JP2007084531A - Muscle damage inhibitory composition - Google Patents
Muscle damage inhibitory composition Download PDFInfo
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- JP2007084531A JP2007084531A JP2006223895A JP2006223895A JP2007084531A JP 2007084531 A JP2007084531 A JP 2007084531A JP 2006223895 A JP2006223895 A JP 2006223895A JP 2006223895 A JP2006223895 A JP 2006223895A JP 2007084531 A JP2007084531 A JP 2007084531A
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- Prior art keywords
- acetic acid
- muscle damage
- vinegar
- organic solvent
- acid bacteria
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Abstract
Description
本発明は、運動時等により発症する筋肉の損傷を抑制する作用を有する組成物に関し、さらに詳細には、酢酸菌または食酢の脂溶性有機溶剤抽出物を有効成分とする筋肉損傷抑制組成物に関する。さらに、本発明は、酢酸菌のアルカリ安定脂質、アルカリ安定脂質中のＮ−アシルスフィンガニン、スフィンガニン、アミノ脂質またはテルペノイド化合物を有効成分とする筋肉損傷抑制組成物に関する。 The present invention relates to a composition having an action of suppressing muscle damage caused by exercise or the like, and more particularly, to a composition for suppressing muscle damage comprising a fat-soluble organic solvent extract of acetic acid bacteria or vinegar as an active ingredient. . Furthermore, the present invention relates to a composition for inhibiting muscle damage, comprising an alkali-stable lipid of acetic acid bacteria, N-acyl sphinganine, sphinganine, aminolipid or terpenoid compound in the alkali-stable lipid as an active ingredient.
家事、庭仕事、通勤のための歩行などの日常生活の活動、余暇に行なう趣味・レジャー活動などの全ての身体活動が健康を維持する上で有用ではあるが、特にスポーツなどの運動は、より健康維持向上に有益であると考えられ、競技者だけでなく、一般人にも普及している。 Daily physical activities such as housework, garden work, walking for commuting, and all physical activities such as hobbies and leisure activities during leisure are useful for maintaining health. It is considered beneficial for health maintenance and is popular not only for athletes but also for the general public.
しかしながら、過度のスポーツなどの運動を行うと筋肉が損傷する場合があり、時には逆効果となる場合が起こり得るとされている。
このような筋肉の損傷は運動過負荷によって引き起こされ、一部の筋肉に損傷が起きると、損傷した筋肉の細胞がサイトカインやケモカインを分泌し、好中球やマクロファージの浸潤を促すことが知られている。そして、好中球が増加して生成する活性酸素種や、また、好中球から放出されるミエロペルオキシダーゼ酵素（以下、ＭＰＯと称する場合もある）の作用によって生成する次亜塩素酸などが、周辺の筋肉に損傷を与えるといった増幅作用によって、炎症を伴う症状が発症すると考えられている。このことから、ＭＰＯ活性を調べることによって、運動過負荷による筋肉の損傷の程度を知ることができる。
However, it is said that if exercise such as excessive sports is performed, muscles may be damaged and sometimes adverse effects may occur.
It is known that such muscle damage is caused by exercise overload, and when some muscles are damaged, damaged muscle cells secrete cytokines and chemokines and promote infiltration of neutrophils and macrophages. ing. And the reactive oxygen species produced by neutrophil increase, hypochlorous acid produced by the action of myeloperoxidase enzyme (hereinafter also referred to as MPO) released from neutrophils, etc. It is believed that symptoms accompanied by inflammation develop due to amplification effects such as damage to surrounding muscles. From this, it is possible to know the degree of muscle damage due to exercise overload by examining the MPO activity.
また、サイトカインのひとつであるインターロイキン−６（以下、ＩＬ−６と称する場合もある）や、ケモカインのひとつである好中球の遊走に関与する好中球走化性因子−１（以下、ＣＸＣＬ−１と称する場合もある）などの分泌量や遺伝子発現量を調べることによっても、筋肉の損傷の程度を知ることができる。 In addition, interleukin-6 (hereinafter also referred to as IL-6), which is one of cytokines, and neutrophil chemotactic factor-1 (hereinafter, which is involved in the migration of neutrophils, which is one of chemokines). The degree of muscle damage can also be determined by examining the secretion amount and gene expression level (sometimes referred to as CXCL-1).
このような筋肉の損傷に伴う炎症に対しては、フェニルブタゾン、サリチル酸誘導体、インドメタシン、フェニル酢酸等の非ステロイド消炎物質などが知られている。 Non-steroidal anti-inflammatory substances such as phenylbutazone, salicylic acid derivatives, indomethacin, and phenylacetic acid are known for inflammation associated with such muscle damage.
しかしながら、該非ステロイド消炎物質の作用は、炎症が起きてからの回復治癒を助けるものであって、筋肉の損傷を抑制防止する作用は有していない。
しかも、該非ステロイド消炎物質は、筋粘膜の損傷や胃潰瘍などの消化器系への副作用を有している。
そこで、これらの副作用を抑制するために、該非ステロイド消炎物質に燐脂質を配合した組成物が提唱されたりもしている（例えば、特許文献１参照）が、必ずしも満足しうるものではなかった。
However, the action of the non-steroidal anti-inflammatory substance helps recovery and healing after inflammation occurs, and does not have the action of suppressing and preventing muscle damage.
Moreover, the non-steroidal anti-inflammatory substance has side effects on the digestive system such as muscular mucosa damage and gastric ulcer.
Therefore, in order to suppress these side effects, a composition in which a phospholipid is blended with the non-steroidal anti-inflammatory substance has been proposed (for example, see Patent Document 1), but it is not always satisfactory.
より有効な作用を期待するには、筋肉の損傷を防ぐ機能を有するものであって、かつ、副作用などが極力少ない安全性の高い物質が求められる中で、例えばビタミンＥは運動過負荷によって作られた活性酸素種を消去することによって筋肉損傷の増幅作用を抑制できると考えられている（例えば、非特許文献１参照）。
また、リコピンなども筋蛋白分解を抑制し、筋損傷予防作用を有することが提唱されている（例えば、特許文献２参照）。
In order to expect more effective action, vitamin E is produced by exercise overload, for example, because it has a function of preventing muscle damage and a highly safe substance with as few side effects as possible. It is considered that the amplification effect of muscle damage can be suppressed by eliminating the generated reactive oxygen species (see, for example, Non-Patent Document 1).
In addition, lycopene and the like have been proposed to suppress muscle protein degradation and have a muscle damage preventing action (see, for example, Patent Document 2).
しかしながら、これらの物質は安全性がより高いと考えられるものの、筋肉の損傷を抑制予防する作用は充分満足できるものではなかった。
このため、安全性が高く、より効果の強い筋肉損傷抑制組成物を開発することが求められていた。
However, although these substances are considered to be safer, the action of suppressing and preventing muscle damage has not been fully satisfactory.
For this reason, it has been required to develop a composition for suppressing muscle damage that is highly safe and more effective.
本発明は、上記した従来技術の問題点を解消し、安全性が高く、より筋肉損傷抑制効果の強い筋肉損傷抑制組成物を提供することを目的とするものである。 An object of the present invention is to solve the above-mentioned problems of the prior art, and to provide a muscle damage suppressing composition having high safety and a stronger muscle damage suppressing effect.
上記課題に鑑み、本発明者は、食酢製造菌などとして古来より使用されており、安全性の高いことが認知されている酢酸菌に着目した。なお、酢酸菌は、古来から人の食生活と深く係わっており、食酢製造以外にも、例えばヨーロッパの伝統食であるカスピ海ヨーグルトに酢酸菌が含まれ、歴史的な食経験のあることも知られている。
なお、酢酸菌は、食酢発酵工程中の発酵終了後において、濾過され、廃棄されているので、安価に入手することも可能である。
さらに、本発明者は、酢酸菌の発酵によって製造される食酢についても着目したが、食酢は古来から親しまれている酸味調味料であり、米酢、穀物酢、黒酢、玄米酢、リンゴ酢やぶどう酢といった果実酢、アルコール酢などがある。
In view of the above problems, the present inventor has focused on acetic acid bacteria that have been used since ancient times as vinegar-producing bacteria and are recognized as having high safety. In addition, acetic acid bacteria have been deeply related to human diet since ancient times, and in addition to vinegar production, for example, the Caspian Sea yogurt, a traditional European food, contains acetic acid bacteria and may have a historical dietary experience. Are known.
In addition, since acetic acid bacteria are filtered and discarded after the fermentation in the vinegar fermentation process, it is also possible to obtain it at low cost.
Furthermore, although this inventor paid attention also to the vinegar produced by fermentation of acetic acid bacteria, a vinegar is a sour seasoning loved from ancient times, and rice vinegar, grain vinegar, black vinegar, brown rice vinegar, apple vinegar There are fruit vinegars such as yam vinegar, alcohol vinegars and so on.
本発明者は、鋭意研究の結果、上記の酢酸菌または食酢から脂溶性有機溶剤で抽出して得られる脂溶性有機溶剤抽出物に筋肉損傷抑制作用が認められることを確認し、本発明を完成するに至った。
さらに、本発明者は、酢酸菌の脂溶性有機溶剤抽出物に含まれるアルカリ安定脂質に、さらにアルカリ安定脂質を構成するＮ−アシルスフィンガニン、スフィンガニン、アミノ脂質、テルペノイド化合物などに筋肉損傷抑制作用が認められることを確認して、本発明を完成するに至った。
As a result of diligent research, the present inventor confirmed that the fat-soluble organic solvent extract obtained by extraction from the above acetic acid bacteria or vinegar with a fat-soluble organic solvent has a muscle damage inhibitory action, and completed the present invention. It came to do.
Furthermore, the present inventor has found that the alkali-stable lipid contained in the fat-soluble organic solvent extract of acetic acid bacteria further suppresses muscle damage to N-acyl sphinganine, sphinganine, aminolipid, terpenoid compound and the like constituting the alkali-stable lipid. After confirming that the action is recognized, the present invention has been completed.
