suspension hydrated so that the solid content becomes 10% by weight is 700 kg / cm. 2 After performing a high-pressure homogenizer treatment while cooling under a pressure of 50 ° C., the suspension was autolyzed under the reaction conditions of 50 ° C. for 7 hours, and then a protease was further added and the enzyme reaction was performed at 50 ° C. for 18 hours. The soluble bacterial components were removed by centrifugation, and the autolysis / enzyme reaction residue obtained here was used as a yeast cell wall fraction. The protein content of the yeast cell wall fraction was 21.3%, and the dietary fiber content (AOAC method) was 62.7%.
[0031]
Preparation Example 3
After accurately weighing the fermented brewer's yeast slurry obtained as a by-product from the beer brewing process, water was added so that the solid content was 10% by weight. Sodium hydroxide was added until the pH became 9, and the mixture was stirred at 10 ° C., centrifuged, washed by adding water to the precipitate fraction, and then centrifuged again. A suspension hydrated so that the solid content becomes 10% by weight is 700 kg / cm. 2 After performing a high-pressure homogenizer treatment while cooling under a pressure of 50 ° C., the suspension was autolyzed under the reaction conditions of 50 ° C. for 7 hours, and then a protease was further added and the enzyme reaction was performed at 50 ° C. for 18 hours. The soluble cell components were removed by centrifugation. The operation of adding water so that the solid content of this fraction was 10% by weight, washing and centrifuging after washing was repeated twice, and the precipitate fraction obtained here was used as a yeast cell wall fraction. The protein content of the yeast cell wall fraction was 14.0%, and the dietary fiber content (AOAC method) was 67.3%.
[0032]
Preparation Example 4
After accurately weighing the fermented brewer's yeast slurry obtained as a by-product from the beer brewing process, water was added so that the solid content was 10% by weight. Sodium hydroxide was added until the pH became 9, and the mixture was stirred at 10 ° C., centrifuged, washed by adding water to the precipitate fraction, and then centrifuged again. A suspension hydrated so that the solid content becomes 10% by weight is 700 kg / cm. 2 After performing a high-pressure homogenizer treatment while cooling under a pressure of 50 ° C., the suspension was autolyzed under the reaction conditions of 50 ° C. for 7 hours, and then a protease was further added and the enzyme reaction was performed at 50 ° C. for 18 hours. The soluble cell components were removed by centrifugation. The obtained autolysis / enzyme reaction residue was washed with water and centrifuged again, and then a suspension hydrated so that the solid content became 10% by weight was 700 kg / cm. 2 The mixture was subjected to a high-pressure homogenizer treatment while being cooled under the pressure described above to obtain a yeast cell wall fraction. Table 1 shows the component analysis values of the yeast cell wall fraction.
[0033]
[Table 1]
[0034]
Test example 1
In order to compare the swelling ability of the yeast cell wall fraction obtained in Preparation Example 2 (hereinafter referred to as “the present invention product 1”) in water with the swelling ability of other typical dietary fiber materials, The settling volume in water in an artificially reproduced environment was measured. In addition to the product 1 of the present invention, commercially available cellulose, wheat bran, corn fiber, beet fiber, and yeast cell wall ("Imsel BF" manufactured by Tanabe Seiyaku Co., Ltd .; hereinafter, referred to as "comparative product") as a sample, each of which is 1 g. Each in a 100 mL medium bottle, and a 1/15 M phosphate buffer (Na 2 HPO 4 4.7 g, KH 2 PO 4 Was taken up, and distilled water was added thereto to add 50 mL of constant volume to 1 L, pH 6.8), followed by stirring. After sonication and degassing were performed for 1 minute, and sonication was further continued for 3 minutes, the solution was transferred to a 100 mL measuring cylinder, and the above buffer was added to make the volume to 100 mL. After standing for 24 hours, the settling volume (mL / g) of each sample was measured. Table 2 shows the results. As can be seen from Table 2, it was found that the product 1 of the present invention had a higher swelling ability in water than other typical dietary fiber materials and a comparative example product which was a similar yeast cell wall material. From the above, the product of the present invention, which exerts a remarkable effect on suppressing increase and decrease in bone density, has a very high swelling property, that is, extremely high dispersibility in water, despite containing water-insoluble dietary fiber. It became clear, and it was confirmed that it is possible to provide a pharmacological composition as a more easily ingestible material.
