JP2017203030A - Probiotic composition and method for preventing arteriosclerosis - Google Patents
Probiotic composition and method for preventing arteriosclerosis Download PDFInfo
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Abstract
Description
本発明は、動脈硬化症を予防するためのプロバイオティクス組成物および方法に関する。本発明はまた、血管内皮機能を向上または改善するためのプロバイオティクス組成物および方法に関する。 The present invention relates to probiotic compositions and methods for preventing arteriosclerosis. The present invention also relates to probiotic compositions and methods for enhancing or improving vascular endothelial function.
心疾患や脳血管疾患等の原因となる動脈硬化症は、生活習慣に起因することが殆どで治療よりも予防することが好ましい。 Arteriosclerosis, which causes heart disease, cerebrovascular disease and the like, is mostly caused by lifestyle habits and is preferably prevented rather than treated.
動脈硬化は、高血圧、高脂血症、糖尿病、喫煙などの危険因子の存在下で生じた血管内皮障害をトリガーとした炎症反応に起因する。障害を受けた血管内皮は炎症性サイトカインや接着因子を発現し、これにより白血球の血管壁への接着が起こり、単球などの炎症細胞が血管壁へ湿潤する。その後、単球はマクロファージに分化し、血管壁に湿潤した低比重リポタンパク質（LDL）コレステロールが酸化された酸化LDLを取り込み泡沫細胞を形成し、さらに炎症性サイトカインを放出し、血管局所で慢性炎症を惹起し、動脈硬化は増悪する。 Arteriosclerosis is caused by an inflammatory reaction triggered by vascular endothelial injury in the presence of risk factors such as hypertension, hyperlipidemia, diabetes, and smoking. Damaged vascular endothelium expresses inflammatory cytokines and adhesion factors, which causes leukocytes to adhere to the vascular wall and moisten inflammatory cells such as monocytes to the vascular wall. After that, monocytes differentiate into macrophages, take up oxidized LDL with oxidized low density lipoprotein (LDL) cholesterol moistened in the blood vessel wall, form foam cells, release inflammatory cytokines, and chronic inflammation locally in blood vessels And atherosclerosis is exacerbated.
プロバイオティクスを利用した動脈硬化予防に関しては、動脈硬化の原因となるファクターの一つである血中脂質を低下させることをターゲットとしたヒト試験が検討されてきた（非特許文献１〜３）。しかしながら、動脈硬化発症に本質的役割を担う、血管内皮機能の維持・改善や慢性炎症の抑制をターゲットとした試みは殆どなされておらず、有効性を示した報告はこれまでにない。 Regarding the prevention of arteriosclerosis using probiotics, human trials aimed at reducing blood lipid, which is one of the factors causing arteriosclerosis, have been studied (Non-Patent Documents 1 to 3). . However, almost no attempt has been made to target maintenance and improvement of vascular endothelial function and suppression of chronic inflammation, which play an essential role in the development of arteriosclerosis, and there have been no reports showing its effectiveness.
本発明は、動脈硬化症を予防するためのプロバイオティクス組成物および方法を提供する。本発明はまた、血管内皮機能を向上または改善するためのプロバイオティクス組成物および方法を提供する。 The present invention provides probiotic compositions and methods for preventing arteriosclerosis. The present invention also provides probiotic compositions and methods for enhancing or improving vascular endothelial function.
以上に鑑み、本件の発明者は、プロバイオティクスを利用した動脈硬化予防に注目し、研究を開始した。鋭意検討の結果、プロバイオティクス微生物、あるいはプロバイオティクス微生物及びアルギニンの組合せが、血管内皮機能の向上および／または改善効果、あるいは炎症の抑制効果を有し、ひいては動脈硬化症の予防に有用であることを見いだした。当該知見に基づいて、本発明は完成された。 In view of the above, the inventors of the present case have focused on prevention of arteriosclerosis using probiotics and have started research. As a result of intensive studies, probiotic microorganisms or a combination of probiotic microorganisms and arginine have an effect of improving and / or improving vascular endothelial function, or an effect of suppressing inflammation, and thus useful for preventing arteriosclerosis. I found something. Based on this finding, the present invention has been completed.
すなわち、一態様において、本発明は以下のとおりであってよい。 That is, in one aspect, the present invention may be as follows.
［１］動脈硬化症を予防するための、プロバイオティクス微生物を含む組成物。 [1] A composition comprising a probiotic microorganism for preventing arteriosclerosis.
［２］血管内皮機能を向上または改善するための組成物であって、プロバイオティクス微生物を含む、前記組成物。 [2] A composition for improving or improving vascular endothelial function, comprising a probiotic microorganism.
［３］血管内皮機能を向上または改善するための組成物であって、プロバイオティクス微生物及びアルギニンの組合せを含む、前記組成物。 [3] A composition for improving or improving vascular endothelial function, comprising a combination of a probiotic microorganism and arginine.
［４］プロバイオティクス微生物が、ビフィドバクテリウム属（Bifidobacterium）に属する微生物、ラクトバチルス属（Lactobacillus）に属する微生物、ストレプトコッカス属（Streptococcus）に属する微生物、ラクトコッカス属（Lactococcus）に属する微生物、エンテロコッカス属（Enterococcus）に属する微生物、及びペディオコッカス属（Pediococcus）に属する微生物、バチルス（Bacillus）属に属する微生物、クロストリジウム（Clostridium）属に属する微生物、及びサッカロミセス属（Saccharomyces）に属する微生物、からなる群より選択される、上記［１］〜［３］のいずれか１項に記載の組成物。 [4] A probiotic microorganism, a microorganism belonging to the genus Bifidobacterium, a microorganism belonging to the genus Lactobacillus, a microorganism belonging to the genus Streptococcus, a microorganism belonging to the genus Lactococcus, From microorganisms belonging to the genus Enterococcus, microorganisms belonging to the genus Pediococcus, microorganisms belonging to the genus Bacillus, microorganisms belonging to the genus Clostridium and microorganisms belonging to the genus Saccharomyces The composition according to any one of [1] to [3], selected from the group consisting of:
［５］プロバイオティクス微生物が以下：ビフィドバクテリウム・アニマリス亜種ラクティス（B. animalis subsp.lactis）、ビフィドバクテリウム・アニマリス亜種アニマリス（B. animalis subsp. animalis）、ビフィドバクテリウム・シュードカテニュラタム（B. pseudocatenulatum）、ビフィドバクテリウム・カテニュラタム（B. catenulatum）、ビフィドバクテリウム・ビフィダム（B. bifidum)、ビフィドバクテリウム・ロンガム（B. longum）、ビフィドバクテリウム・ブレーベ（B. breve）、ビフィドバクテリウム・インファンティス（B. infantis）及びビフィドバクテリウム・アドレスセンティス（B. adolescentis）からなる群より選択される、上記［１］〜［３］のいずれか１項に記載の組成物。 [5] Probiotic microorganisms are: B. animalis subsp. Lactis, B. animalis subsp. Animalis, Bifidobacterium・ Pseudocatenatum (B. pseudocatenulatum), Bifidobacterium catenulatum (B. catenulatum), Bifidobacterium bifidum (B. bifidum), Bifidobacterium longum (B. longum), Bifidobacterium [1] to [3] selected from the group consisting of B. breve, B. infantis and B. adolescentis ] The composition of any one of these.
［６］プロバイオティクス微生物が、ビフィドバクテリウム・アニマリス亜種ラクティスである、上記［１］〜［３］のいずれか１項に記載の組成物。 [6] The composition according to any one of [1] to [3], wherein the probiotic microorganism is Bifidobacterium animalis subspecies lactis.
［７］プロバイオティクス微生物が、ビフィドバクテリウム・アニマリス亜種ラクティス ＬＫＭ５１２株（寄託番号ＦＥＲＭ Ｐ−２１９９８）である、上記［１］〜［３］のいずれか１項に記載の組成物。 [7] The composition according to any one of [1] to [3] above, wherein the probiotic microorganism is Bifidobacterium animalis subsp. Lactis LKM512 strain (deposit number FERM P-21998).
［８］１回用量あたり、プロバイオティクス微生物を１×１０６〜１×１０１１ｃｆｕ含む、上記［１］又は［２］に記載の組成物。 [8] The composition according to [1] or [2] above, which contains 1 × 10 6 to 1 × 10 11 cfu of probiotic microorganism per dose.
［９］１回用量あたり、プロバイオティクス微生物を１×１０６〜１×１０１１ｃｆｕ含み、アルギニンを１０ｍｇ〜１０００ｍｇ含む、上記［３］に記載の組成物。 [9] The composition according to [3] above, comprising 1 × 10 6 to 1 × 10 11 cfu of probiotic microorganism and 10 mg to 1000 mg of arginine per one dose.