すなわち、請求項１に係る本発明は、酢酸菌の脂溶性有機溶剤抽出物を有効成分として含有することを特徴とする筋肉損傷抑制組成物を提供するものである。
また、請求項２に係る本発明は、脂溶性有機溶剤が酢酸エチルであることを特徴とする請求項１に記載の筋肉損傷抑制組成物を提供するものである。
また、請求項３に係る本発明は、脂溶性有機溶剤抽出物がアルカリ安定脂質であることを特徴とする請求項１に記載の筋肉損傷抑制組成物を提供するものである。
また、請求項４に係る本発明は、アルカリ安定脂質がＮ−アシルスフィンガニンであることを特徴とする請求項３に記載の筋肉損傷抑制組成物を提供するものである。
また、請求項５に関する本発明は、アルカリ安定脂質がスフィンガニンであることを特徴とする請求項３に記載の筋肉損傷抑制組成物を提供するものである。
また、請求項６に係る本発明は、アルカリ安定脂質がアミノ脂質であることを特徴とする請求項３に記載の筋肉損傷抑制組成物を提供するものである。
また、請求項７に係る本発明は、アルカリ安定脂質がテルペノイド化合物であることを特徴とする請求項３に記載の筋肉損傷抑制組成物を提供するものである。
さらに、請求項８に係る本発明は、食酢の脂溶性有機溶剤抽出物を有効成分として含有することを特徴とする筋肉損傷抑制組成物を提供するものである。
さらにまた、請求項９に係る本発明は、脂溶性有機溶剤が酢酸エチルであることを特徴とする請求項８に記載の筋肉損傷抑制組成物を提供するものである。
That is, the present invention according to
The present invention according to claim 2 provides the composition for inhibiting muscle damage according to
The present invention according to claim 3 provides the composition for inhibiting muscle damage according to
The present invention according to claim 4 provides the composition for inhibiting muscle damage according to claim 3, wherein the alkali-stable lipid is N-acyl sphinganine.
The present invention relating to claim 5 provides the composition for inhibiting muscle damage according to claim 3, wherein the alkali-stable lipid is sphinganine.
The present invention according to
The present invention according to
Furthermore, the present invention according to claim 8 provides a composition for inhibiting muscle damage, comprising a fat-soluble organic solvent extract of vinegar as an active ingredient.
Furthermore, the present invention according to claim 9 provides the composition for suppressing muscle damage according to claim 8, wherein the fat-soluble organic solvent is ethyl acetate.
本発明の酢酸菌または食酢の脂溶性有機溶剤抽出物は、経口摂取することにより、強い筋肉損傷抑制作用を発揮させることができる。さらに、本発明の酢酸菌の脂溶性有機溶剤抽出物に含まれるアルカリ安定脂質、さらにアルカリ安定脂質を構成するＮ−アシルスフィンガニン、スフィンガニン、アミノ脂質、テルペノイド化合物も、経口摂取することにより、強い筋肉損傷抑制作用を発揮させることができる。
従って、本発明によれば、安全性が高く、しかもより筋肉損傷抑制効果の強い筋肉損傷抑制組成物を提供することができ、本発明に係る筋肉損傷抑制組成物を、食品などとして経口摂取することによって、スポーツや激しい運動における筋肉損傷及び日常生活中の身体的活動に伴う軽度の筋肉損傷を予防できる効果が期待できる。
The fat-soluble organic solvent extract of acetic acid bacteria or vinegar according to the present invention can exert a strong muscle damage-inhibiting action when taken orally. Furthermore, by ingesting orally ingesting the alkali-stable lipid contained in the fat-soluble organic solvent extract of the acetic acid bacterium of the present invention, and further the N-acyl sphinganine, sphinganine, aminolipid, and terpenoid compound constituting the alkali-stable lipid, It can exert a strong muscular damage inhibiting action.
Therefore, according to the present invention, it is possible to provide a muscle damage suppressing composition having high safety and a stronger muscle damage suppressing effect, and the muscle damage suppressing composition according to the present invention is taken orally as food or the like. Therefore, it is possible to expect an effect of preventing muscle damage in sports or intense exercise and mild muscle damage accompanying physical activity in daily life.
以下、本発明を詳細に説明する。
本発明において用いられる酢酸菌としては、特に制限はなく、例えば、グルコンアセトバクター属（Gluconacetobacter）、グルコノバクター属（Gluconobacter）、アセトバクター属（Acetobacter）、アサイア属（Asaia）またはアシドモナス属（Acidomonas）などに属する酢酸菌が例示される。
Hereinafter, the present invention will be described in detail.
The acetic acid bacterium used in the present invention is not particularly limited. ) And the like.
さらに詳細には、まずグルコンアセトバクター属（Gluconacetobacter）の酢酸菌としては、グルコンアセトバクター・ハンゼニイ（Gluconacetobacter hansenii）、グルコンアセトバクター・ジアゾトロフィカス（Gluconacetobacter diazotrophicus）、グルコンアセトバクター・インタメデイウス（Gluconacetobacter intermedius）、グルコンアセトバクター・サッカリ（Gluconacetobacter sacchari）、グルコンアセトバクター・ザイリナス（Gluconacetobacter xylinus）、グルコンアセトバクター・ヨーロッパエウス（Gluconacetobacter europaeus）、グルコンアセトバクター・オボエディエンス（Gluconacetobacter oboediens）などが例示される。 More specifically, first, as acetic acid bacteria of the genus Gluconacetobacter, Gluconacetobacter hansenii, Gluconacetobacter diazotrophicus, Gluconacetobacter intermedius (Gluconacetobacter) intermedius), Gluconacetobacter sacchari, Gluconaacetobacter xylinus, Gluconacetobacter europaeus, Gluconaacetobacter oboediens (Gluconaedobacter) .
次に、グルコノバクター属（Gluconobacter）の酢酸菌としては、グルコノバクター・フラトウリ（Gluconobacter frateurii）、グルコノバクター・セリナス（Gluconobacter cerinus）などが例示される。 Next, examples of acetic acid bacteria belonging to the genus Gluconobacter include Gluconobacter frateurii, Gluconobacter cerinus, and the like.
また、アセトバクター属（Acetobacter）の酢酸菌としては、アセトバクター・トロピカリス（Acetobacter tropicalis）、アセトバクター・インドネンシス（Acetobacter indonesiensis）、アセトバクター・シジギイ（Acetobacter syzygii）、アセトバクター・シビノンゲンシス（Acetobacter cibinongensis）、アセトバクター・オリエンタリス（Acetobacter orientalis）、アセトバクター・パスツリアヌス（Acetobacter pasteurianus）、アセトバクター・オルレアネンシス（Acetobacter orleanensis）、アセトバクター・ロバニエンシス（Acetobacter lovaniensis）、アセトバクター・アセチ（Acetobacter aceti）、アセトバクター・ポモラム（Acetobacter pomorum）などが例示される。 Acetobacter acetic acid bacteria include Acetobacter tropicalis, Acetobacter indonesiensis, Acetobacter syzygii, Acetobacter cibinongensis (Acetobacter cibinongensis). , Acetobacter orientalis, Acetobacter pasteurianus, Acetobacter orleanensis, Acetobacter lovaniensis, Acetobacter lovaniensis, Acetobacter acetiacter -Pomoram (Acetobacter pomorum) etc. are illustrated.
さらに、アサイア属（Asaia）の酢酸菌としては、アサイア・ボゴレンシス（Asaia bogorensis）、アサイア・シアメンシス（Asaia siamensis）などが例示される。 Furthermore, as acetic acid bacteria of the genus Asaia (Asaia), Asaia bogorensis (Asaia bogorensis), Asaia siamensis (Asaia siamensis), etc. are illustrated.
また、アシドモナス属（Acidomonas）の酢酸菌としては、アシドモナス・メタノリカ（Acidomonas methanolica）が例示される。 Examples of acetic acid bacteria belonging to the genus Acidmonas include acidomonas methanolica.
さらに、酢酸菌としては、上記の他、食酢製造やヨーグルトなどの発酵食品に用いられている酢酸菌や、自然界より分離されたもの、また既存の微生物保存機関に保存されていて分譲可能な保存菌株などが適宜利用可能である。 Furthermore, as acetic acid bacteria, in addition to the above, acetic acid bacteria used in fermented foods such as vinegar production and yogurt, those isolated from the natural world, and preserved in existing microorganism storage institutions that can be distributed Strains and the like can be used as appropriate.
酢酸菌は酢酸発酵に用いられており、また、ナタデココ、カスピ海ヨーグルト、紅茶キノコなどで食経験があるので安全性が高いことが認知されており、特に、食酢発酵においては発酵後に濾過により分別されて廃棄されているので、安価に大量に入手することも可能である。 Acetic acid bacteria are used for acetic acid fermentation, and it has been recognized that they are highly safe because they have food experience with natadecoco, caspian sea yogurt, black tea mushrooms, etc. Especially in vinegar fermentation, it is separated by filtration after fermentation Since it is disposed of, it can be obtained in large quantities at a low cost.
また、本発明において用いられる食酢としては特に制限はなく、例えば米酢、穀物酢、黒酢、玄米酢、リンゴ酢やぶどう酢といった果実酢、アルコール酢等が挙げられるが、中でも穀物酢は、小麦、コーン、米などの穀物を原料として製造され汎用されており、比較的安価であることから好ましい。 The vinegar used in the present invention is not particularly limited, and examples include rice vinegar, grain vinegar, black vinegar, brown rice vinegar, fruit vinegar such as apple vinegar and grape vinegar, alcohol vinegar, etc. It is preferable because it is manufactured using cereals such as wheat, corn and rice as a raw material and is widely used.
本発明においては、請求項１または８に記載したように、上記の酢酸菌または食酢から、脂溶性有機溶剤を用いて抽出した有効成分を含む抽出物を用いて筋損傷抑制組成物が構成され、さらに請求項３〜７に記載したように、脂溶性有機溶剤抽出物から抽出されたアルカリ安定脂質や、該アルカリ安定脂質中のＮ−アシルスフィンガニン、スフィンガニン、アミノ脂質、テルペノイド化合物などを用いて筋損傷抑制組成物が構成されるが、例えば、これらの成分を有効成分として含有するものであれば良く、破砕処理した酢酸菌などを使用しても良い。
In the present invention, as described in
なお、酢酸菌の破砕処理は、常法に従えばよいが、例えば、超音波式破砕機や、フレンチプレスなどの高圧式破砕機を用いて実施される。 The acetic acid bacteria crushing treatment may be carried out in accordance with a conventional method, but is performed using, for example, an ultrasonic crusher or a high-pressure crusher such as a French press.
本発明で使用される脂溶性有機溶剤としては、例えば、酢酸エチル、フェノール、ｎ−ブチルアルコールなどの疎水性有機溶剤、またはこれらの疎水性有機溶剤とアセトン、エタノール、メタノール、プロパノール、イソプロパノール、ブタノールなどの親水性有機溶剤を組み合わせた混合溶剤、またはそれらを含有する水溶液と定義される。これらの中では、請求項２または９に記載したように、極性が中性である酢酸エチル、あるいはその混合溶剤が好ましい。 Examples of the fat-soluble organic solvent used in the present invention include hydrophobic organic solvents such as ethyl acetate, phenol, and n-butyl alcohol, or these hydrophobic organic solvents and acetone, ethanol, methanol, propanol, isopropanol, and butanol. It is defined as a mixed solvent combining hydrophilic organic solvents such as or an aqueous solution containing them. Among these, as described in claim 2 or 9, ethyl acetate having a neutral polarity, or a mixed solvent thereof is preferable.
