JPWO2011025019A1 - Lactic acid bacteria having thioredoxin-inducing activity, and foods and beverages and pharmaceuticals for preventing and / or improving biological injury mediated by thioredoxin - Google Patents
Lactic acid bacteria having thioredoxin-inducing activity, and foods and beverages and pharmaceuticals for preventing and / or improving biological injury mediated by thioredoxin Download PDFInfo
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Abstract
飲食品や医薬品に適した、チオレドキシンを誘導し、酸化ストレスに起因する様々な生体傷害を予防および／または改善することが可能な乳酸菌を提供する。また、当該乳酸菌を用いた生体傷害を予防および／または改善する飲食品や医薬品を提供する。チオレドキシン誘導活性を有するラクトバシルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）属、特にラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株である乳酸菌である。Provided is a lactic acid bacterium that can induce thioredoxin and can prevent and / or improve various biological injuries caused by oxidative stress, which are suitable for foods and drinks and pharmaceuticals. Also provided are foods and beverages and pharmaceuticals that prevent and / or improve biological injuries using the lactic acid bacteria. Lactobacillus genus Lactobacillus having thioredoxin-inducing activity, particularly Lactobacillus delbrueckii subsp. Bulgaricus OLLL1224 strain.
Description
本発明は、チオレドキシン誘導活性を有する乳酸菌およびその利用方法に関し、また当該乳酸菌を含む、チオレドキシンを介する生体傷害の予防および／または改善用の飲食品および医薬品に関する。 The present invention relates to a lactic acid bacterium having a thioredoxin-inducing activity and a method for using the lactic acid bacterium, and to a food and drink and a pharmaceutical for preventing and / or improving a biological injury involving thioredoxin, including the lactic acid bacterium.
最近、生活習慣病や老化など、生体傷害の原因の一つとして、酸化ストレスについての重要性が注目を集めており、様々な検討がなされている。特に、酸化ストレスによって生じる生体傷害を防ぐことが生活習慣病の予防につながる可能性が見いだされ始めた。そのため、酸化ストレスに対して有効とされる様々な抗酸化物質の研究開発が進められており、それを含有する抗酸化食品、すなわち酸化ストレス予防食品により、老化やがんに至る種々の疾病を予防することができる可能性のもとに研究が進んでいる。例えば、ゴマリグナン類、ウコンに特異的なクルクミンやアントシアニン類のような化合物に着目して酸化ストレスを予防するための研究が進められているが、未だ不十分であった。 Recently, the importance of oxidative stress has attracted attention as one of the causes of biological injuries such as lifestyle-related diseases and aging, and various studies have been made. In particular, it has begun to be found that the prevention of living-related diseases can be prevented by preventing injuries caused by oxidative stress. For this reason, research and development of various antioxidants that are effective against oxidative stress are being promoted, and anti-oxidant foods containing them, that is, oxidative stress-preventing foods, prevent various diseases that lead to aging and cancer. Research is progressing with the possibility of prevention. For example, research for preventing oxidative stress has been carried out by focusing on compounds such as sesame lignans, curcumin specific to turmeric, and anthocyanins, but it is still insufficient.
その中で、近年注目されている生体内物質として、チオレドキシンがある。チオレドキシンは、活性部位に二つのシスチン残基を有し、酵素のジスルフィド結合の酸化還元を通して、その活性を調節することが可能なタンパク質として知られている。特にその酸化還元反応により種々の酸化ストレスを除去し、それによる生体傷害を防ぐことが可能であることが判明している（例えば非特許文献１、２および３）。 Among them, thioredoxin is an in vivo substance that has been attracting attention in recent years. Thioredoxin is known as a protein having two cystine residues in the active site and capable of regulating its activity through redox of an enzyme disulfide bond. In particular, it has been found that various oxidative stresses can be removed by the oxidation-reduction reaction, thereby preventing biological damage (for example, Non-Patent Documents 1, 2, and 3).
したがって、生体内においてチオレドキシンを発現させ、有効に利用することができれば、酸化ストレスを防御し、また予防することが可能となり、よって種々の生体傷害をも防ぐことが可能となると考えられている。 Therefore, it is considered that if thioredoxin can be expressed and used effectively in a living body, it is possible to prevent and prevent oxidative stress and thus prevent various injuries.
これまでチオレドキシンの発現を誘導する物質については、あまり検討が進んでおらず、いくつかの化合物が知られているのみである。そのような物質として、例えば、α，β−不飽和結合を有するアルデヒド化合物を含有するチオレドキシン誘導剤（特許文献１）、アブラナ科植物由来のスルフォラファン（特許文献２）、ヨモギ、ヨモギ抽出物、青ジソおよび青ジソ抽出物（特許文献３）、イソプレノイド系化合物、ゲラニルゲラニルアセトン（ＧＧＡ）（特許文献４）等が挙げられる。 So far, substances that induce the expression of thioredoxin have not been studied much, and only a few compounds are known. As such substances, for example, a thioredoxin inducer containing an aldehyde compound having an α, β-unsaturated bond (Patent Document 1), a sulforaphane derived from a cruciferous plant (Patent Document 2), mugwort, mugwort extract, blue Examples include diso and blue diso extracts (Patent Document 3), isoprenoid compounds, geranylgeranylacetone (GGA) (Patent Document 4), and the like.