[0035]
[Table 2]
[0036]
Test example 2
An experiment was performed on the effect of the yeast cell wall fraction obtained in Preparation Example 3 (hereinafter referred to as “the present invention product 2”) on Ca absorption from the digestive tract and bone density using healthy rats. Wistar male rats (7 weeks old, 150 to 170 g) were used as test animals, and were preliminarily reared on a control diet shown in Table 3 for one week before administration of the test diet, so as to acclimate to the experimental environment. After the test, these rats were used after being divided into eight rats in each group. As test feeds (test samples), two groups of the product of the present invention prepared according to Preparation Example 3 whose composition is shown in Table 3, a control group containing no dietary fiber component, and yeast cell wall fractions in the same amount Using a group of comparative examples containing a yeast cell wall (Imsel BF), a test feed was given to rats by free intake and bred for 2 weeks. The amounts of casein and soybean oil were corrected so that the protein content and the lipid content of the feed were equal by measuring the protein content and the lipid content of the product 2 of the present invention or the comparative product. Two weeks after administration of the test feed, feces, right femur and abdominal aortic blood were collected, and fecal excretion, fecal Ca excretion, and serum Ca concentration were measured. The collected stool was immediately lyophilized, the dry weight was measured, and the collected blood was immediately centrifuged to separate serum. For the femur, the bone density and the amount of bone mineral were measured by the pQCT method.
[0037]
Bone is roughly divided into cortical bone and cancellous bone, but the turnover is faster in the cancellous bone inside, and the strength of the whole bone decreases as the cancellous bone in the center becomes brittle, so the bone density of cancellous bone Since the increase in osteoporosis is one of the important points in the prevention of osteoporosis, the effect was determined using total bone density, trabecular bone density, total bone mineral density and cancellous bone mineral density as indices. Table 4 shows the results. Ingestion of the product of the present invention obtained in the present invention significantly reduced the amount of Ca excreted in feces, indicating that the rate of absorption of Ca into the body was improved. Furthermore, in the comparison of total bone density, trabecular bone density, total bone mineral density and trabecular bone mineral density, the two groups of the present invention showed significantly higher values than the other two groups. From the above, it is clear that the product 2 of the present invention has an effect of increasing bone density, which is obtained when the dietary fiber component including the product of the present invention is not taken, or when the product of the comparative example is taken. The effect was remarkably higher than that.
[0038]
[Table 3]
[0039]
[Table 4]
[0040]
Test example 3
An experiment was conducted on the effect of the yeast cell wall fraction obtained in Preparation Example 4 (hereinafter referred to as “the present invention product 3”) on Ca absorption from the digestive tract and bone density using healthy rats. As test animals, male Wistar rats (7 weeks old, 150 to 170 g) were pre-bred for 1 week before administration of the test feed with the composition feed of the control group shown in Table 5 to acclimate to the experimental environment. After the test, these rats were used after being divided into eight rats in each group. As test feeds (test samples), yeasts of the present invention group prepared according to Preparation Example 4 whose composition is shown in Table 5, a control group containing no dietary fiber, and yeast having the same amount as the yeast cell wall fraction Using a group of comparative examples containing a cell wall (Imsel BF), test feed was given to rats by free intake, and the rats were bred for 2 weeks. By measuring the protein content and the lipid content of the product 3 of the present invention or the comparative product, the amounts of casein and soybean oil were corrected so that the protein content and the lipid content in the feed became equal. Two weeks after administration of the test feed, feces, right femur and abdominal aortic blood were collected, and fecal excretion, fecal Ca excretion, and serum Ca concentration were measured. The collected stool was immediately lyophilized, the dry weight was measured, and the collected blood was immediately centrifuged to separate serum. For the femur, the bone density and the amount of bone mineral were measured by the pQCT method.