なお、酢酸菌または食酢からの脂溶性有機溶剤抽出物の調製は、上記の脂溶性有機溶剤を用いて直接抽出すればよいが、特に、酢酸菌からの脂溶性有機溶剤抽出物の調製の際には、脂溶性有機溶剤での抽出作業に先立って、予め酢酸菌懸濁液をアセトンなどの親水性有機溶剤で処理することによって析出してくる物質を除去する工程を加えておく方が好ましい。 In addition, preparation of the fat-soluble organic solvent extract from acetic acid bacteria or vinegar may be performed directly using the above-mentioned fat-soluble organic solvent, but in particular when preparing the fat-soluble organic solvent extract from acetic acid bacteria. It is preferable to add a step of removing the precipitated substance by treating the acetic acid bacteria suspension with a hydrophilic organic solvent such as acetone in advance prior to the extraction with the fat-soluble organic solvent. .
酢酸菌由来のアルカリ安定脂質は、エタノール、アセトン、ヘキサン、クロロホルム、メタノール、酢酸エチル等の１種又は２種以上からなる有機溶媒を用いて酢酸菌から脂質類を抽出した脂質画分に対して、弱アルカリ分解処理を施してリン脂質を除去することにより調製することができる。食品用途での安全性、低極性溶媒が望ましいことを考慮すると、抽出溶媒としてはヘキサンやアセトンなどが適している。 Alkali-stable lipids derived from acetic acid bacteria are obtained by extracting lipids from acetic acid bacteria using an organic solvent consisting of one or more of ethanol, acetone, hexane, chloroform, methanol, ethyl acetate, etc. It can be prepared by applying a weak alkaline decomposition treatment to remove phospholipids. Considering safety in food use and the desire for a low polarity solvent, hexane, acetone or the like is suitable as the extraction solvent.
この様にして得られる酢酸菌のアルカリ安定脂質には、含有成分としてＮ−アシルスフィンガニン、スフィンガニン、アミノ脂質、テルぺノイド化合物、脂肪酸などが含まれる。これらの化合物は酢酸菌の菌体由来の膜脂質として一般的なものであり、アセトバクター属（Acetobacter）、グルコノバクター属（Gluconobacter）、グルコンアセトバクター属（Gluconacetobacter）などに属する酢酸菌に含有されているものであり、その化合物の詳細は以下の通りである。 The alkali-stable lipids of acetic acid bacteria thus obtained include N-acyl sphinganine, sphinganine, amino lipid, terpenoid compound, fatty acid and the like as contained components. These compounds are commonly used as membrane lipids derived from cells of acetic acid bacteria, and are contained in acetic acid bacteria belonging to the genus Acetobacter, Gluconobacter, Gluconacetobacter, etc. The details of the compounds are as follows.
すなわち、Ｎ−アシルスフィンガニンとしてはＮ−２’−ヒドロキシパルミトイル−スフィンガニン、Ｏ−１−グルクロニル−Ｎ−２’−ヒドロキシパルミトイル−スフィンガニン、Ｎ−シス−バクセノイル−スフィンガニン、Ｏ−１−グルクロニル−Ｎ−シス−バクセノイル−スフィンガニンなどを含む。 That is, as N-acyl sphinganine, N-2′-hydroxypalmitoyl-sphinganine, O-1-glucuronyl-N-2′-hydroxypalmitoyl-sphinganine, N-cis-bacenoyl-sphinganine, O-1-glucuronyl- N-cis-bexenoyl-sphinganine and the like.
アミノ脂質としては、オルニチルタウリン脂質３−（パルミトイル）−ヒドロキシパルミトイル−オルニチル−タウリン、オルニチン脂質３−（パルミトイル）−ヒドロキシパルミトイル−オルニチン、リゾオルニチン脂質３−ヒドロキシパルミトイル−オルニチン等を含む。 Amino lipids include ornithyl taurolipid 3- (palmitoyl) -hydroxypalmitoyl-ornithyl-taurine, ornithine lipid 3- (palmitoyl) -hydroxypalmitoyl-ornithine, lysoornithine lipid 3-hydroxypalmitoyl-ornithine and the like.
さらに、テルぺノイド化合物（ホパノイド化合物）としては、テトラヒドロキシバクテリオホパン（Ｃ３５−ペンタサイクリックテルペンアルコール）、ホパン−２２−オール、ホプ−２２（２９）−エンを含む。
そして、脂肪酸としては、例えば、シス−バクセン酸などが含まれる。
Furthermore, examples of the terpenoid compound (hopanoid compound) include tetrahydroxybacteriohopane (C35-pentacyclic terpene alcohol), hopan-22-ol, and hop-22 (29) -ene.
Examples of the fatty acid include cis-vaccenic acid.
Ｎ−アシルスフィンガニンは、アルカリ安定脂質に含まれるスフィンゴ脂質の一部を指し、スフィンゴイド塩基であるスフィンガニンに脂肪酸が結合した一般的にセラミドと総称される化合物であり、一般的に以下の化学式（１）で示される。なお、式（１）において、−ＣＯ−Ｒはアシル基を示す。式（１）において、アシル基を構成する脂肪酸は、ヒドロキシ脂肪酸またはノルマル脂肪酸、飽和炭化水素または不飽和炭化水素、直鎖状または分岐鎖状であり、炭素原子数は２から３０以下のものが知られている。 N-acyl sphinganine refers to a part of sphingolipids contained in alkali-stable lipids, and is a compound generally referred to as ceramide in which a fatty acid is bound to sphinganine, which is a sphingoid base. It is represented by chemical formula (1). In the formula (1), —CO—R represents an acyl group. In the formula (1), the fatty acid constituting the acyl group is a hydroxy fatty acid or normal fatty acid, a saturated hydrocarbon or an unsaturated hydrocarbon, a linear or branched chain, and has 2 to 30 carbon atoms. Are known.
なお、酢酸菌由来のＮ−アシルスフィンガニンとしては、アシル基を構成する脂肪酸としてミリスチン酸、パルミチン酸、ステアリン酸、パルミトレイン酸、オレイン酸、バクセン酸、リノール酸、２−ヒドロキシミリスチン酸、２−ヒドロキシパルミチン酸などが確認されており、特に、Ｎ−２’−ヒドロキシパルミトイル−スフィンガニン、Ｎ−パルミトイル−スフィンガニン、Ｎ−シス−バクセノイル−スフィンガニンがその組成比の多くを占める（例えば、「岩手大学大学院連合農学研究科博士論文」、後藤英嗣著、ｐ．１１〜４１、２００１年参照）。 As N-acyl sphinganine derived from acetic acid bacteria, myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, 2-hydroxymyristic acid, 2 as fatty acids constituting the acyl group -Hydroxypalmitic acid has been confirmed, and in particular, N-2'-hydroxypalmitoyl-sphinganine, N-palmitoyl-sphinganine, N-cis-buxenoyl-sphinganine occupies most of the composition ratio (for example, "Iwate University Graduate School of Agricultural Science Doctoral Dissertation ", Goto Hideaki, p. 11-41, 2001).
以上の化合物は酢酸菌の種類により、その含量比は異なるが、酢酸菌が属するグラム陰性菌においてのみ膜脂質の主要構成成分として存在するものであり、グラム陽性菌や真核生物など他の生物種の脂質成分としては存在しないか、または極微量の化合物類である。 Although the content ratio of these compounds varies depending on the type of acetic acid bacteria, it exists as a major component of membrane lipid only in Gram-negative bacteria to which acetic acid bacteria belong, and other organisms such as Gram-positive bacteria and eukaryotes. It does not exist as a lipid component of the species, or is a trace amount of compounds.
酢酸菌または食酢から、有機溶剤を用いて筋肉損傷抑制物質を抽出して筋肉損傷抑制組成物を製造するのは常法に従えばよく、例えば、酢酸菌を適当に超音波破砕後、有機溶剤溶液で筋肉損傷抑制物質を抽出後、濃縮する。
食酢も同様に、有機溶剤溶液で筋肉損傷抑制物質を抽出後、濃縮する。これらの濃縮物をそのまま筋肉損傷抑制組成物として用いることもできる。また、液体クロマトグラフィーや向流分配法などで筋肉損傷抑制物質を分離精製し、また、それを採取して、筋肉損傷抑制組成物として用いることができる。
Extracting a muscle damage inhibitory substance from an acetic acid bacterium or vinegar using an organic solvent to produce a muscle damage inhibiting composition may be carried out according to a conventional method. For example, after acetic acid bacteria are appropriately sonicated, an organic solvent Extract the muscle damage inhibitor with the solution and concentrate.
Similarly, vinegar is concentrated after extracting the muscle damage inhibitor with an organic solvent solution. These concentrates can also be used as a muscle damage inhibiting composition as they are. In addition, a muscle damage inhibitory substance can be separated and purified by liquid chromatography, countercurrent distribution, or the like, and can be collected and used as a muscle damage inhibitory composition.
さらに、例えば、ビタミンＥ，コエンザイムＱ１０などの抗酸化物質、アミノ酸、大豆プロテイン、乳プロテイン等を併用して、本発明の筋肉損傷抑制組成物とすることもできる。 Furthermore, for example, antioxidant compositions such as vitamin E and coenzyme Q10, amino acids, soy protein, milk protein, and the like can be used in combination to form the muscle damage inhibiting composition of the present invention.
また、本発明の筋肉損傷抑制組成物の形態は、水、またはアルコール、含水アルコールなどに溶解した溶液の形態、並びにこれを乾燥して得られる粉末の形態、またはそれを成形して得られる錠剤の形態などとして用いることができる。さらに、運動補助食品、飲食品、医薬品など、その形態は特に限定されるものではない。
飲食品の形態としては、具体的にはトマト，にんじん等を原料とした野菜ジュース、リンゴ，パイナップル，グレープフルーツ，オレンジ，桃等を原料とした果物ジュース、または野菜ジュースと果物ジュースの混合品、アルコール飲料、牛乳，ヨーグルト等の乳製品、スポーツ飲料、コーヒー、紅茶，緑茶，ウーロン茶などの茶製品、または黒酢、穀物酢、米酢、玄米酢、黒酢、アルコール酢、リンゴ酢やぶどう酢など果実を原料に含む果実酢、野菜を原料に含む野菜酢などの食酢製品、キャンデイ，ガム，ゼリー，アイスクリーム，クッキー等の菓子類、食パン，米飯，麺等の主食品等に混合することが挙げられる。
In addition, the form of the composition for inhibiting muscle damage of the present invention is in the form of a solution dissolved in water, alcohol, hydrous alcohol or the like, and in the form of a powder obtained by drying it, or a tablet obtained by molding it. It can be used as a form. Furthermore, the form is not specifically limited, such as an exercise supplement, food / beverage products, and pharmaceuticals.
The form of food and drink specifically includes vegetable juices made from tomatoes, carrots, etc., fruit juices made from apples, pineapples, grapefruits, oranges, peaches, etc., or a mixture of vegetable juices and fruit juices, alcohol Beverages, milk products such as milk, yogurt, sports drinks, coffee products such as coffee, tea, green tea, oolong tea, black vinegar, grain vinegar, rice vinegar, brown rice vinegar, black vinegar, alcohol vinegar, apple vinegar, grape vinegar, etc. Can be mixed with fruit vinegar containing fruits, vinegar products such as vegetable vinegar containing vegetables, sweets such as candy, gum, jelly, ice cream and cookies, main foods such as bread, cooked rice and noodles Can be mentioned.