このように種々の化合物がチオレドキシン誘導物質として探索されているが、チオレドキシン誘導活性を有する微生物はこれまでに見出されていなかった。本発明は、チオレドキシン誘導活性を有する微生物の提供を課題とする。 Thus, various compounds have been searched as thioredoxin inducers, but no microorganism having thioredoxin induction activity has been found so far. An object of the present invention is to provide a microorganism having thioredoxin-inducing activity.
上記従来の問題点に鑑み、本発明者等は鋭意研究を進めたところ、チオレドキシン誘導活性を有する乳酸菌を見出し、本発明を完成するに至った。 In view of the above-mentioned conventional problems, the present inventors have conducted extensive research and found a lactic acid bacterium having a thioredoxin-inducing activity and completed the present invention.
本発明は、飲食品や医薬品に適した、チオレドキシンを誘導し、酸化ストレスに起因する様々な生体傷害を予防および／または改善することが可能な乳酸菌に関する。またさらに、本発明は、上記乳酸菌を用いた生体傷害を予防および／または改善する飲食品や医薬品に関する。 The present invention relates to a lactic acid bacterium suitable for foods and beverages and pharmaceuticals, capable of inducing thioredoxin and preventing and / or improving various biological injuries caused by oxidative stress. Furthermore, this invention relates to the food-drinks and pharmaceutical which prevent and / or improve the biological injury using the said lactic acid bacteria.
即ち、本発明の一態様は、チオレドキシン誘導活性を有するラクトバシルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ属の乳酸菌である。 That is, one embodiment of the present invention is a lactic acid bacterium belonging to the genus Lactobacillus having thioredoxin-inducing activity.
上記乳酸菌としては好ましくはラクトバシルス・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｌｇａｒｉｃｕｓ）属菌であり、さらに好ましくはラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株である。 The lactic acid bacterium is preferably a bacterium belonging to the genus Lactobacillus bulgaricus, and more preferably, Lactobacillus delbrueckii subsp. Bulgaricus 24 strain OL12.
また、本発明の別の態様は、上記本発明の乳酸菌、またはその乳酸菌含有物および／もしくは乳酸菌処理物を含む、チオレドキシンを介する生体傷害の予防および／または改善用の飲食品である。 Another aspect of the present invention is a food or drink for the prevention and / or improvement of thioredoxin-mediated biological injury comprising the lactic acid bacterium of the present invention, or a lactic acid bacterium-containing product and / or a lactic acid bacterium-treated product.
また、本発明のさらに別の態様は、上記本発明の乳酸菌、またはその乳酸菌含有物および／もしくは乳酸菌処理物を含む、チオレドキシンを介する生体傷害の予防および／または改善用の飲食品組成物である。 Still another embodiment of the present invention is a food or beverage composition for preventing and / or improving biological injury through thioredoxin, comprising the lactic acid bacterium of the present invention, or a lactic acid bacterium-containing product and / or a processed product of lactic acid bacterium. .
また、本発明のさらに別の態様は、上記本発明の乳酸菌、またはその乳酸菌含有物および／もしくは乳酸菌処理物を含む、チオレドキシンを介する生体傷害の予防および／または改善用の医薬品である。 Still another embodiment of the present invention is a pharmaceutical product for preventing and / or improving biological injury through thioredoxin, comprising the lactic acid bacterium of the present invention, or a lactic acid bacterium-containing product and / or a processed product of lactic acid bacterium.
本明細書は本願の優先権の主張の基礎となる日本国特願2009-199157の内容を包含する。 This specification includes the contents of Japanese Patent Application No. 2009-199157, which is the basis for the priority claim of the present application.
本発明によるチオレドキシン誘導活性を有する乳酸菌の投与により、生体内でチオレドキシンを誘導することができる。本発明の一態様では、好適には、本発明によるチオレドキシン誘導活性を有する乳酸菌を経口摂取することにより、酸化ストレスに起因する様々な生体傷害を予防および／または改善することができる。また本発明のさらなる態様として、本発明による乳酸菌は、菌含有物、菌処理物などの種々の形態にて提供することが可能なため、これを含有することで簡便に酸化ストレスに起因する様々な生体傷害を予防および／または改善する飲食品、飲食品組成物および医薬品として提供することができる。 By administering a lactic acid bacterium having thioredoxin-inducing activity according to the present invention, thioredoxin can be induced in vivo. In one aspect of the present invention, it is possible to prevent and / or improve various biological injuries caused by oxidative stress by orally ingesting lactic acid bacteria having thioredoxin-inducing activity according to the present invention. Further, as a further aspect of the present invention, the lactic acid bacteria according to the present invention can be provided in various forms such as bacteria-containing substances and bacteria-treated substances. It can be provided as a food / beverage product, a food / beverage product composition, and a pharmaceutical that prevent and / or improve various biological injuries.