［１０］食品組成物又は医薬組成物である、上記［１］〜［９］のいずれか１項に記載の組成物。 [10] The composition according to any one of [1] to [9], which is a food composition or a pharmaceutical composition.
本発明のプロバイオティクス組成物は、血管内皮機能の維持および／または改善効果、あるいは炎症の抑制効果を有する点で有用である。また、本発明のプロバイオティクス組成物は動脈硬化症の予防に有用である。 The probiotic composition of the present invention is useful in that it has an effect of maintaining and / or improving vascular endothelial function, or an effect of suppressing inflammation. The probiotic composition of the present invention is useful for preventing arteriosclerosis.
以下に本発明を具体的に説明するが、本発明はこれらに限定されるものではない。本明細書で特段に定義されない限り、本発明に関連して用いられる科学用語及び技術用語は、当業者によって一般に理解される意味を有するものとする。 The present invention will be specifically described below, but the present invention is not limited thereto. Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.
本発明は、プロバイオティクス微生物を含む、動脈硬化症を予防するための組成物に関する。そして、本発明は動脈硬化症の予防に使用するためのプロバイオティクス微生物に関する。別の態様において、本発明は動脈硬化症を予防するためのプロバイオティクス微生物を含む組成物を投与する又は摂取させる方法に関する。また別の態様において、本発明は、動脈硬化症の予防のための組成物を製造するためのプロバイオティクス微生物の使用に関する。 The present invention relates to a composition for preventing arteriosclerosis comprising a probiotic microorganism. And this invention relates to the probiotic microorganisms used for prevention of arteriosclerosis. In another aspect, the invention relates to a method of administering or ingesting a composition comprising a probiotic microorganism for preventing arteriosclerosis. In yet another aspect, the present invention relates to the use of probiotic microorganisms for the manufacture of a composition for the prevention of arteriosclerosis.
「動脈硬化を予防する」とは、動脈硬化の発症または進行を抑制することを意味する。動脈硬化の予防に関する具体的な指標としては、血管内皮機能の向上または改善、炎症の低減または改善、肥満度の低下、動脈硬化発症に関わる腸内常在菌数の低下、等が挙げられるが、これに限定されない。 “Preventing arteriosclerosis” means inhibiting the onset or progression of arteriosclerosis. Specific indicators for the prevention of arteriosclerosis include improvement or improvement of vascular endothelial function, reduction or improvement of inflammation, reduction of obesity, reduction of the number of intestinal resident bacteria involved in the development of arteriosclerosis, etc. However, the present invention is not limited to this.
本明細書において、「血管内皮機能」とは、血管内皮細胞の作用であって、血管の健康状態を維持するために重要な作用であり、血管の収縮・拡張の調節、炎症細胞の血管壁への接着や血小板の粘着・凝集を抑制する事による血管の保護の作用を意味する。血管内皮機能の評価は、例えば、ストレインゲージ式プレチスモグラフィ―（プレチスモグラフィ）、血流介在血管拡張反応（flow-mediated dilatation：ＦＭＤ）、ＲＨ−ＰＡＴ（reactive hyperemia peripheral arterial tonometry）により評価することができる。また、「循環器病の診断と治療に関するガイドライン（２０１１−２０１２年度合同研究班報告）血管機能の非侵襲的評価法に関するガイドライン」、循環器病の診断と治療に関するガイドライン（2186-5973) 2013巻、3-112頁（2014年1月）に記載の方法により評価してもよい。 In the present specification, “vascular endothelial function” is an action of vascular endothelial cells, which is an important action for maintaining the state of vascular health, regulation of vasoconstriction / dilation, vascular wall of inflammatory cells It means the action of protecting blood vessels by inhibiting adhesion to platelets and adhesion / aggregation of platelets. Vascular endothelial function can be evaluated by, for example, strain gauge plethysmography (plethysmography), blood flow-mediated dilatation (FMD), or RH-PAT (reactive hyperemia peripheral arterial tonometry). it can. In addition, “Guidelines on Cardiovascular Disease Diagnosis and Treatment (2011-2012 Joint Research Group Report) Guidelines on Noninvasive Evaluation of Vascular Function”, Guidelines on Cardiovascular Disease Diagnosis and Treatment (2186-5973) Volume 2013 , Page 3-112 (January 2014).
炎症は、炎症性サイトカイン（例えば、ＴＮＦ−α）の濃度および発現量により評価することができる。 Inflammation can be evaluated by the concentration and expression level of inflammatory cytokines (eg, TNF-α).
本発明は特に、プロバイオティクス微生物及びアルギニンの組合せを含む、血管内皮機能を向上または改善するための組成物に関する。そして、本発明は血管内皮機能の向上若しくは改善に使用するためのプロバイオティクス微生物及びアルギニンの組合せに関する。別の態様において、本発明は、血管内皮機能を向上または改善するための、プロバイオティクス微生物及びアルギニンの組合せを含む組成物を投与する又は摂取させる方法に関する。また別の態様において、本発明は、血管内皮機能の向上または改善のための組成物を製造するためのプロバイオティクス微生物及びアルギニンの組合せの使用に関する。 The invention particularly relates to a composition for enhancing or improving vascular endothelial function comprising a combination of probiotic microorganisms and arginine. The present invention relates to a combination of probiotic microorganisms and arginine for use in improving or improving vascular endothelial function. In another aspect, the present invention relates to a method of administering or ingesting a composition comprising a combination of probiotic microorganisms and arginine for enhancing or improving vascular endothelial function. In yet another aspect, the invention relates to the use of a combination of probiotic microorganisms and arginine to produce a composition for enhancing or improving vascular endothelial function.
本発明におけるプロバイオティクス微生物は、ビフィドバクテリウム属（Bifidobacterium）に属する微生物、ラクトバチルス属（Lactobacillus）に属する微生物、ストレプトコッカス属（Streptococcus）に属する微生物、ラクトコッカス属（Lactococcus）に属する微生物、エンテロコッカス属（Enterococcus）に属する微生物、及びペディオコッカス属（Pediococcus）に属する微生物、バチルス（Bacillus）属に属する微生物、クロストリジウム（Clostridium）属に属する微生物、及びサッカロミセス属（Saccharomyces）に属する微生物、からなる群より選択される微生物の少なくとも一つであってよい。 Probiotic microorganisms in the present invention, microorganisms belonging to the genus Bifidobacterium, microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Streptococcus, microorganisms belonging to the genus Lactococcus, From microorganisms belonging to the genus Enterococcus, microorganisms belonging to the genus Pediococcus, microorganisms belonging to the genus Bacillus, microorganisms belonging to the genus Clostridium and microorganisms belonging to the genus Saccharomyces It may be at least one microorganism selected from the group consisting of:
好ましい態様において、プロバイオティクス微生物はビフィドバクテリウム属に属する微生物であり、以下：ビフィドバクテリウム・アニマリス亜種ラクティス（B. animalis subsp.lactis）、ビフィドバクテリウム・アニマリス亜種アニマリス（B. animalis subsp. animalis）、ビフィドバクテリウム・シュードカテニュラタム（B. pseudocatenulatum）、ビフィドバクテリウム・カテニュラタム（B. catenulatum）、ビフィドバクテリウム・ビフィダム（B. bifidum)、ビフィドバクテリウム・ロンガム（B. longum）、ビフィドバクテリウム・ブレーベ（B. breve）、ビフィドバクテリウム・インファンティス（B. infantis）及びビフィドバクテリウム・アドレスセンティス（B. adolescentis）からなる群より選択される微生物の少なくとも一つであってよい。特に好ましいプロバイオティクス微生物は、ビフィドバクテリウム・アニマリス亜種ラクティスであり、さらに好ましくはビフィドバクテリウム・アニマリス亜種ラクティスＬＫＭ５１２株である。ビフィドバクテリウム・アニマリス亜種ラクティスＬＫＭ５１２株は、受託番号FERM P-21998として２０１０年８月１０日に独立行政法人 製品評価技術基盤機構 ＮＩＴＥ特許微生物寄託センター（ＮＩＴＥ−ＩＰＯＤ：日本国千葉県木更津市かずさ鎌足２−５−８；独立行政法人 産業技術総合研究所 特許生物寄託センターより２０１２年４月に業務承継）に寄託されている。 In a preferred embodiment, the probiotic microorganism is a microorganism belonging to the genus Bifidobacterium and includes the following: B. animalis subsp. Lactis, Bifidobacterium animalis subspecies animalis ( B. animalis subsp. Animalis), B. pseudocatenulatum, B. catenulatum, B. bifidum, Bifidobacteria The group consisting of B. longum, B. breve, B. infantis and B. adolescentis It may be at least one of more selected microorganisms. A particularly preferred probiotic microorganism is Bifidobacterium animalis subsp. Lactis, more preferably Bifidobacterium animalis subsp. Lactis LKM512. The Bifidobacterium animalis subspecies Lactis LKM512 strain was received on August 10, 2010 under the accession number FERM P-21998. NITE-IPOD: Kisarazu Kisarazu, Chiba, Japan 2-5-8, Kazusa-shi, Kazusa-shi; Deposited by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center in April 2012.