上記形態に本発明の筋肉損傷抑制組成物をあわせることで、その他の天然物由来の健康機能成分が含有され、相加効果乃至相乗効果が期待できる。 By combining the muscle damage-suppressing composition of the present invention with the above form, other natural product-derived health functional ingredients are contained, and an additive effect or a synergistic effect can be expected.
本発明の筋肉損傷抑制組成物としての酢酸菌脂溶性有機溶剤抽出物、酢酸菌アルカリ安定脂質、Ｎ−アシルスフィンガニン、スフィンガニン、アミノ脂質またはテルぺノイド化合物または食酢脂溶性有機溶剤抽出物の投与量は、成人１日当たり０．００１ｍｇ〜１００ｇ、好ましくは０．１ｍｇ〜１０ｇである。ここで成人１日当たり０．００１ｍｇ未満の投与量では、筋肉損傷を十分に抑制することはできない。 Acetic acid bacterium fat-soluble organic solvent extract, acetic acid bacterium alkali-stable lipid, N-acyl sphinganine, sphinganine, amino lipid or terpenoid compound or vinegar-fat soluble organic solvent extract as the muscle damage inhibiting composition of the present invention The dosage is 0.001 mg to 100 g, preferably 0.1 mg to 10 g per day for an adult. Here, at a dose of less than 0.001 mg per day for an adult, muscle damage cannot be sufficiently suppressed.
本発明の筋肉損傷抑制組成物は、酢酸菌脂溶性有機溶剤抽出物、酢酸菌アルカリ安定脂質、Ｎ−アシルスフィンガニン、スフィンガニン、アミノ脂質またはテルぺノイド化合物または食酢脂溶性有機溶剤抽出物を有効成分として含有するが、それ以外の各種原料とを混合・均一化した後に、必要に応じて界面活性剤を混合して、安定化を図ることもできる。
これらの界面活性剤としては、ソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、プロピレングリコール脂肪酸エステル、ショ糖脂肪酸エステルおよびレシチンなどを用いることができる。
The composition for inhibiting muscle damage of the present invention comprises an acetic acid bacterium fat-soluble organic solvent extract, an acetic acid bacterium alkali-stable lipid, an N-acyl sphinganine, a sphinganine, an amino lipid, a terpenoid compound, or a vinegar fat-soluble organic solvent extract. Although it is contained as an active ingredient, it can be stabilized by mixing and homogenizing other various raw materials and then mixing a surfactant as necessary.
As these surfactants, sorbitan fatty acid ester, glycerin fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester, lecithin and the like can be used.
本発明の筋肉損傷抑制組成物の適用箇所は、筋肉であれば特に制限はなく、ヒラメ筋，ヒフク筋などの骨格筋、平滑筋、呼吸筋、心筋などが例示されるが、特に骨格筋のヒラメ筋，ヒフク筋や心筋が好ましい。 The application site of the composition for inhibiting muscle damage of the present invention is not particularly limited as long as it is a muscle, and examples thereof include skeletal muscles such as soleus and hifuku muscles, smooth muscles, respiratory muscles, and cardiac muscles. Soleus, hifuku and myocardium are preferred.
本発明の筋肉損傷抑制組成物が効果を発揮し得る運動量には特に制限はなく、ヒトの最大摂取酸素量（ＶＯ２ｍａｘ）８０％以下の運動量でよいが、特に、４０〜６０％の運動量が好ましい。 There is no particular limitation on the amount of exercise that can exert the effect of the composition for suppressing muscle damage of the present invention, and the amount of exercise of human maximum intake oxygen amount (VO 2 max) may be 80% or less, and in particular, the amount of exercise is 40-60%. Is preferred.
本発明における筋肉損傷抑制効果については、下記試験法によって確認することができる。
すなわち、ラットをトレッドミルにより走行させて運動させる系を用いた。トレッドミルは、ヒトがランニングやウオ−キングを目的として使用するランニングマシーンのことで、本発明においては、ラット専用に小型化された機械のことを言う。このトレッドミルは、従来の強制水泳による運動方法と異なり、物理的に筋肉への負荷を行うことができるため、より厳密に運動による効果を検証できる。
The effect of inhibiting muscle damage in the present invention can be confirmed by the following test method.
That is, a system was used in which rats were moved by a treadmill and exercised. A treadmill is a running machine that humans use for the purpose of running and walking. In the present invention, the treadmill is a machine that is miniaturized exclusively for rats. Unlike the conventional method of exercise by forced swimming, this treadmill can physically apply a load on muscles, so that the effect of exercise can be verified more strictly.
本発明では、運動時の筋肉の損傷の指標として、トレッドミルにより走行させて運動させる系では食細胞の１つである好中球の顆粒にあるＭＰＯを用いた。この理由としては、運動負荷により筋肉の損傷が起こると、好中球が増加し、好中球中のＭＰＯが細胞外へ放出され、生体内の過酸化水素と反応して、反応性の高い次亜塩素酸を生成することで、さらに周辺組織に傷害を起こすことが知られており、ＭＰＯ活性を測定することによって運動負荷による損傷の程度を知ることができることによる。 In the present invention, as an index of muscle damage during exercise, MPO in neutrophil granule, which is one of phagocytic cells, was used in a system in which exercise was performed by running on a treadmill. The reason for this is that when muscle damage occurs due to exercise load, neutrophils increase, MPO in neutrophils is released out of the cell, reacts with hydrogen peroxide in the living body, and is highly reactive It is known that the generation of hypochlorous acid causes further damage to surrounding tissues, and the degree of damage due to exercise load can be determined by measuring the MPO activity.
さらに、細胞レベルでの本発明における損傷抑制効果を、筋管細胞を用いる系で検証した。筋管細胞とは筋芽細胞を分化誘導させた細胞で筋肉繊維様の形態を成す。なお、この方法は、動物を用いた試験方法よりも直接的に筋肉の損傷に対する効果を検証できる。 Furthermore, the damage suppression effect in the present invention at the cell level was verified by a system using myotube cells. Myotube cells are cells in which myoblasts are induced to differentiate and form a muscle fiber-like form. This method can directly verify the effect on muscle damage more than the test method using animals.
当該筋管細胞を用いる系では、筋肉の損傷の指標として一般的な炎症マーカーとして用いられているサイトカインＩＬ−６、好中球の遊走に関与するケモカインＣＸＣＬ−１を測定した。この理由としては、運動時における筋の損傷は機械的な筋肉の損傷だけでなく、その損傷に誘発された炎症反応が大きく関与していることが知られており、損傷した筋肉細胞がサイトカインやケモカインを分泌して、好中球やマクロファージの浸潤を促すと考えられているからである。このことから、筋肉の損傷の指標としてＩＬ−６、ＣＸＣＬ−１を測定した。 In the system using myotube cells, cytokine IL-6, which is used as a general inflammatory marker as an indicator of muscle damage, and chemokine CXCL-1, which is involved in neutrophil migration, were measured. The reason for this is that muscle damage during exercise is known not only to mechanical muscle damage, but also to the inflammatory response induced by that damage. This is because chemokines are secreted to promote infiltration of neutrophils and macrophages. Therefore, IL-6 and CXCL-1 were measured as indicators of muscle damage.
次に、本発明を実施例等により詳しく説明するが、本発明の範囲はこれらにより何ら限定されるものではない。 EXAMPLES Next, although an Example etc. demonstrate this invention in detail, the scope of the present invention is not limited at all by these.
製造例１（酢酸菌脂溶性有機溶剤抽出物の大量調製）
酢酸菌株としてはグルコンアセトバクター・ザイリナスNBRC15237（Gluconacetobacter xylinus NBRC15237）株を用いた。この株は、独立行政法人製品評価技術基盤機構バイオテクノロジー本部生物遺伝資源部門（千葉県木更津市かずさ鎌足２−５−８）に保存されており、譲受可能な酢酸菌である。
Production Example 1 (Manufacture of Acetic Acid Bacteria Fat-soluble Organic Solvent Extract)
Gluconacetobacter zylinus NBRC15237 (Gluconacetobacter xylinus NBRC15237) strain was used as the acetic acid strain. This strain is an acetic acid bacterium that is preserved in the Biological Genetic Resources Department of Biotechnology Headquarters, National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashichi, Chiba Prefecture).
まず、酢酸菌脂溶性有機溶剤抽出物は、以下の方法で調製した。酢酸菌乾燥菌体１．８kｇを水３５リットルに懸濁し、フレンチプレス圧力式細胞破砕機を用い、１０，０００ｐｓｉの破砕圧力で破砕後、アセトンと混合した。その後、混合によってできた夾雑物を除去するため濾過を行った。再度アセトンを混合して夾雑物を除去し、これを２回繰り返した。得られた濾過液を減圧濃縮し、濃縮物を酢酸エチルおよび水を加えて混合した後、分液を行い、有機層を得た。再度水層に酢酸エチルを加えて、同様に分液し有機層を回収した。有機層を減圧濃縮し、エタノールに溶解し、再度減圧濃縮して、酢酸菌脂溶性有機溶剤抽出物１０８ｇを得た。 First, an acetic acid bacteria-soluble organic solvent extract was prepared by the following method. 1.8 kg of dried acetic acid bacterial cells were suspended in 35 liters of water, crushed at a crushing pressure of 10,000 psi using a French press pressure cell crusher, and then mixed with acetone. Thereafter, filtration was performed to remove impurities formed by mixing. Acetone was mixed again to remove impurities, and this was repeated twice. The obtained filtrate was concentrated under reduced pressure, and the concentrate was mixed with ethyl acetate and water, followed by liquid separation to obtain an organic layer. Ethyl acetate was added to the aqueous layer again, and the mixture was separated in the same manner to recover the organic layer. The organic layer was concentrated under reduced pressure, dissolved in ethanol, and concentrated again under reduced pressure to obtain 108 g of acetic acid bacteria-soluble organic solvent extract.
製造例２（食酢脂溶性有機溶剤抽出物の大量調製）
穀物酢１０００リットルに対して、酢酸エチル１２００リットルを混合し、静置して分液後、有機層を回収して減圧濃縮を行った。１５リットル程度の容量になった時点でエタノールを同量投入し、攪拌し、さらに減圧濃縮を行い、食酢脂溶性有機溶剤抽出物２３４ｇを得た。
Production Example 2 (Manufacture of vinegar fat-soluble organic solvent extract)
To 1000 liters of grain vinegar, 1200 liters of ethyl acetate was mixed, allowed to stand and separated, and then the organic layer was recovered and concentrated under reduced pressure. When the volume reached about 15 liters, the same amount of ethanol was added, stirred, and concentrated under reduced pressure to obtain 234 g of a vinegar-fat-soluble organic solvent extract.