以下、本発明を詳細に説明するが、本発明は以下に述べる個々の形態には限定されない。 Hereinafter, the present invention will be described in detail, but the present invention is not limited to the individual forms described below.
本発明のチオレドキシン誘導活性を有する乳酸菌としては、ラクトバシルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）属に属する菌類が例示でき、例えばラクトバシルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｇａｓｓｅｒｉ）株、ラクトバシルス・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｌｇａｒｉｃｕｓ）株等を具体的に挙げることができる。しかし、本発明はこれらの種に限定されるものではなく、またこれらの菌株については、単独あるいは２以上を組み合わせて使用することができる。 Examples of lactic acid bacteria having thioredoxin-inducing activity of the present invention include fungi belonging to the genus Lactobacillus, such as Lactobacillus gasseri strain, Lactobacillus bulgaricus strain, and the like. Can do. However, the present invention is not limited to these species, and these strains can be used alone or in combination of two or more.
上記のチオレドキシン誘導活性を有する乳酸菌の中でも特に好ましくはラクトバシルス・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｌｇａｒｉｃｕｓ）株である。更に好ましくはラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株である。 Among the lactic acid bacteria having the thioredoxin-inducing activity described above, Lactobacillus bulgaricus strain is particularly preferable. More preferred is Lactobacillus delbrueckii subsp. Bulgaricus OLL1224 strain.
本発明で用いるラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株は下記の条件で寄託申請され、ブダペスト条約に基づいて寄託されている。 The Lactobacillus delbrueckii subsp. Bulgaricus OLL1224 strain used in the present invention has been deposited under the following conditions and deposited under the Budapest Treaty.
（１） 受領機関名（国際寄託機関の名称）：独立行政法人製品評価技術基盤機構 特許微生物寄託センター
（２） 連絡先（国際寄託機関の住所）：〒２９２−０８１８ 千葉県木更津市かずさ鎌足２−５−８
（３） 受託番号：ＮＩＴＥ ＢＰ−７７８
（４） 識別の表示：Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ ＯＬＬ１２２４
（５） 受領日（受託日）： ２００９年 ７月 ２日
本発明のラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株は、以下の菌学的性質を有するものである。(1) Recipient organization name (name of international depositary organization): National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact (address of international depositary organization): Kazusa Kamazuchi, Kisarazu City, Chiba Prefecture 292-0818 2-5-8
(3) Accession number: NITE BP-778
(4) Identification display: Lactobacillus delbrueckii subsp. bulgaricus OLL1224
(5) Date of receipt (date of commission): July 2, 2009 The Lactobacillus delbruecki subspice bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus) OLL1224 strain of the present invention has the following mycological properties. .
ラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株は、植物由来のグラム陽性偏性嫌気性長桿菌である。Ｌａｃｔｏｂａｃｉｌｌｉ ＭＲＳ Ｂｒｏｔｈ（ＢＤ）に本菌を接種し、ＡｎａｅｒｏＰａｃｋ・ケンキ（三菱ガス化学製）使用による嫌気状態にて３７℃１８時間培養すると、円形、白色、小〜中型、Ｒｏｕｇｈ型（一部はＳｍｏｏｔｈ型）、扁平状（一部は円錐状）コロニーが観察される。 Lactobacillus delbrueckii subsp. Bulgaricus OLL1224 strain is a plant-derived Gram-positive obligate anaerobic bacterium. Lactobacilli MRS Broth (BD) is inoculated with this bacterium and cultured at 37 ° C. for 18 hours under anaerobic conditions using AnaeroPack Kenki (manufactured by Mitsubishi Gas Chemical). Type), flat (partially conical) colonies are observed.
なお本発明のチオレドキシン誘導活性を有する乳酸菌は、当該乳酸菌の含有物またはその処理物として調製してもよい。本発明の含有物またはその処理物としては、以下には限定されないが、具体的には、チオレドキシン誘導活性を有する乳酸菌の培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物、破砕物などが挙げられる。 In addition, you may prepare the lactic acid bacteria which have the thioredoxin induction activity of this invention as the content of the said lactic acid bacteria, or its processed material. The content of the present invention or the processed product thereof is not limited to the following, and specifically, a culture, concentrate, pasted product, spray-dried product, freeze-dried product, vacuum-dried product of lactic acid bacteria having thioredoxin-inducing activity. Product, drum dried product, liquid product, diluted product, and crushed product.
本発明のチオレドキシン誘導活性を有する乳酸菌は、チオレドキシン遺伝子を始めとするチオレドキシン関連遺伝子の発現を誘導または増加させることができる。この本発明の乳酸菌は、チオレドキシンの発現を誘導または増加させることにより、生体障害、好ましくは、酸化ストレスに起因する様々な生体傷害を阻止又は軽減することができる。 The lactic acid bacterium having thioredoxin-inducing activity of the present invention can induce or increase the expression of thioredoxin-related genes including thioredoxin gene. This lactic acid bacterium of the present invention can prevent or reduce biological damage, preferably various biological damages caused by oxidative stress, by inducing or increasing the expression of thioredoxin.
