JP7467804B2 - Composition for improving intestinal flora - Google Patents
Composition for improving intestinal flora Download PDFInfo
- Publication number
- JP7467804B2 JP7467804B2 JP2018541079A JP2018541079A JP7467804B2 JP 7467804 B2 JP7467804 B2 JP 7467804B2 JP 2018541079 A JP2018541079 A JP 2018541079A JP 2018541079 A JP2018541079 A JP 2018541079A JP 7467804 B2 JP7467804 B2 JP 7467804B2
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- JP
- Japan
- Prior art keywords
- derived
- luminacoid
- cocoa
- faecalibacterium
- cacao
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Description
本願は、先行する日本国出願である特願２０１６－１８４９０６（出願日：２０１６年９月２１日）の優先権の利益を享受するものであり、その開示内容全体は引用することにより本明細書の一部とされる。This application benefits from the priority of an earlier Japanese application, Patent Application No. 2016-184906 (filing date: September 21, 2016), the entire disclosure of which is incorporated herein by reference.
本発明は、腸内菌叢改善促進するための組成物に関し、詳細には、カカオ由来ルミナコイドを含んでなるフィーカリバクテリウム属菌の産生を促進するための組成物に関する。The present invention relates to a composition for promoting the improvement of intestinal flora, and more particularly to a composition for promoting the production of bacteria of the genus Faecalibacterium comprising a cocoa-derived luminacoid.
フィーカリバクテリウム属菌（Faecalibacterium）は、クロストリジウム クラスター（Clostridium cluster IV、C. leptum subgroup）に属し、ヒトやヒト以外の哺乳類（ブタ、マウス、子牛など）や家禽でも検出される。フィーカリバクテリウム属菌は、主として大腸上皮粘膜上に分泌された粘液層に付着する短鎖脂肪酸産生菌（主に酪酸を一次代謝産物として産生）である（非特許文献１）。また、フィーカリバクテリウム属菌は、制御性Ｔ細胞（Treg）の誘導能を示すことから、抗炎症・抗アレルギー作用が見出されている（非特許文献２）。さらに、フィーカリバクテリウム属菌は、ヒトの腸内菌叢における占有率が数％と高く、フィーカリバクテリウム属菌の減少が、腸内菌共生バランス失調（Dysbiosis）に起因すると考えられる疾病（クローン病など）と関連する可能性が示唆されている（非特許文献３）。Faecalibacterium belongs to the Clostridium cluster IV (C. leptum subgroup) and is found in humans, non-human mammals (pigs, mice, calves, etc.), and poultry. Faecalibacterium is a short-chain fatty acid-producing bacterium (mainly producing butyric acid as a primary metabolic product) that mainly adheres to the mucus layer secreted on the large intestine epithelial mucosa (Non-Patent Document 1). Faecalibacterium has also been found to have anti-inflammatory and anti-allergic effects because it exhibits the ability to induce regulatory T cells (Treg) (Non-Patent Document 2). Furthermore, Faecalibacterium occupies a high percentage of the human intestinal flora, at several percent, and it has been suggested that a decrease in Faecalibacterium may be associated with diseases (such as Crohn's disease) that are thought to be caused by intestinal dysbiosis (Non-Patent Document 3).
カカオ豆は発芽に必要なアミノ酸源やエネルギー源としてたんぱく質や脂質を貯蔵しており、また細胞壁の構造物質として食物繊維を豊富に含んでいる。これらはカカオ豆から調製された菓子原料であるカカオマスにも含まれている。以前より多糖類やリグニン等の食物繊維が整腸やコレステロール排泄等の機能性を示すことはよく知られていたが、最近、プロテアーゼ耐性を示す一部のたんぱく質（難消化性たんぱく質）が、便秘の緩解などの、食物繊維や難消化性デキストリン等と似た生理的機能を示すことが明らかになってきた（非特許文献４）。しかし、カカオ豆由来のルミナコイドに関しての詳細な知見は得られていない。一方で、カカオ豆からアルカリ抽出および酸沈殿にて調製したたんぱく質画分をマウスに摂取させると糞便量が増大したことが報告されている（特許文献１）。 Cacao beans store proteins and lipids as sources of amino acids and energy necessary for germination, and are also rich in dietary fiber as a structural material of the cell wall. These are also contained in cacao mass, a confectionery ingredient prepared from cacao beans. It has long been well known that dietary fiber such as polysaccharides and lignin has functions such as intestinal regulation and cholesterol excretion, but recently it has become clear that some proteins that are resistant to proteases (resistant proteins) have physiological functions similar to those of dietary fiber and resistant dextrin, such as relieving constipation (Non-Patent Document 4). However, detailed knowledge about luminacoids derived from cacao beans has not been obtained. On the other hand, it has been reported that the amount of feces increased when mice were fed a protein fraction prepared from cacao beans by alkaline extraction and acid precipitation (Patent Document 1).
本発明は、新規なフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤を提供することを目的とする。 The present invention aims to provide a novel composition for promoting the production of Faecalibacterium genus bacteria and a production promoter for Faecalibacterium genus bacteria.
本発明者らは、今般、カカオ豆由来ルミナコイドを高濃度で含有する油脂加工組成物が、ヒト腸内菌叢中のフィーカリバクテリウム属菌の占有率（％）を有意に増加させることを見出した。本発明はこの知見に基づくものである。The present inventors have now discovered that an oil and fat processing composition containing a high concentration of cocoa bean-derived luminacoid significantly increases the occupancy rate (%) of Faecalibacterium genus bacteria in the human intestinal flora. The present invention is based on this finding.
本発明によれば以下の発明が提供される。
［１］カカオ由来ルミナコイドを含んでなる、フィーカリバクテリウム属菌の産生促進のための組成物（以下、「本発明の組成物」ということがある）およびフィーカリバクテリウム属菌の産生促進剤（以下、「本発明の用剤」ということがある）。
［１－１］カカオ由来ルミナコイドが、カカオ豆由来ルミナコイドである、前記［１］に記載のフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤
［１－２］カカオ由来ルミナコイドを０．５ｇ以上含んでなるものである、前記［１］または［１－１］に記載のフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤。
［２］フィーカリバクテリウム属菌の産生促進に有効な１日分の摂取量のカカオ由来ルミナコイドを含んでなる、前記［１］、［１－１］または［１－２］に記載のフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤。
［３］１日分の有効摂取量ごとに包装されてなる、前記［２］に記載のフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤。
［４］フィーカリバクテリウム属菌の産生促進に有効な１日分の摂取量が０．５～１１．９ｇ［カカオ由来ルミナコイド量］である、前記［２］または［３］に記載のフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤。
［５］カカオ由来ルミナコイドを２週間以上摂取させることを特徴とする、前記［１］、［１－１］、［１－２］および［２］～［４］のいずれかに記載のフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤。
［５－１］カカオ由来ルミナコイドを、１日当たり０．５～１１．９ｇで１週間以上摂取させることを特徴とする、前記［１］、［１－１］、［１－２］および［２］～［５］のいずれかに記載のフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤。
［６］油脂加工組成物の形態である、前記［１］、［１－１］、［１－２］、［２］～［５］および［５－１］のいずれかに記載のフィーカリバクテリウム属菌の産生促進用組成物およびフィーカリバクテリウム属菌の産生促進剤。
［７］フィーカリバクテリウム属菌の産生により治療、予防または改善しうる疾患または症状の治療、予防または改善に用いるための、前記［１］、［１－１］、［１－２］、［２］～［５］、［５－１］および［６］のいずれかに記載の組成物。
［８］有効量のカカオ由来ルミナコイドを摂取させることを含んでなる、フィーカリバクテリウム属菌の産生促進方法。
［９］カカオ由来ルミナコイドをフィーカリバクテリウム属菌の産生促進に有効な１日分の摂取量で１週間以上摂取させることを含んでなる、前記［８］に記載のフィーカリバクテリウム属菌の産生促進方法。
［１０］フィーカリバクテリウム属菌の産生促進に有効な１日分の摂取量が０．５～１１．９ｇ（カカオ由来ルミナコイド）である、前記［９］に記載のフィーカリバクテリウム属菌の産生促進方法。
［１１］カカオ由来ルミナコイドを油脂加工組成物の形態で摂取させることを特徴とする、前記［８］～［１０］のいずれかに記載のフィーカリバクテリウム属菌の産生促進方法。
［１２］有効量のカカオ由来ルミナコイドを摂取させるか、或いは投与することを含んでなる、フィーカリバクテリウム属菌の産生により治療、予防または改善しうる疾患または症状の治療、予防または改善方法。
［１３］フィーカリバクテリウム属菌の産生促進剤の製造のための、フィーカリバクテリウム属菌の産生により治療、予防または改善しうる疾患または症状の治療剤、予防剤または改善剤の製造のための、フィーカリバクテリウム属菌の産生促進剤としての、あるいは、フィーカリバクテリウム属菌の産生により治療、予防または改善しうる疾患または症状の治療剤、予防剤または改善剤としての、カカオ由来ルミナコイドの使用。
［１４］フィーカリバクテリウム属菌の産生促進用の、あるいは、フィーカリバクテリウム属菌の産生により治療、予防または改善しうる疾患または症状の治療、予防または改善用の、カカオ由来ルミナコイド。
According to the present invention, the following inventions are provided.
[1] A composition for promoting the production of Faecalibacterium genus bacteria (hereinafter sometimes referred to as the "composition of the present invention") and a production promoter for Faecalibacterium genus bacteria (hereinafter sometimes referred to as the "agent of the present invention") comprising cocoa-derived luminacoid.
[1-1] A composition for promoting the production of Faecalibacterium genus bacteria described in [1] above, and a production promoter for Faecalibacterium genus bacteria, wherein the cocoa-derived luminacoid is a cocoa bean-derived luminacoid. [1-2] A composition for promoting the production of Faecalibacterium genus bacteria and a production promoter for Faecalibacterium genus bacteria described in [1] or [1-1] above, which contain 0.5 g or more of cocoa-derived luminacoid.
[2] A composition for promoting the production of Faecalibacterium genus bacteria and a production promoter for Faecalibacterium genus bacteria described in [1], [1-1] or [1-2], comprising a daily intake amount of cocoa-derived luminacoid effective for promoting the production of Faecalibacterium genus bacteria.
[3] A composition for promoting the production of Faecalibacterium genus bacteria and a production promoter of Faecalibacterium genus bacteria described in [2], packaged in units of a daily effective intake amount.
[4] A composition for promoting the production of Faecalibacterium bacteria and a production promoter for Faecalibacterium bacteria described in [2] or [3], wherein the daily intake amount effective for promoting the production of Faecalibacterium bacteria is 0.5 to 11.9 g [amount of cocoa-derived luminacoid].
[5] A composition for promoting the production of Faecalibacterium spp. and a production promoter for Faecalibacterium spp. described in any of [1], [1-1], [1-2] and [2] to [4], characterized in that cocoa-derived luminacoid is ingested for more than two weeks.
[5-1] A composition for promoting the production of Faecalibacterium genus bacteria and a production promoter for Faecalibacterium genus bacteria described in any of [1], [1-1], [1-2] and [2] to [5], characterized in that 0.5 to 11.9 g of cocoa-derived luminacoid is ingested per day for one week or more.
[6] A composition for promoting the production of Faecalibacterium spp. and a production promoter for Faecalibacterium spp. described in any of [1], [1-1], [1-2], [2] to [5] and [5-1], in the form of an oil or fat processing composition.
[7] A composition described in any of [1], [1-1], [1-2], [2] to [5], [5-1] and [6] for use in the treatment, prevention or amelioration of a disease or symptom that can be treated, prevented or ameliorated by production of a bacterium of the genus Faecalibacterium.
[8] A method for promoting production of Faecalibacterium sp., comprising ingesting an effective amount of cocoa-derived luminacoid.
[9] A method for promoting the production of Faecalibacterium bacteria described in [8], comprising ingesting a cocoa-derived luminacoid at a daily intake amount effective for promoting the production of Faecalibacterium bacteria for one week or more.
[10] A method for promoting the production of Faecalibacterium bacteria described in [9], in which the daily intake amount effective for promoting the production of Faecalibacterium bacteria is 0.5 to 11.9 g (cocoa-derived luminacoid).
[11] A method for promoting the production of Faecalibacterium sp. described in any of [8] to [10], characterized in that cocoa-derived luminacoid is ingested in the form of an oil or fat processed composition.