製造例３（酢酸菌アルカリ安定脂質の調製）
製造例１と同様に、酢酸菌株としてはグルコンアセトバクター・ザイリナスＮＢＲＣ１５２３７（Gluconacetobacter xylinus NBRC15237）株を用いた。
Production Example 3 (Preparation of alkali-stable acetic acid lipid)
As in Production Example 1, the strain Gluconacetobacter xylinus NBRC15237 (Gluconacetobacter xylinus NBRC15237) was used as the acetic acid strain.
まず、酢酸菌アルカリ安定脂質は以下の方法で調製した。すなわち、上記酢酸菌株の乾燥菌体２０００ｇを、クロロホルム−メタノール系の溶媒（クロロホルム：メタノール：水＝１：２：０．８）で抽出し、その後、２層分離を行い、下層を脂質粗画分として回収した。次に、０．４Ｎ ＮａＯＨにより脂質粗画分を弱アルカリ分解してリン脂質を除去し、フォルチの組成（クロロホルム：メタノール：水＝８：４：３）に従って再抽出を行い、濃縮乾固し、アルカリ安定脂質を得た。 First, acetic acid bacterium alkali-stable lipid was prepared by the following method. That is, 2000 g of dry cells of the above acetic acid strain was extracted with a chloroform-methanol solvent (chloroform: methanol: water = 1: 2: 0.8), then separated into two layers, and the lower layer was a crude lipid fraction Collected as minutes. Next, the crude lipid fraction is weakly alkaline-degraded with 0.4N NaOH to remove phospholipids, re-extracted according to the composition of forti (chloroform: methanol: water = 8: 4: 3), and concentrated to dryness. An alkali stable lipid was obtained.
得られた酢酸菌由来のアルカリ安定脂質に含まれる脂質成分を、シリカゲルプレートを用いた薄層クロマトグラフィーにて確認した。該薄層クロマトグラフィーにおける展開溶媒としては、脂肪酸、テルペノイド化合物、Ｎ−アシルスフィンガニンに関してはクロロホルム：メタノール＝９６：４を用い、また、スフィンガニンやアミノ脂質に関してはクロロホルム：メタノール：水＝６５：１６：２を用いた。 The lipid component contained in the obtained alkali-stable lipid derived from acetic acid bacteria was confirmed by thin layer chromatography using a silica gel plate. As a developing solvent in the thin layer chromatography, chloroform: methanol = 96: 4 is used for fatty acids, terpenoid compounds, and N-acyl sphinganine, and chloroform: methanol: water = 65: for sphinganine and aminolipids. 16: 2 was used.
薄層クロマトグラフィーの指標として、テルペノイド化合物、Ｎ−アシルスフィンガニン、アミノ脂質などを含むことが確認されている酢酸菌からの精製品（例えば、「帯大研報」、２３巻、ｐ．９１７〜９２５、１９７８年および「岩手大学大学院連合農学研究科博士論文」、後藤英嗣著、ｐ．１１〜４１、２００１年参照）ならびに市販のスフィンガニン（SIGMA社製）を用いた。薄層クロマトグラフィーの結果、アルカリ安定脂質中には、指標とした化合物が全て存在していることが確認された。 As an indicator of thin-layer chromatography, purified products from acetic acid bacteria that have been confirmed to contain terpenoid compounds, N-acyl sphinganine, aminolipids and the like (for example, “Obihiro Kenho”, Vol. 23, p. 917-925, 1978 and "Doctoral Dissertation of Iwate University Graduate School of Agricultural Sciences" by Eigo Goto, p.11-41, 2001) and commercially available sphinganine (manufactured by SIGMA) were used. As a result of thin layer chromatography, it was confirmed that all of the compounds used as indicators were present in the alkali-stable lipid.
また、アルカリ安定脂質中の主な成分の含有量を高速液体クロマトグラフィーによって確認した。すなわち、アルカリ安定脂質を無水ピリジン及び塩化ベンゾイル存在下にて７０℃で１０分間反応させ、テルペノイド化合物やＮ−アシルスフィンガニンを含んだベンゾイル誘導体を精製した。このベンゾイル誘導体を高速液体クロマトグラフィーに供し、紫外吸光２３０ｎｍの波長を検出した。上記、各化合物の市販標準品または構造が確認されている酢酸菌からの精製品を用いた標準曲線から、アルカリ安定脂質中に含まれる含量を算出した。高速液体クロマトグラフィーの移動相にはヘキサン：イソプロパノール＝１００：１の溶媒を用い、流速は１ｍｌ／分とした。 In addition, the content of main components in the alkali-stable lipid was confirmed by high performance liquid chromatography. That is, an alkali-stable lipid was reacted at 70 ° C. for 10 minutes in the presence of anhydrous pyridine and benzoyl chloride to purify a benzoyl derivative containing a terpenoid compound or N-acyl sphinganine. This benzoyl derivative was subjected to high performance liquid chromatography, and a wavelength of 230 nm in ultraviolet absorption was detected. The content contained in the alkali-stable lipid was calculated from the above-mentioned standard curve using commercially available standard products or purified products from acetic acid bacteria whose structures were confirmed. A solvent of hexane: isopropanol = 100: 1 was used as the mobile phase for high performance liquid chromatography, and the flow rate was 1 ml / min.
このようにして分析された酢酸菌由来のアルカリ安定脂質中の主な成分を下記の表１に示した。
なお、アミノ脂質の存在は確認（組成比は未確認）できたが、ホスファチジルコリン、ホスファチジルセリン、ホスファチジルグリセロールなどのリン脂質類の存在は確認されなかった。
The main components in the alkali-stable lipid derived from acetic acid bacteria analyzed in this way are shown in Table 1 below.
Although the presence of aminolipids was confirmed (composition ratio was not confirmed), the presence of phospholipids such as phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol was not confirmed.
製造例４（テルぺノイド化合物、Ｎ−アシルスフィンガニン、アミノ脂質、スフィンガニンの精製）
上記アルカリ安定脂質１０ｇを、クロロホルムで平衡化したシリカゲルカラムに供し、クロロホルム：酢酸＝９９：１の容量比で混合した溶媒で洗浄後、クロロホルム：メタノール＝９７：３の溶媒で溶出して得られた画分を乾固して、画分１を得た。
Production Example 4 (Purification of terpenoid compound, N-acyl sphinganine, amino lipid, sphinganine)
10 g of the above alkali-stable lipid is applied to a silica gel column equilibrated with chloroform, washed with a solvent mixed with a volume ratio of chloroform: acetic acid = 99: 1, and then eluted with a solvent of chloroform: methanol = 97: 3. The obtained fraction was dried to obtain a
続いて、クロロホルム：メタノール＝９６：４、９５：５の溶媒で溶出して得られた画分を乾固して、画分２を得た。さらに、クロロホルム：メタノール＝９２：８、９：１の溶媒で脂質を溶出して得られた画分を乾固して画分３を得た。さらにクロロホルム：メタノール＝２：１の溶媒で溶出して得られた画分を乾固して、画分４を得た。 Subsequently, the fraction obtained by elution with a solvent of chloroform: methanol = 96: 4, 95: 5 was dried to obtain fraction 2. Further, the fraction obtained by eluting the lipids with a solvent of chloroform: methanol = 92: 8, 9: 1 was dried to obtain a fraction 3. Further, the fraction obtained by elution with a solvent of chloroform: methanol = 2: 1 was dried to obtain a fraction 4.
得られた画分１について、シリカゲルプレートを用いた薄層クロマトグラフィーにて構成脂質を確認した。該薄層クロマトグラフィーにおける展開溶媒としては、クロロホルム：メタノール＝９６：４を用いた。その結果、画分１の構成脂質は、実施例１で用いたＮ−アシルスフィンガニンが確認されている酢酸菌からの精製品と同一の移動度である単一のスポットであった。さらに画分１を常法に従い含水メタノール性塩酸で分解し、薄層クロマトグラフィーにてスフィンゴイド塩基と脂肪酸に分離、精製した。
About the obtained
これらのスフィンゴイド塩基と脂肪酸それぞれについて、常法に従いトリメチルシリル化反応を行い、ガスクロマトグラフィー−マススペクトロメトリーにより構造分析したところ、Ｎ−アシルスフィンガニンとして、Ｎ−２’−ヒドロキシパルミトイル−スフィンガニン、Ｎ−パルミトイル−スフィンガニン、Ｎ−シス−バクセノイル−スフィンガニンを含んでいることが確認された。
以上の結果から、画分１の薄層クロマトグラフィーで検出される単一のスポットがＮ−アシルスフィンガニンであることが確認された。
For each of these sphingoid bases and fatty acids, trimethylsilylation was performed according to a conventional method, and structural analysis was performed by gas chromatography-mass spectrometry. As a result, N-2'-hydroxypalmitoyl-sphinganine, It was confirmed that N-palmitoyl-sphinganine and N-cis-buxenoyl-sphinganine were contained.
From the above results, it was confirmed that the single spot detected by thin layer chromatography of
さらに、画分２、画分３の構成脂質を薄層クロマトグラフィーにより確認した。薄層クロマトグラフィーにおける展開溶媒として、画分２はクロロホルム：メタノール＝９６：４の溶媒を用い、画分３、画分４はクロロホルム：メタノール：水＝６５：１６：２の溶媒を用いた。指標として、製造例３で用いたテルペノイド化合物、アミノ脂質の構造が確認されている酢酸菌からの精製品、ならびに市販のスフィンガニン（SIGMA社製）を用いた。 Furthermore, the constituent lipids of fraction 2 and fraction 3 were confirmed by thin layer chromatography. As a developing solvent in thin layer chromatography, a solvent of chloroform: methanol = 96: 4 was used for fraction 2, and a solvent of chloroform: methanol: water = 65: 16: 2 was used for fraction 3 and fraction 4. As indicators, the terpenoid compound used in Production Example 3, a purified product from acetic acid bacteria whose amino lipid structure was confirmed, and a commercially available sphinganine (manufactured by SIGMA) were used.
薄層クロマトグラフィーの結果、画分２はテルペノイド化合物の指標とスポットが一致し、画分３はスフィンガニン、画分４はアミノ脂質の指標と主なスポットが一致した。
以上の結果から、画分２で検出される薄層クロマトグラフィーのスポットがテルペノイド化合物を含むこと、画分３で検出される薄層クロマトグラフィーのスポットがスフィンガニンを含むこと、画分４で検出される薄層クロマトグラフィーのスポットがアミノ脂質を含むことが、それぞれ確認された。
As a result of thin-layer chromatography, fraction 2 matched the terpenoid compound index with the spot, fraction 3 matched sphinganine, and fraction 4 the aminolipid index matched the main spot.