本発明は、本発明のチオレドキシン誘導活性を有する乳酸菌、その乳酸菌の含有物もしくは処理物、またはそれらを含む医薬組成物（医薬品）を動物（ヒトを含んでも含まなくてもよい）に投与することにより、生体傷害、好ましくは、酸化ストレスに起因する様々な生体傷害を、予防および／または改善することができる。本発明は、このような生体傷害の予防および／または改善方法にも関する。本発明はまた、生体傷害の予防および／または改善に使用するための、本発明のチオレドキシン誘導活性を有する乳酸菌、その乳酸菌の含有物もしくは処理物、またはそれらを含む医薬組成物（医薬品）にも関する。 The present invention comprises administering a lactic acid bacterium having thioredoxin-inducing activity of the present invention, a content or processed product of the lactic acid bacterium, or a pharmaceutical composition (medicine) containing them to an animal (which may or may not include humans). Thus, it is possible to prevent and / or improve biological injuries, preferably various biological injuries caused by oxidative stress. The present invention also relates to a method for preventing and / or improving such a biological injury. The present invention also relates to a lactic acid bacterium having thioredoxin-inducing activity of the present invention, a content or treated product of the lactic acid bacterium, or a pharmaceutical composition (pharmaceutical product) containing them for use in the prevention and / or improvement of biological injury. Related.
本発明のチオレドキシン誘導活性を有する乳酸菌の摂取量あるいは投与量は、投与経路、ヒトを含む投与対象動物の年齢、体重、症状など、種々の要因を考慮して、適宜設定することができる。本発明はこれに限定されないが、成人一日あたり、当該乳酸菌を好ましくは１×１０５〜１×１０１０ｃｆｕ／ｇ、より好ましくは１×１０６〜１×１０９ｃｆｕ／ｇの用量を摂取あるいは投与する。しかしながら、長期間に亘って予防および／または治療の目的で摂取する場合には、上記範囲よりも少量であってもよいし、また本有効成分は、安全性について問題がないので、上記範囲よりも多量に使用してもさしつかえない。The intake or dose of lactic acid bacteria having thioredoxin-inducing activity of the present invention can be appropriately set in consideration of various factors such as the route of administration, the age, weight, and symptoms of animals to be administered including humans. The present invention is not limited to this, but the daily dose of the lactic acid bacteria is preferably 1 × 10 5 to 1 × 10 10 cfu / g, more preferably 1 × 10 6 to 1 × 10 9 cfu / g. Ingest or administer. However, when ingested for the purpose of prevention and / or treatment over a long period of time, the amount may be smaller than the above range, and since this active ingredient has no safety problem, Can be used in large quantities.
また本発明のチオレドキシン誘導活性を有する乳酸菌は、経口投与が好ましい。本発明のチオレドキシン誘導活性を有する乳酸菌は、その乳酸菌の含有物もしくは処理物、またはそれらを含む医薬組成物（医薬品）の形態で投与（好ましくは経口投与）することもできる。 Moreover, oral administration is preferable for the lactic acid bacteria which have the thioredoxin induction activity of this invention. The lactic acid bacterium having thioredoxin-inducing activity of the present invention can be administered (preferably orally administered) in the form of a lactic acid bacterium-containing product or processed product, or a pharmaceutical composition (pharmaceutical product) containing them.
本発明のチオレドキシン誘導活性を有する乳酸菌、その乳酸菌含有物および処理物は、酸化ストレスに起因する様々な生体傷害を予防および／または改善する医薬品もしくは医薬組成物として、またはその製造に、用いることができる。医薬品の投与形態としては、例えば錠剤、被覆錠剤、カプセル剤、顆粒剤、散剤、溶液、シロップ剤、乳液等による経口投与をあげることができる。これらの各種製剤は、常法に従って本発明のチオレドキシン誘導活性を有する乳酸菌、該乳酸菌含有物および／またはその処理物に、賦形剤、結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤、溶解補助剤、懸濁剤、コーティング剤などの医薬の製剤技術分野において通常使用しうる既知の補助剤を用いて製剤化することができる。 The lactic acid bacterium having thioredoxin-inducing activity of the present invention, the lactic acid bacterium-containing product and the processed product thereof can be used as a pharmaceutical or a pharmaceutical composition for preventing and / or improving various biological injuries caused by oxidative stress, or for its production. it can. Examples of the administration form of pharmaceuticals include oral administration by tablets, coated tablets, capsules, granules, powders, solutions, syrups, emulsions and the like. These various preparations are prepared by adding lactic acid bacteria having thioredoxin-inducing activity according to the present invention, the lactic acid bacteria-containing product and / or processed products thereof to excipients, binders, disintegrating agents, lubricants, coloring agents, flavoring agents, and the like. It can be formulated using known adjuvants that can be generally used in the pharmaceutical formulation technical field, such as agents, solubilizers, suspensions, and coating agents.
本発明のチオレドキシン誘導活性を有する乳酸菌、その乳酸菌含有物および処理物は、酸化ストレスに起因する様々な生体傷害を予防および／または改善する飲食品、またはその製造に用いることができる。 The lactic acid bacterium having thioredoxin-inducing activity, the lactic acid bacterium-containing product, and the treated product of the present invention can be used in foods and drinks that prevent and / or improve various biological injuries caused by oxidative stress, or in the production thereof.