[0041]
The effect was determined using the total bone density, trabecular bone density, total bone mineral density, and trabecular bone mineral density as an index of the effect, as in Test Example 2. Table 6 shows the results. Ingestion of the product 3 of the present invention obtained in the present invention significantly reduced the amount of Ca excreted in feces, indicating that the rate of absorption of Ca into the body was improved. Furthermore, in the comparison of the bone mineral density of cancellous bone, the three groups of the present invention showed significantly higher values than the group of the comparative example. Further, when the canine bone density was compared between the product group of the present invention and the comparative product group, the p-value was not significant at the level of 5%, but the p-value was as significant as p = 0.07. The high effect of the invention product 3 on the comparative product was found. From the above, it is clear that the product 3 of the present invention has a bone tissue enhancing effect, which is due to the case where the dietary fiber component including the product 3 of the present invention is not taken, or the case where the product of the comparative example is taken. The effect was significantly higher than that of.
[0042]
[Table 5]
[0043]
[Table 6]
[0044]
Test example 4
Effect of the yeast cell wall fraction obtained in Preparation Example 2 (hereinafter referred to as "the present invention product 1") on bone density using a bone mineral density model rat (osteoporosis model) produced by reducing the amount of Ca in the diet The experiment about was performed. Male SD rats (3 weeks old, 45-55 g) were used as test animals, and reared on a Low Ca diet (0.003% Ca) shown in Table 7 for 2 weeks to produce a bone density reduction model. Thereafter, these rats were divided into eight rats in each group. Cellulose (Abicel, manufactured by Asahi Kasei Co., Ltd.), a typical dietary fiber material, was used as a feed to be subsequently tested in rats so that the dietary fiber content of the 1-combined feed of the present invention and the control feed were equal. A control feed compounded as a product (hereinafter referred to as “Comparative Product 2”) was set. As a test feed, the composition shown in Table 8 is composed of a normal Ca-containing comparative product 2 blended feed (0.5% Ca), a normal Ca-containing 10% present product 1 blended feed (0.5% Ca), High Ca The comparative feed containing 2 comparative examples (1.2% Ca) and the 10% high Ca-containing feed containing 1% of the present invention (1.2% Ca) were freely taken for 2 weeks. The Ca content in each feed was corrected by adjusting the Ca content in a mineral mixture composition based on AIN-93G, which is a feed composition. Two weeks after the administration of the test feed, blood from the right femur and abdominal aorta was collected. The collected blood was immediately centrifuged to collect serum, and then the Ca concentration in serum was measured. For the femur, the bone density and the amount of bone mineral were measured by the pQCT method.
[0045]
Bone is roughly divided into cortical bone and cancellous bone.If bone density decreases due to reduction of dietary Ca content, bone resorption first proceeds from cortical bone, resulting in a decrease in bone density. From, the effect was determined using the total bone density and cortical bone density as indices. The results are shown in Tables 9 and 10. No significant difference was observed in the effect on the bone density due to the combination of the product of the present invention 1 in the normal Ca diet, but the intake of the product 1 of the present invention in the High Ca diet resulted in a total bone density and cortical bone as compared with the control. The decrease in density was significantly suppressed. From the above, it has been clarified that the product 1 of the present invention has an effect of suppressing osteoporosis-like symptoms caused by a decrease in bone density caused by a decrease in the amount of Ca in a diet. This effect was remarkably higher than that in the case of ingesting Comparative Example Product 2, which was a dietary fiber material.
[0046]
[Table 7]
[0047]
[Table 8]
[0048]
[Table 9]
[0049]
[Table 10]
[0050]
Test example 5
Using a rat model of menopausal osteoporosis caused by estrogen deficiency by performing ovariectomy, using yeast cell wall fraction obtained in Preparation Example 2 (hereinafter referred to as "the present invention 1") to evaluate the effect on bone density. An experiment was performed.