From the above results, the thin-layer chromatography spot detected in fraction 2 contains a terpenoid compound, the thin-layer chromatography spot detected in fraction 3 contains sphinganine, and is detected in fraction 4. Each of the thin-layer chromatography spots was confirmed to contain amino lipids.
実施例１（軽程度運動量下での筋肉損傷抑制効果）
Ｃｒｌ:ＣＤ（ＳＤ）ラットを用い、１週間、１０分／日、トレッドミルによる運動負荷（ベルト勾配：６度、速度：１０ｍ／ｍｉｎ）をかけ、運動負荷を行った後、経口摂取による試料の投与を一週間行った。
餌は、固形飼料ＣＲＦ−１（オリエンタル酵母工業株式会社）を基本飼料とし、試料は基本飼料のみ（未投与区）、基本飼料に製造例１で作製した酢酸菌脂溶性有機溶剤抽出物を５００ｍｇ／ｋｇラット体重となるよう強制投与したもの（酢酸菌抽出物区）、製造例２で作製した食酢脂溶性有機溶剤抽出物を５００ｍｇ／ｋｇラット体重となるよう強制投与したもの（食酢抽出物区）の３群（ｎ＝８）とした。また、強制投与には、ディスポーザブルラット用経口ゾンデを取り付けたポリプロピレン製ディスポーザブル注射筒を用いた。
Example 1 (Inhibition effect on muscle damage under light exercise)
Using Crl: CD (SD) rats, applying a treadmill exercise load (belt gradient: 6 degrees, speed: 10 m / min) for 1 week, 10 minutes / day, and after exercise exercise, samples by oral ingestion Was administered for one week.
The bait is solid feed CRF-1 (Oriental Yeast Co., Ltd.) as the basic feed, the sample is only the basic feed (untreated section), and 500 mg of the acetic acid / bacteria-soluble organic solvent extract prepared in Production Example 1 is used as the basic feed. / Kg rat body weight (acetic acid bacteria extract group), forcibly administered edible fat-soluble organic solvent extract prepared in Production Example 2 to 500 mg / kg rat body weight (vinegar extract group) 3 groups (n = 8). For forced administration, a disposable syringe made of polypropylene equipped with an oral sonde for disposable rats was used.
なお、投与７日目に剖検を行い、心臓、ヒラメ筋、ヒフク筋のＭＰＯ活性を測定した。ＭＰＯ活性の測定は、Myeloperoxidase Assay kit （Cytostore社製）を用いて行った。
すなわち、各組織片約０．５ｇに、ヘキサデシルトリメチルアンモニウムブロミド（Hexadecyltri‐methylammouniumbromide）溶液を２５０μｌ加え、乳鉢を用いて破砕し、１００００ｒｐｍで遠心後、上清を回収した。回収した上清を組織タンパク質濃度２０ｍｇ／ｍｌとなるように調整し、ＭＰＯ活性測定用組織破砕液とした。なお、組織タンパク質濃度の測定は、Bio Rad DC‐protein Assay（Bio‐Rad Laboratories社製）を用いた。
次に、反応液の調整を行うため、ｏ−ジアニシジンジヒドロクロライド（ｏ−dianisidine dihydrochloride）／リン酸緩衝液１００ｍｌに対して０．５ｍｌの１％過酸化水素を加え、これを反応液とした。各組織のＭＰＯ活性測定用組織破砕液２０μｌに反応液２００μｌを加え、吸光度計を用いて、直後と１分後の４５０ｎｍの吸光度を測定した。
なお、ＭＰＯ活性１Ｕは、組織タンパク質１ｇ当り過酸化水素１Ｍが反応する際に上昇する１分あたりの吸光度（４５０ｎｍ）と定義した。
以上の結果を図１に示した。なお、各器官の未投与区に対して、酢酸菌抽出物区、食酢抽出物区それぞれを、有意水準５％としてｔ検定を行い、有意差が認められた試験区を※印で表した。
On the 7th day after administration, necropsy was performed, and the MPO activity of the heart, soleus and cypress muscles was measured. The measurement of MPO activity was performed using Myeloperoxidase Assay kit (manufactured by Cytostore).
That is, 250 μl of hexadecyltri-methylammounium bromide solution was added to about 0.5 g of each tissue piece, crushed using a mortar, centrifuged at 10,000 rpm, and the supernatant was collected. The collected supernatant was adjusted to a tissue protein concentration of 20 mg / ml, and used as a tissue disruption solution for MPO activity measurement. The tissue protein concentration was measured using Bio Rad DC-protein Assay (Bio-Rad Laboratories).
Next, in order to adjust the reaction solution, 0.5 ml of 1% hydrogen peroxide was added to 100 ml of o-dianisidine dihydrochloride / phosphate buffer, and this was used as the reaction solution. . 200 μl of the reaction solution was added to 20 μl of the tissue disruption solution for measuring the MPO activity of each tissue, and the absorbance at 450 nm was measured immediately and after 1 minute using an absorptiometer.
MPO activity 1U was defined as absorbance per minute (450 nm) that increases when 1 M hydrogen peroxide reacts per gram of tissue protein.
The above results are shown in FIG. In addition, the t-test was performed with the acetic acid bacteria extract group and the vinegar extract group each having a significance level of 5% with respect to the non-administered group of each organ, and the test group in which a significant difference was recognized was indicated by *.
図１の結果から、未投与区に比べて、酢酸菌抽出物区および食酢抽出物区においては、心臓、ヒラメ筋、ヒフク筋でいずれもＭＰＯ活性が有意に減少し、酢酸菌脂溶性有機溶剤抽出物および食酢脂溶性有機溶剤抽出物どちらにも筋肉の損傷を抑制する作用があることが確認できた。 From the results shown in FIG. 1, in the acetic acid bacteria extract group and the vinegar extract group, MPO activity was significantly decreased in the heart, soleus muscle, and hifuku muscle, compared with the non-administered group. It was confirmed that both the extract and the vinegar-fat-soluble organic solvent extract have an effect of suppressing muscle damage.
実施例２（高程度運動量下での筋肉損傷抑制効果）
運動の種類による効果の違いを検証するため、１週間、１０分／日、トレッドミルによる運動負荷（ベルト勾配：６度、速度：１０ｍ／ｍｉｎ）を行った後、強制経口摂取による餌の投与を行いながら、６日間運動なし、７日目にトレッドミルによる運動負荷（ベルト勾配：１０度、速度：２０ｍ／ｍｉｎ）を行った。
餌は、固形飼料ＣＲＦ−１（オリエンタル酵母工業株式会社）を基本飼料とし、試料は基本飼料のみ（未投与区）、基本飼料に製造例１で作製した酢酸菌脂溶性有機溶剤抽出物を５００ｍｇ／ｋｇラット体重となるよう強制投与したもの（酢酸菌抽出物区）、製造例２で作製した食酢脂溶性有機溶剤抽出物を５００ｍｇ／ｋｇラット体重となるよう強制投与したもの（食酢抽出物区）の３群（ｎ＝８）とした。投与７日目、運動終了２時間後に剖検を行い、心臓のＭＰＯ活性を測定した。測定方法は実施例１と同様とした。
以上の結果を図２に示した。なお、未投与区に対して、酢酸菌抽出物区、食酢抽出物区それぞれを有意水準５％としてｔ検定を行い、有意差が認められた試験区を※印で表した。
Example 2 (Inhibition effect on muscle damage under high momentum)
In order to verify the difference in effect depending on the type of exercise, one week, 10 minutes / day, exercise exercise with a treadmill (belt gradient: 6 degrees, speed: 10 m / min), then administration of food by gavage No exercise was performed for 6 days, and exercise load (belt gradient: 10 degrees, speed: 20 m / min) by a treadmill was performed on the 7th day.
The bait is solid feed CRF-1 (Oriental Yeast Co., Ltd.) as the basic feed, the sample is only the basic feed (untreated section), and 500 mg of the acetic acid / bacteria-soluble organic solvent extract prepared in Production Example 1 is used as the basic feed. / Kg rat body weight (acetic acid bacteria extract group), forcibly administered edible fat-soluble organic solvent extract prepared in Production Example 2 to 500 mg / kg rat body weight (vinegar extract group) 3 groups (n = 8). On
The above results are shown in FIG. In addition, the t-test was performed with the acetic acid bacteria extract group and the vinegar extract group each having a significance level of 5% with respect to the non-administered group, and the test group in which a significant difference was recognized was indicated by *.
図２の結果から、未投与区に比べて酢酸菌抽出物区および食酢抽出物区においては、いずれもＭＰＯ活性が有意に減少し、酢酸菌脂溶性有機溶剤抽出物および食酢脂溶性有機溶剤抽出物のどちらも筋肉の損傷を抑制することが確認できた。また、運動量を増加させても効果があることを確認できた。 From the results shown in FIG. 2, MPO activity was significantly decreased in the acetic acid bacteria extract group and the vinegar extract group compared with the non-administered group, and the acetic acid bacteria fat soluble organic solvent extract and the vinegar fat soluble organic solvent extraction were obtained. Both of these things were confirmed to suppress muscle damage. It was also confirmed that increasing the amount of exercise was effective.
実施例３（筋管細胞を用いた筋損傷抑制効果）
本評価系にはマウス由来Ｃ２Ｃ１２筋芽細胞を用いた。この細胞は理化学研究所に保存されており，譲受可能な細胞株である。また分化誘導することで単核の筋芽細胞から多核の筋管細胞へと分化し、筋繊維様の形態をとる。本細胞をコラーゲンIコーティングした３５ｍｍシャーレに播種し、ＤＭＥＭ（11965-092；GIBCO社製）＋１０％ＦＢＳ（以下、増殖培地と称する場合もある）にて、３７℃、５％ＣＯ２の条件下で９０％コンフルエントになるまで培養した後にＤＭＥＭ（11885-084；GIBCO社製）＋５％ＨＳ培地（以下、分化培地と称する場合もある）にて３７℃、５％ＣＯ２の条件下で７２時間培養し，筋管細胞に分化させた。なお、培地は４８時間後に一度交換した。
Example 3 (Inhibition of muscle damage using myotube cells)
In this evaluation system, mouse-derived C2C12 myoblasts were used. This cell is stored in RIKEN and is a transferable cell line. In addition, differentiation-induced differentiation from mononuclear myoblasts to multinucleated myotubes takes a muscle fiber-like form. The cells were seeded in a 35 mm Petri dish coated with collagen I, and DMEM (11965-092; manufactured by GIBCO) + 10% FBS (hereinafter sometimes referred to as growth medium) at 37 ° C. and 5% CO 2 . And then cultured in DMEM (11885-084; manufactured by GIBCO) + 5% HS medium (hereinafter sometimes referred to as differentiation medium) at 37 ° C. and 5% CO 2 for 72 hours. Cultured and differentiated into myotube cells. The medium was changed once after 48 hours.