[12] A method for treating, preventing or ameliorating a disease or symptom that can be treated, prevented or ameliorated by the production of bacteria of the genus Faecalibacterium, comprising ingesting or administering an effective amount of a cocoa-derived luminacoid.
[13] Use of cocoa-derived luminacoid as a production promoter of Faecalibacterium genus bacteria for the manufacture of a therapeutic, preventive or ameliorating agent for diseases or symptoms that can be treated, prevented or ameliorated by the production of Faecalibacterium genus bacteria, or as a therapeutic, preventive or ameliorating agent for diseases or symptoms that can be treated, prevented or ameliorated by the production of Faecalibacterium genus bacteria.
[14] A cocoa-derived luminacoid for promoting the production of Faecalibacterium sp. bacteria, or for treating, preventing or ameliorating a disease or symptom that can be treated, prevented or ameliorated by the production of Faecalibacterium sp. bacteria.
本発明の組成物および用剤は、長年食品の原料として用いられてきたカカオに含まれる成分を利用するものであることから、本発明の組成物および用剤は、長期間にわたって服用しても副作用が少なく、安全性が高い点において有利である。 The compositions and agents of the present invention utilize ingredients contained in cacao, which has been used as a food ingredient for many years, and therefore have the advantage that they have few side effects and are highly safe, even when taken over a long period of time.
本発明において「カカオ由来ルミナコイド」は、カカオに含まれるルミナコイドを意味し、典型的には、カカオ豆由来ルミナコイドである。従って、本発明においては、カカオの植物体またはその加工品から抽出（粗抽出を含む）あるいは精製（粗精製を含む）したカカオのルミナコイドを本発明の有効成分として使用することができる。In the present invention, "cacao-derived luminacoid" refers to a luminacoid contained in cacao, typically a luminacoid derived from cacao beans. Therefore, in the present invention, cacao luminacoid extracted (including crude extraction) or purified (including crude purification) from the cacao plant or a processed product thereof can be used as an active ingredient of the present invention.
ここで、「ルミナコイド」とは、小腸で消化吸収されず大腸に到達する全ての食品成分の総称で、非デンプン性のルミナコイドとデンプン性のルミナコイドに分類される。非デンプン性のルミナコイドは、難消化性タンパク質（レジスタントプロテイン、RP）、食物繊維、オリゴ糖、糖アルコール、その他に分類される。食物繊維はさらに、リグニンと多糖類に分類される。多糖類には、植物性多糖類、動物性多糖類、微生物性多糖類、化学修飾性多糖類がある。デンプン性ルミナコイドは、難消化性デンプン、難消化性デキストリンがある。カカオ由来ルミナコイドとしては、難消化性たんぱく質および食物繊維が挙げられる。 Here, "luminacoids" is a general term for all food ingredients that reach the large intestine without being digested and absorbed in the small intestine, and are classified into non-starch luminacoids and starchy luminacoids. Non-starch luminacoids are classified into resistant proteins (RP), dietary fiber, oligosaccharides, sugar alcohols, and others. Dietary fiber is further classified into lignin and polysaccharides. Polysaccharides include plant polysaccharides, animal polysaccharides, microbial polysaccharides, and chemically modified polysaccharides. Starchy luminacoids include resistant starch and resistant dextrin. Cocoa-derived luminacoids include resistant proteins and dietary fiber.
本発明において、カカオ由来ルミナコイドの原料となりうるカカオの植物体またはその加工品としては、カカオ樹皮、カカオ葉、カカオ豆、カカオシェル、カカオマス、脱脂カカオマス、ココアパウダー等、植物体の各種部位またはカカオ豆加工品を挙げることができる。カカオマスはカカオ豆を磨砕したものであり、脱脂カカオマスはカカオマスから油脂を除去することにより得ることができる。油脂の除去方法は特に制限されず、圧搾などの公知の方法に従って行うことができる。脱脂カカオマスを粉砕すればココアパウダーとなる。また、カカオの植物体またはその加工品を原料として抽出を行う場合は、抽出効率の観点から、磨砕、粉砕等の微粒化処理が施されているカカオマスやココアパウダーを用いるのが好ましい。なお、カカオの植物体には、意図してないしは意図せずにカカオの植物体以外の物も含めることができる。また、カカオの植物体またはその加工品を原料として抽出を行う際にも、意図してないしは意図せずにカカオの植物体以外の物も含めることができる。さらに、カカオマスやココアパウダーにも、意図してないしは意図せずにカカオの植物体以外の物も含めることができる。In the present invention, examples of the cacao plant or its processed products that can be used as the raw material for cacao-derived luminacoids include various parts of the plant or processed products of cacao beans, such as cacao bark, cacao leaves, cacao beans, cacao shells, cacao mass, defatted cacao mass, and cocoa powder. Cacao mass is obtained by grinding cacao beans, and defatted cacao mass can be obtained by removing fats and oils from cacao mass. The method for removing fats and oils is not particularly limited, and can be performed according to known methods such as squeezing. Cocoa powder can be obtained by crushing the defatted cacao mass. In addition, when extracting using the cacao plant or its processed products as the raw material, it is preferable to use cacao mass or cocoa powder that has been subjected to a micronization process such as grinding or pulverization from the viewpoint of extraction efficiency. Note that the cacao plant can also include things other than the cacao plant, whether intentionally or unintentionally. In addition, when extracting cacao plants or their processed products as raw materials, substances other than cacao plants may be included, either intentionally or unintentionally. Furthermore, cacao mass and cocoa powder may also contain substances other than cacao plants, either intentionally or unintentionally.
カカオの植物体またはその加工品を原料とする抽出方法は公知であり、例えば、特開２００９－１８３２２９号公報や特開２０１１－９３８０７号公報の記載に従ってカカオ由来ルミナコイド含有組成物を調製することができる。抽出溶媒は、特に限定されるものではないが、水またはアルカリ性の水溶液を用いることが好ましい。また、カカオの植物体またはその加工品を原料とする精製方法は、合成吸着剤、イオン交換樹脂、限外ろ過、活性白土処理等の公知の方法を使用することができ、特に限定されるものではない。 Extraction methods using the cacao plant or its processed products as raw materials are known, and for example, a cacao-derived luminacoid-containing composition can be prepared according to the description in JP 2009-183229 A or JP 2011-93807 A. The extraction solvent is not particularly limited, but it is preferable to use water or an alkaline aqueous solution. In addition, purification methods using the cacao plant or its processed products as raw materials can use known methods such as synthetic adsorbents, ion exchange resins, ultrafiltration, activated clay treatment, etc., and are not particularly limited.
本発明においてカカオ由来ルミナコイド含量は、難消化性たんぱく質含量と食物繊維含量により求められる。難消化性たんぱく質含量は、実施例に記載の難消化性たんぱく質の抽出方法により抽出した後、ケルダール法（たんぱく質定量の公定法）により定量できる。また、実施例に記載の人工消化試験を行うことにより、タンパク質が難消化性たんぱく質であることが確認できる。また、食物繊維含量は、酵素－重量法（エーオーエーシ―インターナショナルAOAC Internationalによる公定法、AOAC Method 985.29、別名：プロスキー法）により定量できる。In the present invention, the cacao-derived luminacoid content is determined by the resistant protein content and dietary fiber content. The resistant protein content can be quantified by the Kjeldahl method (official method for quantifying protein) after extraction using the resistant protein extraction method described in the Examples. In addition, it is possible to confirm that the protein is a resistant protein by conducting the artificial digestion test described in the Examples. In addition, the dietary fiber content can be quantified by the enzymatic-gravimetric method (official method by AOAC International, AOAC Method 985.29, also known as the Prosky method).
カカオ由来ルミナコイドはカカオの植物体を原料にして調製することができるため、本発明の組成物および用剤は、カカオ由来ルミナコイド以外のカカオ豆由来の成分を含んでいてもよい。そのような成分としては、ポリフェノール、テオブロミン、カフェイン、アミノ酸類、ペプチド、脂肪酸等が挙げられる。また、本発明の組成物および用剤は、カカオに由来しない成分を含んでいてもよい。 Because cacao-derived luminacoids can be prepared using the cacao plant as a raw material, the compositions and preparations of the present invention may contain components derived from cacao beans other than cacao-derived luminacoids. Such components include polyphenols, theobromine, caffeine, amino acids, peptides, fatty acids, etc. The compositions and preparations of the present invention may also contain components not derived from cacao.
本発明の組成物および用剤は、カカオ由来ルミナコイド単独で提供することができ、あるいは、カカオ由来ルミナコイドと他の成分とを混合して提供することもできる。本発明の組成物および用剤におけるカカオ由来ルミナコイドの配合量は、その目的、用途、形態、剤型、症状、体重等に応じて任意に定めることができ、本発明はこれに限定されない。例えば、本発明の組成物および用剤におけるカカオ由来ルミナコイドの含量は、固形分中の乾燥質量割合で２％以上とすることができ、好ましくは、２～９０質量％、より好ましくは２～５０質量％、より一層好ましくは５～３５質量％、さらに好ましくは１０～２１質量％とすることができる。本発明においては、本発明の用剤をカカオ由来ルミナコイドからなるものとし、本発明の組成物をカカオ由来ルミナコイドと他の成分とを含んでなるものとすることができる。The composition and preparation of the present invention can be provided as cacao-derived luminacoid alone, or as a mixture of cacao-derived luminacoid and other ingredients. The amount of cacao-derived luminacoid in the composition and preparation of the present invention can be determined arbitrarily depending on the purpose, use, form, dosage form, symptoms, body weight, etc., and the present invention is not limited thereto. For example, the content of cacao-derived luminacoid in the composition and preparation of the present invention can be 2% or more in terms of dry mass ratio in the solid content, preferably 2 to 90% by mass, more preferably 2 to 50% by mass, even more preferably 5 to 35% by mass, and even more preferably 10 to 21% by mass. In the present invention, the preparation of the present invention can be made of cacao-derived luminacoid, and the composition of the present invention can be made to contain cacao-derived luminacoid and other ingredients.
後記実施例に示されるように、カカオ由来ルミナコイドは腸内菌叢改善促進作用、具体的には、フィーカリバクテリウム属菌の産生促進作用を有する。従って、カカオ由来ルミナコイド（特に、カカオ豆由来ルミナコイド）は腸内菌叢改善促進剤およびフィーカリバクテリウム属菌の産生促進剤として使用することができるとともに、腸内菌叢改善促進方法およびフィーカリバクテリウム属菌の産生促進方法に使用することができる。また、カカオ由来ルミナコイド（特に、カカオ豆由来ルミナコイド）は腸内菌叢改善促進のための組成物およびフィーカリバクテリウム属菌の産生促進のための組成物としても使用することができる。As shown in the examples below, the cocoa-derived luminacoid has an effect of promoting the improvement of the intestinal flora, specifically, an effect of promoting the production of the genus Faecalibacterium. Therefore, the cocoa-derived luminacoid (particularly, the cocoa bean-derived luminacoid) can be used as an agent for promoting the improvement of the intestinal flora and an agent for promoting the production of the genus Faecalibacterium, and can be used in a method for promoting the improvement of the intestinal flora and a method for promoting the production of the genus Faecalibacterium. The cocoa-derived luminacoid (particularly, the cocoa bean-derived luminacoid) can also be used as a composition for promoting the improvement of the intestinal flora and a composition for promoting the production of the genus Faecalibacterium.
ここで、「フィーカリバクテリウム属菌の産生促進」とは、フィーカリバクテリウム属菌の発現や増殖を促進することを意味し、フィーカリバクテリウム属菌の産生促進の程度は、腸内細菌の占有率を指標にして評価することができる（実施例参照）。具体的には、カカオ由来ルミナコイド（特に、カカオ豆由来ルミナコイド）の摂取または投与後の腸内細菌に占めるフィーカリバクテリウム属菌の割合が、摂取または投与前の腸内菌叢中のフィーカリバクテリウム属菌を上回る場合に、好ましくは約１．１倍以上である場合に、より好ましくは約１．２倍以上である場合に、より一層好ましくは約１．３倍以上、さらに好ましくは１．４倍以上、特に好ましくは１．５倍以上、さらに特に好ましくは１．６倍以上、さらに特に好ましくは１．８倍以上、最も好ましくは２倍以上である場合に、フィーカリバクテリウム属菌の産生を促進したと判定することができる。Here, "promotion of the production of Faecalibacterium" means promoting the expression and proliferation of Faecalibacterium, and the degree of promotion of the production of Faecalibacterium can be evaluated using the occupancy rate of intestinal bacteria as an index (see Examples). Specifically, when the proportion of Faecalibacterium in the intestinal bacteria after ingestion or administration of cacao-derived luminacoid (particularly, cacao bean-derived luminacoid) exceeds that of Faecalibacterium in the intestinal flora before ingestion or administration, preferably about 1.1 times or more, more preferably about 1.2 times or more, even more preferably about 1.3 times or more, even more preferably 1.4 times or more, particularly preferably 1.5 times or more, even more particularly preferably 1.6 times or more, even more particularly preferably 1.8 times or more, and most preferably 2 times or more, it can be determined that the production of Faecalibacterium has been promoted.