本発明の飲食品は、カテゴリーや種類に制限はなく、機能性食品、特定保健用食品、健康食品、介護用食品でもよく、また、菓子、乳酸菌飲料、チーズやヨーグルト等の乳製品、調味料等であってもよい。飲食品の形状についても制限はなく、固形、液状、流動食状、ゼリー状、タブレット状、顆粒状、カプセル状など、通常流通し得るあらゆる飲食品形状をとることができる。上記飲食品の製造は、当業者の常法によって行うことができるが、乳酸菌の生育を妨げない限り、糖質、タンパク質、脂質、食物繊維、ビタミン類、生体必須微量金属（硫酸マンガン、硫酸亜鉛、塩化マグネシウム、炭酸カリウム等）、香料やその他の配合物を添加することもできる。 The food and drink of the present invention are not limited in category and type, and may be functional foods, foods for specified health use, health foods, foods for nursing care, dairy products such as confectionery, lactic acid bacteria beverages, cheese and yogurt, and seasonings Etc. There is no restriction | limiting also about the shape of food / beverage products, It can take all the food / beverage product forms which can distribute | circulate normally, such as solid, liquid, liquid food form, jelly form, tablet form, granule form, and capsule form. The above food and drink can be produced by ordinary methods of those skilled in the art. However, as long as the growth of lactic acid bacteria is not hindered, carbohydrates, proteins, lipids, dietary fibers, vitamins, biologically essential trace metals (manganese sulfate, zinc sulfate) , Magnesium chloride, potassium carbonate, etc.), perfumes and other blends can also be added.
本発明のチオレドキシン誘導活性を有する乳酸菌、その乳酸菌含有物および処理物は、上記の通り乳製品・発酵乳を含む一般飲食品に加工できる他、ヨーグルトやチーズ等の乳製品・発酵乳の製造用スターターとして利用することも可能である。スターターとする場合は、本発明のラクトバシルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）属乳酸菌の生息・増殖に支障がない限り、また、乳製品製造に支障がない限り、他の微生物を混合してもよい。例えば、ヨーグルト用乳酸菌として主要な菌種であるストレプトコッカス・サーモフィラス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｔｈｅｒｍｏｐｈｉｌｕｓ）株、ラクトバシルス・アシドフィラス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ）株等と混合してもよく、その他、一般にヨーグルト用やチーズ用として用いられる菌種と混合してスターターとすることができる。上記スターターによる乳製品、発酵乳の製造は、常法に従って行うことができる。例えば、加温・混合・均質化・殺菌処理後に冷却した乳または乳製品に、上記スターターを混合し、発酵・冷却することにより、プレーンヨーグルトを製造することができる。 The lactic acid bacteria having thioredoxin-inducing activity of the present invention, lactic acid bacteria-containing products and processed products thereof can be processed into general foods and drinks including dairy products and fermented milk as described above, as well as for producing dairy products and fermented milk such as yogurt and cheese. It can also be used as a starter. In the case of a starter, other microorganisms may be mixed as long as there is no hindrance to the inhabiting and growth of Lactobacillus lactic acid bacteria of the present invention, and there is no hindrance to dairy production. For example, it may be mixed with Streptococcus thermophilus strains, Lactobacillus acidophilus strains, which are the main bacterial species as yogurt lactic acid bacteria, and other bacterial species generally used for yogurt and cheese Can be used as a starter. Production of dairy products and fermented milk using the starter can be performed according to a conventional method. For example, plain yogurt can be produced by mixing the above starter with milk or a dairy product cooled after heating, mixing, homogenizing, and sterilizing, and then fermenting and cooling.
また本発明の飲食品は、具体的には、各種飲食品、飲食品組成物（例えば、牛乳、清涼飲料、発酵乳、ヨーグルト、チーズ、パン、ビスケット、クラッカー、ピッツァクラスト、調製粉乳、流動食、病者用食品、幼児用粉乳等食品、授乳婦用粉乳等食品、栄養食品等）に本発明のチオレドキシン誘導活性を有する乳酸菌、ならびにその乳酸菌含有物および／または乳酸菌処理物を添加し、これを摂取してもよい。また、その性状についても、通常用いられる飲食品の状態、例えば、固体状（粉末、顆粒状その他）、ペースト状、ゲル状、液状ないし懸濁状のいずれでもよい。 In addition, the food and drink of the present invention specifically includes various foods and drinks, food and drink compositions (for example, milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, prepared milk powder, and liquid foods. Lactic acid bacteria having the thioredoxin-inducing activity of the present invention, and lactic acid bacteria-containing products and / or lactic acid bacteria-treated products, which are added to foods for patients, infant milk powders, lactated milk powders, nutritional foods, etc. May be taken. Moreover, about the property, the state of the food / beverage products used normally, for example, any of solid (powder, granule, etc.), paste, gel, liquid or suspension may be sufficient.