Wistar female rats (8 weeks old, 180-210 g) were acclimated for 1 week with solid feed (CE-2, manufactured by CLEA Japan), and the rats were divided into 10 rats per group. As shown in Table 11, a 10% High Ca-containing feed containing 1% of the present invention (2.5% Ca) and a High Ca feed not containing the present product (2.5% Ca) were administered as control feeds for 1 week, Bilateral ovariectomy was performed. Subsequently, after the same test feed was freely ingested for 3 weeks, the right femur and abdominal aortic blood were collected. The collected blood was immediately centrifuged to collect serum, and then the serum Ca concentration was measured. For the femur, the bone density was measured by the pQCT method.
[0051]
In the case of a model of menopausal osteoporosis caused by estrogen deficiency, increasing the bone density of trabecular bone is one of the important points in preventing osteoporosis, because the bones become weaker from the central cancellous bone and the strength of the whole bone decreases. Therefore, the effect was determined using the total bone density and the cancellous bone density as indices. In addition, a reduction in bone density of ovariectomized rats was confirmed by separately setting a sham operation group ingesting the non-combined feed of the product 1 of the present invention. Table 12 shows the results. The ingestion of the product 1 of the present invention significantly reduced the decrease in trabecular bone density as compared with the control. From the above, it was clarified that ingestion of the product of the present invention promoted Ca absorption or bone formation and contributed to improvement or prevention of osteoporosis symptoms.
[0052]
[Table 11]
[0053]
[Table 12]
[0054]
Test Example 6
After being ingested, the yeast cell wall fraction of the present invention reaches the lower gastrointestinal tract almost without being affected by digestive enzymes and physical actions in the stomach, where it becomes a fermentation substrate for intestinal bacteria, thereby producing acetic acid and propion. It is converted into short-chain fatty acids such as acid and butyric acid, but the action of the short-chain fatty acids keeps the gastrointestinal tract in an acidic region, thereby increasing the solubilization rate of calcium and other minerals present on the mucosal side of the gastrointestinal tract. Therefore, the mineral absorption is promoted, and the effect of increasing the bone density and the effect of suppressing the decrease in the bone density are exerted. Although the yeast cell wall fraction in the present invention contains abundant indigestible components such as dietary fiber which can be a substrate for producing short-chain fatty acids, Preparation Example 2 was used to examine the abundance of the dietary fiber component. , The yeast cell wall fraction obtained in Preparation Example 3 (hereinafter referred to as “Product 2 of the present invention”), the yeast cell obtained in Preparation Example 4 The AOAC official Prosky method was used to determine the dietary fiber content present in the wall fraction (hereinafter referred to as "the present product 3") and similar yeast cell walls ("Imsel BF" manufactured by Tanabe Seiyaku Co., Ltd .; hereinafter referred to as "comparative sample"). Was measured. As a result (Table 13), it was found that the product 1, the product 2 and the product 3 of the present invention contained 60% or more of dietary fiber. On the other hand, it was found that the amount of glucose contained in the comparative example product was 54.5%, and that sufficient mineral absorption promotion, increase in bone density and suppression of bone density decrease were caused by the presence of 60% or more of dietary fiber. Was required.
[0055]
[Table 13]
[0056]
Test example 7
Test Example 6 revealed that the yeast cell wall fraction of the present invention contained abundant indigestible components such as dietary fiber which could be a substrate for producing short-chain fatty acids. Among them, the yeast cell wall fraction obtained in Preparation Example 3 (hereinafter referred to as "the present invention 2") was used in order to examine the abundance of β-1,3-glucan and β-1,6-glucan, which are the main components. Present in the yeast cell wall fraction obtained in Preparation Example 4 (hereinafter referred to as "the present invention product 3") and a similar yeast cell wall ("Imcel BF" manufactured by Tanabe Seiyaku Co., Ltd .; hereinafter referred to as "comparative example product"). The glucose content derived from β-glucan was determined by decomposing the product of the present invention or the comparative product by an ordinary acid hydrolysis method, and then by an absolute calibration curve method using high performance liquid chromatography.