本発明のフィーカリバクテリウム属菌の産生促進方法は、有効量のカカオ由来ルミナコイドをヒトまたは非ヒト動物に摂取させるか、あるいは投与することにより実施することができる。The method for promoting the production of Faecalibacterium of the present invention can be carried out by ingesting or administering an effective amount of cocoa-derived luminacoid to a human or non-human animal.
なお、本発明におけるカカオ由来ルミナコイドの使用はヒトおよび非ヒト動物並びにこれらに由来する試料における使用であってもよく、治療的使用と非治療的使用のいずれもが意図される。ここで、「非治療的」とはヒトを手術、治療または診断する行為（すなわち、ヒトに対する医療行為）を含まないことを意味し、具体的には、医師または医師の指示を受けた者がヒトに対して手術、治療または診断を行う方法を含まないことを意味する。 Note that the use of the cocoa-derived luminacoids in the present invention may be in humans and non-human animals, as well as in samples derived therefrom, and both therapeutic and non-therapeutic uses are intended. Here, "non-therapeutic" means that it does not include the act of performing surgery, treatment or diagnosis on a human (i.e., medical procedures on a human), and specifically means that it does not include methods in which a physician or a person under the direction of a physician performs surgery, treatment or diagnosis on a human.
本発明ではフィーカリバクテリウム属菌の産生促進がその治療、予防または改善に有効である疾患および症状の治療、予防または改善にカカオ由来ルミナコイドを使用することができる。 In the present invention, cocoa-derived luminacoids can be used to treat, prevent or ameliorate diseases and symptoms for which promoting the production of bacteria of the genus Faecalibacterium is effective in treating, preventing or ameliorating them.
フィーカリバクテリウム属菌の産生促進により治療、予防または改善しうる疾患および症状としては、炎症性疾患（例えば、潰瘍性大腸炎やクローン病などの炎症性腸疾患）、アレルギー性疾患（例えば、食品アレルギー、花粉症、喘息、アレルギー性鼻炎、薬物アレルギー、アトピー性皮膚炎、ダニアレルギー）および腸内菌共生バランス失調（Dysbiosis）に起因すると考えられる疾病（例えば、クローン病、大腸がん、炎症性腸疾患、過敏性腸症候群、小腸細菌異常増殖症（SIBO）、偽膜性大腸炎（クロストリジウム・ディフィシル腸炎）、抗菌薬起因性下痢、アルコール性脂肪性肝炎（ASH）や非アルコール性脂肪性肝炎（NASH）などの肝臓疾患、肥満、２型糖尿病、アレルギー疾患、自閉症などの精神疾患、多発性硬化症）が挙げられる。後記実施例に示される通り、カカオ豆由来ルミナコイドはフィーカリバクテリウム属菌の産生を促進することができ、これまでにフィーカリバクテリウム属菌の増加が、抗炎症・抗アレルギー作用に繋がることや、フィーカリバクテリウム属菌の減少が腸内菌共生バランス失調（Dysbiosis）に起因すると考えられる疾病（クローン病など）と関連することが示されている（非特許文献２および３並びにProc. Natl. Acad. Sci. USA. 2013, 105(43): 16731-16736）。従って、カカオ由来ルミナコイドは、炎症性疾患、アレルギー性疾患および腸内菌共生バランス失調（Dysbiosis）に起因すると考えられる疾病（クローン病など）の治療用、予防用または改善用組成物あるいは治療剤、予防剤または改善剤として使用できるとともに、炎症性疾患、アレルギー性疾患および腸内菌共生バランス失調（Dysbiosis）に起因すると考えられる疾病（クローン病など）の治療方法、予防方法および改善方法に使用することができる。Diseases and symptoms that can be treated, prevented, or ameliorated by promoting the production of Faecalibacterium bacteria include inflammatory diseases (e.g., inflammatory bowel diseases such as ulcerative colitis and Crohn's disease), allergic diseases (e.g., food allergies, hay fever, asthma, allergic rhinitis, drug allergies, atopic dermatitis, and dust mite allergies), and diseases thought to be caused by an imbalance of intestinal bacteria (dysbiosis) (e.g., Crohn's disease, colon cancer, inflammatory bowel disease, irritable bowel syndrome, small intestinal bacterial overgrowth (SIBO), pseudomembranous colitis (Clostridium difficile colitis), antibiotic-induced diarrhea, liver diseases such as alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH), obesity,
本発明の組成物および用剤、本発明の治療剤、予防剤および改善剤並びに本発明の治療用、予防用および改善用組成物は、医薬品、医薬部外品、飲食品、飼料などの形態で提供することができ、下記の記載に従って実施することができる。また本発明のフィーカリバクテリウム属菌の産生促進方法並びに本発明の治療方法、予防方法および改善方法は下記の記載に従って実施することができる。本発明の治療用、予防用および改善用組成物が医薬品を目的としている場合には、これらは治療用医薬組成物、予防用医薬組成物および改善用医薬組成物とすることができる。The compositions and agents of the present invention, the therapeutic, preventive and ameliorating agents of the present invention, and the therapeutic, preventive and ameliorating compositions of the present invention can be provided in the form of pharmaceuticals, quasi-drugs, foods and beverages, feeds, etc., and can be carried out according to the description below. Furthermore, the method for promoting the production of Faecalibacterium spp. of the present invention, and the therapeutic, preventive and ameliorating methods of the present invention can be carried out according to the description below. When the therapeutic, preventive and ameliorating compositions of the present invention are intended as pharmaceuticals, they can be therapeutic pharmaceutical compositions, preventive pharmaceutical compositions and ameliorating pharmaceutical compositions.
本発明の有効成分であるカカオ由来ルミナコイドをヒトおよび非ヒト動物に投与ないし摂取させる場合、カカオ豆に由来するルミナコイドの含有率を高めた組成物を使用することができ、このような組成物は、カカオ豆に由来する脂肪分をほとんど含まず、苦味および収斂味をほとんど有しないため、種々の飲食品、医薬品、医薬部外品、飼料、化成品等の素材として有用である。When the active ingredient of the present invention, cocoa-derived luminacoid, is administered or ingested by humans and non-human animals, a composition having a high content of luminacoid derived from cocoa beans can be used. Such compositions contain almost no fat derived from cocoa beans and have almost no bitter or astringent taste, making them useful as ingredients for various foods and beverages, pharmaceuticals, quasi-drugs, feed, chemical products, etc.
本発明の有効成分であるカカオ由来ルミナコイドはヒトおよび非ヒト動物に経口投与することができる。経口剤としては、顆粒剤、散剤、錠剤（糖衣錠を含む）、丸剤、カプセル剤、シロップ剤、乳剤、懸濁剤が挙げられる。これらの製剤は、当分野で通常行われている手法により、薬学上許容される担体を用いて製剤化することができる。薬学上許容される担体としては、賦形剤、結合剤、希釈剤、添加剤、香料、緩衝剤、増粘剤、着色剤、安定剤、乳化剤、分散剤、懸濁化剤、防腐剤等が挙げられる。The cacao-derived luminacoid, which is the active ingredient of the present invention, can be orally administered to humans and non-human animals. Oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be formulated using pharma- ceutical acceptable carriers by methods commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc.
本発明の有効成分であるカカオ由来ルミナコイドはヒトおよび非ヒト動物に経管投与、経鼻管投与、点滴、座薬等の経口投与以外の体内への投与も本発明の組成物および用剤の形状に応じて可能である。例えば、カカオ由来ルミナコイドを含む粘性を有する液状の組成物、または、カカオ由来ルミナコイドを含む半固形状の組成物とすることで、咀嚼や嚥下の機能が低下し、経口摂取ないしは経口投与ができないヒトおよび非ヒト動物に対しても投与することができる。本発明の組成物および用剤を経口摂取以外で摂取させるか、あるいは投与することにより、咀嚼や嚥下の機能が加齢などにより低下したとしても、フィーカリバクテリウム属菌の産生促進が期待でき、これらのヒトおよび非ヒト動物の炎症性疾患、アレルギー性疾患、腸内菌共生バランス失調（Dysbiosis）に起因すると考えられる疾病（クローン病など）の治療、予防および改善が期待できる。The active ingredient of the present invention, cacao-derived luminacoid, can be administered to humans and non-human animals by tube feeding, nasogastric tube administration, drip infusion, suppository, or other methods other than oral administration, depending on the form of the composition and preparation of the present invention. For example, by making it into a viscous liquid composition containing cacao-derived luminacoid or a semi-solid composition containing cacao-derived luminacoid, it can be administered to humans and non-human animals who have reduced chewing and swallowing functions and cannot take orally or are unable to take orally. By having the composition and preparation of the present invention ingested or administered by a method other than oral ingestion, it is expected that the production of Faecalibacterium genus bacteria will be promoted, even if the chewing and swallowing functions have decreased due to aging, etc., and it is expected that the treatment, prevention, and improvement of inflammatory diseases, allergic diseases, and diseases (such as Crohn's disease) that are thought to be caused by intestinal bacterial symbiotic imbalance (dysbiosis) in these humans and non-human animals can be expected.
本発明の有効成分であるカカオ由来ルミナコイドはヒトおよび非ヒト動物に経口摂取させることができる。カカオ由来ルミナコイドを経口摂取させる場合にカカオ由来ルミナコイドは単離、精製または粗精製された形態のものであっても、カカオ由来ルミナコイドを含む食品あるいは食品の原料の形態であってもよい。また、カカオ由来ルミナコイドはヒトおよび非ヒト動物に経口摂取させるにあたり、常温の状態、温かい状態、冷たい状態等から任意に選択することができる。 The cacao-derived luminacoid, which is the active ingredient of the present invention, can be orally ingested by humans and non-human animals. When cacao-derived luminacoid is orally ingested, the cacao-derived luminacoid may be in an isolated, purified or crudely purified form, or in the form of a food or food ingredient containing the cacao-derived luminacoid. Furthermore, when cacao-derived luminacoid is orally ingested by humans and non-human animals, it can be in any state selected from room temperature, warm, cold, etc.
本発明の有効成分であるカカオ由来ルミナコイドを食品として提供する場合には、カカオ由来ルミナコイドをそのまま食品に含有させることができ、該食品はカカオ由来ルミナコイドを有効量含有した食品である。ここで、カカオ由来ルミナコイドを「有効量含有した」とは、個々の食品において通常喫食される量を摂取した場合に後述するような範囲でカカオ由来ルミナコイドが摂取されるような含有量をいう。また「食品」とは、健康食品、機能性食品、保健機能食品（例えば、特定保健用食品、栄養機能食品、機能性表示食品）、特別用途食品（例えば、幼児用食品、妊産婦用食品、病者用食品）を含む意味で用いられる。「食品」の形態は特に限定されるものではなく、例えば、飲料の形態であっても、半液体やゲル状の形態であっても、固形状の形態であってもよい。When the cacao-derived luminacoid, which is the active ingredient of the present invention, is provided as a food product, the cacao-derived luminacoid can be directly contained in the food product, and the food product contains an effective amount of the cacao-derived luminacoid. Here, "containing an effective amount" of the cacao-derived luminacoid refers to a content such that the cacao-derived luminacoid is ingested in the range described below when the amount normally consumed in each food product is ingested. In addition, the term "food" is used to include health foods, functional foods, health functional foods (e.g., foods for specified health uses, foods with nutritional functions, foods with functional claims), and foods for special uses (e.g., foods for infants, foods for pregnant women, foods for sick people). The form of the "food" is not particularly limited, and may be, for example, a beverage, a semi-liquid or gel-like form, or a solid form.