その他の成分についても特に限定されないが、本発明のチオレドキシン誘導活性を有する乳酸菌、その乳酸菌含有物および／または乳酸菌処理物を飲食品または飲食品組成物に含ませて用いる場合には、水、タンパク質、糖質、脂質、ビタミン類、ミネラル類、有機酸、有機塩基、果汁、フレーバー類等を主成分として使用することができる。タンパク質としては、例えば全脂粉乳、脱脂粉乳、部分脱脂粉乳、カゼイン、ホエイ粉、ホエイタンパク質、ホエイタンパク質濃縮物、ホエイタンパク質分離物、α−カゼイン、β−カゼイン、κ−カゼイン、β−ラクトグロブリン、α−ラクトアルブミン、ラクトフェリン、大豆タンパク質、鶏卵タンパク質、肉タンパク質等の動植物性タンパク質、これら加水分解物、バター、乳清ミネラル、クリーム、ホエイ、非タンパク態窒素、シアル酸、リン脂質、乳糖等の各種乳由来成分などが挙げられる。糖質としては一般の糖類、加工澱粉（デキストリンのほか、可溶性澱粉、ブリティッシュスターチ、酸化澱粉、澱粉エステル、澱粉エーテル等）、食物繊維などが挙げられる。脂質としては、例えば、ラード、魚油等、これらの分別油、水素添加油、エステル交換油等の動物性油脂；パーム油、サフラワー油、コーン油、ナタネ油、ヤシ油、これらの分別油、水素添加油、エステル交換油等の植物性油脂などが挙げられる。ビタミン類としては、例えば、ビタミンＡ、カロチン類、ビタミンＢ群、ビタミンＣ、ビタミンＤ群、ビタミンＥ、ビタミンＫ群、ビタミンＰ、ビタミンＱ、ナイアシン、ニコチン酸、パントテン酸、ビオチン、イノシトール、コリン、葉酸などが挙げられ、ミネラル類としては、例えば、カルシウム、カリウム、マグネシウム、ナトリウム、銅、鉄、マンガン、亜鉛、セレン、乳清ミネラルなどが挙げられる。有機酸としては、例えば、リンゴ酸、クエン酸、乳酸、酒石酸などが挙げられる。これらの成分は、単独でも２種以上を組み合わせても使用することができ、合成品および／またはこれらを多く含む食品を用いてもよい。 Other components are not particularly limited, but when using the lactic acid bacterium having thioredoxin-inducing activity of the present invention, the lactic acid bacterium-containing product and / or the lactic acid bacterium-treated product in a food or drink or a food or drink composition, water, protein Sugars, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like can be used as main components. Examples of the protein include whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and vegetable proteins, hydrolysates thereof, butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, lactose, etc. And various milk-derived components. Examples of the saccharide include general saccharides, modified starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used alone or in combination of two or more, and a synthetic product and / or a food containing a lot of these may be used.
本発明のチオレドキシン誘導活性を有する乳酸菌、その乳酸菌含有物および／または処理物は、その目的、用途に応じて含有量を任意に定めることができ。本発明はこれに限定されないがその含量としては、１×１０５〜１×１０１０ｃｆｕ／ｇが好ましく、より好ましくは１×１０６〜１×１０９ｃｆｕ／ｇである。The content of the lactic acid bacterium having thioredoxin-inducing activity, the lactic acid bacterium-containing product and / or the treated product of the present invention can be arbitrarily determined according to the purpose and application. As is content that the present invention is not limited to, 1 × 10 5 ~1 × 10 10 cfu / g and is more preferably 1 × 10 6 ~1 × 10 9 cfu / g.
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
以下、本発明を実験例、実施例を挙げて説明するが、本発明はこれにより限定されるものではない。なお、本明細書において％表示は明示しない場合には重量％を示す。 Hereinafter, the present invention will be described with reference to experimental examples and examples, but the present invention is not limited thereto. In addition, in this specification,% display shows weight%, when not showing clearly.
［実施例１］
＜乳酸菌培養液のメタノール抽出物の調製法＞
本発明の乳酸菌（ラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株は表１に示すＴＰＹＧ培地５ｍｌに接種し、３７℃、１８時間、アネロパック（三菱ガス化学）にて嫌気培養した。培養終了時の６６０ｎｍにおける培養液の吸光度は４．５３０であった（ＴＰＹＧ培地のみでは０．０４４）。培養終了後、培養液をステンレスバットに移して、凍結乾燥を行った。凍結乾燥物の重量は０．６１ｇであった。凍結乾燥前の培養液と等量のメタノールを、凍結乾燥物に加え、よく攪拌した後、５℃にて４０時間静置することで抽出した。その後、よく攪拌し、遠心分離（１２０００Ｇ，１０分）して上清を回収し、これを試料とした。また、乳酸菌を接種しない培養液を対照（Ｃｏｎｔｒｏｌ）とした。[Example 1]
<Method for preparing methanol extract of lactic acid bacteria culture solution>
The lactic acid bacterium (Lactobacillus delbrueckii subsp. Bulgaricus) OLL1224 strain of the present invention was inoculated into 5 ml of TPYG medium shown in Table 1 at 37 ° C. for 18 hours by aneropack (Mitsubishi Gas Chemical). The absorbance of the culture solution at 660 nm at the end of the culture was 4.530 (0.044 for the TPYG medium alone) After the culture, the culture solution was transferred to a stainless steel vat and freeze-dried. The weight of the dried product was 0.61 g, and an equal amount of methanol to the culture solution before lyophilization was added to the lyophilized product, stirred well, and then extracted by allowing to stand at 5 ° C. for 40 hours. Then, stir well and collect the supernatant by centrifugation (12000G, 10 minutes) , Which was used as a sample. Also, the medium without inoculation with lactic acid bacteria as a control (Control).