アルギニンは、天然アミノ酸の一種で、特に明示しない限りＬ−アルギニンを指す。 Arginine is a kind of natural amino acid, and refers to L-arginine unless otherwise specified.
本発明の組成物が、プロバイオティクス微生物及びアルギニンの組合せを含む場合における「組合せ」とは、プロバイオティクス微生物とアルギニンが同一剤中に存在する態様を意味してもよく、又は、プロバイオティクス微生物を含む剤とアルギニンを含む別の剤を含む組合せ組成物の態様を意味してもよい。プロバイオティクス微生物とアルギニンの組合せを投与することは、プロバイオティクス微生物とアルギニンを同時に投与する又は摂取させること、プロバイオティクス微生物を投与する又は摂取させた後にアルギニンを投与する又は摂取させること、または、アルギニンを投与する又は摂取させた後にプロバイオティクス微生物を投与する又は摂取させることを意味する。 When the composition of the present invention contains a combination of a probiotic microorganism and arginine, the “combination” may mean an embodiment in which the probiotic microorganism and arginine are present in the same agent, or It may mean an embodiment of a combined composition comprising an agent comprising a tics microorganism and another agent comprising arginine. Administering a combination of probiotic microorganisms and arginine includes administering or ingesting probiotic microorganisms and arginine simultaneously, administering or ingesting arginine after administering or ingesting probiotic microorganisms, Or it means administering or ingesting probiotic microorganisms after administering or ingesting arginine.
プロバイオティクス微生物、または、プロバイオティクス微生物及びアルギニンの組合せは、有効量で投与されることが好ましい。ここでいう有効量とは、動脈硬化症の予防のために必要な量であれば特に限定されない。例えば、１回用量あたり、プロバイオティクス微生物は、１×１０６〜１×１０１１ｃｆｕ、好ましくは１×１０８〜１×１０１１ｃｆｕ、１×１０８〜１×１０１０ｃｆｕ、１×１０９〜１×１０１１ｃｆｕ、１×１０９〜１×１０１０ｃｆｕである。プロバイオティクス微生物とアルギニンを組み合わせる場合のアルギニンの量は、１回用量あたり、１０〜１０００ｍｇ、好ましくは１０ｍｇ〜８００ｍｇ、５０ｍｇ〜８００ｍｇ、２００ｍｇ〜８００ｍｇ、５０ｍｇ〜６００ｍｇ、２００ｍｇ〜６００ｍｇである。 Preferably, the probiotic microorganism or the combination of probiotic microorganism and arginine is administered in an effective amount. The effective amount herein is not particularly limited as long as it is an amount necessary for preventing arteriosclerosis. For example, per dose, the probiotic microorganism is 1 × 10 6 to 1 × 10 11 cfu, preferably 1 × 10 8 to 1 × 10 11 cfu, 1 × 10 8 to 1 × 10 10 cfu, 1 × 10 9 to 1 × 10 11 cfu, 1 × 10 9 to 1 × 10 10 cfu. The amount of arginine when combining a probiotic microorganism and arginine is 10 to 1000 mg, preferably 10 mg to 800 mg, 50 mg to 800 mg, 200 mg to 800 mg, 50 mg to 600 mg, 200 mg to 600 mg per dose.
本発明の組成物は、１日１回、１日２回、または１日３回の投与または摂取としてもよい。投与・摂取期間は、随時、３日以上、１週間以上、４週間以上、または８週間以上、としてもよい。また、本発明の組成物は、いずれの対象に適用してもよい。対象には哺乳動物、特にヒトが含まれる。好ましい態様において対象は、動脈硬化予備軍と判定されたヒトである。動脈硬化予備軍の判定は、例えば、血液生化学的検査による中性脂肪値が１５０ｍｇ／ｄＬ以上、ＬＤＬコレルテロールが１４０ｍｇ／ｄＬ以上、ＨＤＬコレルテロールが４０ｍｇ／ｄＬ未満、総コレステロール値が２２０ｍｇ／ｄＬ以上、収縮期血圧が１３０ｍｍＨｇ以上、拡張期血圧が８５ｍｍＨｇ以上、又はＢＭＩが２５以上、のいずれか１項目を満たすという基準により行うことができる。あるいは、動脈硬化予備軍の判定は、血管機能検査、例えば反応性充血指数（Reactive hyperemia index: RHI）、脈波伝播速度（PWV）、心臓足首血管指数（CAVI）、中心血圧、増大係数（AI）、又は足関節上腕血圧比（ABI）において正常値範囲との比較により行ってもよい。血管機能の評価は、「循環器病の診断と治療に関するガイドライン（２０１１−２０１２年度合同研究班報告）血管機能の非侵襲的評価法に関するガイドライン」、循環器病の診断と治療に関するガイドライン（2186-5973) 2013巻、3-112頁（2014年1月）に記載の方法により評価してもよく、あるいは、上述した血管内皮機能評価方法により評価してもよい。 The composition of the present invention may be administered or ingested once a day, twice a day, or three times a day. The administration / intake period may be 3 days or longer, 1 week or longer, 4 weeks or longer, or 8 weeks or longer as needed. Moreover, you may apply the composition of this invention to any object. Subjects include mammals, particularly humans. In a preferred embodiment, the subject is a human who has been determined to be atherosclerotic reserve. The determination of the arteriosclerosis reserve is, for example, a triglyceride level of 150 mg / dL or more by blood biochemical examination, LDL corelterol is 140 mg / dL or more, HDL corelterol is less than 40 mg / dL, and total cholesterol value is 220 mg / dL or more In addition, it can be performed based on the criteria that systolic blood pressure is 130 mmHg or more, diastolic blood pressure is 85 mmHg or more, or BMI is 25 or more. Alternatively, arteriosclerosis reserves can be determined by vascular function tests such as reactive hyperemia index (RHI), pulse wave velocity (PWV), cardiac ankle vascular index (CAVI), central blood pressure, and increase factor (AI ), Or an ankle brachial blood pressure ratio (ABI) may be compared with a normal value range. Evaluation of vascular function is based on “Guidelines on diagnosis and treatment of cardiovascular disease (reported by the 2011-2012 Joint Research Group) Guidelines on non-invasive evaluation of vascular function”, Guidelines on diagnosis and treatment of cardiovascular disease (2186- 5973) Evaluation may be made by the method described in 2013, page 3-112 (January 2014), or may be evaluated by the vascular endothelial function evaluation method described above.
本発明の組成物は、食品組成物または医薬組成物であってもよい。本発明の組成物は、好ましくは経口摂取または経口投与される。本発明の組成物は、医薬品又は食品（例えば、機能性食品、健康食品、サプリメントなど）として継続的な摂取が行いやすいように、例えば顆粒剤（ドライシロップを含む）、カプセル剤（軟カプセル剤、硬カプセル剤）、錠剤（チュアブル剤などを含む）、散剤（粉末剤）、丸剤などの各種の固形製剤、又は内服用液剤（液剤、懸濁剤、シロップ剤を含む）などの液状製剤などの形態で調製することができるが、特に限定されるものではない。プロバイオティクス微生物およびアルギニンは腸溶コーティングされていてもよい。しかしながら、胃内環境に対して耐性を有するプロバイオティクス微生物を用いる場合、プロバイオティクス微生物に対する腸溶コーティングは必ずしも必要ない。 The composition of the present invention may be a food composition or a pharmaceutical composition. The composition of the present invention is preferably taken orally or administered orally. The composition of the present invention can be used for example as granules (including dry syrup), capsules (soft capsules, soft capsules, etc.) so as to facilitate continuous ingestion as pharmaceuticals or foods (for example, functional foods, health foods, supplements, etc.). Hard capsules), tablets (including chewables), powders (powder), various solid preparations such as pills, or liquid preparations such as liquids for internal use (including liquids, suspensions, and syrups) However, it is not particularly limited. Probiotic microorganisms and arginine may be enteric coated. However, when a probiotic microorganism having resistance to the gastric environment is used, an enteric coating for the probiotic microorganism is not always necessary.
腸溶コーティングは、当業者に公知のものを使用でき、特に限定されない。例えば、腸溶コーティングされた錠剤として、有用な成分を含む素錠の表面に、アンダーコート層と、大腸崩壊性基剤層と、第１の腸溶性基剤層と、第２の腸溶性基剤層とを、この順に被覆してなり、前記第１の腸溶性基剤層がゼインを含み、前記第２の腸溶性基剤層がシェラックを含む、大腸ドラッグデリバリーシステム錠剤を使用することができる。 As the enteric coating, those known to those skilled in the art can be used, and are not particularly limited. For example, as an enteric-coated tablet, an undercoat layer, a colon-disintegrating base layer, a first enteric base layer, and a second enteric group are formed on the surface of an uncoated tablet containing useful components. Using a large intestine drug delivery system tablet in which the first enteric base layer contains zein and the second enteric base layer contains shellac. it can.