カカオ由来ルミナコイドはフィーカリバクテリウム属菌の産生促進作用を有するため、日常摂取する食品や、サプリメントとして摂取する食品に含有させて提供することができる。本発明で提供される食品としては、その形態や形状に特に制限はないが、好ましくは、カカオ豆を主原料とする食品が挙げられ、より好ましくは、油脂加工組成物であり、より一層好ましくはチョコレートおよびココアなどの油脂加工食品である。 Since cacao-derived luminacoids have the effect of promoting the production of Faecalibacterium bacteria, they can be provided by being contained in foods consumed daily or foods consumed as supplements. The food provided by the present invention is not particularly limited in its form or shape, but preferably includes foods whose main ingredient is cacao beans, more preferably oil-processed compositions, and even more preferably oil-processed foods such as chocolate and cocoa.
前記の通り、カカオ豆由来ルミナコイドを効率よく摂取させるためには濃縮されたカカオ豆由来ルミナコイド組成物を本発明に使用することができる。従って、カカオ豆を主原料とする食品およびサプリメントは、例えば、カカオ豆由来ルミナコイドを高濃度で含むものであることが好ましく、より好ましくはカカオ豆由来ルミナコイドを高濃度で含む油脂加工組成物であり、より一層好ましくはカカオ豆由来ルミナコイドを高濃度で含む油脂加工食品（具体的には、チョコレートおよびココア）である。As described above, in order to efficiently ingest cocoa bean-derived luminacoids, a concentrated cocoa bean-derived luminacoid composition can be used in the present invention. Therefore, foods and supplements whose main ingredient is cocoa beans are preferably those that contain a high concentration of cocoa bean-derived luminacoids, more preferably oil-processed compositions that contain a high concentration of cocoa bean-derived luminacoids, and even more preferably oil-processed foods (specifically, chocolate and cocoa) that contain a high concentration of cocoa bean-derived luminacoids.
ここで、食品およびサプリメント中のカカオ豆由来ルミナコイドの含有量はカカオ豆由来ルミナコイドの摂取が可能である限り特に限定されるものではないが、カカオ豆由来ルミナコイドの効率的な摂取の観点から、油脂加工組成物中の含有量は組成物の固形分当たり、例えば、２～９０質量％とすることができ、好ましくは２～５０質量％、より好ましくは５～３５質量％、より一層好ましくは１０～２１質量％である。また、食品およびサプリメント中のカカオ豆由来ルミナコイドの含有量はカカオ豆由来ルミナコイドの摂取が可能である限り特に限定されるものではないが、カカオ豆由来ルミナコイドの効率的な摂取の観点から、油脂加工組成物中の含有量は組成物の固形分２５ｇ当たり、例えば、好ましくは０．５～１１．９ｇ、より好ましくは１．４～８．８ｇ、さらに好ましくは２．６～５．２ｇである。Here, the content of the cocoa bean-derived luminacoid in the food and supplement is not particularly limited as long as the cocoa bean-derived luminacoid can be ingested, but from the viewpoint of efficient ingestion of the cocoa bean-derived luminacoid, the content in the oil-processed composition can be, for example, 2 to 90% by mass, preferably 2 to 50% by mass, more preferably 5 to 35% by mass, and even more preferably 10 to 21% by mass, per solid content of the composition. In addition, the content of the cocoa bean-derived luminacoid in the food and supplement is not particularly limited as long as the cocoa bean-derived luminacoid can be ingested, but from the viewpoint of efficient ingestion of the cocoa bean-derived luminacoid, the content in the oil-processed composition is, for example, preferably 0.5 to 11.9 g, more preferably 1.4 to 8.8 g, and even more preferably 2.6 to 5.2 g, per 25 g of solid content of the composition.
ここで、食品およびサプリメント中のカカオ豆由来食物繊維の含有量はカカオ豆由来難消化性タンパク質の摂取が可能である限り特に限定されるものではないが、カカオ豆由来食物繊維の効率的な摂取の観点から、油脂加工組成物中の含有量は組成物の固形分当たり、例えば、１～４０質量％とすることができ、好ましくは４～２８質量％、より好ましくは６～２０質量％、より一層好ましくは８～１６質量％である。また、食品およびサプリメント中のカカオ豆由来食物繊維の含有量はカカオ豆由来難消化性タンパク質の摂取が可能である限り特に限定されるものではないが、カカオ豆由来食物繊維の効率的な摂取の観点から、油脂加工組成物中の含有量は組成物の固形分２５ｇ当たり、例えば、好ましくは０．３～９．４ｇ、より好ましくは１．０～７．０ｇ、さらに好ましくは２．０～４．０ｇである。Here, the content of the dietary fiber derived from cocoa beans in the food and supplement is not particularly limited as long as the ingestion of the indigestible protein derived from cocoa beans is possible, but from the viewpoint of efficient ingestion of the dietary fiber derived from cocoa beans, the content in the oil-processed composition can be, for example, 1 to 40 mass% per solid content of the composition, preferably 4 to 28 mass%, more preferably 6 to 20 mass%, and even more preferably 8 to 16 mass%. In addition, the content of the dietary fiber derived from cocoa beans in the food and supplement is not particularly limited as long as the ingestion of the indigestible protein derived from cocoa beans is possible, but from the viewpoint of efficient ingestion of the dietary fiber derived from cocoa beans, the content in the oil-processed composition is, for example, preferably 0.3 to 9.4 g, more preferably 1.0 to 7.0 g, and even more preferably 2.0 to 4.0 g per 25 g of solid content of the composition.
ここで、食品およびサプリメント中のカカオ豆由来難消化性タンパク質の含有量はカカオ豆由来難消化性タンパク質の摂取が可能である限り特に限定されるものではないが、カカオ豆由来難消化性タンパク質の効率的な摂取の観点から、油脂加工組成物中の含有量は組成物の固形分当たり、例えば、０．５～１５質量％とすることができ、好ましくは１～１０質量％、より好ましくは１．５～８質量％、より一層好ましくは２～５質量％である。また、食品およびサプリメント中のカカオ豆由来難消化性タンパク質の含有量はカカオ豆由来難消化性タンパク質の摂取が可能である限り特に限定されるものではないが、カカオ豆由来難消化性タンパク質の効率的な摂取の観点から、油脂加工組成物中の含有量は組成物の２５ｇ固形分当たり、例えば、好ましくは０．２～２．５ｇ、より好ましくは０．４～１．８ｇ、さらに好ましくは０．６～１．２ｇである。Here, the content of the cocoa bean-derived resistant protein in the food and supplement is not particularly limited as long as the cocoa bean-derived resistant protein can be ingested, but from the viewpoint of efficient ingestion of the cocoa bean-derived resistant protein, the content in the oil-processed composition can be, for example, 0.5 to 15 mass% per solid content of the composition, preferably 1 to 10 mass%, more preferably 1.5 to 8 mass%, and even more preferably 2 to 5 mass%. In addition, the content of the cocoa bean-derived resistant protein in the food and supplement is not particularly limited as long as the cocoa bean-derived resistant protein can be ingested, but from the viewpoint of efficient ingestion of the cocoa bean-derived resistant protein, the content in the oil-processed composition is, for example, preferably 0.2 to 2.5 g, more preferably 0.4 to 1.8 g, and even more preferably 0.6 to 1.2 g per 25 g solid content of the composition.
本発明で提供される食品としては、チョコレートやココアのようにカカオ豆を主原料とする食品はもちろんのこと、カカオ由来ルミナコイドを含有させることができる食品であれば特に限定されない。例えば、パン類、ビスケット類、麺類、クラッカー、栄養補給バー等の澱粉系食品；キャンディー類、ガム類、グミ、スナック等の各種菓子類；牛乳、加工乳、アイスクリーム類、発酵乳（ヨーグルト等）、乳飲料、チーズ類、バター類、クリーム類等の乳および乳製品；プリン、ゼリー、ババロア、ムース等のデザート類；非アルコール飲料、アルコール飲料等の飲料類；ハム、ソーセージ等の畜肉加工品；カマボコ、竹輪、魚肉ソーセージ等の魚肉加工品；ジャム、ピューレ等の果実加工品；ルウ、ソース等の調味料類等が挙げられる。カカオ豆由来ルミナコイドは各食品の特性、目的に応じ、適当な製造工程の段階で適宜配合することができる。The foods provided by the present invention include, of course, foods whose main ingredient is cacao beans, such as chocolate and cocoa, but are not particularly limited as long as they can contain cacao-derived luminacoids. Examples include starch-based foods such as breads, biscuits, noodles, crackers, and nutritional bars; various confectioneries such as candies, gums, gummies, and snacks; milk and dairy products such as milk, processed milk, ice cream, fermented milk (yogurt, etc.), milk drinks, cheeses, butters, and creams; desserts such as puddings, jellies, bavarois, and mousses; beverages such as non-alcoholic beverages and alcoholic beverages; processed meat products such as ham and sausages; processed fish products such as kamaboko, chikuwa, and fish sausages; processed fruit products such as jams and purees; and seasonings such as roux and sauces. The cacao bean-derived luminacoids can be appropriately blended at an appropriate stage of the manufacturing process depending on the characteristics and purpose of each food.
本発明の医薬品および食品は、食品として古くから重用されていたカカオに含まれる成分を利用することから、それを必要とする哺乳動物（例えば、ヒト、マウス、ラット、ウサギ、イヌ、ネコ、ウシ、ウマ、ブタ、サル等）に対し安全に用いることができる。カカオ由来ルミナコイド（特に、カカオ豆由来ルミナコイド）の投与量または摂取量は、受容者の性別、年齢および体重、症状、投与時間、剤形、投与経路並びに組み合わせる薬剤等に依存して決定できる。例えば、カカオ由来ルミナコイド（特に、カカオ豆由来ルミナコイド）を医薬として経口投与する場合、成人１日当たり、０．５～１１．９ｇ、好ましくは１．４～８．８ｇ、より好ましくは２．６～５．２ｇの範囲となるように、投与することができる。また、カカオ由来ルミナコイド（特に、カカオ豆由来ルミナコイド）を食品として摂取させる場合には、成人１日当たり、０．５～１１．９ｇ、好ましくは１．４～８．８ｇ、より好ましくは２．６～５．２ｇの範囲となるように、投与することができる。 The pharmaceuticals and foods of the present invention utilize ingredients contained in cacao, which has long been used as a food, and therefore can be safely used in mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, etc.) that require them. The dosage or intake of cacao-derived luminacoid (particularly cacao bean-derived luminacoid) can be determined depending on the recipient's sex, age, and weight, symptoms, administration time, dosage form, administration route, and drugs to be combined, etc. For example, when cacao-derived luminacoid (particularly cacao bean-derived luminacoid) is orally administered as a pharmaceutical, it can be administered in the range of 0.5 to 11.9 g, preferably 1.4 to 8.8 g, and more preferably 2.6 to 5.2 g per day for an adult. Furthermore, when cacao-derived luminacoid (particularly cacao bean-derived luminacoid) is ingested as a food product, it can be administered in the range of 0.5 to 11.9 g, preferably 1.4 to 8.8 g, and more preferably 2.6 to 5.2 g per day to an adult.
本発明の組成物および用剤は、他の経口摂取できる組成物と併用することに制限はない。例えば、抗炎症・抗アレルギー作用、腸内菌共生バランス失調（Dysbiosis）に起因すると考えられる疾病（クローン病など）の予防、治療または改善効果が期待できる素材や組成物と併用することで、フィーカリバクテリウム属菌の産生促進効果をさらに高めることができる。There is no restriction on the use of the composition and agent of the present invention in combination with other compositions that can be orally ingested. For example, the effect of promoting the production of Faecalibacterium can be further enhanced by using the composition and agent in combination with materials or compositions that are expected to have anti-inflammatory and anti-allergic effects, and to prevent, treat, or improve diseases (such as Crohn's disease) that are thought to be caused by an imbalance of intestinal bacterial symbiosis (dysbiosis).