ＴＰＹＧ培地の組成
［実施例２］
＜アレイサンプル＞
マウス線維芽細胞３Ｔ３−Ｌ１細胞を９ｃｍディッシュに播種した。十分な生育を確認した後、デキサメタゾン（１μＭ）、インシュリン（２μＭ）、３−イソブチル−１−メチルキサンテン（０．２５ｍＭ）を含む培地に交換し、分化誘導した。さらに３、５、７日後に、インシュリン（２μＭ）を含む培地に交換し、分化促進をした。分化開始より９日後に培地を棄て、さらに本発明の乳酸菌（ラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカスＯＬＬ１２２４株（ＮＩＴＥ ＢＰ−７７８）、以下「本発明の乳酸菌ＯＬＬ１２２４株」と称する）の培養物１％を含む試料を添加した培地中（５００μＬ）にて３時間培養した（実験群）。尚、乳酸菌培養の工程において、マウス線維芽細胞３Ｔ３−Ｌ１細胞のみの場合を無添加群、本発明の乳酸菌培養物は無く、表１の培地のみを添加した群をＣｏｎｔｒｏｌ群とした。この培地を棄て滅菌したＰＢＳで２回洗浄した後、ＴＮＦ−α（１０ｎｇ／ｍＬ）を含む培地にて２０時間培養した。この上清を回収し、適宜ＰＢＳで希釈したのち、ＥＬＩＳＡにてＩＬ−６の量を測定した。細胞についてはＰＢＳによる２回の洗浄後Ｔｒｉｚｏｌに溶解し、常法によりマイクロアレイサンプル用のＲＮＡを回収した。[Example 2]
<Array sample>
Mouse fibroblast 3T3-L1 cells were seeded in 9 cm dishes. After confirming sufficient growth, the medium was replaced with a medium containing dexamethasone (1 μM), insulin (2 μM), and 3-isobutyl-1-methylxanthene (0.25 mM) to induce differentiation. Further, after 3, 5, and 7 days, the medium was replaced with a medium containing insulin (2 μM) to promote differentiation. Nine days after the start of differentiation, the medium was discarded, and the culture 1 of the lactic acid bacterium of the present invention (Lactobacillus delbruecki subspecies bulgaricus OLL1224 strain (NITE BP-778), hereinafter referred to as “lactic acid bacterium OLL1224 strain of the present invention”). The cells were cultured in a medium (500 μL) to which a sample containing% was added for 3 hours (experimental group). In the lactic acid bacteria culture step, the mouse fibroblast 3T3-L1 cells alone were used as the non-added group, and the group containing only the culture medium shown in Table 1 was used as the control group. The medium was discarded and washed twice with sterilized PBS, and then cultured for 20 hours in a medium containing TNF-α (10 ng / mL). The supernatant was collected and appropriately diluted with PBS, and the amount of IL-6 was measured by ELISA. The cells were washed twice with PBS and then dissolved in Trizol, and RNA for microarray samples was collected by a conventional method.
その後、酸化ストレス、炎症反応、抗酸化酵素、解毒酵素、または肥満に関与する遺伝子２２１種類を搭載した抗体チップ（ジェノパール、三菱レイヨン社製）を作成して用い、上記で回収したＲＮＡを被験試料として、これらの遺伝子発現データを取得した。 Thereafter, an antibody chip (Genopal, manufactured by Mitsubishi Rayon Co., Ltd.) carrying 221 types of genes involved in oxidative stress, inflammatory reaction, antioxidant enzyme, detoxification enzyme, or obesity was prepared and used to test the RNA recovered above. These gene expression data were obtained as samples.