製剤化のための添加物としては、例えば、賦形剤、滑沢剤、結合剤、崩壊剤、流動化剤、分散剤、湿潤剤、防腐剤、粘稠剤、ｐＨ調整剤、着色剤、矯味矯臭剤、界面活性剤、溶解補助剤などが挙げられる。また、液剤の形態にする場合は、ペクチン、キサンタンガム、グアガムなどの増粘剤を配合することができる。また、コーティング剤を用いてコーティング錠剤にしたり、ペースト状の膠剤とすることもできる。さらに、他の形態に調製する場合であっても、従来の方法に従えばよい。 As additives for formulation, for example, excipients, lubricants, binders, disintegrants, fluidizers, dispersants, wetting agents, preservatives, thickeners, pH adjusters, colorants, Examples include flavoring agents, surfactants, and solubilizing agents. Moreover, when making it into the form of a liquid agent, thickeners, such as pectin, xanthan gum, and guar gum, can be mix | blended. Moreover, it can also be set as a coating tablet using a coating agent, or it can also be set as a paste-form glue. Furthermore, even if it is a case where it prepares in another form, what is necessary is just to follow the conventional method.
本発明の組成物は、必要に応じ、従来公知の着色剤、保存剤、香料、風味剤、コーティング剤などの成分を配合して調製することもできる。 The composition of the present invention can be prepared by blending components such as conventionally known colorants, preservatives, fragrances, flavoring agents, and coating agents, if necessary.
本発明の組成物が食品組成物である場合、以下のような形態であってもよい。例えば、飲料、発酵食品、菓子類、パン類、スープ類等の各種食品又はその添加成分として；又はドッグフード、キャットフードなどの各種ペットフード又はその添加成分として使用することができる。これらの食品の製造方法は、本発明の効果を損なわないものであれば特に限定されず、各用途で当業者に使用されている方法に従えばよい。本発明の組成物を適用できる食品は、例えば、対象が日常的に摂取する食品だけでなく、特定保健用食品、栄養機能食品、特別用途食品等の機能性食品にも適用できる。本発明の組成物が適用可能な食品の具体例としては、牛乳、ヨーグルト、発酵乳、乳酸菌飲料、チーズ、プリン、アイスクリーム、氷菓、清涼飲料水（フルーツジュース、緑茶、紅茶、烏龍茶、及びコーヒー等を含む）、サプリメント、及び豆乳等が挙げられる。固形食品に本発明の組成物を加える場合には、例えば顆粒剤、及び粉剤等の形態で加えることが好ましい。また、飲料、ペースト状食品に本発明の組成物を加える場合には、例えばマイクロカプセルの形態で加えることが好ましい。 When the composition of the present invention is a food composition, the following form may be employed. For example, it can be used as various foods such as beverages, fermented foods, confectionery, breads and soups or their additive components; or as various pet foods such as dog foods and cat foods or their additive components. The method for producing these foods is not particularly limited as long as the effects of the present invention are not impaired, and may be a method used by those skilled in the art for each application. The food to which the composition of the present invention can be applied is applicable not only to foods that the subject daily ingests, but also to functional foods such as foods for specified health use, nutritional functional foods, and special-purpose foods. Specific examples of foods to which the composition of the present invention can be applied include milk, yogurt, fermented milk, lactic acid bacteria beverages, cheese, pudding, ice cream, ice confectionery, soft drinks (fruit juice, green tea, tea, oolong tea, and coffee Etc.), supplements, and soy milk. When the composition of the present invention is added to a solid food, it is preferably added in the form of granules, powders, and the like. Moreover, when adding the composition of this invention to a drink and a paste-form foodstuff, adding in the form of a microcapsule, for example is preferable.
本発明の組成物は、血管内皮機能の向上または改善、あるいは炎症の低減または改善に有効である。血管内皮機能が向上または維持されること、あるいは炎症が低減または改善されることは、動脈硬化の予防、動脈硬化の進行の予防、さらには心筋梗塞や脳卒中の予防に有効である。したがって、本発明の組成物は、動脈硬化の予防、動脈硬化の進行の予防、さらには心筋梗塞や脳卒中の予防に用いることもできる。 The composition of the present invention is effective in improving or improving vascular endothelial function or reducing or improving inflammation. Improvement or maintenance of vascular endothelial function or reduction or improvement of inflammation is effective for prevention of arteriosclerosis, prevention of progression of arteriosclerosis, and prevention of myocardial infarction and stroke. Therefore, the composition of the present invention can also be used for prevention of arteriosclerosis, prevention of progression of arteriosclerosis, and prevention of myocardial infarction and stroke.
本発明について全般的に記載したが、さらに理解を得るために参照する特定の実施例をここに提供する。しかし、これらは例示目的とするものであって、本発明の範囲はこれに限定されるものではない。 Although the present invention has been generally described, specific examples are provided herein for reference for further understanding. However, these are for illustrative purposes, and the scope of the present invention is not limited thereto.
実施例１〜３では、被験者の選定に際して、薬物による脂質異常症の治療を受けている患者、コレステロール値に影響のある薬剤を服用している方、重篤な心疾患、肝疾患、腎疾患などの患者は除外した。また、被験者には担当医師より本研究の意義や目的を十分に説明し、インフォームド・コンセントを得た上で、ヘルシンキ宣言の精神に則り実施した。 In Examples 1 to 3, when selecting subjects, patients undergoing treatment for dyslipidemia with drugs, those taking drugs that affect cholesterol levels, severe heart disease, liver disease, kidney disease Patients were excluded. In addition, we explained the significance and purpose of this study to the subjects from the doctor in charge, obtained informed consent, and carried out in accordance with the spirit of the Declaration of Helsinki.
実施例１
実験方法
・被験者
募集期間中に集まった被験者候補を対象に、試験開始２週間前に血液生化学的検査を実施し、中性脂肪値（１００〜２５０ｍｇ／ｄＬ）でBMIが25程度の被験者を中心に、医師が動脈硬化予備軍と判定した３０名を被験者として登録した。登録された被験者はプロトコルに従い、割付責任者が試験前に無作為に試験食群２０名、プラセボ群１０名に群分けした。しかしながら、直前の血液検査で試験食群の３名の中性脂肪が１００ｍｇ／ｄｌ未満となったため除外した。結果的に、試験食群１７名（男性９名、女性８名、平均年齢４１．７歳）とプラセボ群１０名（男性６名、女性４名、平均年齢４１．７歳）で実施した。 また、被験者はアンケートによる食生活から、総摂取カロリーは平均２０００kcal／日以上と推測された。
・試験食
試験食は、ビフィドバクテリウム・アニマリス亜種ラクティス ＬＫＭ５１２株（寄託番号ＦＥＲＭ Ｐ−２１９９８）凍結乾燥粉末を１ｇ（生菌数は約６×１０９ｃｆｕ）ずつ分包したスティックを１セットとし、１日に２包（朝、晩に一包ずつ）１２週間摂取した。プラセボ用粉末は、賦形剤のみのスティックを作製して用いた。
・試験スケジュール
摂取期間１２週間の無作為化二重盲検並行群間比較試験を実施した。摂取前及び摂取１２週後に、血清中のＴＮＦ−αを測定した。また、試験開始前及び摂取１２週後に糞便を回収した。なお、試験期間２週間前から終了時まで、乳酸菌およびビフィズス菌を含むヨーグルト、乳酸菌飲料、およびサプリメント、また腸内菌叢解析結果に著しい影響を及ぼす納豆の摂取は禁止したが、その他の食事は制限しなかった。
・血清中のＴＮＦ−αの測定
摂取前及び摂取１２週後に、各被験者より採血して血清を調製後、速やかに凍結し、−８０℃に保存して検体とした。血清中ＴＮＦ−αは、Human TNF-α Chemiluminescent Immunoassay（QuantiGlo（登録商標））を使用し、化学発光酵素免疫測定法にて測定した。
Example 1
Test method・Subject candidates who gathered during the recruitment period will undergo a blood biochemical test 2 weeks before the start of the test, and subjects with a triglyceride level (100-250 mg / dL) and a BMI of about 25 At the center, 30 doctors who were determined to be atherosclerotic reserves were registered as subjects. The enrolled subjects were randomly divided into 20 test meal groups and 10 placebo groups by the assignment manager before the test according to the protocol. However, it was excluded because the triglyceride of 3 people in the test meal group was less than 100 mg / dl in the last blood test. As a result, 17 test meal groups (9 men, 8 women, average age 41.7 years) and 10 placebo groups (6 men, 4 women, average age 41.7 years) were used. Moreover, the test subject estimated that the total calorie intake was 2000 kcal / day or more on average based on the dietary habits.