本発明の組成物および用剤は、フィーカリバクテリウム属菌の産生促進に有効な１日分の摂取量のカカオ由来ルミナコイドを含んでなる組成物で提供することができる。この場合、本発明の組成物および用剤は、１日分の有効摂取量を摂取できるように包装されていてもよく、１日分の有効摂取量が摂取できる限り、包装形態は一包装であっても、複数包装であってもよい。包装形態で提供する場合、１日分の有効摂取量が摂取できるように摂取量に関する記載が包装になされているか、または該記載がなされた文書を一緒に提供することが望ましい。また、１日分の有効摂取量を複数包装で提供する場合には、摂取の便宜上、１日分の有効摂取量の複数包装をセットで提供することもできる。The composition and agent of the present invention can be provided as a composition comprising a daily intake amount of cocoa-derived luminacoids effective for promoting the production of Faecalibacterium. In this case, the composition and agent of the present invention may be packaged so that one day's effective intake amount can be ingested, and the packaging form may be a single package or multiple packages as long as one day's effective intake amount can be ingested. When provided in a packaged form, it is desirable that the package has a description regarding the intake amount so that one day's effective intake amount can be ingested, or that a document with such description is provided together. In addition, when one day's effective intake amount is provided in multiple packages, multiple packages of one day's effective intake amount can be provided as a set for convenience of intake.
本発明の組成物および用剤を提供するための包装形態は一定量を規定する形態であれば特に限定されず、例えば、包装紙、袋、ソフトバック、紙容器、缶、ボトル、カプセルなどの収容可能な容器などが挙げられる。The packaging form for providing the compositions and preparations of the present invention is not particularly limited as long as it specifies a certain amount, and examples include wrapping paper, bags, soft bags, paper containers, cans, bottles, capsules, and other containers that can accommodate the contents.
本発明の組成物および用剤は、その効果をよりよく発揮させるために、１週間以上継続的に投与または摂取させることが好ましく、投与および摂取期間はより好ましくは１～２週間、特に好ましくは１～４週間である。ここで、「継続的に」とは毎日投与または摂取を続けることを意味する。本発明の組成物および用剤を包装形態で提供する場合には、継続的摂取のために一定期間（例えば、１週間）の有効摂取量をセットで提供してもよい。In order to better exert the effects of the compositions and agents of the present invention, it is preferable to administer or ingest them continuously for one week or more, and the administration and ingestion period is more preferably one to two weeks, and particularly preferably one to four weeks. Here, "continuously" means to administer or ingest them daily. When the compositions and agents of the present invention are provided in a packaged form, they may be provided as a set containing an effective intake amount for a certain period (e.g., one week) for continuous ingestion.
本発明の別の面によれば、有効量のカカオ由来ルミナコイドをヒトまたは非ヒト動物に摂取させるか、或いは投与することを含んでなる、腸内菌叢改善促進方法およびフィーカリバクテリウム属菌の産生促進方法が提供される。本発明の促進方法は、本発明の組成物および用剤に関する記載に従って実施することができる。According to another aspect of the present invention, there is provided a method for promoting the improvement of intestinal flora and a method for promoting the production of bacteria of the genus Faecalibacterium, comprising ingesting or administering an effective amount of a cocoa-derived luminacoid to a human or non-human animal. The promotion method of the present invention can be carried out according to the description of the composition and preparation of the present invention.
本発明のさらに別の面によれば、有効量のカカオ由来ルミナコイドをヒトまたは非ヒト動物に摂取させるか、或いは投与することを含んでなる、フィーカリバクテリウム属菌の産生促進により治療、予防または改善しうる疾患または症状の治療、予防または改善方法が提供される。本発明の治療、予防または改善方法は、本発明の組成物および用剤に関する記載に従って実施することができる。 According to yet another aspect of the present invention, there is provided a method for treating, preventing or ameliorating a disease or symptom that can be treated, prevented or ameliorated by promoting the production of Faecalibacterium spp., comprising ingesting or administering to a human or non-human animal an effective amount of a cocoa-derived luminacoid. The present method for treating, preventing or ameliorating can be carried out in accordance with the description of the compositions and preparations of the present invention.
本発明のさらにまた別の面によれば、腸内菌叢改善促進剤またはフィーカリバクテリウム属菌の産生促進剤の製造のための、カカオ由来ルミナコイドの使用と、腸内菌叢改善促進剤またはフィーカリバクテリウム属菌の産生促進剤としての、カカオ由来ルミナコイドの使用が提供される。本発明によればまた、フィーカリバクテリウム属菌の産生促進により治療、予防または改善しうる疾患または症状の治療剤、予防剤または改善剤の製造のための、カカオ由来ルミナコイドの使用と、フィーカリバクテリウム属菌の産生促進により治療、予防または改善しうる疾患または症状の治療剤、予防剤または改善剤としての、カカオ由来ルミナコイドの使用が提供される。本発明の使用は、本発明の組成物および用剤に関する記載に従って実施することができる。According to yet another aspect of the present invention, there is provided the use of cocoa-derived luminacoid for the manufacture of an agent for promoting the improvement of intestinal flora or the production of Faecalibacterium spp., and the use of cocoa-derived luminacoid as an agent for promoting the improvement of intestinal flora or the production of Faecalibacterium spp. The present invention also provides the use of cocoa-derived luminacoid for the manufacture of a therapeutic, preventive or ameliorating agent for a disease or symptom that can be treated, prevented or ameliorated by promoting the production of Faecalibacterium spp., and the use of cocoa-derived luminacoid as a therapeutic, preventive or ameliorating agent for a disease or symptom that can be treated, prevented or ameliorated by promoting the production of Faecalibacterium spp. The use of the present invention can be carried out according to the description of the composition and agent of the present invention.
本発明のさらにまた別の面によれば、腸内菌叢改善促進に用いるための、あるいは、フィーカリバクテリウム属菌の産生促進に用いるための、カカオ由来ルミナコイドが提供される。本発明によればまた、フィーカリバクテリウム属菌の産生促進により治療、予防または改善しうる疾患または症状の治療、予防または改善に用いるための、カカオ由来ルミナコイドが提供される。上記のカカオ由来ルミナコイドは、本発明の組成物および用剤に関する記載に従って実施することができる。According to yet another aspect of the present invention, there is provided a cocoa-derived luminacoid for use in promoting the improvement of the intestinal flora or for use in promoting the production of Faecalibacterium spp. The present invention also provides a cocoa-derived luminacoid for use in the treatment, prevention or amelioration of a disease or symptom that can be treated, prevented or ameliorated by promoting the production of Faecalibacterium spp. The above-mentioned cocoa-derived luminacoid can be used in accordance with the description of the compositions and preparations of the present invention.
以下の例に基づいて本発明をより具体的に説明するが、本発明はこれらの例に限定されるものではない。The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
試験例１：カカオ豆由来ルミナコイドによるフィーカリバクテリウム属菌の産生促進効果の検討（１）
カカオ豆由来ルミナコイドがフィーカリバクテリウム属菌の産生に与える影響を検証するために以下の試験を行った。 Test Example 1: Examination of the effect of luminacoid derived from cocoa beans on promoting the production of Faecalis bacterium (1)
The following test was conducted to verify the effect of cocoa bean-derived luminacoids on the production of Faecalibacterium sp.
２０歳以上５０歳未満の健康な日本人女性で、スクリーニングにより選抜した４０名を被験者とし、評価者盲検並行群間比較試験を行った。途中で試験を中断した９名を除いて、試験食品群１６名、対照食品群１５名の結果が得られた。試験期間中は、食事制限およびその他の制限（食事制限およびその他の制限の詳細は、下記に示す。）を行った。 Forty healthy Japanese women aged 20 to 50 years were selected through screening as subjects in an evaluator-blinded parallel group comparative study. Excluding nine subjects who discontinued the study midway, results were obtained for 16 subjects in the test food group and 15 subjects in the control food group. Dietary and other restrictions were imposed during the study period (details of dietary and other restrictions are given below).
スクリーニングは、摂取開始７日前（－７日目、－１Ｗ）に、生活習慣アンケート（年齢、既往歴、合併症、食物アレルギーの有無、医薬品や健康食品の摂取状況、飲酒等の確認）、自覚アンケート（整腸に関する自覚症状の調査）、理学的検査（身長、体重、血圧、脈拍数、ＢＭＩ）を実施し、排便の状態（排便回数、便性、便量）、年齢、食習慣（カカオ製品、納豆、乳製品等の日常的な摂取状況）による除外基準を満たした（除外基準の詳細は、下記に示す。）、排便回数が週平均４回以下の４０名を選抜した。Screening took place 7 days prior to the start of intake (-7th day, -1 week) with a lifestyle questionnaire (confirming age, medical history, complications, presence or absence of food allergies, intake of medicines and health foods, alcohol consumption, etc.), a subjective questionnaire (survey of subjective symptoms related to intestinal regulation), and a physical examination (height, weight, blood pressure, pulse rate, BMI). 40 people were selected based on their bowel movement status (frequency of bowel movements, stool quality, stool volume), age, and dietary habits (daily intake of cocoa products, natto, dairy products, etc.) (details of the exclusion criteria are given below), and who had an average bowel movement frequency of 4 times per week or less.
スクリーニングにおける除外基準
以下の者をスクリーニング対象から除外した。
・現在、何らかの慢性疾患を有し、薬物治療を受けている者
・チョコレート、牛乳、大豆にアレルギーを有する者
・乳糖不耐症を有する者
・スクリーニング前３か月間に、カカオ製品を週４回以上摂取している者
・スクリーニング前３か月間に、納豆を週４回以上摂取している者
・スクリーニング前３か月間に、ヨーグルト、乳酸菌飲料、整腸剤等の乳酸菌含有食品を週４回以上摂取している者
・本試験結果に影響する可能性がある医薬品、医薬部外品、サプリメント、健康食品、特定保健用食品（トクホ）を日常において服用・摂取している者、または試験中に服用・摂取する予定のある者
・スクリーニング前１か月間に、他の臨床試験およびモニター試験に参加した者、あるいは本臨床試験同意後に他の臨床試験に参加する予定のある者
・妊娠している者、試験期間中に妊娠の予定、希望がある者
・授乳中の者
・アルコール多飲用者（純アルコール換算で、１日平均６０ｇ以上を超える飲酒者）
・その他、被験者として不適当と判断した者Those who met the screening exclusion criteria were excluded from screening.
-Those who currently have any chronic disease and are receiving drug treatment -Those who are allergic to chocolate, milk, or soy -Those who are lactose intolerant -Those who have consumed cocoa products more than four times a week in the three months prior to screening -Those who have consumed natto more than four times a week in the three months prior to screening -Those who have consumed lactic acid bacteria-containing foods such as yogurt, lactic acid bacteria drinks, and intestinal regulators more than four times a week in the three months prior to screening -Those who take or consume medicines, quasi-drugs, supplements, health foods, or foods for specified health uses (FOSHU) on a daily basis that may affect the results of this study, or those who plan to take or consume them during the study -Those who have participated in other clinical trials or monitor studies in the month prior to screening, or those who plan to participate in other clinical trials after agreeing to this clinical trial -Those who are pregnant, or who plan to become pregnant during the study period -Those who are breastfeeding -Those who drink a lot of alcohol (those who drink an average of more than 60g of pure alcohol per day)
-Others who are deemed inappropriate as subjects
試験期間中の食事制限およびその他の制限
被験者には、試験期間中は通常通りの生活を送らせるとともに、次の制限事項を遵守するよう指導した。
食事、飲み物および嗜好品に関する事項
・食事は、これまでの食生活（食事量、食事内容）を大きく変えず、日常範囲を大きく逸脱する過度な節食や過食は控える。但し、スクリーニングから摂取２週後の採便が終了するまで、納豆の摂取を禁止する。
・飲料の摂取は、緑茶やコーヒー等、摂取量や回数は制限しないが、これまでの摂取の程度（量、種類等）を大きく変えることは控える。
・間食は、原則として自由とするが、通常摂取している量を超えないようにする。但し、スクリーニングから摂取２週後の採便が終了するまで、試験食品以外のカカオを含む飲食品の摂取を禁止する。
・アルコールの摂取は、原則として制限しないが、これまでのアルコール摂取の程度（量、種類等）を大きく変えることは控える。
・本試験に影響を及ぼす可能性がある医薬品（一般用医薬品を含む）、医薬部外品、サプリメント、特定保健用食品または健康食品は、スクリーニング時から、摂取２週後の採便が終了するまで使用や摂取を禁止する。抗生物質、下剤、便秘薬、整腸剤、便秘や、下痢、消化不良を改善する作用を有する芍薬、黄柏、大黄、大棗、桂皮、人蔘等を含む漢方処方（桂枝加芍薬大黄湯、人蔘湯、五苓散料、半夏瀉心湯、加味逍遥散、大柴胡湯、乙字湯、桃核承気湯、防風通聖散）および生薬（芍薬、黄柏、大黄、大棗、桂皮、人蔘等）を含有する医薬品（一般用医薬品を含む）、医薬部外品、サプリメント、特定保健用食品（トクホ）または健康食品が該当する。
・試験期間中、生活日誌は毎日記録する。
・スクリーニング時から、摂取２週後の採便が終了するまで、大きな生活の変化は、可能な限り控える。 Dietary and other restrictions during the study Subjects were instructed to continue their normal lifestyle during the study and to adhere to the following restrictions:
Food, drink and luxury items
- Do not make any major changes to your eating habits (amount of food eaten, food content), and refrain from excessive dieting or overeating that deviates significantly from your normal range. However, you will be prohibited from eating natto until stool collection is completed two weeks after the screening.