製造例１で作製した酢酸菌脂溶性有機溶剤抽出物、製造例３で作製した酢酸菌アルカリ安定脂質、製造例４で作製したテルぺノイド化合物、Ｎ−アシルスフィンガニン、アミノ脂質およびスフィンガニンをそれぞれＤＭＳＯに溶解し、終濃度１０μｇ／ｍｌになるように分化培地に添加した。ＤＭＳＯの細胞への影響も考慮し、培地添加後のＤＭＳＯは終濃度５μｌ/ｍｌになるよう調整した。添加後、３７℃、５％ＣＯ２存在下で２４時間培養した。 Acetic acid bacteria-soluble organic solvent extract produced in Production Example 1, Acetic acid bacteria alkaline stable lipid produced in Production Example 3, terpenoid compound produced in Production Example 4, N-acyl sphinganine, amino lipid and sphinganine Each was dissolved in DMSO and added to the differentiation medium to a final concentration of 10 μg / ml. Considering the influence of DMSO on cells, DMSO after addition of the medium was adjusted to a final concentration of 5 μl / ml. After the addition, the cells were cultured at 37 ° C. in the presence of 5% CO 2 for 24 hours.
各脂質を添加して２４時間培養した筋管細胞をＰＢＳで３回洗浄し、１ｍＭ Ｈ２Ｏ２＋分化培地にて３７℃、５％ＣＯ２存在下で２時間培養した後に培地を棄て、筋管細胞を回収した。尚、すぐに回収した細胞を用いない場合は液体窒素を用いて瞬間凍結し、−８０℃のフリーザーで使用するまで保存した。 Myotubes cultured for 24 hours after adding each lipid were washed 3 times with PBS, cultured in 1 mM H 2 O 2 + differentiation medium at 37 ° C. in the presence of 5% CO 2 for 2 hours, and the medium was discarded. Myotube cells were collected. When the cells collected immediately were not used, they were snap-frozen using liquid nitrogen and stored in a freezer at −80 ° C. until use.
回収した細胞のＩＬ−６及びＣＸＣＬ−１の遺伝子発現を解析する為に細胞からのｔｏｔａｌ ＲＮＡの抽出、逆転写反応によるｃＤＮＡの合成、定量ＰＣＲによる遺伝子の発現解析を行った。すなわち、回収した細胞をフェノール、グアニジンイソチオシアネートを含むＴｒｉｚｏｌ溶液（invitrogen社製）を用いて粗精製した後に、SV total RNA isolation system（promega社製）を用いて付属のプロトコールに従ってｔｏｔａｌ ＲＮＡの抽出・精製を行い、逆転写反応に用いた。抽出したｔｏｔａｌ ＲＮＡは吸光度（２６０ｎｍ、２８０ｎｍ）の測定結果に基づいてＲＮＡ濃度を決定した。 In order to analyze the gene expression of IL-6 and CXCL-1 in the collected cells, extraction of total RNA from the cells, synthesis of cDNA by reverse transcription reaction, and gene expression analysis by quantitative PCR were performed. That is, after the collected cells were roughly purified using a Trizol solution (manufactured by Invitrogen) containing phenol and guanidine isothiocyanate, total RNA was extracted and extracted using the SV total RNA isolation system (manufactured by promega) according to the attached protocol. Purification was performed and used for reverse transcription reaction. The RNA concentration of the extracted total RNA was determined based on the measurement result of absorbance (260 nm, 280 nm).
逆転写反応における反応液はTaqman Reverse Transcription Reagentを用いて、付属のプロトコールに基づいて調製した。尚、１サンプル辺りｔｏｔａｌ ＲＮＡの量は２００ｎｇとした。逆転写反応は２５℃（１０分）→４８℃（３０分）→９５℃（５分）→４℃の条件でＰＣＲ反応を行い、ｃＤＮＡを得た。 A reaction solution in the reverse transcription reaction was prepared using Taqman Reverse Transcription Reagent based on the attached protocol. The amount of total RNA per sample was 200 ng. The reverse transcription reaction was performed under the conditions of 25 ° C. (10 minutes) → 48 ° C. (30 minutes) → 95 ° C. (5 minutes) → 4 ° C. to obtain cDNA.
得られたｃＤＮＡを用いて定量ＰＣＲを行った。定量ＰＣＲは、反応試薬としてqPCR super mix-UDG with ROXを用い、プライマーとしてＩＬ−６用にはＭｍ００４４６１９０（AB社製）を、ＣＸＣＬ−１用にはＭｍ００４３３８５９（AB社製）を、さらに内部標準として用いたグリセロール３燐酸脱水素酵素（以下、ＧＡＰＤＨと称する場合もある）用としてはＭｍ９９９９９９１５（AB社製）を用い、プロトコールに従ってＰＣＲ反応液を調製した。定量ＰＣＲはABI prism 7000を用いて、５０℃ ２分→９５℃ １０分→＜９５℃ １５秒→６０℃ １分＞（４０サイクル）→４℃の反応条件下で行い、ＩＬ−６およびＣＸＣＬ−１の各測定値を、ＧＡＰＤＨの測定値を１００とした相対値で表した。 Quantitative PCR was performed using the obtained cDNA. Quantitative PCR uses qPCR super mix-UDG with ROX as a reaction reagent, Mm00446190 (manufactured by AB) for IL-6 as a primer, Mm00433859 (manufactured by AB) for CXCL-1, and an internal standard As a glycerol 3-phosphate dehydrogenase (hereinafter also referred to as GAPDH) used as a Mm999999915 (manufactured by AB), a PCR reaction solution was prepared according to the protocol. Quantitative PCR was carried out using ABI prism 7000 under the reaction conditions of 50 ° C. 2 minutes → 95 ° C. 10 minutes → <95 ° C. 15 seconds → 60 ° C. 1 minute> (40 cycles) → 4 ° C., IL-6 and CXCL Each measured value of -1 was expressed as a relative value with the measured value of GAPDH as 100.
結果を図３および図４に示す。なお、過酸化水素処理と比較して、酢酸菌脂溶性抽出物、酢酸菌アルカリ安定脂質、Ｎ−アシルスフィンガニン、スフィンガニン、アミノ脂質およびテルペノイド化合物処理区それぞれを有意水準５％としてｔ検定を行い、有意差が認められた試験区を※印で表した。 The results are shown in FIG. 3 and FIG. In addition, compared with hydrogen peroxide treatment, t-test was conducted with each of the treatment groups treated with fat-soluble extract of acetic acid bacteria, alkaline stable lipid of acetic acid bacteria, N-acyl sphinganine, sphinganine, aminolipid and terpenoid compound treated with a significance level of 5%. The test plots with significant differences were marked with *.
図３の結果より、酢酸菌脂溶性抽出物、酢酸菌アルカリ安定脂質にＩＬ−６の遺伝子発現抑制効果が認められた。
図４の結果より、Ｎ−アシルスフィンガニン、スフィンガニン、アミノ脂質、テルペノイド化合物にＩＬ−６、ＣＸＣＬ−１の各遺伝子発現抑制効果が認められた。
以上の結果より、本発明の酢酸菌脂溶性抽出物、酢酸菌アルカリ安定脂質、スフィンゴ脂質、スフィンガニン、アミノ脂質およびテルペノイド化合物に筋肉損傷抑制効果があることが確認された。
From the results shown in FIG. 3, the IL-6 gene expression inhibitory effect was observed in the acetic acid bacteria fat-soluble extract and the acetic acid bacteria alkali-stable lipid.
From the results of FIG. 4, N-acyl sphinganine, sphinganine, amino lipid, and terpenoid compound were confirmed to have IL-6 and CXCL-1 gene expression inhibitory effects.
From the above results, it was confirmed that the acetic acid bacterium fat-soluble extract, acetic acid bacterium alkali-stable lipid, sphingolipid, sphinganine, aminolipid, and terpenoid compound of the present invention have an effect of suppressing muscle damage.
実施例４（錠剤の製造）
（１）酢酸菌脂溶性有機溶剤抽出物
酢酸発酵液１０キロリットルを高速遠心機器（８０００ｒｐｍ、２０分）で集菌し、湿菌体１０kｇを得た。得られた湿菌体１０kｇを蒸留水にて洗浄後に大型凍結乾燥機で凍結減圧乾固し、乾燥菌体１．８kｇを得た。
Example 4 (Manufacture of tablets)
(1) Acetic acid bacteria fat-soluble organic solvent extract 10 kiloliters of acetic acid fermentation broth was collected with a high-speed centrifugal device (8000 rpm, 20 minutes) to obtain 10 kg of wet cells. 10 kg of the obtained wet bacterial cells were washed with distilled water and then freeze-dried under reduced pressure using a large freeze dryer to obtain 1.8 kg of dried bacterial cells.
得られた乾燥菌体１kｇをアセトン１０リットルと共にソックスレー抽出器に仕込み、２０時間加熱還流した。得られた抽出液を減圧乾固し、１０リットルの酢酸エチルと１０リットルの蒸留水を添加し、分液して有機層をロータリーエバポレーターで蒸発し、１リットル程度の容量になった時点でエタノールを同量投入し、攪拌し、ロータリーエバポレーターで蒸発乾固した後、淡黄褐色の酢酸菌脂溶性有機溶剤抽出物を約５０ｇ得た。得られた酢酸菌脂溶性有機溶剤抽出物１ｇ（０．７重量％）、結晶セルロース３５ｇ（２６．９重量％）、乾燥コーンスターチ６７ｇ（５１．５重量％）、乳糖２２ｇ（１６．９重量％）、ステアリン酸カルシウム２ｇ（１．５重量％）、および結合剤としてポリビニルピロリドン３ｇ（２．３重量％）を加え、混合粉末化した後に、ゼラチン硬カプセルに充填した。 1 kg of the obtained dried cells was charged into a Soxhlet extractor together with 10 liters of acetone and heated to reflux for 20 hours. The obtained extract was evaporated to dryness under reduced pressure, 10 liters of ethyl acetate and 10 liters of distilled water were added, liquids were separated, and the organic layer was evaporated with a rotary evaporator. When the volume reached about 1 liter, ethanol was obtained. The same amount was added, stirred, and evaporated to dryness on a rotary evaporator to obtain about 50 g of a light yellowish brown acetic acid bacteria-soluble organic solvent extract. 1 g (0.7% by weight) of acetic acid bacteria-soluble organic solvent extract obtained, 35 g (26.9% by weight) crystalline cellulose, 67 g (51.5% by weight) dried corn starch, 22 g (16.9% by weight) lactose ), 2 g (1.5% by weight) of calcium stearate, and 3 g (2.3% by weight) of polyvinylpyrrolidone as a binder, mixed powdered, and filled into gelatin hard capsules.