-Test meal The test meal consists of 1 stick of Bifidobacterium animalis subspecies Lactis LKM512 strain (deposit number FERM P-21998) lyophilized powder, each containing 1 g of lyophilized powder (viable cell count is approximately 6 × 10 9 cfu). As a set, 2 capsules per day (one package each morning and evening) were taken for 12 weeks. For the placebo powder, a stick made only of an excipient was used.
• A randomized, double-blind, parallel group comparison study with a study schedule intake period of 12 weeks was conducted. Serum TNF-α was measured before and 12 weeks after ingestion. Feces were collected before the start of the test and 12 weeks after ingestion. In addition, from 2 weeks before the test period to the end, yogurt containing lactic acid bacteria and bifidobacteria, lactic acid bacteria drinks and supplements, and natto, which significantly affects the results of intestinal flora analysis, were prohibited, but other meals I did not limit.
-Measurement of TNF-α in serum Before and 12 weeks after ingestion, blood was collected from each subject to prepare serum, immediately frozen, stored at -80 ° C, and used as a specimen. Serum TNF-α was measured by chemiluminescent enzyme immunoassay using Human TNF-α Chemiluminescent Immunoassay (QuantiGlo (registered trademark)).
＜ＢＭＩ、γーＧＴＰの測定＞
接種前及び摂取１２週間後に、各被験者のＢＭＩ（Body Mass Index）を測定した。ＢＭＩ値は、次式：［体重（kg)／（身長（cm)）２］によって計算される。また、摂取前及び摂取１２週後に、各被験者より得た血液について、血中γ−ＧＴＰを測定した。
・腸内菌叢解析
（糞便サンプル）
糞便サンプルは、試験開始前と摂取１２週後に回収した。採便シート「ナガセール」（オザックス株式会社製）を用いて、排便後、直ちに糞便を採便管に回収し、冷蔵条件下で輸送し、排便後１２時間以内に−８０℃で保存し、腸内菌叢解析用に処理した。すなわち、２００−３００ｍｇの糞便を９倍量のダルベッコリン酸バッファー（ｐＨ７．２）（D-PBS, GIBCO社製）に均一に懸濁し、１分間の激しい撹拌後、遠心分離を行い（16000×g、１０分間）、上清を除去し沈殿を得た。さらに再びD-PBSを１ｍｌ添加し、同様の作業を二度繰り返し、得られた沈殿物を菌叢解析用試料として解析時まで−８０℃にて保存した。
（糞便サンプルからのＤＮＡ抽出）
ＤＮＡの抽出はMatsukiら（Matsuki, T., et al., Appl. Environ. Microbiol, 2004, 70:7220-7228）の方法の一部を改変して行った。改変点は、抽出ＤＮＡの回収をエタ沈メイト（Takara）で実施し、冷７０％エタノール（1 ml）を用いて２回の遠心洗浄（15000×g、５分間）後、風乾した点である。
（ＩｏｎＰＧＭによる菌叢解析）
16S rRNA遺伝子の可変領域V1-V2をフュージョンプライマー法で増幅した。フォワードプライマーはIon Aアダプターやキー配列、バーコード配列、アダプター配列（GT）、ならびに16S rRNA特異的な27Fmodプライマー配列を有するものを使用し、リバースプライマーはIon truncated P1アダプター配列、アダプター配列（CC）、ならびに338Rプライマー配列を有するものを使用した（Kim, S. W., et al., DNA Res., 2013, 20:241-253）。PCRの反応液の組成はPlatinum（登録商標） PCR SuperMix High Fidelity (Invitrogen, Carlsbad, CA, USA)を23.5μｌ、各５μＭのプライマー混合液を0.5μｌ、ＤＮＡ溶液を１μｌを含む合計２５μｌの系を用いた。サーマルサイクラーの温度条件は、最初のＤＮＡ変性を９４℃で３分間行い、その後、変性(９４℃で３０秒)、アニーリング(５５℃で４５秒)、伸長(６８℃で１分間)のサイクルを２５サイクル繰り返した。ＰＣＲ産物はPureLink PCR Purification Kit (Invitorogen)を用いて精製した。サンプルのDNA濃度はQuant dsDNA HS Assay Kit (Invitrogen)とQubit 2.0 Flourometer (Invitrogen)を用いて定量し、各サンプルが等量となるように混合した。ＤＮＡ混合液は２．０％TAEアガロースゲルにて電気泳動し、目的産物のバンドを切り出し精製した。精製したＤＮＡはBioanalyzer（Agilent）を用いて精製産物のピークと濃度を確認した。エマルジョンＰＣＲとライブラリービーズの回収はIon PGM Template OT2 400 Kit（ThermoFisher Scientific）を用い、シークエンスはIon PGM Sequencing 400 Kit （ThermoFisher Scientific）とIon 318 Chip V2（ThermoFisher Scientific）を用いた。いずれもThermoFisher Scientificのプロトコールに従って行った。シークエンスはIon PGM System（ThermoFisher Scientific）を用いて行った。
（シークエンスデータの解析方法）
Ion PGMのシークエンスデータはfastq形式で取得し、Qiimeソフトウェアー（Caporaso, J. G., et al., Bioinformatics, 2010, 26:266-267）を用いて解析した。出力された配列データより、バーコード、フォワードプライマー、リバースプライマー配列部位のミスマッチが無く、かつ平均のクオリティースコアが２０以上の配列を抽出した。抽出した配列は、uclust方法（Edgar, R. C., Bioinformatics, 2010, 26:2460-2461）とfarthest neighbor アルゴリズムを用いて９７％の相同性となるようにoperational taxonomic units（OTUs）にクラスター化した。各OTUの内、最も頻度の高い配列を代表配列として抽出した。代表配列はPyNASTアルゴリズム（Caporaso, J. G., et al., Bioinformatics, 2010, 26:266-267）を用いてアライメントした。キメラ配列はChimeraSlayerアルゴリズムを用いて確認し、解析に用いる配列から削除した。配列の系統分類はRDP classifier（Wang, Q., et al., Appl. Environ. Microbiol., 2007, 73: 5261-5267）を用い、confidence cutoff値を８０に設定して行った。
・統計解析
ＴＮＦ−αの測定値についての試験食群とプラセボ群の比較はSAS 9.3（SAS Institute Inc.）によるスチューデントのｔ検定（Student’s t-test）あるいはウィルコクソンの順位和検定（Wilcoxon rank-sum test）で実施し、各群内の摂取前後の結果の比較は対応のあるｔ検定（paired t-test）で実施した。糞便菌叢の検出菌群の相対存在量の比較はSPSS（IBM）を用いてKolmogorov-Smirnov の正規性検定を行い、正規分布が否定された菌群の独立2群比較についてはマン・ホイットニーのＵ検定、各群内の摂取前後の結果の比較についてはウィルコクソンの符号順位検定を行った。正規分布している菌群の独立２群比較は、Ｆ検定後、スチューデントのｔ検定、あるいはウェルチのｔ検定にて解析し、各群内の摂取前後の結果の比較は対応のあるｔ検定（paired t-test）で実施した。
<Measurement of BMI and γ-GTP>
Before inoculation and 12 weeks after ingestion, the BMI (Body Mass Index) of each subject was measured. The BMI value is calculated by the following formula: [weight (kg) / (height (cm)) 2 ]. In addition, blood γ-GTP was measured for blood obtained from each subject before ingestion and 12 weeks after ingestion.
・Intestinal flora analysis (fecal sample)
Fecal samples were collected before the start of the study and 12 weeks after ingestion. Using fecal collection sheet “Nagasale” (manufactured by Ozax Co., Ltd.), immediately after defecation, stool is collected in a stool collection tube, transported under refrigerated conditions, stored at −80 ° C. within 12 hours after defecation, Processed for internal flora analysis. That is, 200-300 mg of stool was uniformly suspended in 9 volumes of Dulbecco's phosphate buffer (pH 7.2) (D-PBS, manufactured by GIBCO), and after 1 minute of vigorous stirring, centrifuged (16000 × g, 10 minutes), the supernatant was removed to obtain a precipitate. Furthermore, 1 ml of D-PBS was added again, the same operation was repeated twice, and the obtained precipitate was stored at −80 ° C. as a sample for analyzing the flora until the time of analysis.
(DNA extraction from stool samples)
DNA extraction was performed by modifying a part of the method of Matsuki et al. (Matsuki, T., et al., Appl. Environ. Microbiol, 2004, 70: 7220-7228). The modification point is that the extracted DNA was collected with Eta Precipitate (Takara), and then centrifuged twice with cold 70% ethanol (1 ml) (15000 × g, 5 minutes) and then air-dried. .