- There are no restrictions on the amount or frequency of intake of beverages such as green tea or coffee, but you should refrain from making any major changes to your current level of intake (amount, type, etc.).
In principle, participants are free to eat snacks between meals, but should not exceed the amount they normally consume. However, they are prohibited from consuming any food or drink that contains cocoa other than the test food until the completion of stool collection two weeks after the screening test.
- As a general rule, there will be no restrictions on alcohol intake, but you should refrain from making any major changes to your current level of alcohol intake (amount, type, etc.).
- Drugs (including over-the-counter drugs), quasi-drugs, supplements, foods for specified health uses, or health foods that may affect this study are prohibited from use or intake from screening until stool collection is completed two weeks after intake. This includes antibiotics, laxatives, constipation medicines, intestinal regulators, herbal prescriptions containing peony root, Phellodendron bark, Rhubarb, Daijube, cinnamon, ginseng, etc. that have the effect of improving constipation, diarrhea, and indigestion (Kei Shi Ka Shakuyaku Daio Tang, Ninjin Tang, Gorei Sanryo, Hangesha Shin Tang, Kamishakuyo San, Daisaiko Tang, Otsuji Tang, Touke Joki Tang, Bofu Tsusho San), and herbal medicines (peony root, Phellodendron bark, Rhubarb, Daijube, cinnamon, ginseng, etc.) that contain herbal medicines (including over-the-counter drugs), quasi-drugs, supplements, foods for specified health uses (Tokuho), or health foods.
-During the test period, record your daily diary every day.
- Refrain from making any major changes to your lifestyle as much as possible from the time of screening until stool collection is completed two weeks after intake.
試験食品は、市販のカカオ豆由来ルミナコイド高含有油脂加工組成物（「チョコレート効果カカオ７２％」株式会社明治）を毎日２５ｇ（１枚５ｇの油脂加工組成物を５枚）ずつ２週間継続して摂取させた。上記油脂加工組成物は１枚当たりカカオポリフェノールを１２７ｍｇ（総ポリフェノール量）含んでいることから、被験者が１日に摂取するカカオポリフェノールは６３５ｍｇとなる。また、上記油脂加工組成物は１枚当たり食物繊維０．６ｇ、難消化性たんぱく質０．１８ｇを含んでいることから、被験者が１日に摂取する食物繊維量は３．０ｇとなり、難消化性たんぱく質量は０．８８ｇとなる。すなわち、被験者が１日に摂取するカカオ豆由来ルミナコイド量は３．８８ｇとなる。対照食品は、市販のチョコレート（「ホワイトチョコレート」株式会社明治）を毎日２５ｇ（１枚４．２ｇの油脂加工組成物を６枚）ずつ２週間継続して摂取させた。対照食品は、ポリフェノール、食物繊維、難消化性たんぱく質を含まない。The test food consisted of a commercially available cocoa bean-derived luminacoid-rich oil-processed composition ("Chocolate Effect Cocoa 72%" Meiji Co., Ltd.) that was consumed daily at 25 g (five slices of 5 g oil-processed composition) for two consecutive weeks. The oil-processed composition contains 127 mg of cocoa polyphenols (total polyphenol content) per slice, so the subjects consumed 635 mg of cocoa polyphenols per day. The oil-processed composition also contains 0.6 g of dietary fiber and 0.18 g of resistant protein per slice, so the subjects consumed 3.0 g of dietary fiber and 0.88 g of resistant protein per day. In other words, the subjects consumed 3.88 g of cocoa bean-derived luminacoids per day. The control food consisted of a commercially available chocolate ("White Chocolate" Meiji Co., Ltd.) that was consumed daily at 25 g (six slices of 4.2 g oil-processed composition) for two consecutive weeks. The control food contained no polyphenols, dietary fiber, or resistant proteins.
また、試験食品における、１日に摂取する１～４量体のポリフェノールの含量は７６ｍｇであった。主な成分の内訳として、カテキン（単量体）が１０．４ｍｇ、エピカテキン（単量体）が２７．８ｍｇ、プロシアニジンＢ２（２量体）が１５．８ｍｇ、プロシアニジンＢ５（２量体）が４．４ｍｇ、プロシアニジンＣ１（３量体）が９．６ｍｇ、シンナムタンニンＡ２（４量体）が７．１ｍｇであった。なお、各成分については、ＨＰＬＣを用いて測定した。カラムは、Ｄｅｖｅｒｏｓｉｌ－ＯＤＳ－ＨＧ５（４．６ｍｍ×２５０ｍｍ、φ５μ、野村化学株式会社製)を使用した。溶離液は、Ａ液とＢ液で構成され、Ａ液は０．１%トリフルオロ酢酸水溶液、Ｂ液は０．１％トリフルオロ酢酸／アセトニトリル溶液を使用した。カラムへ通す溶離液の流速は０．８ｍｌ／分、グラジェントの条件は、溶離液全体に占めるＢ液の割合を、開始時点で１０％、開始５分後で１０％、開始３５分後で２５％、開始４０分後で１００％、開始４５分後で１００％とした。サンプルインジェクション量は１０μＬであり、エピカテキンを標準品として、各成分をエピカテキン当量で定量した。また、ポリフェノール含量は、プルシアンブルー法により測定した。具体的には、Martin L. Price and Larry G. Butler, J. Agric Food Chem. 1977, 25(6): 1268-1273に記載の方法に従い、市販のエピカテキンを標準物質としてポリフェノール含量を算出した。The content of mono- to tetramer polyphenols in the test food per day was 76 mg. The main components were catechin (monomer) 10.4 mg, epicatechin (monomer) 27.8 mg, procyanidin B2 (dimer) 15.8 mg, procyanidin B5 (dimer) 4.4 mg, procyanidin C1 (trimer) 9.6 mg, and cinnamtannin A2 (tetramer) 7.1 mg. Each component was measured using HPLC. The column used was Deverosil-ODS-HG5 (4.6 mm x 250 mm, φ5μ, manufactured by Nomura Chemical Co., Ltd.). The eluent consisted of solution A and solution B, with solution A being a 0.1% aqueous solution of trifluoroacetic acid, and solution B being a 0.1% trifluoroacetic acid/acetonitrile solution. The flow rate of the eluent passing through the column was 0.8 ml/min, and the gradient conditions were as follows: the ratio of solution B to the entire eluent was 10% at the start, 10% 5 minutes after the start, 25% 35 minutes after the start, 100% 40 minutes after the start, and 100% 45 minutes after the start. The sample injection amount was 10 μL, and each component was quantified in epicatechin equivalents using epicatechin as a standard. The polyphenol content was measured by the Prussian blue method. Specifically, the polyphenol content was calculated using commercially available epicatechin as a standard according to the method described in Martin L. Price and Larry G. Butler, J. Agric Food Chem. 1977, 25(6): 1268-1273.
また、試験食品における、１日に摂取する食物繊維の含量は３．０ｇであった。具体的には（エーオーエーシ―インターナショナルAOAC Internationalによる公定法）である酵素－重量法により算出した。The dietary fiber content of the test food to be ingested per day was 3.0 g. Specifically, this was calculated using the enzymatic-gravimetric method (official method set by AOAC International).
また、試験食品における、１日に摂取する難消化性タンパク質の含量は０．８８ｇであった。具体的には、下記、難消化性タンパク質の抽出方法に従い抽出したたんぱく質量を、ケルダール法（たんぱく質定量の公定法）により算出した。また、下記、人工消化試験により、抽出したたんぱく質が難消化性タンパク質であることを確認した。The amount of resistant protein ingested per day in the test food was 0.88 g. Specifically, the amount of protein extracted according to the resistant protein extraction method described below was calculated using the Kjeldahl method (the official method for quantifying protein). The extracted protein was also confirmed to be resistant protein by the artificial digestion test described below.
難消化性たんぱく質の抽出方法
チョコレートを湯煎にて溶解し、チョコレートの５倍量のヘキサンを添加して３時間撹拌した。１０，０００ｒｐｍ、４℃で１０分遠心し、沈殿を回収した。回収した沈殿を、初発チョコレート量の３倍量のヘキサンに懸濁し１時間撹拌した。１０，０００ｒｐｍ、４℃で１０分遠心し、沈殿を一晩風乾した（チョコパウダーという）。前述のチョコパウダーを２０倍量の脱イオン水に懸濁し、１０分ほど撹拌した。ｐＨを確認し、ＮａＯＨでｐＨ１２に調整した。再び１０分撹拌した後、ｐＨを確認し、ＮａＯＨでｐＨ１２に調整した。５０℃に加温し、到達したらｐＨを確認し、ＮａＯＨでｐＨ１２に調整した。７０℃に加温し、到達したら３０分抽出した。１０，０００ｒｐｍ、１０分、室温で遠心し、上清を回収した。得られた上清にリン酸を添加してｐＨを２．０に調整し、一晩４℃で静置した。１０，０００ｒｐｍ、で１０分、室温で遠心し、沈殿を回収した。沈殿に原料（チョコパウダー重量）の５倍量の脱イオン水を加えて懸濁し、１０，０００ｒｐｍ、で１０分、２５℃で遠心し上清を除去した。洗浄は２回実施した。沈殿を回収し８０℃で７２時間減圧乾燥した。 Extraction method of indigestible protein Chocolate was dissolved in a hot water bath, and hexane was added in an
人工消化試験
（１）ペプシン消化
被験サンプルに水を加えて１０％とした後、塩酸でｐＨを２．０に調整した。そこにペプシン（１６３－００６４２、和光純薬工業社）を採取量の１／１００を添加して３７℃で４時間反応させた。 Artificial digestion test (1) Pepsin digestion After adding water to the test sample to make it 10%, the pH was adjusted to 2.0 with hydrochloric acid. Pepsin (163-00642, Wako Pure Chemical Industries, Ltd.) was added in an amount of 1/100 of the collected amount and reacted at 37°C for 4 hours.
（２）パンクレアチン消化
上記（１）の反応物を水酸化ナトリウムでｐＨを８～１０に調整し、パンクレアチン（１６３－００１４２、和光純薬工業社）を採取量の１／５０を添加して３７℃で１６時間反応させた。酵素反応終了後、１００℃、５分間加熱して酵素反応を止めた。また、（１）、（２）ともに、酵素に代えて酵素添加量と等量（重量）の水を加え、他を酵素処理と同様に操作したブランク区を検体と同時に処理した。
(2) Pancreatin digestion The pH of the reaction product in (1) was adjusted to 8-10 with sodium hydroxide, and 1/50 of the collected amount of pancreatin (163-00142, Wako Pure Chemical Industries, Ltd.) was added and reacted at 37°C for 16 hours. After completion of the enzyme reaction, the enzyme reaction was stopped by heating at 100°C for 5 minutes. In addition, in both (1) and (2), a blank group was treated simultaneously with the specimen, in which water was added instead of the enzyme in an amount (weight) equal to the amount of enzyme added, and the rest of the operations were the same as in the enzyme treatment.