[0057]
As a result (Table 14), it was found that the present invention product 2 and the present invention product 3 contained 49% or more of glucose. On the other hand, the amount of glucose contained in the comparative example product was found to be 40.1%, and 41% or more of glucose, that is, glucose, was sufficient for promoting sufficient mineral absorption, increasing bone density and suppressing bone density reduction. It was confirmed that the presence of β-glucan, a polysaccharide having glucose as a constituent sugar, was important. In comparison of the total content of glucose and mannose, which are constituent polysaccharides that can be assimilated by intestinal bacterial cells, the product 2 of the present invention and the product 3 of the present invention contain 75% or more of a polysaccharide. There was found. On the other hand, it was found that the total amount of polysaccharides contained in the comparative example product was 61.8%, and that the sufficient effect of promoting mineral absorption, increasing bone density and suppressing bone density reduction was not less than 63%. It was confirmed that the presence of the components, that is, the presence of a polysaccharide component having glucose and mannose as constituent sugars was important.
[0058]
[Table 14]
[0059]
Test Example 8
The yeast cell wall fraction in the present invention is converted into short-chain fatty acids such as acetic acid, propionic acid and butyric acid by being taken up and reaching the digestive tract, and then becoming a fermentation substrate for intestinal bacteria. By the action, the solubilization rate of minerals such as calcium present in the digestive tract is increased, thereby promoting the absorption of minerals, and furthermore, the effect of increasing bone density and the effect of suppressing bone density decrease are brought about. The yeast cell wall fraction obtained in Preparation Example 3 (hereinafter referred to as “the present invention product 2”) was used to examine how much the yeast cell wall fraction is fermented by intestinal bacteria to produce short-chain fatty acids. The yeast cell wall fraction obtained in Preparation Example 4 (hereinafter referred to as "the present invention product 3") and a similar yeast cell wall ("Imcel BF" manufactured by Tanabe Seiyaku Co., Ltd .; hereinafter referred to as "comparative example product") were used in normal rats. After administration, an experiment was performed to measure the amount of short-chain fatty acids produced in the cecum after ingestion. As a test animal, a Wistar male rat (7 weeks old, 150 to 170 g) was preliminarily reared with a composition feed of the control group shown in Table 3 of Test Example 2 for one week before administration of the test feed. After acclimation, rats were divided into eight groups and used.
[0060]
As a test group, a group of the present invention 2 prepared by Preparation Example 3 whose composition is shown in Table 3 of Test Example 2, and a preparation group prepared by Preparation Example 4 whose composition is shown in Table 5 of Test Example 3 In addition, three groups of the present invention product and a comparative example product group containing a comparative example product having the same amount as the yeast cell wall fraction were set, and the test feed was given to rats by free intake, and the rats were bred for 2 weeks. A control group to which a feed containing no dietary fiber material was administered was also set. By measuring the protein content and the lipid content of the product of the present invention 2, the product of the present invention 3 or the comparative product, the amounts of casein and soybean oil were corrected so that the protein content and the lipid content in the feed became equal. Dissection was performed 2 weeks after administration of the test feed, and after the fermentation product in the cecum was collected, water was immediately added and sufficiently dispersed, and short-chain fatty acids were extracted by centrifugation and filtration. It was fractionated as a cecal content extract. The content of short-chain fatty acids present in the extract was measured by an absolute calibration method using high performance liquid chromatography. As a result (Table 15), significantly higher production of short-chain fatty acids was observed by ingesting the product of the present invention 2 or the product of the present invention 3 as compared with the control and comparative example products. In addition, this effect was significantly higher than when the comparative product was ingested. From the above, it has been clarified that ingestion of the product of the present invention promotes Ca absorption or bone formation and is effective for amelioration or prevention of osteoporosis symptoms.
[0061]
[Table 15]
[0062]
【The invention's effect】
By the mineral absorption enhancer of the present invention, including the suppression of bone loss and prevention of osteoporosis in the elderly, the improvement of symptoms of osteoporosis patients, and the maintenance and improvement of bone mass and bone density in general women and pregnant women, such as Ca It is possible to provide a pharmacologically active composition that effectively promotes the absorption of minerals, and is effective for increasing bone density and preventing osteoporosis and improving symptoms, with fewer side effects and safe. The active ingredient of the present invention has a high dispersibility in water and can be taken as a more easily ingestible material, as a dosage form as various pharmacologically active compositions, or as a food or drink containing the same.