(Bion flora analysis by IonPGM)
The variable region V1-V2 of 16S rRNA gene was amplified by the fusion primer method. Use forward primer with Ion A adapter, key sequence, barcode sequence, adapter sequence (GT) and 16S rRNA-specific 27Fmod primer sequence, reverse primer with Ion truncated P1 adapter sequence, adapter sequence (CC) As well as those with the 338R primer sequence (Kim, SW, et al., DNA Res., 2013, 20: 241-253). The composition of the PCR reaction solution is a total of 25 μl containing Platinum (registered trademark) PCR SuperMix High Fidelity (Invitrogen, Carlsbad, Calif., USA) 23.5 μl, 0.5 μl each 5 μM primer mixture, and 1 μl DNA solution. Using. The temperature condition of the thermal cycler is that the first DNA denaturation is performed at 94 ° C. for 3 minutes, and then a cycle of denaturation (94 ° C. for 30 seconds), annealing (55 ° C. for 45 seconds), and extension (68 ° C. for 1 minute) is performed. 25 cycles were repeated. The PCR product was purified using the PureLink PCR Purification Kit (Invitorogen). The DNA concentration of the sample was quantified using a Quant dsDNA HS Assay Kit (Invitrogen) and a Qubit 2.0 Flourometer (Invitrogen), and mixed so that each sample was equal. The DNA mixture was electrophoresed on a 2.0% TAE agarose gel, and the target product band was cut out and purified. The purified DNA was confirmed for the peak and concentration of the purified product using Bioanalyzer (Agilent). Eon PCR and library beads were collected using Ion PGM Template OT2 400 Kit (ThermoFisher Scientific), and Ion PGM Sequencing 400 Kit (ThermoFisher Scientific) and Ion 318 Chip V2 (ThermoFisher Scientific). All were performed according to the ThermoFisher Scientific protocol. The sequence was performed using the Ion PGM System (ThermoFisher Scientific).
(Sequence data analysis method)
Ion PGM sequence data was acquired in fastq format and analyzed using Qiime software (Caporaso, JG, et al., Bioinformatics, 2010, 26: 266-267). From the output sequence data, a sequence having no mismatch between the barcode, forward primer and reverse primer sequence sites and having an average quality score of 20 or more was extracted. The extracted sequences were clustered into operational taxonomic units (OTUs) to 97% homology using the uclust method (Edgar, RC, Bioinformatics, 2010, 26: 2460-2461) and the farthest neighbor algorithm. Of each OTU, the most frequent sequence was extracted as a representative sequence. Representative sequences were aligned using the PyNAST algorithm (Caporaso, JG, et al., Bioinformatics, 2010, 26: 266-267). The chimeric sequence was confirmed using the ChimeraSlayer algorithm and deleted from the sequence used for analysis. The phylogenetic classification of the sequences was performed using the RDP classifier (Wang, Q., et al., Appl. Environ. Microbiol., 2007, 73: 5261-5267) and setting the confidence cutoff value to 80.
・Statistical analysis Comparison of test food group and placebo group for TNF-α measurements is based on Student's t-test or Wilcoxon rank-sum test by SAS 9.3 (SAS Institute Inc.) The comparison of the results before and after ingestion in each group was performed by a paired t-test. For comparison of relative abundance of detected bacteria in fecal flora, Kolmogorov-Smirnov's normality test was performed using SPSS (IBM), and Mann-Whitney The Wilcoxon sign rank test was performed for comparison of the U test and the results before and after ingestion in each group. Independent two-group comparison of normally distributed fungal groups is analyzed by Student's t-test or Welch's t-test after F test, and comparison of results before and after ingestion in each group is a corresponding t-test ( paired t-test).
結果
・血清ＴＮＦ−α、ＢＭＩ、γ−ＧＴＰ
摂取１２週目の群間比較及び摂取前後の比較を行い、数値は平均値±標準誤差で表した。
Results・Serum TNF-α, BMI, γ-GTP
Comparison was made between groups at the 12th week of ingestion and before and after ingestion, and the values were expressed as mean ± standard error.
血清ＴＮＦ−αは、試験食群では、摂取前０．９６２±０．０９４ｐｇ／ｍｌ、摂取後０．７２２±０．０７０ｐｇ／ｍｌと有意な減少（ｐ＜０．０１）が認められたのに対し、プラセボ群では摂取前０．９９７±０．１２４ｐｇ／ｍｌ、摂取後０．８７３±０．１３３ｐｇ／ｍｌで有意な変化は認められなかった。 Serum TNF-α was significantly decreased (p <0.01) in the test meal group, 0.962 ± 0.094 pg / ml before ingestion and 0.722 ± 0.070 pg / ml after ingestion. In contrast, in the placebo group, no significant change was observed at 0.997 ± 0.124 pg / ml before ingestion and 0.873 ± 0.133 pg / ml after ingestion.
ＴＮＦ−αが試験食群で有意に減少したことは、ビフィドバクテリウム・アニマリス亜種ラクティスが、動脈硬化予備軍の動脈硬化症への進行を抑制する効果を有していることを示している。プロバイオティクス投与による動脈硬化予備軍に対する抗炎症効果はこれまでに報告がない。 A significant decrease in TNF-α in the test meal group indicates that Bifidobacterium animalis subsp. Lactis has the effect of inhibiting the progression of arteriosclerosis reserves to arteriosclerosis. Yes. The anti-inflammatory effect on the arteriosclerotic reserve by probiotic administration has not been reported so far.
ＢＭＩ値の変動は、摂取１２週後において試験食群０．２６±０．１７減少したのに対し、プラセボ群は０．２２±０．０９の増加と有意差が認められた（ｐ＜０．０５）。血中γ−ＧＴＰの変動は、摂取１２週後において試験食群０．８２±２．３１Ｕ／Ｌ減少したのに対し、プラセボ群は１４．９±７．１５Ｕ／Ｌの増加と有意差が認められた（ｐ＜０．０５）。 The change in BMI value decreased 0.26 ± 0.17 in the test meal group 12 weeks after ingestion, whereas the placebo group showed a significant difference with an increase of 0.22 ± 0.09 (p <0). .05). The fluctuation in blood γ-GTP decreased 0.82 ± 2.31 U / L in the test food group 12 weeks after ingestion, whereas the placebo group showed a significant difference from an increase of 14.9 ± 7.15 U / L. Found (p <0.05).
ビフィドバクテリウム・アニマリス亜種ラクティスの摂取によりＢＭＩ値、血中γ−ＧＴＰが減少したことは、間接的に動脈硬化への発展を抑制することにつながる。
・糞便菌叢の変化
動脈硬化症と腸内菌叢に関する研究は少ないが、最近、食事由来フォスファチジルコリン、コリンやカルニチンが腸内常在菌によりトリメチルアミン（TMA）に変換され吸収後、肝臓でトリメチルアミン−Ｎ−オキシド（TMAO）に変換され、このTMAOがアテローム性動脈硬化を促進することが注目されている（Wang, Z., et al., Nature, 2011, 472: 57-63；Koeth, R. A., et al., Nat. Med., 2013, 19:576-585）。このコリンからTMAへの変換は、コリンが炭素−窒素結合（C-N bond）切断によりTMAとアセトアルデヒドが生じる反応に起因し、それに関与しているcholine utilization（cut）遺伝子クラスターがデスルフォビブリオ・デスルフリカンス（Desulfovibrio desulfuricans）ゲノムから発見されている（Craciun, S. and Balskus, E. P., Proc. Natl. Acad. Sci. U.S.A, 2012, 109:21307-21312）。この遺伝子と相同性が高い遺伝子を有する可能性があるとリストアップされた菌（Craciun, S. and Balskus, E. P., 同上）と本実験で変動した腸内細菌を比較した。その結果、試験１２週目に試験食群がプラセボ群と比較し有意に低くかったクロストリジア（Clostridia）綱およびクロストリジアレス（Clostridiales）目（共に試験食群６６．６％、プラセボ群７７．５％、ｐ＜０．０１）に属する細菌が２４菌種、ラクノスピラセアエ（Lachnospiraceae）科（試験食群５３．７％、プラセボ群６０．９％、ｐ＜０．０５）に属する細菌が４菌種含まれていた。また、試験期間中にプラセボ群でのみ有意に増加した（試験前０．０７％、１２週目０．４７％）クロストリジウム（Clostridium）（ハンガテラ（Hungatella）含む）属（クロストリジアセアエ（Clostridiaceae）科）に属する６菌種、有意差は認められなかったが試験期間中に試験食群で減少傾向を示したクレブシエラ（Klebsiella）属の細菌が６菌種含まれていた。
Decrease in BMI and blood γ-GTP by ingestion of Bifidobacterium animalis subspecies lactis indirectly suppresses the development of arteriosclerosis.