無添加群での遺伝子発現を１とし、Ｃｏｎｔｒｏｌ群、実験群の遺伝子発現の割合を算出した。表２は、アレイサンプルの１例を示す。この結果、チオレドキシン関連遺伝子である、Ｔｘｎｄｃ２、Ｔｘｎｌ１およびＴｘｎｄｃ１７の遺伝子発現において、Ｃｏｎｔｒｏｌ群と比較して本発明の乳酸菌培養物を添加した群での発現が高まった。従って、本発明の乳酸菌にはチオレドキシンを誘導する活性を有していることが明らかとなった。
なお、表２に記載されている遺伝子の名称は、表３の通りである。
また、肥満の脂肪細胞ではＴＮＦ−α、ＩＬ−６といった炎症性サイトカインの産生が亢進することが知られている。そこで、ＥＬＩＳＡにてＩＬ−６の量を測定したところ、サンプル無添加群では、１４３９ｐｇ／ｍＬ、Ｃｏｎｔｒｏｌ群では、１３７３ｐｇ／ｍＬ、本発明の乳酸菌（ＮＩＴＥ ＢＰ−７７８）を含む群では８７６ｐｇ／ｍＬであり、本発明の乳酸菌はＩＬ−６の産生亢進を抑制することも確認された。 Further, it is known that production of inflammatory cytokines such as TNF-α and IL-6 is enhanced in obese adipocytes. Therefore, when the amount of IL-6 was measured by ELISA, 1439 pg / mL in the sample-free group, 1373 pg / mL in the Control group, and 876 pg / mL in the group containing the lactic acid bacteria of the present invention (NITE BP-778). It was also confirmed that the lactic acid bacterium of the present invention suppresses the enhanced production of IL-6.
［実施例３］
上記実施例２にてチオレドキシン誘導活性を有することが確認された乳酸菌、ラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株から、常法により乳酸菌飲食品としてヨーグルトを製造したところ、本発明の乳酸菌を含有する所望のチオレドキシンを介する生体傷害の予防および／または改善用のヨーグルトを得ることができた。[Example 3]
From the lactic acid bacteria confirmed to have thioredoxin-inducing activity in Example 2 above, Lactobacillus delbrueckii subsp. Bulgaricus OLLL1224 strain, yogurt was produced as a lactic acid bacteria food and drink by a conventional method. Thus, it was possible to obtain a yogurt for preventing and / or ameliorating a biological injury via a desired thioredoxin containing the lactic acid bacterium of the present invention.
［実施例４］
上記実施例２にてチオレドキシン誘導活性を有することが確認された乳酸菌、ラクトバシルス・デルブリュッキ・サブスピーシーズ・ブルガリカス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＯＬＬ１２２４株から、常法により乳酸菌濃縮物を作成し、これを用いて常法により適宜の医薬品用添加剤を併用してカプセル剤を製造することにより、本発明の乳酸菌を含有する所望のチオレドキシンを介する生体傷害の予防および／または改善用のカプセル剤を得ることができた。[Example 4]
From the lactic acid bacterium, Lactobacillus delbruecki subsp. Bulgaricus OLLL1224 strain, which was confirmed to have thioredoxin-inducing activity in Example 2 above, a lactic acid bacterium concentrate was prepared by a conventional method. To obtain a capsule for preventing and / or ameliorating a desired thioredoxin-containing biological injury containing the lactic acid bacterium of the present invention by producing a capsule using an appropriate pharmaceutical additive in combination with a conventional method. I was able to.
本発明によるチオレドキシン誘導活性を有する乳酸菌を経口摂取することにより、酸化ストレスに起因する様々な生体傷害を予防および／または改善することができる。また本発明による乳酸菌は種々の形態にて提供することが可能なため、簡便に酸化ストレスに起因する様々な生体傷害を予防および／または改善する飲食品または医薬品として提供することができる。乳酸菌を用いる技術は簡便であり、また人体に対する安全性についても問題がないために、その優れた効果を簡便かつ有効に活用することができ、その利用価値は高い。 By ingesting lactic acid bacteria having thioredoxin-inducing activity according to the present invention orally, various biological injuries caused by oxidative stress can be prevented and / or improved. Moreover, since the lactic acid bacteria according to the present invention can be provided in various forms, they can be provided as foods and drinks or pharmaceuticals that can easily prevent and / or improve various biological injuries caused by oxidative stress. Since the technique using lactic acid bacteria is simple and there is no problem with respect to safety to the human body, the excellent effect can be easily and effectively utilized, and its utility value is high.
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| PCT/JP2010/064756 WO2011025019A1 (en) | 2009-08-31 | 2010-08-31 | Lactic acid bacterium with thioredoxin-inducing activity, and foods, beverages, and medicines for preventing and/or ameliorating biological damage via thioredoxin |
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| JPH07228536A (en) * | 1994-02-16 | 1995-08-29 | Meiji Milk Prod Co Ltd | Immunopotentiator using lactic acid bacterium |
| JPH107577A (en) * | 1996-06-17 | 1998-01-13 | Yakult Honsha Co Ltd | Hypoglycemic agent |
| JP2007269737A (en) * | 2006-03-31 | 2007-10-18 | Morinaga Milk Ind Co Ltd | Interleukin production regulator, pharmaceutical composition and food and drink comprising the interleukin production regulator and method for producing the same |
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| JPH07228536A (en) * | 1994-02-16 | 1995-08-29 | Meiji Milk Prod Co Ltd | Immunopotentiator using lactic acid bacterium |
| JPH107577A (en) * | 1996-06-17 | 1998-01-13 | Yakult Honsha Co Ltd | Hypoglycemic agent |
| JP2007269737A (en) * | 2006-03-31 | 2007-10-18 | Morinaga Milk Ind Co Ltd | Interleukin production regulator, pharmaceutical composition and food and drink comprising the interleukin production regulator and method for producing the same |
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