（２）食酢脂溶性有機溶剤抽出物
食酢５リットルを酢酸エチル６リットルと混合し、有機層をロータリーエバポレーターで蒸発し、１リットル程度になったところでエタノールを同量加えて、さらにロータリーエバポレーターで蒸発乾固した後、茶褐色の食酢脂溶性有機溶剤抽出物約１ｇを得た。
得られた食酢脂溶性有機溶剤抽出物１ｇ（０．７重量％）、結晶セルロース３５ｇ（２６．９重量％）、乾燥コーンスターチ６７ｇ（５１．５重量％）、乳糖２２ｇ（１６．９重量％）、ステアリン酸カルシウム２ｇ（１．５重量％）、および結合剤としてポリビニルピロリドン３ｇ（２．３重量％）を加え、混合粉末化した後に、ゼラチン硬カプセルに充填した。
(2) Vinegar fat-soluble organic solvent extract Mix 5 liters of vinegar with 6 liters of ethyl acetate, evaporate the organic layer with a rotary evaporator, add the same amount of ethanol when it becomes about 1 liter, and evaporate with a rotary evaporator. After drying, about 1 g of a brownish brown vinegar-soluble organic solvent extract was obtained.
1 g (0.7 wt%) of the obtained vinegar and fat-soluble organic solvent extract, 35 g (26.9 wt%) of crystalline cellulose, 67 g (51.5 wt%) of dried corn starch, 22 g (16.9 wt%) of lactose Then, 2 g (1.5% by weight) of calcium stearate and 3 g (2.3% by weight) of polyvinyl pyrrolidone as a binder were added to form a mixed powder, which was then filled into a gelatin hard capsule.
上記（１）及び（２）で調製された錠剤は、筋肉損傷抑制組成物として、有効に経口摂取可能であることが期待できる。 It can be expected that the tablets prepared in the above (1) and (2) can be effectively taken orally as a muscle damage inhibiting composition.
実施例５（ゼリーの製造）
（１）酢酸菌破砕粉末
酢酸発酵液キロリットルを高速遠心機器（８０００ｒｐｍ、２０分）で集菌し、湿菌体１０kｇを得た。得られた湿菌体１０kｇを等量の蒸留水に分散させた。分散させた２０ｋｇの酢酸菌分散液を高圧ホモジナイザー（２００００ｐｓｉ）に３回通過させ細胞破壊処理を施した後に、大型凍結乾燥機で凍結減圧乾固し、乾燥菌体粉末１．５kｇを得た。
Example 5 (Production of jelly)
(1) Acetic acid bacteria pulverized powder A kiloliter of acetic acid fermentation broth was collected with a high-speed centrifuge (8000 rpm, 20 minutes) to obtain 10 kg of wet cells. 10 kg of the obtained wet microbial cells were dispersed in an equal amount of distilled water. The dispersed 20 kg of acetic acid bacteria dispersion was passed through a high-pressure homogenizer (20000 psi) three times and subjected to cell disruption treatment, and then freeze-dried with a large freeze dryer to obtain 1.5 kg of dried cell powder.
得られた乾燥菌体粉末８ｇ（０．８重量％）、砂糖２５０ｇ（２５重量％）、カラギーナン１．６ｇ（０．１６重量％）、ローカストビーンガム０．８ｇ（０．０８重量％）、キサンタンガム０．８ｇ（０．０８重量％）を混合均一化し、粉体混合物を得た。鍋に３００ｇの水を計量し、黒糖３０ｇ（３．０重量％）を溶解し、さらに還元水あめ１５０ｇ（１５重量％）を混合し、先に得た粉体混合物をダマができないように攪拌しながら、更に混合した。それらを均一に攪拌し、残りの水２５８．８ｇを加え、加温した。溶液温度が８５℃となるように１０分間加熱溶解後、流水で冷却し、酢酸菌体含有ゼリー食品を得た。 8 g (0.8 wt%) of the obtained dry cell powder, 250 g (25 wt%) sugar, 1.6 g (0.16 wt%) carrageenan, 0.8 g (0.08 wt%) locust bean gum, Xanthan gum 0.8 g (0.08 wt%) was mixed and homogenized to obtain a powder mixture. Weigh 300 g of water in a pan, dissolve 30 g (3.0% by weight) of brown sugar, mix 150 g (15% by weight) of reduced water candy, and stir the powder mixture obtained previously so that it does not become lumpy. Further mixing was performed. They were stirred uniformly and the remaining 258.8 g of water was added and warmed. After heating and dissolving for 10 minutes so that the solution temperature was 85 ° C., the solution was cooled with running water to obtain acetic acid bacterium-containing jelly food.
（２）食酢脂溶性有機溶剤抽出物
食酢５０リットルを酢酸エチル６０リットルと混合し、有機層をロータリーエバポレーターで蒸発させ、１０リットル程度になったところでエタノールを同量加えて、さらにロータリーエバポレーターターで蒸発乾固した後、茶褐色の食酢脂溶性有機溶剤抽出物約１０ｇを得た。
(2) Vinegar fat-soluble organic solvent extract Mix 50 liters of vinegar with 60 liters of ethyl acetate, evaporate the organic layer with a rotary evaporator, add the same amount of ethanol when it reaches about 10 liters, and further with a rotary evaporator. After evaporating to dryness, about 10 g of brownish brown vinegar-soluble organic solvent extract was obtained.
得られた食酢脂溶性有機溶剤抽出物８ｇ（０．８重量％）、砂糖２５０ｇ（２５重量％）、カラギーナン１．６ｇ（０．１６重量％）、ローカストビーンガム０．８ｇ（０．０８重量％）、キサンタンガム０．８ｇ（０．０８重量％）を混合均一化し、粉体混合物を得た。鍋に３００ｇの水を計量し、黒糖３０ｇ（３．０重量％）を溶解し、さらに還元水あめ１５０ｇ（１５重量％）を混合し、先に得た粉体混合物をダマができないように攪拌しながら、更に混合した。それらを均一に攪拌し、残りの水２５８．８ｇを加え、加温した。溶液温度が８５℃となるように１０分間加熱溶解後、流水で冷却し、食酢脂溶性有機溶剤抽出物含有ゼリー食品を得た。 8 g (0.8% by weight) of the obtained vinegar-soluble organic solvent extract, 250 g of sugar (25% by weight), 1.6 g (0.16% by weight) carrageenan, 0.8 g of locust bean gum (0.08% by weight) %) And 0.8 g (0.08 wt%) of xanthan gum were mixed and homogenized to obtain a powder mixture. Weigh 300 g of water in a pan, dissolve 30 g (3.0% by weight) of brown sugar, mix 150 g (15% by weight) of reduced water candy, and stir the powder mixture obtained previously so that it does not become lumpy. Further mixing was performed. They were stirred uniformly and the remaining 258.8 g of water was added and warmed. After heating and dissolving for 10 minutes so that the solution temperature became 85 ° C., the solution was cooled with running water to obtain a jelly-fat-soluble organic solvent extract-containing jelly food.
上記（１）及び（２）で調製されたゼリー食品は、筋肉損傷抑制組成物として、有効に経口摂取可能であることが期待できる。 It can be expected that the jelly food prepared in the above (1) and (2) can be effectively taken orally as a muscle damage inhibiting composition.
実施例６（飲料の製造）
（１）酢酸菌体含有食酢
酢酸発酵液１０キロリットルを高速遠心機器（８０００ｒｐｍ、２０分）で集菌し、湿菌体１０kｇを得た。得られた湿菌体１０kｇを食酢１００リットルに分散させた。分散させた酢酸菌分散液を高圧ホモジナイザー（２００００ｐｓｉ）に３回通過させ、細胞破壊処理を施した。その溶液を５００ｍｌ容の瓶に分注し、７５℃まで加温により殺菌し、酢酸菌体含有食酢を得た。
Example 6 (Manufacture of beverages)
(1) Acetic acid bacterium-containing vinegar 10 kg of acetic acid fermentation broth was collected with a high-speed centrifuge (8000 rpm, 20 minutes) to obtain 10 kg wet cells. 10 kg of the obtained wet microbial cells were dispersed in 100 liters of vinegar. The dispersed acetic acid bacteria dispersion was passed through a high-pressure homogenizer (20000 psi) three times to perform cell destruction treatment. The solution was dispensed into a 500 ml bottle and sterilized by heating to 75 ° C. to obtain acetic acid bacterium-containing vinegar.
（２）食酢脂溶性有機溶剤抽出物高濃度含有食酢
食酢５００リットルを酢酸エチル６００リットルと混合し、有機層をロータリーエバポレーターで蒸発させ、５０リットル程度になったところでエタノールを同量加えて、さらにロータリーエバポレーターターで蒸発乾固した後、茶褐色の食酢脂溶性有機溶剤抽出物（食酢脂溶性画分）約１００ｇを得た。得られた食酢脂溶性画分１００ｇを食酢１リットルに分散させた。その溶液を５００ｍｌ容の瓶に分注し、７５℃まで加温により殺菌し、食酢脂溶性有機溶剤抽出物高濃度含有食酢を得た。
(2) Vinegar fat-soluble organic solvent extract high-concentration vinegar Mix 500 liters of vinegar with 600 liters of ethyl acetate, evaporate the organic layer with a rotary evaporator, add the same amount of ethanol when it reaches about 50 liters, After evaporating to dryness with a rotary evaporator, about 100 g of brown brown vinegar fat-soluble organic solvent extract (dietary fat-soluble fraction) was obtained. 100 g of the obtained vinegar fat-soluble fraction was dispersed in 1 liter of vinegar. The solution was dispensed into a 500 ml bottle and sterilized by heating to 75 ° C. to obtain vinegar containing a high concentration of edible fat-soluble organic solvent extract.
上記（１）及び（２）で調製された飲料は、筋肉損傷抑制組成物として、有効に経口摂取可能であることが期待できる。 It can be expected that the beverage prepared in the above (1) and (2) can be effectively taken orally as a muscle damage inhibiting composition.
本発明は、人体への安全性が高く、且つ筋肉損傷抑制効果の経口物を安価に提供することを可能にする。従って、本発明に係る筋肉損傷抑制組成物を、食品などとして経口摂取することによって、スポーツや激しい運動における筋肉損傷及び日常生活中の身体的活動に伴う軽度の筋肉損傷を予防できる効果が期待される。 The present invention makes it possible to provide an oral product that is highly safe for the human body and has an effect of suppressing muscle damage at low cost. Therefore, by orally ingesting the muscle damage-suppressing composition according to the present invention as a food or the like, an effect of preventing muscle damage in sports or intense exercise and mild muscle damage associated with physical activity in daily life is expected. The
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| JP2012180311A (en) * | 2011-03-02 | 2012-09-20 | Kao Corp | Endurance improver |
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| JP2006199624A (en) * | 2005-01-20 | 2006-08-03 | Mitsukan Group Honsha:Kk | Acetobacter-containing in vivo antioxidative composition |
| JP2007070342A (en) * | 2005-08-09 | 2007-03-22 | Mitsukan Group Honsha:Kk | Composition for ameliorating cerebral function |
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| JP2006199624A (en) * | 2005-01-20 | 2006-08-03 | Mitsukan Group Honsha:Kk | Acetobacter-containing in vivo antioxidative composition |
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