・Changes in fecal flora There are few studies on arteriosclerosis and intestinal flora, but recently, dietary phosphatidylcholine, choline and carnitine are converted to trimethylamine (TMA) by intestinal resident bacteria and then absorbed into the liver. Is converted to trimethylamine-N-oxide (TMAO), which is noted to promote atherosclerosis (Wang, Z., et al., Nature, 2011, 472: 57-63; Koeth , RA, et al., Nat. Med., 2013, 19: 576-585). This choline-to-TMA conversion is caused by a reaction in which choline generates TMA and acetaldehyde by carbon-nitrogen bond (CN bond) cleavage, and the choline utilization (cut) gene cluster involved in the reaction is desulfobibrio desulfuricans ( Desulfovibrio desulfuricans) (Craciun, S. and Balskus, EP, Proc. Natl. Acad. Sci. USA, 2012, 109: 21307-21312). We compared the bacteria listed in this experiment (Craciun, S. and Balskus, EP, supra) with the possibility of having a gene highly homologous to this gene, and the intestinal bacteria changed in this experiment. As a result, the Clostridia and Clostridiales eyes (both in the test food group 66.6% and the placebo group 77.5) were significantly lower in the test food group than in the placebo group at the 12th week of the test. %, P <0.01) bacteria belonging to 24 species, Lachnospiraceae family (test food group 53.7%, placebo group 60.9%, p <0.05) Contained 4 bacterial species. In addition, it increased significantly only in the placebo group during the test period (0.07% before the test, 0.47% at the 12th week), the genus Clostridium (including Hungatella) (Clostridiaceae) 6 strains belonging to the family), 6 species of bacteria belonging to the genus Klebsiella, which showed no significant difference but showed a decreasing tendency in the test food group during the test period, were included.
これらの結果は、平均約2000 kcal／日以上の生活をしている被験者は１２週間の試験期間中においてもプラセボ群はTMAを産生し易い菌叢に変化しているのに対し、同様の食事をしていてもプロバイオティクス微生物摂取がコリンからTMAへの変換に関与する腸内細菌の増加を抑えたことを示している。 These results show that subjects who live on average about 2000 kcal / day or more changed to a flora that tends to produce TMA in the placebo group even during the 12-week study period, while the same diet. This shows that the intake of probiotic microorganisms suppressed the increase of intestinal bacteria involved in the conversion of choline to TMA.
同様に、カルニチンからTMAが合成される腸内細菌の研究も始まっており、Rieske-type oxygenase/reductase（CntAB）およびその関連遺伝子を有する菌グループの関連が示唆されている（Zhu, Y., Proc. Natl. Acad. Sci. U.S.A, 2014, 111:4268-4273）。これらの遺伝子を有していると推測される菌として本実験の被験者からも検出されたガンマプロバクテリア（Gammaproteobacteria）綱に属するクレブシエラ（Klebsiella）属、エシェリキア（Escherichia）属、シトロバクター（Citrobacter）属等が挙げられており、この内クレブシエラ属に関しては、プラセボ群では有意な変動が無かったが試験食群で減少傾向（p＝0.087）が示された。この結果は、プロバイオティクス摂取はカルニチンからのTMA合成に関与する腸内細菌の増加を抑えたことを示している。 Similarly, research on enterobacteria in which TMA is synthesized from carnitine has also begun, suggesting an association between Rieske-type oxygenase / reductase (CntAB) and its related genes (Zhu, Y., Proc. Natl. Acad. Sci. USA, 2014, 111: 4268-4273). The genus Klebsiella, the genus Escherichia, and the genus Citrobacter belonging to the Gammaproteobacteria class, which were also detected by the subjects of this experiment as bacteria presumed to have these genes Among these, the Klebsiella genus showed no significant change in the placebo group, but showed a decreasing trend (p = 0.087) in the test food group. This result indicates that the intake of probiotics suppressed the increase of enteric bacteria involved in TMA synthesis from carnitine.
したがって、プロバイオティクス微生物の摂取は、アテローム性動脈硬化症と関連がある腸内菌叢の改善に有用である。 Thus, ingestion of probiotic microorganisms is useful for improving the intestinal flora associated with atherosclerosis.
実施例２
実験方法
・被験者
４０歳以上７５歳以下でＢＭＩが２５．０付近である成人（性別は問わない）を公募した。募集期間中に集まった４６名の被験者候補を対象に、試験開始２週間前に前観察期を設け、EndoPAT値および血液生化学的検査を行い、医師が動脈硬化予備軍と判定した４０名（男性２１名、女性１９名、平均年齢４９．３歳）を被験者として登録した。登録された被験者はプロトコルに従い、割付責任者が試験前に無作為に試験食群２０名、プラセボ群２０名に群分けした。
・試験食
試験食は、ビフィドバクテリウム・アニマリス亜種ラクティス ＬＫＭ５１２株（寄託番号ＦＥＲＭ Ｐ−２１９９８）生菌粉末（約６×１０９ｃｆｕ／包×１包）とアルギニン錠剤（１００ｍｇ／錠×３錠）を１セットとし、１日に２回、朝・夕食後に８週間摂取した。プラセボ用粉末は賦形剤のみで作製し、プラセボ用錠剤はアルギニンの代わりにデンプンを用いた。
・試験スケジュール
摂取期間８週間の無作為化二重盲検並行群間比較試験を実施した。摂取直前および摂取８週後に、EndoPATにて血管内皮機能の測定を行った。なお、試験期間２週間前から終了時まで、乳酸菌およびビフィズス菌を含むヨーグルト、乳酸菌飲料、およびサプリメント、また腸内菌叢解析結果に著しい影響を及ぼす納豆の摂取は禁止したが、その他の食事は制限しなかった。
・EndoPATを用いた血管内皮機能の測定
血管内皮細胞は血管の収縮・拡張を調節するほか、血小板の粘着、凝集を抑制し血管の保護をしている。この作用を血管内皮機能と表現し、本試験では、この内皮細胞が健康な状態に保たれているかをEndoPATで検査した。
Example 2
Experimental method - Subjects 40 to 75 years old and adults (regardless of gender) with a BMI of around 25.0 were recruited. Forty-six candidate candidates who gathered during the recruitment period had a pre-observation period 2 weeks before the start of the study, performed EndoPAT values and blood biochemical examinations, and 40 ( 21 males, 19 females, and an average age of 49.3 years) were registered as subjects. The enrolled subjects were randomly divided into 20 test meal groups and 20 placebo groups by the assignment manager before the test according to the protocol.
Test meal The test meal consists of Bifidobacterium animalis subspecies Lactis LKM512 strain (deposit number FERM P-21998) live bacterial powder (about 6 × 10 9 cfu / pack × 1) and arginine tablet (100 mg / tablet × 3 tablets) were taken as a set, and were taken twice a day for 8 weeks after morning and dinner. The placebo powder was made only with excipients, and the placebo tablets used starch instead of arginine.
• A randomized, double-blind, parallel group comparison study with a study schedule intake period of 8 weeks was conducted. The vascular endothelial function was measured with EndoPAT immediately before ingestion and 8 weeks after ingestion. In addition, from 2 weeks before the test period to the end, yogurt containing lactic acid bacteria and bifidobacteria, lactic acid bacteria drinks and supplements, and natto, which significantly affects the results of intestinal flora analysis, were prohibited, but other meals I did not limit.
・Measurement of vascular endothelial function using EndoPAT In addition to regulating the contraction and expansion of blood vessels, vascular endothelial cells protect blood vessels by inhibiting platelet adhesion and aggregation. This action is expressed as vascular endothelial function. In this study, EndoPAT was used to test whether the endothelial cells were kept healthy.
EndoPAT測定は、摂取直前および8週目に実施した。被験者は、試験前日は午後9時に食事を終え、試験当日は朝食抜きの状態で測定に臨んだ。被験者は横になり、両手は体側に伸ばした状態で、両手の指にプローブを装着した。５分間の安静ののち、片腕を５分間駆血し、その後、駆血を外し、５分間動脈の拡張機能を測定した。左右の指尖脈は同時計測により駆血解放前と解放後の動脈血液量を求め、その比を動脈の血管内皮機能（RHI：反応性充血指数）として評価した。測定にはイタマー・メディカル・ジャパン株式会社製EndoPAT（登録商標）2000（医療機器承認番号22500BZI00008000）を用いた。 EndoPAT measurement was performed immediately before ingestion and at 8 weeks. The subjects finished the meal at 9:00 pm the day before the test and went to the measurement without breakfast on the day of the test. The subject lay down and both hands extended to the side of the body, and the probe was attached to the fingers of both hands. After resting for 5 minutes, one arm was driven for 5 minutes, and then the blood was removed to measure the diastolic function of the artery for 5 minutes. For the left and right finger veins, the arterial blood volume before and after the release of blood was determined by simultaneous measurement, and the ratio was evaluated as the arterial vascular endothelial function (RHI: reactive hyperemia index). For the measurement, EndoPAT (registered trademark) 2000 (medical device approval number 22500BZI00008000) manufactured by Itamar Medical Japan Co., Ltd. was used.