（３）未消化物（残渣）の回収
上記（２）の反応物を塩酸でｐＨを２．０に調整し、１０，０００ｒｐｍ、１０分、４℃で遠心し、沈殿を回収した。回収した沈殿物に、試料採取量の５倍量の水を加えて沈殿物を溶解・洗浄して再度遠心分離（１０，０００ｒｐｍ、１０分、４℃）を行った。この操作を合計２回行った。
(3) Recovery of undigested matter (residue) The pH of the reaction product of (2) above was adjusted to 2.0 with hydrochloric acid, and centrifuged at 10,000 rpm for 10 minutes at 4°C to recover the precipitate. Water was added to the recovered precipitate in an
（４）乾燥
回収した沈殿は凍結乾燥した。その際、凍結乾燥前後における重量（以下、残渣回収量とする）を測定した。なお成分量はケルダール法にて求めた。
以下の計算式にて消化率を算出した。
(4) Drying The collected precipitate was freeze-dried. The weights before and after freeze-drying (hereinafter referred to as the collected residue) were measured. The amounts of the components were determined by the Kjeldahl method.
The digestibility was calculated using the following formula.
結果、難消化性たんぱく質含有物中のタンパク質の消化率は２０％、同時に試験を実施したカゼインは９９．３％であった。As a result, the digestibility of protein in the resistant protein-containing material was 20%, while that of casein, which was tested at the same time, was 99.3%.
以上のことからチョコレートより抽出したたんぱく質が難消化性であることが確認できた。 From the above, it was confirmed that the protein extracted from chocolate is resistant to digestion.
評価および結果
摂取開始日を１日目とし、摂取開始７日前（－７日目、－１Ｗ）から摂取最終日（１４日目、２Ｗ）までの間、生活日誌（試験食品摂取の確認、自覚症状の記録、摂取開始日７日前よりの排便状況（排便回数、便性、便量）、性周期等、医薬品の服用状況等の記録）による記録を行った。摂取開始７日前（－７日目、－１Ｗ）および摂取最終日（１４日目、２Ｗ）に、生活習慣アンケート、自覚アンケート、理学的検査（身長、体重、血圧、脈拍数、ＢＭＩ）採便を実施した。また、便中菌叢解析（Nagashima法による便中菌叢のT-RFLP解析）、便中ビフィズス菌数および総菌数の定量・ビフィズス菌占有率の測定（リアルタイムＰＣＲ法）、便の網羅的菌叢解析（ＤＧＧＥ法、次世代アンプリコンシーケンス法：メタゲノム解析による被験者腸内菌叢（属レベル）の評価を行った。得られたデータは平均値±標準誤差で示した。統計解析は以下の通り行った。便色・便量などの群間比較：「独立サンプルのt検定」（解析ソフトウェア：「IBM SPSS Statistics 23」）。排便回数の群間比較：「Mann-WhitneyのU検定」（解析ソフトウェア：「IBM SPSS Statistics 23」）。網羅的菌叢解析の群間比較：エクセル統計(SSRI)を用い、対照食品群と試験食品群の比較をｔ－ｔｅｓｔで評価した（有意水準は５％未満とした）。 Evaluation and Results The day of ingestion was counted as
排便回数は、試験食品群において顕著な経時的増加を示すとともに、－７日目～－１日目（－１Ｗ～０Ｗ）に対して、０日目～７日目（０Ｗ～１Ｗ）、８日目～１４日目（１Ｗ～２Ｗ）ともに有意差（危険率（ｐ値）５％未満）があった。対照食品群に比べ試験食品群において明らかな排便回数の増加が認められた（図１）。
The number of defecations showed a significant increase over time in the test food group, and there were significant differences (risk rate (p value) less than 5%) on
便性は、便色と排便量により評価した。便色は５段階の便色スコアにより評価した。具体的には、便色スコア値は、１：黄色～黄褐色、２：褐色、３：茶褐色、４：暗褐色、５：黒褐色～黒色とし（腸内細菌学雑誌.2004, 18(2); 107-115参照）、被験者には、目視にて比色するため、上記５段階の便色スコア値および名称が表記され、対応する便色がカラー印刷された便色スケールを渡し、便の色を被験者自身に観測させた。その結果、試験食品群の摂取１４日後の便色スコア値が、対照食品群の便色スコア値よりも有意に低い値を示していた（図２）。すなわち、便色において試験食品摂取群に摂取１４日後には改善傾向がみられたが、対照食品摂取群では、ほとんど変化がみられなかった（図２）。便量については、－７日目～－１日目（－１Ｗ～０Ｗ）の摂取前１週間の全排便量(前)に対する０日目～７日目（０Ｗ～１Ｗ）、または８日目～１４日目（１Ｗ～２Ｗ）までのそれぞれ１週間の全排便量の比を求めた。その結果、試験食品群の便量が経時的に増加する傾向がみられた（データ示さず）。Stool quality was evaluated based on stool color and volume. Stool color was evaluated using a 5-level stool color score. Specifically, stool color score values were 1: yellow to yellowish brown, 2: brown, 3: dark brown, 4: dark brown, and 5: blackish brown to black (see Intestinal Microbiology Journal, 2004, 18(2); 107-115). The subjects were given a stool color scale with the above 5-level stool color score value and name written on it and the corresponding stool color printed in color so that they could visually compare the colors, and were asked to observe the color of the stool themselves. As a result, the stool color score value of the
図１および図２の結果から、カカオ豆由来ルミナコイド摂取により排便回数、排便量が増加すること、便色が改善することが確認された。 The results in Figures 1 and 2 confirm that intake of cocoa bean-derived luminacoids increases bowel movement frequency and stool volume, and improves stool color.
また、次世代アンプリコンシーケンス法（Proc Natl Acad Sci U S A. 2011, 108(Suppl 1): 4516-4522参照）を用いたメタゲノム解析をMiSeqシステム（イルミナ社製）により実施した。菌叢を構成する各菌の分類は、16SrDNA配列情報に従って行った（Nucl Acids Res. 2007, 35:18. e120参照）。その結果、被験者腸内菌叢（属レベル）では、腸内菌叢全菌数を１００％とした場合のフィーカリバクテリウム属菌の占有率が、試験食品摂取群において、摂取前（０日目）と比較して摂取後（１４日目）で有意に増加した（図３）。すなわち、排便回数の増加、排便量の増加、便色の改善は、フィーカリバクテリウム属菌の増加によるものであることが確認された。 In addition, metagenomic analysis was performed using the MiSeq system (Illumina) with next-generation amplicon sequencing (see Proc Natl Acad Sci U S A. 2011, 108(Suppl 1): 4516-4522). The classification of each bacterium that constitutes the microbiota was performed according to 16S rDNA sequence information (see Nucl Acids Res. 2007, 35:18. e120). As a result, in the subjects' intestinal microbiota (genus level), the occupancy rate of Faecalibacterium spp., when the total number of bacteria in the intestinal microbiota was taken as 100%, was significantly increased after ingestion (day 14) compared to before ingestion (day 0) in the test food intake group (Figure 3). In other words, it was confirmed that the increase in defecation frequency, increase in defecation volume, and improvement in stool color were due to an increase in Faecalibacterium spp.
以上の結果から、カカオ豆由来ルミナコイドはフィーカリバクテリウム属菌の産生を促進することが確認された。従って、カカオ豆由来ルミナコイドは、炎症性疾患、アレルギー疾患、腸内菌共生バランス失調（Dysbiosis）に起因すると考えられる疾病（クローン病など）などのフィーカリバクテリウム属菌の産生促進により改善される疾患や病状の改善効果を有することが示された。 These results confirm that cocoa bean-derived luminacoids promote the production of Faecalibacterium spp. Therefore, it has been shown that cocoa bean-derived luminacoids have the effect of improving diseases and conditions that are improved by promoting the production of Faecalibacterium spp., such as inflammatory diseases, allergic diseases, and diseases thought to be caused by intestinal bacterial symbiotic imbalance (dysbiosis) (e.g., Crohn's disease).
試験例２：カカオ豆由来ルミナコイドによるフィーカリバクテリウム属菌の産生促進効果の検討（２）
カカオ豆由来ルミナコイドがフィーカリバクテリウム属菌の産生に与える影響を検証するために、チョコレートから抽出したルミナコイドを用いて以下の試験を行った。 Test Example 2: Examination of the effect of luminacoid derived from cocoa beans on promoting the production of Faecalis bacterium (2)
In order to verify the effect of cocoa bean-derived luminacoids on the production of Faecalibacterium sp., the following test was conducted using luminacoids extracted from chocolate.
（１）ルミナコイドの調製
市販のカカオ豆由来ルミナコイド高含有油脂加工組成物（「チョコレート効果カカオ７２％」株式会社明治）１６０３ｇを湯煎で溶かし、チョコレートの５倍量のヘキサン（８Ｌ）を添加後、３時間撹拌することで脱脂した。遠心分離後（１００００ｒｐｍ、１０分、４℃）、上清を除去し沈殿物を回収した。沈殿物にチョコレートの３倍量のヘキサン（４．８Ｌ）を添加後、１時間撹拌した。遠心分離後、上清を除去し沈殿物を回収後、ドラフト内で一晩乾燥させた。乾燥させた沈殿物をミルサーで粉砕することでチョコレートパウダー９８１ｇを得た。
(1) Preparation of Luminacoid 1603 g of a commercially available cocoa bean-derived luminacoid-rich oil-and-fat composition ("Chocolate Effect Cacao 72%" Meiji Co., Ltd.) was melted in a hot water bath, and hexane (8 L) was added in an amount five times the amount of chocolate, followed by stirring for 3 hours to defatt the mixture. After centrifugation (10,000 rpm, 10 minutes, 4°C), the supernatant was removed and the precipitate was collected. Hexane (4.8 L) in an amount three times the amount of chocolate was added to the precipitate, followed by stirring for 1 hour. After centrifugation, the supernatant was removed and the precipitate was collected, followed by drying overnight in a fume hood. The dried precipitate was ground in a mill to obtain 981 g of chocolate powder.
上記のようにして得たチョコレートパウダー（９８１ｇ）を２０倍量の水（１９．６Ｌ）に懸濁し、８Ｎ ＮａＯＨを用いてｐＨ１２．０に調整後、７０℃に加温し３０分間抽出を行った。遠心分離後（１００００ｒｐｍ、１０分、２５℃）、上清を回収した。The chocolate powder (981 g) obtained as described above was suspended in 20 times the amount of water (19.6 L), adjusted to pH 12.0 with 8N NaOH, heated to 70°C, and extracted for 30 minutes. After centrifugation (10,000 rpm, 10 minutes, 25°C), the supernatant was collected.
回収した上清をリン酸でｐＨ２．０に調整し、４℃で一晩静置した。遠心分離後（１００００ｒｐｍ、１０分、２５℃）、上清を除去し沈殿物を回収した。The collected supernatant was adjusted to pH 2.0 with phosphoric acid and allowed to stand overnight at 4°C. After centrifugation (10,000 rpm, 10 min, 25°C), the supernatant was removed and the precipitate was collected.
沈殿物に、チョコレートパウダーの５倍量の水（４．９Ｌ）を加え水洗を行い、遠心分離後（１００００ｒｐｍ、１０分、２５℃）、上清を捨てた。水洗を２回繰り返して得られた沈殿物を８０℃で６時間、減圧乾燥機にて乾燥させ、カカオプロテイン含有物１２９ｇを得た（たんぱく質：４６．６％、食物繊維：４３．３％）。得られたカカオプロテイン含有物に含まれるたんぱく質と食物繊維は難消化性であることから、カカオプロテイン含有物はカカオ豆由来ルミナコイドに相当する。The precipitate was washed with water (4.9 L) in an amount five times the amount of chocolate powder, and centrifuged (10,000 rpm, 10 minutes, 25°C), after which the supernatant was discarded. The precipitate obtained after two water washes was dried in a vacuum dryer at 80°C for six hours, yielding 129 g of cocoa protein-containing material (protein: 46.6%, dietary fiber: 43.3%). As the protein and dietary fiber contained in the obtained cocoa protein-containing material are indigestible, the cocoa protein-containing material corresponds to luminacoid derived from cocoa beans.
（２）試験方法
試験は、上記（１）で調製したカカオ豆由来ルミナコイド（カカオプロテイン含有物）を配合した飼料（試験飼料）を摂取させる試験飼料群と、カカオ豆由来ルミナコイドを配合しない飼料（対照飼料）を摂取させる対照飼料群の２群に対して行った。試験飼料としては、ＡＩＮ９３Ｇの５％セルロース分を５％カカオプロテイン含有物に置換した飼料を用いた。対照飼料としては、ＡＩＮ９３Ｇのうち不溶性食物繊維である５％セルロース分を５％コーンスターチに置換した飼料を用いた。ＡＩＮ９３Ｇからセルロース分を除いた飼料は、オリエンタル酵母工業社製のものを用いた。
(2) Test method The test was conducted on two groups: a test group fed with a feed (test feed) containing the cacao bean-derived luminacoid (cacao protein-containing material) prepared in (1) above, and a control group fed with a feed (control feed) not containing the cacao bean-derived luminacoid. The test feed was a feed in which 5% of the cellulose content of AIN93G was replaced with 5% of the cacao protein-containing material. The control feed was a feed in which 5% of the cellulose content of AIN93G, which is an insoluble dietary fiber, was replaced with 5% corn starch. The feed in which the cellulose content was removed from AIN93G was manufactured by Oriental Yeast Co., Ltd.
ＢＡＬＢ／ｃＡマウス（メス、６週齢、日本クレア社）１２匹を対照飼料にて１週間予備飼育した後、平均体重が同等となるように群分けを行った。群分けは各群６匹となるようにした。12 BALB/cA mice (female, 6 weeks old, CLEA Japan) were pre-fed with control feed for 1 week, and then divided into groups with the same average body weight. Each group contained 6 mice.
試験開始時から各飼料および水は自由摂取とした。週に２回の体重測定および摂餌量測定を行った。また、試験開始後１１～１３日目の２日分の糞便を回収し、凍結乾燥後、乾燥重量を測定した。From the start of the study, the animals were allowed free access to all the feed and water. Body weight and food intake were measured twice a week. In addition, feces for two days, from the 11th to 13th day after the start of the study, were collected, freeze-dried, and the dry weight was measured.
試験開始後１４日目にイソフルラン麻酔下にて腹部下行大静脈より全採血を行った。盲腸内容物を採取後、重量を測定し－８０℃にて保存した。また、盲腸内短鎖脂肪酸濃度の測定は、以下の測定機器および条件で行った。
測定機器：島津有機酸分析システム
カラム：Shim-Pack SCR-102(H),300mm×8mm ID、２本直列で使用
ガードカラム：Shim-Pack SCR-102(H),50mm×6mm ID
溶離液：５ｍｍｏｌ／Ｌ ｐ－トルエンスルホン酸
反応液：５ｍｍｏｌ／Ｌ ｐ－トルエンスルホン酸、１００μｍｏｌ／Ｌ ＥＤＴＡ、２０ｍｍｏｌ／Ｌ Ｂｉｓ－Ｔｒｉｓ
流速：０．８ｍＬ／分
オーブン温度：４５℃
検出器：電気伝導度検出器ＣＤＤ－１０Ａ
参照文献：Advances in Microbiology. 2015, 5(7):531-540およびBenef Microbes. 2016 Jun, 7(3):337-344
On the 14th day after the start of the study, whole blood was collected from the abdominal inferior vena cava under isoflurane anesthesia. The cecal contents were collected, weighed, and stored at −80° C. The concentration of short-chain fatty acids in the cecum was measured using the following measuring equipment and conditions.
Measurement equipment: Shimadzu organic acid analysis system Column: Shim-Pack SCR-102(H), 300mm x 8mm ID, two columns used in series Guard column: Shim-Pack SCR-102(H), 50mm x 6mm ID
Eluent: 5 mmol/L p-toluenesulfonic acid Reaction solution: 5 mmol/L p-toluenesulfonic acid, 100 μmol/L EDTA, 20 mmol/L Bis-Tris
Flow rate: 0.8 mL/min Oven temperature: 45° C.
Detector: Electrical conductivity detector CDD-10A
References: Advances in Microbiology. 2015, 5(7):531-540 and Benef Microbes. 2016 Jun, 7(3):337-344
得られたデータは平均値±標準誤差で示した。統計解析はエクセル統計 (SSRI)を用い、対照飼料群と試験飼料群の比較をｔ－ｔｅｓｔで評価した。有意水準は５％未満とした。なお、試験開始時および終了時の体重と総摂餌量については、いずれも各群間に有意な差はなかった（データ示さず）。The data obtained are shown as mean values ± standard error. Statistical analysis was performed using Excel Statistics (SSRI), and comparisons between the control and test feed groups were evaluated using t-tests. The significance level was set at less than 5%. There were no significant differences between the groups in body weight and total food intake at the start and end of the study (data not shown).
（３）結果
試験開始後１１～１３日目の乾燥糞便重量は図４に、解剖時の盲腸内容物重量は図５にそれぞれ示す。また、盲腸内の短鎖脂肪酸量（酪酸、プロピオン酸、酢酸）は図６Ａ、図６Ｂおよび図６Ｃに示す。
(3) Results The weights of dried feces on days 11 to 13 after the start of the test are shown in Figure 4, and the weights of the cecal contents at the time of dissection are shown in Figure 5. The amounts of short-chain fatty acids (butyric acid, propionic acid, and acetic acid) in the cecum are shown in Figures 6A, 6B, and 6C.
図４に示されるように、対照飼料群と比較して、試験飼料群において糞便重量の有意な増加が認められた。この結果は、試験例１における「チョコレート効果カカオ７２％」の摂取による便通改善効果の有効成分がカカオプロテイン含有物（ルミナコイド）であることを示している。As shown in Figure 4, a significant increase in fecal weight was observed in the test feed group compared to the control feed group. This result indicates that the active ingredient in the bowel movement improvement effect of ingestion of "Chocolate Effect Cocoa 72%" in Test Example 1 is a cocoa protein-containing substance (luminacoid).
また、図５に示されるように、対照飼料群と比較して、試験飼料群において盲腸内容物重量の有意な増加が認められた。さらに、図６Ａ、図６Ｂおよび図６Ｃに示されるように、対照飼料群と比較して、試験飼料群において盲腸内の短鎖脂肪酸量の有意な増加が認められた。これらのことから、カカオプロテイン含有物（ルミナコイド）は、便の嵩増し効果による糞便量増加に加えて、盲腸内の短鎖脂肪酸の産生による糞便量増加効果を有することが示された。盲腸内の短鎖脂肪酸は、フィーカリバクテリウム属菌により産生され、フィーカリバクテリウム属菌の占有率の変化量とヒト糞便中の短鎖脂肪酸濃度の変化量との間には強い正の相関が見られることから（例えば、Brit J Nutr. 2010, 104(5): 693-700参照）、試験例１の結果と併せると、カカオプロテイン含有物（ルミナコイド）は、フィーカリバクテリウム属菌の産生を促進し、腸内菌叢の改善を促進する効果を有することが示された。 As shown in FIG. 5, the weight of the cecal contents was significantly increased in the test feed group compared to the control feed group. Furthermore, as shown in FIG. 6A, FIG. 6B, and FIG. 6C, the amount of short-chain fatty acids in the cecum was significantly increased in the test feed group compared to the control feed group. These results indicate that the cocoa protein-containing material (Luminacoid) has an effect of increasing the amount of feces by producing short-chain fatty acids in the cecum, in addition to increasing the amount of feces by increasing the stool bulk. Short-chain fatty acids in the cecum are produced by the genus Faecalibacterium, and there is a strong positive correlation between the change in the occupancy rate of Faecalibacterium and the change in the concentration of short-chain fatty acids in human feces (see, for example, Brit J Nutr. 2010, 104(5): 693-700). In combination with the results of Test Example 1, it was shown that the cocoa protein-containing material (Luminacoid) has the effect of promoting the production of Faecalibacterium and improving the intestinal flora.
試験例３：カカオ豆由来ルミナコイドによるフィーカリバクテリウム属菌の産生促進効果の検討（３）
カカオ豆由来ルミナコイドが糞便中の短鎖脂肪酸の産生に与える影響を検証するために以下の試験を行った。 Test Example 3: Examination of the effect of luminacoid derived from cocoa beans on promoting the production of Faecalis bacterium (3)
The following test was conducted to verify the effect of cocoa bean-derived luminacoid on the production of short-chain fatty acids in feces.
２０歳以上５０歳未満の健康な日本人女性から被験者をスクリーニングにより選抜し、評価者盲検並行群間比較試験を行った（試験食品群、対照食品群、各３０名）。試験期間中は、食事制限およびその他の制限を行った。試験期間中の食事制限およびその他の制限、並びに食事、飲み物および嗜好品に関する事項については、試験例１に記載された基準と同じ基準を採用した。Healthy Japanese women aged 20 to 50 years were selected through screening to conduct an evaluator-blinded parallel group comparative study (30 subjects in each of the test food group and control food group). Dietary and other restrictions were imposed during the study period. The same criteria as those described in Test Example 1 were adopted for dietary and other restrictions during the study period, as well as matters related to food, beverages, and luxury items.
スクリーニングは、摂取開始７日前（－７日目、－１Ｗ）に、生活習慣アンケート（年齢、既往歴、合併症、食物アレルギーの有無、医薬品や健康食品の摂取状況、飲酒等の確認）、自覚アンケート（整腸に関する自覚症状の調査）、理学的検査（身長、体重、血圧、脈拍数、ＢＭＩ）を実施し、排便の状態（排便回数、便性、便量）、年齢、食習慣（カカオ製品、納豆、乳酸菌含有食品等の日常的な摂取状況）による所定の除外基準を満たした、排便回数が週平均４回の６０名を選抜した。スクリーニングにおける除外基準は、試験例１に記載される基準と同じ基準を採用した。Screening was carried out 7 days prior to the start of intake (-7th day, -1 week) by completing a lifestyle questionnaire (confirming age, medical history, complications, presence or absence of food allergies, intake of medicines and health foods, alcohol intake, etc.), a subjective questionnaire (survey of subjective symptoms related to intestinal regulation), and a physical examination (height, weight, blood pressure, pulse rate, BMI). 60 participants who met the specified exclusion criteria based on bowel movement status (stool frequency, stool quality, stool volume), age, and dietary habits (daily intake of cocoa products, natto, foods containing lactic acid bacteria, etc.) and had an average bowel movement frequency of 4 times per week were selected. The same exclusion criteria used in screening were as those described in Test Example 1.
試験食品と対照食品は、試験例１に記載された手順と同様の手順で調製し、評価したものを用いた。The test and control foods were prepared and evaluated using procedures similar to those described in Test Example 1.
評価は以下の通り行った。摂取開始日を１日目とし、摂取開始７日前（－７日目、－１Ｗ）から摂取最終日（１４日目、２Ｗ）までの間、生活日誌（試験食品摂取の確認、自覚症状の記録、摂取開始日７日前よりの排便状況（排便回数、便性、便量）、性周期等、医薬品の服用状況等の記録）による記録を行った。摂取開始７日前（－７日目、－１Ｗ）および摂取最終日（１４日目、２Ｗ）に、生活習慣アンケート、自覚アンケート、理学的検査（身長、体重、血圧、脈拍数、ＢＭＩ）、採便を実施した。
Evaluation was carried out as follows. The day intake began was counted as
また、便中の短鎖脂肪酸濃度を測定した。便中短鎖脂肪酸濃度は、試験例２に記載された測定機器を用いて、試験例２に記載された条件に従って測定した。その結果、試験食品を摂取させることにより便中短鎖脂肪酸濃度が増大することが確認された（データ示さず）。大腸内の短鎖脂肪酸は、フィーカリバクテリウム属菌により産生され、フィーカリバクテリウム属菌の占有率の変化量とヒト糞便中の短鎖脂肪酸濃度の変化量との間には強い正の相関が見られることから（例えば、Brit J Nutr. 2010, 104(5): 693-700参照）、試験例１の結果と併せると、カカオプロテイン含有物（ルミナコイド）は、フィーカリバクテリウム属菌の産生を促進し、腸内菌叢の改善を促進する効果を有することが本試験例でも示された。 In addition, the concentration of short-chain fatty acids in the stool was measured. The concentration of short-chain fatty acids in the stool was measured using the measuring device described in Test Example 2 and under the conditions described in Test Example 2. As a result, it was confirmed that the concentration of short-chain fatty acids in the stool increased by ingestion of the test food (data not shown). Short-chain fatty acids in the large intestine are produced by bacteria of the genus Faecalibacterium, and a strong positive correlation is observed between the change in the occupancy rate of Faecalibacterium and the change in the concentration of short-chain fatty acids in human stool (see, for example, Brit J Nutr. 2010, 104(5): 693-700). In combination with the results of Test Example 1, this test example also showed that the cocoa protein-containing material (Luminacoid) has the effect of promoting the production of Faecalibacterium and promoting the improvement of the intestinal flora.
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