結果
試験食群の血管内皮機能（EndoPAT測定値）は、試験前１．６４４±０．３９３ ＲＨＩ、試験後１．９７６±０．５２８ ＲＨＩと有意な上昇（ｐ＝０．０１、１標本Wilcoxon検定）を示したのに対し、プラセボ群は、試験前１．５１７±０．３８９ ＲＨＩ、試験後１．６８３±０．０．３４０ ＲＨＩとなり有意差は認められなかった。すなわち、ビフィドバクテリウム・アニマリス亜種ラクティスとアルギニンの併用摂取により、血管内皮機能が向上することが確認された。
Results The vascular endothelial function (measured by EndoPAT) in the test meal group was significantly increased (p = 0.01, 1 specimen Wilcoxon) to 1.644 ± 0.393 RHI before the test and 1.976 ± 0.528 RHI after the test. On the other hand, the placebo group had 1.517 ± 0.389 RHI before the test and 1.683 ± 0.0.340 RHI after the test, and no significant difference was observed. That is, it was confirmed that the combined use of Bifidobacterium animalis subspecies lactis and arginine improves vascular endothelial function.
実施例３
実験方法
・被験者
３０歳以上６５歳以下でＢＭＩが３０未満である成人（性別は問わない）を公募した。募集期間中に集まった４４名の被験者（男性２１名、女性２３名、平均年齢４４．２歳）を対象に実施した。登録された被験者はプロトコルに従い、割付責任者が試験前に無作為に試験食群２２名、プラセボ群２２名に群分けした。
・試験食
試験食は、通常の乳酸菌で発酵させたヨーグルトにビフィドバクテリウム・アニマリス亜種ラクティス ＬＫＭ５１２株（寄託番号ＦＥＲＭ Ｐ−２１９９８）とアルギニンを添加したヨーグルト１００ｇ（ＬＫＭ５１２数：約１×１０１0ｃｆｕ／カップ、アルギニン：６００ ｍｇ／カップ）（以後、ＬＫＭ５１２＆Ａｒｇヨーグルト）とし、毎日昼食後に1カップを１２週間摂取した。プラセボはＬＫＭ５１２とアルギニンを除いた通常ヨーグルトを用いた。
・試験スケジュール
摂取期間１２週間の無作為化二重盲検並行群間比較試験を実施した。その結果、試験食群は男性１０名、女性１２名、平均年齢４４．２歳、プラセボ群は男性１１名、女性１１名、平均年齢４４．２歳となった。摂取直前および摂取１２週後に、EndoPATにて血管内皮機能の測定を行った。なお、試験期間２週間前から終了時まで、乳酸菌およびビフィズス菌を含むヨーグルト、乳酸菌飲料、およびサプリメント、また腸内菌叢解析結果に著しい影響を及ぼす納豆の摂取は禁止したが、その他の食事は制限しなかった。
・EndoPATを用いた血管内皮機能の測定
実施例２と同様にEndoPATにより血管内皮機能を測定した。EndoPAT測定は、摂取直前および１２週目に実施した。
・糞便中ビフィドバクテリウム・アニマリス亜種ラクティス菌数の定量
試験最終週に回収した糞便より、細菌由来ＤＮＡをMatsukiら（Matsuki, T., et al., Appl. Environ. Microbiol, 2004, 70:7220-7228）の方法の一部を改変して行った。改変点は、菌体破砕をMicro Smash MS-100 (トミー精工）を用い、菌体抽出ＤＮＡの回収をエタ沈メイト（Takara）で実施し、冷７０％エタノール（1 ml）を用いて２回の遠心洗浄（15000×g、５分間）後、風乾した点である。ビフィドバクテリウム・アニマリス亜種ラクティスの定量は、Matsumotoら（Matsumoto, M., et al., Microbiology and Immunology, 2009, 53:421-432）の本菌種特異的プライマーを用いたStepOne Realtime PCR systemで定量した。
Example 3
Experimental Method・Subjects were recruited for adults (any gender) with a BMI of 30 to 65 and a BMI of less than 30. The test was conducted on 44 subjects (21 men, 23 women, average age 44.2 years) who gathered during the recruitment period. The enrolled subjects were randomly divided into 22 test food groups and 22 placebo groups by the assignment manager before the test according to the protocol.
Test food The test food was yoghurt obtained by adding Bifidobacterium animalis subspecies lactis LKM512 strain (deposit number FERM P-21998) and arginine to yoghurt fermented with ordinary lactic acid bacteria (number of LKM512: about 1 × 10 ×) 10 cfu / cup, arginine: 600 mg / cup) (hereinafter LKM512 & Arg yogurt), and 1 cup was ingested after lunch every day for 12 weeks. As the placebo, normal yogurt excluding LKM512 and arginine was used.
• A randomized, double-blind, parallel group comparison study with a study schedule intake period of 12 weeks was conducted. As a result, the test meal group had 10 males and 12 females with an average age of 44.2 years, and the placebo group had 11 males and 11 females with an average age of 44.2 years. Immediately before ingestion and 12 weeks after ingestion, vascular endothelial function was measured with EndoPAT. In addition, from 2 weeks before the test period to the end, yogurt containing lactic acid bacteria and bifidobacteria, lactic acid bacteria drinks and supplements, and natto, which significantly affects the results of intestinal flora analysis, were prohibited, but other meals I did not limit.
Measurement of vascular endothelial function using EndoPAT Similar to Example 2, vascular endothelial function was measured by EndoPAT. EndoPAT measurement was performed immediately before ingestion and at 12 weeks.
・Quantitative test of the number of Bifidobacterium animaris subspecies lactis in feces From the feces collected in the last week, bacterial DNA was collected from Matsuki et al. (Matsuki, T., et al., Appl. Environ. Microbiol, 2004, 70 : 7220-7228) was partially modified. Modifications were made using Micro Smash MS-100 (Tomy Seiko) for cell disruption, recovery of cell-extracted DNA using Ekara Precipitate (Takara), and twice using cold 70% ethanol (1 ml). This is the point of air-drying after centrifugal washing (15000 × g, 5 minutes). Quantification of Bifidobacterium animalis subspecies lactis was performed using StepOne Realtime PCR using this strain-specific primer of Matsumoto et al. (Matsumoto, M., et al., Microbiology and Immunology, 2009, 53: 421-432). Quantified with system.
結果
摂取前の中性脂肪値が低過ぎると医師が判断した３名と試験最終週の糞便中のビフィドバクテリウム・アニマリス亜種ラクティス菌数でプロトコル違反と判断された６名の合計９名（試験食群４名、プラセボ群５名）を除去して層別解析を行った。その結果、EndoPAT測定値の摂取前後の差（摂取後から摂取前の値を引いた値）において、試験食群（０．３１±０．５１）がプラセボ群（-０．１７±０．７９）より有意に（ｐ ＜ ０．０５）高い値を示した。すなわち、ビフィドバクテリウム・アニマリス亜種ラクティスとアルギニンの併用摂取により、血管内皮機能が向上することが確認された。
Results A total of 9 patients, 3 of whom doctors determined that the triglyceride level before intake was too low and 6 that were determined to violate the protocol due to the number of Bifidobacterium animalis subspecies lactis in the stool in the last week of the study (4 test meal groups, 5 placebo groups) were removed and stratified analysis was performed. As a result, in the difference between before and after ingestion of EndoPAT measurement values (value obtained by subtracting the value before ingestion from that after ingestion), the test meal group (0.31 ± 0.51) was placed in the placebo group (−0.17 ± 0.79). ) Significantly higher (p <0.05). That is, it was confirmed that the combined use of Bifidobacterium animalis subspecies lactis and arginine improves vascular endothelial function.
血管内皮機能が低下した状態が続くと、将来的に、動脈硬化が進行し心筋梗塞や脳卒中などを発症するリスクが高くなることから、実施例２および３の結果は、プロバイオティクス微生物とアルギニンの併用摂取は動脈硬化の進行、さらに心筋梗塞や脳卒中の予防に繋がることを示すものである。 If the state in which the vascular endothelial function continues to decline continues, the risk of developing arteriosclerosis and developing myocardial infarction, stroke, etc. in the future increases, so the results of Examples 2 and 3 Concomitant use of is shown to lead to progression of arteriosclerosis and further prevention of myocardial infarction and stroke.
本発明のプロバイオティクス組成物は、血管内皮機能を維持および／または改善する、または炎症を低減および／または改善するものであり、ひいては動脈硬化症の予防において有用である。 The probiotic composition of the present invention maintains and / or improves vascular endothelial function or reduces and / or improves inflammation, and thus is useful in preventing arteriosclerosis.
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